(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0194758 A1
`
`
` Lebreton (43) Pub. Date: Aug. 31, 2006
`
`IJSZOO60194758A1
`
`(54) CROSS-LINKING 0F LOW AND HIGH
`MOLECULAR WEIGHT
`
`(30)
`
`Foreign Application Priority Data
`
`POLYSACCHARIDES PREPARATION OF
`INJECTABLE MONOPHASE HYDROGELS
`AND POLYSACCHARIDES AND
`DYDROGELS THUS OBTAINED
`
`(76)
`
`Inventor:
`
`Pierre Lebreton, Annecy Le Vieux
`(FR)
`
`Correspondence Address:
`COHEN, PONTANI, LIEBERMAN & PAVANE
`551 FIFTH AVENUE
`SUITE 1210
`
`NEW YORK, NY 10176 (US)
`
`(21)
`
`Appl. No.:
`
`10/552,309
`
`(22)
`
`PCT Filed:
`
`Apr. 8, 2004
`
`PCT No.:
`
`PCT/FR04/00870
`
`(86)
`
`Apr. 10, 2003
`
`(FR) .............................................. 0304444
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`(2006.01)
`A61K 31/728
`(2006.01)
`C083 37/00
`(52) use. ................................................. 514/54; 536/53
`
`(57)
`
`ABSTRACT
`
`least one polymer
`A process for the crosslinking of at
`selected from polysaccharides and derivatives thereof,
`which is carried out in an aqueous solvent by the action of
`an effective and non-excessive amount of at
`least one
`
`crosslinking agent, characterized in that it is carried out on
`a mixture containing at
`least one low-molecular weight
`polymer and at least one high-molecular weight polymer. A
`process for the preparation of an injectable monophase
`hydrogel of at least one crosslinked polymer selected from
`polysaccharides and derivatives thereof is also disclosed.
`Crosslinked polymers and injectable monophase hydrogels,
`respectively, are obtainable by each of said processes.
`
`Page 1
`
`Teoxane S.A.
`
`
`Exhibit 1027
`
`Page 1
`
`

`

`Patent Application Publication Aug. 31, 2006
`
`US 2006/0194758 A1
`
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`Page 2
`
`Page 2
`
`

`

`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`CROSS-LINKING OF LOW AND HIGH
`MOLECULAR WEIGHT POLYSACCHARIDES
`PREPARATION OF INJECTABLE MONOPHASE
`HYDROGELS AND POLYSACCHARIDES AND
`DYDROGELS THUS OBTAINED
`
`[0001] The present invention relates to:
`
`a novel process for the crosslinking of at least one
`[0002]
`polymer selected from polysaccharides and derivatives
`thereof;
`
`a process for the preparation of an injectable
`[0003]
`monophase hydrogel of at least one such polymer; and
`
`injectable
`and
`polymers
`crosslinked
`the
`[0004]
`monophase hydrogels respectively obtainable by each of
`said processes.
`
`in question, based on said
`[0005] The hydrogels
`crosslinked polymers, have numerous outlets, especially as
`filling materials in plastic, cosmetic and dental surgery, in
`ophthalmology, in orthopedics, etc., as products for prevent-
`ing tissue adhesions, in general surgery, in urology, etc. Said
`hydrogels are particularly suitable for repairing vocal cords.
`The outlets indicated above for products of this type, without
`implying any limitation, are familiar to those skilled in the
`art.
`
`[0006] The invention is the result of a genuine effort to
`optimize the operation of crosslinking the polymers in
`question with a view to obtaining injectable monophase
`hydrogels that are of particular value in respect of the
`following compromise: on the one hand mechanical prop-
`erties and remanence, and on the other hand injectability
`(with acceptable injection forces and injection needle diam-
`eters).
`
`It is pointed out here that the term “inj ectable”
`[0007]
`employed in the present text, with reference to both the
`hydrogels of the prior art and the hydrogels of the invention,
`denotes manual injectability by means of syringes equipped
`with conventional needles (having a diameter of between 0.1
`and 0.5 mm). Within the framework of the present invention,
`it is possible in particular to formulate hydrogels that can be
`injected through hypodermic needles of 30 Gl/z, 27 Gl/z, 26
`Gl/z and 25 G.
`
`[0008] According to the prior art, hydrogels, especially
`injectable hydrogels, have already been prepared from
`polysaccharides and derivatives thereof%specially hyalu-
`ronic acid saltsihaving a zero,
`low or high degree of
`crosslinking.
`
`[0009] With reference to the specific problem of inject-
`ability, biphase compositions have been proposed whose
`continuous phase, in particular, is based on such hydrogels.
`The continuous phase serves as a plasticizer,
`injection
`vehicle for a disperse phase. This disperse phase is more or
`less solid and more or less differentiated from the continuous
`
`phase. Thus:
`
`the biphase compositions described in patent appli-
`[0010]
`cation EP-A-0 466 300 consist of two bioabsorbable
`
`phases%ontinuous and disperseiand take the form of
`slurries. Said two phases are advantageously prepared from
`fibers of Hylan (natural hyaluronic acid chemically modified
`in situ in order to facilitate its extraction from the tissues);
`
`the biphase compositions described in patent appli-
`[0011]
`cation WO-A-96 337 51 also have two bioabsorbable phases
`with a better separation, the disperse phase consisting of
`insoluble fragments of a highly crosslinked polymer hydro-
`gel (selected from hyaluronic acid and its salts);
`
`the biphase compositions described in patent appli-
`[0012]
`cation WO-A-OO 014 28 contain a non-bioabsorbable dis-
`
`perse phase (particles of at least one hydrogel of a (co)poly-
`mer obtained by the polymerization and crosslinking of
`acrylic acid and/or methacrylic acid and/or at least one
`derivative of said acids) suspended in an aqueous solution of
`a crosslinked or non-crosslinked polymer selected from
`proteins, polysaccharides and derivatives thereof.
`
`[0013] These biphase systems are not fully satisfactory
`insofar as they are associated with justifiable fears of uneven
`flow during injection and particularly after injection, a more
`rapid disappearance of the continuous phase (having a zero
`or low degree of crosslinking) and hence an at least partial
`loss of the desired effect, especially filling effect.
`
`[0014] Monophase hydrogels, developed from the same
`types of polymers, were therefore also proposed in parallel.
`
`In patent applications WO-A-98 356 39 and WO-
`[0015]
`A-98 356 40, the product in question is not an injectable
`hydrogel but a product of solid consistency. Said patent
`applications in fact describe ocular implants used to tempo-
`rarily fill a surgically created void. The hydrogel developed
`in US. Pat. No. 4,716,154 is proposed as a substitute for the
`vitreous body. The polymer in question (sodium hyalur-
`onate) has a very low degree of crosslinking in order to
`obtain an injectable hydrogel. The monophase hydrogel
`described in patent application WO-A-02 057 53 is laden
`with an antiseptic that is effective in protecting it from free
`radicals after implantation. Patent application WO-A-02 063
`50 describes a process capable of generating this type of
`hydrogel that is very homogeneous throughout.
`
`these monophase hydrogels were obtained
`[0016] All
`from high-molecular weight polymers crosslinked using an
`effective and non-excessive amount of at least one crosslink-
`
`ing agent, in an aqueous solvent.
`
`In the light of this prior art, the inventors wished to
`[0017]
`improve the efficacy of crosslinking of the polymer in
`question, especially in order to improve the degradation
`resistance (remanence) of the implanted hydrogel while at
`the same time preserving the possibility of injecting said
`hydrogel under acceptable conditions.
`
`To improve the crosslinking efficacy, the inventors
`[0018]
`initially considered using more crosslinking agent. This
`approach was quickly discarded on the grounds that
`it
`inescapably causes denaturation of the polymer in question
`and chemical contamination of the crosslinked product
`obtained.
`
`Said inventors then considered increasing the con-
`[0019]
`centration of polymer in the reaction mixture. In the same
`way, this second approach had to be discarded, a priori,
`because of the polymers conventionally used hitherto,
`namely high-molecular weight polymers. Thus
`sodium
`hyaluron6ate is always used with high molecular weights
`(Mw>10 Da, z2.106 Da, 3.106 Da) at concentrations close to
`the maximum concentration, which is about 105-110 mg/g.
`Using it at a higher concentration is difficult (the viscosity of
`
`Page 3
`
`Page 3
`
`

`

`US 2006/0194758 Al
`
`Aug. 31, 2006
`
`the reaction mixture becomes too high) and inescapably
`causes problems of solubility, poor homogeneity, etc.
`
`[0020] Concentrating the reaction medium, on the other
`hand, is found to be possible with low-molecular weight
`polymers (sodium hyaluronate of molecular weight 300,000
`Da, having an intrinsic. Viscosity of 600 ml/g (those skilled
`in the art are perfectly familiar with the relationship between
`these two parameters: molecular weight (M) and intrinsic
`Viscosity (n), which is given by the Mark-Houwink formula:
`M=kn°‘, the values of k and (X depending on the nature of the
`polymer in question), can be concentrated from 110 to 200
`mg/g). Unfortunately the crosslinked polymer obtained gen-
`erates an inhomogeneous, injectable biphase hydrogel under
`these conditions.
`
`In such a context, the inventors surprisingly estab-
`[0021]
`lished that associating low-molecular weight polymer(s)
`with high-molecular weight polymer(s) affords an excellent
`compromise, namely the possibility of generating, for a
`non-excessive degree of crosslinking (equivalent to that of
`the prior art), an injectable monophase hydrogel which has
`improved mechanical and remanence properties. This low-
`molecular weight/high-molecular weight association makes
`it possible to obtain a hydrogel that more than satisfies the
`following specifications:
`
`[0022]
`
`monophase;
`
`better mechanical properties and remanence than
`[0023]
`the equivalent products of the prior art;
`
`unaffected or even improved injectability that is
`[0024]
`still possible with conventional injection forces using con-
`ventional injection devices.
`
`[0025] The key factor of the crosslinking process of the
`invention therefore lies in the concentration of the reactants
`
`(which is greater than that of the reaction mixtures of the
`prior art due to the use of low-molecular weight polymer(s)),
`although the crosslinking of said concentrated reactants is
`“governed” by the use of high-molecular weight polymer(s),
`which guarantee the homogeneity of the crosslinked product
`obtained and then of the hydrogel obtained.
`
`[0026] According to its first subject, the present invention
`therefore relates to a process for the crosslinking of at least
`one polymer selected from polysaccharides and derivatives
`thereof, which is carried out in an aqueous solvent by the
`action of an effective and non-excessive amount of at least
`
`one crosslinking agent, said process being improved in that
`it
`is carried out on a mixture containing at
`least one
`low-molecular weight polymer and at least one high-mo-
`lecular weight polymer.
`
`Said mixture of course contains said low-molecular
`[0027]
`weight polymer(s) in a sufficient amount to guarantee a
`relatively high concentration of polymer(s) in the reaction
`medium, and said high-molecular weight polymer(s) in a
`sufficient amount to guarantee that said crosslinked polymer
`obtained has a homogeneous consistency.
`
`[0028] The crosslinking process of the invention is a
`process for the crosslinking of polymers selected from
`polysaccharides and derivatives thereof. The polymer(s) in
`question can therefore be natural or synthetic. Examples of
`natural polymers are hyaluronic acid and its salts, other
`glycosaminoglycans such as chondroitin sulfates, keratan
`sulfate, heparin and heparan sulfate, alginic acid and its
`
`biologically acceptable salts, starch, amylose, dextran, xan-
`than, pullulan, etc. Examples of synthetic derivatives of
`natural polysaccharides are carboxy cellulose, carboxym-
`ethyl cellulose, alkyl celluloses such as hydroxyethyl cellu-
`lose and hydroxypropyl methyl cellulose (HPMC), oxidized
`starch, etc.
`
`[0029] The process of the invention is suitable for the
`crosslinking of any one of these polymers insofar as said
`polymer is used with low and high molecular weights.
`
`[0030] The process of the invention is suitable for the
`crosslinking of mixtures of such polymers, said mixtures
`containing at least one low-molecular weight polymer and at
`least one high-molecular weight polymer.
`
`[0031] The terms “low” and “high” applied to the molecu-
`lar weights in question obviously cannot be defined more
`precisely at this stage of the description of the invention
`since they depend on the mixture in question and the nature
`of the polymer(s) present. Likewise,
`it
`is not generally
`possible to indicate the relative proportions in which the
`polymer(s) present is(are) used. However, those skilled in
`the art have a perfect understanding of the spirit of the
`invention, which is to concentrate the reaction medium
`containing the low-molecular weight polymer(s), but
`to
`introduce at least one high-molecular weight polymer to
`moderate and control the crosslinking in question. The aim
`is to obtain a coherent crosslinked product
`that
`is the
`precursor of a monophase hydrogel. It is desirable to avoid
`the formation of lumps that may be coherent when crosslink-
`ing has ended, but capable of losing their coherence when
`the injectable hydrogel is prepared.
`
`[0032] The above explanations are given a posteriori. The
`result obtained was in no way predictable.
`
`[0033] Within the framework of one advantageous variant,
`the reaction medium contains a single polymer which is used
`with at least two differentiated molecular weights, at least
`one being low and at
`least one being high. Within the
`framework of this advantageous variant, the same polymer
`is preferably used with a single low molecular weight and a
`single high molecular weight.
`
`[0034] The polymer in question is advantageously a hyalu-
`ronic acid salt. It is very advantageously selected from the
`sodium salt,
`the potassium salt and mixtures thereof. It
`preferably consists of the sodium salt (NaHA).
`
`In the context of the crosslinking of this type of
`[0035]
`those skilled in the art understand that said
`polymer,
`crosslinking is carried out in a basic aqueous solvent. In
`general, said crosslinking is obviously carried out under pH
`conditions that favor the dissolution of the polymer in
`question.
`
`In the context of the crosslinking of this type of
`[0036]
`polymer (hyaluronic acid salt(s)), in one preferred variant of
`carrying out the crosslinking, the reaction mixture contains:
`
`at least one hyaluronic acid salt of low molecular
`[0037]
`weight m, where m§9.9.105 Da, advantageously 104
`Da§m§9.9.105 Da; and
`
`at least one hyaluronic acid salt of high molecular
`[0038]
`weight M, where M2106 Da,
`advantageously
`106
`DaéMélO8 Da
`and
`very
`advantageously
`1.1.106
`Da§M§5.106Da,
`
`Page 4
`
`Page 4
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`

`

`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`said low-molecular weight and high-molecular weight salts
`advantageously being of the same nature and very adVan-
`tageously consisting of sodium hyaluronate (NaHA).
`
`In such a context, said reaction mixture adVanta-
`[0039]
`geously has an intrinsic Viscosity of less than 1900 ml/g, i.e.
`2mi[ni]0§1900 ml/g, where mi
`is the mass fraction of
`polymer fraction i, haVing an intrinsic Viscosity [11],, in the
`reaction mixture. Those skilled in the art are familiar with
`the intrinsic Viscosity parameter and are aware of the laws of
`additiVity of said parameter.
`
`[0040] The condition stated ab0Ve makes it possible to
`obtain a monophase hydrogel that is optimized in respect of
`its remanence and injectability properties. It fixes the rela-
`tiVe proportions of the salts of low molecular weight (m) and
`high molecular weight (M).
`
`In the context referred to here (NaHA of molecular
`[0041]
`weights m and M),
`the reaction mixture adVantageously
`contains more than 50% by weight, Very adVantageously
`more than 70% by weight, of at least one hyaluronic acid salt
`of low molecular weight m, and hence, logically, adVanta-
`geously less than 50% by weight, Very adVantageously less
`than 30% by weight, of at least one hyaluronic acid salt of
`high molecular weight M.
`
`In general, to obtain the expected effect, there is at
`[0042]
`least 5% by weight of at least one hyaluronic acid salt of
`high molecular weight M in the reaction mixture.
`
`[0043] The crosslinking process of the inVention is adVan-
`tageously carried out with the sodium salt of hyaluronic acid
`used with one low molecular weight m and one high
`molecular weight M, said parameters then Very adVanta-
`geously being as follows: mz3.105 Da and Mz3.106 Da.
`
`[0044] Any agent known for crosslinking polysaccharides
`and deriVatiVes thereof Via its hydroxyl groups can be used
`as the crosslinking agent with all types of polymer, said
`crosslinking agent being at least bifunctional in order to
`ensure crosslinking, an epoxy compound or deriVatiVes
`thereof being used in particular.
`
`It is recommended to use bifunctional crosslinking
`[0045]
`agents, by themselVes or in a mixture. It
`is particularly
`recommended to use epichlorohydrin, diVinyl sulfone, 1,4-
`bis(2,3-epoxypropoxy)butane (or 1,4-bisglycidoxybutane or
`1,4-butanediol diglycidyl ether (BDDE)), 1,2-bis(2,3-ep-
`oxypropoxy)ethylene, 1-(2,3-epoxypropyl)-2,3-epoxycyclo-
`hexane, and aldehydes such as formaldehyde, glutaralde-
`hyde and crotonaldehyde,
`taken by themselVes or in a
`mixture. It is Very particularly recommended to use 1,4-
`bis(2,3-epoxypropoxy)butane (BDDE).
`
`[0046] Those skilled in the art will know how to determine
`the effectiVe and non-excessiVe amount of crosslinking
`agent(s) to use. It is recommended to use an effectiVe and
`non-excessiVe amount such that the degree of crosslinking
`(‘5), defined by the following ratio:
`
`Total number of reactive groups
`
`in said crosslinking agent
`_
`T _ Total number of disaccharide X
`units in the polymer molecules
`
`100
`
`’
`
`[0047] The crosslinking process of the inVention is n0Vel
`by Virtue of the forms in which the polymers in question are
`used. In other respects it
`is carried out
`in conVentional
`manner with at least one crosslinking agent. It is noted that
`said crosslinking agent is generally reacted with the dis-
`solVed polymer(s), but reacting it with said polymer(s)
`during hydration, by the process described in WO-A-02 06
`350, is in no way ruled out.
`
`[0048] The crosslinked product obtained after carrying out
`the crosslinking process of the inVention is generally for-
`mulated for generating the desired injectable monophase
`hydrogel. If necessary, it is neutralized beforehand. It has
`been seen that
`the hyaluronic acid salts are actually
`crosslinked in a basic medium. The formulation is carried
`out in a solution buffered to a pH compatible with the human
`body (since the hydrogel in question is generally intended
`for injection into the human body), said pH being between
`6.5 and 7.5, adVantageously between 7 and 7.4 and Very
`adVantageously between 7.1 and 7.3. The crosslinked poly-
`mer is in equilibrium in said solution. It also acquires an
`osmolarity compatible with that of the human body. Sur-
`prisingly, after this formulation step, the diluted crosslinked
`polymers of the inVention are monophase hydrogels.
`
`In one preferred Variant of carrying out the inVen-
`[0049]
`tion, an injectable hydrogel of the inVention is prepared by
`crosslinking a mixture of at least one polymer consisting of
`hyaluronic
`acid salt(s)
`(see
`ab0Ve), neutralizing the
`crosslinked product obtained, and then formulating it into a
`solution buffered to a pH of between 7.1 and 7.3, at a
`concentration of between 10 and 40 mg/g, adVantageously
`of between 20 and 30 mg/g.
`
`[0050] The process for the preparation of the injectable
`monophase
`hydrogel
`from the
`crosslinked polymer
`(obtained by the crosslinking process constituting the first
`subject of the present
`inVention) constitutes the second
`subject of the present inVention.
`
`[0051] We now come to the third and fourth subjects,
`which respectiVely consist of the crosslinked polymer
`obtainable after carrying out the crosslinking process (first
`subject), and the injectable monophase hydrogel obtainable
`by the formulation (second subject) of said crosslinked
`polymer, as stated ab0Ve.
`
`Said polymer and hydrogel adVantageously contain
`[0052]
`low-molecular weight sodium hyaluronate and high-mo-
`lecular weight sodium hyaluronate, the proportion of said
`low-molecular weight sodium hyaluronate Very adVanta-
`geously being more than 50% by weight.
`
`[0053] The structure of the injectable monophase hydro-
`gelifourth subject of the present inVentioniis n0Vel. Its
`consistency is resistant to degradation. This resistance of the
`hydrogel is far greater than that of the equiValent products of
`the prior art.
`
`[0054] Those skilled in the art are aware that one of the
`methods of estimating the consistency of a hydrogel, espe-
`cially of this type, is to measure the following parameter:
`
`r;
`/ _
`tan-delta = — - f(stressing frequency).
`
`theoretically between 0.5 and 70%, adVantageously
`is
`between 4 and 50%.
`
`[0055] The hydrogels of the inVention haVe the outlets
`indicated in the introduction of the present text. They are
`found to be particularly efficient for these purposes.
`
`Page 5
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`

`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`It is now proposed to illustrate the invention in its
`[0056]
`various features by means of the Examples below. More
`precisely:
`
`[0057] Example 1 illustrates the prior art (crosslinking of
`a polymer of high molecular weight);
`
`[0058] Example 2 illustrates the remarks made in the
`introduction of the present text (crosslinking of the same
`polymer of low molecular weight); and
`
`invention
`the
`illustrate
`and 4
`3
`[0059] Examples
`(crosslinking of the same polymer of low and high molecu-
`lar weight, used in different relative amounts).
`
`[0060] These are preceded by a description of a few
`methods of measurement used to characterize the products
`in question.
`
`Measurement of the Intrinsic Viscosity
`
`[0061] The intrinsic viscosity of sodium hyaluronate
`(NaHA) (in ml/g) is determined according to the European
`Pharmacopeia for NaHA (2.2.9) using a capillary viscometer
`of the Ubbelohde type.
`
`Measurement of the Ejection Force (No Specific Standard
`for This Test)
`
`[0062] The injectability of the gel based on NaHA is
`determined by measuring the force (in Newtons, N) required
`to eject the gel contained in a standard syringe, through a
`needle of 27 Gl/z, at a rate of 12.5 mm/min. The tests were
`performed on a Verstatet® tensile device marketed by Mec-
`mesin.
`
`Measurement of the Remanence
`
`[0063] The consistency of the gel is characterized at 25° C.
`by rheological measurement of the moduli of elasticity (G')
`and viscosity (G") as a function of the frequency (from 0.05
`to 10 Hz), in the constant deformation domains, using a
`controlled stress rheometer (Carrimed CSL 500 from TA
`Instruments) and a cone-and-plate geometry of 4 cm 2°. This
`rheometer is checked and calibrated regularly. Degradation
`of the crosslinked gel results in a change in its consistency,
`which is measured by the increase in the parameter tangent
`delta (tan .delta=G"/G') as a function of time, at a frequency
`of 1 Hz. The gels are degraded by being heated to a
`temperature of 93° C. The time after which tan .delta reaches
`a value of 0.65 (corresponding to a degraded gel state) is
`measured at
`this temperature. A remanence index of 1
`(corresponding to said time) was arbitrarily set for the gel of
`Example 1. The remanence index values indicated for the
`other gels are relative values.
`
`Appearance of the Hydrogel
`
`Monophase
`
`apparent
`no
`appearance:
`[0064] Microscopic
`phaseifine fragmentation of the gel into facets
`
`liquid
`
`[0065] Macroscopic appearance: soft and free-flowing
`
`Biphase
`
`EXAMPLE 1
`
`High-Molecular Weight Fibers
`
`3.5 g of sodium hyaluronate (NaHA) fibers of
`[0068]
`intrinsic viscosity 2800 ml/g and moisture content 8.7% are
`weighed out and 25.6 g of 0.25 N NaOH are added.
`Hydration of the fibers takes 2 h with regular manual
`homogenization using a spatula. 0.96 g of a solution of
`1,4-butanediol diglycidyl ether (BDDE) diluted to 1/s in 0.25
`N sodium hydroxide solution is added to the reaction
`medium, this being followed by mechanical homogenization
`for 15 min before immersion in a thermostatically controlled
`bath at 50° C.=1° C.
`
`[0069] R=[BDDE]O/[NaHA]O=6%; [NaHA]i=105 mg/g
`
`[0070] The reaction takes 2 h. The crosslinked product is
`neutralized to pH 7.2 in a phosphate buffer solution and then
`dialyzed. The concentration of the resulting hydrogel is then
`adjusted ([NaHA]f=26 mg/g) and the hydrogel is mechani-
`cally homogenized before being packed into syringes and
`sterilized in an autoclave by means of moist heat.
`
`[0071]
`
`lnjection force after sterilization: 25 N
`
`[0072] Remanence index of the hydrogel: 1.0
`
`[0073] Monophase hydrogel
`
`EXAMPLE 2
`
`Low-Molecular Weight Fibers
`
`1.56 g of sodium hyaluronate (NaHA) fibers of
`[0074]
`intrinsic viscosity 600 ml/g and moisture content 5.5% are
`weighed out and 7.15 g of 0.25 N NaOH are added.
`Hydration of the fibers takes 2 h with regular manual
`homogenization using a spatula. 0.31 g of a solution of
`1,4-butanediol diglycidyl ether (BDDE) diluted to 1/s in 0.25
`N sodium hydroxide solution is added to the reaction
`medium, this being followed by mechanical homogenization
`for 15 min before immersion in a thermostatically controlled
`bath at 50° C.=1° C.
`
`[0075] R=[BDDE]O/[NaHA]O=6.8%; [NaHA]i=174 mg/g
`
`[0076] The reaction takes 2 h. The crosslinked product is
`neutralized to pH 7.2 in a phosphate solution and then
`dialyzed. The concentration of the resulting hydrogel is then
`adjusted ([NaHA]f=26 mg/g) and the hydrogel is mechani-
`cally homogenized before being packed into syringes and
`sterilized in an autoclave.
`
`[0077]
`
`lnjection force after sterilization: 24 N
`
`[0078] Remanence index of the hydrogel: 6.0
`
`[0079] Biphase hydrogel
`
`EXAMPLE 3
`
`Mixture of Fibers
`
`[0066] Microscopic appearance: gel fragments bathed in a
`low-viscosity liquid medium
`
`[0067] Macroscopic appearance: “puree” that fragments
`very easilyino cohesion of the gel and no free-flowing
`appearance
`
`0.763 g of sodium hyaluronate (NaHA) fibers of
`[0080]
`intrinsic viscosity 600 ml/g and moisture content 5.5% and
`0.237 g of sodium hyaluronate fibers of intrinsic viscosity
`2800 ml/g and moisture content 9.3% are weighed out.
`Proportions by weight
`in the mixture: 600/2800:77/23
`(w/w).
`
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`[0081] The procedure remains identical to that of Example
`2.
`
`[0082] R=[BDDE]O/[NaHA]O=7%;
`[NaHA]f=26 mg/g
`
`[NaHA]i=140 mg/g;
`
`[0083]
`
`Injection force after sterilization: 15 N
`
`[0084] Remanence index of the hydrogel: 3.6
`
`[0085] Monophase hydrogel
`
`EXAMPLE 4
`
`Mixture of Fibers
`
`[0086] The experiment of Example 3 is repeated, modi-
`fying the proportions by weight. Proportions by weight in
`the mixture: 600/2800:90/ 10 (w/w).
`
`[0087] The procedure is identical to that of Example 2.
`
`[0088] R=[BDDE]O/[NaHA]O=6.5%; [NaHA]i=140 mg/g;
`[NaHA]f=26 mg/g
`
`[0089]
`
`Injection force after sterilization: 14 N
`
`[0090] Remanence index of the hydrogel: 7.7
`
`[0091] Monophase hydrogel
`
`[0092]
`
`Said Examples are summarized in the Table below.
`
`TABLE
`
`least one low-molecular weight
`a mixture containing at
`polymer and at least one high-molecular weight polymer.
`2. Process according to claim 1, characterized in that said
`mixture contains a single polymer with at least two different
`molecular weights, at least one being low and at least one
`being high, and advantageously with two different molecular
`weights, one low and one high.
`3. Process according to claim 1 or 2, characterized in that
`said polymer is a hyaluronic acid salt.
`4. Process according to claim 3, characterized in that said
`hyaluronic acid salt is selected from the sodium salt, the
`potassium salt and mixtures thereof, and advantageously
`consists of the sodium salt.
`
`5. Process according to any one of claims 1 to 4, char-
`acterized in that said mixture contains:
`
`at least one hyaluronic acid salt of low molecular weight
`m, where m§9.9.105 Da,
`advantageously
`104
`Da§m§9.9.105 Da; and
`
`at least one hyaluronic acid salt of high molecular weight
`M, where M2106 Da, advantageously 106 Da é M§108
`Da, and very advantageously 1.1.106 Daé Mé 5.106
`Da,
`
`said low-molecular weight and high-molecular weight
`salts advantageously being of the same nature and very
`advantageously consisting of sodium hyaluronate.
`
`[NaHA]0 = concentration of NaHA in the reaction medium at to
`[NaHA]f = concentration of NaHA in the final hydrogel after
`reaction and dilution with a sufficient amount of phosphate buffer
`G': modulus of elasticity of the final hydrogel (Pa - s)
`G”: modulus of viscosity of the final hydrogel (Pa - s)
`Tan - delta = G”/G’
`mm: intrinsic viscosity of the NaHA fiber/Ubbelohde viscometer
`F: ejection force of the gel in N through a 27 Gl/é needle/100 N dynamometer
`
`Carrimed CSL 500 rheometer
`
`711m. (ml/g)
`% = proportion by
`weight in mixture
`
`R =
`mBDDE/mNaHA
`
`[NaHA]O
`mg/g
`
`[NaHA]f in
`final gel
`mg/g
`
`6%
`(100%) 2800
`6.8%
`(100%) 600
`7
`(77%) 600 + (23%) 2800
`(90%) 600 + (10%) 2800 6.5
`
`105
`174
`140
`140
`
`26
`26
`26
`26
`
`n°
`
`l
`2
`3
`4
`
`G', G”,
`tan - delta
`Appearance* (1 Hz)
`M
`143/65/0.40
`B
`1300/100/0.08
`M
`262/27/0.10
`M
`571/41/0.07
`
`FaP Sm
`27 G1/2
`25
`24
`15
`14
`
`Remanence
`index
`1
`6
`3.6
`7.7
`
`*M = monophase
`B = biphase
`
`[0093] The attached FIGURE shows the following curve:
`Tan .delta=f (stressing frequency)
`
`for each of the four hydrogels prepared according to
`Examples 1 to 4.
`
`[0094] The rheological behavior of the hydrogels of the
`invention (Examples 3 and 4) is different from that of the
`hydrogel of the prior art (Example 1).
`
`the hydrogels of the invention are
`[0095] Furthermore,
`monophase and thus very different from the hydrogel of
`Example 2 (biphase).
`
`1. Process for the crosslinking of at least one polymer
`selected from polysaccharides and derivatives thereof,
`which is carried out in an aqueous solvent by the action of
`an effective and non-excessive amount of at
`least one
`
`crosslinking agent, characterized in that it is carried out on
`
`6. Process according to claim 5, characterized in that said
`mixture has an intrinsic viscosity of less than 1900 ml/g.
`7. Process according to claim 5 or 6, characterized in that
`said mixture contains more than 50% by weight, advanta-
`geously more than 70% by weight, of at least one hyaluronic
`acid salt of low molecular weight m, and less than 50% by
`weight, advantageously less than 30% by weight, of at least
`one hyaluronic acid salt of high molecular weight M.
`8. Process according to any one of claims 5 to 7, char-
`acterized in that said mixture contains at least 5% by weight
`of at least one high-molecular weight hyaluronic acid salt.
`9. Process according to any one of claims 5 to 8, char-
`acterized in that said mixture contains about 90% by weight
`of the sodium salt of hyaluronic acid having a molecular
`weight of about 3.105 Da, and about 10% by weight of the
`sodium salt of hyaluronic acid having a molecular weight of
`about 3.106 Da.
`
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`
`to 9,
`10. Process according to any one of claims 1
`characterized in that said crosslinking agent is selected from
`bifunctional crosslinking agents and mixtures thereof,
`is
`advantageously selected from epichlorohydrin, divinyl sul-
`fone,
`1,4-bis(2,3-epoxypropoxy)butane,
`1,2-bis(2,3-ep-
`oxypropoxy) ethylene, 1-(2,3-epoxypropyl)-2,3-epoxycy-
`clohexane, aldehydes such as formaldehyde, glutaraldehyde
`and crotonaldehyde, and mixtures thereof, and very advan-
`tageously consists of 1,4-bis(2,3-epoxypropoxy)butane.
`11. Process according to any one of claims 1
`to 10,
`characterized in that
`said effective and non-excessive
`
`amount of at least one crosslinking agent is such that the
`degree of crosslinking, defined by the ratio: 100><(total
`number of reactive groups in said crosslinking agent/total
`number of disaccharide units in the polymer molecules
`present),
`is theoretically between 0.5 and 70%, advanta-
`geously between 4 and 50%.
`12. Process
`for
`the preparation of an injectable
`monophase hydrogel of at least one crosslinked polymer
`selected from polysaccharides and derivatives thereof, char-
`acterized in that it comprises:
`
`the crosslinking of a mixture according to any one of
`claims 1 to 11; and
`
`the formulation of said crosslinked mixture, neutralized if
`necessary, into a solution buffered to a pH of between
`
`6.5 and 7.5, advantageously of between 7 and 7.4 and
`very advantageously of between 7.1 and 7.3.
`13. Process according to claim 12, characterized in that it
`comprises:
`
`the crosslinking of a mixture according to any one of
`claims 3 to 11; and
`
`the formulation of said crosslinked mixture, neutralized,
`into a solution buffered to a pH of between 7.1 and 7.3,
`at a concentration of between 10 and 40 mg/g, advan-
`tageously of between 20 and 30 mg/g.
`14. Crosslinked polymer obtainable after a crosslinking
`process according to any one of claims 1
`