TOPICALLY APPLICABLE PREPARATION COMPRISING PEPTIDES AS ALLERGEN PROTECTION FOR THE SKIN

Abstract

Allergenic substances, allergens, are captured by topically applicable preparations comprising peptides that consist of 2-10 amino acids, at least one amino acid having one or more reactive side chains. The preparation comprises antioxidants and/or the pH of the preparation is less than 7. This barrier effect of the preparation allows for a novel anti-allergic form of therapy.

Claims

1.-15. (canceled)

16. A preparation, wherein the preparation is topically applicable and comprises one or more peptides having 2 to 10 amino acids, at least one of these amino acids having one or more reactive side chains, and (i) further comprises one or more antioxidants and/or stabilizers and/or (ii) has a pH of less than 7.

17. The preparation of claim 16, wherein the one or more antioxidants and/or stabilizers comprise one or more of α-glycosylrutin, isoquercitin, tocopherol, citric acid, citrate buffer, BHT, EDTA.

18. The preparation of claim 16, wherein the preparation comprises from 0.01% to 2% by weight of the one or more antioxidants and/or stabilizers, based on a total weight of the preparation.

19. The preparation of claim 18, wherein the preparation comprises from 0.05% to 1.5% by weight of the one or more antioxidants and/or stabilizers.

20. The preparation of claim 16, wherein the preparation has a pH of less than 6.

21. The preparation of claim 16, wherein the preparation has a pH of from 4 to 7.

22. The preparation of claim 16, wherein at least one of the amino acids of the one or more peptides has one or more free thiol groups.

23. The preparation of claim 16, wherein at least one of the amino acids of the one or more peptides is histidine, cysteine, lysine, tyrosine, serine, selenocysteine, asparagine, aspartic acid, glutamine, glutamic acid, methionine, phenylalanine or pyrrolysine.

24. The preparation of claim 16, wherein at least one of the one or more peptides is GSH, HHHHHH, Ac-RFAACAA, Ac-RFAALAA, RFAALAA, RFAACAA, Ac-RAACAA, RAACAA, Ac-RFACAA, RFACAA, Ac-RFACA or RFACA.

25. The preparation of claim 16, wherein the preparation comprises from 0.01% to 10% by weight of the one or more peptides, based on a total weight of the preparation.

26. The preparation of claim 25, wherein the preparation comprises from 0.1% to 5% by weight of the one or more peptides.

27. The preparation of claim 25, wherein the preparation comprises from 0.2% to 5% by weight of the one or more peptides.

28. The preparation of claim 16, wherein the preparation is present in the form of at least one of an emulsion, a gel or a spray.

29. The preparation of claim 16, wherein the preparation is capable of preventing or reducing allergic reactions in or on skin.

30. The preparation of claim 16, wherein the preparation is capable of forming a skin barrier against foreign substances and/or allergens.

31. The preparation of claim 16, wherein the preparation is capable of preventing or reducing a penetration and/or accumulation of foreign substances and/or allergens on skin and/or in skin.

32. The preparation of claim 16, wherein the preparation is capable of attenuating atopic dermatitis and/or allergic contact dermatitis.

33. A method of protecting skin against allergens, wherein the method comprises applying onto skin the preparation of claim 16.

34. A method of preventing or attenuating allergic reactions in or on skin, wherein the method comprises applying onto the skin the preparation of claim 16.

35. A method of preventing or attenuating atopic dermatitis and/or allergic contact dermatitis, wherein the method comprises applying onto skin the preparation of claim 16.

Description

[0129] FIG. 2 shows a peptide of the invention (0.5 mM AcRFAACAA) on its own, 50 mM DNCB, and 50 mM DNCB plus peptide (0.5 mM AcRFAACAA).

[0130] FIG. 3 shows the peptide on its own, 2.5 mM CA (cinnamaldehyde), and 2.5 mM cinnamaldehyde plus peptide.

[0131] In FIG. 4, the interleukin 6 (IL6) concentration in pg/mL is plotted on the y-axis and the substances applied to the skin model (peptide on its own, 2.5 mM DNCB, and 2.5 mM DNCB plus peptide) are plotted on the x-axis.

[0132] DNCB (dinitrochlorobenzene) is a strong allergen. CA (cinnamaldehyde) is a moderate allergen. Eugenol is a phenylpropanoid with an intense cloves-like odor. Eugenol is both an antioxidant and a pro-oxidant. Its skin-irritant and allergy-promoting effect on skin and mucous membranes is based on the latter. All three allergens serve as model allergens for demonstrating the barrier effect and prevention or attenuation of allergic reactions of the peptides of the invention.

[0133] The unchanged skin model is clearly recognizable in FIGS. 2 to 4 (left bar in each case). By applying the allergens, a strong corrosive effect due to the allergens DNCB and CA (middle bar) is evident. Both IL 8 and IL 6 have been produced and released by the skin cells, which represents an allergic skin reaction. However, when the peptide was applied in combination with the allergen (right bar), the situation was surprisingly unchanged. The effect of the allergen was completely nullified by the peptide.

[0134] Further test results (immunohistochemistry) are shown in FIGS. 5 and 6.

[0135] For this, skin biopsies or skin models were embedded in OCT medium and frozen in the gas phase of liquid nitrogen. The frozen models were on the cryostat cut into sections with a layer thickness of 7 μm and applied to microscope slides. After air-drying for 1 to 24 h, they were fixed for 20 min in 3.7% paraformaldehyde at RT. After 3 PBS wash steps of 5 min each, the sections were blocked for 90 min at RT in a solution containing 0.2% Triton X-100. Primary antibodies (e.g., DNP, rabbit from Sigma (1:200)) were diluted appropriately and incubated with the sections for at least 4 h at RT. This was followed by three more PBS wash steps of 5 min each. The secondary antibody (1:800) and Hoechst Fluorescent Stain (1:2000) were incubated with the sections for 1-2 h at RT in the dark. After washing three more times, the sections were mounted with Fluoromount-G mounting medium (Sothern BioTech) and covered with a coverslip. Fluorescence microscopy was carried out using a Zeiss Axio Observer Z1.

[0136] The fluorescence images were analyzed with ImageJ. The DNP-stained areas were measured with the Threshold Color tool.

[0137] FIG. 5 shows the hematoxilin and eosin (H&E) staining of the cryosections, here in black and white, in the original in color.

[0138] FIG. 5 shows the H&E-stained cryosections of the Phenion skin models, A) after incubating with PBS (control), B) after incubating with 0.5 mM AcRFAACAA peptide (control), C) after incubating with 2.5 mM DNCB (strong allergen), and D) after incubating with DNCB and peptide.

[0139] The unchanged skin model in A) and B) and also a strong corrosive effect of DNCB in C) can be seen clearly. In addition to a dissolving stratum corneum, a strong loss of cell nuclei in the epidermis due to the effect of the allergen can be seen (arrows). In D) the situation is as in A) and B). The effect of the allergen was completely nullified by the peptide.

[0140] In further investigations, frozen sections were thawed and incubated for 10 min in 3.7% paraformaldehyde at RT. After three PBS wash steps and one minute in distilled water, they are stained for 3 min in hematoxylin solution. After washing with water and 0.1% HCl, the sections are stained for 3 min in eosin solution and dehydrated in an ethanol series of 70%, 95% and 100%. The sections were mounted and coverslipped in Leica CV Ultra mounting medium. The images were acquired using a Pannoramic Scan II (3DHistech).

[0141] FIG. 6 shows immunohistological analyses of cryosections from Phenion skin models, [0142] A) after incubating with PBS (control), [0143] B) after incubating with 2.5 mM DNCB (strong allergen), and [0144] C) after incubating with DNCB and peptide (0.5 mM AcRFAACAA peptide).

[0145] The cell nucleus staining with DAPI (4,6-diamine-2-phenylindole) in blue (here gray dots) and proteins haptenized by DNCB in red (here white area).

[0146] FIG. 6D shows the quantification of the red intensity of A, B, and C. The y-axis shows the red area above a threshold value in relation to the total area under consideration, with the substances applied to the skin model (peptide on its own, 2.5 mM DNCB, and 2.5 mM DNCB plus peptide) shown on the x-axis.

[0147] What can be seen clearly is the absence of red staining in A) (no allergen used), and in B) an intense red staining both of the epidermis and of parts of the dermis, which indicates a very strong reaction of DNCB with the proteins of the skin and thus strong haptenization. In C) extremely reduced red staining can be seen, which shows that the effect of the allergen was almost completely nullified by the use of the peptide. This is quantified in D). (Red, or white in black/white copy).

[0148] FIGS. 7A-E, 8, and 9 show investigations into the reactivity of peptides of the invention with allergens over time (x-axis) and after 24 h. The absorbance is plotted on the y-axis. The peptide-allergen reactions were realized according to a direct peptide reactivity assay. The test is referred to as the in-chemico method, that is to say no cells are used, but a chemical reaction is carried out.

[0149] The direct peptide reaction assay (DPRA) is an OECD-validated test method.

[0150] The native, still-unbound peptide is detected by reaction with Ellman's reagent and corresponding absorbance measurement. The direct peptide reactivity assay (DPRA) is a chemical method for predicting epidermal protein binding. DPRA uses for example HPLC to measure the depletion of peptides in solution after exposure to test chemicals.

[0151] In the investigations carried out (FIG. 7), DPRA examines the reactivity of the test chemicals eugenol and DNCB with the peptides of the invention (in each case at 0.5 mM) and incubation for up to 24 hours. The measurement employed in the test is the rate of depletion of these peptides and is evaluated by means of a widely used HPLC-UV method.

[0152] RFAACAA, RAACAA, RFACAA, RFACA, and GSH were selected as exemplary representatives of the peptides of the invention, which represent the inventive use over the entire range of the peptides of the invention.

[0153] The reductions in absorbance due to the reaction of the peptide with allergen, eugenol (black), and DNCB (dashed) can be clearly seen in FIGS. 7A to E, which demonstrates the inventive allergen-scavenging function of the peptides of the invention.

[0154] In the DPRA method, the absorbance at 415 nm is determined as the measured variable.

[0155] The allergen concentration is in the investigations carried out a standard 5 mM and thus 10 times the concentration of the peptide used (0.5 mM).

[0156] The reaction courses shown in FIGS. 8 and 9 were determined colorimetrically through the reaction of the peptide with Ellmann's reagent in a photometer. In this reaction, DTNB ((5,5′-dithiobis-2-nitrobenzoic acid, Ellmann's reagent) is cleaved and attached to the SH residue of the amino acid cysteine of the peptides of the invention (Ac-RFAACAA, RFAACAA).

[0157] The SH radical is at the same time the reactive binding site of the peptide with the allergens. The color reaction with DTNB (color change from colorless to yellow) competes with the allergen reaction. This means that the better the allergen reacts with the peptide, the less peptide remains available for the reaction with the detection reagent, i.e. the less the resulting yellow coloration. This measurement at 415 nm is shown in FIG. 9.

[0158] Alternatively, the decrease in the peptide in the allergen reaction can also be determined using a UV detector after HPLC separation, as shown in FIG. 8.

[0159] FIG. 8 shows the decrease (depletion n in %) of the allergen eugenol (5 mM Eu) in the presence of the peptide Ac-RFAACAA (0.5 mM) over time (24 h) compared to the peptide on its own.

[0160] FIG. 9 shows the allergen reduction (eugenol, urushiols) in the direct peptide reaction assay (DPRA) after a 24 h reaction, n=3.

[0161] FIG. 9 shows the fall in peptide against eugenol and against various urushiol derivatives. The control is the peptide in the buffer system without allergen, i.e. instead of allergen only its solvent (ethanol) is added.

[0162] Urushiols of the structure below were used.

TABLE-US-00003 Urushiol I Uruishiol II Urushiol III Urushiol IV Urushiol V [00001] —(CH.sub.2).sub.14CH.sub.3 —(CH.sub.2).sub.7CH═CH(CH.sub.2).sub.5CH.sub.3 —(CH.sub.2).sub.7CH═CHCH.sub.2CH═CH(CH.sub.2).sub.2CH.sub.3 —(CH.sub.2).sub.7CH═CHCH.sub.2CH═CHCH═CHCH.sub.3 —(CH.sub.2).sub.7CH═CHCH.sub.2CH═CHCH.sub.2CH═CH.sub.2

[0163] The investigated urushiols differ in the saturation in the side chain R.

[0164] Urushiols where R=C15 singly (C15:1), doubly (C15:2) or triply (C15:3) unsaturated were investigated.

[0165] The results demonstrate very high reactivity and thus protective activity of the peptides of the invention against urushiols too. The peptides, especially Ac-RFAACAA and RFAACAA, show very high reactivity and thus protective activity against urushiol structures I-V in particular.

[0166] Urushiols are the strongest naturally occurring allergen, which is in addition responsible for many sensitizations, as explained above. Urushiols are the main sensitizing constituent of the sumac plants, such as poison sumac (Toxicodendron quercifolium), including, in particular, in the various Rhus species such as poison ivy (Toxicodendron radicans), poison oak (Toxicodendron diversilobum), and lacquer tree (Rhus verniciflua), and also in Toxicodendron rydbergii, Toxicodendron toxicarium, and Toxicodendron vernix.

[0167] Allergy to urushiols, which requires prior sensitization, can be triggered in approx. 50-75% of all Americans. This contact dermatitis affects 10-50 million Americans every year.

[0168] Contact allergy to urushiols is a serious occupational disease, for example in agricultural and forestry workers and firefighters, but also in all recreation seekers who move around in nature. In the USA they are responsible for 7.1 million doctor visits and 430,000 hospital inpatient stays. For example, this contact allergy is the cause of 10% of all work time lost to injury at US Forest Services, and approximately one-third of forestry workers in California, Oregon, and Washington are unfit for work during the wildfire season because of these reactions.

[0169] The invention now provides a possible remedy here.

[0170] The preparations of the invention comprising the short-chain peptides of the invention result in alleviation of the allergic skin reaction to constituents of poison ivy.

[0171] All investigations impressively demonstrate the allergen barrier effect of the peptides of the invention in preventing or reducing the penetration and/or accumulation of foreign substances and/or allergens on and/or in the skin.

[0172] The numerous investigations of the various peptides demonstrate by way of example the barrier effect and the prevention or attenuation of allergic reactions for all peptides of the invention having a total of 2 to 10 amino acids, wherein at least one amino acid has one or more reactive side chains.

[0173] In addition to the pure demonstrations of efficacy, tests were also carried out with various concentrations of the allergens and peptides.

[0174] The allergens, such as for example DNCB, were tested in the range of 10; 5; 2.5; 0.5; 0.05; 0.005; 0.0005 mM, cinnamaldehyde in the range of 5; 2.5; 0.5; 0.05; 0.0005 mM, and eugenol in the range of 5, 2.5; 0.05; 0.0005 mM. The peptide concentrations were varied in the range of 4, 2, 0.5; 0.25 mM.

[0175] Significant barrier effects were achieved in all content ranges and ratios.

[0176] The peptides of the invention can advantageously be incorporated into typical cosmetic and dermatological preparations, which can take different forms. For instance, examples of preferred preparation forms are a solution, a water-in-oil (W/O) type emulsion or an oil-in-water (0/W) type emulsion, or multiple emulsions, for example a water-in-oil-in-water (W/O/W) type or oil-in-water-in-oil (O/W/O) type, a hydrodispersion or lipodispersion, a gel, a solid stick or even an aerosol.

[0177] The content of one or more peptides of the invention in preparations for topical application is advantageously to be selected within a range from 0.01% to 10% by weight, especially within a range from 0.1% to 5% by weight, more preferably within a range from 0.2% to 5% by weight, based on the total mass of the preparation.

[0178] Preferred application forms of the peptides of the invention are emulsion preparations, gels, and spray formulations.

[0179] Emulsions produced according to the invention, for example in the form of a cream, a lotion or a cosmetic milk, are advantageous and comprise, for example, fats, oils, waxes, and/or other fatty substances, and also water and one or more emulsifiers as are customarily used for formulations of this type.

[0180] The preparations of the invention advantageously comprise one or more film formers.

[0181] In addition, the peptides can be chemically modified to improve the physicochemical properties. For example, derivatization with fatty acid residues (for example acetyl, caproyl, undecenoyl, or palmitoyl) increases the lipophilicity. Secondly, chemical modifications can help make the peptide more stable and thus protect it for example from degradation by peptidases.

[0182] So-called penetration enhancers, solubilizers and physical methods are able to increase the permeability of the skin and also further boost distribution in the skin.

[0183] An addition of solubilizers is therefore advantageous.

[0184] It is of course known to those skilled in the art that sophisticated cosmetic compositions are generally not conceivable without the customary auxiliaries and additives. These include for example consistency enhancers, fillers, dyes, emulsifiers, additional active substances such as vitamins or proteins, light stabilizers, stabilizers, insect repellents, alcohol, water, salts, EDTA, antimicrobial, proteolytic or keratolytic substances, etc., the addition of potential allergens being of course preferably avoided.

[0185] Mutatis mutandis, corresponding requirements apply to the formulation of medicinal preparations.

[0186] Allergy-triggering substances, allergens, are intercepted by the peptides of the invention consisting of 2-10 amino acids, wherein at least one amino acid has one or more reactive side chains. This barrier effect of the peptides, particularly in preparations for topical application, results in a novel form of antiallergic therapy.

EXAMPLE PREPARATIONS

[0187] The numerical values are contents by weight based on the total mass of the preparation.

TABLE-US-00004 1 2 3 4 INCI m [%] m [%] m [%] m [%] Caprylic/Capric 4.00 4.00 4.00 4.00 Triglyceride Cetyl Alcohol 3.00 3.00 3.00 3.00 Aqua + 1.00 1.00 1.00 1.00 Trisodium EDTA BHT 0.05 0.05 0.05 0.05 Phenoxyethanol 0.80 0.80 0.80 0.80 Hydrogenated 2.00 2.00 2.00 2.00 Coco-Glycerides Glycerol 7.50 7.50 7.50 7.50 Aqua + Sodium 0.01 0.01 0.01 0.01 Hydroxide Alcohol Denat. + 4.00 4.00 4.00 4.00 Aqua Xanthan Gum 0.30 0.30 0.30 0.30 Acrylates/C10-30 0.20 0.20 0.20 0.20 Alkyl Acrylate Crosspolymer Glyceryl Stearate 2.00 2.00 2.00 2.00 Citrate Dicaprylyl Ether 4.00 4.00 4.00 4.00 Aqua to 100 to 100 to 100 to 100 Ac-RFAACAA 0.25 — — 0.2  GSH — 1   — — Ac-RFAALAA — — 0.4  0.1