(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0194758 A1
`Lebreton
`(43) Pub. Date:
`Aug. 31, 2006
`
`US 2006O194758A1
`
`(54) CROSS-LINKING OF LOW AND HIGH
`MOLECULAR WEIGHT
`POLYSACCHARDES PREPARATION OF
`NECTABLE MONOPHASE HYDROGELS
`AND POLYSACCHARDES AND
`DYDROGELS THUS OBTANED
`
`(76)
`
`Inventor:
`
`Pierre Lebreton, Annecy Le Vieux
`(FR)
`
`Correspondence Address:
`COHEN, PONTANI, LIEBERMAN & PAVANE
`SS1 FIFTHAVENUE
`SUTE 1210
`NEW YORK, NY 10176 (US)
`
`(21)
`
`Appl. No.:
`
`10/552,309
`
`(22)
`
`PCT Fed:
`
`Apr. 8, 2004
`
`(86)
`
`PCT No.:
`
`PCTAFRO4/OO870
`
`(30)
`
`Foreign Application Priority Data
`
`Apr. 10, 2003 (FR).............................................. O3O4444
`
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`A6II 3L/728
`(2006.01)
`COSB 37/00
`(52) U.S. Cl. ................................................. 514/54; 536/53
`
`ABSTRACT
`(57)
`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, 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.
`
`Exhibit 1029
`Prollenium v. Allergan
`
`

`

`Patent Application Publication Aug. 31, 2006
`
`US 2006/0194758A1
`
`- - - - - - example 1
`-e-example 2
`-0- example 3
`-- example 4
`"- .
`. . ."
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`--"
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`. .
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`. "
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`...
`-- " .
`--"
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`0,6
`
`0,5
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`0,4
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`0,3
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`0,2
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`0,1
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`O,01
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`0,1
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`1
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`10
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`Frequency (Hz)
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`

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`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`CROSS-LINKING OF LOW AND HIGH
`MOLECULAR WEIGHT POLYSACCHARDES
`PREPARATION OF INUECTABLE MONOPHASE
`HYDROGELS AND POLYSACCHARDES AND
`DYDROGELS THUS OBTANED
`0001. The present invention relates to:
`0002 a novel process for the crosslinking of at least one
`polymer selected from polysaccharides and derivatives
`thereof
`0003 a process for the preparation of an injectable
`monophase hydrogel of at least one such polymer; and
`0004 the
`crosslinked polymers and injectable
`monophase hydrogels respectively obtainable by each of
`said processes.
`0005 The hydrogels in question, based on said
`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).
`0007. It is pointed out here that the term “injectable”
`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 G/3, 27 G4, 26
`G/2 and 25 G.
`0008 According to the prior art, hydrogels, especially
`injectable hydrogels, have already been prepared from
`polysaccharides and derivatives thereof especially hyalu
`ronic acid salts—having 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:
`0010 the biphase compositions described in patent appli
`cation EP-A-0 466 300 consist of two bioabsorbable
`phases—continuous and disperse—and 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);
`
`0011 the biphase compositions described in patent appli
`cation WO-A-96 33751 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);
`0012 the biphase compositions described in patent appli
`cation WO-A-00014 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.
`0015. In patent applications WO-A-98 356 39 and WO
`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 U.S. 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-02063
`50 describes a process capable of generating this type of
`hydrogel that is very homogeneous throughout.
`0016 All these monophase hydrogels were obtained
`from high-molecular weight polymers crosslinked using an
`effective and non-excessive amount of at least one crosslink
`ing agent, in an aqueous solvent.
`0017. In the light of this prior art, the inventors wished to
`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.
`0018 To improve the crosslinking efficacy, the inventors
`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.
`0019 Said inventors then considered increasing the con
`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
`hyaluronate is always used with high molecular weights
`(Mws10 Da, s2.10 Da, 3.10°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
`
`

`

`US 2006/0194758 A1
`
`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 (m), which is given by the Mark-Houwink formula:
`M=km, the values of k and C. 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.
`0021. In such a context, the inventors surprisingly estab
`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:
`monophase;
`0022)
`0023 better mechanical properties and remanence than
`the equivalent products of the prior art;
`0024 unaffected or even improved injectability that is
`still possible with conventional injection forces using con
`ventional injection devices.
`0.025 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.
`0027 Said mixture of course contains said low-molecular
`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.
`0.031) 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).
`0035) In the context of the crosslinking of this type of
`polymer, those skilled in the art understand that said
`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.
`0036). In the context of the crosslinking of this type of
`polymer (hyaluronic acid salt(s)), in one preferred variant of
`carrying out the crosslinking, the reaction mixture contains:
`0037 at least one hyaluronic acid salt of low molecular
`weight m, where ms 9.9.10 Da, advantageously 10
`Dasms 9.9.10 Da; and
`0038 at least one hyaluronic acid salt of high molecular
`weight M, where Me 10° Da, advantageously 10°
`Das Ms 10 Da and very advantageously 1.1.10°
`Das Ms 5.10 Da.
`
`

`

`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).
`0039. In such a context, said reaction mixture advanta
`geously has an intrinsic viscosity of less than 1900 ml/g, i.e.
`X.comos 1900 ml/g, where () is the mass fraction of
`polymer fraction i, having an intrinsic viscosity mo, 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 above 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).
`0041. In the context referred to here (NaHA of molecular
`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.
`0042. In general, to obtain the expected effect, there is at
`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: ms3.10 Da and Ms3.10 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.
`0045. It is recommended to use bifunctional crosslinking
`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
`(t), defined by the following ratio:
`
`Total number of reactive groups
`100
`in said crosslinking agent
`Total number of disaccharide X I () (),
`units in the polymer molecules
`
`0047 The crosslinking process of the invention is novel
`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.
`0049. In one preferred variant of carrying out the inven
`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 above), 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 above.
`0052 Said polymer and hydrogel advantageously contain
`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
`gel—fourth Subject of the present invention is novel. 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:
`
`tan delta = , = f(stressing frequency).
`, -
`
`is theoretically between 0.5 and 70%, advantageously
`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.
`
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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);
`0.058 Example 2 illustrates the remarks made in the
`introduction of the present text (crosslinking of the same
`polymer of low molecular weight); and
`0059 Examples 3 and 4 illustrate the invention
`(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 G/2, at a rate of 12.5 mm/min. The tests were
`performed on a Verstatet(R) 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
`0064 Microscopic appearance: no apparent
`phase—fine fragmentation of the gel into facets
`0065 Macroscopic appearance: soft and free-flowing
`Biphase
`0.066 Microscopic appearance: gel fragments bathed in a
`low-viscosity liquid medium
`0067 Macroscopic appearance: “purée’ that fragments
`very easily—no cohesion of the gel and no free-flowing
`appearance
`
`liquid
`
`EXAMPLE 1.
`
`High-Molecular Weight Fibers
`0068 3.5 g of sodium hyaluronate (NaHA) fibers of
`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 /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/NaHA]=6%; NaHA]= 10 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=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.
`Injection force after sterilization: 25 N
`0071)
`0072 Remanence index of the hydrogel: 1.0
`0.073 Monophase hydrogel
`
`EXAMPLE 2
`
`Low-Molecular Weight Fibers
`0074) 1.56 g of sodium hyaluronate (NaHA) fibers of
`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 /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/NaHA]=6.8%; NaHA]= 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=26 mg/g) and the hydrogel is mechani
`cally homogenized before being packed into Syringes and
`sterilized in an autoclave.
`Injection force after sterilization: 24 N
`0.077
`0078 Remanence index of the hydrogel: 6.0
`0079 Biphase hydrogel
`
`EXAMPLE 3
`
`Mixture of Fibers
`0080) 0.763 g of sodium hyaluronate (NaHA) fibers of
`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).
`
`

`

`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`0081. The procedure remains identical to that of Example
`2.
`0082) R=BDDE/NaHA]=7%; NaHA)=140 mg/g:
`NaHA=26 mg/g
`Injection force after sterilization: 15 N
`0083)
`0084. Remanence index of the hydrogel: 3.6
`0085 Monophase hydrogel
`
`EXAMPLE 4
`
`Mixture of Fibers
`0.086 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)/NaHA]=6.5%: NaHA)=140 mg/g:
`NaHA=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.
`
`a mixture containing at least one low-molecular weight
`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 ms 9.9.10
`Da, advantageously 10
`Dasms 9.9.10 Da; and
`at least one hyaluronic acid salt of high molecular weight
`M, where Me 10 Da, advantageously 10° Das Ms 10
`Da, and very advantageously 1.1.10 Das Ms 5.10
`Da,
`said low-molecular weight and high-molecular weight
`salts advantageously being of the same nature and very
`advantageously consisting of Sodium hyaluronate.
`
`TABLE
`
`NaHA = concentration of NaHA in the reaction medium at to
`NaHA = 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'
`m: intrinsic viscosity of the NaHA fiber?Ubbelohde viscometer
`F: ejection force of the gel in N through a 27 G/3 needle/100 N dynamometer
`
`Carrimed CSL 500 rheometer
`
`nint. (ml/g)
`% = proportion by R =
`weight in mixture
`mBDDE/mNaHA
`
`NaHA
`mgg
`
`NaHA in
`final gel
`mgg
`
`G', G",
`tan delta
`Appearance' (1 Hz)
`
`n
`
`Faster Remanence
`27 GA,
`index
`
`6%
`(100%) 2800
`1
`6.8%
`(100%) 600
`2
`3 (77%) 600 + (23%) 2800 7
`4 (90%) 600 + (10%) 2800 6.5
`
`105
`174
`140
`140
`
`26
`26
`26
`26
`
`M 143,6S.O.40
`B
`1300,100.0.08
`M 262,27 O.10
`M 571,41 O.O7
`
`25
`24
`15
`14
`
`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).
`0.095. Furthermore, the hydrogels of the invention are
`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 3.10 Da, and about 10% by weight of the
`Sodium salt of hyaluronic acid having a molecular weight of
`about 3.10 Da.
`
`

`

`US 2006/0194758 A1
`
`Aug. 31, 2006
`
`10. Process according to any one of claims 1 to 9,
`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: 100x(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 4