PTEROSTILBENE (PTER) FOR USE IN THE PREVENTION AND/OR TREATMENT OF SKIN DISEASES, DAMAGES OR INJURIES

Abstract

The present invention refers to the use of PTER, or any acceptable salt thereof, optionally in combination with QUER, or any acceptable salt thereof, for the manufacture of pharmaceutical compositions for preventing and/or treating skin diseases, damages or injuries. Those methods of prevention and/or treatment comprise the administration of an effective amount of a composition comprising PTER as active compound, or any acceptable salt thereof, optionally in combination with QUER, or any acceptable salt thereof, to the subject. The present invention also refers to the compositions, per se, comprising PTER as active compound, or any acceptable salt thereof, optionally in combination with QUER, or any acceptable slat thereof, characterized by being formulated in the form of liposomes, and to the method for preparing said liposome formulations.

Claims

1-29. (canceled)

30. A method of preventing and/or treating skin disease, damage, or injuries caused by inflammatory dermatitis, comprising administering an effective amount of PTER or any acceptable salt thereof, thereby preventing and/or treating skin disease, damage, or injuries caused by the inflammatory dermatitis.

31. The method of claim 30, further comprising administering an effective amount of QUER or any acceptable salt thereof.

32. The method of claim 30, further comprising administering a polyphenol.

33. The method of claim 32, wherein the effective amount of PTER and/or QUER is administered to an affected skin area.

34. The method of claim 31, wherein the PTER and/or QUER is in a dermatologically acceptable excipient.

35. The method of claim 34, wherein the dermatologically excipient is a topical excipient.

36. The method of claim 35, wherein the topical excipient is in the form of liposomes.

37. The method of claim 30, further comprising an emollient to prevent or relieve dryness.

38. The method of claim 30, wherein the inflammatory dermatitis is an autoimmune dermatitis, a sebaceous gland disorder, papulosquamous dermatoses, allergic dermatoses, pruritic dermatoses, vascular dermatoses, bacterial dermatoses, viral dermatoses, mycolic skin infections, granulomatous dermatoses, parasitic skin dermatoses, exfoliative dermatitis, bullous dermatoses, pigmented dermatoses, photosensitive dermatoses, dermatoses caused by collagen diseases, and dermatoses due to internal diseases.

39. The method of claim 38, wherein the inflammatory dermatitis is an autoimmune dermatitis.

40. The method of claim 39, wherein the autoimmune dermatitis is at least one of atopic dermatitis, mast cell disease, bullous pemphigoid, pemphigus vulgaris, necrotizing vasculitis, discoid lupus erythematosus, systemic lupus erythematosus, or dermatitis herpetiformis.

41. The method of claim 40, wherein the autoimmune dermatitis is atopic dermatitis.

42. The method of claim 40, wherein the inflammatory dermatitis is allergic dermatoses.

43. The method of claim 42, wherein the allergic dermatoses are at least one of contact dermatitis, photoallergic dermatitis, industrial dermatitis, atopic eczema, and dermatoses caused by drugs and nummular eczema.

44. The method of claim 38, wherein the inflammatory dermatosis is a papulosquamous dermatosis.

45. The method of claim 44, wherein the papulosquamous dermatosis is at least one of psoriasis, Pityriasis rosea, tinea versicolor, or lichen planus.

46. The method of claim 45, wherein the papulosquamous dermatosis is psoriasis.

47. The method of claim 38, wherein the sebaceous gland disorder is an acneform disorder.

48. The method of claim 47, wherein the acneform disorder is at least one of acne vulgaris, acne conglombata, hidradenitis supportiva, acne rosacea, seborrhea, seborrheic dermatitis, gram negative folliculitis, pyoderma faciale, steatocystoma multiplex, sebaceous hyperplasia, or rhinophyma.

49. The method of claim 48, wherein the acneform disorder is acne vulgaris.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0073] FIG. 1. It shows the skinfold thickness (%) of mice treated with different compositions before (left column) or after (right column) being exposed to UVB. Commercial creams (Vichy, Heliocare -Helio- and Isdin). C+UVB=Control irradiated without any treatment; C Lipo+UVB=Control irradiated wherein only liposomes (without containing any active compound) applied on. RESV=Resveratrol; P+Q=PTER+QUER.

[0074] FIG. 2. It shows the thickness (μm) of the epidermis treated with different compositions before (left column) or after (right column) being exposed to irradiation (24 hours). Commercial creams (Vichy, Heliocare -Helio- and Isdin). Control=No irradiation, no treatment; C+UVB=Control irradiated without any treatment; C Lipo+UVB=Control irradiated wherein only liposomes (without containing any active compound) applied on. RESV=Resveratrol; P+Q=PTER+QUER.

[0075] FIG. 3. It shows the thickness (μm) of the epidermis treated with different compositions before (left column) or after (right column) being exposed to irradiation (1 week). Commercial creams (Vichy, Heliocare -Helio- and Isdin). C Lipo+UVB=Control irradiated wherein only liposomes (without containing any active compound) applied on. RESV=Resveratrol; P+Q=PTER+QUER.

[0076] In all FIGS. 1-3 the asterisk * shown in the bars histogram represents a difference statistically significant (P<0.05), when compared with control values.

[0077] FIG. 4. It shows the increase in the skinfold with respect to day 0 (1 week after pre-exposure to TNCB, and the start of continues exposure on alternate days) in the trial regarding the induction of experimental atopic dermatitis with TNCB.

[0078] FIG. 5. It shows the loss of transepidermal fluid 24 hours after the 4° exposure to TNCB, in the trial regarding the induction of experimental atopic dermatitis with TNCB.

[0079] FIG. 6. It shows the lesions 24 hours after the 4° exposure to TNCB, in the trial regarding the induction of experimental atopic dermatitis with TNCB.

[0080] FIG. 7. It illustrates the protocol developed in the trial regarding the induction of experimental atopic dermatitis with TNCB.

[0081] FIG. 8. It shows the increase of skinfold in reference to day 0 of TPA treatment, in the trial regarding the induction of experimental psoriasis with TPA.

[0082] FIG. 9. It shows the loss of transepidermal fluid (TEWL) 24 hours after the 7° exposure to TPA, in the trial concerning the induction of experimental psoriasis with TPA.

[0083] FIG. 10. It shows the evolution of the lesions on the exposure to TPA, in the trial concerning the induction of experimental psoriasis with TPA. [0084] A. Image of a mouse 24 hours after 3 daily exposures of 2 nmoles of TPA; [0085] B. Image of the same mouse 24 hours after 6 daily exposures of 2 nmoles of TPA; [0086] C. Image of real psoriasis on a patient's finger (Image from the Dermatology Image Atlas); [0087] D. Images of real psoriasis on a patient's knee (Image from the Dermatology Image Atlas).

[0088] FIG. 11. It shows the protocol developed in the trial concerning the induction of experimental psoriasis with TPA.

[0089] FIG. 12. It shows the protocol developed in the trial concerning the induction of experimental hypersensitivity by contact to oxazolone.

[0090] FIG. 13. It shows the absorption of polyphenols, specifically the absorption spectrum of different 1 mM solutions of each of the polyphenols (PTER, Resveratrol and QUER), in pure ethanol, in the trial regarding the assessment of the mechanical filter capacity of polyphenols.

[0091] FIG. 14. It shows the condition of the mice after chronic exposure to UV-B in the trial regarding the assessment of the photoprotector role of the polyphenols. It is a photograph of the different experimental groups after 25 weeks of chronic exposure to 180 mJ/cm.sup.2 (three times per week). Both the reference compounds and the ones subject of this study (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) were applied to the back of the mice for 20 minutes prior to their exposure to UV-B.

[0092] FIG. 15. It shows the evolution of the development of lesions in the trial regarding the assessment of the photoprotector role of the polyphenols. First signs of precancerous lesions of more than 1 mm of diameter in the different experimental groups after 30 weeks of chronic exposure to 180 mJ/cm.sup.2 (three times per week). Both the reference compounds and the ones subject of this study (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) were applied to the back of the mice for 20 minutes prior to their exposure to UV-B.

[0093] FIG. 16. It shows the anatomopathological study of the skin after chronic exposure to UV-B. Anatomopathological evaluation of histological sections of mice after 30 weeks of chronic exposure to 180 mJ/cm.sup.2 (three times per week). Both the reference compounds and the ones subject of this study (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) were applied to the back of the mice for 20 minutes prior to their exposure to UV-B.

[0094] FIG. 17. It shows the skinfold after the continue administration of treatments, in the trial regarding the assessment of the effect of the different polyphenols on dermatitis. The application of the treatments 10 μmoles/mouse of polyphenols and 0.5 μmoles/mouse of dexamethasone was performed daily on a third of the back of the mice for 6 consecutive days. The skinfold was measured with the help of a vernier caliper.* p>0.001 with regards to the control. ANOVA followed by a Tukey test.

[0095] FIG. 18. It shows the histological sections representing the different experimental conditions in the trial regarding the assessment of the protection from severe exposure to radiation UV-B. The application of treatments (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) was performed 20 minutes before or after irradiation with 360 mJ/cm.sup.2 of UV-B. The mice were sacrificed 24 hours after irradiation to UV-B. This figure indicates the changes in the thickness of the epidermis.

[0096] FIG. 19. It shows the increase in the skinfold 24 hours after the first administration of TNCB in the trial regarding the protection in an experimental model of atopic dermatitis induced by TNCB. The application of treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 24 hours before (day 0) to the application of 100 μl of 1% TNCB on mice already sensitized to 1 TNCB. The skinfold was measured with a vernier caliper and the increase with respect to day 0 was calculated. * p>0.05 with respect to the control; .sup.+p>0.05 with respect to the control treated with TNCB. ANOVA followed by a Tukey test.

[0097] FIG. 20. It shows the evaluation of the lesions in the model of atopic dermatitis in the trial regarding the macroscopic assessment of the lesions. The application of treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 24 hours before the application of 100 μl of 1% TNCB on mice already sensitized to 1 TNCB. The measure of the loss of water for epidemic transmission (TEWL) was evaluated with the Tewameter system TM 210 (CK electronics, Germany). * p>0.05 with respect to the control; .sup.+p>0.05 with respect to the control treated with empty Liposomes. ANOVA followed by a Tukey test.

[0098] FIG. 21. It shows the evaluation of the mice in the model of atopic dermatitis in the trial regarding the macroscopic assessment of the lesions. The application of treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 24 hours before the application of 100 μl of 1% TNCB every 2 days on mice already sensitized to 1 TNCB, and up to 4 applications of TNCB.

[0099] FIG. 22. It shows the increase of skinfold 24 hours of administration of Oxazolone, in the trial regarding the protection against a model of contact hypersensivity induced by Oxazolone. The application of treatments (1 μmoles/mouse of polyphenols, 20 mg of diprosone) was performed 24 hours prior (day 0) of the application of 20 μL of 2.5% oxazolone in ethanol on the mice's ears, previously sensitized to Oxazolone. The skinfold was measured with a vernier caliper and the increase with respect to day 0 was calculated. * p>0.05 with respect to the control treated with Oxazolone; .sup.+p>0.05 with respect to the control treated with diprosone before the application of Oxazolone. ANOVA followed by a Tukey test.

[0100] FIG. 23. It shows the evolution of the increase of the skinfold in reference to day 0 throughout the procedure, in the trial regarding the protection against a model of psoriasis induced by TPA. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA. This administration guideline was repeated over 6 consecutive days. The skinfold was measured with a vernier caliper and the increase with respect to day 0 was calculated.

[0101] FIG. 24. It shows the increase in the skinfold 24 hours after the first administration of TPA in the trial regarding the protection against a model of psoriasis induced by TPA. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA. The skinfold was measured with a vernier caliper and the increase with respect to day 0 was calculated. * p>0.05 with regards to the control. ANOVA followed by a Tukey test.

[0102] FIG. 25. It shows the skinfold 24 hours after the third administration of TPA in the trial regarding the protection against a model of psoriasis induced by TPA. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA. The skinfold was measured with a vernier caliper and the increase with respect to day 0 was calculated. * p>0.05 with respect to the control; .sup.+p>0.05 with respect to the group TNCB. ANOVA followed by a Tukey test.

[0103] FIG. 26. It shows the macroscopic view of the mice in the psoriasis model in the trial regarding the protection against a model of psoriasis induced by TPA. The photos were taken (A) 24 hours after the administration of TPA and (B) 24 hours after the 7.sup.a TPA administration. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA.

[0104] FIG. 27. It shows the evaluation of descaling 24 hours after seven administrations of TPA in the trial regarding the protection against a model of psoriasis induced by TPA. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA. The descaling aspect of the skin was quantified in a scale of 0-3, being 0, nothing; 1, mild; 2, moderate; 3, severe. And the medium of each group indicated the degree of inflammation.

[0105] FIG. 28. It shows the histological analysis of the psoriasis procedure. The application of the treatments (10 μmoles/mice of polyphenols, 0.5 μmoles/mice of dexamethasone) was performed 30 minutes prior the application of 2 nmol/mouse of TPA. Image of a microscopic view of a cut of 5 μm with staining of hematoxylin-eosin from mice at 24 hours (A.) and 72 hours (B.) after being treated with 2 nmoles of TPA daily over 7 days.

DETAILED DESCRIPTION OF THE INVENTION

[0106] Studies for evaluating the capacity of the compositions of the invention of preventing skin diseases (such as skin cancer) induced by ultraviolet B radiation were carried out by acute irradiation at a dose of 360 mJ/cm.sup.2.

[0107] Protocol for Exposure to UVB Radiation.

[0108] To make the exposure to UVB a specially upgraded camera (Crosslinker equipped with 5 tubes which emit 80% of the radiation as UVB, with a maximum emission at 312 nm) was used.

[0109] UVB Acute exposure protocols were used: Mice underwent a single exposure of 360 mJ/cm2. The study was conducted at 24 hours after exposure. This protocol was used to evaluate the photoprotective effect of the compounds studied. With the same acute-phase protocol mice from all groups were irradiated. Subsequently mice remained in the animal storage area for a week to see the evolution of the inflammatory response and edema.

[0110] Administration and Dosage of the Compounds Under Study.

[0111] For the UVB exposure studies (chronic or acute) a topical administration to the back of the mouse was conducted. The commercial sunscreens were applied directly on the mouse. The polyphenols in the study were administered in a liposomal preparation diluted 1:1 with Carbopol polymer which will be the vehicle for these liposomes. Each polyphenol dose administered was 10 μmol/mouse, as free acid.

[0112] Parameters Indicators of Skin Damage. [0113] 1. Edema formation: skinfold thickness was determined with a caliper 24 hours after the first exposure. [0114] 2. Study of the thickness of the epidermis after exposure to acute irradiation (360 mj/cm.sup.2 24 h hours and 7 days after irradiation). [0115] 3. Histological studies: measuring, in the pictures obtained from histological sections, the thickness of the epidermis.

[0116] Histological Studies.

[0117] For studies of acute exposure skin samples were taken from the back of the mouse. These samples were fixed with 4% paraformaldehyde (24 hours) and embedded in paraffin. Following cutting the samples were stained with hematoxylin and eosin.

[0118] Study of the Absorption Spectrum of the Different Compounds.

[0119] A solution of 1 mM of each polyphenol was prepared (PTER, Resveratrol and QUER) in absolute ethanol (HPLS quality; Quima) and the absorption spectrum was studied between 200 and 800 nm using a Multiskan Spectrum (ThermoScientific).

[0120] Procedures of Photo Carcinogenesis (Chronic Exposure to Radiation UV-B).

[0121] The role of UV-B radiation in tumor formation is well known. The continuous exposure to UV-B promotes the formation of oxidative/nitrosative stress, along with alterations in DNA, proteins, etc. that activates various transcription factors (among them NF-κB and AP-1), increase in the telomerase activity, stress, etc. which in turn induces carcinogenesis.

[0122] Furthermore, the exposure to UV-B increases the expression and levels of antiapoptotic proteins, which correlate with the increase in proliferation and certain inflammation markers.

[0123] The mice chosen for this study were SKH-1. These mice have a greater risk of developing tumors following chronic exposure to low doses of UV radiation (comparable to the exposure that a human being could have on a daily basis).

[0124] The protocol used was the standardized protocol, whereby several mice were irradiated three times per week with a dose of 180 mJ/cm.sup.2 with exposures for 24 weeks; however, during the trial the protocol was modified by lengthening the time of exposure to up to 28 weeks, in order to obtain more evidence of the photoprotector effect on the different treatments used.

[0125] The dose of exposure was 180 mJ/cm2, since it had been determined in previous trials that this was the minimal erythemic dose (MED) for this type of rodent.

[0126] A range of different commercial photoprotectors were included in this study: [0127] Vichy: Capital Soleil SPF-50+ (Vichy Laboratoires). [0128] Isdin: Photoprotector Isdin Extrem (SPF-40 (Isdin SA) [0129] Heliocare: Heliocare gel Protection Ultra 90 (Difa Cooper S.P.A.)

[0130] Both the reference compounds and the ones subject of this study were applied to the back of the mice for 20 minutes prior to their exposure to UV-B.

[0131] The groups, of 20 mice, for this assessment were: [0132] Not treated. [0133] Reference compound Vichy prior: 200 μl of Isdin applied 20 minutes prior to irradiation. [0134] Reference compound Isdin prior: 200 μl of Isdin applied 20 minutes prior to irradiation. [0135] Reference compound Heliocare prior: 200 μl of Heliocare applied 20 minutes prior to irradiation. [0136] Medium prior: 200 μl of vacuumed Liposomes applied 20 minutes prior to irradiation. [0137] Resveratrol prior: 200 μL of Resveratrol Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0138] PTER prior: 200 μL of PTER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0139] QUER prior: 200 μL of QUER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0140] PTER+QUER prior: 200 μL of PTER+QUER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation.

[0141] UV-B induces edema and erythema in the short-term. This inflammation is critical for the hyperproliferation process.

[0142] Skin cells exposed to UV have higher levels of the nuclear transcription NF-κB factor, this factor control the output of COX-2 and iNOS, among others, a reason why UV-B is a clear cause of skin inflammation.

[0143] With the purpose of assessing the effects of the photoprotective compounds studied, female mice SKH-1 of between 6-8 weeks old were subjected to a single exposure of 360 mJ/cm.

[0144] For the ultraviolet B (UVB) irradiation exposure, a camera specifically prepared, available at our laboratory was used (Crosslinker; Bio-Link BLX 254) equipped with 5 tubes that produce 80% of the UVB radiation, with a maximum emission of 312 nm.

[0145] The two protocols of UV-B exposure used were: [0146] Intense exposure after a single exposure of 360 mJ/cm.sup.2. The study was carried out 24 hours after the exposure. This protocol was used to assess the photoprotective effects of the compounds studied. [0147] Intense exposure after a single exposure of 360 mJ/cm.sup.2. The study was carried out 1 week after the exposure. The mice were kept in the shed for a week to observe the evolution of the inflammatory and edematous response.

[0148] As reference, several commercial photoprotective products were used (Vichy, Isdin and Heliocare). The reference compounds as well as the products object of this study, were applied on the back of the mice for 20 minutes after or 20 minutes before the irradiation with UV-B, to determine if the reaction observed was due to an effect of the solar filter or not.

[0149] The groups, of 5 mice, for this assessment were: [0150] Not treated. [0151] Reference compound Vichy prior: 200 μl of Isdin applied 20 minutes prior to irradiation. [0152] Reference compound Vichy after: 200 μl of Isdin applied 20 minutes after irradiation. [0153] Reference compound Isdin prior: 200 μl of Isdin applied 20 minutes prior to irradiation. [0154] Reference compound Isdin after: 200 μl of Isdin applied 20 minutes after irradiation. [0155] Reference compound Heliocare prior: 200 μl of Heliocare applied 20 minutes prior to irradiation. [0156] Reference compound Heliocare after: 200 μl of Heliocare applied 20 minutes after irradiation. [0157] Medium prior: 200 μl of vacuumed Liposomes applied 20 minutes prior to irradiation. [0158] Medium after: 200 μl of vacuumed Liposomes applied 20 minutes after irradiation. [0159] Resveratrol prior: 200 μL of Resveratrol Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0160] Resveratrol after: 200 μL of Resveratrol Liposomes (10 μmol in total) applied 20 minutes after irradiation. [0161] PTER prior: 200 μL of PTER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0162] PTER after: 200 μL of PTER Liposomes (10 μmol in total) applied 20 minutes after irradiation. [0163] QUER prior: 200 μL of QUER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0164] QUER after: 200 μL of QUER Liposomes (10 μmol in total) applied 20 minutes after irradiation. [0165] PTER+QUER prior: 200 μL of PTER+QUER Liposomes (10 μmol in total) applied 20 minutes prior to irradiation. [0166] PTER+QUER after: 200 μL of PTER+QUER Liposomes (10 μmol in total) applied 20 minutes after irradiation.

[0167] At 24 hours or 1 week after the administration of the different treatments the mice were sacrificed and their skin fixed with 4% PFA.

[0168] Ear Thickness.

[0169] The edema was calculated as the difference between the thickness that was produced in the ears, before the outcomes of the reaction, and their thickness 24 hours later, and it was measured with a vernier caliper.

[0170] The change was expressed as the relative increase in relation to day 0.

[0171] Macroscopic Assessment of the Skin.

[0172] The mice were observed daily to detect any changes on the appearance of the skin or any general changes.

[0173] Also, on certain specific days prior to sacrificing them, the mice were assessed by a veterinarian who analyzed the skin lesions, qualifying them in function of: [0174] Erythema: redness of the skin conditioned by inflammation due to the excess of blood irrigation by the dilation of blood vessels. [0175] Excoriation: or irritation of the skin with flaky aspect, increase in the descaling of the skin which could result in hyperkeratosis. [0176] The depth of the lesion that could reach the dermis producing soars or ulcers.

[0177] Each variable was evaluated on a scale of 0-4, 0, for none; 1, for mild; 2, for moderate; 3, marked; and 4, very marked. The addition of the 3 values of each mouse indicated the degree of inflammation.

[0178] During the photocarcinogenesis process, the presence of different tumors was followed and quantified throughout the process. Any lesion of more than 1 mm of diameter was considered a lesion.

[0179] Microscopic Analysis.

[0180] Histochemical techniques were used to study the skin tissues of the different procedures, at a microscopic level.

[0181] The tissues were fixed in 4% formaldehyde in PBS over 24 h at 22° C. After the tissues were fixed, they had to be dehydrated in large amounts of ethanol and placed in a hydrophobic and xylene mixture to allow the paraffin to penetrate the tissues to provide sufficient consistency for slicing them thinly in a microtome.

[0182] The samples in paraffin were processed with the microtome obtaining slices of about 5-10 μm. The samples were spread over a drop of gelatin at 37° C. and were collected in gelatinized slides.

[0183] For other procedures it was necessary to eliminate the paraffin over temperatures of 60° C. and by washing the samples with xylene, and by rehydrating them with ethanol.

[0184] Hematoxylin is a cationic colorant while eosin is an anionic colorant belonging to the xanthens. Therefore the nucleus is stained in blue and the cytoplasm in pink.

[0185] The protocol used for staining the samples was: [0186] 1. Incubation with Mayer's Hematoxylin for 10-15 minutes. [0187] 2. Wash under running water until bluish. [0188] 3. Incubate with Eosin (1%) for 2-4 minutes. [0189] 4. Eliminate the remaining eosin in ethanol 70° [0190] 5. Dehydration. [0191] 6. Rinse with xylol for 3 minutes. [0192] 7. Let the trays dry out on the stove at 37°.

[0193] Statistical Analysis.

[0194] The results were expressed with the means±standard deviation. The standard deviation was represented in the form of error bars, in some cases only positive semi bars were shown to simplify the graphics.

[0195] The data was analyzed based on the variations (ANOVA) of one or more factors or tests as appropriate. The homogeneity of the variants was analyzed with the Levene test. The null hypothesis was accepted for all the values of the tests in which value F was not significant with a value of p greater than 0.05. The data, for which the value F was significant, was analyzed with the Turkey test with a p value of 0.05.

[0196] The computer programs used were Excel for the calculations and graphics and for the statistics SPSS version 14.0 for Windows (SPSS Inc) was used, the University of Valencia has a user's license for this.

EXAMPLES

Example 1

Experimental Animal Model

[0197] Strain SKH-1 mice provided by Charles River Company were used as experimental animals. They are hairless immuno competent mice. The experimental groups were formed with female mice aged 3 to 4 weeks as showed in Table 2. Each experimental group of photoprotection consisted of between 5 and 6 animals. In these studies, each experimental group consisted of 20 mice.

TABLE-US-00002 TABLE 2 GROUP NAME TREATMENT UVB EXPOSURE I Negative control Liposomal vehicle No IIt UVBa Liposomal vehicle 20 min after treatm. IIa UVBb Liposomal vehicle 20 min before treatm. IIIt RESa RES liposomes 20 min after treatm. IIIa RESb RES liposomes 20 min before treatm. IVt PTERa PTER liposomes 20 min after treatm. IVa PTERb PTER liposomes 20 min before treatm. Vt QUERa QUER liposomes 20 min after treatm. Va QUERb QUER liposomes 20 min before treatm. VIt (PTER + QUER)a PTER + QUER 20 min after treatm. liposomes VIa (PTER + QUER)b PTER + QUER 20 min before treatm. liposomes VIIt ISDINa 20 min after treatm. VIIa ISDINb 20 min before treatm. VIIIt HELICAREa 20 min after treatm. VIIIa HELIOCAREb 20 min before treatm. IXt VICHYa 20 min after treatm. IXa VICHYb 20 min before treatm. b refers to “before” a refers to “after”

[0198] Moreover, in vivo trials were performed on 4 week-old naked female mice SKH1-E from Charles River, or on 6-8 week BALB/c mice.

[0199] The studies took place at the University of Valencia, Central Support Service of Experimental Research (CSSER) in the area of animal experimentation from the animal production service, excepting the studies performed by TNO Quality of Life (Leiden, Netherlands), of hypersensitivity to oxazoline.

[0200] Once the animals were acclimatized after a period of 2 weeks, the experiments were performed in rooms equipped with protective barriers, under controlled conditions, with temperatures of: 22±2° C., relative humidity: 65-80%, 12 changes of air/hour and a photoperiod of 12/12 hours, in compliance with the current international regulation [European Community Council Directive (86/609, OJ L 358. 1, Dec. 12, 1987), National Health Institute (Guide for the Care and Use of Laboratory Animals, NIH Publ. No. 85-23, 1985, USA)].

[0201] The animals were sacrificed by cervical dislocation. Samples were taken from the hindquarters for histology, and were fixed in 4% formaldehyde overnight. Parts of the samples were kept in liquid N.sub.2 for the intense UV-B exposure procedures, and for subsequent evaluation for inflammation and oxidative/nitrosative stress.

[0202] Subsequently, 20 μl of blood were taken and added to 80 μl of N-ethylmaleimide (NEM).

[0203] 100 μl perchloric acid (PCA) 4% were added after incubation for about 10 min. Samples were preserved in ice after vigorous shacking. Finally all samples were centrifuged at 13000 rpm for 15 minutes at 4° C. Supernatants were collected and stored at −20° C. until analysis of the GSH (reduced form of glutathione)/GSSG (oxidized form of glutathione) ratio. The GSH/GSSG ratio is a parameter that reflects the intracellular redox status, and the intracellular thiol redox status in particular. Therefore changes in this ratio are used to evaluate oxidative stress.

[0204] Four samples were taken from skin of the hindquarters. One for histology (was fixed overnight in 4% formaldehyde) and 3 were frozen with liquid nitrogen for biochemical analysis.

Example 2

Preparation of Liposomes

[0205] Regardless of the polyphenol(s) the methodology of preparation of liposomes were equally used: soy lecithin; dichloromethane as organic solvent in the case of PTER and/or QUER, and ethyl ether as organic solvent for resveratrol due to its inability to dissolve in dichloromethane. The evaporation of organic solvent was performed using a rotary evaporator connected to vacuum and a temperature of 40° C. After removal of organic solvent, liposomes were resuspended in PBS diluted to 1 mM. In the final liposome concentration, before adding the Carbopol, the concentration of each polyphenol was of 20 μM polyphenols as determined by HPLC/MS-MS.

[0206] The steps for preparing the liposomes can be summarized as follows:

TABLE-US-00003 1. Weight polyphenols and lecithin PTER QUER RESV LECITHIN 0.512 gr 0.676 gr 0.458 gr 2.376 [0207] 2. Dissolve the polyphenol and lecithin in 40 ml of dichloromethane (in the case of PTER and/or QUER), or 140 ml of ethyl ether (in the case of resveratrol). [0208] 3. Introduce the solution into a rotary evaporator flask and attached to the device. Once all the solvent evaporated cut the expected vacuum and wait for 15 min. to eliminate all traces of solvent which may have become. [0209] 4. Add 20 ml of diluted PBS (1 mM) in the rotary evaporator flask and re-attach the device but this time without vacuum. [0210] 5. The liposomes formed are collected in a 50 ml tube. [0211] 6. Sonication of liposomes (6 seconds, 25 cycles, with an interval of 3 seconds between each cycle; high intensity). [0212] 7. Dilution 1:1 with Carbopol (final concentration of 10 μmoles/200 μl) [0213] 8. To avoid possible contamination is added Phenonip (6 ml of Phenonip per 1000 ml of preparation).

[0214] Control liposomes were made in the same manner as those of PTER and QUER, but only contain lecithin (2.376 g).

[0215] The procedures to obtain liposomes were performed in our laboratories in co-operation with the Colloids Research Unit (Dept. of Chemistry and Physics; Univ. Valencia).

[0216] The liposomes can be loaded with a variety of active principles, such as polyphenols. Due to their ability to penetrate the skin, they can carry these active principles for long distances within the skin, and stop at a determined spot. This is the reason why liposomes can be used in dermatological therapy. There is a variety of products that contain polyphenols that come from green tea (Replenix; Kaplan cosmetic Surgery Centers), and resveratrol that comes from grapes (Resveraderm; SesDERMA Laboratorios).

[0217] A mixture of soy lecithin was used to obtain liposomes; this is a natural mixture of about 90% phosphatidylcholine and other fatty acids. The soy lecithin (99% pure) was provided by GUINAMA S.L (Valencia, Spain).

[0218] Polar organic solvents were used for solubility, such as dichloromethane (Sigma) for the PTER (CML Europe BV) and QUER (Sigma), and ether (Sigma) for the Resveratrol (Sigma).

[0219] The ideal mixture to obtain liposomes contained: [0220] 5.9 (w/v) of lecithin, 1.28 (w/v) of PTER and 1.69 (w/v) of QUER, dissolved in dichloromethane. [0221] 1.69 (w/v) of lecithin and 1.15 (w/v) of Resveratrol dissolved in ether.

[0222] The microscopic packaging of the different polyphenols was achieved by the dehydration of the soy lecithin at 40° C. in a vacuum rotary evaporator, which was later hydrated with a solution of 1 mM phosphate pH 7 at 20° C. for a final concentration of 0.1 mM per compound in the final solution.

[0223] The liposomes were standardized in relation to the size of the particles by observing the morphology via microscope. The concentration of the polyphenols in the liposomes suspension was quantified by HPLC-MS/MS.

[0224] The solution was stabilized Carbopol 940 (Guinama) to a dilution 1:1, adding 0.6% (v/v) of antimicrobial preservative Phenonip (Guinama), with which was obtained a solution of liposomes with 10 μmoles for each polyphenol/200 μl.

[0225] The administration of the liposomes was done by applying and smoothing a homogeneous film of the cream to the back of the mouse with an automatic micropipette.

[0226] The compounds were applied in accordance with the administration protocol, described in each procedure, which were: [0227] Empty liposomes: liposomes without charge. [0228] PTER Liposomes: 1 μmol/20 μL of cream. [0229] QUER Liposomes: 1 μmol/20 μL of cream. [0230] PTER+QUER Liposomes: 1 μmol of each compound/20 μL of cream. [0231] Resveratrol Liposomes: 1 μmol/20 μL of cream.

Example 3

Determination of Skinfold 24 H After Irradiation

[0232] The skinfold was measured by making a fold on the skin from the back of the mice, from the axillae to the hips; the fold was measured with a vernier caliper.

[0233] The increase in the fold was calculated by the difference between the initial point, before the effects of the reaction, and the thickness in each point.

[0234] FIG. 1 shows the results obtained in measuring the thickness of the skin of the hindquarters mice (where the various treatments have been applied). The results were statistically analyzed by ANOVA and contrasted by the Tukey test and grouped according to the significant differences. Standard deviations are incorporated in FIG. 1. As shown in FIG. 1, the combination of PTER+QUER and also PTER separately, are more efficient as photoprotector, when applied before the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin) and that RESV. Therefore either the combination of PTER+QUER or PTER separately could be used efficiently in the prevention of skin diseases induced by ultraviolet B radiation.

[0235] Moreover, FIG. 1 also shows that the combination of PTER+QUER or PTER separately are more efficient for the treatment of skin diseases, damages or injuries due to radiation, when applied after the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin) and that RESV. Therefore the combination of PTER+QUER could be used efficiently in the treatment of skin diseases, damages or injuries induced by ultraviolet B radiation.

Example 4

Measuring the Thickness of the Epidermis After Irradiation (24 Hours)

[0236] To measure the thickness of the epidermis to evaluate possible hyperplasia, a computer program was used, Image J, calibrated with the assistance of a Neubauer camera which allowed the measuring of the epidermis. Results obtained were statistically analyzed by ANOVA and contrasted by the Tukey test and grouped according to the significant differences. As shown in FIG. 2, the use of PTER alone is more efficient as photoprotector, when applied before the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin). Therefore PTER alone could be used efficiently in the prevention of skin diseases, injuries or damages induced by ultraviolet B radiation.

[0237] Moreover, FIG. 2 also shows that the combination of PTER+QUER is also more efficient for the treatment of skin diseases, damages or injuries caused by radiation, when applied after the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin) and that RESV. Therefore the combination of PTER+QUER, as well as PTER separately, could be used efficiently in the treatment of skin diseases, damages or injuries induced by ultraviolet B radiation. Control in this experiment means no radiation and no treatment.

Example 5

Measuring the Thickness of the Epidermis After Irradiation (1 Week)

[0238] The evaluation of skin thickness was carried out by using computer software (IMAGE J). Results obtained by ANOVA were contrasted by the Tukey test and grouped according to the significant differences. As shown in FIG. 3, the use of PTER separately is more efficient as photoprotector, when applied before the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin). Therefore PTER separately could be used efficiently in the prevention of skin diseases, damages or injuries induced by ultraviolet B radiation.

[0239] Moreover, FIG. 3 also shows that the combination of PTER+QUER is more efficient too, for the treatment of skin diseases, damages or injuries caused by radiation, when applied after the exposure to UVB, than the commercial creams (Vichy, Heliocare and Isdin) and that RESV. Therefore the combination of PTER+QUER, as well as PTER separately could be used efficiently in the treatment of skin diseases induced by ultraviolet B radiation.

Example 6

Induction of Experimental Atopic Dermatitis with TNCB

[0240] As to agent “2-chloro-1,3,5-trinitrobenzene or 2,4,6-trinitrochlorobenzene” (TNCB), also commonly known as Pycril chloride, this is a chemical agent that induces an allergic process combined with the inflammation of the skin of the mice. Furthermore, the continuous administration of TNCB ointment increased the production of T-lymphocytes, mastocytes and IgE in the serum, as it occurs among human patients with atopic dermatitis.

[0241] To produce atopic dermatitis lesions, the protocol described by Matsumoto was used, applying 100 μL of a solution of 1% TNCB in acetone [3 applications of 33 μL (with the aid of a dispenser of an automatic micropipette) to prevent the volume from spreading and to maintain the lesion area more localized] on the back of the SKH-1 mice.

[0242] In contrast to the protocol proposed by Matsumoto, where the treatment with TNCB lasted 32 days following a prior testing period with TNCB, a protocol consisting of one test with TNCB and a week later 4 doses followed by TNCB every 2 days was developed. Shorter protocols produced an increase in the skinfold as shown in FIG. 4, as in the loss of transepidermal fluid as shown in FIG. 5, these lesions being obvious at macroscopic level as shown in FIG. 6.

[0243] Therefore, the protocol developed in this procedure was the one shown in FIG. 7. As a standard treatment, the following was used Dexamethasone (from Sigma Solution 25 mM in acetone). The administration of the products to be tested and the standard treatment were given in alternate days, beginning 24 hours prior to the first continuous application of TNCB and after the mice were exposed to TNCB (1 week prior).

[0244] The groups, of 5 mice, for this assessment were: [0245] Not treated. [0246] Reference compound: 0.5 μmoles of Dexamethasone (20 μl of one solution 25 mM in acetone). [0247] Medium: 200 μl of vacuumed Liposomes. [0248] Resveratrol: 200 μL of Resveratrol Liposomes (10 μmol in total). [0249] PTER: 200 μL of PTER Liposomes (10 μmol in total). [0250] QUER: 200 μL of QUER Liposomes (10 μmol in total). [0251] PIER+QUER: 200 μL of PTER Liposomes+QUER (10 μmol in total).

[0252] At 24 hours (day 9) after the last administration of the different treatments the mice were sacrificed and their skin fixed with 4% PFA.

Example 7

Experimental Induction of Psoriasis with TPA

[0253] For the induction of psoriasis, the agent “12-O-tetradecanoylphorbol-13 acetate” (TPA) was used. In relation to the agent “12-O-tetradecanoylphorbol-13 acetate” (TPA), also commonly known as “phorbol 12-myristate 13-acetate” (PMA), this is the diester of phorbol and a strong promoter of tumors usually used in biomedical research to activate the transduction of signals via the kinase enzyme of protein C (PKC).

[0254] In reference to the dose of TPA needed to develop the disease on the hairless mice SKH-1, some authors have used elevated doses of 10-17 nmoles/mouse, the achieved reproduction of hyperplasia is similar to how it occurs in Balb/C mice, previously shaved, with psoriasis, and this model is widely used in the studies of psoriasis. However, these models were used to evaluate biochemical or molecular parameters after only one administration of TPA. Since our objective was to assess the progress of the lesions caused by psoriasis in combination with the use of polyphenols, a model of continuous exposure and a weaker concentration of TPA were used, on a daily dose of 2 nmoles of TPA over a period of a week, which allowed us to observe the progress of the psoriasis.

[0255] The induction was performed with 2 nmol TPA/mouse on an area of 1 cm.sup.2, applying 10 μL of the 200 μM solution in acetone. This application protocol was carried out taking into account that when applying 20 μL as stated in the model, the volume increased considerably and it lost its concentration, therefore it was decided to make it more concentrated.

[0256] Shorter protocols produced an increase in the skinfold as shown in FIG. 8, as in the loss of transepidermal fluid (TEWL) as shown in FIG. 9, these lesions being obvious at macroscopic level as shown in FIG. 10.

[0257] Therefore, the protocol developed in this procedure was the one shown in FIG. 11. As a standard treatment, Dexamethasone was used, (from Sigma Solution 25 mM in acetone). The administration of the standard treatment and the products to be tested were given 30 minutes prior to the application of TPA, both beginning on the same day.

[0258] The groups, of 6 mice, for this assessment were: [0259] Not treated. [0260] Reference compound: 0.5 μmoles of Dexamethasone (20 μl of one solution 25 mM in acetone). [0261] Medium: 200 μl of vacuumed Liposomes. [0262] Resveratrol: 200 μL of Resveratrol Liposomes (10 μmol in total). [0263] PTER: 200 μL of PTER Liposomes (10 μmol in total). [0264] QUER: 200 μL of QUER Liposomes (10 μmol in total). [0265] PTER+QUER: 200 μL of PTER Liposomes+QUER (10 μmol in total).

[0266] At 24 hours (day 7) after the last administration of the different treatments and TPA, 3 mice from each group were sacrificed and their skin fixed with 4% PFA. The remaining mice were kept under the treatment with no exposure to TPA, to verify if the skin recovered faster on the treatment than on its own. At 24 hours (day 9) after the last administration of the different treatments the rest of the mice were sacrificed and their skin fixed with 4% PFA.

Example 8

Experimental Induction of Hypersensitivity by Contact to Oxazolone

[0267] The application of oxazolone after sensitization to the same substance produces a reaction of hypersensitivity by contact, which appears as an eczematous reaction, with infiltration of lymphocytes and macrophages, with edema of the epidermis.

[0268] The BALB/c mice are sensitized on day 0 through the application, on the shaved abdomen, of 100 μL of oxazolone at 1.5% in acetone (w/v). Prior to the induction for hypersensitization, the thickness of the ear was measured with the help of a vernier caliper (Mitutoyo micrometer). The reaction was caused on day 7 by applying 20 μL of 2.5% oxazolone in ethanol (10 μL to each side of the right ear).

[0269] Therefore, the protocol developed in this procedure was the one shown in FIG. 12. As a control measure the left ear was used, to which 20 μL of ethanol was applied. And a standard treatment, Diprosone® cream was used: (Schering Plough B.V., Utrecht, The Netherlands), that contains 0.5 mg de betamethasone/g.

[0270] The groups, of 8 mice, for this assessment were: [0271] Not treated. [0272] Reference compound: 10 mg of Diprosone® cream to each side of the ear, applied 30 minutes after applying oxazolone on day 7. [0273] Medium: 10 μl of empty Liposomes to each side of the ear applied 30 minutes prior to the application of oxazolone on day 7. [0274] Resveratrol prior: 10 μl of Resveratrol Liposomes to each side of the ear (1 μmol in total) applied 30 minutes prior to the application of oxazolone on day 7. [0275] Resveratrol after: 10 μl of Resveratrol Liposomes to each side of the ear (1 μmol in total) applied 3-4 hours after the application of oxazolone on day 7. [0276] PTER prior: 10 μl of PTER Liposomes to each side of the ear (1 μmol in total) applied 30 minutes prior to the application of oxazolone on day 7. [0277] PTER after: 10 μl of PTER Liposomes to each side of the ear (1 μmol in total) applied 3-4 hours after the application of oxazolone on day 7. [0278] QUER prior: 10 μl of QUER Liposome to each side of the ear (1 μmol in total) applied 30 minutes prior to the application of oxazolone on day 7. [0279] QUER after: 10 μl of QUER Liposomes to each side of the ear (1 μmol in total) applied 3-4 hours after the application of oxazolone on day 7. [0280] PTER+QUER prior: 10 μl of PTER+QUER Liposomes to each side of the ear (1 μmol in total) applied 30 minutes prior to the application of oxazolone on day 7. [0281] PTER+QUER after: 10 μl of PTER+QUER Liposomes to each side of the ear (1 μmol in total) applied 3-4 hours after the application of oxazolone on day 7. [0282] 24 hours after the administration of the oxazolone (day 1), the mice were sacrificed and both ears were fixed with 4% PFA.

Example 9

Transepidermal Water Loss

[0283] Some skin conditions are characterized by compromising the mechanical properties of the skin, such as in scleroderma and psoriasis. [0284] Nonetheless, the objective evaluation of these characteristics is complex, and the measuring of the loss of transepidermal water (PTEA or TEWL) assessed through the Tewameter TM 210 system (CK electronics, Germany) is considered an indicator of the functions of cutaneous “barriers” (g/h/cm2).

[0285] The physical foundation of this type of measurement is based in the law of diffusion established by Adolf Fick in 1885:

[00001]$\frac{d.Math..Math.m}{\mathrm{dt}}=-D\times A\times \frac{\mathrm{dp}}{\mathrm{dx}}$

[0286] whereas: [0287] A=surface in m.sub.2 [0288] m=transportation of water (in g) [0289] t=time (h) [0290] D=constant of diffusion (=0.0877 g/m.Math.h.Math.mm Hg) [0291] p=pressure of atmospheric vapor (mm Hg) [0292] x=distance from the skin to the measuring point (m)

[0293] The law of diffusion dm/dt indicates the mass by cm.sup.2 transported in an interval of time, which is proportional to area A and the variation of concentration by the distance dc/dx. D represents the coefficient of diffusion of water vapor in the air. This law is valid only within a zone of homogeneous diffusion, which has the approximate shape of an empty cylinder. The resulting gradient density is registered indirectly by two sets of detectors (temperature and relative humidity) analyzed by a microprocessor.

[0294] In the probe of the Tewameter TM 210 system, the temperature and hydration detectors, as well as the circuitry for measuring and data for calibration, are located in the interior of the sensor. The head of the sensor is made by a hollow and narrow cylinder, (diameter=10 mm, length=20 mm) and its function is to reduce the air turbulence in the sensor.

[0295] After immobilizing the mice, the probe was place on their back, in the same area treated with different chemical agents. TEWL readings were performed continuously until the metrics were stabilized, which can produce between 30-60 seconds of readings.

Example 10

Photoprotector and Anticarcinogenic Effect During Chronic Exposure to UV-B Irradiation

[0296] Mechanical Filter Capacity of Polyphenols.

[0297] With the purpose of studying the capacity of the polyphenols used in this study, we decided to evaluate the absorption of light throughout the ultraviolet light spectrum as shown in FIG. 13.

[0298] It was confirmed that Resveratrol as well as PTER have peaks or maximum absorption in the range of Ultraviolet B, in contrast QUER is absorbed in larger proportion in the wavelengths within the UV-A.

[0299] Photoprotector Role of Polyphenols.

[0300] The following phase of the study was to evaluate the photoprotector component (adding the polyphenols before exposure to UV-B) against the possible repairing capability of the polyphenols against photocarcinogenesis (adding polyphenols after exposure to UV-B).

[0301] For this purpose, the photoprotector capacity was evaluated after chronic exposure to UV-B as shown in FIG. 14. The mice were subject to 3 exposures per week over 28 weeks to a dose of 180 mJ/cm.sup.2 of UV-B per exposure. With this protocol neoplasias are induced which begin to show starting on the twelfth week. Throughout the procedure the precancerous lesions were evaluated and quantified as shown in the FIG. 15.

[0302] If we compare the effects of the different treatments in relation to the signs of lesions, we will be able to appreciate than the control group as well as the one treated with empty liposomes, beginning on week 12 of continuous exposure to UV-B, over 50% of the mice had tumors. In contrast, the treatment with polyphenols delayed the showing of tumors until approximately the 20th week in the case of Resv and QUER. However, under Pter and after 30 weeks of chronic exposure to UV-B, only 10% of the mice presented lesions, with Pter alone or in combination with Quer. This protective effect was superior to the one found in the commercial photoprotectors.

[0303] In relation to the number of lesions per mouse, Pter and the one combined with Quer were the ones that provided the most protection, since the number of lesions per mouse were null in comparison to any of the other treatments. The Table 3 below shows the lesions per mouse: first signs of precancerous lesions of more than 1 mm of diameter in the different experimental groups after 30 weeks of chronic exposure to 180 mJ/cm2 (three times per week). Both the reference compounds and the ones subject of this study (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) were applied to the back of the mice for 20 minutes prior to their exposure to UV-B. Group of 20 mice.

TABLE-US-00004 TABLE 3 no of group(mean) tumor/mouse x weeks 20 24 30 Control 4.4 9.2 13.8 Free liposomes 5.7 8.2 12.9 PTER liposomes 0.0 0.1 0.0 QUER liposomes 0.3 2.2 4.6 Resveratrol liposomes 0.3 1.2 3.8 Pter + Quer liposomes 0.0 0.1 0.1 Vichy: 0.0 0.3 0.7 Heliocare: 0.0 0.5 1.06 Isdin: 0.1 0.5 0.6

[0304] Anatomopathological Studies.

[0305] In relation to the histopathological exam of the tumors induced by chronic irradiation to UV in the SKH1 mice, it was confirmed that the treatment with UV-B produced a strong acanthosis due primarily to the inflammatory process; this gave way to the first signs of tumors, mostly of carcinoma type and not melanoma.

[0306] The great majority of the two non-melanoma skin cancers are: basal cell carcinoma and flaky cell carcinoma.

[0307] The use of commercial photoprotectors protected mainly from inflammation, however only Vichy diminished the possible development of tumors.

[0308] The use of polyphenols had anti-inflammatory and anti-tumor formation effects; PTER was the compound with greatest benefits. However, when compared with its counterpart resveratrol, a great difference was observed at the level of anti-inflammatory and anti-tumor formation. It also presented better results when compared to different commercial photoprotectors.

[0309] About the combination of Pter and Quer, no differences were found between the uses of Pter alone or combined in relation to its photoprotector effect, but there was a difference in relation to its anti-inflammatory effect as it will be discussed later. As it is represented in FIG. 16, the Table 4 below shows the anatomopathological study of the skin after chronic exposure to UV-B, specifically after 30 weeks of chronic exposure to 180 mJ/cm2 (three times per week). Both the reference compounds and the ones subject of this study (10 μmoles/mouse of polyphenols, or 200 μl of each photoprotector) were applied to the back of the mice for 20 minutes prior to their exposure to UV-B. Medium of groups of 20 mice. Every parameter was measured according to the degree of seriousness (+) light-(+++) serious.

TABLE-US-00005 TABLE 4 Resv + Isdin + Heliocare + UV PQ + UV UV UV UV Tumor (no/mouse × group) 13.8 0.1 3.8 0.7 1.1 Stroma Dysplasia + ++ ++ ++ Fibrosis Reactive Acanthosis /+/+++ +++/++ ++ +++/++ Acantholysis Parakeratosis +++ + + Inflammation +++ ++ ++ ++

Example 11

Effect on Dermatitis

[0310] Prior to evaluating the results of different treatments on different models of dermatitis, the possible side effects of continues administration of polyphenols on the skin of the mice had to be evaluated as shown in FIG. 17. For this purpose, the dose for this daily evaluation was administered over 6 consecutive days.

[0311] As it can be observed, the treatment alone did not affect the skinfolds after a chronic treatment, however, the chronic use of corticoids (such as dexamethasone) did have side effects on the structure of the skin.

[0312] Protection from Acute Exposure to Radiation UV-B.

[0313] Having seen the anti-inflammatory characteristic of the polyphenols as protection against photocarcinogenesis, the anti inflammatory role was studied after severe exposure to UV-B. The results obtained with measures of the thickness of the skin of the four mice's backs are shown in FIG. 1.

[0314] The polyphenols Pter and Resv individually and the combination with Pter+Quer, showed a beneficial effect after the exposure to UVB irradiation since the formation of the edema was reduced.

[0315] This increase in the thickness of the skin could be due to the volume caused primarily by some inflammatory process, or by the hyperproliferation of the cells of the epidermis. Therefore it was needed to measure the thickness of the epidermis as shown in FIG. 2.

[0316] A significant difference in the thickness of the epidermis of the mice treated with polyphenols was noticed with respect to the group treated only with UV-B. In fact, the treatment with PTER with or without QUER produced better results than even commercial photoprotectors and Resveratrol.

[0317] In relation to the time the treatment was applied, there were no important differences between applications before or after exposure to UV-B, except for Resveratrol, which showed a different result in relation to the time of application with regards to its good photoprotection role but not as a repairer.

[0318] Also, when the thickness of the epidermis was analyzed a week after irradiation of 360 mJ/cm.sup.2 of UV-B, it was noticed that the mice treated with polyphenols before UV-B exposure were protected from the damage of UV-B, they maintained the same results of the thickness of the epidermis obtained 24 hours after irradiation, there were no statistical differences. If the polyphenols were applied after irradiation the same results were obtained, except for the QUER that gave a worst prognosis if applied after the exposure to UV-B, the results were similar to the results observed in commercial photoprotectors as shown in FIG. 3 and FIG. 18.

[0319] Protection in an Experimental Model of Atopic Dermatitis Induced by TNCB.

[0320] The increase in the skinfolds 24 hours after the first administration of TNCB was studied as shown in FIG. 19.

[0321] In this model it was impossible to measure the skinfold, since there were lesions produced throughout the process that did not allow the correct measurement of the thickness of the skin. However, it was noticed that there were increases in the skinfold 24 hours after the first exposure to TNCB, 1 week after the previous sensitization with TNCB. As it can be observed in the figure, the dexamethasone as well as the PTER (alone or combines with QUER) decreased their inflammatory effect due to the TNCB agent.

[0322] It should also be observed that the empty liposomes had no effect on the skinfolds, showing that the liposomes on their own do not work as mechanical filters, disallowing the TNCB to reach their target producing the expected lesions.

[0323] Macroscopic Assessment of the Lesions.

[0324] The lesions were evaluated with the purpose of quantifying the differences between the different treatments, as shown in FIG. 20 and FIG. 21.

[0325] It was observed that from the beginning of the first continuous application of TNCB, there were changes in the aspect of the skin, there were changes in the level of hydration from the start of the first exposure to TNCB and throughout the process.

[0326] In short term, the analysis of the TEWL values showed that for the treatment with TNCBA, as well as for the pre-treatment with dexamethasone or empty liposomes there was a significant statistical increase of the hydration of the epidermis in relation to the basal values.

[0327] However, what was also showed by the analysis of the results was that in the case of treatments with polyphenols, especially in the combination Pter−Quer, there were no significant statistical differences with relation to untreated skin (basal value). This shows that the polyphenols have a hydrating effect on the epidermis, more so than water alone, or a protective effect towards aggression produced by TNCB.

[0328] At this time, the differences stopped being statistically significant with relation to the values obtained with TNCB, possibly because there were lesions in all the groups, including the group treated with dexamethasone

[0329] In relation to the lesions, there is indication that the low levels of degree of the lesion was because in contrast with other models, the protocol for administering the TNCB was of a lower dose, which also produced minor lesions but more similar to real lesions.

[0330] The most important fact to remark on will be in regards to the possible prevention or cure of atopic dermatitis, the dexamethasone did not produce any beneficial effect in relation to the index of lesions or in relation to the increase of the skinfold. Te PTER produced less serious lesions than with the use of dexamethasone or with respect to the lack of treatment.

[0331] Protection Against a Model of Contact Hypersensitivity Induced by Oxazolone.

[0332] Skin disorders such as contact hypersensitivity, atopic dermatitis and psoriasis, are characterized by hyperproliferative and inflammatory disorders of the skin.

[0333] The current treatment for skin disorders includes the use of anti-inflammatory agents such as glucocorticoids and antiproliferative agents. In this study it is stated that the use of natural polyphenols produces a clinical improvement of dermatitis as shown in FIG. 22.

[0334] The effects of PTER on skin disorders was established according to procedures performed on animals that showed a similar hypersensitivity to contact on the inflammation of the dermis, measured by use of oxazolane in the prevention of the edema of the ear.

[0335] The results of this pharmacological trial in vivo that emulated skin immune-related inflammatory disorders indicates that the polyphenols in particular PTER, applied on the lesion (applied after the administration of oxazolane) could prevent inflammatory changes as a result of inflammation induced by oxazolone. Similar results can be observed with diprosone.

Example 12

Effect on Psoriasis Induced by TPA

[0336] Study of Edema in Psoriasis

[0337] Since the application of TPA on the skin produces vasodilatation, infiltration of leukocytes and edema, it is interesting to measure not just the variation of the skinfold as the weight of the tissue but as a perimeter of the cutaneous inflammation, as shown in FIG. 23.

[0338] While evaluating the increase of skinfold throughout the procedure (6 continues days) 3 different groups were observed. The first group corresponds to the Control group and the one treated with dexamethasone (0.5 μmoles/mouse 30 minutes prior to administering TPA), there were no differences among these through the treatment, indicating that the dexamethasone prevented the development of psoriasis. The second group would be the one formed by TPA, empty liposomes and the Resv, where at least throughout the first half of the procedure the evolution was similar with an increase in the formation of edema.

[0339] And lastly, the third group formed by the group of Pter, Quer and Pter+Quer, where an important decrease in the increase of skinfolds was noticed, under the TPA group throughout, and significantly different at least after 24 hours of the first administration as shown in FIG. 24.

[0340] As it can be observed by the increase of the skinfold, the application of TPA on the mice' skin produces edema, a significant strong inflammation, 24 hours after the administration. The application of the product in reference, the dexamethasone (0.5 μmoles/mouse) 30 minutes prior to the topical administration of TPA, reduced significantly the increase of the skinfold.

[0341] At the same time, treatments with PTER, QUER and the combination of both also significantly diminished the appearance of edema. It is important to note that the use of empty liposomes (not charged) had no effect upon the appearance of edema, indicating that the effect observed by the Pter, Quer or Pter+Quer, was not due to the composition of the liposomes but instead it was due to a specific effect of the polyphenols. This effect was corroborated in comparisons with liposomes charged with the polyphenol in question, resveratrol; this did not produce any effect on the increase of the skinfold in comparison to the TPA group.

[0342] If the skinfold is compared after 3 administrations of TPA, where the lesions at a macroscopic level are obvious, it is obtained the FIG. 25.

[0343] At this time, it was obvious how the use of Pter, Quer and a combination of both produce a diminishing effect on the inflammation produced by TPA.

[0344] Macroscopic Assessment of the Lesions.

[0345] The lesions were evaluated with the purpose of quantifying the differences between the different treatments as shown in FIG. 26.

[0346] A numeric value was given to the lesions according to the excoriation of the lesions 24 hours after the 7 administrations of TPA, since this was the only alteration that was noticed, there were no indications of erythema or ulcers.

[0347] The flaky aspect of the skin was quantified in a scale of 0-3, being 0, nothing; 1, mild; 2, moderate; 3, severe. And the medium of each group indicated the degree of inflammation as shown in FIG. 2.

[0348] It can be observed that only the groups with PTER on its own or in combination with QUER, were the groups with fewer indexes of excoriation or descaling.

[0349] Histological Analysis of the Lesions.

[0350] This analysis is illustrated in FIG. 28.

[0351] Histologically, there is elongation of the rete ridge with thickness of the inferior part (psoriasiform hyperplasia), elongation and edema of the papillae, suprapapillary epidermal atrophy with spongiform intracorneal pustules (of Kogoj), lack of granular stratum or hypogranulosis, parakeratosis, Munro's intraepidermal microabscess, dilated and tortuous papillary capillaries, perivascular lymphohistiocytic infiltration and few eosinophils and plasmocytes.

[0352] Psoriasis is chronic inflammatory dermatosis, characterized clinically by lesions in the form of papules and scaling erythema, with abundant pearly-white-descaling. Its course is fussy although generally it has a chronic evolution with periods of exacerbation and remission. There have been different clinical descriptions, although generally there is not a set frame since there can be variations within the same patient. We can see many of these during examination or during different evolution stages of the illness.

[0353] For years it has been known that the hyperplasia of the epidermis in psoriasis is secondary to the hyperproliferation of keratinocytes, which is characterized by the number of germinating cells in the proliferation stage and of the fragmentation of growth in the epidermis. An increase in the index of mitosis and the number of cells during the clinical phase has been detected.

[0354] The typical histopathological characteristic of a stable psoriasis lesion is the “epidermal psoriasiform hyperplasia” (Lever's Histopathology of the Skin. 10th edition). It is characterized by a regular epidermal acanthosis with rete ridges wide at the base and narrow on the upper part, where there is usually a thinning of the suprapapillary epidermis.

[0355] In our model with induction of psoriasis in the group with TPA this papillomatosis was observed; however, there was no narrowing of the suprapapillary epidermis. Also hyperkeratosis parakeratosis was observed.

[0356] When the cuts were made to the mice that were retired from the treatment, there was no evidence of clear lesions at a macroscopic level; therefore we did not analyze the level of excoriation since it could have led to errors. However, the histological analysis indicated that the skin was not healthy skin. Therefore when thoroughly analyzed it, it was confirmed that when psoriasis lesions enter a period of involution, the normal cronification tends to be recovered, the parakeratosis begins to disappear and the epidermis begins to thin out. Also, the papillomatosis disappears although the morphology did not reach the level to be that of normal skin.

[0357] At comparing the different groups of treatment, it was observed that the group treated with dexamethasone presented very thin epidermis without papillomatosis or hyperkeratosis. In some points there was also evidence of lesions to the sebaceous glands.

[0358] In reference to the group treated with PTER, the most important difference was that there were no signs of papillomatosis, but there was acanthosis due to a massive proliferation. And if we compared with the group treated only with TPA or with empty liposomes, the acanthosis was less pronounced, same as with the parakeratosis.

[0359] This did not occur with the group treated with Resveratrol, a similar structure of PTER, since in this group the proliferation and acanthosis was less sharp than in the group treated with PTER. Besides, comparing the different treatments, the group treated with Resveratrol resembles more the group treated with TPA, than the group with PTER.

[0360] Also, the combination of PTER with QUER found a phenotype similar to the one found with the PTER alone