NOVEL COMPOSITIONS ACTIVE ON ADIPOCYTES

Abstract

An aqueous composition of chitosan including between 0.1 and 5 wt.-% chitosan relative to the total weight of the composition, for use in increasing the volume and/or number of adipocytes. The increase in the volume and/or number of adipocytes allows for use in the treatment of or combating against ageing of the skin, for filling in skin defeats, or the repair or reconstruction of continuous or subcutaneous tissues.

Claims

1. Aqueous chitosan composition comprising between 0.1% and 5% by weight chitosan, with respect to the total weight of the composition.

2. A method for treating or combating against skin ageing, for filling in skin defects such as wrinkles or for repairing and reconstructing cutaneous or subcutaneous tissues comprising administering to a subject in need thereof an effective amount of the aqueous chitosan composition of claim 1.

3. The method according to claim 2, wherein the aqueous composition comprises between 0.2% and 3% by weight, preferably between 0.5% and 2% by weight, more preferentially between 0.5% and 1.9% by weight chitosan, with respect to the total weight of the composition.

4. The method according to claim 2, wherein the aqueous composition has a pH of less than 8.5, advantageously between 4 and 7.6, more advantageously between 5.5 and 7.5, preferably between 6.2 and 6.8.

5. The method according to claim 2, wherein the chitosan has a degree of acetylation of less than 30%, advantageously less than 15%, and preferably less than 10%.

6. The method according to claim 2, wherein the aqueous composition further comprises at least one component of the extracellular matrix, and/or at least one substituent of the extracellular matrix.

7. The method according to claim 6, wherein the component of the extracellular matrix is chosen from proteins and the glycosylated derivatives thereof, in particular chosen from collagen, elastin, fibronectin, laminin or mixtures thereof.

8. The method according to claim 6, wherein the composition contains between 0.01% and 5% by weight, with respect to the total weight of the composition, of at least one component of the extracellular matrix and/or of at least one substituent of the extracellular matrix, advantageously between 0.1% and 4% by weight, more advantageously between 0.2% and 3% by weight, even more advantageously between 0.3% and 2% by weight, or even between 0.5% and 1% by weight.

9. The method according to claim 2, wherein the aqueous composition is liquid or in gel form.

10. The method according to claim 2, wherein the aqueous composition further comprises at least one active compound such as an analgesic compound, a local anaesthetic, such as lidocaine, mepivacaine, bupivacaine or ropivacaine, an angiogenic compound, or an active compound of the growth factor or bioactive oligosaccharide type.

11. The method according to claim 2, wherein the aqueous composition is formulated so as to be administered by intradermal injection, by subcutaneous or intraperitoneal injection.

12. A dermatological composition comprising the aqueous chitosan composition of claim 1.

13. A bioresorbable or partially resorbable implant comprising the aqueous chitosan composition of claim 1.

14. A method for reducing the toxicity with regard to adipocytes of an aqueous chitosan composition comprising between 0.1% and 5% by weight chitosan with respect to the total weight of the composition to said composition comprising adding at least one component of the extracellular matrix, and/or at least one substituent of the extracellular matrix.

15. A method for increasing the volume and/or number of adipocytes comprising administering to a subject in need thereof an effective amount of the aqueous chitosan composition of claim 1.

16. A method device comprising the aqueous chitosan composition of claim 1.

Description

DESCRIPTION OF THE FIGURES

[0097] FIGS. 1A and 1B: effect on the proliferation of 3T3L1s. FIG. 1 shows that, for all concentrations, the 3T3L1 cells are capable of proliferating and reaching confluence in 5 days although, two days after seeding, the morphology of the cells suggests an aggressive or even toxic medium for the 2% condition. FIG. 1B specifies where, on the experimental images obtained, the signal corresponding to AdipoRed could be seen (green experimentally, versus red for Hoecht).

[0098] FIGS. 2A, 2B and 2C: these figures show the influence of the chitosan concentration on the storage of oleic acid by the 3T3L1 cells during adipocyte differentiation. The AdipoRed marking (FIG. 2A) shows an accumulation of fatty acids when the cells are in the presence of chitosan at 0.5%, 0.75% and 1% compared with the conditions not containing chitosan. FIG. 2B specifies where, on the experimental images obtained, the signal corresponding to Adipored could be seen (green experimentally). Quantification of this accumulation (FIG. 2C) demonstrates that it is maximum when the cells are cultivated in the presence of chitosan at 0.5%.

[0099] FIG. 3: this figure shows a distribution of the lipid droplets (n>10000) of the 3T3L1s set out a composition of chitosan and at a formulation comprising chitosan combined with collagen and fibronectin. FIG. 3 shows that (Col 2+FN1)+Chitosan 2%) are the best formulations for increasing the number and average size (volume) of the lipid droplets.

EXAMPLES

Example 1: Preparation of the Solutions

[0100] Two strategies for preparing a range of concentrations of gelling chitosan solutions are used.

By Final Dilution

[0101] 4 g of chitosan is stirred in 95 g of saline solution containing 150 mM of NaCl (equivalent to 300 mOsmol.Math.l.sup.1). Once the chitosan is completely dispersed, 1 g of acetic acid is added. Stirring is maintained until the chitosan is completely dissolved. A clear solution of 4% concentrated chitosan is then obtained with a pH that may lie between 3 and 4.5 according to the origin of the chitosan.

[0102] A 4% (w/w) chitosan solution is obtained in a saline solution (NaCl at 300 mOsmol.Math.l.sup.1) containing 1% (w/w) of acetic acid.

[0103] The pH of the acidic chitosan solution is increased until it is between 6 and 6.5. This step is carried out by dialysis against a PBS buffer having a pH of between 6.5 and 7. During this step, the swelling of the chitosan solution is controlled in order to reach a final concentration of 3% (w/w) (degree of swelling 33%). The chitosan solutions are generally sterilised (121 C. for 15 minutes in an autoclave) in order to be stored before use.

[0104] This solution is dialysed in order to obtain a pH close to 6 and a concentration of 3% (w/w).

[0105] Before use, the 3% chitosan solution can be diluted in a saline solution at 150 mM of NaCl+1% acetic acid in order to access lower concentrations.

[0106] The solutions obtained can gel in the presence of the physiological medium or a culture medium.

By Initial Dilution

[0107] 4 g of chitosan is stirred in 95 g of saline solution containing 150 mM of NaCl (equivalent to 300 mOsmol.Math.l.sup.1). Once the chitosan is completely dispersed, 1 g of acetic acid is added. Stirring is maintained until the chitosan is completely dissolved. A clear 4% concentrated chitosan solution is then obtained with a pH that can be between 3 and 4.5 according to the origin of the chitosan.

[0108] A 4% (w/w) chitosan solution is obtained in a saline solution (NaCl at 300 mOsmol.Math.l.sup.1) containing 1% (w/w) of acetic acid.

[0109] This mother solution (4%) is next diluted in a saline solution (NaCl at 300 mOsmol.Math.l.sup.1) containing 1% (w/w) of acetic acid in different concentrations ranging from 4% to 0.1%. Each solution is then dialysed against a PBS buffer having a pH of between 6.5 and 7. The degree of swelling is measured in order to control the final concentrations of each solution. Dialysis is stopped when the pH of the solutions is between 6.0 and 7.4. Each solution is sterilised by autoclave (121 C. for 15 minutes). The solutions are used as they are without any other form of dilution, which preserves their pH.

[0110] The solutions obtained can gel in the presence of the physiological medium or a culture medium.

Example 2: Effect of Various Dilutions of Chitosan on the Proliferation, Differentiation and Capture of Fatty Acid by the 3T3L1 Murine Adipocyte Line

Equipment and Methods

[0111] Various chitosan solutions (2%, 1%, 0.75% and 0.5%) are prepared by dilution of the solutions obtained in example 1.

[0112] For example, a 2% (w/w) chitosan preparation obtained in example 1 (DA: 2%, Mw: 450000 g.Math.mol.sup.1, Bioxis Pharmaceuticals S.A.S.) is diluted in a saline solution (NaCl at 300 mOsmol.Math.l.sup.1) containing 1% (w/w) of acetic acid in a well (in 24-well plates) and then distributed over half of the other wells, dried, rinsed with PBS and then with the culture medium before cell seeding (3T3L1 murine adipocyte line ATCC CL-173). When the cells are at confluence, differentiation is induced by adding a differentiation medium. The cells are then treated with a small dose of oleic acid (OA, 2.5 M) for 6 hours. The cells are put back in a culture medium without OA until the next day in order to avoid any background noise from the OA. Proliferation is analysed by phase contrast microscopy (20): the images are taken at various times (2 days and 5 days post seeding), at an identical position for each well. The differentiation and the storage of oleic acid are analysed in fluorescence microscopy: the cells are fixed and then marked with AdipoRed (as identified in the figures) revealing the presence of fatty acids in the form of triglycerides (TGs) stored in a plurality of droplets during differentiation: fusing in a single droplet when the adipocytes are mature) and with Hoechst 3352 (red experimentally) for marking the nuclei. The marking is observed in fluorescence microscopy and quantification carried out by imaging. The imaging and the fluorescence quantification are carried out on a real-time imaging station. The controls are fixed before the induction of differentiation (confluence, DO).

Results

[0113] Two days after seeding, for all concentrations, the 3T3L1 cells are capable of proliferating and achieving confluence in 5 days (FIGS. 1A and 1B). Thus, at the concentrations tested, the adipocytes increase in number and volume. The morphology of the cells does however suggest an aggressive or even toxic medium for the 2% condition.

[0114] FIG. 2 shows the influence of the chitosan concentration on the storage of oleic acid for the 3T3L1 cells during adipocyte differentiation. Thus the same cells as those tested in FIG. 1 are used in the presence of oleic acid (fatty acid that the adipocyte cells have an ability to capture). The AdipoRed marking (FIGS. 2A and 2B) show an accumulation of fatty acids when the cells are in the presence of chitosan at 0.5%, 0.75% and 1% compared with the conditions not containing chitosan. Thus, in the presence of chitosan, the 3T3L1 cells capture more oleic acid than the reference. Quantification of this accumulation demonstrates that it is maximum when the cells are cultivated in the presence of chitosan at 0.5% (FIG. 2C).

Conclusion

[0115] 3T3L1 cells are capable of proliferating in the presence of chitosan. After two days in culture, the presence of chitosan does however appear to have a toxic effect on the 3T3L1 line for concentrations greater than or equal to 2%. Chitosan assists the capture of fatty acids in a dose-dependent fashion. Among the solutions tested, it appears that it is the 0.5% solutions that have the best efficacy. This capture would allow an increase in the volume of the adipocytes and therefore, by extrapolation, of the fatty tissues in vivo.

Example 3: Effect of Various Dilutions of Chitosan on the Capture of Fatty Acid in the Presence of Fibronectin and Collagen by the 3T3L1 Murine Adipocyte Line

Equipment and Methods

[0116] Various chitosan solutions (2%, 1% 0.75% and 0.5%) are prepared by diluting the solutions obtained in example 1.

[0117] For example, a 2% (w/w) chitosan preparation obtained in example 1. For this purpose, a 2% (w/w) chitosan preparation (DA: 2%, Mw: 450000 g.Math.mol.sup.1, Bioxis Pharmaceuticals S.A.S.). The solutions were used as they are without undergoing any predilution in acid saline solution. In addition, a solution based on 2 mg/ml collagen I+1 mg/ml fibronectin I+2% chitosan (denoted Col FN Chi2) is also evaluated. The compositions are distributed in a 96-well plate after cell seeding (3T3L1 murine adipocyte line, ref ATCC CL-173). When the cells are at confluence, differentiation is induced by adding a differentiation medium. The cells are then treated with a low dose of oleic acid (OA, 2.5 VM) for 6 hours. The cells are put back in a culture medium without oleic acid until the next day before observation on Cytation 3. The size distribution of the lipid droplets (the volume of the adipocytes) is observed.

Results

[0118] FIG. 3 shows that the 2 mg/ml collagen I+1 mg/ml fibronectin I+2% chitosan solution (denoted Col FN Chi2) has fewer adipocytes with a size below 15 m and more adipocytes with a size of between 15 and 55 m than the 2% chitosan solution. Adding compounds of the extracellular matrix such as collagen or fibronectin therefore promotes the increase in the number and average size (and therefore of the volume) of the lipid droplets.

Conclusion

[0119] Chitosan alone increases the size of lipid droplets. This effect is further increased in the presence of fibronectin and collagen.