AMINOACID-BASED COMPOSITION FOR FIBROELASTIN RECOVERY IN DERMAL CONNECTIVE TISSUES

Abstract

The present invention relates to compositions containing, as active ingredient, a mixture of amino acids able to stimulate the biosynthesis of elastin and collagen.

Claims

1. Compositions containing, as active ingredient, a mixture of amino acids consisting of glycine, L-proline, L-alanine, L-valine, L-leucine and L-lysine hydrochloride in the following weight ratios: Glycine 1; L-proline: 0.7-0.8; L-alanine: 0.47-0.76; L-valine: 0.35-0.56; L-leucine: 0.13-0.27; L-lysine hydrochloride: 0.10-0.12.

2. Compositions containing, as active ingredient, a mixture of amino acids consisting of glycine, L-proline, L-alanine, L-valine, L-leucine L-lysine hydrochloride and L-cysteine or N-acetyl-L-cysteine in the following weight ratios: Glycine 1; L-proline: 0.7-0.8; L-alanine: 0.47-0.76; L-valine: 0.35-0.56; L-leucine: 0.13-0.27; L-lysine hydrochloride: 0.10-0.12 L-cysteine or N-acetyl-L-cysteine in percentages by weight ranging between 1 and 20% of the total amino-acid mixture.

3. Compositions as claimed in claim 1 wherein the weight ratios of the amino acids are: Glycine 1; L-proline: 0.75; L-alanine: 0.48-0.51; L-valine: 0.35-0.37; L-leucine: 0.13-0.15; L-lysine hydrochloride: 0.10-0.11.

4. Compositions as claimed in claim 1 wherein the weight ratios of the amino acids are: Glycine 1; L-proline: 0.75; L-alanine: 0.75-0.76; L-valine: 0.54-0.56; L-leucine: 0.13-0.14; L-lysine hydrochloride: 0.10-0.11.

5. (canceled)

6. Compositions as claimed in claim 1 wherein the weight ratios of the amino acids are: Glycine 1; L-proline: 0.75; L-alanine: 0.49-0.51; L-valine: 0.35-0.36; L-leucine: 0.26-0.27; L-lysine hydrochloride: 0.10-0.11.

7. Compositions as claimed in claim 1 for oral use.

8. Compositions as claimed in claim 7 in the form of solutions, granules, dispersible powder, tablets or capsules.

9. Compositions containing, as active ingredient, a mixture of amino acids consisting of glycine, L-proline, L-alanine, L-valine, L-leucine and L-lysine hydrochloride in the following weight ratios: Glycine 1; L-proline: 0.7-0.8; L-alanine: 0.47-0.76; L-valine: 0.35-0.56; L-leucine: 0.13-0.27; L-lysine hydrochloride: 0.10-0.12 and hyaluronic acid or salts thereof, with an average molecular weight ranging between 500,000 and 3,000,000 Da, in percentages ranging between 0.01 and 3% by weight of the total composition.

10. Compositions as claimed in claim 9 for topical or injective use.

11. Compositions as claimed in claim 10 in the form of gels, ointments, emulsions, transdermal patches, sterile solutions or sterile amino-acid powders designed to be reconstituted with sterile aqueous solutions of hyaluronates.

12. Method of treating elastosis and dermoepidermal atrophy resulting from photoaging, skin disorders with a dermoatrophic and iatrogenic cause, burns, radiation burns, skin lesions, bedsores, dermal aplasia caused by drug administration, tendon and joint lesions in subjects in need thereof with the compositions according to claim 1, said method comprising; administering to said subjects a pharmaceutical effective amount of the compositions according to claim 1.

13. Compositions as claimed in claim 9, wherein said hyaluronic acid salt is sodium hyaluronate.

Description

DESCRIPTION OF THE INVENTION

[0015] The invention relates to compositions containing a mixture of amino acids that selectively trigger the expression of genes encoding tropoelastin (ELN), lysine oxidase (LOXL-1) and Type IV collagen (COL4A1) and inhibit the expression of genes encoding metalloelastases.

[0016] The mixture of amino acids according to the invention consists of glycine, L-proline, L-alanine, L-valine, L-leucine and L-lysine hydrochloride in the following weight ratios: [0017] Glycine 1; [0018] L-proline: 0.7-0.8; [0019] L-alanine: 0.47-0.76; [0020] L-valine: 0.35-0.56; [0021] L-leucine: 0.13-0.27; [0022] L-lysine hydrochloride: 0.10-0.12.

[0023] L-cysteine or N-acetyl-L-cysteine may also be present in percentages by weight ranging between 1 and 20% of the total amino acid mixture.

[0024] The preferred weight ratios are: [0025] Glycine 1; [0026] L-proline: 0.75; [0027] L-alanine: 0.48-0.51; [0028] L-valine: 0.35-0.37; [0029] L-leucine: 0.13-0.15; [0030] L-lysine hydrochloride: 0.10-0.11, or: [0031] Glycine 1; [0032] L-proline: 0.75; [0033] L-alanine: 0.75-0.76; [0034] L-valine: 0.54-0.56; [0035] L-leucine: 0.13-0.14; [0036] L-Lysine hydrochloride: 0.10-0.11, or [0037] Glycine 1; [0038] L-proline: 0.75; [0039] L-alanine: 0.75-0.76; [0040] L-valine: 0.54-0.56; [0041] L-leucine: 0.13-0.14; [0042] L-Lysine hydrochloride: 0.10-0.11, or [0043] Glycine 1; [0044] L-proline: 0.75; [0045] L-alanine: 0.49-0.51; [0046] L-valine: 0.35-0.36; [0047] L-leucine: 0.26-0.27; [0048] L-lysine hydrochloride: 0.10-0.11.

[0049] Said compositions may be in a form suitable for oral administration, such as solutions, granules, dispersible powder, tablets or capsules.

[0050] The compositions according to the invention may also contain hyaluronic acid or salts thereof, in particular sodium hyaluronate, with an average molecular weight ranging between 500,000 and 3,000,000 Da, in percentages ranging between 0.01 and 3% by weight of the total composition. Said compositions containing the mixture of amino acids described above and hyaluronic acid are. suitable for topical or injectable use. Examples of usable formulations include gels, ointments, emulsions, transdermal patches, sterile solutions and sterile amino-acid powders designed to be reconstituted with sterile aqueous solutions of hyaluronates.

[0051] In the case of oral formulations, the unit doses of glycine range between 100 and 1500 mg.

[0052] In the case of injectable formulations, the unit dose of glycine ranges between 10 and 50 mg, and those of hyaluronic acid or its sodium salt between 10 and 100 mg.

[0053] In the case of topical formulations, the glycine concentration can range from 0.1 to 2% mg/ml.

[0054] The compositions according to the invention are useful in the treatment of elastosis and dermoepidermal atrophy resulting from photoaging, skin disorders with a dermoatrophic and iatrogenic base [17], burns (including radiation burns), skin lesions, bedsores, dermal aplasia caused by drugs (antiretrovirals, anti-HIV drugs, corticosteroids or chemotherapy), tendon and joint lesions.

[0055] Using in vitro transcriptomics and proteomics, it has been found that the mixture of amino acids induces an increase in expression of the genes encoding tropoelastin (ELN) and Type IV collagen (COLIV) after 120 hours' incubation of human fibroblasts. Surprisingly, said effect is no longer present if even one of the amino acids is eliminated or the weight ratios are varied.

[0056] The delivery of amino acids in the form of aqueous solution gelled with hyaluronic acid in the form of sodium salt restores the dermal matrix plasticity and guarantees the retention of the amino acids in the dermoepidermal area for a sufficient time to induce the desired biological effect.

[0057] Injectable formulations can be prepared by dissolving the amino acids in the form of sterile powder in the sterile solution of hyaluronic acid sodium salt by introducing the hyaluronic acid (sodium salt) gel directly (e.g. with a dermal implant syringe) into the vial containing the powder. When completely dissolved, the resulting gel solution is injected into the dennoepidermal region.

[0058] The sterile aqueous solution of hyaluronic acid sodium salt may contain pH-correcting buffer agents (e.g. phosphate buffer) or osmolarity correctors (e.g. sodium chloride) and other technological adjuvants able to guarantee the physicochemical and tissue compatibility characteristics required for sterile injectable pharmaceutical forms. The invention is illustrated in detail in the following examples.

Example 1

[0059] Sterile Solution of Sodium Hyaluronate

TABLE-US-00001 Phase Raw material mg/3 ml A SODIUM 30,000 HYALURONATE (MW = 1,000,000 Daltons) A Phosphate buffer q.s. pH = 7.2 D Sodium chloride q.s. 250 < osm. < 300 D Water for injection q.s. for 3 ml

[0060] Sterile Amino-Acid Powder

TABLE-US-00002 Phase Raw material mg/3 ml A Glycine 30.200 A L-leucine  4.200 A L-valine 16.800 A L-proline 22.700 A L-alanine 22.800 A L-lysine HCl (hydrochloride)  3.300

Example 2

[0061] Sterile Solution of Sodium Hyaluronate

TABLE-US-00003 Phase Raw material mg/3 ml A SODIUM HYALURONATE 30,000 (MW = 1000,000 Daltons) A Phosphate buffer q.s. pH = 7.2 D Sodium chloride q.s. 250 < osm. < 300 D Water for injection q.s. for 3 ml

[0062] Sterile Amino-Acid Powder

TABLE-US-00004 Phase Raw material mg/3 ml A L-proline 25.100 A L-lysine hydrochloride  3.700 A L-valine 12.300 A L-alanine 16.800 A Glycine 33.400 A L-leucine  8.700

Example 3

[0063] Sterile Solution of Sodium Hyaluronate

TABLE-US-00005 Phase Raw material mg/3 ml A SODIUM HYALURONATE 30,000 (MW = 1,000,000 Daltons) A Phosphate buffer q.s. pH = 7.2 D Sodium chloride q.s. 250 < osm. < 300 D Water for injection q.s. for 3 ml

[0064] Sterile Amino-Acid Powder

TABLE-US-00006 Phase Raw material mg/3 ml A L-proline 25.100 A L-lysine hydrochloride  3.700 A L-valine 12.300 A L-alanine 16.800 A Glycine 33.400 A L-leucine  8.700 A N-acetylcysteine 12.500

Example 4

[0065] Topical Hydrogel Based on Amino Acids and Sodium Hyaluronate

TABLE-US-00007 Phase Raw material g/100 g A Glycerine  3.000 A Water q.s. 100 g A Glycine  0.915 A L-proline  0.688 A L-alanine  0.691 A L-leucine  0.127 A L-valine  0.509 A L-lysine hydrochloride  0.100 A N-acetylcysteine  0.600 B Potassium sorbate  0.200 B Sodium benzoate  0.200 B Sodium hyaluronate (mw = 3000000 d)  1.200 from Streptococcus equi 100.00000

Example 5

[0066] Sachets (Stickpack) for Oral Solution

TABLE-US-00008 mg/20 ml stickpack Glycine 1208 mg L-proline  908 mg L-leucine  168 mg L-lysine HCl  132 mg L-valine  672 mg L-alanine  912 mg

[0067] Excipients: preservatives: sodium benzoate, potassium sorbate; acidity regulators: citric acid, sodium citrate.

[0068] Efficacy Tests

[0069] The efficacy of the mixtures of amino acids according to the invention was evaluated by comparison with control mixtures and hyaluronic acid mixtures in stimulating production of the structural ingredients of the extracellular matrix, especially neosynthesis of elastin, and facilitating a more efficient deposit of elastic fibres (elastogenesis), while at the same time maintaining collagen stimulation.

[0070] A primary culture of standardised human fibroblasts was used. The trial design was structured to evaluate the gene expression of elastin and collagen. Gene expression was evaluated by RT-qPCR at the following times: 24, 72 and 120 h. The production of said matrix proteins was evaluated by the Western Blot technique at 120 h.

[0071] A preliminary evaluation of the cytotoxicity of the amino acid and hyaluronic acid mixtures was conducted to identify the maximum concentration tested that was not cytotoxic. The concentration of 1000 μg/ml was selected on the basis of the data obtained.

[0072] Results of Transcriptional Study

[0073] This study included a negative control (NC), corresponding to untreated fibroblasts (physiological response). Mixture A, consisting only of the amino acids constituting collagen (Gly, L-Pro, L-Lys, L-Leu), did not induce significant gene expression. Conversely, the two mixtures containing six amino acids tested, C and D, consisting of the amino acids most expressed in both collagen and elastin (Gly, L-Pro, L-Lys, L-Leu, L-Ala and L-Val), induced significant modulation of the ELN (elastin) and COLIV (collagen IV) genes after 120 h.

[0074] The compositions of the mixtures tested are set out in the table below.

TABLE-US-00009 Mixture A Mixture C Mixture D Glycine 50.0 33.4 30.2 L-Proline 37.5 25.1 22.7 L-Leucine  7.0  8.7  4.2 L-lysine HCl  5.5  3.7  3.3 L-valine — 12.3 16.8 L-alanine — 16.8 22.8

[0075] Gene Expression of Elastin (ELN) and Collagen IV (COLIV) Quantified by Comparison with the Control.

TABLE-US-00010 NC A C D ELN 1 1.478 2.121 2.164 COLIV 1 1.484 2.464 3.197

[0076] FIG. 1 Shows the Results of the Post-Translational Study

[0077] As shown in the graph, mixture A induces an increase in the production of collagen only, while mixtures C and D significantly modulate the production of both elastin and collagen IV.

[0078] Relative Quantitation of the Proteins Collagen IV and Elastin Using NC as Reference.

TABLE-US-00011 NC A C D Elastin 1 0.805 2.403 2.00 Collagen IV 1 1.685 2.547 2.278

[0079] Discussions

[0080] The graph in FIG. 2 shows the results of single-layer human fibroblast studies conducted on the basis of a transcriptomic approach, demonstrating that only certain mixtures of amino acids, optionally combined with hyaluronic acid, can modulate the biosynthesis of the extracellular matrix proteins, and especially promote elastogenesis. Only mixtures of amino acids combined according to the ratios disclosed in this patent increase the gene and protein expression of elastin as well as boosting the stimulation of collagen (especially collagen IV).

[0081] It should be noted that the integrity and quality of the matrix is not restricted to collagen, but to the production and physiological interaction of all the structural proteins produced by the fibroblasts, especially elastin.

[0082] In fact, collagen and elastin maintain the anisotropy in the matrix, i.e. the ability of the fibres produced to propagate the tensile forces, a characteristic which is lost with age.

[0083] It has also been demonstrated that the mixtures of amino acids in question cause physiological elastin gene induction with kinetics very different from the response to UV radiation, wherein the appearance of elastin is rapid, disorderly and, by inducing elastase, actually causes degradation.

CONCLUSIONS

[0084] The results of this study demonstrate that only the mixtures according to the invention promote significant production of the two proteins. It has therefore been demonstrated that not only does an inductive mechanism exist, but the mRNA produced encodes a functional protein.

REFERENCES

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