COLLAGENOUS EXTRACTS FOR USE AS A MEDICAMENT

Abstract

The present invention relates to a collagenous marine invertebrate extract for use in the treatment of Epidermolysis Bullosa, pharmaceutical compositions comprising said collagenous marine invertebrate extract, and methods for manufacturing the collagenous marine invertebrate extract of the invention.

Claims

1. A collagenous marine invertebrate extract for use in the treatment of Epidermolysis Bullosa, wherein the collagenous extract comprises collagen in a hydrolysate form.

2. The collagenous marine invertebrate extract according to claim 1, wherein the collagen in hydrolysate form is produced by heat treatment of the extract.

3. The collagenous marine invertebrate extract for use according to claim 1, wherein the Epidermolysis Bullosa to be treated is selected from Epidermolysis bullosa simplex (EBS), Junctional epidermolysis bullosa (JEB), Dystrophic epidermolysis bullosa (DEB), Epidermolysis bullosa acquisita (EBA), and/or Kindler syndrome (KS).

4. The collagenous marine invertebrate extract for use according to claim 1, wherein the collagenous marine invertebrate extract: (a). supports improved dermal cohesion and basal layer structuring of cells via the activation of the genes COL7A1, LAMA5, KRT5; (b) downregulates MMP-3 activity supporting better treatment outcomes for severe RDEB; (c) supports improved cell adhesion, growth, migration, and differentiation via the activation of the genes FN1; (d) promotes extracellular matrix synthesis, collagen fibre organisation and structuring via activation of the genes COL3A1, FBN1, FBN2, SPARC, and LUM; (e). supports improved dermal cohesion and basal layer structuring of cells via the activation of the genes COL7A1, LAMA5 and CDC42; (f). supports dermis renewal and protection via the activation of Epithelial Growth Factor (EGF), SIRT4 and SIRT6; (g). supports improved dermal cellular junction structure via activation of the genes EPPK and LOR, and/or (h) supports improved extracellular matrix structure by reducing MMP3 activation involved in EB blistering.

5. The collagenous marine invertebrate extract for use according to claim 1, wherein the source of the extract is jellyfish.

6. The collagenous marine invertebrate extract for use according to claim 5, wherein the jellyfish is from the class Scyphozoa.

7. The collagenous marine invertebrate extract for use according to claim 5, wherein the jellyfish is one or more selected from the group comprising: Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Aurelia sp., Nemopilema nomurai, Cassiopea andromeda, Nemopilema nomurai, Chrysaora sp., or any combination thereof.

8. The collagenous marine invertebrate extract for use according to claim 7, wherein the jellyfish is Rhizostomas pulmo.

9. The collagenous marine invertebrate extract for use according to claim 1, wherein the collagenous marine invertebrate extract is in the form of a solution, powder, sponge matrix, nano-fibre electrospun matrix, a hydrogel or in a lyophilised form.

10. The collagenous marine invertebrate extract for use according to claim 1, wherein the collagenous marine invertebrate extract is administered in combination with at least one growth factor, preferably chosen among Platelet Rich Plasma (PRP), Epithelial Growth Factor (EGF), Transforming Growth Factor-Beta (TGF-B, TGF-B2, TGF-B3), Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), Granulocyte-Monocyte Colony Stimulating Growth Factor, Platelet Derived Growth Factor, Insulin-like Growth Factor 1 (IGF1), basic Fibroblast Growth Factor (bFGF), and/or Vascular Endothelial Growth Factor (VEGF), and/or at least one antimicrobial compound, preferably selected from nano siliver, penicillin, ofloxacin, tetracycline, aminoglycosides and erythromycin, flucloxacillin, clarithromycin, doxycycline, gentamicin, metronidazole, co-amoxiclav, co-trimoxazole (in penicillin), ceftriaxone, piperacillin with tazobactam, clindamycin, ciprofloxacin, vancomycin, teicoplanin, linezolid, and/or the standard of care antimicrobial agent.

11. The collagenous marine invertebrate extract for use according to claim 1, wherein the collagenous marine invertebrate extract is administered at a dose from 0.01 g/L to 200 g/L, preferably 1 g/L to 20 g/L per administration, more preferably 1 g/L to 50 g/L per administration.

12. A pharmaceutical composition comprising the collagenous marine invertebrate extract defined in claim 1.

13. The pharmaceutical composition according to claim 12, wherein the composition further comprises at least one pharmaceutically acceptable compound.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable compound is one or more selected from the group comprising: a growth factor, an antimicrobial, an antibiotic, and/or another Epidermolysis Bullosa therapeutic.

15. The pharmaceutical composition according to claim 12, wherein the composition is formulated in any form normally used for topical application, preferably a cream, ointment, mask, serum, milk, lotion, paste, foam, aerosol, stick, shampoo, conditioner, patch, hydroalcoholic or oily aqueous solution, an oil-in-water or water-in-oil or multiple emulsion, an aqueous or oily gel, a liquid, pasty or solid anhydrous product, an electrospun collagen nano-fibre matrix, a membrane and/or an oil dispersion in an aqueous phase using spherules, these spherules being polymeric nanoparticles such as nanospheres and nanocapsules or lipid vesicles of ionic and/or non-ionic type, more preferably an electrospun collagen nano-fibre matrix and/or a membrane.

16. A method for manufacturing a collagenous marine invertebrate extract, comprising the steps of: (a) Heating marine invertebrate soluble collagen between 40? C.-100? C. for between 10 minutes to 120 minutes, preferably between 60? C.-80? C. for 45 minutes to 90 minutes, so as to hydrolyse the collagenous material; (b) Recovering the hydrolysed material.

17. The method according to claim 16, wherein the method further comprises purification of the material obtained after step (a) prior to step (b).

18. The method according to claim 17, wherein the purification is by membrane filtration or chromatographic purification.

Description

DESCRIPTION OF THE FIGURES

[0111] FIG. 1 shows the results of the immunolabeling of Loricrin. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0112] FIG. 2 shows the results of the immunolabeling of SIRT-4. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0113] FIG. 3 shows the results of the immunolabeling of SIRT-6. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0114] FIG. 4 shows the results of the immunolabeling of collagen VII. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0115] FIG. 5 shows the results of the immunolabeling of collagen III. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0116] FIG. 6 shows the results of the immunolabeling of collagen V. (A) shows the result of the negative control without the primary antibody; (B) shows the control batch at Day 0 (J0); (C) shows the control batch at Day 8 (J8); (D) shows the P1 batch at day 8 (J8); (E) shows the P2 batch at day 8 (J8).

[0117] FIG. 7 shows a representative SDS PAGE image (3-8% Tris acetate gel) demonstrating the effect of heat-treatment on the size distribution of proteins in collagenous jellyfish extracts. Lane 1: molecular weight marker; Lanes 2-4: native collagenous jellyfish extracts; Lane 5: empty; Lanes 6-8: heat-treated collagenous jellyfish extracts.

[0118] FIG. 8 shows the results of treating healthy human skin explants with heat-treated collagenous jellyfish extract. (A) Representative immunofluorescence image of explant slices stained for collagen 7 with and without heat-treated collagenous jellyfish extract treatment. (B) Graph showing the average percentage area of collagen 7 staining in untreated and treated explant slices (n=10). The percentage area of collagen 7 staining is normalised to the untreated control (100%) and the error bars represent standard error of the mean.

[0119] FIG. 9 shows COL7 gene expression in healthy human epidermal keratinocytes (NHEKs) following treatment with heat-treated collagenous jellyfish extract, as determined by semi-quantitative RT-PCR (Applied Biosystems 7300 Real Time PCR System). The average expression level of COL7 mRNA in treated NHEKs (n=3) is shown relative to untreated control cells (1.0) and the error bars represent standard error of the mean.

[0120] FIG. 10 shows a representative image immunofluorescence image of explant slices stained for collagen 7 with and without heat-treated collagenous jellyfish extract treatment in accordance with the analysis described in Example 10. Analogous images were generated for explant slices stained for laminin 5 and keratin 5.

EXAMPLES

Example 1: Characterisation of R. Pulmo Collagen

[0121] The physicochemical characteristics of R. pulmo collagens (native or hydrolysed by heat-treatment) are shown in Table 1.

TABLE-US-00001 TABLE 1 Physicochemical characteristics of R. pulmo collagens Collagenous extract characteristics Collagen hydrolysate Aspect Liquid Liquid Colour Colourless to pale yellow Colourless to pale yellow Solvent Acetic acid 0.1M Acetic acid 0.5M Concentration 3-6 mg .Math. mL.sup.?1 3-10 mg .Math. mL.sup.?1 (8.25 mg .Math. mL.sup.?1) Turbidity Clear to slightly cloudy Clear to slightly cloudy Protein Level >80% >80% pH 2.8-3.2 2.8-3.5

[0122] Jellyfish collagen was also tested for cytotoxicity. The biocompatibility of jellyfish collagen (3.7 mg.Math.mL-1) was evaluated by a measurement of cytotoxicity on a cell culture (murine fibroblasts L929), according to the ISO 10993-5 2009 method by Contact Indirect. The results indicate that neither the native nor the hydrolysed collagenous jellyfish extract resulted in detectable cytotoxicity under the test conditions used.

Example 2: Evaluation of the Jellyfish Collagenous Hydrolysate by Immunolabeling of the Synthesis of Loricrin, Sirtuin-4 and 6, and Collagen III, V and VII

[0123] Twelve explants of about 1 cm in diameter were prepared from an abdominoplasty surgery of a 51-year-old woman with dark skin. The explants were placed in a BEM (BIO-EC's Explants Medium) environment at 37? C. in a humid atmosphere, enriched with 5% CO2. Table 2 below shows the treatment conditions used on the obtained explants.

TABLE-US-00002 TABLE 2 Treatment conditions of explants Lots Names Concentration No explants End T0 Abdominoplasty / 3 J0 control T Untreated / 3 J8 control P1 Product 1 0.1 mg/mL 3 J8 P2 Product 2 1 mg/mL 3 J8

[0124] P1 and P2 are R. Pulmo collagen hydrolysates at different concentrations (as shown in Table 3 below). J0 and J8 mean Day 0 and 8 respectively.

TABLE-US-00003 TABLE 3 Concentrations and names of the products tested: Product Concentrations P1 0.1 mg/mL P2 1 mg/mL

[0125] The purpose of this study was to carry out the following immunolabeling on mounted slices from the treated explants: [0126] Immunolabeling of loricrin; [0127] Immunolabeling of sirtuin-4; [0128] Immunolabeling of sirtuin-6; [0129] Immunolabeling of collagen VII; [0130] Immunolabeling of collagen III; [0131] Immunolabeling of collagen V.

[0132] The products P1 (0.1 mg/mL) and P2 (1 mg/mL) were applied to the explants described above, and compared after 8 days with an abdominoplasty control (TO) and a non-treated control (T).

[0133] Histological Treatments

[0134] Cuts of 5 ?m were made using a microtome type Minot, Leica RM2125 and mounted on Superfrost? histological glass slides. Cuts of 7 ?m were made using a Leica CM 3050 cryostat and mounted on Superfrost? Plus silanised histological glass slides.

[0135] Microscopic observations were made by optical microscopy, using a Leica microscope such as DMLB or Olympus BX43. The shots were taken with an Olympus DP72 camera and CellD software.

[0136] a. Immunolabeling of Loricrin

[0137] Loricrin was labelled on formalinised paraffin sections with a polyclonal antibody against loricrin (Covance, ref. PRB-14P), diluted to 1/1600th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, with a biotin amplifier/streptavidin system, revealed in VIP, a violet peroxidase substrate (Vector, ref. SK 4600). The labeling was carried out using an immunolabeling automaton (Autostainer, Dako) and was evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0138] b. Immunolabeling of Sirtuin-4 (SIRT-4)

[0139] Sirtuin-4 was labelled on formalin paraffin sections with a polyclonal antibody against sirtuin-4 (GeneTex, ref. GTX51798), diluted 1/100th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, with a biotin amplifier/streptavidin system, revealed in VIP, a violet peroxidase substrate (Vector, ref. SK 4600). The labeling was carried out using an immunolabeling automaton (Autostainer, Dako) and was evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0140] c. Immunolabeling of Sirtuin-6 (SIRT-6)

[0141] Sirtuin-6 was labelled on formalin paraffin sections with a polyclonal antibody against sirtuin-6 (Novus Biologicals, ref. NB100-2522), diluted to 1/100th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, with a biotin/streptavidin amplifier system, revealed in VIP, a purple peroxidase substrate (Vector, ref. SK 4600). The labeling was carried out using an immunolabeling automaton (Autostainer, Dako) and was evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0142] d. Immunolabeling of Collagen VII

[0143] Collagen VII was labelled on frozen sections with a monoclonal antibody against collagen VII (Santa Cruz, ref. sc-53226, clone LH 7.2), diluted 1/100th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, and revealed in AlexaFluor 488 (Lifetechnologies, ref. A11008). The labeling was carried out using an immunolabeling automaton (Autostainer, Dako) and was evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0144] e. Immunolabeling of Collagen III

[0145] Collagen III was labelled on frozen sections with a polyclonal antibody against collagen III (SBA, ref. 1330-01), diluted to 1/100th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, with a biotin/streptavidin amplifier system, revealed in VIP, a purple peroxidase substrate (Vector, ref. SK 4600). The marking was done manually and evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0146] f. Immunolabeling of Collagen V

[0147] Collagen V was labelled on formalin paraffin sections with a polyclonal antibody against collagen V (Novotec, ref. 20511), diluted to 1/100th in PBS-BSA 0.3%-Tween 20 to 0.05% for 1 h at room temperature, with a biotin/streptavidin amplifier system, revealed in VIP, a violet peroxidase substrate (Vector, ref. SK 4600). The labeling was carried out using an immunolabeling automaton (Autostainer, Dako) and was evaluated by microscopic examination. Lots concerned: T0, TJ8, P1J8 and P2J8.

[0148] Results and Conclusions

[0149] The results for Loricrin are shown in FIG. 1. At Day 0, on the control batch T0, the loricrin marking is fairly clear to clear in the granular layer. At Day 8, on the control batch TJ8, loricrin expression is moderate in the granular layer. Relative to control lot TJ8, both P1 and P2 induce a slight increase in loricrin expression.

[0150] The results for SIRT-4 are shown in FIG. 2. At Day 0, on the control batch T0, the marking of sirtuin-4 is fairly clear in the epidermis. At Day 8, on the control lot TJ8, the expression of sirtuin-4 is quite clear to clear in the epidermis. Relative to control lot TJ8, P1 induces a slight or moderate decrease in sirtuin-4 expression and P2 induces a moderate decrease in sirtuin-4 expression.

[0151] The results for SIRT-6 are shown in FIG. 3. At Day 0, on the control batch T0, the marking of sirtuin-6 is fairly clear to clear in the epidermis. At Day 8, on the control lot TJ8, the expression of sirtuin-6 is clear in the epidermis. Relative to control lot TJ8, P1 induces a slight decrease in sirtuin-6 expression and P2 does not induce any observable change in expression of sirtuin-6.

[0152] The results for Collagen VII are shown in FIG. 4. At Day 0, on the control batch T0, the labeling of collagen VII is weak along the dermal-epidermal junction. At Day 8, on the control lot TJ8, collagen VII expression is low to moderate along the dermo-epidermal junction. Relative to control lot TJ8, both P1 and P2 induce a moderate increase in Collagen VII expression.

[0153] The results for Collagen III are shown in FIG. 5. At Day 0, on the control batch T0, the marking of collagen III is quite clear in the dermis papillary. At day 8, on the control lot TJ8, collagen III expression is clearly low in the papillary dermis. Relative to control lot TJ8, P1 and P2 did not induce an observable change in expression of Collagen III.

[0154] The results for Collagen V are shown in FIG. 6. At day 0, on the control batch T0, the marking of collagen V is clear to very clear in the papillary dermis. At day 8, on the control batch TJ8, the expression of collagen V is clear in the papillary dermis. Relative to control lot TJ8, P1 induces a moderate decrease in Collagen V expression and P2 induces a very clear decrease in Collagen V expression.

[0155] A summary of the results obtained in the above-described immunolabeling experiments is shown in Table 4.

TABLE-US-00004 TABLE 4 Immunolabelling results for P1 and P2 products Variation vs TJ8 P1 P2 Loricrin Slight Increase Slight decrease Sirtuin-4 Slight decrease Moderate decrease Sirtuin-6 Slight decrease No Change Collagen VII Moderate Increase Moderate Increase Collagen III No Change No Change Collagen V Moderate decrease Very clear decrease Scale = High Decrease < Very clear < clear < Fairly clear < Moderate < Slight Decrease < No Change < Slight Increase < Moderate < Fairly clear < clear < Very clear < Strong Increase

Example 3: In Vitro Study of the Effect of Rhizostoma Pulmo Collagenous Extract on a Primary Culture of Human Fibroblast

[0156] After application of hydrolysed collagenous extracts according to the invention to human fibroblast cells for 24 hours, intracellular and extracellular collagen was quantified with the red dye Sirius (Direct red 80), which has a particular affinity for the triple helix structure (Gly-X-Y)n of native collagen (collagen type I to V). The absorbance of the dye and collagen complex was measured at 540 nm. The total protein fraction was evaluated, after sonication, using the Bradford method (Bradford et al. Anal Biochem 1976; 72:248-54).

[0157] Test System

[0158] Primary human fibroblasts were prepared and the cells were confirmed as being free of mycoplasma.

[0159] The cells were cultured in DMEM medium 4.5 g/L glucose, 2 mM stabilised L-glutamine or glutamine, 10% heat inactivated foetal calf serum (FSC), 50 IU/ml penicillin, 50 ?g/ml streptomycin. The cells were kept in a humid atmosphere (37? C., 5% CO2). The cells were used at passage 6.

[0160] Reference Elements

[0161] Negative control: culture medium with 1% SVF.

[0162] Positive control: Transforming growth factor ?1 (TGF?) at 10 ng/ml in culture medium with 1% SVF

[0163] Definition of the Series

[0164] Five concentrations of collagenous extracts according to the invention were tested. The initial concentration of hydrolysed collagenous extract (8.25 mg/ml) was reduced to 500, 100, 10, 10, 5 and 1 ?g/ml. Each condition and reference element was tested in triplicate.

[0165] Test Protocol:

[0166] Cell seeding: The cells were seeded at 50,000 cells/cm.sup.2 in culture plates of 24 wells and then incubated overnight (37? C., 5% CO2).

[0167] Contact between the cells and the test item or reference items: Dilutions of the test items or reference items were performed in culture medium at 1% SVF. The culture medium was aspirated and replaced by 500 ?l of the different concentrations of the test items or reference items. The plates were incubated for 24 hours t 1 hour (37? C., 5% CO2).

[0168] Assessment of collagen synthesis and cell density: The culture medium of each well was then sampled and the cell layer was rinsed with 500 ?l of concentrated protease inhibitor cocktail 2 times. The entire set, culture medium and inhibitor, was pooled in the same tube.

[0169] The cell layer of each well was scraped off in 500 ?l of concentrated protease inhibitor cocktail 1 time and the wells were rinsed again with 500 ?l of cocktail 1 time. The two volumes, which constitute the extracellular matrix, were combined in a single tube and treated with an ultrasound probe for 40 seconds.

[0170] The collagen of each fraction was complexed with the red dye Sirius and, after centrifugation and washing with 0.1M HCl, the pellet was solubilised in 0.5M NaOH. The absorbance of the dye and collagen complex was measured at 540 nm. A calibration range has been established under the same conditions between 0 and 10 g per collagen tube.

[0171] The amount of total protein was evaluated using the Bradford method (Bradford et al. Anal Biochem 1976; 72:248-54) against a calibration range established from a BSA solution at 400 ?g/ml in PBS. 30 ?l of each sample was mixed with 280 ?l of Bradford reagent in a 96-well plate. The plate was incubated for about 15 minutes at room temperature in the dark. Absorbance was measured at 620 nm against a reagent blank consisting of the Bradford reagent.

[0172] Results

[0173] The results of the above-described experiments are summarised in Table 5 below.

TABLE-US-00005 TABLE 5 Stimulation of collagen synthesis by the Rhizostoma pulmo collagenous extract. Collagen cell matrix Collagen culture medium Protein % % Concentration Average ?g Stimulation/ Average ?g Stimulation/ ?g protein/ Standard collagen/ Negative Standard collagen/ Negative Concentration wells/ deviation well/ control deviation well/ control Rhizostoma pulmo collagen hydrolysate 500 ?g/ml 54.78 0.256 2.60 73 0.233 2.37 0 3.8 0.015 0.023 100 ?g/ml 64.64 0.251 2.55 70 0.250 2.54 7 9.0 0.042 0.040 10 ?g/ml 62.17 0.272 2.77 84 0.252 2.56 0 3.5 0.022 0.050 5 ?g/ml 56.81 0.205 2.09 39 0.226 2.30 0 3.9 0.020 0.028 1 ?g/ml 50.58 0.195 1.99 32 0.225 2.29 0 7.0 0.043 0.032 Positive 65.87 0.210 2.14 42 0.217 2.20 0 control 2.8 0.031 0.029 TGF-? 10 ng/ml Negative 50.14 0.148 1.50 control 4.1 0.029

[0174] Treatment with the hydrolysed collagenous jellyfish extract resulted in a significant increase in collagen synthesis in the cellular matrix with a maximum increase of 84% when 10 ?g/ml of that collagenous product was used.

Example 4: Comparative Data Against Hydrolysed Fish Collagen (Peptan SR Marine)

[0175] The same experiments as those performed in Example 3 were performed using hydrolysed fish collagen (Peptan SR Marine) at 500, 100, and 10 ?g/ml. The results obtained when using hydrolysed jellyfish and fish collagen are compared in Table 6 below.

TABLE-US-00006 TABLE 6 Stimulation of collagen synthesis by the hydrolysed jellyfish collagenous extract and hydrolysed fish collagen. Protein Collagen cell matrix Collagen culture medium Concentration Average ?g ?g Average ?g ?g protein/ Standard collagen/ protein/ Standard collagen/ Concentration wells/ deviation well/ Concentration wells/ deviation well/ Rhizostoma pulmo collagen hydrolysate 500 ?g/ml 54.78 0.256 2.60 73 0.233 2.37 0 3.8 0.015 0.023 100 ?g/ml 64.64 0.251 2.55 70 0.250 2.54 7 9. 0.042 0.040 10 ?g/ml 62.17 0.272 2.77 84 0.252 2.56 0 3.5 0.022 0.050 5 ?g/ml 56.81 0.205 2.09 39 0.226 2.30 0 3.9 0.020 0.028 1 ?g/ml 50.58 0.195 1.99 32 0.225 2.29 0 7.0 0.043 0.032 Hydrolysed fish collagen 500 ?g/ml 95.22 0.144 1.47 0 0.254 2.58 9 1.2 0.019 0.021 100 ?g/ml 66.09 0.214 2.18 45 0.212 2.15 0 0.6 0.030 0.015 10 ?g/ml 50.87 0.249 2.53 68 0.222 2.26 0 6.1 0.012 0.031 Positive 65.87 0.210 2.14 42 0.217 2.20 0 control 2.8 0.031 0.029 TGF-? 10 ng/ml Negative 50.14 0.148 1.50 control 4.1 0.029

[0176] Hydrolysed fish collagen has a maximum effect of 68% at a concentration of 10 ?g/ml. The response appears to be inversely proportional to the concentration, and remains lower than the values obtained with hydrolysed jellyfish collagenous extract.

Example 5: Evaluation of Hydrolysed Collagenous Extracts on Gene Expression in Human Skin Explants

[0177] Biological Model

[0178] NativeSkin? skin explant has been used, which is a biopsy of human normal skin embedded in a solid and nourishing matrix. The epidermal surface is maintained in contact with air to allow topical application.

[0179] The explants used were prepared from a 32-year-old woman's abdominoplasty, corresponding to a pigmentation of 3 on the Fitzpatrick scale.

[0180] The hydrolysed collagenous extract used in this example was concentrated at 100 ?g/ml (0.1 g/L).

[0181] The experiment was carried out in triplicate in the following conditions: [0182] Skin explant (control) untreated (n=3) [0183] Skin explant+Hydrolysed jellyfish collagenous extract (BT-10R&D) 0.1 g/L (n=3) [0184] Skin explant+Hydrolysed fish collagen (2132415-1/28052018) 0.1 g/L (n=3)

[0185] The explants were treated with the products for 2 days with a new application of the product in the morning and in the evening.

[0186] NB: Hydrolysed fish collagen seems to weaken the epidermis. Indeed, after treatment the explants were prepared for RNA extraction by punching them to remove skin unexposed to the product. It was observed that the epidermis was slightly detached from the dermis.

[0187] At the end of the culture phase, the explants were removed from their inserts and microfluidic RT-qPCR was used to characterise the gene expression of skin explants.

[0188] Results/Conclusions

[0189] The specificity of each primer pair was verified for all conditions by dissociation curve analysis and the problematic points with double peaks are removed from the analysis. Each CT was then normalised using the reference genes and the control condition in order to calculate the ??CT and the relative expression was calculated as a function of ??CT using the formula 2-??CT.

[0190] For each condition, the standard error to the mean (SEM) was calculated and the points with too large an SEM value (>40% difference between the SEM and the average of the 3 values) were removed from the analysis. The results are expressed as a percentage of overexpression or subexpression relative to the reference condition.

[0191] Changes in the expression of the genes of interest induced by the treatments are shown in Table 7 (heat-treated collagenous jellyfish extract) and Table 8 (hydrolysed fish collagen peptides) below.

TABLE-US-00007 TABLE 7 Effect of the R. Pulmo hydrolysed collagenous extract on the gene expression of human skin explants. Genes % increase or decrease in expression COL3A1 +60% COL5A1 +110% DCN +140% FBN1 +80% FBN2 +490% LOX +80% LUM +270% SPARC +100% LOXL1 ?90% CDC42 +60% COL7A1 +170% LAMA5 +70% PI3 ?90% EGF +130% SIRT4 +340% SIRT6 +60% HBEGF ?60% MGST1 +230% TNFA +70% EPPK1 +100% EVPL +80% FLG +60% LOR +130% MMP3 ?80% POMC +140% DKK1 +230%

[0192] Hydrolysed R. Pulmo collagenous extract was able to increase the synthesis of COL3A1, COL5A1, DCN, FBN1, FBN2, LOX, LUM, SPARC, CDC42, COL7A1, LAMA5, PI3, EGF, SIRT4, SIRT6, MGST1, TNFA, EPPK1, EVPL, FLG, LOR, POMC, DKK1, and decrease the synthesis of MMP3, HBEGF, PI3, and LOXIL1.

TABLE-US-00008 TABLE 8 Effect of the fish hydrolysed collagen according to the invention on the gene expression of human skin explants. Genes % increase or decrease in expression LOXL1 +180% P4HA1 +120% COL1A1 ?90% COL5A1 ?60% DCN ?70% SPARC ?70% CD44 +60% CDC42 +80% COL4A1 +120% CTNNA1 +90% ITGA2 +170% LAMC2 +430% PXN +200% SDC4 +440% COL17A1 ?70% COL7A1 ?90% FN1 ?60% LAMA5 ?90% SDC1 ?60% MMP9 ?80% PI3 +3230% TIMP1 +130% MMP1 +2390% MMP3 +180% AQP1 ?60% AQP3 ?60% HBEGF +1000% SIRT6 +140% TINF2 +70% IGFBP6 +140% GLRX +70% SIRT3 +80% SOD2 +570% CAT ?70% MSRB2 ?90% COX2 +240% IL1A +4350% IL6 +380% IL8 +9280% S100A7 +790% TNFA +1150% IVL +540% TGM1 +520% EVPL ?60% FLG ?90% LOR ?60% POMC +90% DKK1 +200%

[0193] Hydrolysed fish collagen is able to increase the synthesis of LOXL1, P4HA1, CD44, CDC42, COL4A1, CTNNA1, ITGA2, LAMC2, PXN, SDC4, PI3, TIMP1, MMP1, MMP3, HBEGF, SIRT6, TINF2, IGFBP6, GLRX, SIRT3, SOD2, COX2, IL1A, IL6, IL8, S100A7, TNFA, IVL, TGM1, POMC, DKK1, and decrease the synthesis of COL1A1, COL5A1, DCN, SPARC, COL17A1, COL7A1, FN1, LAMA5, SDC1, MMP9, AQP1, AQP3, CAT, MSRB2, EVPL, FLG, LOR.

[0194] These results indicate that the R. Pulmo collagenous extract is capable of effectively activating expression of the genes COL3A1, FBN1, FBN2, SPARC, LUM, COL7A1, LAMA5, CDC42, EGF, SIRT4, SIRT6, EPPK, LOR, and TNFA and also decreasing expression of MMP3

[0195] As noted above, the R. Pulmo collagenous extract increases the expression of COL7A1 by +170%. A study by Izmiryan et al (Ex vivo COL7A1 correction for recessive dystrophic epidermolysis bullosa using CRISPR/Cas9 and homology-directed repair (2018). Molecular Therapy-Nucleic Acids, 12, 554-567.), indicated that re-expression of COL7A1 transcripts estimated to be up to 15% in RDEB cells and up to 19% in skin grafts was sufficient to allow anchoring fibril formation with no dermal-epidermal separation. Accordingly, the effects seen with the R. Pulmo collagenous extract represent a promising opportunity for treating EB. That increase in the synthesis of COL7A1 suggests that the R. Pulmo collagenous extract has therapeutic relevance in the context of EB treatment.

[0196] In contrast, the fish collagen hydrolysate decreases the synthesis of many EB relevant genes, including COL7A1.

Example 6: A Study to Determine the Effect of Heat Treated Jellyfish Collagenous Extract on the Activation of Genes Ex-Vivo Associated with EB in Human Skin Biopsies

[0197] Methodology

[0198] Collagenous jellyfish extracts and control samples were tested ex vivo on healthy skin explants NativeSkin?, a full-thickness skin biopsy embedded in a solid and nourishing matrix while its epidermal surface is left in contact with air.

[0199] Hydrolysis of collagenous extracts was carried out by heating the extract to 80? C. for 60 minutes. FIG. 7 shows a representative SDS-PAGE demonstrating the effect of heat-treatment on the size distribution of the proteins present in the collagenous jellyfish extracts.

[0200] Explants were tested under the following conditions: [0201] No extract treatment; [0202] Exposure to untreated collagenous jellyfish extract (0.1 mg/mL); [0203] Exposure to heat-treated collagenous jellyfish extract (0.1 mg/mL); [0204] Exposure to heat-treated collagenous mammalian extract (0.1 mg/mL).

[0205] The explants were exposed to the test extracts for 24 hours. After that incubation, RNA from the skin explants was extracted and then reverse-transcribed into cDNA. The expression of 90 genes was assessed in triplicate by microfluidic technology RT-qPCR (Biomark?Fluidigm) and normalised to the expression of reference genes. In order to confirm an activating or inhibitory effect, values were compared to the untreated control. The results were expressed in terms of a expression rate relative to the untreated control.

[0206] Results

[0207] Significant changes in EB-associated gene expression in human explants following different treatments are summarised in Table 9 below (as measured by microfluidic RT-qPCR).

TABLE-US-00009 TABLE 9 Summary of significantly changed expression of EB-associated genes under in differently treated human explants EB- Increase in expression associated relative to untreated Explant gene control Untreated collagenous jellyfish ITGA6 +150% extract 1O (negative control) Heat-treated collagenous jellyfish COL7A +60% extract 1O DSP +160% ITGA6 +460% ITGB4 +260% KRT5 +240% LAMB3 +240% LAMC2 +170% Heat-treated collagenous jellyfish COL7A +80% extract 2U DSP +70% ITGA6 +200% ITGB4 +50% KRT5 +50% Heat-treated collagenous jellyfish DSP +50% extract 3WU ITGB4 +100% KRT5 +70% LAMB3 +70% LAMC2 +110% Heat-treated collagenous jellyfish COL7A +100% extract 1OF DSP +50% ITGA6 +180% Heat-treated collagenous jellyfish ITGA6 +240% extract 1O2H ITGB4 +60% LAMB3 +80% LAMC2 +70% Heat-treated collagenous ITGA6 +60% mammalian extract 1OF

[0208] The results in Table 9 illustrate that increased expression of key EB-associated genes is closely associated with the treatment of explants with heat-treated collagenous jellyfish extracts and was generally absent in mammalian extracts treated in the same way. Non-hydrolysed collagenous jellyfish extracts did not activate EB-associated gene expression in the same as heat-treated extracts, strongly suggesting that the observed effects are specific to hydrolysed collagenous jellyfish extracts.

[0209] In some explants exposed to heat-treated collagenous jellyfish extract increases in the expression of other EB associated genes was observed. Specifically, increases in the expression of COL3A1, COL5A1, DCN, FN1. FBN1, FBN2, ITGA2, LOX, LUM, SPARC, and CDC42 were observed. A decrease in MMP3 gene expression was also observed. MMP3 whose upregulation is associated with severe forms of RDEB and so decreasing its expression would potentially be beneficial to sufferers of that disease.

Example 7: A Study to Evaluate Ex-Vivo the Expression of Collagen Type VII Protein in Healthy Human Skin Explant

[0210] Methodology

[0211] Heat-treated collagenous jellyfish extracts were tested ex vivo on healthy skin explants NativeSkin?, a full-thickness skin biopsy embedded in a solid and nourishing matrix while its epidermal surface is left in contact with air. The experiment was carried out on 5 donors (N=5) and explants were exposed to the test items for 5 days with heat-treated collagenous jellyfish extract.

[0212] After treatment, skin explants were fixed with formalin, embedded in paraffin and cut to allow the labeling. Collagen VII was labelled by immunofluorescence and observed by fluorescence microscopy (DM5000BLeica Microsystems). The fluorescence intensity is measured by relative quantification compared to the control using Image J software. The means of % area of labeling at the level of basal layer measured on 10 pictures/condition?standard error of mean (SEM) was represented, and the results obtained for products were compared to untreated condition.

[0213] Results

[0214] The results of the immunofluorescent staining of fixed explants shown in FIG. 8A indicate an increase in the level of collagen VII protein present in explants treated with heat-treated collagenous jellyfish extract compared to untreated controls. The increase seen in the treated explants was quantified and expressed as a percentage increase in area of labeling of collagen VII protein relative to the untreated control (FIG. 8B).

Example 8: An In Vitro Study to Evaluate the Gene Expression of Collagen Type VII in Healthy Human Keratinocyte (NHEK)

[0215] Methodology

[0216] Healthy human epidermal keratinocytes (NHEK) were cultured with appropriate culture media containing heat-treated collagenous jellyfish extract. Treatment was carried out for 48 hours at 37? C. The expression of the COL7A1 mRNA was measured by semi-quantitative PCR on the Applied Biosystems 7300 Real Time PCR System. The expression level of the mRNAs calculated and normalised with reference gene (GAPDH). The values were reported to the untreated control so as to indicate an activating or inhibitory effect (n=3) and error bars represent standard error of the mean.

[0217] Results

[0218] The results shown in FIG. 9 indicate that treatment of NHEK with heat-treated collagenous jellyfish extract leads to a significant increase in the expression of COL7A1 mRNA relative to an untreated control. This observation is consistent with the immunostaining data described in Example 7 above.

Example 9: Ex-Vivo Study to Evaluate Jellyfish Collagenous Extract on the Expression of Collagen Type VII, Keratin 5 and Laminin 5 in Healthy Human Skin Explant Biopsies

[0219] Methodology

[0220] In this study the effect of heat-treated collagenous jellyfish extract on the protein expression of collagen 7, laminin 5, and keratin 5 in an ex vivo healthy human skin model. The study was carried out on skin explants NativeSkin?, a full-thickness skin biopsy embedded in a solid and nourishing matrix while its epidermal surface is left in contact with air. The skin biopsy is firmly embedded in the matrix that prevents any lateral diffusion of topically applied formulations. The study was carried out on 10 donors (n=10) and explants were exposed to the test items for 5 days with heat-treated collagenous jellyfish extract.

[0221] After treatment, skin explants were fixed with formalin, embedded in paraffin and cut to allow the labeling. Biomarkers of interest were labelled by immunofluorescence and observed by fluorescence microscopy (DM5000BLeica Microsystems). The fluorescence intensity is measured by relative quantification compared to the control using Image J software (Biomarkers quantified=collagen 7, keratin 5 and laminin 5).

[0222] The means of fluorescence intensity of protein labeling were measured on 10 pictures/condition and t standard error of the mean (SEM) is represented. The results obtained for the heat-treated collagenous jellyfish extract were compared to untreated condition. FIG. 10 shows representative images used in the immunostaining analysis.

[0223] Results

[0224] The global results for all 10 donors in respect to laminin 5, collagen 7 and keratin 5 protein expression in ex vivo healthy human skin are shown in Tables 10, 11, and 12 below. Those results indicate that the expression of all the tested proteins is increased in explants treated with heat-treated collagenous jellyfish extract. An increase of almost 25% is observed for collagen 7, which is strongly implicated in the disease mechanism of a number of types of EB.

TABLE-US-00010 TABLE 10 Summary of laminin 5 protein expression results in healthy human skin explants Untreated Heat-treated collagenous Value control jellyfish extract Fluorescence intensity of 100.0% 104.3% laminin 5 staining (%) SEM 1.2 1.6 Significance relative to control N/A 0.01 < p < 0.05 P-value N/A 0.03

TABLE-US-00011 TABLE 11 Summary of collagen 7 protein expression results in healthy human skin explants Untreated Heat-treated collagenous Value control jellyfish extract Fluorescence intensity of 100.0% 124.3% collagen 7 staining (%) SEM 2.1 4.7 Significance relative to control N/A p < 0.001 P-value N/A 0.0001

TABLE-US-00012 TABLE 12 Summary of keratin 5 protein expression results in healthy human skin explants Untreated Heat-treated collagenous Value control jellyfish extract Fluorescence intensity of 100.0% 102.1% keratin 5 staining (%) SEM 0.3 0.7 Significance relative to control N/A 0.001 < p < 0.01 P-value N/A 0.007

Example 10: In Vitro Study to Determine the Effect of Heat-Treated Jellyfish Collagenous Extract on Gene Activation and Protein Expression of Laminin-5, Collagen IV and Collagen VII in Human Diseased Fibroblast Cell Lines of DEB, EBS and JEB

[0225] Experimental Set-Up

[0226] This study evaluated the effect of heat-treated collagenous jellyfish extract on the mRNA and protein expression of Collagen 7, Laminin 5 and Collagen 4 in EB-disease cell lines. The test was carried out on human fibroblasts from three donors suffering from EB.

[0227] Donor 1=D1=male, 6 years, Simplex EB (SEB)

[0228] Epidermolysis bullosa simplex (EBS), is a disorder resulting from mutations in the genes encoding Keratin 5 or Keratin 14.

[0229] Donor 2=D2=female, 12 years, dystrophic EB (DEB)

[0230] Dystrophic epidermolysis bullosa (DEB) is a form of inherited epidermolysis bullosa (EB) characterised by cutaneous and mucosal fragility resulting in blisters and superficial ulcerations. DEB is caused by mutations in the COL7A1 (3p21.31) gene encoding the type VII collagen protein. Mutations in this gene alter the function, reduce or disrupt the production of collagen VII.

[0231] Donor 3=D3=male, 8 months, Junctional EB (JEB)

[0232] Junctional epidermolysis bullosa is a skin condition characterised by blister formation within the lamina lucida of the basement membrane zone. Junctional epidermolysis bullosa most commonly results from mutations in the LAMA3, LAMB3, LAMC2, and COL17A1 genes.

[0233] In order to allow comparison with healthy skin, an NHDF (Normal human dermal fibroblast), female, 20 years old, was used.

[0234] mRNA Analysis

[0235] The cells were treated with appropriate media with or without the test extracts and incubated for 48 hours at 37? C. After treatment, RNA was extracted from the cells and reverse-transcribed into cDNA. The expression of the COL4A1, COL7A1, LAMC2 mRNA was measured by semi-quantitative PCR on the Applied Biosystems 7300 Real Time PCR System. The expression level of the mRNAs was calculated and normalised with reference genes (GAPDH). The values were reported to the untreated control to express an activating or inhibitory effect and the results are expressed as an induction factor. The results are considered significant when the mRNA expression is +/?40% (n=3).

[0236] Protein Expression Analysis

[0237] The cells were treated with appropriate media with or without the test extracts and incubated for 48 hours at 37? C. After treatment, the cells were washed and fixed with Formalin. After permeabilisation, the cells were incubated with an anti-collagen 4, anti-collagen 7 or anti-laminin5 antibody and a relevant fluorescently-labelled secondary antibody (ALEXA). Finally, the nuclei were labelled with DAPI.

[0238] The labelled proteins were observed and quantified by automated fluorescence microscopy Cellinsight CX7 High-Content Screening Platform (Thermofisher). The fluorescence intensity t standard error of the mean (SEM) is represented (n=3). Values were compared to the control. The significance is correlated with the p-value: ns=no significant, * p-value<0.5, ** p-value<0.01, *** p-value<0.001.

[0239] Results

[0240] The results of the mRNA and protein expression analysis is summarised in Tables 13, 14, and 15 below.

TABLE-US-00013 TABLE 13 Effect of heat-treated collagenous jellyfish extract on the mRNA and protein expression in simplex EB (SEB) fibroblasts (Donor 1) (n = 3) Gene/protein Gene expression Protein expression Collagen 4 + ? Collagen 7 = + Laminin 5 = ? Representation of variation trend (mRNA +/?40%; protein +/?10%)

TABLE-US-00014 TABLE 14 Effect of heat-treated collagenous jellyfish extract on the mRNA and protein expression in dystrophic EB (DEB) fibroblasts (Donor 2) (n = 3) Gene/protein Gene expression Protein expression Collagen 4 + ? Collagen 7 = = Laminin 5 + + Representation of variation trend (mRNA +/?40%; protein +/?10%)

TABLE-US-00015 TABLE 15 Effect of heat-treated collagenous jellyfish extract on the mRNA and protein expression in junctional EB (JEB) fibroblasts (Donor 3) (n = 3) Gene/protein Gene expression Protein expression Collagen 4 + = Collagen 7 = + Laminin 5 = + Representation of variation trend (mRNA +/?40%; protein +/?10%)

[0241] The mRNA quantification shows that treatment with heat-treated collagenous jellyfish extract led to significant increases in COL4A1 gene expression in all types of EB fibroblasts tested. In addition, LAMC2 gene expression was significantly increased in DEB cells treated with heat-treated collagenous jellyfish extract.

[0242] In relation to protein expression, the immunostaining analysis indicates that SEB and JEB cells treated with heat-treated collagenous jellyfish extract had significant increases in the level of laminin 5 protein present. In addition, treated SEB and JEB cells showed increased levels of collagen 7 protein.

[0243] This study demonstrates new positive effects of heat-treated collagenous jellyfish extract on fibroblasts from EB skin on the basal lamina-related proteins collagen 4, collagen 7 and laminin 5.