1111111111111111 IIIIII IIIII 11111 1111111111 1111111111 1111111111 1111111111 1111111111 11111111
`US 20050136122Al
`
`(19) United States
`(12) Patent Application Publication
`Sadozai et al.
`
`(10) Pub. No.: US 2005/0136122 Al
`Jun. 23, 2005
`(43) Pub. Date:
`
`(54) CROSSLINKED HYALURONIC ACID
`COMPOSITIONS FOR TISSUE
`AUGMENTATION
`
`(75)
`
`Inventors: Khalid K. Sadozai, Shrewsbury, MA
`(US); Tamera B. Gooding, Jamaica
`Plain, MA (US); Kyle Bui, North
`Andover, MA (US); Charles H.
`Sherwood, Sudbury, MA (US)
`
`Correspondence Address:
`HAMILTON, BROOK, SMITH & REYNOLDS,
`P.C.
`530 VIRGINIA ROAD
`P.O. BOX 9133
`CONCORD, MA 01742-9133 (US)
`
`(73)
`
`Assignee: Anika Therapeutics, Inc., Woburn, MA
`(US)
`
`(21)
`
`Appl. No.:
`
`10/743,557
`
`(22)
`
`Filed:
`
`Dec. 22, 2003
`
`Publication Classification
`
`(51)
`
`Int. Cl.7
`
`(52) U.S. CI .
`
`....................... A61K 48/00; A61K 39/395;
`A61K 31/24
`......................... 424/493; 514/304; 514/536;
`514/2; 514/44; 424/130.1; 536/53
`
`(57)
`
`ABSTRACT
`
`A hyaluronic acid (HA) composition includes crosslinked,
`water-insoluble, hydrated HA gel particles. The HA includes
`crosslinks represented by the following structural formula:
`
`HA-U-R2-U-HA
`The variables are defined herein.
`
`A method of augmenting tissue in a subject includes insert(cid:173)
`ing a needle into a subject at a location in the subject that is
`in need of tissue augmentation, wherein the needle is
`coupled to a syringe loaded with the HA composition, and
`applying force to the syringe, to deliver the HA composition
`into the subject.
`
`A method of preparing the HA composition, includes form(cid:173)
`ing water-insoluble, dehydrated crosslinked HA particles,
`separating the water-insoluble, dehydrated particles by aver(cid:173)
`age diameter, selecting a subset of particles by average
`diameter, and hydrating the subset of dehydrated particles
`with a physiologically compatible aqueous solution.
`
`Another method of preparing the crosslinked HA composi(cid:173)
`tion includes crosslinking a precursor of the crosslinked HA
`with a biscarbodiimide in the presence of a pH buffer and
`dehydrating the crosslinked HA.
`
`Also included is a method of augmenting tissue in a subject
`that is in need of tissue augmentation.
`
`A method of stabilizing crosslinked HA includes hydrating
`water-insoluble, dehydrated crosslinked HA with a physi(cid:173)
`ologically compatible aqueous solution that includes a local
`anesthetic, wherein the value of storage modulus G' for the
`stabilized composition is at least about 110% of the value of
`G' for a non-stabilized composition,.
`
`Also included is the stabilized HA composition.
`
`Exhibit 1030
`Prollenium v. Allergan
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 1 of 7
`
`US 2005/0136122 Al
`
`UV absorption of cross linked Hyaluronic acid with different
`degree of cross linking
`1.8 - - - - - - - - - - - - - - - - - - - ,
`
`----------------------------------------------------------------
`
`• • • • • • • • • • • • • • • • • • • • • • • • • - • • • • • • • • • • • - • • •T • • • • • • •
`
`Ii W rrax 249 nm
`
`1.6
`1.4
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`-E
`~ -= :&
`
`C
`en
`
`~
`
`0.4
`
`0.2
`
`0
`
`75%
`
`100%
`
`125%
`
`FIG. I
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 2 of 7
`
`US 2005/0136122 Al
`
`UV absorption of cross linked products obtained from
`different pH reactions
`1.4 - . - - - - - - - - - - - - - - - - - - - - .
`
`l!J lN rmx 249 nm
`
`1.2
`
`1
`
`-
`E
`
`C I o.a -f.1 0.6
`
`:E
`~ 0.4
`
`0.2
`
`0
`
`pHS.5
`
`pH6.0
`
`pH6.5
`
`FIG. 2
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 3 of 7
`
`US 2005/0136122 Al
`
`Effect of Particle Size Distribution on Storage Modulus (G')
`..--------.
`Ii Average Initial G'
`
`1400
`
`1200
`
`1000
`
`';'800
`
`a. -c, 600
`
`400
`
`200
`
`0
`
`0
`I
`N
`u,
`
`N
`u,
`.....
`'
`u,
`
`(JI
`
`.....
`.....
`'
`N
`u,
`
`N
`UI
`....,
`I
`ex,
`0
`X-llnked powder particle size range (µm)
`
`ex,
`c:>
`N
`'
`u,
`0
`
`FIG. 3
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 4 of 7
`
`US 2005/0136122 Al
`
`Effect of Particle Size on Extrusion Force
`
`Ill Average Extrusion
`Force
`
`X-linked Powder Particle Size Range (µm)
`
`FIG. 4
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 5 of 7
`
`US 2005/0136122 Al
`
`Degradation Profiles of Tissue Augmentation Products Exposed to Hyaluronldase
`500 ~-----------_.;:;E;.;_;n=-..;;.m;.;..e;;_.;s;..._ ________ ~
`
`450
`
`400
`
`350
`
`0
`
`0
`
`Invention
`
`• G' RESTYLANE®
`
`PERLANE®
`
`- G' -PERLANE®
`
`., G' HYLAFORM®
`
`• G' Invention
`
`10000
`
`20000
`
`30000
`Tima (Sac)
`
`40000
`
`50000
`
`60000
`
`FIG. 5
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 6 of 7
`
`US 2005/0136122 Al
`
`1200
`
`1000
`
`--
`
`800.0
`
`600.0
`
`400.0
`
`200.0
`
`0
`0
`
`q
`-=o
`QI ._
`
`•Gel I
`
`/
`
`+Gel2
`............
`
`t1Qel 4
`/"'
`
`•Gels/
`
`/
`
`~Gel3
`
`10000
`
`20000
`
`30000
`time (s)
`
`40000
`
`50000
`
`60000
`
`FIG. 6
`
`

`

`Patent Application Publication Jun. 23, 2005 Sheet 7 of 7
`
`US 2005/0136122 Al
`
`900.0
`
`800.0 ,-:
`
`700.0
`
`600.0
`
`,.....
`
`500.0
`
`400.0
`
`300.0~
`200.0 , ..
`:
`100.0
`
`0
`0
`
`Ci}
`-=a
`
`Dl --
`
`8 0.3% Lidocaine Buffer
`
`•0.2% Lidocaine Buffer
`
`+ No Lidocaine Buffer
`
`10000
`
`20000
`
`30000
`time (s)
`
`40000
`
`50000
`
`60000
`
`FIG. 7
`
`

`

`US 2005/0136122 Al
`
`Jun.23,2005
`
`1
`
`CROSSLINKED HYALURONIC ACID
`COMPOSITIONS FOR TISSUE AUGMENTATION
`
`BACKGROUND OF THE INVENTION
`
`[0001] Currently, all tissue augmentation fillers approved
`by the United States Food and Drug Administration are
`derived from collagen. Approximately 3-5% of human sub(cid:173)
`jects show serious allergic reactions to bovine collagen, thus
`requiring careful testing before using these fillers in any
`particular subject.
`[0002] Hyaluronic acid, also referred to as "HA," is a
`naturally occurring, water soluble polysaccharide that is a
`major component of the extra-cellular matrix and is widely
`distributed in animal tissues. Naturally occurring HA gen(cid:173)
`erally has a molecular weight range of about between 6x104
`to about 8x106 Daltons. It has excellent biocompatibility and
`does not give a foreign body or allergic reaction when
`implanted into a subject.
`[0003] Methods of preparing commercially available
`hyaluronan are well known. Also known are various meth(cid:173)
`ods of coupling HA and cross-linking HA to reduce the
`water solubility and diffusibility of HA, and to increase the
`viscosity of HA. See, for example, U.S. Pat. Nos. 5,356,883
`and 6,013,679, the entire teachings of which are incorpo(cid:173)
`rated herein by reference. Further, many forms of HA have
`been employed, e.g., as surgical aids to prevent post opera(cid:173)
`tive adhesions of tissues, as adjuncts to synovial fluid in
`joints, as fluid replacement and/or surgical aids in oph(cid:173)
`thalmic surgery, as a scaffold for tissue engineering in vitro
`or guided tissue regeneration or augmentation in vivo, and
`the like.
`[0004] The use of such HA products suffers several
`defects, however, in particular, a tradeoff between in vivo
`properties and surgical usability. For example, HA that is
`sufficiently chemically modified or crosslinked to have
`desirable in vivo mechanical and biostability properties can
`be so highly viscous that injection through fine needles is
`difficult or impossible. Conversely, HA that is injectable can
`have inferior in vivo biostability and mechanical properties.
`[0005] Further, there is high current interest in employing
`chemically modified HA for vehicle-assisted time release
`delivery of bioactive agents including, for example, thera(cid:173)
`peutic agents or drugs and biological probes. A major
`challenge is the development of a delivery vehicle that will
`provide the appropriate level of bioavailability of a thera(cid:173)
`peutic agent at the affected area to achieve a desired clinical
`result, yet also have a desirable balance of in vivo mechani(cid:173)
`cal and biostability properties balanced with surgical/admin(cid:173)
`istrative usability. The bioavailability of a drug depends
`upon the nature of the drug, the drug delivery vehicle used,
`and the route of delivery, for example, oral, topical, trans(cid:173)
`dermal, mucosal, administration by injection, administration
`by inhalation, or administration by a combination of two or
`more of these routes. The bioavailability may be low as a
`result of, for example, the degradation of the drug by
`metabolic processes, rapid or uneven degradation of the
`delivery vehicle, rapid or uneven release of the drug from
`the delivery vehicle, and the like. These can be accompanied
`by similar problems of frequency of administration, diffi(cid:173)
`culty of administration, e.g., difficulty of injection, biodeg(cid:173)
`radation, and the like. In addition to the difficulties noted
`above, frequent administration of insufficiently stable drug
`
`delivery vehicles can lead to variations in drug delivery,
`leading to an increase in the occurrence of damaging side
`effects, a decrease in therapeutic benefit, and the like.
`
`[0006] Therefore, there is a need for a HA composition
`that overcomes or minimizes the above referenced prob(cid:173)
`lems.
`
`SUMMARY OF THE INVENTION
`
`[0007] The invention is directed to a hyaluronic acid (HA)
`composition and a method of making and using a HA
`composition that is effective for tissue augmentation and/or
`drug delivery.
`
`[0008] A hyaluronic acid (HA) composition includes
`crosslinked, water-insoluble, hydrated HA gel particles. The
`HA includes crosslinks represented by the following struc(cid:173)
`tural formula:
`
`HA'-U-R2-U-HA'
`[0009] Each HA' is the same or different crosslinked HA'
`molecule, e.g., the crosslinks can be intramolecular or
`intermolecular.
`
`[0010] Each U is independently an optionally substituted
`O-acyl isourea or N-acyl urea.
`
`[0011] R2 is optionally substituted alkyl, alkenyl, alkynyl,
`alkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl aryl, het(cid:173)
`eroaryl, heterocyclyl, cycloaliphaticalkyl, aralkyl, het(cid:173)
`eroaralkyl, or heterocyclylalkyl.
`
`[0012] A method of augmenting tissue in a subject that is
`in need of tissue augmentation includes the step of inserting
`a needle into a subject at a location in the subject that is in
`need of tissue augmentation, wherein the needle is coupled
`to a syringe loaded with the HA composition. Also included
`is applying force to the syringe, whereby at least a portion
`of the HA composition is delivered into the subject.
`
`[0013] A method of preparing the HA composition,
`includes forming water-insoluble, dehydrated crosslinked
`HA particles, separating the water-insoluble, dehydrated
`particles by average diameter, selecting a subset of particles
`by average diameter, and hydrating the subset of dehydrated
`particles with a physiologically compatible aqueous solu(cid:173)
`tion, thereby forming the HA composition.
`
`[0014] Another method of preparing the crosslinked HA
`composition includes crosslinking a precursor of the
`crosslinked HA with a biscarbodiimide in the presence of a
`pH buffer that is at a pH between about 4 and about 8, and
`dehydrating the crosslinked HA to produce the dehydrated,
`crosslinked HA.
`
`[0015] A method of stabilizing crosslinked HA includes
`hydrating water-insoluble, dehydrated crosslinked HA with
`a physiologically compatible aqueous solution, thereby
`forming the stabilized HA composition, wherein the physi(cid:173)
`ologically compatible aqueous solution includes at least
`about 0.1 % by weight of a local anesthetic, wherein the
`value of storage modulus G' for the stabilized composition
`is at least about 110% of the value of G' measured for a
`non-stabilized composition, when measured at 37° C. and 1
`Hz frequency using a 4 cm flat geometry.
`
`[0016] Also included is the stabilized HA composition.
`
`

`

`US 2005/0136122 Al
`
`Jun.23,2005
`
`2
`
`[0017] The embodiments disclosed herein are effective for
`preparing and using crosslinked, water-insoluble, hydrated
`HA gel particles, wherein the crosslinks in the HA include
`linking group R2, that have improved combinations of
`in-vivo biostability and mechanical properties, while at the
`same time having improved usability, e.g., improved ease of
`injection through fine needles. For example, as shown in the
`Exemplification, the HA compositions have improved val(cid:173)
`ues for storage modulus G' and kinematic viscosity, while
`exhibiting improved in vitro and in vivo biostability to
`hyaluronidase enzyme. The disclosed embodiments are
`effective for employing crosslinked HA in tissue augmen(cid:173)
`tation while reducing the frequency of implantation needed.
`The embodiments are also effective
`for employing
`crosslinked HA as a drug delivery vehicle that exhibits the
`surprising and unexpected effect of increasing biostability
`along with effective drug release properties and effective
`administrative properties.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`[0018] FIG.1: UV absorbance of the crosslinked products
`obtained by reacting HA and p-phenylene-bis(ethylcarbodi(cid:173)
`imide) in a molar equivalent ratio of 75%, 100%, and 125%.
`[0019] FIG. 2: UV absorbance of the crosslinked products
`obtained by reacting HA and p-phenylene-bis( ethylcarbodi(cid:173)
`imide) in MES buffer of pH 5.5, 6.0, and 6.5.
`[0020] FIG. 3: Effect of the particle average diameter
`distribution on the storage modulus (G') of the gel.
`[0021] FIG. 4: Effect of the particle average diameter
`distribution in the gel on the force required to extrude the gel
`from a 30-gauge needle.
`[0022] FIG. 5: Degradation of crosslinked HA product in
`the presence of enzyme hyaluronidase, compared to the
`other tissue augmentation products RES1YLANE®, PER(cid:173)
`LANE® and HYLAFORM®
`[0023] FIG. 6: Degradation profile of the gel of different
`initial G', prepared from the crosslinked HA of the invention.
`[0024] FIG. 7: Storage Modulus G' and degradation pro(cid:173)
`file of the gels prepared in phosphate buffer containing no
`lidocaine, 0.2% lidocaine, and 0.3% lidocaine.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0025] The invention is directed to crosslinked HA com(cid:173)
`positions, their preparation, and their methods of use.
`[0026] Uncrosslinked HA, e.g, the precursor to
`the
`crosslinked HA of the invention, typically comprises disac(cid:173)
`charide units of D-glucuronic acid (GlcUA) and N-acetyl(cid:173)
`D-glucosamine (GlcNAc), which are alternately linked,
`forming a linear polymer. HA often occurs naturally as the
`sodium salt, sodium hyaluronate. HA, sodium hyaluronate,
`and preparations of either HA or sodium hyaluronate are
`often referred to as "hyaluronan." As used herein, the terms
`"HA" and "hyaluronan" also refer to any of the other
`hyaluronate salts, including, for example, potassium hyalu(cid:173)
`ronate, magnesium hyaluronate, calcium hyaluronate, and
`the like. The uncrosslinked HA used as precursors for the
`crosslinking typically has an average molecular weight
`range of from between about 6xl04 to about 8xl06 Daltons,
`or 150 to 20,000 disaccharide repeat units. HA from any of
`
`a variety of sources, including HA extracted from animal
`tissues or harvested as a product of bacterial fermentation,
`can be used as a starting material. Alternatively, the
`uncrosslinked HA used to make the composites of this
`invention can be produced in commercial quantities by
`bioprocess technology, as described, for example, in Nimrod
`et al., PCT Publication No. WO 86/04355, the entire teach(cid:173)
`ings of which are incorporated herein by reference.
`
`[0027] Crosslinked HA can be formed by reacting
`uncrosslinked HA with a crosslinking agent under suitable
`reaction conditions. The crosslinked HA is prepared by
`reacting uncrosslinked HA with a biscarbodiimide in the
`presence of a pH buffer, wherein the buffer is at a pH
`between about 4 and about 8. The pH of the buffer can be
`between about 4 and about 7, typically between 5 and about
`6.5, or more typically between about 5 and about 6. In a
`preferred embodiment, the pH is about 5.5.
`
`[0028] The pH buffer can include any buffer agent known
`to one skilled in the art, e.g., 2-(N-morpholino)ethane(cid:173)
`sulfonic acid (MES); 2,2-bis(hydroxymethyl)-2,2',2"-nitrot(cid:173)
`riethanol; succinate/succinic acid; KH2PO4 ; N-tris(hy(cid:173)
`droxymethyl-2-aminoethanesulfonic acid; triethanolamine;
`diethylbarbituate;
`tris(hydroxymethyl)aminoethane; N-tr(cid:173)
`is(hydroxy)methylglycine; and N,N-bis(2-hydroxyethyl)g(cid:173)
`lycine. The buffer agent can be employed with an additional
`acid or base, e.g., 2-(N-morpholino)ethanesulfonic acid with
`NaOH; 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriethanol with
`HCl; succinate with succinic acid; KH2PO4 with borax;
`N-tris(hydroxymethyl-2-aminoethanesulfonic
`acid with
`NaOH; triethanolamine with HCl; diethylbarbituate with
`HCl; tris(hydroxymethyl)aminoethane with HCl; N-tris(hy(cid:173)
`droxy)methylglycine with HCl; and N,N-bis(2-hydroxyeth(cid:173)
`yl)glycine with HCI. Preferably, the buffer includes 2-(N(cid:173)
`morpholino)ethanesulfonic acid and NaOH.
`
`[0029] Typically, the buffer agent is mixed in aqueous
`media, in a concentration between about 5 mM (millimolar)
`and about 250 mM, typically between about 10 mM and
`about 150 mM, more typically between about 25 and about
`100 mM, and preferably about 75 mM.
`
`[0030] Typically, the uncrosslinked HA is mixed in aque(cid:173)
`ous media, e.g., the pH buffer solution, in a concentration
`between about 1 mM (millimolar) and about 100 mM,
`typically between about 10 mM and about 50 mM, more
`typically between about 25 and about 50 mM, and preferably
`about 37 mM. The particular concentration employed can
`vary depending on
`the molecular weight of
`the
`uncrosslinked HA precursor. At lower concentrations, the
`reactions can be slower. At higher concentrations, the prod(cid:173)
`uct can be difficult to handle due to the increase in viscosity.
`Examples of acceptable concentrations of uncrosslinked HA
`for other crosslinking reactions are described in U.S. Pat.
`No. 5,356,883, to Kuo et al., the teachings of which are
`incorporated herein by reference in their entirety.
`
`[0031] The reaction can be carried out at a temperature
`range of between about 0° C. and about 60° C., typically
`between about 10° C. and about 40° C., more typically
`between about 15° C. and about 30° C., and preferably about
`25° C. Exemplary reaction conditions can be found in
`Examples 1-9.
`
`[0032] The biscarbodiimide can be combined with the
`uncrosslinked HA solution alone, or more typically as a
`
`

`

`US 2005/0136122 Al
`
`Jun.23,2005
`
`3
`
`heteroaryl, heterocyclyl, cycloaliphaticalkyl, aralkyl, het(cid:173)
`eroaralkyl, heterocyclylalkyl, groups, and the like. Suitable
`optional substituents are those that do not substantially
`interfere with the properties of the resulting crosslinked HA
`composition and are described herein in the section describ(cid:173)
`ing each of the respective groups. R2 can optionally include
`or be interrupted by other groups, e.g, carbonyl, amide, oxy,
`sulfide, disulfide, and the like. In other embodiments, R2 is
`a cycloaliphatic, aryl, heteroaryl, or heterocyclyl group. In
`still other embodiments, R2 is 1,6-hexamethylene octam(cid:173)
`ethylene,
`decamethylene,
`dodecamethylene,
`PEG,
`-CH2 CH2-S-S-CH2 CH2- , para-phenylene-S-S-para(cid:173)
`phenylene, meta-phenylene-S-S-meta-phenylene, meta-phe(cid:173)
`nylene or para-phenylene. More preferably, R2 is phenylene.
`Preferably, R2 is para-phenylene.
`
`[0040]
`In one embodiment, the biscarbodiimide is selected
`from 1,6-hexamethylene bis( ethylcarbodiimide ), 1,8-octam(cid:173)
`ethylene bis( ethylcarbodiimide ), 1,10 decamethylene bis(cid:173)
`( ethylcarbodiimide ), 1,12 dodecamethylene bis( ethylcarbo(cid:173)
`diimide ),
`PEG-bis(propyl( ethylcarbodiimide )),
`2,2'(cid:173)
`dithioethyl bis(ethylcarbodiimde), 1,1'-dithio-p-phenylene
`bis( ethylcarbodiimide ); para-phenylene-bis( ethylcarbodiim(cid:173)
`ide ), and 1,1'-dithio-m-phenylene bis(ethylcarbodiimide). In
`a preferred embodiment, the biscarbodiimide is para-phe(cid:173)
`nylene-bis( ethylcarbodiimide ). Methods of preparing bis(cid:173)
`carbodiimides are described in U.S. Pat. Nos. 6,013,679;
`2,946,819; 3,231,610; 3,502,722; 3,644,456; 3,972,933;
`4,014,935; 4,066,629; 4,085,140; 4,096,334; 4,137,386,
`6,548,081, and 6,620,927 the teachings of which are incor(cid:173)
`porated herein by reference in their entireties.
`
`[0041] The reaction of HA with a biscarbodiimide
`crosslinking reagent, in the presence of an available proton,
`is believed to comprise protonation in the first step. The acid
`anion can then attach to the carbon atom of the cation
`formed, resulting in the formation of an O-acyl isourea
`intermediate. The acyl group in the intermediate can migrate
`from the oxygen atom to a nitrogen atom to produce a
`N-acyl isourea derivative of the HA. It is believed that the
`O-to-N migration can be incomplete, resulting in a product
`reaction mixture that can include both the N-acyl urea and
`the O-acyl isourea. Thus, a crosslink resulting from reaction
`of a biscarbodiimide with the crosslinked HA precursor
`typically can contain two O-acyl isoureas connected through
`R2, as represented in the following structural formula:
`
`[0042] or an O-acyl isourea and an N-acyl urea connected
`through R2, as represented in the following structural for(cid:173)
`mula:
`
`R1
`
`solution in a water-miscible organic solvent, e.g., acetone,
`methyl ethyl ketone, dimethyformamide, dimethyl sulfox(cid:173)
`ide, methanol, ethanol, 2-propanol, acetonitrile, tetrahydro(cid:173)
`furan, N-methyl pyrrolidone, and the like. More typically,
`the solvent is acetone, and the biscarbodiimide is at a
`concentration of between about 0.1 mg/mL and about 100
`mg/mL, typically between about 1 mg/mL and about 50
`mg/mL, more typically between about 5 mg/mL and about
`25 mg/mL, and preferably about 15 mg/mL.
`
`[0033] The uncrosslinked HA and the biscarbodiimide can
`be combined in any molar equivalent ratio, e.g., between
`about 1 % and about 200%, typically between about 10% and
`about 150%, more typically between about 18% and about
`125%. In various embodiments, the molar equivalent ratio is
`about 18%; or about 38%, or about 50%, or about 75%, or
`about 100%, or about 125%.
`[0034] A HA composition crosslinked with the biscarbo(cid:173)
`diimide can include crosslinks characterized by a linking
`group R2 of the biscarbodiimide agent included in the
`crosslink, e.g., the linking group connecting through a group
`U at each end to a HA' molecule, as shown in the following
`structural formula:
`HA'-U-R2-U-HA'
`[0035] Each HA' in the preceding formula can be different
`or the same HA' molecule, e.g., the crosslink can be an
`intermolecular or intramolecular crosslink. Each U can be
`the same or different and is an optionally substituted N-acyl
`urea or O-acyl isourea, as shown in the bracketed fragments
`in the following structural formulas:
`
`[0-acyl isourea]
`
`[N-acyl urea]
`
`[0036] These crosslinks can be generated in the reaction of
`HA' with a biscarbodiimide crosslinking reagent, repre(cid:173)
`sented in the following structural formula:
`
`R1-N=C=N-R2-N=C=N-R1
`[0037] wherein two carbodiimides, substituted with Rl,
`are connected through the linking group R2.
`[0038]
`In the preceding structural formulas, each Rl can
`be the same or different and is an optionally substituted
`group selected from hydrogen, aliphatic (e.g., alkyl, alkenyl,
`alkynyl), alkoxy, cycloaliphatic (e.g., cycloalkyl, cycloalk(cid:173)
`enyl, and cycloalkynyl), aryl, heteroaryl, heterocyclyl,
`cycloaliphaticalkyl, aralkyl, heteroaralkyl, heterocyclyla(cid:173)
`lkyl, and the like. Suitable optional substituents are those
`that do not substantially interfere with the properties of the
`resulting crosslinked HA composition and are described
`herein in the section describing each of the respective
`groups. In other embodiments, Rl is an optionally substi(cid:173)
`tuted aliphatic group. More preferably, Rl is alkyl, e.g.,
`Cl-C6 linear or branched alkyl, e.g., methyl, ethyl, propyl,
`butyl, 2-propyl, tert-butyl, and the like. Preferably, each Rl
`is ethyl.
`[0039] Each R2 is an optionally substituted linking group
`including one or more of aliphatic, cycloaliphatic, aryl,
`
`~)=N-R -NJ_NJ_HA
`
`H
`
`2 H
`
`I
`
`R1
`
`

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`Jun.23,2005
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`4
`
`[0043] or two N-acyl ureas connected through R2, as
`represented in the following structural formula:
`
`~N~N -R -N~N~HA .
`I
`I
`H
`2 H
`
`R1
`
`R1
`
`[0044] The mixed products can be used separately or
`together to prepare the compositions according to embodi(cid:173)
`ments of the invention.
`
`[0045] The crosslinked HA can be precipitated by pouring
`into a water-miscible organic solvent, e.g., acetone, methyl
`ethyl ketone, dimethyformamide, dimethyl sulfoxide,
`methanol, ethanol, 2-propanol, acetonitrile, tetrahydrofuran,
`N-methyl pyrrolidone, and the like, preferably an alcohol,
`e.g., ethanol. The precipitate can be collected and dried, e.g.,
`under reduced pressure.
`[0046] The dried crosslinked HA can be formed into
`particles by any means well known to one in the art, e.g.,
`abrading, grinding, fracturing, and the like, preferably by
`grinding in a cryogenic mill. Alternatively, the undried
`crosslinked HA can be cryoprecipitated to form small par(cid:173)
`ticles, which can then be dried, or the undried crosslinked
`HA can be ground in a cryogenic mill and then the resulting
`particles can be dried.
`
`[0047] As used herein, "water-insoluble" and like terms
`refers to compositions, e.g., the water-insoluble dehydrated
`particles, or the water insoluble hydrated particles that are
`heterogeneous when suspended in a sufficient amount of
`water at room temperature. In one embodiment, "water(cid:173)
`insoluble" means that upon placing the particles of the HA
`composition in water at neutral pH and 25° C. for at least
`about 2 weeks, the HA in the particles is essentially undis(cid:173)
`solved, e.g., essentially no HA from the particles becomes
`freely dissolved in the water. In other embodiments, "water(cid:173)
`insoluble" means that the HA in the particles is essentially
`undissolved after generally at least about 4 weeks under the
`preceding conditions, typically at least about 6 weeks, more
`typically at least about 8 weeks, or preferably at least about
`12 weeks. In one embodiment, "water-insoluble" means that
`the HA in the particles is essentially undissolved after at
`least about 26 weeks under the preceding conditions. As
`used herein, "freely dissolved" means salvation of HA
`molecules in the water separately from the hydrated, water(cid:173)
`swelled particles.
`
`[0048] Moreover, a cross-linked HA derivative can be a
`hydrogel. As the term is used herein, a "hydrogel" is a
`cross-linked macromolecular network that can swell in
`water or biological fluids, and can retain a significant portion
`of water within its structure without dissolving. As used
`herein, the term "swelling" refers to the taking up of a liquid,
`for example water, by a gel with an increase in volume,
`typically with the addition of heat and pressure. Hydrogels
`have a large molecular weight that generally cannot be
`measured by conventional methods and are composed of a
`polymer backbone and cross-links.
`
`[0049] The crosslinked HA particles can be characterized
`by particle average diameter distribution. The average diam-
`
`eter can be measured as the average diameter of the hydrated
`particles and/or the average diameter of the dehydrated
`particles. Typically, the particle average diameter is selected
`from the group consisting of a hydrated particle average
`diameter between about 20 µm (micrometers) and about
`1000 µm and a dehydrated particle average diameter
`between about 10 µm and about 500 µm. In other embodi(cid:173)
`ments, the hydrated particle average diameter is between
`about 40 µm and about 600 µm and a dehydrated particle
`average diameter between about 20 µm and about 300 µm,
`or more preferably, the hydrated particle average diameter is
`between about 50 µm and about 500 µm and the dehydrated
`particle average diameter is between about 25 µm and about
`250 µm.
`
`[0050]
`In a particular embodiment, the HA in the compo(cid:173)
`sition consists essentially of the crosslinked, water-in(cid:173)
`soluble, hydrated HA gel particles. For example, the HA
`composition in this embodiment can be considered to be a
`single hydrated particle phase, e.g., any liquid in the com(cid:173)
`position is essentially contained in the hydrated particles,
`e.g., there is essentially no free liquid phase. In other
`embodiments wherein the HA in the composition consists
`essentially of the crosslinked, water-insoluble, hydrated HA
`gel particles, certain forms of HA is excluded from the
`composition, e.g., typically excluded are HA particles or
`molecules that have an average diameter smaller than about
`1 µm, more typically excluded are HA particles or molecules
`that have an average diameter smaller than about 10 µm, and
`preferably excluded are HA particles or molecules that have
`an average diameter smaller than about 20 µm.
`
`[0051] As used herein, a "subset" of particles by average
`diameter means that a collection of particles are character(cid:173)
`ized by average diameter, and at least some fraction of
`particles is rejected, e.g., not included in the subset.
`
`[0052]
`In still other embodiments, the particle average
`diameters are selected, e.g., by sieving or other methods well
`known to the art, so that particular average diameter distri(cid:173)
`butions can be chosen to result in particular properties in the
`final composition, for example, as shown in the Examples.
`Thus, the dehydrated particles can be sieved to separate by
`average diameter fractions, e.g., as in Example 16, where
`five average diameter fractions of particles were collected:
`0-25 µm, 25-75 µm, 75-125 µm, 125-180 µm, and 180-250
`µm.
`
`[0053] The various average diameter fractions of a par(cid:173)
`ticular composition can be employed to determine a com(cid:173)
`bination of average diameter fractions in various proportions
`that will result in particular combined properties. Thus, in
`one embodiment, the water-insoluble, dehydrated particles
`can be separated into at least two average diameter fractions
`and the fractions can be combined in a ratio to adjust the
`properties of the combination, e.g., in Example 17, two
`fractions containing 125-250 µm, and 0-125 µm are com(cid:173)
`bined in a 1:1 ratio. The resulting composition has a average
`diameter distribution that is different from the ground par(cid:173)
`ticles before sizing, for example, the average diameter
`distribution can be a multimodal average diameter distribu(cid:173)
`tion, e.g., a bimodal average diameter distribution when two
`average diameter fractions are selected for the composition.
`The properties of the multimodal composition are built from
`the properties of the individual average diameter fractions
`and their amounts in the composition. In another embodi-
`
`

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`Jun.23,2005
`
`5
`
`ment, the water-insoluble, dehydrated particles can be sepa(cid:173)
`rated into at least three average diameter fractions, where at
`least two average diameter fractions are selected and at least
`one average diameter fraction is rejected.
`
`[0054] The dehydrated particles are typically hydrated in
`the presence of the physiologically acceptable solution ( e.g.,
`a saline solution, or a phosphate buffer as provided in the
`Examples) under conditions including a temperature of at
`least about 100° C., a pressure of at least about 120 kPa
`(kilopascals); and a duration of at least about 15 min. Such
`conditions can be achieved in an autoclave, for example, and
`can also serve to sterilize the particles. Other conditions
`include: temperatures of from about 100° C. to about 150°
`C., typically from about 110° C. to about 140° C., or
`preferably from about 120° C. to about 140° C.; pressures
`from about 120 kPa to about 200 kPa, typically from about
`120 kPa to about 160 kPa, or preferably from about 130 kPa
`to about 140 kPa; and durations from about 15-75 min, more
`typically from about 20 to about 60 min.
`
`[0055] Additional contemplated sterilization/hydration
`techniques include the following. In one embodiment, prod(cid:173)
`uct can be contacted with a clean steam supply at a tem(cid:173)
`perature of 118-133° C. (typically about 121 ° C.) and the
`corresponding steam pressure at saturation ( about 103 kPa to
`about 186 kPa). Evaporative cooling can occur during
`vacuum or "natural" cooling, e.g., slow venting of steam
`pressure. In another embodiment, a product can be sterilized
`at a temperature of 118-133° C. (typicall