`
`PCT/FR96/00636
`WO 96/33751
`
`Injectable two-phase compositions containing hyaluronic
`acid, notably useful in reconstructive and cosmetic
`surgery
`
`The present invention relates to:
`
`
`– two-phase compositions containing a polymer
`selected from hyaluronic acid and its salts,
`
` –
`
` a method of preparing said compositions,
`
` –
`
` a filling material, useful in reconstructive
`surgery and in cosmetic surgery, based on said
`two-phase compositions.
`
`
`The present invention notably proposes a solution to
`the technical problem of permanent filling of volume
`defects of the skin, such as wrinkles or scars, notably
`on the face. More generally, it proposes an original
`formulation based on hyaluronic acid.
`
`Said hyaluronic acid is a glycosaminoglycan or
`mucopolysaccharide of high molecular weight that occurs
`in animal tissues such as the umbilical cord, vitreous
`humor, synovial fluid, cock's combs, the skin,
`connective tissues (joints, tendons etc.) etc. Said
`acid can thus be obtained naturally by extraction from
`certain of said animal tissues (notably from cock's
`combs and umbilical cords). It can also be obtained by
`bacterial fermentation.
`
`The chemical structure of said acid is that of a
`polymer having disaccharide monomers of N-acetyl-D-
`glucosamine and of D-glucuronic acid, said amine and
`said acid being joined together by a β1 → 3 glycosidic
`bond. The disaccharide monomers themselves are joined
`together by β1 → 4 glycosidic bonds to generate the
`unbranched, noncrosslinked polysaccharide chain.
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`PCT/FR96/00636
`WO 96/33751
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`However, said chain has, at the level of its monomers,
`functions that allow it to be crosslinked chemically to
`create a network of varying density.
`
`It is generally known that any molecule is much more
`resistant to degradation and to heat when it is
`crosslinked. Thus, it is known that there is interest
`in crosslinking crosslinked hyaluronic acid. Said
`crosslinked hyaluronic acid is much more stable in the
`body than the hyaluronic acid molecule. It is also more
`resistant to autoclave sterilization.
`
`Hyaluronic acid is known for its viscoelastic
`properties as well as its very great tendency to absorb
`water. Its properties largely explain the elasticity of
`the skin. Its biocompatibility, tolerance and toxicity
`have been investigated extensively, since this molecule
`has been used in the medical and cosmetic fields for
`more than 10 years. Its use is notably known in
`ophthalmological surgery, for treating osteoarthritis,
`and for treating patients with third-degree burns.
`
`According to the prior art, numerous compositions of
`various types, containing hyaluronic acid, have
`therefore already been described. Notably the following
`have been described:
`
`
`– in patent US-A-5,137,875: injectable collagen
`solutions or dispersions containing hyaluronic
`acid in solution as well as use thereof for
`filling voids in soft tissues;
`
`– in patent US-A-4,716,154: a gel of crosslinked
`hyaluronic acid, as a substitute for the vitreous
`humor;
`
`– in application EP-A-0 466 300: a viscoelastic gel
`comprising a gelatinous phase (that has undergone
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`PCT/FR96/00636
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`a low degree of crosslinking) dispersed in a
`liquid
`phase
`(that
`has
`not
`undergone
`crosslinking); said two phases advantageously
`having been prepared from fibers of Hylan
`(natural hyaluronic acid modified chemically in
`situ for the purpose of facilitating its
`extraction from tissues). It is recommended to
`use said compositions in many contexts in the
`medical field.
`
`
`According to the first aim of the invention, as stated
`above, two-phase compositions are proposed that contain
`hyaluronic acid or a salt thereof (called the polymer)
`and that have an original structure. Said compositions
`consist of an injectable suspension whose dispersed
`phase consists of insoluble fragments of a hydrogel of
`said highly crosslinked polymer and whose continuous
`phase consists of an aqueous solution of said polymer
`and/or of another biocompatible polymer, selected from
`proteins, polysaccharides and derivatives thereof,
`slightly crosslinked or not crosslinked.
`
`The term hyaluronic acid is used hereinafter as a
`generic name to denote both hyaluronic acid per se and
`its salts and notably the hyaluronate salts. The two-
`phase compositions of the invention advantageously
`contain as polymer selected from hyaluronic acid and
`its salts, at least in their dispersed phase, sodium
`hyaluronate. It is to be noted here that said sodium
`hyaluronate used is advantageously of bacterial origin.
`
`The two-phase compositions of the invention are
`injectable compositions. They have been formulated for
`this purpose. This is notably why they contain a
`continuous phase; said phase serves as injection
`vehicle for the fragments of the dispersed phase.
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`The qualification injectable, used in the present text,
`signifies injectable manually by means of syringes
`equipped with conventional needles. The two-phase
`compositions of the invention are particularly
`interesting in that they can be formulated to be
`injectable by means of very fine needles (with a
`diameter between 0.3 and 0.5 mm). A person skilled in
`the art will understand that the determining parameter
`is the largest dimension of the fragments in
`suspension. In the context of the present invention, it
`is notably possible to formulate compositions,
`containing
`hyaluronic
`acid,
`injectable
`through
`hypodermic needles of 30 G, 26 G1/2, 25 G. Said
`compositions constitute the most advantageous variant
`of the compositions of the invention.
`
`The main characteristics of each of the phases of the
`two-phase compositions of the invention are discussed
`in greater detail below.
`
`The continuous phase, which, as already stated above,
`serves as injection vehicle, consists of an aqueous
`solution which, characteristically, contains hyaluronic
`acid (or a salt thereof) and/or another biocompatible
`polymer selected from proteins, polysaccharides and
`derivatives thereof. Said hyaluronic acid and/or the
`other polymer used are not crosslinked or are slightly
`crosslinked (by means of a crosslinking agent). The
`viscosity of said continuous phase should remain
`compatible with its function of injection vehicle. It
`should be noted that said continuous phase also fulfils
`another function. After injection and implantation of
`the two-phase composition, it protects the dispersed
`phase, and slows down its degradation.
`
`Said continuous phase can notably be in the form of a
`solution or in the form of a gel.
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`PCT/FR96/00636
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`It is not excluded from the scope of the present
`invention that said continuous phase contains a mixture
`of polymers.
`
`According to an advantageous embodiment of the
`invention, it contains the same polymer as the
`dispersed
`phase,
`namely
`hyaluronic
`acid;
`said
`hyaluronic acid being used, however, in said continuous
`phase with a far lower degree of crosslinking, or even
`not crosslinked at all. The use of hyaluronic acid in
`the two phases is widely preferred in view of the
`advantageous properties of this product, which can
`notably be obtained by a bacterial route, by a cellular
`route (therefore free from any contaminant of the virus
`or prion type) and which has both a highly gelatinous
`character, appreciable lubricity, good biocompatibility
`as well as good behavior in the body.
`
`The other polymers that may be used in the continuous
`phase of the compositions of the invention are, as
`stated
`above,
`proteins
`or
`polysaccharides
`and
`derivatives thereof. We may mention, non-exhaustively,
`among the proteins whose use is recommended: collagen,
`albumin, elastin etc.;
`
`among the polysaccharides and derivatives thereof,
`whose use is also recommended (other than hyaluronic
`acid): chondroitin sulfates, keratan sulfates, heparin,
`alginic acid, starch, carboxymethylcellulose, etc.
`
`it,
`within
`contains
`phase
`continuous
`Said
`characteristically, insoluble fragments of a hydrogel
`of highly crosslinked hyaluronic acid. These insoluble
`fragments (of variable geometry) constitute real
`entities that can be separated from the diluted
`continuous phase by decanting or centrifugation. Within
`said fragments, the hyaluronic acid is highly
`crosslinked: a network of a relative density was
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`PCT/FR96/00636
`WO 96/33751
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`constituted from chains of hyaluronic acid and of a
`crosslinking agent. This is explained and quantified
`further in the present text.
`
`The use of fragments (or particles) of this type
`containing hyaluronic acid, as dispersed phase in a
`continuous phase as described above, endows the two-
`phase compositions of the invention with their original
`structure.
`It
`notably
`makes
`said
`compositions
`precursors of permanent filling materials or implants,
`useful in reconstructive surgery and in cosmetic
`surgery, that are original and have particularly high
`performance.
`
`The injectable two-phase compositions of the invention
`are quite particularly intended for dermal injection
`(superficial, medium or deep) for implantation in the
`dermis. For this purpose, with the aim of eliminating
`any unpleasant sensation or even any pain during
`injection thereof and during implantation thereof, they
`are advantageously buffered at a pH between 6.5 and
`7.5, preferably between 7 and 7.4, even more preferably
`between 7.2 and 7.3.
`
`Thus, the aqueous solution (continuous phase) on the
`one hand and the hydrogel (dispersed phase) on the
`other hand are advantageously buffered at these pH
`values.
`
` A
`
` phosphate buffer is generally used.
`
`The advantageous characteristics of the fragments
`constituting the dispersed phase of the two-phase
`compositions of the invention are noted below.
`
`Said fragments can be included in said compositions, at
`equilibrium, under- or over-hydrated. When they are
`injected,
`under
`or
`over-hydrated,
`they
`reach
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`PCT/FR96/00636
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`equilibrium, after implantation. In any case, they are
`used with dimensions (and in an amount) compatible with
`the
`means
`envisaged
`for
`their
`injection.
`Advantageously, more than half of said fragments have
`their largest dimension between 40 and 280 µm,
`preferably
`between
`75
`and
`250 µm.
`Even
`more
`advantageously, almost all of said fragments have that
`characteristic. We are then dealing with compositions
`that are injectable by means of fine needles, with a
`diameter between 0.3 and 0.5 mm.
`
`The term largest dimension of said fragments has been
`used; the term equivalent diameter could also have been
`used. The insoluble fragments of hydrogel of the two-
`phase compositions of the invention are obtained, as
`will be stated later in the present text, by shearing
`of a mass and therefore do not generally consist of
`spheres.
`
`The hydrogel constituting said fragments was obtained
`from hyaluronic acid that has been highly crosslinked
`by means of a crosslinking agent. For obtaining
`fragments containing a reasonable amount of said
`crosslinking agent, it is recommended to use, as
`starting material, a hyaluronic acid whose molecular
`weight is greater than or equal to 1 million dalton.
`According to an advantageous embodiment, it is
`recommended to use a hyaluronic acid whose molecular
`weight is between 1 and 3 million dalton. It is,
`moreover, recommended to effect said crosslinking via
`the hydroxyl functions of the hyaluronic acid, by means
`of a crosslinking agent, in conditions which lead to a
`degree of crosslinking of said hyaluronic acid
`(starting material) characterized by the ratio: total
`number of reactive functions of said crosslinking
`agent/total number of disaccharide units of the
`molecules of hyaluronic acid present, between 0.8 and
`1.
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`PCT/FR96/00636
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`
`In fact, the network of insoluble fragments of the two-
`phase compositions of the invention is based on
`molecules of hyaluronic acid joined together by bridges
`of molecules of crosslinking agent; each of the
`disaccharide units of said molecules of hyaluronic acid
`advantageously having between 0.8 and 1 of its hydroxyl
`functions used in said bridges.
`
`The range indicated for said degree of crosslinking is
`an optimal range. The use, in compositions of the
`invention, of fragments having said degree of
`crosslinking below the value 0.8 (while making sure
`that said fragments still have their intrinsic
`properties of insolubility) or beyond the value 1 (then
`taking care not to alter excessively the nature of the
`fragments based on hyaluronic acid, which will contain
`more and more crosslinking agent), is not excluded.
`
`As crosslinking agent, it is possible to use, for
`generating the hydrogel constituting the insoluble
`fragments of the two-phase compositions of the
`invention, any agent known to crosslink hyaluronic acid
`via its hydroxyl functions – at least bifunctional
`crosslinking agent – and notably a polyepoxide or
`derivatives thereof. As said crosslinking agent, it is
`notably
`possible
`to
`use
`epichlorohydrin,
`divinylsulfone,
`1,4-bis(2,3-epoxypropoxy)butane
`(or
`1,4-bis(glycidyloxy)butane or 1,4-butanediol diglycidyl
`ether = BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene, 1-
`(2,3-epoxypropyl)-2,3-epoxy cyclohexane etc. The use of
`several crosslinking agents is not excluded from the
`scope of the invention.
`
`Moreover, the insoluble fragments of hydrogel of the
`compositions of the invention can be characterized by
`other parameters, such as their dry matter content or
`their optical properties.
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`
`The dry matter content of said fragments was measured
`in the context of the invention, with said fragments
`buffered (at a pH between 6.5 and 7.5), at equilibrium.
`In these conditions, said fragments of the invention
`advantageously have a proportion of dry matter between
`1.5 and 20%, even more advantageously between 5 and
`15%.
`
`Said fragments, in the same conditions, transmit less
`than 5% of light at 400 nm.
`
`The two-phase compositions of the invention generally
`contain from 10 to 200 mg/ml, advantageously from 20 to
`150 mg/ml, of said insoluble fragments in suspension in
`their continuous phase. Introducing an excessive amount
`of dispersed phase in said continuous phase can
`compromise
`the
`injectable
`character
`of
`said
`compositions. Introducing an insufficient amount of
`said dispersed phase in said continuous phase may
`greatly reduce the advantages of said compositions.
`They have in fact been designed for allowing the
`placement by injection of an effective amount of highly
`crosslinked hyaluronic acid (therefore resistant to
`degradation). It is in fact necessary to optimize the
`ratio: active mass transported/mass of injection
`vehicle.
`
`Advantageous characteristics of the aqueous solution
`constituting the continuous phase of the two-phase
`compositions of the invention are noted below. Said
`continuous phase contains hyaluronic acid and/or
`another biocompatible polymer, slightly crosslinked or
`not crosslinked.
`
`Said continuous phase can be characterized by its
`intrinsic viscosity [η]. This parameter is given by the
`formula:
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`[η] = limc→0
`
`
`
`η - η0
`η0 c
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`PCT/FR96/00636
`
`
`in which η0 is the viscosity of the solvent,
`
`
`η is the measured viscosity of the solution,
`
`
`c is the concentration of said solution.
`
`Said continuous phase of the two-phase compositions of
`the invention advantageously has an intrinsic viscosity
`between 1500 and 3200 ml/g. More preferably the
`intrinsic viscosity is between 2000 and 2700 ml/g.
`
`Said intrinsic viscosity of said continuous phase
`depends of course on the nature of the polymer used,
`its concentration and its degree of crosslinking.
`
`Said viscosity is to be optimized, in the context of a
`compromise, in that said continuous phase is to
`constitute the injection vehicle of the insoluble
`fragments on the one hand, and is to slow down the
`degradation of said implanted fragments, on the other
`hand.
`
`Generally, this involves optimization at the level of
`the composition of said continuous phase; the
`parameters available being the nature of the polymer(s)
`used, the concentration of said polymer(s), and
`optionally the degree of crosslinking of said
`polymer(s).
`
`Whatever the nature of said polymer used in the
`continuous phase, a person skilled in the art is able
`to control its crosslinking, when the latter is
`possible.
`
`We have seen that according to an advantageous
`embodiment of the invention, hyaluronic acid is also
`used at the level of said continuous phase. Said
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`PCT/FR96/00636
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`hyaluronic acid used here can be noncrosslinked or
`slightly crosslinked. It is also recommended here to
`employ a hyaluronic acid whose molecular weight is
`greater than or equal to 1 million dalton and even more
`advantageously a hyaluronic acid whose molecular weight
`is between 1 and 3 million dalton. When said hyaluronic
`acid is slightly crosslinked, it is recommended to
`effect said crosslinking, via its hydroxyl functions,
`by means of a crosslinking agent, in conditions that
`lead to a degree of crosslinking of said hyaluronic
`acid characterized by the ratio: total number of
`reactive functions of said crosslinking agent/total
`number of disaccharide units of the hyaluronic acid
`molecules present, between 0.01 and 0.4. It can be seen
`that said degree of crosslinking for the hyaluronic
`acid used in the continuous phase is generally always
`less than half that of the hyaluronic acid used in the
`dispersed phase.
`
`during
`effected
`generally
`is
`crosslinking
`The
`preparation of the continuous phase in the same way as
`it is effected during preparation of the dispersed
`phase; when hyaluronic acid is used in both said
`phases.
`
`Thus, said crosslinking of the continuous phase also
`advantageously uses a polyepoxide and notably 1,4-
`bis(2,3-epoxypropoxy)butane as crosslinking agent.
`
`In this case said polyepoxide is attached by ether
`bridges to the disaccharide units of the chains of
`hyaluronic acid, present in the continuous and the
`dispersed phases.
`
`Said hyaluronic acid, present in the dispersed phase of
`the two-phase compositions of the invention, or even
`advantageously
`in
`both
`phases,
`dispersed
`and
`continuous, of said compositions (to the exclusion of
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`PCT/FR96/00636
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`any other polymer according to a particularly preferred
`variant) is preferably a hyaluronic acid that was
`obtained by a bacterial route.
`
`According to the preferred variant of the invention, an
`injectable suspension is proposed as defined above
`containing hyaluronic acid, obtained by bacterial
`fermentation, in both its phases. By means of such a
`suspension, which is highly biocompatible, insoluble
`fragments are injected very easily and said fragments
`are particularly resistant to degradation, once
`implanted, on account of their intrinsic structure and
`because they are protected by the continuous phase with
`which they were injected.
`
`According to the second aim of the present invention, a
`method is proposed for preparing the two-phase
`compositions described above. Said method comprises,
`characteristically:
`
`
`– preparing and purifying an insoluble hydrogel of
`highly crosslinked hyaluronic acid;
`
`– fragmenting said hydrogel by shearing;
`
`– suspending the fragments of said sheared hydrogel
`in a suitable continuous phase.
`
`
`During the first of the above steps, a hydrogel of
`highly crosslinked hyaluronic acid should be prepared.
`For this purpose, generally the raw material used
`(often fibers of sodium hyaluronate) is dissolved and
`it is reacted with a suitable crosslinking agent in
`suitable proportions. A hyaluronic acid whose molecular
`weight is greater than or equal to 1 million dalton,
`preferably between 1 and 3 million dalton, is
`advantageously used as starting material, and it is
`crosslinked so that the ratio: total number of reactive
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`PCT/FR96/00636
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`functions of said crosslinking agent/total number of
`disaccharide units of the hyaluronic acid molecules
`present, is between 0.8 and 1. At the end of the
`crosslinking reaction, the reaction mixture is purified
`to remove the unreacted reactants. This purification
`can be carried out by extraction with deionized water
`in Soxhlet apparatus. The purified hydrogel obtained is
`then fragmented by shearing. Said shearing generates
`fragments of nonuniform geometry and size. A person
`skilled in the art will be able to optimize this
`operation to obtain suitable fragments.
`
`Said fragments are then suspended in the continuous
`phase prepared beforehand or in parallel.
`
`Said continuous phase is prepared in a similar manner
`if crosslinking is to be effected. Advantageously, said
`continuous phase is prepared from a hyaluronic acid
`whose molecular weight is greater than or equal to 1
`million dalton, preferably between 1 and 3 million
`dalton, and said hyaluronic acid is crosslinked so that
`the ratio: total number of reactive functions of the
`crosslinking agent/total number of disaccharide units
`of the hyaluronic acid molecules present, is between
`0.01 and 0.4. If such crosslinking is effected to a
`lesser degree, purification of the crosslinked product
`must generally be effected by techniques other than
`extraction. It is notably possible to carry out
`successive cycles of solubilization/precipitation, or
`even dialysis.
`
`If no crosslinking is carried out, the continuous phase
`is prepared by simple mixing of the raw material in an
`aqueous solution.
`
`The phases obtained at the end of their preparation
`process are more or less hydrated.
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`For mixing them, for suspending the fragments of
`hydrogel in the continuous phase, notably one or other
`of the following variants can be adopted, which are not
`in any way exhaustive:
`
`
`– notably the suitably hydrated fragments can be
`mixed in a continuous phase prepared with the
`desired degree of hydration,
`
`– it is also possible to mix said dry fragments as
`well as a dry product that is a precursor of the
`continuous phase and suitably hydrate said dry
`mixture,
`
`– it is also possible to envisage mixing of the two
`phases, one in the dry state, and the other in
`the hydrated state, and adjust the degree of
`hydration of the mixture if necessary.
`
`
`The first of these variants is preferred.
`
`The compositions of the invention thus prepared can be
`packaged, notably in syringes, and then sterilized, in
`an autoclave for example.
`
`Earlier in the present text it was seen that they are
`recommended for injection in the skin as filling
`material, more precisely as precursor of such a filling
`material, which records its long-term efficacy.
`
`According to its last aim, the invention therefore
`relates to a filling material useful in reconstructive
`surgery and in cosmetic surgery, based on the two-phase
`compositions as described above.
`
`Said material, characteristically, has a structure
`which, after injection and implantation of the latter
`in the dermis, evolves.
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`Following absorption of the continuous phase (provided
`for protecting the dispersed phase), the fragments of
`the dispersed phase unite and generate a stable film.
`This film is a completely original structure.
`
`It is recommended to use this filling material notably
`for filling wrinkles on the face such as glabellar
`wrinkle, perioral wrinkles, the nasomental furrows, for
`attenuating crow's-feet etc.
`
`The invention is illustrated by the following examples.
`
`Example 1
`
`A two-phase composition of the invention is prepared
`from a single polymer: fibers of sodium hyaluronate (of
`molecular weight: Mw = 2.106), of bacterial origin.
`
`Two solutions, at 11.8 wt% of said fibers, in 0.25 M
`soda are prepared first.
`
`.430 µl of 1,4-bis(2,3-epoxypropoxy)butane (butanediol
`diglycidyl ether: BDDE) is added to one of said
`solutions. The homogenized mixture is put in a water
`bath at 50°C for 2 hours. The hydrogel obtained is a
`solid. It is purified (by extraction with deionized
`water in Soxhlet apparatus) in order to remove both the
`crosslinking agent (BDDE) and the unreacted polymer
`from its structure. In said hydrogel, the ratio: total
`number of reactive functions of said crosslinking
`agent/total number of disaccharide units of the
`molecules of the polymer present, is 0.84. At
`equilibrium in phosphate buffer, said hydrogel - highly
`crosslinked - contains 9.7 wt% of dry matter. In this
`state, it transmits less than 5% of light at 400 nm.
`
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`PCT/FR96/00636
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`Said purified hydrogel is then sheared to obtain solid
`fragments of it with an average size between 75 and
`250 µm. These fragments are suspended in a phosphate
`buffer at pH 7.2, at a rate of 6 g per 100 ml of
`phosphate buffer.
`
`In parallel, 31 µl of 1,4-bis(2,3-epoxypropoxy)butane
`(butanediol diglycidyl ether: BDDE) is added to the
`other of said solutions. The homogenized mixture is
`also put in a water bath at 50°C for 2 hours. At the
`end of this heating, a very viscous fluid is obtained,
`and it too must be purified. In this viscous fluid, the
`ratio: total number of reactive functions of said
`crosslinking agent/total number of disaccharide units
`of the molecules of the polymer present, is 0.12. The
`continuous phase of the desired two-phase composition
`is prepared from said fluid. Said fluid is in fact
`purified by solubilization/precipitation. At the end of
`said purification, the precipitate is dried and then
`re-hydrated with a phosphate buffer at pH 7.2, at a
`rate of 6 g of said precipitate per 100 ml of said
`buffer.
`
`The suspension containing the fragments and the viscous
`aqueous solution are then mixed in proportions of 1/1.
`An
`injectable
`suspension
`is
`obtained,
`notably
`injectable through needles from 26 G 1/2 to 30 G.
`
`Example 2
`
`Generally the procedure in example 1 is followed,
`except that:
`
`1) for preparing the dispersed phase, only 250 µl of
`BDDE is used;
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`PCT/FR96/00636
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`2) for preparing the continuous phase, the fibers of
`hyaluronic acid are dissolved directly in the phosphate
`buffer at pH 7.2 (crosslinking is not carried out);
`
`3) during final mixing, dispersed phase/continuous
`phase, said two phases are used in a ratio of 2/1.
`
`The suspensions (prepared according to example 1 and
`example 2) were injected for filling facial wrinkles in
`10 volunteers. In fact, less than a milliliter of these
`suspensions was injected each time. The product,
`injected in the medium or deep dermis, did not cause
`any undesirable reaction; notably no inflammatory
`reaction, no redness, and no pain. After three months,
`the implant is still present and provides effective
`filling of the cutaneous defect treated. The
`suspensions - two-phase compositions - of the invention
`are effective for long-lasting treatment of cutaneous
`hollows.
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`PCT/FR96/00636
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`Claims
`
`1. A two-phase composition containing a polymer
`selected from hyaluronic acid and its salts,
`characterized in that it consists of an injectable
`suspension whose dispersed phase consists of
`insoluble fragments of a hydrogel of said highly
`crosslinked polymer and whose continuous phase
`consists of an aqueous solution of said polymer
`and/or of another biocompatible polymer, selected
`from proteins, polysaccharides and derivatives
`thereof, slightly crosslinked or not crosslinked.
`
`
`2. The two-phase composition as claimed in claim 1,
`characterized in that said suspension is buffered
`at a pH between 6.5 to 7.5.
`
`
`3. The two-phase composition as claimed in one of
`claims 1 or 2, characterized in that more than
`half of said fragments have their largest
`dimension between 40 and 280 µm and advantageously
`between 75 and 250 µm.
`
`
`4. The two-phase composition as claimed in any one of
`claims 1 to 3, characterized in that the hydrogel
`constituting said fragments is obtained from said
`polymer whose molecular weight is greater than or
`equal to 1 million dalton, advantageously between
`1 and 3 million dalton and which was crosslinked,
`via the hydroxyl functions of said polymer, by
`means of a crosslinking agent, in a ratio: total
`number of reactive functions of said crosslinking
`agent/total number of disaccharide units of the
`molecules of the polymer present, between 0.8 and
`1.
`
`
`5. The two-phase composition as claimed in any one of
`claims 2 to 4, characterized in that said
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`PCT/FR96/00636
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`fragments, at equilibrium, contain between 1.5 and
`20 wt% of dry matter, advantageously between 5 and
`15 wt%.
`
`
`6. The two-phase composition as claimed in any one of
`claims 1 to 5, characterized in that it contains
`from 10 to 200 mg/ml, advantageously from 20 to
`150 mg/ml, of said fragments in suspension in said
`continuous phase.
`
`
`7. The two-phase composition as claimed in any one of
`claims 1 to 5, characterized in that said
`continuous phase has an intrinsic viscosity
`between 1500 and 3200 ml/g.
`
`
`8. The two-phase composition as claimed in any one of
`claims 1 to 7, characterized in that said
`continuous phase is an aqueous solution of said
`polymer whose molecular weight is greater than or
`equal to 1 million dalton, advantageously between
`1 and 3 million dalton and which has optionally
`been crosslinked, via the hydroxyl functions of
`said polymer, by means of a crosslinking agent, in
`a ratio: total number of reactive functions of
`said
`crosslinking
`agent/total
`number
`of
`disaccharide units of the molecules of the polymer
`present between 0.01 and 0.4.
`
`
`9. The two-phase composition as claimed in any one of
`claims 1 to 8, characterized in that the dispersed
`phase, and optionally the continuous phase,
`was/were crosslinked with a polyepoxide and
`notably 1,4-bis(2,3-epoxypropoxy)butane.
`
`
`10. The two-phase composition as claimed in any one of
`claims 1 to 9, characterized in that the
`hyaluronic acid or a salt thereof, present in the
`suspension, i.e. in the dispersed phase and
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`optionally the continuous phase, was obtained by a
`bacterial route.
`
`- 20 -
`
`
`11. A method of preparing a two-phase composition as
`claimed in any one of the preceding claims,
`characterized in that it comprises:
`
`
`12. A filling material useful in reconstructive
`surgery and in cosmetic surgery, characterized in
`that it is based on a two-phase composition as
`claimed in any one of claims 1 to 9, before
`injection and in that, after injection, its
`structure evolves, following absorption of the
`continuous phase, toward that of a stable film
`resulting from the regrouping of the fragments of
`the dispersed phase.
`
`{00578302;v1}
`
`preparing and purifying an insoluble hydrogel
`of said highly crosslinked polymer,
`
`fragmenting said hydrogel by shearing,
`
`suspending the fragments of said hydrogel in
`a suitable continuous phase.
`
`–
`
`
`–
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` –
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`
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