WO1996033751A1

WO1996033751A1 - Injectable hyaluronic acid-containing dual-phase compositions, particularly useful in corrective and plastic surgery

Info

Publication number: WO1996033751A1
Application number: PCT/FR1996/000636
Authority: WO
Inventors: Yves Debacker; Franck Villain; Valérie JALLET
Original assignee: W.K. Et Associes
Priority date: 1995-04-25
Filing date: 1996-04-25
Publication date: 1996-10-31
Also published as: FR2733426A1; AU5766796A; FR2733426B1

Abstract

Dual-phase compositions containing a polymer selected from hyaluronic acid and its salts, method for preparing the compositions, and filler material useful in corrective and plastic surgery based on said dual-phase compositions. The compositions comprise an injectable suspension with a dispersed phase composed of insoluble fragments of a hydrogel of said strongly cross-linked polymer and a continuous phase composed of an aqueous solution of said polymer and/or another biocompatible polymer, selected from proteins, polysaccharides and derivatives thereof, non cross-linked or weakly cross-linked.

Description

Biphasic injectable compositions containing hvaluronic acid. especially useful in restorative and cosmetic surgeries. The subject of the present invention is:

- biphasic compositions containing a polymer chosen from hyaluronic acid and its salts,

- a process for the preparation of said compositions,

- a filling material, useful in reconstructive surgery and in cosmetic surgery, based on said two-phase compositions.

The present invention in particular provides a satisfactory solution to the technical problem of lasting filling of skin volume defects, such as wrinkles or scars, in particular on the face. More generally, it offers an original formulation based on hyaluronic acid.

Said hyaluronic acid is a high molecular weight glycosaminoglycan or mucopolysaccharide found in animal tissues such as umbilical cords, vitreous humor, synovial fluid, rooster crests, skin, connective tissue (joints, tendons ...) ... Said acid can thus be obtained naturally by extraction from some of said animal tissues (rooster combs and umbilical cords in particular). 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-glucoronic acid, said amine and said acid being linked by a glucosidic bond βl - * 3. The monomers disaccharides are themselves linked together by glucosidic bonds βl - * 4 to generate the non-crosslinked polysaccharide chain, without branching.

Said chain has, however, at the level of its monomers, functions which make it possible to crosslink chemically in order to create a more or less dense network.

It is generally known that any molecule is much more resistant to degradation and to heat when it is crosslinked. Thus the advantage of crosslinking crosslinked hyaluronic acid is known. 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 great propensity to absorb water. Its properties largely explain the elasticity of the skin. Its biocompatibility, tolerance and toxicity have been widely studied since, for more than 10 years, this molecule has applications in the medical and cosmetic fields. It is notably known to use it in ophthalmological surgery, to treat osteoarthritis, to treat burn victims. According to the prior art, numerous compositions of different types have therefore already been described, containing hyaluronic acid. We have notably described:

- In patent US-A-5,137,875: injectable solutions or dispersions of collagen containing hyaluronic acid in solution as well as their use for filling gaps in soft tissues; - In patent US-A-4,716,154: a cross-linked hyaluronic acid gel, a substitute for vitreous humor;

- in patent application EP-A-0 466 300: a viscoelastic gel comprising a gelatinous phase (having undergone low level crosslinking) dispersed in a liquid phase (not having undergone crosslinking); said two phases having advantageously been prepared from Hylan fibers (natural hyaluronic acid chemically modified in situ in order to facilitate its extraction from the tissues). It is recommended to use said compositions in many contexts in the medical field.

According to the first subject of the invention, as specified above, two-phase compositions are proposed which contain hyaluronic acid or one of its salts (called the polymer) and which have an original structure. Said compositions consist of an injectable suspension, the dispersed phase of which consists of insoluble fragments of a hydrogel of said highly crosslinked polymer and the continuous phase of which consists of an aqueous solution of said polymer and / or of another biocompatible polymer, chosen from proteins, polysaccharides and their derivatives, weakly or not crosslinked (s).

The term hyaluronic acid is used in the remainder of this text as a generic name to designate both hyaluronic acid per se and its salts and in particular the hyaluronate salts. The two-phase compositions of the invention advantageously contain, as polymer chosen from hyaluronic acid and its salts, at least in their dispersed phase, sodium hyaluronate. It is already specified that said intervening sodium hyaluronate is advantageously of bacterial origin.

The biphasic compositions of the invention are injectable compositions. They were formulated with this in mind. It is especially for this purpose that they contain a continuous phase; said phase serving as an injection vehicle for the fragments of the dispersed phase.

The qualifier injectable, used in the present text, means manually injectable by means of syringes fitted with conventional needles. The biphasic compositions of the invention are particularly advantageous in that they can be formulated to be injectable by means of very fine needles (with a diameter of between 0.3 and 0.5 mm). Those skilled in the art understand that the determining parameter is that of the largest dimension of the fragments in suspension. In the context of the present invention, it is in particular possible to formulate compositions, containing hyaluronic acid, injectable through hypodermic needles of 30 G, 26 Gl 2, 25 G. Said compositions constitute the most advantageous variant compositions of the invention.

The main characteristics of each of the phases of the two-phase compositions of the invention are developed below. The continuous phase which, as already specified above, serves as an injection vehicle consists of an aqueous solution which, typically, contains hyaluronic acid (or one of its salts) and / or a other biocompatible polymer chosen from proteins, polysaccharides and their derivatives. Said hyaluronic acid and / or the other polymer intervenes uncrosslinked or weakly crosslinked (via a crosslinking agent). The viscosity of said continuous phase should remain compatible with its function as an injection vehicle. It should be noted that said continuous phase also provides another function. After injection and implantation of the biphasic composition, it protects the dispersed phase and slows down its degradation. Said continuous phase may in particular exist in the form of a solution or in that of a gel.

It is not excluded from the scope of the present invention that said continuous phase contains a mixture of polymers.

According to an advantageous variant of the invention, it contains the same polymer as the dispersed phase, namely hyaluronic acid; said hyaluronic acid intervening, however, within said continuous phase at a much lower rate of crosslinking, or even in no way crosslinked. The intervention of hyaluronic acid in the two phases is widely preferred in view of the advantageous properties of this product which can in particular be obtained by bacterial route, by cellular route (therefore free of any contaminant of the virus or prion type) and which presents at the same time a strong gelatinous character, an appreciable lubricating power, a good biocompatibility as well as a good behavior in the organism.

The other polymers capable of intervening in the continuous phase of the compositions of the invention are, as indicated above, proteins or polysaccharides and their derivatives. Mention will be made, by way of non-limiting example, among the proteins whose intervention is recommended: collagen, albumin, elastin ... among the polysaccharides and their derivatives, whose intervention is also recommended (other than hyaluronic acid): sulfates of chondroitin, keratan, heparin, alginic acid, starch, carboxymethylcellulose ...

Said continuous phase typically contains within it insoluble fragments of a highly crosslinked hyaluronic acid hydrogel. These insoluble fragments (of variable geometry) constitute real entities which are separable from the continuous phase diluted by decantation or centrifugation. Within said fragments, hyaluronic acid is highly crosslinked: a network of relative density has been formed from chains of hyaluronic acid and a crosslinking agent. This is explained, quantified, later in this text.

The intervention of this type of fragments (or particles) containing hyaluronic acid, as a dispersed phase in a continuous phase as described above, gives the biphasic compositions of the invention their original structure. It makes in particular said compositions, precursors of filling materials or durable implants, useful in restorative surgery and in cosmetic surgery, original and particularly effective.

The biphasic injectable compositions of the invention are very particularly intended for a dermal injection (surface, medium or deep) for implantation in the dermis. To this end, in order to eliminate any unpleasant sensation or even any pain during their injection and during their implantation, they are advantageously buffered at a pH of 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 they advantageously buffered at these pHs. A phosphate buffer is generally used. Advantageous characteristics of the fragments constituting the dispersed phase of the two-phase compositions of the invention are specified below. Said fragments can intervene within said compositions, at equilibrium, under or over-hydrated. When they are injected, under or over-hydrated, they balance, after implantation. In any event, they come in dimensions (and in quantity) compatible with the means provided 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 this characteristic there. We are then dealing with injectable compositions using fine needles, with a diameter of between 0.3 and 0.5 mm. We are talking about the largest dimension of said fragments, we could also talk about their equivalent diameter. The insoluble hydrogel fragments of the two-phase compositions of the invention are obtained, as will be specified later in the present text, by shearing of a mass and therefore generally do not consist of spheres. The hydrogel constituting said fragments was obtained from highly crosslinked hyaluronic acid via a crosslinking agent. It is recommended, to obtain fragments containing a reasonable amount of said crosslinking agent, to use as starting material a hyaluronic acid whose molecular mass is greater than or equal to 1 million Daltons. According to an advantageous variant, it is recommended to use a hyaluronic acid whose molecular mass is between 1 and 3 million Daltons. It is also recommended to use said crosslinking, via the hydroxyl functions of hyaluronic acid, by means of a crosslinking agent, under conditions which lead to a crosslinking rate of said hyaluronic acid (starting material) characterized by ratio: total number of reactive functions of said crosslinking agent / total number of disaccharide units of the hyaluronic acid molecules present between 0.8 and 1.

In fact, the network of insoluble fragments of the biphasic compositions of the invention is based on molecules of hyaluronic acid linked by bridges of molecules of crosslinking agent; each of the disaccharide units of said hyaluronic acid molecules advantageously having between 0.8 and 1 of its hydroxyl functions engaged in such bridges.

The range indicated for said crosslinking rate is an optimal range. It is not excluded to involve in compositions of the invention fragments having such a rate of crosslinking below the value 0.8 (care will however be taken to keep said fragments their intrinsic insolubility properties) or beyond the value 1 (care will therefore be taken not to alter the nature of the fragments based on hyaluronic acid too much which will contain more and more crosslinking agent).

As crosslinking agent, it is possible to use, to generate the hydrogel constituting the insoluble fragments of the biphasic compositions of the invention, any agent known for crosslinking hyaluronic acid via its hydroxyl functions - crosslinking agent at less bifunctional - and in particular a polyέpoxide or its derivatives. As such crosslinking agent, it is possible in particular to use epichlorohydrin, divinylsulfone, 1,4-bis (2,3-epoxypropoxy) butane (or 1,4-bis (glycidyloxy) butane or 1,4- butanediol diglycidyl ether = BDDE), l, 2-bis (2,3-epoxypropoxy) ethylene, l- (2,3- epoxypropyl) -2,3-epoxy cyclohexane ... It is not excluded from the framework of the invention to involve several crosslinking agents ...

It is also possible to characterize the insoluble hydrogel fragments of the compositions of the invention by other parameters, such as their dry matter content or their optical properties.

The dry matter content of said fragments was measured in the context of the invention, with said buffered fragments (at a pH between 6.5 and 7.5), at equilibrium. Under these conditions, said fragments of the invention advantageously have a dry matter content of between 1.5 and 20%, even more advantageously between 5 and 15%.

Said fragments, under the same conditions, transmit less than 5% of the light at 400 nm.

The biphasic 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. By introducing too much dispersed phase into said continuous phase, the injectable nature of said compositions can be compromised. By introducing too small a quantity of said dispersed phase into said continuous phase, the advantage of said compositions can be greatly reduced. They have in fact been designed to allow the establishment by injection of an effective amount of highly crosslinked hyaluronic acid (therefore resistant to degradation). In fact, the ratio should be optimized: active mass transported / mass of injection vehicle, etc.

Advantageous characteristics of the aqueous solution constituting the continuous phase of the two-phase compositions of the invention are specified below. Said continuous phase contains hyaluronic acid and / or another biocompatible polymer, weakly or not crosslinked.

Said continuous phase can be characterized by its intrinsic viscosity [η]. This parameter is given by the formula: M ^≈ ^ -o ² ^ in which η ₀ is the viscosity of the solvent, η the measured viscosity of the solution, c the concentration of said solution. Said continuous phase of the two-phase compositions of the invention advantageously has its intrinsic viscosity of between 1,500 and 3,200 ml / g. More preferably, the intrinsic viscosity is between 2000 and 2700 ml / g.

Said intrinsic viscosity of said continuous phase obviously depends on the nature of the polymer involved, its concentration and its crosslinking rate. Said viscosity is to be optimized, as part of a compromise, insofar as it is desired that said continuous phase constitutes the vehicle for injecting the insoluble fragments, on the one hand and slows down the degradation of said implanted fragments, somewhere else.

In general, we can speak of an optimization in terms of the composition of said continuous phase; the parameters available being the nature of the polymer (s) involved, the concentration of the said polymer (s), and possibly the crosslinking rate of the said polymer (s) ( s).

Whatever the nature of said polymer involved in the continuous phase, a person skilled in the art knows how to control its crosslinking, when it is possible.

We have seen that according to an advantageous variant of the invention, hyaluronic acid also intervenes at the level of said continuous phase. Said hyaluronic acid can intervene here uncrosslinked or weakly crosslinked It is also recommended here to involve a hyaluronic acid whose molecular mass is greater than or equal to 1 million Daltons and even more advantageously a hyaluronic acid whose molecular mass is between 1 and 3 million Daltons. When said hyaluronic acid is weakly crosslinked, it is recommended to use said crosslinking, via its hydroxyl functions, by means of a crosslinking agent, under conditions which lead to a crosslinking rate of said hyaluronic acid characterized by the ratio: total number reactive functions of said crosslinking agent / total number of disaccharide units of the molecules of hyaluronic acid present, between 0.01 and 0.4. It is noted that said crosslinking rate for the hyaluronic acid intervening in the continuous phase is generally always less than half that of the hyaluronic acid intervening in the dispersed phase.

Crosslinking is generally carried out during the preparation of the continuous phase in the same way as it is carried out during the preparation of the dispersed phase; when hyaluronic acid intervenes in said two phases. Thus said crosslinking of the continuous phase also advantageously involves it as a crosslinking agent for a polyepoxide and in particular 1,4-bis (2,3-epoxypropoxy) butane.

We find, in this hypothesis, said polyepoxide attached by ether bridges to the disaccharide units of the chains of hyaluronic acid, present in the continuous and dispersed phases.

Said hyaluronic acid, present in the dispersed phase of the biphasic compositions of the invention, or even, advantageously in the two dispersed and continuous phases of said compositions (excluding any other polymer according to a particularly preferred variant) is preferably a hyaluronic acid which was obtained by bacteria.

According to the preferred variant of the invention, an injectable suspension as defined above containing hyaluronic acid, obtained by bacterial fermentation, is proposed in its two phases. By means of such a highly biocompatible suspension, insoluble fragments are injected with great ease and said fragments are particularly resistant to degradation, once implanted, by their intrinsic structure and by the fact that they are protected by the continuous phase with which they were injected.

According to the second object of the present invention, there is provided a process for preparing the two-phase compositions described above. Said method typically comprises:

- preparation and purification of an insoluble hydrogel of highly cross-linked hyaluronic acid;

- the fragmentation of said hydrogel by shearing;

- suspending the fragments of said sheared hydrogel in an adequate continuous phase. In the first of the above steps, a highly cross-linked hyaluronic acid hydrogel should be prepared. To this end, the raw material used is generally dissolved (often sodium hyaluronate fibers) and a suitable crosslinking agent is reacted thereon in adequate proportions. A hyaluronic acid whose molecular mass is greater than or equal to one million Daltons, preferably between 1 and 3 million Daltons, is advantageously used as starting material, and it is crosslinked so that the ratio: total number of reactive functions of said crosslinking agent / total number of disaccharide units of the molecules of hyaluronic acid present, ie between 0.8 and 1. At the end of the crosslinking reaction, the reaction mixture is purified to remove the reagents which do not have not reacted. This purification can be carried out by extraction with ionized water in a soxhlet. The purified hydrogel obtained is then fragmented by shearing. Such shearing generates fragments of non-uniform geometry and size. Those skilled in the art will know how to optimize its implementation for obtaining adequate fragments.

Said fragments are then suspended in the continuous phase prepared beforehand or in parallel.

Said continuous phase is prepared in an analogous manner if crosslinking is to be carried out. Advantageously, said continuous phase is prepared from a hyaluronic acid whose molecular mass is greater than or equal to 1 million Daltons, preferably between 1 and 3 million Daltons, 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 molecules of hyaluronic acid present, ie between 0.01 and 0.4. Insofar as such crosslinking is carried out to a lesser degree, the purification of the crosslinked product must generally be carried out by other techniques than extraction. One can in particular proceed by successive solubilization / precipitation cycles or even by 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.

For their mixing, for the suspension of the hydrogel fragments in the continuous phase, one can in particular proceed according to one or other of the variants below, which are by no means exhaustive: - it is possible in particular to mix the suitably hydrated fragments in a continuous phase developed at the desired hydration rate,

- it is also possible to mix said dry fragments and a dry product which is a precursor of the continuous phase and to hydrate said dry mixture properly, - it is also possible to mix the two phases, one in a dry state, the other at hydrated state and adjust the rate of hydration of the mixture if necessary.

The first of said variants is preferred.

The compositions of the invention thus prepared can be packaged, in particular in syringes, then sterilized, in an autoclave for example.

We have seen above in the present text that their injection into the skin is recommended as a filling material, more precisely as a precursor of such a filling material, which registers its effectiveness over time.

According to its last object, the invention therefore relates to a filling material useful in restorative surgery and in cosmetic surgery, based on two-phase compositions as described above.

Said material, typically, has a structure which, after injection and implantation thereof in the dermis, evolves.

Following resorption of the continuous phase (intended to protect the dispersed phase), the fragments of the dispersed phase come together to generate a stable film. The said film is a completely original structure.

We recommend the use of such a filling material to fill in particular facial wrinkles such as the glabellar wrinkle, perioral wrinkles, nasolabial folds, to reduce crow's feet ...

The invention is illustrated by the examples below.

Example 1

A biphasic composition of the invention is prepared from a single polymer: fibers of sodium hyaluronate (of molecular mass: M - 2.1θ6), of bacterial origin.

Two solutions, at 11.8% by mass of said fibers, in 0.25 M sodium hydroxide are first prepared. . 430 μl of 1,4-bis (2,3-epoxypropoxy) butane (butanediol diglycidyl ether: BDDE) are added to one of said solutions. The homogenized mixture is placed 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 a soxhlet) in order to remove from its structure both the crosslinking agent (BDDE) and the polymer which have not reacted. Within such a 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% by mass, of dry matter. In this state, it transmits less than 5% of the light at 400 nm.

Said purified hydrogel is then sheared to obtain solid fragments of an average size of between 75 and 250 μm. Said 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) are added to the other of the said solutions. The mixture, homogenized, 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 which must also be purified. Within 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. From said fluid, the continuous phase of the desired biphasic composition is prepared. Said fluid is in fact purified by solubilization / precipitation. After such 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.

. Then mixed in proportions of 1/1 the suspension containing the fragments and the viscous aqueous solution. An injectable suspension is obtained, in particular through 26 G 1/2 to 30 G needles.

Example 2

The procedure is generally as in Example 1, except that:

1) for the preparation of the dispersed phase, only 250 μl of BDDE is used;

2) for the preparation of the continuous phase, the fibers of hyaluronic acid are directly dissolved in the phosphate buffer at pH 7.2 (no crosslinking is used); 3) during the final mixing, dispersed phase / continuous phase, said two phases are involved in a 2/1 ratio.

Such suspensions (prepared according to Example 1 and Example 2) were injected to fill facial wrinkles in 10 volunteers. In fact, less than one milliliter of such suspensions has been injected each time. The product, injected into the middle or deep dermis, did not cause any adverse reactions, including no inflammatory reaction, no redness, and no pain. After three months, the implant is still present and achieves an effective filling of the treated skin defect. The suspensions - two-phase compositions - of the invention are effective for the long-term treatment of skin depressions.

Claims

- Claims -

1. Biphasic composition containing a polymer chosen 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, chosen from proteins, polysaccharides and their derivatives, weakly or not crosslinked (s).

2. Biphasic composition according to claim 1, characterized in that said suspension is buffered at a pH between 6.5 to 7.5.

3. Biphasic composition according to one of claims 1 or 2, characterized in that more than half of said fragments have their largest dimension between 40 and 280 microns and advantageously between 75 and 250 microns.

4. Biphasic composition according to any one of claims 1 to

3, characterized in that the hydrogel constituting said fragments is obtained from said polymer whose molecular mass is greater than or equal to 1 million Daltons, advantageously between 1 and 3 million Daltons and which has been crosslinked, via the functions hydroxyls 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. Biphasic composition according to any one of claims 2 to

4, characterized in that said fragments, at equilibrium, contain between 1.5 and 20% by mass of dry matter, advantageously between 5 and 15% by mass.

6. biphasic composition according to 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. biphasic composition according to any one of claims 1 to 5, characterized in that said continuous phase has an intrinsic viscosity between 1,500 and 3,200 ml / g.

8. Biphasic composition according to any one of claims 1 to 7, characterized in that said continuous phase is an aqueous solution of said polymer whose molecular mass is greater than or equal to 1 million Daltons, advantageously between 1 and 3 million Daltons and which has optionally been crosslinked, via the hydroxyl functions of the said polymer, using an agent crosslinking, 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. biphasic composition according to any one of claims 1 to 8, characterized in that the dispersed phase, as well as possibly the continuous phase, has been crosslinked with a polyepoxide and in particular 1,4-bis (2,3- epoxypropoxy) butane.

10. Biphasic composition according to any one of claims 1 to 9, characterized in that the hyaluronic acid or its salt, present in the suspension, ie in the dispersed phase and optionally the continuous phase, was obtained by bacterial route.

11. Process for the preparation of a biphasic composition according to any one of the preceding claims, characterized in that it comprises:

the preparation and the purification of an insoluble hydrogel of said highly crosslinked polymer,

- the fragmentation of said hydrogel by shearing,

- suspending the fragments of said hydrogel in an adequate continuous phase.

12. Filling material useful in restorative surgery and in cosmetic surgery characterized in that it is based on a biphasic composition according to any one of claims 1 to 9, before injection and that, after injection, its structure changes , following resorption of the continuous phase, towards that of a stable film resulting from the regrouping of the fragments of the dispersed phase.