Use of polymers

Abstract

The present invention relates to the use of a thiol-group-containing polymer for preparing an implant for tissue augmentation, wherein the basis polymer is a polysaccharide.

Claims

1. An implant for dermal, muscle, or connective tissue augmentation comprising a thiol-group-containing polymer, wherein the basis polymer is a polysaccharide; wherein the implant stays for at least two weeks at or in the vicinity of the site of application, and wherein the thiol-groups of the thiol-group-containing polymer are partially involved in crosslinking of the polymers via disulfide bridges and partially are available as free thiol-groups and wherein the implant comprises the thiol-group containing polymer at a concentration of from 0.1 to 20% by weight and wherein the implant is formulated for subcutaneous or intradermal administration.

2. The implant of claim 1, wherein the thiol-group-containing polymer has a molecular mass of at least 10,000 g/mol.

3. The implant of claim 2, wherein the thiol-group-containing polymer has a molecular mass of at least 25,000 g/mol.

4. The implant of claim 3, wherein the thiol-group-containing polymer has a molecular mass of at least 50,000 g/mol.

5. The implant of claim 1, wherein thiol-groups within the polymer form inter- and/or intra-molecular disulfide bonds.

6. The implant of claim 1, wherein the thiol-groups comprise more than 20 mol/g of polymer.

7. The implant of claim 6, wherein the thiol-groups comprise more than 50 mol/g of polymer.

8. The implant of claim 7, wherein the thiol-groups comprise more than 100 mol/g of polymer.

9. The implant of claim 1, wherein the thiol-group-containing polymer is thiol-group-containing hyaluronic acid, thiol-group-containing chitosan, or a combination thereof.

10. The implant of claim 1, wherein the thiol-group of the polymer is derived from cysteine, cysteamine, or N-acetyl-cysteine.

11. The implant of claim 1, wherein the implant is provided as a gel or aqueous preparation.

12. The implant of claim 11, wherein the preparation is a single phase preparation.

13. The implant of claim 1, further comprising chitosan and/or hyaluronic acid.

14. The implant of claim 1, further defined as comprising at least one active substance.

15. The implant of claim 14, wherein the at least one active substance is an antiphlogistic agent, antirheumatic agent, analgesic, anti-infective agent, antiviral agent, antibiotic, antimycotic, antiseptic agent, chemotherapeutic agent, spasmolytic, vitamin, cytostatic agent, local anesthetic, antiallergic agent, antihistamine, anti-inflammatory agent, antivaricosic agent, agent for treating hemorrhoids, therapeutic agent for treating the skin, gynecologic agent, ophthalmic agent, urologic agent, rhinologic agent, or otologic agent.

16. The implant of claim 14, further defined as comprising at least one pharmaceutical excipient.

17. The implant of claim 16, wherein the at least one pharmaceutical excipient is a buffer salt, preserving agent, excipient for adjusting the desired osmolality, excipient for adjusting the desired viscosity, stabilizer, softener, coating material, flow agent, binder, lubricant, filler, desiccant, disintegrating agent, solvent, solubilizer, or excipient for enhancing the compatibility of the formulation.

18. A method for the cosmetic augmentation of the skin comprising: obtaining an implant of claim 1; and applying the implant subcutaneously and/or intradermally.

19. The method of claim 18, wherein the implant reduces inflammation and/or oxidation of the skin at the site of implantation.

Description

(1) The invention will be explained in more detail by way of the following examples and figures to which, of course, it is not restricted.

(2) FIG. 1 shows a schematic representation of the antioxidant active mechanism of the thiol-group-containing polymers.

(3) FIG. 2 shows the inhibition of the oxidating effect of hydroxyl radicals by 0.1% (m/v) aqueous solutions of thiolated hyaluronic acid in comparison with the unmodified polymers (n3; SD).

(4) FIG. 3 shows the inhibition of the oxidating effect of hydroxyl radicals by 0.1% (m/v) aqueous solutions of thiolated hyaluronic acid, modified to various degrees (200 M of thiol-groups/g of polymer and 70 M of thiol-groups/g of polymer) in comparison with unmodified hyaluronic acid (n4; SD).

(5) FIG. 4 shows the inhibition of the lipid oxidation on porcine cornea in vitro by 0.1% (m/v) aqueous solutions of thiolated hyaluronic acid in comparison with unmodified polymers (n3; SD).

EXAMPLES

Example 1

Inhibition of the Oxidating Effect of Hydroxyl Radicals by Inventive Thiol-Group-Containing Polymers

(6) The hydroxyl radical-scavenging properties of the inventive thiol-group-containing polymers were evaluated with the deoxyribose test (Halliwell et al., Food Chem. Toxicol. 33 (1995): 601-617). In this test system, hydroxyl radicals are generated by iron ions which attack deoxyribose. The resultant degradation products react with thiobarbituric acid to a pink colouring agent whose absorption is measured. With this method, both the radical-scavenging and also the complexing properties of the inventive thiol-group-containing polymers are tested.

(7) The hydroxyl radical-scaventing and complexing properties of the inventive thiol-group-containing polymers were also evaluated with the deoxyribose test in dependence on their concentration and their degree of modification. It is important that the inventive thiol-group-containing polymers still unfold their antioxidant activity even at low concentrations because in this wayin combination with the extended dwell time of these thiol-group-containing polymersa long-lasting effect can be achieved. Moreover, in preparations for ocular, intra-articular, intradermal or subcutaneous applications, high-molecular polymeric compounds can be used only at relatively low initial concentrations, since otherwise, due to their high viscosity, an optimum tolerance is no longer ensured.

(8) For this test, polymer solutions were freshly prepared by dissolving the polymer in phosphate buffer, pH 7.4, so that the final concentration of the polymer in the entire sample solution was 0.1% (m/v). If necessary, the pH of the sample solution was adjusted to 7.4 by the addition of NaOH.

(9) Phosphate buffer, pH 7.4 without polymer served as the control. To 0.6 ml of the sample solution, at first 0.1 ml of a 10 mM 2-deoxy-D-ribose solution were added.

(10) Immediately after the addition of 0.1 ml of a freshly prepared 10 mM FeSO.sub.4 solution, the samples were incubated for 120 min at 37 C. The reaction was stopped by the addition of 0.5 ml of 2.5% (v/v) trifluoroacetic acid. After the addition of 0.2 ml of 1% (m/v) thiobarbituric acid, the samples were incubated for 20 min at 95 C. for color development. Then the samples were cooled to room temperature and centrifuged for 5 min. One aliquot of the sample solution was transferred in microphotometric cuvettesi and the absorption was measured at 532 nm.

(11) The antioxidant properties of the inventive polymer were calculated in the form of the inhibition of the deoxyribose oxidation:
Oxidation inhibition in %=(1(A.sub.s/A.sub.c))*100,
wherein A.sub.s is the absorption of the sample, and A.sub.c is the absorption of the control.

(12) The results are illustrated in FIGS. 2 and 3. On the y-axis, in each case the inhibition of the oxidation effect (in %) of hydroxyl radicals by the polymers is illustrated. The values illustrated are the mean values of at least 3 test repetitionsstandard deviation. In FIG. 2 it is shown that by the covalent binding of thiol compounds, the antioxidant effect of hyaluronic acid could be significantly increased. The dependence of the antioxidant effect of the polymers according to the invention on the degree of modification with thiol compounds is illustrated in FIG. 3 by way of the example of thiolated hyaluronic acid. Thiolated hyaluronic acid having a degree of modification of 200 M of thiol-groups/g of polymer thus has a higher antioxidant effect than thiolated hyaluronic acid having a degree of modification of 70 M of thiol-groups/g of polymer.

Example 2

Inhibition of the Lipid Peroxidation in the Skin by Thiol-Group-Containinq Polymers

(13) With an in vitro-test, the potential of the thiol-group-containing polymers to inhibit the oxidation of lipids of the dermis was evaluated. Peroxidation of intracellular lipids caused e.g. by UU radiation leads to damages of the human skin/dermis.

(14) With this method, both the radical-scavenging and the complexing properties of the thiol-group-containing polymers are tested (Halliwell et al., Food Chem. Toxicol. 33 (1995): 601-617). In this in vitro-test system, the lipid peroxidation is accelerated by the addition of iron ions and heating. The oxidated lipid fragments (TBARS; thiobarbituric acid reactive substances) react with thiobarbituric acid to a pink coloring agent whose absorption is measured.

(15) Polymer solutions are freshly prepared in isotonic phosphate buffer so that the final concentration of the polymer in the entire sample solution was 0.05% (m/v). The pH of the sample solutions was adjusted to 7.4 by the addition of NaOH. Isotonic phosphate buffer, pH 7.4 served as the control. To these samples, 100 mg of porcine dermis were added. After the addition of 0.3 ml of a freshly prepared 20 mM FeSO.sub.4 solution, the samples were incubated at 95 C. for 60 min. The reaction was stopped by the addition of 0.3 ml of 20% (v/v) trifluoroacetic acid. After the addition of 0.3 ml of 1% (m/v) thiobarbituric acid, the samples were incubated for further 20 min at 95 C. and, after cooling to room temperature, centrifuged. One aliquot was transferred to microphotometric cuvettes, and the absorption was measured at 532 nm.

(16) The inhibition of the lipid peroxidation was calculated according to the following equation:
Oxidation inhibition in %=(1(A.sub.s/A.sub.c)*100,
wherein A.sub.s is the adsorption of the sample, and A.sub.c is the absorption of the control.

(17) The results are illustrated in FIG. 5. On the y-axis, the oxidation inhibition of the respective polymers is plotted in %. The values shown are the mean values from 3 test repetitions+standard deviation. From these studies it is clearly apparent that on account of their radical-scavenging, complexing and, thus, antioxidant properties, the thiol-group-containing polymers are capable of significantly reducing the oxidation of lipids caused by inflammatory processes.

Example 3

Intradermal Application of the Thiol-Group-Containing Polymers According to the Present Invention

(18) The following preparation for intradermal applications was produced: 2 g thiol-group-containing hyaluronic acid was solved in sterile isotone phosphate buffer, stirred to form a partially crosslinked polymer, filled into syringes and sterilised. 0.1 ml of this formulation was injected intradermally into the back region of rabbits. The application produced a minimal local irritation which disappeared after one day. The depot formed by the thiol-group containing hyaluronic acid was tactually detectable over the whole examination period of two weeks.

Example 4

Production of a Preparation for the Subcutaneous and Intradermal Use for Dermal Augmentation

(19) A preparation for the subcutaneous and intradermal use was manufactured as follows: 1 g sterile thiol-group containing hyaluronic acid was solved under aseptic conditions and in the absence of oxygen in a 100 ml sterile phosphate buffer pH 7.4. The osmality of this preparation was adjusted by the addition of NaCl to give an osmality of the solution between 200 and 400 mosmol/kg. The solution was filled into flasks and packed gas impermeable.

Example 5

Formulation for Augmenting of Scar Tissue

(20) A preparation was produced as follows: 3 g sterile thiol-group containing hyaluronic acid was solved under aseptic conditions in 100 ml sterile phosphate buffer pH 7.4 and partially crosslinked. Afterwards, the osmality for suggested addition of NaCl to give an osmality of the solution between 200 and 400 mosmol/kg. The solution was filled into flasks and closed gas impermeable.

Example 6

Formulation for Augmenting the Anal and Urethral Sphincter Muscles

(21) The thiol-group containing polymers have been used to augment the internal anal sphincter in order to prevent anal incontinence by supporting natural occurring cushioning in the anal canal. The same formulation was used also to augment the urethral sphincter muscles (see also U.S. Pat. No. 5,785,642). Incontinence is a often occurring problem, in particular for women, whose pelvic floor is significantly weakened after pregnancy. If the pelvic floor muscles cannot be forced by conventional methods, the injection of materials for augmentation of the urethra is a good alternative.

(22) A formulation for the augmentation of the sphincter was produced as follows: 5 g sterile thiol-group-containing chitosan was solved under aseptic conditions in 100 ml sterile borate buffer pH 6.5 and partially crosslinked, the osmality was adjusted by addition of NaCl to 200 to 400 mosmol/kg. The solution was filled into syringes.

Example 7

Enzymatic Degradation of a Single Phase Crosslinked Thiol-Group-Containing Hyaluronic Acid Formulation and a Crosslinked Hyaluronic Acid Particle Comprising Formulation

(23) Further advantageous of the thiol-group-containing polymers, in particular of thiol-group-containing hyaluronic acid, is shown in this example. Thiol-group-containing hyaluronic acid and commercially available formulations comprising crosslinked hyaluronic acid particles have been digested with hyaluronidase. In both cases the hyaluronic acid was degraded, whereby the degradation was comparable with an unmodified hyaluronic acid in the case of thiol-group-containing hyaluronic acid and the fragments formed show a much higher molecular weight as the crosslinked hyaluron acid comprising particles. This results from the fact that the disulfide bridges are not degraded by an enzyme and secondly the degree of crosslinking is much higher than in crosslinked hyaluronic acid particles. Therefore, the crosslinked thiol-group-containing hyaluronic acids according to the present invention stay longer at the side of application than crosslinked hyaluronic acid particles.