Sterilized composition comprising at least one hyaluronic acid and magnesium ascorbyl phosphate

Abstract

The invention relates to a sterilized composition comprising at least one hyaluronic acid or biologically acceptable salt thereof, alone or in a mixture, and magnesium ascorbyl phosphate, in a ratio [HA]/[MAP] by mass of the amount of hyaluronic acid or salt thereof [HA] to the amount of magnesium ascorbyl phosphate [MAP] of greater than or equal to 1, the amount of magnesium ascorbyl phosphate being between 0.001% and 1% by weight, relative to the total weight of said composition, with an elastic component G which is retained or improved after sterilization and is between 5 and 400 Pa. It also relates to a composition of the invention which further includes dimethyl sulfone, to methods for producing same, and to uses thereof.

Claims

1. A sterilized composition comprising at least one noncrosslinked hyaluronic acid or biologically acceptable salt thereof, alone or in a mixture, and magnesium ascorbyl phosphate, in a ratio [HA]/[MAP] by mass of the amount of hyaluronic acid or salt thereof [HA] to the amount of magnesium ascorbyl phosphate [MAP] of greater than or equal to 1, with an elastic component G which is retained or increased after sterilization relative to a same composition not comprising the magnesium ascorbyl phosphate and is between 5 and 400 Pa.

2. The composition as claimed in claim 1, further comprising dimethyl sulfone.

3. The composition as claimed in claim 2, wherein the amount of dimethyl sulfone is between 0.001% and 5% by weight, relative to the total weight of the composition.

4. The composition as claimed in claim 2, wherein the amount of dimethyl sulfone is between 0.1% and 1% by weight, relative to the total weight of the composition.

5. The composition as claimed in claim 1, wherein the molecular mass Mw of the hyaluronic acid or salt thereof is between 0.01 and 5 MDa.

6. The composition as claimed in claim 1, wherein the amount of noncrosslinked hyaluronic acid or salt thereof, alone or in a mixture, is between 0.2% and 5% by weight, relative to the total weight of the composition.

7. The composition as claimed in claim 1, wherein the amount of noncrosslinked hyaluronic acid or salt thereof, alone or in a mixture, is greater than or equal to 1% by weight, relative to the total weight of the composition.

8. The composition as claimed in claim 1, the composition further comprising at least one additional agent.

9. The composition as claimed in claim 8, wherein the additional agent is selected from antioxidants and local anesthetics, alone or in a mixture.

10. The composition as claimed in claim 9, wherein the local anesthetics are selected from the group consisting of lidocaine, procaine, mepivacaine, ropivacaine, bupivacaine, or pharmaceutically acceptable salts thereof.

11. The composition as claimed in claim 9, wherein the antioxidants are selected from polyols.

12. The composition as claimed in claim 1, wherein the content of the magnesium ascorbyl phosphate is between 0.7 and 1 mg/g by weight, relative to the total weight of the composition.

13. A composition for filling in wrinkles comprising the composition as claimed in claim 1.

14. A composition for viscosupplementation comprising the composition as claimed in claim 1.

15. A kit comprising a composition as claimed in claim 1, wherein the composition is packaged in sterile syringes.

16. A method for producing a composition as claimed in claim 1, wherein the production method comprises at least: a step of hydrating fibers of at least one hyaluronic acid or salt thereof, alone or in a mixture, to give a hydrogel, a step of mixing a solution of magnesium ascorbyl phosphate with the hydrogel obtained in the preceding step, a step of homogenizing; and a step of sterilizing.

17. The production method as claimed in claim 16 wherein the method further comprises at least one step of mixing a solution of dimethyl sulfone with the hydrogel obtained in the step of hydrating fibers of at least one hyaluronic acid or salt thereof, alone or in a mixture.

Description

(1) FIG. 1 is a diagram illustrating the percentage increase, on the ordinate in the diagram, in the elastic component G, as a function of the amount of magnesium ascorbyl phosphate in mg/g of composition, on the abscissa in the diagram, following sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(2) FIG. 2 is a diagram illustrating the percentage increase, on the ordinate in the diagram, in the viscosity , as a function of the amount of magnesium ascorbyl phosphate in mg/g of composition, on the abscissa in the diagram, following sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(3) The characteristics of the composition according to the present invention and also methods for producing it, and the properties thereof, are illustrated in the examples below.

EXAMPLE 1

(4) This example illustrates a composition according to the invention comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(5) Fibers of sodium hyaluronate (NaHA) of injectable grade (1 g; molecular mass: approximately 2.7 MDa) are weighed out into a container. An aqueous solution of phosphate buffer (32.3 g) is added, and the whole is homogenized for approximately 1 hour using a spatula, at room temperature and under an atmospheric pressure of 900 mmHg.

(6) The noncrosslinked NaHA hydrogel thus obtained has an NaHA content of approximately 30 mg/g.

(7) Magnesium ascorbyl phosphate (MAP) (60 mg or 2.410.sup.4 mol) is dissolved in a phosphate buffer solution (19.94 g) to give an aqueous solution of magnesium ascorbyl phosphate with a content of 3 mg/g.

(8) The NaHA hydrogel obtained in the preceding step is diluted by adding the pre-prepared aqueous solution of magnesium ascorbyl phosphate. The resulting composition is then homogenized.

(9) This gives a composition comprising noncrosslinked NaHA in an amount of 20 mg/g and MAP in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(10) The composition thus obtained is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 2

(11) This example illustrates a composition example according to the invention comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(12) The composition comprising crosslinked hyaluronic acid is obtained by the crosslinking procedure described in WO 2009/071697 (example 1, first part) to VIVACY, starting from fibers of sodium hyaluronate (NaHA) (1 g; molecular mass: approximately 2.7 MDa) and butanediol diglycidyl ether (BDDE) (54 mg). The resulting composition contains approximately 30 mg/g of crosslinked NaHA with a degree of crosslinking X of approximately 0.12.

(13) An aqueous solution of magnesium ascorbyl phosphate with a content of 3 mg/g is prepared as in example 1.

(14) The crosslinked NaHA hydrogel obtained in the preceding step is diluted by adding the pre-prepared aqueous solution of magnesium ascorbyl phosphate. The resulting composition is then homogenized.

(15) This gives a composition comprising crosslinked NaHA in an amount of 20 mg/g and magnesium ascorbyl phosphate in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(16) The composition thus obtained is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 3

(17) This example illustrates a composition example according to the invention, comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and lidocaine.

(18) A composition comprising crosslinked NaHA is prepared by the procedure described in example 2, starting from an NaHA hydrogel with a content of 30 mg/g and from magnesium ascorbyl phosphate with a content of 10 mg/g.

(19) The addition of a lidocaine solution with a content of 13 mg/g to the composition obtained above is made by the procedure described in WO 2009/024670 to ANTEIS or by the procedure described in patent applications U.S. 61/791,977 or FR 13/52971 to VIVACY.

(20) The resulting composition comprises crosslinked hyaluronic acid in an amount of 20 mg/g, lidocaine in an amount of 3 mg/g, and magnesium ascorbyl phosphate in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(21) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 4

(22) This example illustrates a composition example according to the invention, comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone.

(23) The composition comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate is prepared by the procedure of example 1, starting from a hydrogel of hyaluronic acid with a content of 30 mg/g and from a magnesium ascorbyl phosphate solution with a content of 10 mg/g.

(24) A solution of dimethyl sulfone with a content of 4.3 mg/g is also added to the composition obtained above.

(25) The resulting composition is then homogenized.

(26) This gives a composition which comprises noncrosslinked hyaluronic acid in an amount of 20 mg/g, dimethyl sulfone in an amount of 1 mg/g, and magnesium ascorbyl phosphate in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(27) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 5

(28) This example illustrates a composition example according to the invention, comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone.

(29) A composition comprising crosslinked NaHA is prepared by the procedure described in example 2, starting from an NaHA hydrogel with a content of 30 mg/g and from magnesium ascorbyl phosphate with a content of 10 mg/g.

(30) A solution of dimethyl sulfone with a content of 4.3 mg/g is also added to the composition obtained above.

(31) The resulting composition is then homogenized.

(32) This gives a composition which comprises crosslinked hyaluronic acid in an amount of 20 mg/g, dimethyl sulfone in an amount of 1 mg/g, and magnesium ascorbyl phosphate in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(33) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 6

(34) This example illustrates a composition example comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and mannitol.

(35) A composition comprising noncrosslinked NaHA is prepared by the procedure described in example 1, starting from an NaHA hydrogel with a content of 30 mg/g and from magnesium ascorbyl phosphate with a content of 10 mg/g.

(36) A solution of mannitol with a content of 4.3 mg/g is also added to the composition obtained above.

(37) The resulting composition is then homogenized.

(38) This gives a composition which comprises noncrosslinked hyaluronic acid in an amount of 20 mg/g, magnesium ascorbyl phosphate in an amount of 1 mg/g and mannitol in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(39) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 7

(40) This example illustrates a composition example comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and mannitol.

(41) A composition comprising crosslinked NaHA is prepared by the procedure described in example 2, starting from an NaHA hydrogel with a content of 30 mg/g and from magnesium ascorbyl phosphate with a content of 10 mg/g.

(42) A solution of mannitol with a content of 4.3 mg/g is also added to the composition obtained above.

(43) The resulting composition is then homogenized.

(44) This gives a composition which comprises crosslinked hyaluronic acid in an amount of 20 mg/g, magnesium ascorbyl phosphate in an amount of 1 mg/g and mannitol in an amount of 1 mg/g; the ratio [HA]/[MAP] by mass is 20.

(45) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 8

(46) This example illustrates a composition example comprising crosslinked hyaluronic acid and sodium ascorbyl phosphate (SAP).

(47) The composition comprising crosslinked hyaluronic acid and sodium ascorbyl phosphate is prepared by the procedure of example 2, starting from a hyaluronic acid gel with a content of 30 mg/g and from sodium ascorbyl phosphate with a content of 3 mg/g.

(48) The resulting composition comprises crosslinked hyaluronic acid in an amount of 20 mg/g and sodium ascorbyl phosphate in an amount of 1 mg/g; the ratio [HA]/[SAP] by mass is 20.

(49) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 9

(50) This counterexample illustrates a composition example comprising noncrosslinked hyaluronic acid and mannitol.

(51) The composition comprising noncrosslinked hyaluronic acid and mannitol (MAN) is prepared by the procedure of example 1, starting from a hyaluronic acid hydrogel with a content of 30 mg/g and from a solution of mannitol with a content of 3 mg/g.

(52) The resulting composition comprises noncrosslinked hyaluronic acid in an amount of 20 mg/g and mannitol in an amount of 1 mg/g; the ratio [HA]/[MAN] by mass is 20.

(53) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 10

(54) This counterexample illustrates a composition example comprising crosslinked hyaluronic acid and mannitol.

(55) A composition comprising crosslinked NaHA is prepared by the procedure described in example 2, starting from an NaHA hydrogel with a content of 30 mg/g and from mannitol with a content of 3 mg/g.

(56) The resulting composition comprises crosslinked hyaluronic acid in an amount of 20 mg/g and mannitol in an amount of 1 mg/g; the ratio [HA]/[MAN] by mass is 20.

(57) The resulting composition is packaged in syringes which are sterilized by steam autoclaving (T=121 C., 10 min).

EXAMPLE 11

(58) Characterization of the rheological properties before and after sterilization by steam autoclaving of the compositions comprising crosslinked hyaluronic acid that are exemplified above.

(59) The elastic components G of the compositions comprising crosslinked hyaluronic acid, before and after sterilization by steam autoclaving, were measured on a TA Instrument AR 2000 Ex rheometer, in oscillation at 25 C., with the values for the elastic component G being related to a frequency of 1 Hz.

(60) For all of the measurements, a reference composition is formulated, by replacing the aqueous solution of magnesium ascorbyl phosphate with an equivalent quantity of aqueous phosphate buffer solution.

(61) The percentage improvement in the elastic component G is defined as follows:

(62) $\%\mathrm{improvement}\mathrm{in}{G}^{}=\frac{Y-Y^{}}{Y}.Math.100$ where Y=percentage loss in elastic component G on sterilization of the reference composition and Y=percentage loss in elastic component G on sterilization of the composition tested.

(63) Eight compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of between 0.3 and 20 mg/g in the composition are prepared by the procedure described in example 2.

(64) Three compositions comprising crosslinked hyaluronic acid and mannitol in an amount of between 1 and 10 mg/g in the composition are prepared by the procedure described in example 10.

(65) Two compositions comprising crosslinked hyaluronic acid and sodium ascorbyl phosphate in an amount of between 1 and 5 mg/g in the composition are prepared according to example 8.

(66) A reference composition comprising crosslinked hyaluronic acid is prepared by the procedure described in example 2. The aqueous solution of magnesium ascorbyl phosphate is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(67) The elastic components of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 1 below:

(68) TABLE-US-00001 TABLE 1 % improvement in elastic component G Elastic relative to component [MAP] Ratio [MAN] [SAP] reference G (Pa) of Test (mg/g) [HA]/[MAP] (mg/g) (mg/g) composition sterile gels 1 0 n/a 0 0 0 144 2 0.3 67 0 0 19 180 3 0.7 29 0 0 27 192 4 1 20 0 0 26 191 5 1.5 13 0 0 22 185 6 2 10 0 0 21 182 7 3 7 0 0 16 172 8 10 2 0 0 12 160 9 20 1 0 0 0 142 10 0 n/a 1 0 15 163 11 0 n/a 5 0 25 182 12 0 n/a 10 0 29 188 13 0 n/a 0 1 12 105 14 0 n/a 0 5 26 76 n/a: not applicable

(69) The results obtained above for the compositions comprising MAP are also illustrated by FIG. 1, which is a diagram representing the percentage increase in the elastic component G as a function of the magnesium ascorbyl phosphate content after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(70) For the compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of between 0.3 and 20 mg/g it is observed that the elastic component G is improved, relative to the reference composition, when the amount of magnesium ascorbyl phosphate is between 0.3 and 10 mg/g, with a sharp drop when this amount is greater than 10 mg/g.

(71) These results contradict the results obtained for the compositions comprising crosslinked hyaluronic acid and mannitol, which show that, the more the mannitol content of the composition is increased, the more the elastic component G is improved relative to the reference composition.

(72) They also contradict the results obtained for the compositions comprising crosslinked hyaluronic acid and SAP, for which the elastic component G is reduced at the two concentrations tested.

EXAMPLE 12

(73) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid.

(74) The compositions comprising noncrosslinked hyaluronic acid are characterized by their viscosity .

(75) The viscosity of the compositions is measured on a TA Instruments AR 2000 Ex rheometer, in imposed stress at 25 C. The viscosity value is related to a stress of 0.02 s-1.

(76) Six compositions comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of between 0.3 and 3 mg/g in the composition are prepared by the procedure described in example 1.

(77) A reference composition comprising noncrosslinked hyaluronic acid is prepared by the procedure described in example 1. The aqueous solution of magnesium ascorbyl phosphate described in the procedure for example 1 is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(78) The percentage improvement in the viscosity is defined as follows:

(79) $\%\mathrm{improvement}\mathrm{in}\mathrm{the}\mathrm{viscosity}=\frac{Z-Z^{}}{Z}.Math.100$ where Z=percentage loss in viscosity on sterilization of the reference composition and Z=percentage loss in viscosity on sterilization of the composition tested.

(80) The viscosities of the compositions tested are measured before and after sterilization and the percentage improvements in the viscosity are calculated, with the results obtained being presented in table 2 below:

(81) TABLE-US-00002 TABLE 2 % improvement in viscosity relative [MAP] to reference Test (mg/g) composition 15 0 0 16 0.3 4 17 0.7 0 18 1 0 19 1.5 4 20 2 7 21 3 12

(82) The results obtained above are also illustrated in FIG. 2, which is a diagram representing the percentage increase in the viscosity as a function of the magnesium ascorbyl phosphate content after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate.

(83) A retention of the viscosity is observed for the compositions according to the invention having a magnesium ascorbyl phosphate content of between 0.7 and 1 mg/g, relative to the reference composition.

EXAMPLE 12bis a)

(84) A similar test was carried out with rheological monitoring of the elastic component G.

(85) Seven compositions comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of between 0.3 and 1.25 mg/g in the composition are prepared by the procedure described in example 1 (the molecular mass of sodium hyaluronate is approximately 2.7 MDa).

(86) A reference composition comprising noncrosslinked hyaluronic acid is prepared by the procedure described in example 1. The aqueous solution of magnesium ascorbyl phosphate described in the procedure for example 1 is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(87) The elastic components G of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 3 below:

(88) TABLE-US-00003 TABLE 3 % improvement in elastic component G relative to [MAP] reference Elastic component Test (mg/g) composition G (Pa) 15 0 0 134 16 0.3 11 169 40 0.5 12 172 17 0.7 7 161 41 0.85 5 148 18 1 4 153 42 1.25 0 132

(89) An improvement in elasticity is observed for the compositions according to the invention having a magnesium ascorbyl phosphate content of between 0.3 and 1 mg/g relative to the reference composition, and also a retention of elasticity for a magnesium ascorbyl phosphate content of 1.25 mg/g.

EXAMPLE 12bis b)

(90) A test identical to test 12bis a) described above was carried out with compositions comprising noncrosslinked sodium hyaluronate with a molecular mass of approximately 1.4 MDa, and the elastic component G was monitored rheologically. The compositions are prepared by the procedure described in example 1, with substitution of the 2.7 MDa molecular mass sodium hyaluronate by 1.4 MDa molecular mass sodium hyaluronate.

(91) The elastic components G of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 4 below:

(92) TABLE-US-00004 TABLE 4 % improvement in elastic Elastic [MAP] component G relative to component G Test (mg/g) reference composition (Pa) 43 0 0 7 44 0.7 20 10 45 1 16 9 46 3 0 7

(93) An improvement in elasticity is observed for the compositions according to the invention having a magnesium ascorbyl phosphate content of between 0.7 and 1 mg/g relative to the reference composition, and also a retention of the elasticity for a magnesium ascorbyl phosphate content of 3 mg/g.

EXAMPLE 13

(94) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and an additional agent.

(95) The rheological properties before and after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and an additional agent are measured and the percentage improvements in the elastic component G are calculated, according to the protocol described in example 11.

(96) Four compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate in an amount of 1 mg/g, and an additional agent in an amount of 1 mg/g in the composition are prepared by the procedure described in example 7. The additional agents exemplified are mannitol (MAN), sorbitol (SOR), the water-soluble salt of potassium sucrose octasulfate (KSOS), and lidocaine hydrochloride (LIDO).

(97) A composition comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of 1 mg/g is prepared by the procedure described in example 2.

(98) A reference composition comprising crosslinked hyaluronic acid is prepared by the procedure described in example 2. The aqueous solution of magnesium ascorbyl phosphate is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(99) The elastic components of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 5 below:

(100) TABLE-US-00005 TABLE 5 Additional % improvement in Elastic agent elastic component G component G [MAP] in an amount of relative to reference (Pa) of sterile Test (mg/g) 1 mg/g composition gels 22 0 0 0 138 23 1 0 31 196 24 1 MAN 31 197 25 1 SOR 29 197 26 1 KSOS 29 195 27 1 LIDO 8 153

(101) It is found that the improvement in rheological properties that is observed in example 11 for the compositions comprising hyaluronic acid and magnesium ascorbyl phosphate is retained in the presence of all of the additional agents. Only the addition of lidocaine hydrochloride entails a significant reduction in this improvement.

EXAMPLE 13bis

(102) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and lidocaine at 3 mg/g.

(103) The elastic components of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 6 below:

(104) TABLE-US-00006 TABLE 6 % improvement in Elastic elastic component G component [MAP] relative to reference G (Pa) of Test (mg/g) [LIDO] (mg/g) composition sterile gels 47 0 0 0 109 48 1 0 30 144 49 0 3 7 103 50 1 3 5 104 51 3 3 22 138 52 10 3 9 120

(105) The addition of lidocaine alone is found to entail a reduction in the elastic component G of the composition after sterilization. The addition of MAP at 1 mg/g is unable to counter the effect of the lidocaine on the elasticity of the composition. However, the addition of MAP at 3 and 10 mg/g does improve the elasticity of the composition in the presence of lidocaine. These results are unexpected and are contrary to the teachings and effects described in the prior art and especially abovementioned WO2012/104419 to Q-MED.

EXAMPLE 14

(106) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and an additional agent.

(107) The rheological properties before and after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and an additional agent are measured and the percentage improvements in viscosity are calculated, according to the protocol described in example 12.

(108) Three compositions comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate in an amount of 1 mg/g, and an additional agent in an amount of 1 mg/g in the composition are prepared by the procedure described in example 6. The additional agents exemplified are mannitol (MAN), sorbitol (SOR), and the water-soluble salt of potassium sucrose octasulfate (KSOS).

(109) A composition comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of 1 mg/g is prepared by the procedure described in example 1.

(110) A reference composition comprising noncrosslinked hyaluronic acid is prepared by the procedure described in example 1. The aqueous solution of magnesium ascorbyl phosphate described in the procedure for example 1 is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(111) The viscosities of the compositions tested are measured before and after sterilization and the percentage improvements in the viscosity are calculated, with the results obtained being presented in table 7 below:

(112) TABLE-US-00007 TABLE 7 % improvement in Additional agent viscosity relative [MAP] in an amount of to reference Test (mg/g) 1 mg/g composition 28 0 0 0 29 1 0 0 30 1 MAN 0 31 1 SOR 0 32 1 KSOS 0

(113) A retention of the viscosity is observed for all of the compositions.

EXAMPLE 15

(114) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone (DMS).

(115) The rheological properties before and after sterilization by steam autoclaving of compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone (DMS) are measured and the percentage improvements in the elastic component G are calculated, according to the protocol described in example 11.

(116) Two compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate in an amount of 1 mg/g in the composition, and dimethyl sulfone in an amount of between 1 and 10 mg/g in the composition are prepared by the procedure described in example 5.

(117) One composition comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of 1 mg/g is prepared by the procedure described in example 2.

(118) A reference composition comprising crosslinked hyaluronic acid is prepared by the procedure described in example 2. The aqueous solution of magnesium ascorbyl phosphate is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(119) The elastic components of the compositions tested are measured before and after sterilization and the percentage improvements in the elastic component G are calculated, with the results obtained being presented in table 8 below:

(120) TABLE-US-00008 TABLE 8 % improvement in Elastic elastic component G component G [MAP] [DMS] relative to reference (Pa) of sterile Test (mg/g) (mg/g) composition gels 33 0 0 0 185 34 1 0 29 243 35 1 1 30 242 36 1 10 29 246

(121) It is found that the improvement in rheological properties observed in example 11 for the compositions comprising hyaluronic acid and magnesium ascorbyl phosphate is retained in the presence of DMS in an amount of between 1 and 10 mg/g.

EXAMPLE 16

(122) Characterization of the rheological properties before and after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone (DMS).

(123) The rheological properties before and after sterilization by steam autoclaving of compositions comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone (DMS) are measured and the percentage improvements in the viscosity are calculated, according to the protocol described in example 12.

(124) The composition comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate in an amount of 1 mg/g in the composition, and dimethyl sulfone in an amount of 1 mg/g in the composition according to the invention is prepared and sterilized by the procedure described in example 4 and is characterized by rheology according to example 12.

(125) One composition comprising noncrosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of 1 mg/g is prepared by the procedure described in example 1.

(126) A reference composition comprising noncrosslinked hyaluronic acid is prepared by the procedure described in example 1. The aqueous solution of magnesium ascorbyl phosphate described in the procedure for example 1 is replaced with an equivalent amount of an aqueous phosphate buffer solution.

(127) The viscosities of the compositions tested are measured before and after sterilization and the percentage improvements in the viscosity are calculated, with the results obtained being presented in table 9 below:

(128) TABLE-US-00009 TABLE 9 % improvement in [DMS] viscosity relative to Test [MAP] (mg/g) (mg/g) reference composition 37 0 0 0 38 1 0 0 39 1 1 0

(129) A retention in viscosity is found for the composition comprising noncrosslinked hyaluronic acid, magnesium ascorbyl phosphate in an amount of 1 mg/g, and dimethyl sulfone in an amount of 1 mg/g.

EXAMPLE 17

(130) Comparison with the compositions described in the prior art.

(131) In international patent application WO 2011/086458 to ALLERGAN, example 4 describes a composition comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in amounts of between 0.6 and 2% by weight, relative to the total weight of the composition. According to the patentee, these compositions are said not to be stable, since the Tan 1 Hz values of said compositions are greater than 0.1.

(132) The Tan 1 Hz values of the compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in amounts respectively of 0.6% and 1% are not defined.

(133) Only the Tan 1 Hz value of the composition comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of 2% is defined, and is 0.344, a value greater than 0.1.

(134) WO 2011/086458 also teaches that the greater the amount of magnesium ascorbyl phosphate in the composition, the more the Tan 1 Hz value in said composition tends to reduce and tends to approach the value of 0.1.

(135) As in the patent application cited above, the compositions studied earlier are characterized by their Tan 1 Hz, which is the difference between the Tan 1 Hz of the composition tested and the Tan 1 Hz of the reference composition, with Tan 1 Hz being the ratio of the viscous component G to the elastic component G.

(136) The Tan 1 Hz values of the compositions tested, relative to the reference composition, are presented in table 10 below:

(137) TABLE-US-00010 TABLE 10 Test [MAP] (mg/g) [MAN] (mg/g) [SAP] (mg/g) Tan 1 Hz 1 0 0 0 0 2 0.3 0 0 0.0135 3 0.7 0 0 0.0145 4 1 0 0 0.0140 5 1.5 0 0 0.0128 6 2 0 0 0.0136 7 3 0 0 0.0084 8 10 0 0 0.0008 9 20 0 0 0.0240 10 0 1 0 0.0131 11 0 5 0 0.0196 12 0 10 0 0.0210 13 0 0 1 0.0203 14 0 0 5 0.0771

(138) It is found that the Tan 1 Hz values of the compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate in an amount of between 0.3 and 20 mg/g are less than 0.1.

(139) It can also be seen that the Tan 1 Hz values of the compositions comprising crosslinked hyaluronic acid and mannitol in an amount of between 1 and 10 mg/g are lower than 0.1, and also the Tan 1 Hz values of the compositions comprising crosslinked hyaluronic acid and SAP in an amount of between 1 and 5 mg/g.

(140) In conclusion, the results obtained for the compositions comprising crosslinked hyaluronic acid and magnesium ascorbyl phosphate of between 0.3 and 20 mg/g are in contradiction relative to the teaching of WO 2011/086458, since, with amounts of magnesium ascorbyl phosphate that are equivalent to or less than that in WO 2011/086458 in the compositions according to the invention, the Tan 1 Hz values therefore are less than 0.1.

(141) The same determinations of Tan 1 Hz were carried out on compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and an additional agent, and the values obtained are presented in table 11 below:

(142) TABLE-US-00011 TABLE 11 Additional agent in an amount of Test [MAP] (mg/g) 1 mg/g Tan 1 Hz 22 0 0 0 23 1 0 0.0197 24 1 MAN 0.0191 25 1 SOR 0.0181 26 1 KSOS 0.0142 27 1 LIDO 0.0068

(143) It is found, as before, that the Tan 1 Hz values after sterilization by steam autoclaving are less than 0.1.

(144) The same determinations of Tan 1 Hz were carried out on compositions comprising crosslinked hyaluronic acid, magnesium ascorbyl phosphate, and dimethyl sulfone (DMS), and the values obtained are presented in table 12 below:

(145) TABLE-US-00012 TABLE 12 Test [MAP] (mg/g) [DMS] (mg/g) Tan 1 Hz 33 0 0 0 34 1 0 0.0050 35 1 1 0.0061 36 1 10 0.0064

(146) It is found again that the Tan 1 Hz values are less than 0.1.