Growth promoting peptides, and uses thereof

Abstract

A natural peptide comprising a cellular growth promoting fragment of a protein selected from SEQ ID NOs: 1 to 13, and a composition comprising a plurality of growth promoting peptides, is described. Also disclosed is the use of the peptides and compositions in prevention of ageing of human skin, treatment of diseases or conditions characterised by damaged epithelial cells or tissue such as colon cancer and peripheral inflammatory disorders, and wound treatment. Specific pea and rice protein derived peptides are described in SEQ ID NOs: 15 to 505, and 546 to 704.

Claims

1. A composition formulated for topical application to the skin of a human comprising a peptide and a cosmetically acceptable excipient, wherein the peptide is up to 50 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 283.

2. The composition according to claim 1, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 283.

3. The composition according to claim 1, wherein the composition is in the form of a gel, cream, lotion or ointment.

4. The composition according to claim 1, wherein the composition is in the form of a cream, lotion or ointment, and wherein the cosmetically acceptable excipient is selected from an emollient or an emulsifying agent.

5. The composition according to claim 1, comprising 0.1 to 5000 ppm of the peptide.

6. The composition according to claim 1, comprising 0.5 to 500 ppm of the peptide.

7. The composition according to claim 1, wherein the composition is in the form of an emulsion.

8. The composition according to claim 1, wherein the cosmetically acceptable excipient is selected from the group consisting of: a diluent, carrier, binder, lubricant, suspending agent, coating agent, preservative, stabilisers, dyes, vehicle, solubilising agent, base, emollient, emulsifying agent, fragrance, humectant, and surfactants.

9. The composition according to claim 1, wherein the cosmetically acceptable excipient is an emollient or an emulsifying agent.

10. A composition comprising a peptide, wherein the peptide is up to 50 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 283, and wherein the composition is a powder.

11. The composition according to claim 10, wherein the powder is free of un-hydrolysed protein.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1 to 100: Effect of synthetic peptides of the invention on proliferation of Human Dermal Fibroblasts (HDF).

(2) FIG. 101: Effect of synthetic peptide of the invention (SEQ ID NO: 42) on elastin synthesis of Human Dermal Fibroblasts (HDF).

(3) FIG. 102: Effect of synthetic peptide of the invention (SEQ ID NO: 42) on collagen synthesis of Human Dermal Fibroblasts (HDF).

(4) FIG. 103: Effect of synthetic peptide of the invention (SEQ ID NO: 701) on elastin synthesis of Human Dermal Fibroblasts (HDF).

(5) FIG. 104: Effect of synthetic peptide of the invention (SEQ ID NO: 701) on collagen synthesis of Human Dermal Fibroblasts (HDF).

(6) FIG. 105: Effect of synthetic peptide of the invention (SEQ ID NO: 246) on elastin synthesis of Human Dermal Fibroblasts (HDF).

(7) FIG. 106: Effect of synthetic peptide of the invention (SEQ ID NO: 246) on collagen synthesis of Human Dermal Fibroblasts (HDF).

(8) FIG. 107: Effect of synthetic peptide of the invention (SEQ ID NO: 284) on elastin synthesis of Human Dermal Fibroblasts (HDF).

(9) FIG. 108: Effect of synthetic peptide of the invention (SEQ ID NO: 245) on elastin synthesis of Human Dermal Fibroblasts (HDF).

(10) FIG. 109: Effect of synthetic peptide of the invention (SEQ ID NO: 245) on collagen synthesis of Human Dermal Fibroblasts (HDF).

(11) FIG. 110. shows the integrity controls and viability controls for the assay system.

(12) FIG. 111. % of elastin expression in superficial dermis compared to control (water or DMSO) for peptides P1, P2 and P3

(13) *shows significant increases of elastin expression in superficial AND middle dermis.

(14) FIG. 112. % of elastin expression in middle dermis compared to control (water or DMSO) for peptides P1, P2 and P3.

(15) *shows significant increases of elastin expression in superficial AND middle dermis.

(16) FIG. 113. % of cell proliferation in the basal layer of epidermis compared to control (water or DMSO) for peptides P6 and P8, and peptide compositions P9 and P10

(17) *shows significant increases.

(18) FIG. 114. Histological analysis of the elastic fibers (+catechin, 200) FIG. 115. Immunohistochemical evaluation of the mitotic index (Ki67, 400)

DETAILED DESCRIPTION OF THE INVENTION

Example 1Cell Proliferation Assay

(19) BrDu is incorporated into newly synthesised DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, Brdu is detected immunochemically allowing the assessment of the population of cells which are synthesizing DNA.

(20) Human Dermal Fibroblasts (HDFSigma 10605a) were seeded in a 96 well plate at 10,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.

(21) Following the initial 24 h incubation the cells were incubated with 5 g/ml, 0.5 g/ml or 0.05 g/ml synthetic peptide for 24 h respectively.

(22) After 18 h incubation with synthetic peptides 20 l BrDu reagent was added to each well.

(23) At 24 h incubation the cell were fixed and the amount of 2-DG6P was measured using the BrdU Cell Proliferation Assay, all steps were carried out according to the manufacturer's instructions.

(24) Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates greatER incorporation of BrDu and increase cell proliferation.

(25) The results are shown in FIGS. 1-100 and Table 1 below.

(26) TABLE-US-00009 TABLE 1 FIG. SEQ ID INCREASE IN NO NO: PROLIFERATION 1 121 48% 2 105 40% 3 249 30% 4 226 30% 5 84 20% 6 330 18% 7 181 33% 8 83 32% 9 247 28% 10 97 26% 11 74 29% 12 13 168 14 151 15 470 119% 16 257 118% 17 256 117% 18 457 114% 19 499 113% 20 253 112% 21 222 110% 22 272 97% 23 252 111% 24 248 86% 25 472 77% 26 365 58% 27 502 68% 28 496 51% 29 98 49% 30 454 38% 31 85 35% 32 453 25% 33 158 21% 34 464 18% 35 73 16% 36 359 15% 37 124 15% 38 112 15% 39 733 40 728 41 727 42 730 43 731 44 148 45 343 46 345 47 484 48 729 49 456 50 494 51 723 52 722 53 498 54 475 13% 55 718 56 337 8% 57 500 6% 58 717 59 297 60 340 61 719 62 726 63 725 64 724 65 720 66 721 67 503 125% 68 474 121% 69 504 119% 70 114 119% 71 505 118% 72 482 113% 73 479 106% 74 477 81% 75 410 73% 76 475 69% 77 497 58% 78 480 102% 79 463 100% 80 465 96% 81 467 90% 82 461 85% 83 341 83% 84 468 82% 85 285 81% 86 496 81% 87 146 80% 88 478 76% 89 452 76% 90 495 68% 91 403 51% 92 455 47% 93 270 47% 94 501 43% 95 473 41% 96 39% 97 471 38% 98 460 38% 99 93 26% 100 462 15%

(27) FIG. 1 shows an increase in HGF cell proliferation of 48% when incubated with SEQ ID 121.

(28) FIG. 2 shows an increase in HGF cell proliferation of 40% when incubated with SEQ ID 105.

(29) FIG. 3 shows an increase in HGF cell proliferation of 30% when incubated with SEQ ID 249.

(30) FIG. 4 shows an increase in HGF cell proliferation of 30% when incubated with SEQ ID 226.

(31) FIG. 5 shows an increase in HGF cell proliferation of 20% when incubated with SEQ ID 84.

(32) FIG. 6 shows an increase in HGF cell proliferation of 18% when incubated with SEQ ID 330.

(33) FIG. 7 shows an increase in HGF cell proliferation of 33% when incubated with SEQ ID 181.

(34) FIG. 8 shows an increase in HGF cell proliferation of 32% when incubated with SEQ ID 83.

(35) FIG. 9 shows an increase in HGF cell proliferation of 28% when incubated with SEQ ID 247.

(36) FIG. 10 shows an increase in HGF cell proliferation of 26% when incubated with SEQ ID 97.

(37) FIG. 11 shows an increase in HGF cell proliferation of 29% when incubated with SEQ ID 74.

(38) FIG. 15 shows an increase in HGF cell proliferation of 119% when incubated with SEQ ID 470.

(39) FIG. 16 shows an increase in HGF cell proliferation of 118% when incubated with SEQ ID 257.

(40) FIG. 17 shows an increase in HGF cell proliferation of 117% when incubated with SEQ ID 256.

(41) FIG. 18 shows an increase in HGF cell proliferation of 114% when incubated with SEQ ID 457.

(42) FIG. 19 shows an increase in HGF cell proliferation of 113% when incubated with SEQ ID 499.

(43) FIG. 20 shows an increase in HGF cell proliferation of 112% when incubated with SEQ ID 253.

(44) FIG. 21 shows an increase in HGF cell proliferation of 110% when incubated with SEQ ID 222.

(45) FIG. 22 shows an increase in HGF cell proliferation of 97% when incubated with SEQ ID 272.

(46) FIG. 23 shows an increase in HGF cell proliferation of 111% when incubated with SEQ ID 252.

(47) FIG. 24 shows an increase in HGF cell proliferation of 86% when incubated with SEQ ID 248.

(48) FIG. 25 shows an increase in HGF cell proliferation of 77% when incubated with SEQ ID 472.

(49) FIG. 26 shows an increase in HGF cell proliferation of 58% when incubated with SEQ ID 365.

(50) FIG. 27 shows an increase in HGF cell proliferation of 68% when incubated with SEQ ID 502.

(51) FIG. 28 shows an increase in HGF cell proliferation of 51% when incubated with SEQ ID 496.

(52) FIG. 29 shows an increase in HGF cell proliferation of 49% when incubated with SEQ ID 98.

(53) FIG. 30 shows an increase in HGF cell proliferation of 38% when incubated with SEQ ID 454.

(54) FIG. 31 shows an increase in HGF cell proliferation of 35% when incubated with SEQ ID 85.

(55) FIG. 32 shows an increase in HGF cell proliferation of 25% when incubated with SEQ ID 453.

(56) FIG. 33 shows an increase in HGF cell proliferation of 21% when incubated with SEQ ID 158.

(57) FIG. 34 shows an increase in HGF cell proliferation of 18% when incubated with SEQ ID 464.

(58) FIG. 35 shows an increase in HGF cell proliferation of 16% when incubated with SEQ ID 73.

(59) FIG. 36 shows an increase in HGF cell proliferation of 15% when incubated with SEQ ID 359.

(60) FIG. 37 shows an increase in HGF cell proliferation of 15% when incubated with SEQ ID 124.

(61) FIG. 38 shows an increase in HGF cell proliferation of 15% when incubated with SEQ ID 112.

(62) FIG. 54 shows an increase in HGF cell proliferation of 13% when incubated with SEQ ID 475.

(63) FIG. 56 shows an increase in HGF cell proliferation of 8% when incubated with SEQ ID 337.

(64) FIG. 57 shows an increase in HGF cell proliferation of 6% when incubated with SEQ ID 500.

(65) FIG. 67 shows an increase in HGF cell proliferation of 125% when incubated with SEQ ID 503.

(66) FIG. 68 shows an increase in HGF cell proliferation of 121% when incubated with SEQ ID 474.

(67) FIG. 69 shows an increase in HGF cell proliferation of 119% when incubated with SEQ ID 504.

(68) FIG. 70 shows an increase in HGF cell proliferation of 119% when incubated with SEQ ID 114.

(69) FIG. 71 shows an increase in HGF cell proliferation of 118% when incubated with SEQ ID 505.

(70) FIG. 72 shows an increase in HGF cell proliferation of 113% when incubated with SEQ ID 482.

(71) FIG. 73 shows an increase in HGF cell proliferation of 106% when incubated with SEQ ID 479.

(72) FIG. 74 shows an increase in HGF cell proliferation of 81% when incubated with SEQ ID 477.

(73) FIG. 75 shows an increase in HGF cell proliferation of 73% when incubated with SEQ ID 410.

(74) FIG. 76 shows an increase in HGF cell proliferation of 69% when incubated with SEQ ID 475.

(75) FIG. 77 shows an increase in HGF cell proliferation of 58% when incubated with SEQ ID 497.

(76) FIG. 78 shows an increase in HGF cell proliferation of 102% when incubated with SEQ ID 480.

(77) FIG. 79 shows an increase in HGF cell proliferation of 100% when incubated with SEQ ID 463.

(78) FIG. 80 shows an increase in HGF cell proliferation of 96% when incubated with SEQ ID 465.

(79) FIG. 81 shows an increase in HGF cell proliferation of 90% when incubated with SEQ ID 467.

(80) FIG. 82 shows an increase in HGF cell proliferation of 85% when incubated with SEQ ID 461.

(81) FIG. 83 shows an increase in HGF cell proliferation of 83% when incubated with SEQ ID 341.

(82) FIG. 84 shows an increase in HGF cell proliferation of 82% when incubated with SEQ ID 468.

(83) FIG. 85 shows an increase in HGF cell proliferation of 81% when incubated with SEQ ID 485.

(84) FIG. 86 shows an increase in HGF cell proliferation of 81% when incubated with SEQ ID 496.

(85) FIG. 87 shows an increase in HGF cell proliferation of 80% when incubated with SEQ ID 146.

(86) FIG. 88 shows an increase in HGF cell proliferation of 76% when incubated with SEQ ID 478.

(87) FIG. 89 shows an increase in HGF cell proliferation of 76% when incubated with SEQ ID 452.

(88) FIG. 90 shows an increase in HGF cell proliferation of 68% when incubated with SEQ ID 495.

(89) FIG. 91 shows an increase in HGF cell proliferation of 51% when incubated with SEQ ID 403.

(90) FIG. 92 shows an increase in HGF cell proliferation of 47% when incubated with SEQ ID 455.

(91) FIG. 93 shows an increase in HGF cell proliferation of 47% when incubated with SEQ ID 270.

(92) FIG. 94 shows an increase in HGF cell proliferation of 43% when incubated with SEQ ID 501.

(93) FIG. 95 shows an increase in HGF cell proliferation of 41% when incubated with SEQ ID 473.

(94) FIG. 97 shows an increase in HGF cell proliferation of 38% when incubated with SEQ ID 471.

(95) FIG. 98 shows an increase in HGF cell proliferation of 38% when incubated with SEQ ID 460.

(96) FIG. 99 shows an increase in HGF cell proliferation of 26% when incubated with SEQ ID 93.

(97) FIG. 100 shows an increase in HGF cell proliferation of 15% when incubated with SEQ ID 462.

Example 2Collagen Production Assay

(98) Hydroxyproline in tissue preparations is a direct measure of the amount of collagen present. FIRELISA Human Hydroxyproline ELISA kit assay is designed to measure hydroxyproline in tissue or peptide compositions.

(99) Human Dermal Fibroblasts (HDF Sigma 10605a) were seeded in 24 well plates at 50,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.

(100) Following the initial 24 h incubation the cells were incubated with 5 g/ml, 1 g/ml or 0.1 g/ml synthetic peptide for 96 h respectively.

(101) After treatment the cells were lysed using 4 freeze thaw cycles in liquid nitrogen. The lysed cells were centrifuged and 50 l/ml of each supernatant was assayed using the FIRELISA

(102) Human Hydroxyproline ELISA kit. All steps were carried out according to the manufacturer's instructions.

(103) Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates an increase collagen content.

(104) The results are shown in FIGS. 102, 104, 106 and 109

Example 3Elastin Production Assay

(105) Elastin is a highly elastic protein in connective tissue and allows many tissues in the body to resume their shape after stretching or contracting. FIRELISA Human Elastin ELISA kit assay is designed to measure Elastin in tissue or protein/peptide compositions.

(106) Human Dermal Fibroblasts (HDF) were seeded in 24 well plates at 50,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.

(107) Following the initial 24 h incubation the cells were incubated with 5 g/ml, 1 g/ml or 0.1 g/ml synthetic peptide for 96 h respectively.

(108) After treatment the cells were lysed using 4 freeze thaw cycles in liquid nitrogen. The lysed cells were centrifuged and 50 l/ml of each supernatant was assayed using the FIRELISA Human Elastin ELISA kit. All steps were carried out according to the manufacturer's instructions.

(109) Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates an increase collagen content.

(110) The results are shown in FIGS. 101, 103, 105, 107, 108 and 109.

Example 4Elastin and Cell Proliferation Assays

(111) TABLE-US-00010 TABLE 2 Test items. Orange bands correspond to samples dissolved into DMSO 0.3% instead of water. Intertek Item Denomination Concentration Provider Nature reference Solubility Storage Peptide 1 E_280_PJ 20 M Nuritas Peptide 14-CHL- Water 80 C. 0723-01 Peptide 2 I_222two_IN 20 M Nuritas Peptide 14-CHL- Water Ambient 0723-02 Peptide 3 E_134_two_IN 20 M Nuritas Peptide 14-CHL- Water 80 C. 0723-03 Peptide 4 E_30two_IN 20 M Nuritas Peptide 14-CHL- Water 80 C. 0723-04 Peptide 5 E_121two_IN 20 M Nuritas Peptide 14-CHL- Water 80 C. 0723-05 Peptide 6 I_10two_IN 20 M Nuritas Peptide 14-CHL- DMSO 80 C. 0723-06 0.3% Peptide 7 I_41two_IN 20 M Nuritas Peptide 14-CHL- DMSO 80 C. 0723-07 0.3% Peptide 8 E_41_PJ 10 M* Nuritas Peptide 14-CHL- Water 80 C. 0723-08 Composition E_2_IN 500 g/mL Nuritas Composition 14-CHL- Water 80 C. P9 of peptides 0723-09 Composition I_2_IN 500 g/mL Nuritas Composition 14-CHL- Water 80 C. P10 of peptides 0723-10
Equipment

(112) Incubator, Flow Laminar Chamber, Sterile Polished Plastic Rod, Pipettor, Maintenance medium, Plate 6 well, Plate 24 well.

(113) Reagents

(114) MTT, PBS, SDS, Formaldehyde, Xylene, Ethanol absolute, Dulbecco's phosphate-buffered saline (DPBS), Metal Enhanced DAB substrate kit, ABC peroxidase staining kit, Citric acid, Sodium hydroxide 2N, Hydrogen peroxide 30%, Anti-Filaggrin, Anti-rabbit IgG-Biotin, Tween 20.

(115) Test System

(116) Nature: Human skin tissue 5 mm diameter

(117) Batch number: EXP004050B009 and EXP004050B011

(118) Provider: Laboratoire Biopredic International8-18 rue Jean Pecker35000 RennesFrance. Tel: +33 (0)2.99.14.36.14Fax: +33 (0)2.99.54.44.72.

(119) Certificates of analysis are present in Annex 1.

(120) Two batches are used for the assay. Batch EXP004050B005 is used for experiment day 1, and Batch EXP004050B006 is used for experiment day 5.

(121) Maintenance Medium

(122) Maintenance Medium: Batch No: MIL 218C

(123) Provider: Laboratoire Biopredic International8-18 rue Jean Pecker35000 RennesFrance.

(124) Peptides Tested

(125) TABLE-US-00011 P1: SEQIDNO:283 P2: SEQIDNO:246 P3: SEQIDNO:284 P4: (SEQIDNO:776) RPYYSNAPQEIF P5: (SEQIDNO:777) VLLEQQEQEPQH P6: SEQIDNO:245 P7: (SEQIDNO:778) QQYGIAASPFLQSAA P8: SEQIDNO:42
Compositions Tested

(126) P9 (14-CHL-0723-09) is the Pea composition (SEQ ID Numbers: 50, 85, 74, 140, 82, 136, 189, 77, 169, 149, 171, 178, 143, 127, 190, 141, 147, 133, 186, 125, 122, 119, 87, 90, 86, 89, 138, 129, 123, 120, 117, 113, 110, 121, 105, 98, 55, 161, 19, 317, 135, 130, 146, 177, 160, 170, 188, 83, 78, 36, 96, 159, 26, 330, 168, 148, 184, 151, 151, 165, 114, 284)

(127) P10 (14-CHL-0723-010) is the Rice composition (SEQ ID Numbers: 245, 246, 263, 250, 257, 259, 276, 255, 251, 264, 256, 266, 274, 270, 269, 356, 245, 380, 262, 258, 356, 218, 252, 358, 271, 253, 344, 275, 272, 226, 224, 220, 248, 261, 265, 373, 375, 247, 249, 363, 273, 343, 273, 362)

(128) Application Method

(129) Skin explants were prepared from abdominal plastic surgery. Some explants were delipidated with alcohol to obtain a dehydrated skin.

(130) These explants were maintained in maintenance medium supplied by the provider Bioprdic International for 5 days. Test items are applied twice per day with 5 L per explant.

(131) At the end of the test, viabilities controls are realized with the MTT on two explants, the third explant is fixed in the formaldehyde 4% for histology and cell staining.

(132) For each time of analysis (D1 and D5), histologies on delipidated explants, treated explants with test items, the DMSO 0.3% control and water control, are performed.

(133) After receipt in the laboratory, each skin explant in the maintenance medium is delipidated with 5 L alcohol during 3 hours.

(134) After 3 hours, all skin explants are treated two per day with test items, and they are incubated at 37 C.+/2 C., 5% CO2 for 1 day or 5 days.

(135) Integrity of the system is realized at day 1 and day 5 with a viability control with MTT.

(136) Immunostaining

(137) Histology is realized by the laboratory Gredeco and the immunostaining to elastin and Ki67 are realized by the same laboratory. Immunostaining to filaggrin is realized by the laboratory Intertek.

(138) The detection of elastin (rabbit monoclonal antibody, clone P15502, LSBio) is performed using an immunoperoxidase technique two layers (ABC kit, Vector Laboratories) and revealed by AEC (3-amino-9-ethylcarbazole). The immunohistochemical staining intensity in the elastic fibers is evaluated using a semi-quantitative histological score.

(139) Epithelial proliferation was analyzed by immunohistochemistry using anti-Ki67 antibody. Immunodetection was performed using an indirect immunoperoxidase technique three layers, amplified (DAKO kit) and revealed by AEC (3-Amino-9-ethylcarbazole). Counting the number of labeled cells (keratinocytes of the basal layer of the epidermis) is performed and provides the total number of basal cells to calculate the % of labeled cells.

(140) The specific staining of filaggrin is performed with an immunoperoxidase staining (ABC kit, Fisher). The intensity of immunohistochemical marker in the epidermis is evaluated relative to the negative control of the solvent (Water or DMSO 0.3%).

(141) C: Results

(142) Viability Control

(143) The integrity control and the viability control are present in FIG. 1. These controls do allow to validate the assay system. The viability is >50% for test items, and they do not show a cytotoxicity according to the test.

(144) Immunostaining

(145) Elastin Expression

(146) The elastic fibers of the dermis were revealed by staining with the catechin and morphometrically quantified by analysis by computer-assisted image. The percentage area taken up by elastic fibers in the dermis was calculated in the dermis and the average superficial dermis. Results are presented in FIGS. 101, 105, 107, and 108.

(147) TABLE-US-00012 TABLE 3 Morphometric quantification of elastic fibers in the superficial dermis (%) and middle dermis. All samples dissolved in water except where indicated. Morphometric Morphometric quantification quantification of elastic fibers of elastic fibers in the superficial in the middle dermis (%) dermis (%) Conditions D1 D5 D1 D5 Dehydrated with alcohol 4.31 4.9 5 5.83 and hydrated with water Dehydrated 2.38 7.26 4.39 9.59 EGF (Epidermal Growth 3.64 5.61 5.68 6.61 Factor) 10 ng/mL Dehydrated with alcohol 3.76 7.24 6 10.36 and hydrated with DMSO 0.3% 0723.01 4.45 10.21 7.59 10.17 0723.02 6.09 7.59 11.75 9.08 0723.03 3 11.68 4.9 9 0723.04 3.28 8.94 5.22 9 0723.05 6.34 6.26 8.8 6.61 0723.06 (dissolved in DMSO 3.8 4.03 4.54 8.67 0.3% instead of water) 0723.07 (dissoved in DMSO 4 5.15 6.46 5.33 0.3% instead of water) 0723.08 2.7 5.32 3.52 7.27 0723.09 3.26 8.26 5.75 7.92 0723.10 4.1 8 5.73 8.34

(148) Under the experimental conditions of the study, 0723-1 and 0723-3 samples show an increase by twice of elastic fibers in the superficial dermis compared to control water (Figure), and an increase in the middle dermis compared to the water control at D5.

(149) The 0723-2 sample shows an increase doubled in the middle dermis at day 1 compared to control water and an increase at day 5.

(150) Ki67 Expression

(151) The results of the immunohistochemical analysis of Ki67 are reported in Table 4 and expressed as % of labelled at the basal layer of the epidermis. The FIG. 115 shows the percentage of Ki 67 cells compared to negative controls (water or DMSO). Immunohistochemical analysis of mitotic activity is shown in annex 4 with a reminder of the average for each analysed conditions.

(152) TABLE-US-00013 TABLE 4 % of Ki67 positive cells in the basal layer of the epidermis. All samples dissolved in water except where indicated. Conditions D1 D5 Dehydrated with alcohol and hydrated with 19.09 3.53 water Dehydrated 17.05 1.76 EGF (Epidermal Growth Factor) 10 ng/mL 25.11 4.2 Dehydrated with alcohol and hydrated with 17.2 2.61 DMSO 0.3% 0723.01 18.57 3.92 0723.02 19.61 6.73 0723.03 22.01 10.04 0723.04 14.97 11.36 0723.05 9.48 3.08 0723.06 (dissolved in DMSO 31.97 5.04 0.3% instead of water) 0723.07 (dissoved in DMSO 22.22 5.26 0.3% instead of water) 0723.08 27.83 5.72 0723.09 31.02 2.4 0723.10 31.94 3.57

(153) Under the experimental conditions of the study, test item 0723-06, 0723-08, 0723-09 and 0723-010 show an increase in the number of mitotic cells compared to EGF at day 1. A decrease in the mitotic index was observed on day 5 compared to day 1 for all analysed conditions.

(154) The decrease in this cell staining on day 5 is caused by the model. Indeed, after approximately 3 days cell turnover is exhausted on this model.

(155) The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.