COMPOSITION COMPRISING HYALURONIC ACID AND A POLYOL AND/OR CARBOXYMETHYL CELLULOSE

Abstract

The invention relates to a composition comprising at least hyaluronic acid and at least one polyol and/or carboxymethyl cellulose (CMC).

The invention also relates to the use of at least one polyol and/or carboxymethyl cellulose to stabilize hyaluronic acid in a composition.

Claims

1. A composition comprising at least hyaluronic acid and at least one polyol and/or carboxymethyl cellulose (CMC).

2. The composition according to claim 1, characterized in that the hyaluronic acid has a molecular weight between 10.sup.5 and 10.sup.7 Da.

3. The composition according to claim 1, characterized in that the polyol is a saturated or unsaturated, linear, branched or cyclic alkyl compound with at least two —OH functions on the alkyl chain, as well as polymers (polyethers) of these polyhydroxylated alkyl compounds.

4. The composition according to claim 3, characterized in that the polyol is an alkyl compound having from 2 to 12 carbon atoms.

5. The composition according to claim 1, characterized in that the polyol is selected from ethylene glycol [(HOCH.sub.2—CH.sub.2OH)], diethylene glycol [(HOCH.sub.2—CH.sub.2—O—CH.sub.2—CH.sub.2OH)], triethylene glycol [(HOCH.sub.2—CH.sub.2—O—CH.sub.2—CH.sub.2OCH.sub.2—CH.sub.2OH], propylene glycol [(propane-1,2-diol: HOCH.sub.2—CHOH—CH.sub.3)], trimethylene glycol [(propane-1,3-diol: HOCH.sub.2—CH.sub.2—CH.sub.2OH)]; propylene glycol, polymers and copolymers of glycerol, ethylene glycol and propylene glycol, dipropylene glycol, and hexaglycerol, hexylene glycol, pentylene glycol, butyldiglycol, 1,2,3trihydroxyhexane, butylene glycol [(butane-1,3-diol], n-butylene glycol [(butane-1,4-diol], 2,3-butylene glycol [or secbutylene glycol (butane-2,3-diol)], Triols, glycerol; Tetraols, Erythritol, Threitol, Pentols (pentanols), Xylitol, Arabitol (lyxitol), Ribitol (adonitol); Hexols, Sorbitol (Gulitol), Dulcitol (Galactitol), Mannitol, Fucitol, Iditol; Heptols, Volemitol; C.sub.12 Isomalt, C.sub.12 Maltitol, C.sub.12 Isomaltitol, C.sub.12 Lactitol (lactositol), C.sub.18 Maltotriitol, C.sub.24 Maltotetraitol; and Polyglycitol.

6. The composition according to claim 1, characterized in that the hyaluronic acid is present in the composition in an amount comprised between 0.01% and 20% of the total weight of the composition.

7. The composition according to claim 1, characterized in that the polyol is present in the composition in an amount between 0.05 and 90% of the total weight of the composition.

8. The composition according to claim 1, characterized in that the carboxymethyl cellulose is present in the composition in an amount between 0.1% and 72% of the total weight of the composition.

9. The composition according to claim 1, characterized in that the ratio between the hyaluronic acid and the polyol in the composition is between 0.0001 and 400.

10. The composition according to claim 1, characterized in that the ratio between the hyaluronic acid and the carboxymethyl cellulose in the composition is between 0.0001 and 200.

11. The composition according to claim 1, characterized in that the ratio in the composition between the polyol and the carboxymethyl cellulose is between 0.0007 and 900.

12. A method comprising the use of at least one polyol and/or carboxymethyl cellulose to slow down, limit or even eliminate the degradation of hyaluronic acid, in a composition.

13. The method according to claim 12, to slow down, limit or even eliminate the degradation of the hyaluronic acid induced by ionizing radiation.

14. The method according to claim 12, to slow down, limit or even eliminate the degradation of the hyaluronic acid induced by oxidative stress.

15. The composition according to claim 1, comprising hyaluronic acid and at least one polyol and carboxymethyl cellulose.

16. The composition according to claim 2, characterized in that the hyaluronic acid has a molecular weight between 5.10.sup.5 and 2.10.sup.6 Da.

17. The composition according to claim 4, characterized in that the polyol is an alkyl compound having 2 or 3 carbon atoms.

18. The composition according to claim 6, characterized in that the hyaluronic acid is present in the composition in an amount comprised between 0.05% and 1% of the total weight of the composition.

19. The composition according to claim 8, characterized in that the carboxymethyl cellulose is present in the composition in an amount between 1 and 5% of the total weight of the composition.

20. The composition according to claim 10, characterized in that the ratio between the hyaluronic acid and the carboxymethyl cellulose in the composition is between 0.01 and 1.

Description

[0060] Other features of the invention may be apparent from the following specific, but not limiting, examples illustrating the invention, in which

[0061] FIG. 1 presents the results obtained during the study by gel permeation chromatography (GPC) of the degradation of hyaluronic acid in compositions comprising a polyol and carboxymethyl cellulose (CMC), subjected or not subjected to a stress of ionizing radiation type.

[0062] FIG. 1a presents the results of GPC analysis of hyaluronic acid degradation in non-irradiated compositions.

[0063] FIG. 1b presents the molar masses (MW) of HA as a function of their association with polyols like mannitol, sorbitol, glycerol and CMC.

[0064] FIG. 1c presents the results of the analysis of hyaluronic acid degradation under stress conditions induced by an eBeam irradiation dose of 8 to 12 kGy that was or was not previously associated with different polyols or CMC.

[0065] FIG. 1d presents the change in the molar masses (MW) of HA, under the stress condition induced by an eBeam irradiation dose of 8 to 12 kGy and as a function of their association with the polyols of type mannitol, sorbitol, glycerol and CMC. The arrow indicates a shift towards lower molecular weight (higher retention time).

[0066] FIG. 1e presents the results of the hyaluronic acid degradation analysis under eBeam irradiation conditions at 12-25 kGy.

[0067] FIG. 1f presents the change in the molar masses (MW) of HA, under eBeam irradiation stress conditions at a dose of 12 to 25 kGy and as a function of their association with polyols of type mannitol, sorbitol, glycerol and CMC. The arrow indicates a shift towards lower molecular weight (higher retention time).

[0068] FIG. 1g presents the results of the hyaluronic acid degradation analysis under eBeam irradiation conditions at 25-50 kGy.

[0069] FIG. 1h presents the change in the molar masses (MW) of HA, under eBeam irradiation stress conditions at a dose of 25 to 50 kGy and as a function of their association with polyols of type mannitol, sorbitol, glycerol and CMC. The arrow indicates a shift towards lower molecular weight (higher retention time).

[0070] FIG. 2 presents the elution profiles of the reference hyaluronic acid standards in the context of the effect of oxidative stress.

[0071] FIG. 3 prevents the effects of oxidative stress on the molecular weight of hyaluronic acid in the presence or absence of polyols as a function of time.

EXAMPLE 1: COMPARATIVE EVALUATION OF THE EFFECT OF EXCIPIENTS ON HYALURONIC ACID BEFORE AND AFTER IRRADIATION

[0072] The aim of this experimentation is to evaluate comparatively the molecular weight of hyaluronic acid dissolved in different excipient solutions, before and after irradiation at different doses. [0073] 1. Principle

[0074] Hyaluronic acid is dissolved in different solutions of excipients to study their effect on its molecular weight before and after Ebeam irradiation at three different doses (8-12 kGy, 12-25 kGy and 25-50 kGy). [0075] 2. Materials and Methods [0076] a. Materials [0077] 5 mL glass tubes+stoppers+metal caps [0078] 1 mL GPC vials (Agilent ref 5188-6593) [0079] Hyaluronic acid GPC filter (PTFE 13 mm 0.2 μm Agilent 5190 5265) [0080] Sartorius Scale (No. 4) [0081] b. Reagents [0082] Hyaluronic acid (Altergon batch 9998-3M Da) [0083] Mannitol (Merck batch FN 1378803750) [0084] Sorbitol (Roquette batch E090B) [0085] Glycerol (Sigma batch SHBK3676) [0086] CMC (Coluxia batch C161437) [0087] Distilled water [0088] Ultra pure water GPC grade [0089] NaCl (Fisher ref 10274392) [0090] Methanol (Fisher 1851587) [0091] Ultra pure water GPC grade (Merk ref 1.115333.1000) [0092] c. Preparation of the Solutions

[0093] The set of solutions is prepared according to the following table:

TABLE-US-00002 hyaluronic Glycerol Mannitol Sorbitol CMC Volume distilled acid (mg) (g) (mg) (mg) (mg) water (mL) 200 — 1050 — — Qsp 20 200 — — 1050.00 — 200 100 — — — 200 0.2 — — — 200 — — — 1000 200 — — — —

[0094] The solutions thus prepared are then freeze-dried (Cryotec Pilot Compact) according to the protocol comprising a first step of freezing by passing from a temperature of +25° C. to a temperature of −45° C. in 60 minutes, then maintaining at −45° C. for 6 hours; a second step of sublimation by passing from a temperature of −45° C. to a temperature of −20° C. (under a vacuum of 0.16 mBar), for 4 hours, then maintaining at −20° C. for 24 hours (under a vacuum of 0.16 mBar); a third step of secondary drying by passing from a temperature of 20° C. to 25° C. in 4 hours (under a vacuum of 0.007 mBar) and maintaining at the temperature of +25° C. for 15 hours (under a vacuum of 0.007 mBar).

[0095] After freeze-drying, the samples are then subjected to Beta irradiation in a Mevex A29, 34 kW, 10 Mev apparatus at a frequency of 640 Hz, with a scan setting of 2.7A, for a number of revolutions, at a speed of 2.51 m/min, at the following doses: 0, 8-12, 17.5-25 and 25-50 kGy. [0096] 3. GPC Analysis [0097] 3.a Preparation of Samples for Hyaluronic Acid Analysis

[0098] Before and after irradiation, each prepared solution is analyzed with GPC according to the following protocol:

[0099] A dilution in a mixture of 2M NaCl/MeOH 2% is carried out if necessary in order to obtain a final concentration of hyaluronic acid between 0.5 and 1 g/L.

[0100] The samples are then filtered and dispensed into vials for analysis with GPC. [0101] 3.b Analysis of the Hyaluronic Acid:

[0102] The analysis of the molecular mass of the hyaluronic acid (between 64 kDa and 1.3 MDa) was performed in an Agilent SL1200 apparatus according to the following protocol:

[0103] Columns: TSKgel GMPWXL and associated guard column;

[0104] Column/detector temperature: 45° C.;

[0105] Flow rate: 0.5 mL/min 0.2 M NaCl and 2% MeOH;

[0106] Detectors:

[0107] Refractive Index Detector (RID).fwdarw.Temperature: 45° C.

[0108] Diode Array Detectors (DAD).fwdarw.Wavelength: 205 nm

[0109] Injection volume: 50 μL [0110] 3.c Results of GPC Analysis [0111] 3.c.1 Range [0112] The following reference range is established.

TABLE-US-00003 Retention time at the apex of Molecular the peak weight Log Point (mins) (MW) MW % error 1 15.80940 1300000 6.11 −29.42 2 17.16300 720000 5.86 21.25 3 17.82000 390000 5.59 14.25 4 17.82000 1 0.00 −33440718.57 5 18.79920 210000 5.32 27.50 6 19.07820 90000 4.95 −35.19 7 19.57320 77000 4.89 −6.16 8 19.83960 60000 4.78 −9.98

[0113] The coefficient for determining R2 has the value 0.958192; the range is validated. [0114] 3.c.2 Samples

[0115] The table below shows the results obtained on the samples tested.

TABLE-US-00004 Molar Molar mass at mass Molar the as a mass in Irradiation Apex number mass Polydispersity Samples dose Mp Mn Mw PD hyaluronic acid alone not 3955260 1889602 3956345 2.1 irradiated  8-12 kGy 324438 27570 35356 1.28 12-25 kGy 14577 8629 19307 2.2 25-50 kGy 19327 8117 14915 1.84 hyaluronic acid + not 3792786 2045056 3555105 1.74 Mannitol irradiated  8-12 kGy 337323 246524 340051 1.38 12-25 kGy 174929 71073 165084 2.32 25-50 kGy 91903 72292 100650 1.4 hyaluronic acid + not 4276571 2411778 3995109 1.66 Sorbitol irradiated  8-12 kGy 266860 218014 408302 1.87 12-25 kGy 145154 78841 152422 1.93 25-50 kGy 60243 40529 109265 2.7 hyaluronic acid + not 2991852 2089073 3077895 1.47 Glycerol 100 irradiated  8-12 kGy 502827 297841 598552 2.01 12-25 kGy 379800 224544 394409 1.76 25-50 kGy 235647 101000 215774 2.14 hyaluronic acid + not 3898463 2400824 4003304 1.67 Glycerol 0.2 irradiated  8-12 kGy 278292 110160 245240 2.2 12-25 kGy 71869 39110 79587 2.04 25-50 kGy 44015 22988 49331 2.15 hyaluronic acid + not 4053720 1851695 3635442 1.96 CMC irradiated  8-12 kGy 200695 104860 404981 3.86 12-25 kGy 85739 50251 300369 5.9 25-50 kGy 82814 55613 172783 3.1

[0116] These results are also presented in FIGS. 1a to 1f [0117] 4. Conclusions

[0118] It is observed that: [0119] Whatever the solution in which the hyaluronic acid is put back into suspension, prior to irradiation, its molecular weight remains stable. [0120] The higher the dose of irradiation, the more the hyaluronic acid is degraded. [0121] A protective effect by each excipient on the hyaluronic acid during irradiation, whatever the dose, the greatest protective effect being observed in the presence of glycerol 100 and CMC.

EXAMPLE 2: COMPARATIVE EVALUATION OF THE EFFECT OF EXCIPIENTS ON HYALURONIC ACID IN AND NOT IN A CONDITION OF OXIDATIVE STRESS

[0122] The aim of this experiment is to evaluate comparatively the effect of oxidative stress on the molecular weight of the hyaluronic acid dissolved in different excipient solutions. [0123] 1. Principle

[0124] The hyaluronic acid is dissolved in different excipient solutions to study their effect on its molecular weight in the presence or absence of hydrogen peroxide and copper chloride according to the protocol of Chen et al. 2019 (Molecules 2019, 24, 61). [0125] 2. Materials and methods [0126] a. Materials [0127] 5 mL glass tubes+stoppers+metal caps [0128] 1 mL GPC vials (Agilent ref 5188-6593) [0129] Hyaluronic acid GPC filter (PTFE 13 mm 0.2 μm Agilent 5190 5265) [0130] Water bath (Memmert 10 L) [0131] Sartorius Scale (No. 4) [0132] b. Reagents [0133] Hyaluronic acid (Altergon batch 9998-3M Da) [0134] Mannitol (Merck Batch FN 1378803750) [0135] Sorbitol (Roquette batch E090B) [0136] Glycerol (Sigma batch SHBK3676) [0137] CMC (Coluxia batch C161437) [0138] H.sub.2O.sub.2(Fisher ref 15632040) [0139] CuCl.sub.2 (Fisher ref 10093650) [0140] Distilled water [0141] Ultra pure water GPC grade [0142] NaCl (Fisher ref 10274392) [0143] Methanol (Fisher 1851587) [0144] Sodium Chloride (NaCl) [0145] Ultra pure water GPC grade (Merk ref 1.115333.1000) [0146] c Preparation of the solutions [0147] NaCl Solution 0.2M: [0148] The molecular weight of the NaCl being 58.44 g.Math.mol.sup.−1, a 1.17% solution is prepared, i.e. 2.34 g in 200 mL of distilled water. [0149] H.sub.2O.sub.2 solution at 3%: the 30% H.sub.2O.sub.2 stock solution is diluted by a factor of 10. [0150] CuC12 solution at 50 mM: [0151] The molecular weight of CuCl.sub.2 being 170.48 g.Math.mol.sup.−1, 8.5 mg is weighed into 20 mL of distilled water. [0152] Sample Preparations: [0153] All the solutions were prepared according to the following table to obtain a hyaluronic acid concentration of 0.5% w/v according to the protocol of Chen eta/2019:

TABLE-US-00005 Hyaluronic acid Glycerol Mannitol Sorbitol CMC NaCl 0.2M (mg) (g) (mg) (mg) (mg) (mL) 100 — 525 — — Qsp 20 100 — — 525 — 100 50 — — — 100 0.1 — — — 100 — — — 500 100 — — — — [0154] 3. Oxidative Stress Protocol

[0155] Two 1 mL tubes are prepared for each sample and each time: 1 without addition, 1 to which are added 5 mM of H.sub.2O.sub.2 i.e. 5.7 μL/tube of the solution of H.sub.2O.sub.2 at 3% and 5 μM of CuCl.sub.2 i.e. 2 μL/tube of the solution prepared previously.

[0156] The samples under oxidative stress conditions are incubated at 50° C. for 5, 10, 20, 30 and 60 min.

[0157] After each incubation period, the reaction is stopped by quick freezing at −80° C. for GPC analysis. [0158] 4. GPC Analysis [0159] 4.a Preparation of samples for hyaluronic acid analysis

Preparation of the Range of Standards

[0160] The standards (1 to 7) and the samples to be analyzed are prepared in 0.2M NaCl/2% MeOH buffer (which serves as eluent) at concentrations between 0.5 and 1 g/L and then filtered (0.22 μM) before being sampled in vials for GPC analysis.

[0161] Analysis by GPC (Gel Permeation Chromatography)

[0162] The analysis of the molecular mass of the hyaluronic acid (between 64 kDa and 1.3 MDa) was performed in an AGILENT SL 1200 apparatus according to the following protocol:

[0163] Columns: TSKgel GMPWXL and associated guard column

[0164] Column/detector temperature: 45° C.

[0165] Flow rate: 0.5 mL/min 0.2 M NaCl and 2% MeOH

[0166] Detectors:

[0167] RID.fwdarw.Temperature: 45° C.

[0168] DAD.fwdarw.Wavelength: 205 nm

[0169] Injection volume: 50 μL [0170] 4.b Results of GPC Analysis [0171] 4.b.1 Range [0172] The following reference range is established.

TABLE-US-00006 Retention time at the apex of Molecular the peak weight Log Standards (mins) (MW) MW % error 1 18.00540 1300000 6.11 12.81 2 18.29880 720000 5.86 −22.91 3 19.33920 390000 5.59 5.66 4 20.03220 210000 5.32 2.36 5 20.70720 77000 4.89 −50.68 6 21.13920 90000 4.95 10.46 7 21.34260 60000 4.75 −13.13

[0173] Determination coefficient: 0.986936; Coefficient of linear correlation: −0.993446; Standard Y Error E

[0174] The standard range is in accordance with an R.sup.2 of 0.99. The analyses can continue (see FIG. 2) [0175] 4.b.2 Sample

[0176] The table below shows the results obtained on the samples tested.

TABLE-US-00007 Samples Time Mp Mn Mw PD % hyaluronic acid T = 0 1736745 958442 2055581 2.1 100.0 alone hyaluronic T = 5 min 220759 59767 255328 4.3 12.4 acid + 5M T = 10 min 138791 54202 232579 4.3 11.3 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 38177 11575 46620 4 2.3 T = 30 min 44505 18640 58951 3.2 2.9 T = 60 Min 7822 2847 11561 4 0.6 hyaluronic T = 0 2187646 118176 2421190 2 100.0 acid + mannitol hyaluronic T = 5 min 542153 244225 656459 2.7 27.1 acid + mannitol + T = 10 min 546024 249815 767406 3.072 31.7 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 117619 63292 295937 4.7 12.2 T = 30 min 137950 62881 275914 4.4 11.4 T = 60 Min 45738 17750 88586 5 3.7 hyaluronic T = 0 2392690 1281982 2617233 2 100.0 acid + sorbitol hyaluronic T = 5 min 415459 184625 503731 2.7 19.2 acid + sorbitol + T = 10 min 416701 180666 565745 3.1 21.6 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 106726 52670 291150 5.5 11.1 T = 30 min 97580 43554 201977 4.64 7.7 T = 60 Min 34275 13963 107268 7.7 4.1 hyaluronic T = 0 2049366 1046444 2214354 2.1 100.0 acid + glycerol 0.2 hyaluronic T = 5 min 394921 185736 485579 2.6 21.9 acid + glycerol 0.2 + T = 10 min 197106 83178 246580 2.7 11.1 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 76416 31075 102560 3.3 4.6 T = 30 min 88543 35747 170932 4.9 7.7 T = 60 Min 29402 10610 85505 8 3.9 hyaluronic T = 0 2530971 1516634 2743329 1.8 100.0 acid + glycerol 100 hyaluronic T = 5 min 961714 433316 1021851 2.4 37.2 acid + glycerol 100 + T = 10 min 633176 308487 704977 2.3 25.7 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 337409 162026 369177 2.3 13.5 T = 30 min 481365 207910 596919 2.9 21.8 T = 60 Min 164272 88322 239735 2.7 8.7 hyaluronic T = 0 1335506 463913 1351801 2.9 100 acid + CMC hyaluronic T = 5 min 729745 158142 987117 6.2 73.0 acid + CMC + T = 10 min 279924 73608 505860 6.9 37.4 H.sub.2O.sub.2/CuCl.sub.2 T = 20 min 272377 51313 360989 7 26.7 T = 30 min 260247 64023 383282 6 28.4 T = 60 Min 171667 38554 273637 7.1 20.2

[0177] The following table shows the percentage of hyaluronic acid in each sample after incubation compared to the initial amount of hyaluronic acid

TABLE-US-00008 Time HA + 5M HA/glyc HA/glyc (min.) H.sub.2O.sub.2/CuCl.sub.2 HA/mannitol HA/sorbitol 0, 2 100 HA/CMC 0 100 100 100 100 100 100 5 12.4 27.1 19.2 21.9 37.2 73 10 11.3 31.7 21.6 11.1 25.7 37.4 20 2.3 12.2 11.1 4.6 13.5 26.7 30 2.9 11.4 7.7 7.7 21.8 28.4 60 0.6 3.7 4.1 3.9 8.7 20.2 HA: Hyaluronic Acid

[0178] These results are also presented in FIG. 3. [0179] 1. Conclusions

[0180] The following is observed: [0181] Almost complete degradation of hyaluronic acid alone over 60 minutes [0182] At each time, a greater degradation of hyaluronic acid alone compared to hyaluronic acid in the presence of excipient the being the case whatever the excipient studied. [0183] All the excipients used protect the hyaluronic acid from degradation linked to oxidative stress, the greatest protective effect being observed in the presence of glycerol 100 and CMC.