COMPOSITION FOR THE TREATMENT OF SKIN LESIONS

Abstract

The present invention relates to the association between the lactoferrin protein and the mesoglycan, in particular for the use in the therapy of skin lesions of different kinds and degrees.

Claims

1. A pharmaceutical or cosmetic composition comprising lactoferrin and mesoglycan.

2. The composition according to claim 1 wherein lactoferrin and mesoglycan are each contained in a concentration range ranging from 0.1 to 100000 μg/ml.

3. The composition according to claim 1 characterized by the fact of being formulated in a form suitable for oral administration.

4. The composition according to claim 3 wherein said form of oral administration is selected from tablet, capsule, solution, suspension, granules or oily pearl.

5. The composition according to claim 1 characterized by the fact of being formulated in a form suitable for parenteral administration.

6. The composition according to claim 5 wherein said parenteral administration form is selected in the group consisting of aqueous buffer solution and oily suspension.

7. The composition according to claim 5 wherein said parenteral administration is selected in the group consisting of intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal or intrasplenic administration.

8. The composition according to claim 1 characterized by the fact of being formulated in a form suitable for topical administration.

9. The composition according to claim 8 wherein said form of topical administration is selected in the group consisting of cream, pomade, ointment, unguent, gel, bandage and gauze.

10. A method of treating skin lesions skin lesions selected in the group consisting of burns, sunburns, insect bites, frictions, from viral infections, excoriations, ulcers, the method comprising administering the pharmaceutical composition according to claim 1 to a patient in need thereof.

11. The method according to claim 10 where said ulcers are selected in the group consisting of venous ulcers, arterial ulcers, arteriovenous or mixed ulcers, ischemic, neuropathic and mixed diabetic ulcers, pressure ulcers, vasculitic ulcers, neuropathic ulcers not related to diabetes, pressure or decubitus ulcers, ulcers in the course of haematological diseases, ulcers in obesity, ulcers in the course of infectious diseases (generalized and localized), ulcers in the course of skin diseases.

12. A cosmetic method comprising administering of the cosmetic composition according to claim 1 to a person in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1—in vitro cell migration assay

[0033] FIG. 2—in vitro cell invasion assay

[0034] FIG. 3—cell proliferation assay: haemocytometer counting on BJ cells

[0035] FIG. 4—assay of formation of capillary-like structures (in vitro angiogenesis)

[0036] FIG. 5—inhibition of bacterial biofilm formation of P. aeruginosa

[0037] FIG. 6—immunofluorescence characterization of the main protein markers on HaCaT cells

[0038] FIG. 7—immunofluorescence characterization of the main protein markers on HUVEC cells

[0039] FIG. 8—immunofluorescence characterization of the main protein markers on BJ cells

EXPERIMENTAL PART

[0040] Techniques and Materials Used

[0041] The lactoferrin used in this study was bought from the company Sigma Aldrich, a Merck KGaA group, but can be extended to all possible origins. The mesoglycan used in this study was bought by the company LDO (Laboratori Derivati Organici spa) but can be extended to any origin of the aforesaid compound.

[0042] The experiments were conducted in vitro on the following human cell lines: immortalized keratinocytes of dermis, HaCaT; immortalized fibroblasts of epidermis, BJ; primary endothelial cells deriving from the umbilical cord vein, HUVEC (Human Umbilical Vein Endothelial Cell). These cell lines were cultivated following the manufacturer's guidelines and kept sterile at 37° C., in a humidified atmosphere with 5% v/v of CO.sub.2. The HUVECs were used for the experiments up to a step 10.

[0043] Human lactoferrin was resuspended first in sterile deionized water and then diluted in PBS 1× (Phosphate Buffered Saline) or in the cell culture medium. The sodium mesoglycan was solubilized either in PBS 1× or in the cell culture medium before being administered. The two substances were administered both together and individually.

EXAMPLE 1

Cell Migration Analysis (Wound Healing Assay)

[0044] The HaCaT, BJ and HUVEC cells were seeded in 12 well multi-well plates in a number of 5×10.sup.5, 10×10.sup.5 and 3×10.sup.5, respectively, per well. After 24 hours the cells reached 100% confluence and a scratch was created in the middle of the well using a sterile p10 tip. After removing the growth medium and washing the wells twice with a 1× PBS solution, the cells were incubated in the presence of mesoglycan and lactoferrin at the reported concentrations by crossing the concentrations of 100 μg/ml of the first and 200 μg/ml of the second. An untreated control point was maintained for each cell line. All the experimental points were treated with mitomycin C at a concentration of 10 μg/ml to ensure that the mitosis was blocked. The plates containing the cells were then incubated in a chamber that was humidified and balanced at 5% v/v of CO.sub.2 of an Integrated Live Cell Workstation Leica AF-6000 LX microscope. A 10× phase contrast objective was used to capture the images of the cells at a frequency of 10 minutes for at least 10 different positions of each experimental point. The migration rate was measured on individual cells from time 0 to 24 hours using Leica ASF software. At least 10 cells were selected and analysed for each photographed position.

[0045] The HaCaT, BJ and HUVEC cells significantly increase their migration rate when treated with mesoglycan and lactoferrin individually, but this effect is greatly enhanced when the two substances are co-administered (FIG. 1). In detail, the percentage increase compared to the untreated control is expressed as:

[0046] for HaCaT cells

TABLE-US-00001 meso100 Lf200 meso100 + Lf200 23.3 32.8 60.9

[0047] for BJ cells

TABLE-US-00002 meso100 Lf200 meso100 + Lf200 11.4 11.8 29.2

[0048] for HUVEC cells

TABLE-US-00003 meso100 Lf200 meso100 + Lf200 27 31.1 69.6

EXAMPLE 2

Analysis of Cellular Invasion (Invasion Assay)

[0049] The cells were plated on inserts with 8 μm pores, with a total diameter of 12 mm. A solution of matrigel diluted in the culture medium (1:3) of each cell line without growth factors and kept at 37° C. until its complete gelation was previously seeded on these inserts. Matrigel is a mixture of proteins able to mimic the extracellular matrix. The HaCaT, BJ and HUVEC cells were seeded in a number of 5×10.sup.4 in the upper chamber of the insert in 350 μI of growth medium without serum (thus without growth factors with chemo-attracting properties). Instead, the treatments in 1.4 ml of complete growth medium were performed in the lower chamber. The cells were incubated for 24 hours at 37° C., in a sterile humidified atmosphere at 5% v/v of CO.sub.2. At the end of the experimental time, the medium was aspirated from each insert, they were washed twice with PBS 1×, the cells present were then fixed in 4% paraformaldehyde in PBS for 10 minutes, permeabilized with methanol 100% for 20 minutes and stained with a violet crystal solution at 0.5% w/v for 15 minutes. Finally, the inserts were washed twice with PBS 1×, cleaned with a cotton swab to remove excess cells that were not inside the layer of matrigel. All the experimental points were treated with mitomycin C 10 μg/ml to block mitoses. The number of cells within the layer was counted by images captured in at least 12 random fields on the insert under the EVOS microscope, 10× objective.

[0050] The number of HaCaT, BJ and HUVEC cells that are able to invade the layer of matrigel significantly increases in the presence of mesoglycan and lactoferrin alone (FIG. 2). This effect is further increased when the two substances are present together. In detail, the increase compared to the untreated control in percentage terms is quantified as follows:

[0051] for HaCaT cells

TABLE-US-00004 meso100 Lf200 meso100 + Lf200 49.7 30.5 75.7

[0052] for BJ cells

TABLE-US-00005 meso100 Lf200 meso100 + Lf200 27.6 26 46.2

[0053] for HUVEC cells

TABLE-US-00006 meso100 Lf200 meso100 + Lf200 30.7 28.1 47.6

EXAMPLE 3

Haemocytometer Counting

[0054] The BJ cells were plated in a number of 2×10.sup.4 in a 12 well multi-well plate. After 12 hours the growth medium was replaced by a serum-free medium so as to synchronize the cell cycle for all cells. The cells were harvested at 24, 48, 72 hours, 5 and 7 days starting from the treatments performed with the mesoglycan and lactoferrin association by crossing the concentrations of 100 μg/ml of the first and 200 μg/ml of the second. Equal volumes of the stain trypan blue, 0.4% w/v of cell suspension were mixed. About 10 μl of this mixture was placed on the edge of a slide in the Burker chamber. The haemocytometer counting chamber was visualized with an optical microscope (10× objective) and the mean of the number of cells arranged on 5 of the 9 quadrants displayed was performed to calculate the live cells per ml of suspension. This value was multiplied by the dilution factor 2 and by 10.sup.4. The mesoglycan and lactoferrin association, in addition to the single substances, induces the increase of cell proliferation starting from 48 hours of administration (FIG. 3). In detail, compared to the untreated control, the changes in the number of cells in percentage terms are:

TABLE-US-00007 meso100 Lf200 meso100 + Lf200 24 h 28 −3.2 −4.8 48 h 36.5 10.3 18.8 72 h 42 27.4 36.4  5 d 4.9 33.1 39.2  7 d 3.4 22.2 35.4

EXAMPLE 4

In Vitro Angiogenesis (Tube Formation Assay)

[0055] The wells of a 24 well multi-well plate were coated with a layer of matrigel in a 1:1 solution with the culture medium of HUVEC cells on ice. The plate was kept in an incubator at 37° C. until gelation. The HUVEC cells were seeded on the gel in a number of 5×10.sup.4 per well in the presence or not of the selected treatments. The cells were incubated under sterility at 37° C., in a humidified atmosphere with 5% v/v of CO.sub.2 overnight. After about 12 hours, images (10× objective) were taken with the EVOS microscope and the length of each tube and the number of branches were calculated using the Angiogenesis Analyzer software for ImageJ.

[0056] The mesoglycan and lactoferrin association induces the increase in the ability of HUVEC cells to constitute capillary-like structures in 12 hours. It is possible to note from FIG. 4 that the number of branching points and the length of the tubes increase significantly not only with the single treatments but above all when they are present together.

EXAMPLE 5

Analysis of the Effect on Bacterial Biofilm Formation

[0057] In order to determine the ability of the mesoglycan and lactoferrin association to inhibit the formation of Pseudomonas aeruginosa biofilm, a colony of P. aeruginosa PAO1 was inoculated in 7 ml of Mueller Hinton Broth (MHB) and incubated overnight at 37 ° C. under stirring. Following a spectrophotometric reading at 600 nm of the overnight culture, a culture of 1×10.sup.6 cells/ml in Luria-Bertani (LB) Broth was prepared and supplemented with 2% glucose. This culture was used to fill the wells of a 96 well plate (100 μl/well). A well with only bacterial cells was used as a positive control for biofilm formation. In two separate wells, mesoglycan alone (300 μg/ml) and mesoglycan combination 300 μg/ml and lactoferrin 10 mg/ml were added in separate wells and incubated overnight at 37° C. without stirring (static culture). Subsequently, the culture medium was removed from each well, and the not adherent cells of P. aeruginosa were removed by washing the wells with PBS. After this washing, the formed biofilm mass was fixed by incubating the plate at 60° C. for 1 hour, and subsequently highlighted with the violet crystal staining. The biofilms were briefly stained with 3% v/v CV for 10 min. The excess violet crystal was removed with water, while that trapped in the biofilm was recovered by adding 200 μl/ml of 70% ethanol to each well and subjected to spectrophotometric reading (OD600 nm).

[0058] The action of lactoferrin is significant compared to the lack of ability of the mesoglycan to act as an anti-microbial.

[0059] Statistical Analyses

[0060] Statistical analyses were performed on resultants deriving from at least three experiments with similar results in which the experimental points were maintained in technical triplicate. Statistical comparisons were performed by means of tow-tailed Students t-test with different variance. The differences were considered significant if in case of p value at least <0.05 */¥/$; <0.01 **/¥¥/$$; <0.001 ***/¥¥¥/$$$.

EXAMPLE 6-8

Immunofluorescence (Analysis by Confocal Microscopy)

[0061] The HaCaT, BJ and HUVEC cells were plated in a number of 2×10.sup.4 on coverslips 12 mm in diameter and 0.1 mm thick. After 24 hours the treatments with mesoglycan and lactoferrin were performed for 48 hours, by crossing the concentrations of 100 μg/ml of the first and 200 μg/ml of the second. At the end of the experimental time, the cells were fixed with a 4% paraformaldehyde solution in PBS 1× for 5 minutes, permeabilized with 0.5% v/v triton in PBS 1× for more 5 minutes and incubated with goat serum (20% v/v in PBS) for 30 minutes. Subsequently, the cells were incubated overnight at 4° C. with antibodies against VEGF (Vascular Endothelial Growth Factor) 1:100, CD31 (cluster of differentiation 31) 1:100, ICAM-1 (Intercellular Adhesion Molecule-1) 1:100, N-cadherin 1:250, laminin 332 1:250, fibronectin 1:250, integrin β1 1:250, cytokeratin 6 1:500, cytokeratin 10 1:500, E-cadherin 1:1000, vimentin 1:1000, α-SMA (alpha-Smooth Muscle Actin) 1:250, FAP-α (Fibroblast activation protein-alpha) 1:250, vinculin 1:100. On the next day the slides with the cells were washed and incubated for 2 hours at room temperature in the dark in the presence of secondary anti-mouse, anti-rabbit and anti-goat fluorescent antibodies conjugated with fluorochromes 488 and 555. To stain the nuclei, fluorescent Hoechst stain 33342 1:1000 was added to the solution with the secondary antibodies. Finally, FITC 1:500 fluorochrome-conjugated phalloidin was used for 30 minutes in the dark at room temperature in order to highlight the polymerization of F-actin. Subsequently the slides were mounted on coverslips with a transparent mounting solution and kept at 4° C. until confocal analysis. The emission signals were analysed with laser 488 nm Ar (argon) and 555 nm He—Ne (helium-neon). The samples were scanned vertically from the first to the last focal plane for a total distance of 5μm to a 63× immersion objective. The images (FIG. 6-8) were generated using the Zeiss ZEN Confocal software and processed with ImageJ, Adobe Photoshop and Microsoft Power Point software.

[0062] Results

[0063] Lactoferrin and sodium mesoglycan show significant pro-migratory and pro-invasive effects. As evident in example 1, by means of the cell migration assay it was possible to demonstrate the increase in the distance travelled over 24 hours by all the three HaCaT, BJ and HUVEC cell lines when treated with mesoglycan 100 μg/ml and lactoferrin 200 μg/ml individually. When these two treatments are combined together the migration rate undergoes a considerable increase, which is significant compared not only to the untreated control but also to individual experimental points. Instead, the pro-invasive effect is described in FIG. 2 whose histograms show the number of cells that crossed the layer of matrigel over 24 hours. In the presence of the aforesaid treatments, this number increases significantly. Also in this case the effect enhancement on the biological process under examination is evident in the case of the co-administration of mesoglycan and lactoferrin.

[0064] FIG. 3 shows the histogram depicting the BJ human fibroblast count after 24, 48, 72 hours, 5 and 7 days of treatment with sodium mesoglycan 100 μg/ml with lactoferrin 200 μg/ml. The proliferation rate appears to be affected by the presence of mesoglycan and lactoferrin at different times. Therefore, both substances are active in allowing fibroblasts to proliferate more quickly both in the case of single treatments and co-administration. In addition to the processes of cell migration and invasion, proliferation is also a crucial event in the different phases of tissue regeneration in skin lesions. In fact granulation, a moment in which the fibroblasts intervene with collagen production, becomes much more effective if the number of such protagonist cells increases significantly. The result obtained on the BJ cell population represents a strength for the mesoglycan/lactoferrin association due to its ability to positively influence different aspects of epithelial repair.

[0065] As previously mentioned, another essential moment for the correct skin regeneration is the formation of new vascular structures that are able to bring oxygen, nutrients and cells to the damaged site. For this reason, an in vitro angiogenesis assay was also performed in the manner described above. FIG. 4 shows the images of capillary-like structures that HUVEC cells were able to form in 12 hours from treatments. In the presence of sodium mesoglycan and lactoferrin, at their respective concentrations of 100 μg/ml and 200 μg/ml, these cells organize themselves to form not only longer capillary tubes but also with a greater number of branching points, compared to the untreated control point. It is possible to note that the treatment of lactoferrin and mesoglycan brings more pronounced pro-angiogenic properties.

[0066] In literature, the characteristics of lactoferrin as a protein inhibiting bacterial growth and biofilm formation are known, therefore the adhesion of bacterial populations. These anti-bacterial properties guarantee a protective effect of lactoferrin that supports the repair of skin lesions. For this reason, an evaluation assay of the biofilm formation of the P. aeruginosa PAO1 strain was performed. From the histogram shown in FIG. 5 it is possible to notice that, compared to the positive control (the only cells of Pseudomonas where 100% of the possible biofilm has formed), the mesoglycan alone at 300 μg/ml inhibited 14% of the biofilm, therefore it remained at non-significant levels of efficacy. The experimental point with the association between mesoglycan 300 μg/ml and lactoferrin 10 mg/ml, instead, inhibited the biofilm of the Gram-negative bacterium by more than 40%. In this way, among the effects of the mesoglycan/lactoferrin association it was possible to include also an antimicrobial effect which contributes to a better and faster tissue repair in skin lesions. In particular, mesoglycan alone is not able to inhibit bacterial adhesion unlike lactoferrin, which brings about these characteristics in case of co-administration.

[0067] The characterization of the effect of mesoglycan and lactoferrin as single agents but in particular in association was also performed by immunofluorescence assays with which some of the main molecular markers involved in the activation of the three HaCaT, BJ and HUVEC cell lines were evaluated.

[0068] Considering each cell line, FIG. 6 shows some proteins characterizing the biological processes previously examined on human HaCaT keratinocytes. First, cellular differentiation was demonstrated by the increase in the expression of the two cytokeratins 6 and 10. Differentiation represents an important moment for the final phases of re-epithelialization. In fact, these cells pass from a basal layer to the corneum stratum and are able not only to maintain a correct epidermal barrier but also to form it following a lesion. Some important aspects concerning pro-migratory activation are based on the significant organization of the plasma membrane of integrin β1, the dramatic reduction in E-cadherin expression and the cytoplasmic diffusion of laminin 332. In parallel, the decrease in ICAM-1 expression in keratinocytes can be linked not only to differentiation (basal keratinocytes have greater expression than this protein) but also to the acquisition a greater capacity of these cells to resist inflammatory stimuli and external insults.

[0069] The panels shown in FIG. 7 show that on HUVEC cells the association induces the endothelium-mesenchymal transition, i.e. it allows the passage from a “resident” phenotype to one that is able to migrate and invade, aimed therefore at the formation of capillaries. As confirmation of the transition, we analysed fibronectin, N-cadherin, integrin β1, ICAM-1 demonstrating a strong increase in expression for all four of these proteins. On the other hand, the actin cytoskeleton appears strongly organized and laminin 332 diffuses from the perinuclear area to the cell membrane, ready to be secreted and contribute to the formation of the extracellular matrix.

[0070] Finally, the increase in VEGF and CD31 expression further demonstrates angiogenesis induction.

[0071] FIG. 8 shows the fluorescences performed on human BJ fibroblasts. It was possible to demonstrate their activation after 48 hours of treatment with mesoglycan and lactoferrin but more markedly with the association of the two thanks to the strong organization of the F-actin cytoskeleton, of vimentin, of N-cadherin and of α-SMA. Other markers of fibroblast maturation are FAP-α, integrin β1 and vinculin whose expression increases and laminin 332 whose localization passes from the perinuclear zone towards the plasma membrane.

[0072] In all the cases examined, the mesoglycan and lactoferrin association enhances the molecular variations both in terms of expression modulation and protein localization, compared to the compounds administered individually. These aspects highlight the inventive activity of the subject invention. The way in which the mesoglycan and the lactoferrin intervene with different effects in a more or less marked manner, depending on the cellular process examined, makes it possible for the invention to fall within various types of medications for skin regeneration. Examples of these medications are those able to promote (biochemical and/or physicochemical) autolysis and the removal of part of the material (debridement), those granulating, anti-microbial and eudermic. Each of these types are able to intervene in distinct phases of tissue repair. The tested association, instead, tends to promote the main aspects of all these phases, thus acting transversely.