Topical composition

Abstract

The invention relates to a topical composition for maintaining the skin condition in good state or improving the condition of the skin, comprising methyl sulfonyl methane and a peptide with sequence H-DL-Phe-DL-Ala- DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-DL-Leu-DL-Lys-DL-Ala- DL-Leu-DL-Lys-DL-Ala-DL-Leu-NH2.

Description

[0001] TOPICAL COMPOSITION

[0002] ***

[0003] DESCRIPTION

[0004] Field ef the invention

[0005] The present invention relates to a composition for topical use or topical composition, in particular to a composition of the above-mentioned type for maintaining the skin condition in good state or improving the condition of the skin of a mammal, specifically a human being.

[0006] In accordance with the invention, the aforementioned topical composition was found to be particularly effective in the treatment of an inflammatory condition of the skin, whether acute or chronic, and is therefore suitable for use on both injured and uninjured skin for the maintenance, restoration or improvement of the barrier functions of the skin in a therapeutic treatment or in a non-therapeutic cosmetic treatment.

[0007] State of the art

[0008] Inflammation of the skin can be caused by various factors and can manifest in different forms of varying intensity, often characterised by erythema with sensations such as burning, tingling and itching.

[0009] The inflammation may have an irritative or allergic origin (contact dermatitis) in response to environmental factors, such as ultraviolet radiation, cold and humid climates, allergens and irritants due to pollution, for instance, or it may be present in a chronic form, such as for example in rosacea, psoriasis, and atopic dermatitis.

[0010] In any case, in the many forms of skin inflammation there is a significant loss of the barrier functions of the skin.

[0011] Contact dermatitis is an inflammation of the skin caused by direct contact with irritants (irritant contact dermatitis) or allergens (allergic contact dermatitis). Symptoms include itching and sometimes burning pain. Skin changes include erythema, desquamation, skin swelling and sometimes blistering and ulceration, and often loss of the barrier functions of the skin.

[0012] Another significant aspect is the so-called sensitive skin syndrome picture, in which the triggering factor is unspecified but which manifests with high intolerability and the presence of signs such as erythema and discomfort such as tingling, burning and itching. Also in sensitive skin syndrome there is evidence of an increase in certain mediators such as, for example, IL-1 a and loss of the barrier functions of the skin.

[0013] In rosacea, a form of photoaggravated dermatitis, the inflammation mainly affects the face - the cheeks, chin, nose, forehead, and is often characterised by remission and exacerbation, including a range of symptoms such as intermittent erythema, permanent erythema, telangiectasia formation, oedema, papules, pustules and ocular lesions.

[0014] Atopic dermatitis is a chronic recurrent inflammatory skin disease with a complex pathogenesis involving genetic susceptibility, immunological dysfunction, epidermal barrier and environmental factors. Itching is a primary symptom; skin lesions range from mild erythema to severe lichenification to erythrodermia. The diagnosis is given by the medical history and objective examination. Treatments include advice on appropriate skin care, avoidance of triggers, and topical corticosteroids and immunosuppressants. The control of itching and superinfections is also important. Severe cases may require systemic immunosuppressive treatment. Atopic dermatitis in children often disappears or is greatly reduced in adulthood.

[0015] Psoriasis is a chronic, relapsing disease characterised by the appearance of raised, reddened patches with distinct margins, covered with silvery scales that form between patch and normal skin.

[0016] Skin inflammation is characterised by the overproduction of certain chemical mediators, detectable in contact dermatitis, rosacea, atopic dermatitis and to some extent psoriasis.

[0017] A very important role is the effect of free radicals in the skin’s inflammatory process; indeed, reactive oxygen species (either photoinduced or the result of an immunological process) contribute to defining the severity of the skin’s inflammatory picture.

[0018] The specific pathophysiological origin of immunological skin inflammation lies in the induction of well-defined chemical mediators that, in most cases, are over-regulated.

[0019] A broad spectrum of physical (UV, temperature), biological (microbiota, food) and endogenous (genetic, stress) stimuli are ‘triggers’ of most inflammatory states of the skin. So far, pattern recognition receptors such as TLR2, transient receptor potential ion channels (TRPVs), cytokines, chemokines and proteases have been implicated as critical receptors / mediators.

[0020] The main chemical mediators generally over-expressed in the various forms of skin inflammation are: Cytokines (IL-1 a, IL-1 p, IL-6, IL-8, IL-17); TNFa, VEGF, KLK5, KLK7, Peptide LL37, Histamine.

[0021] Among the various dermatitis, a special emphasis has been placed on the aetiopathogenesis of rosacea, a classic inflammatory form of photoaggravated dermatosis. Rosacea is a chronic condition that mainly affects the face - the cheeks, chin, nose, forehead, and is often characterised by remission and exacerbation, including a range of symptoms such as intermittent erythema, permanent erythema, telangiectasia formation, oedema, papules, pustules and ocular lesions.

[0022] Rosacea affects both males and females and normally appears after the age of 30.

[0023] The exact nosology of rosacea has not yet been established. The scientific committee of the United States’ National Rosacea Society has defined 4 subtypes of Rosacea.

[0024] Subtype 1 - erythematotelangiectatic rosacea:

[0025] It is mainly characterised by intermittent or persistent central facial erythema. The appearance of telangiectases i.e. dilations of small blood vessels is common but not essential for a diagnosis of this subtype. Oedema, scaling, roughness, burning and tingling sensation may also be reported. Skin dehydration and changes in the barrier functions of the skin are conspicuous.

[0026] Subtype 2 - papulopustular rosacea:

[0027] This subtype is characterised by the presence of persistent erythema with transient papules and pustules generally but not exclusively localised in the central part of the face. In many respects it can be mistaken for acne vulgaris, except that comedones are absent. Subtype 2 is often associated with subtype 1 .

[0028] Subtype 3 - phymatous rosacea:

[0029] This sub-type of rosacea includes symptoms such as thickening skin, irregular surface nodularities, and enlargement of the nose (rhinophyma), although phymatous rosacea may occur in other locations, including the chin, forehead, cheeks, and ears.

[0030] Subtype 3 also often occurs in combination with subtypes 1 and 2, with the presence of persistent erythema and telangiectasias.

[0031] Subtype 4 - ocular rosacea:

[0032] The diagnosis of ocular rosacea should be considered when a patient’s eyes have one or more of the following signs and symptoms: inter-palpebral and conjunctival hyperaemia, burning, tingling, dryness, light sensitivity, telangiectasias of the conjunctiva and lid margin and periocular erythema. Ocular rosacea is often diagnosed when rosacea symptoms are present.

[0033] Erythematotelanqiectatic rosacea is very often prodromal to evolution into papulopustular rosacea.

[0034] That is why it is important to start the appropriate treatments in phase 1 to control the evolution of the disease.

[0035] Early treatment of rosacea is therefore of paramount importance as it is possible to hinder its course. Rosacea very often worsens when left untreated and the chances of therapeutic success decrease.

[0036] The clinical spectrum of rosacea thus encompasses a huge variability; from erythema (vasodilatation) to papules / pustules (inflammatory infiltrate) to phyma (fibrosis, glandular hyperplasia) with more than plausible interactions between the neurovascular and immune systems.

[0037] The inflammatory cascade affecting the development of rosacea is specifically correlated to the expression of antimicrobial peptides, inflammosome, IL- 1 p, TNF, IFN-y, proteases and neuropeptides.

[0038] Rosacea patients, after activation of TLR2 - Toll-like receptor - (induced e.g. by ultraviolet radiation) show an increase in the amount of cathelicidin and proteolytic enzymes such as KLK5 and KLK7 (kallikrein - serine proteases) capable of hydrolysing cathelicidin (hCAP18) into the antimicrobial peptide LL37.

[0039] Overexpression of kallikreins KLK5 and KLK7 leads to increased proteolytic activity on hCAP18 and subsequent release of LL37.

[0040] Overexpression of peptide LL37, which can be seen in papulopustular rosacea patients, leads to the induction of other chemical mediators, induces a series of pro-inflammatory events including leukocyte chemotaxis, NF-KB activation, and the promotion of angiogenesis. The vascular endothelial growth factor (VEGF), which is expressed by keratinocytes and lymphocytes in rosacea, is also influenced by LL-37 and also contributes to angiogenesis and leukocyte chemotaxis.

[0041] The cathelicidin activation pathway is also related to the activation of inflammasome NALP3 and caspase-1 , together with its downstream target IL-1 (3, which are over-expressed.

[0042] IL-i p activities, although not exclusive to cytokine, may include increased expression of IL-8, tumour necrosis factor (TNF) and cyclooxygenase (COX)-2, all of which are increased in rosacea.

[0043] The pathways associated with cathelicidin and the inflammosome are not mutually exclusive. TLR2 activation is required for NALP3 sensitisation and pro-IL-1 p expression during a microbial insult.

[0044] LL-37 is also able to increase IL-1 p release through inflammasome activation.

[0045] LL-37 and IL-i p are NF-KB responsive, and have demonstrated synergistic inflammatory and angiogenic activity.

[0046] The important role played by IL-17 in the aetiopathogenesis of rosacea has also been demonstrated.

[0047] Altered levels of IL-6 and IL-18, regardless of the clinical type of rosacea, have also been shown to play a role in the development of an inflammatory condition during the course of the disease.

[0048] TRPV1 is a non-selective channel receptor widely expressed in skin tissues, including keratinocytes, peripheral sensory nerve fibres and immune cells. It is activated by a variety of exogenous or endogenous inflammatory mediators, triggering the release of neuropeptides and the neurogenic inflammatory response. TRPV1 receptor activation has been shown to be closely related to the onset and / or development of rosacea and plays a key role especially in erythematotelangiectatic rosacea caused by an overexpression of neurogenic inflammation mediators such as substance P.

[0049] Skin hypersensitivity and the presence and intensity of erythema have been directly linked to neural stimulation in rosacea. The density of sensory neurons is moderately increased in the erythematotelangiectatic (ETR) subtype of rosacea, and an increased density of TRP ion channels on sensory neurons, blood vessels and immune cells has also been reported in all subtypes of cutaneous rosacea.

[0050] Activation of the TRPV1 receptor on sensory nerves causes the release of vasoactive neuropeptides, including substance P and calcitonin gene-related peptide (CGRP), which are elevated in one or more rosacea subtypes. Substance P is likely to be involved in the oedema associated with rosacea, and VEGF induction, hence vasodilation.

[0051] Under normal physiological conditions, unstimulated skin keratinocytes constitutively produce small doses of cytokines. Under specific skin conditions or under the impulse of toxic and stressful stimuli, such as exposure to UV or somewhat aggressive biological and chemical substances, keratinocytes increase the production and release of some of these cytokines, such as lnterleukin-1 a (11-1 a), lnterleukin-1 p (11-1 (3), IL-6, IL-8, IL-17 and Interferon-y (IFN-y). These molecules are soluble mediators involved in the genesis of rosacea.

[0052] Equally important is the release of histamine as a result of neurogenic inflammation.

[0053] Generic therapy of inflammatory processes

[0054] Topical corticosteroids are the background pharmacological therapy in contact dermatitis and other inflammatory skin conditions. The side effects of corticosteroids are an expression of their mechanism of action, but usually occur in the case of prolonged therapy with very powerful steroids. The main and most fearsome is skin atrophy, which can be persistent. It may be diffuse or in the form of striae distensae. In general, the anti-inflammatory potency parallels the atrophogenic potential. However, mometasone furoate combines strong anti-inflammatory activity with a weak atrophogenic potential. Other side effects may include folliculitis, acneiform eruptions, rosaceiform dermatitis, telangiectasias and hypertrichosis. Allergic reactions due to sensitisation to excipients or the active substance are possible, particularly for molecules such as thixocortol pivalate and budesonide followed by hydrocortisone 17- butyrate. The application of topical steroids on the eyelids or periorbital region may cause ocular toxicity with complications such as glaucoma, cataracts or ocular hypertension. The possibility of systemic side effects, although very rare and dependent on the type and dose of topical steroid, should always be taken into account, especially when using more potent drugs on large skin areas. The most significant are Cushing’s syndrome, hypertension, diabetes mellitus, and osteoporosis.

[0055] Additionally , the use of corticosteroids has been shown to impair the ability to restore the barrier functions of the skin, due to their specific effect on horny layer lipids and the ’’brick and mortar” structure of the stratum corneum.

[0056] Therefore, topical corticosteroids, although they have been shown to reduce inflammation through various mechanisms, conversely induce abnormalities in the synthesis of horny layer lipids, which delay the restoration of the skin barrier and may be a source of earlier eczematous flare-ups.

[0057] As far as the treatment of general skin inflammation is concerned, the available tools are mainly related to the use of cortisone drugs, with all the limitations related to their use, especially owing to the fact that the therapy can only be continued for short periods (to avoid thinning of the skin and other side effects as described above).

[0058] The state of the art for counteracting skin irritations is also based on cosmetic treatments with a soothing action, which can have a palliative but not definitive action.

[0059] Therapy of erythema in rosacea

[0060] Brimonidine tartrate is mainly used in erythematotelangiectatic rosacea; this drug acts by binding and activating certain receptors, called alpha2-adrenergic receptors, present on the blood vessel cells of the skin. This causes a narrowing of the blood vessels, which reduces the blood flow to the face, thus decreasing redness. However, the effect of brimonidine tartrate is short-lived (also leading to a rebound effect when the product is discontinued, due to the specific mechanism of action on alpha2-adrenergic receptors) and this is a real limitation for appropriate therapy of erythematotelangiectatic rosacea. One study demonstrated the activity of brimonidine tartrate in reducing facial redness 3 hours after the first application (day 1 ) in 16.3% (21 of 129) of patients treated with brimonidine tartrate compared to 3.1 % (4 of 131 ) treated with placebo. The effect lasted for 12 hours after application, although it began to wear off after 6 hours. On day 29, 31 .5% (40 of 127) of patients exhibited a reduction in facial redness 3 hours after application with brimonidine tartrate compared to a 10.9% (14 of 128) response with placebo.

[0061] In another study, a reduction in facial redness was observed 3 hours after the first application (day 1 ) in 19.6% (29 of 148 patients) of patients treated with brimonidine tartrate, compared to 0% (none of 145 patients) treated with placebo. Again, the positive effect lasted 12 hours after application, although it began to wear off after 6 hours. On day 29, 25.4% (36 of 142) of the patients showed a reduction in facial redness 3 hours after application with brimonidine tartrate, compared to a 9.2% response (13 of 142) with placebo.

[0062] Other treatments, more cosmetic than pharmaceutical, involve the generic use of substances with antioxidant and mildly soothing activity such as bioflavonoids. Other products used involve the use of simple moisturisers capable of counteracting the skin dryness that rosacea patients very often experience.

[0063] Summary of the mvention

[0064] The technical problem underlying the present invention was to provide a composition capable of overcoming one or more of the aforementioned drawbacks with reference to the state of the art.

[0065] According to the invention, the aforementioned problem is solved by a composition for topical use, i.e. intended for application on the skin, hereinafter also identified as topical composition, comprising methyl sulfonyl methane and a peptide with sequence:

[0066] H-DL-Phe-DL-Ala-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu- DL-Leu-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-NH2.

[0067] The aforementioned peptide, of artificial origin, already known for its specific antibacterial properties towards Propionibacterium Acne, hereinafter referred to as RO-peptide-23, has molecular formula (brute formula) C78H140N20O15 and can be identified more specifically at https: / / pubchem.ncbi.nlm.nih.gov / compound / Oligopeptide-10 of the PubChem chemical database at the National Institutes of Health (NIH), 9000 Rockville Pike, Bethesda, Maryland 20892 - US, of which the following main data are provided:

[0068] - PubChem CID 155886002 - PubChem Compound Identification, a non-zero integer PubChem accession identifier for a unique chemical structure.

[0069] - CAS number: 466691 -40-7 - The CAS number is a numerical identifier that uniquely identifies a chemical substance.

[0070] - IUPAC base name:

[0071] H-DL-Phe-DL-Ala-DL-Lys-DL~Ala-DL~Leu-DL-Lys-DL-Ala-DL-Leu- DL-Leu-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-NH2

[0072] Which corresponds to the sequence of the aforementioned peptide as indicated above.

[0073] - IUPAC complete name:

[0074] DL-phenylalanyl-DL-alanyl-DL-lysyl-DL-alanyl-DL-leucyl-DL-lysyl- DL-alanyl-DL-leucyl-DL-leucyl-DL-lysyl-DL-alanyl-DL-leucyl-DL-lysyl-DL- alanyl-DL-leucinamide

[0075] - Sequence: FAKALKALLKALKAL

[0076] - PLN: H-FAKALKALLKALKAL-[NH2]

[0077] - Molecular weight: 1598.1 g / mol

[0078] RO-peptide-23 can therefore be considered an active ingredient of the topical composition according to the present invention which, as mentioned above, also includes methyl sulfonyl methane, hereinafter also referred to as MSM, which may be considered a further active ingredient of this topical composition.

[0079] Methyl sulfonyl methane, IUPAC name dimethyl sulfone, has brute formula C2H6O2S, molecular weight 94.14 g / mol and CAS number: 67-71 - 0.

[0080] The topical composition according to the present invention has been shown to be particularly effective in protecting the skin, in particular in maintaining in good state the condition of the skin or improving the condition of the skin by maintaining, restoring or improving the barrier functions of the skin, whether detectable in the presence of an inflammatory condition of the skin or prodromal thereto.

[0081] The topical composition according to the present invention was found to be particularly effective in the treatment of an inflammatory condition of the skin, whether acute or chronic, and is therefore suitable for use on both injured and uninjured skin for the improvement of the barrier functions of the skin in a therapeutic treatment or in a non- therapeutic cosmetic treatment.

[0082] This topical composition can be conveyed in different chemicalphysical forms for the alleviation, control and therapy of the symptoms of general skin inflammation, for use as non-therapeutic or therapeutic functional aids.

[0083] Bearing the above in mind and remembering that skin and cute are used here as synonyms, it must be said that by preference, the aforementioned topical composition comprises the aforementioned peptide in a concentration between 0.000005% w / w and 1 % w / w, more preferably between 0.00005% w / w and 0.075% w / w, more preferably still between 0.0005% w / w and 0.005% w / w, where “% w / w” indicates the percentage by weight of the compound or component (active ingredient) considered, relative to the total weight of the topical composition.

[0084] The aforementioned topical composition preferably comprises methyl sulfonyl methane in a concentration between 0.005% w / w and 25% w / w, more preferably between 0.05% w / w and 7.5% w / w, more preferably still between 0.075% w / w and 5% w / w.

[0085] Preferably, the aforementioned topical composition comprises at least one of: 4-t-butylcyclohexanol, niacinamide (vitamin PP), silymarin, xylitol, acetylsalicylic acid, salicylic acid and at least one solar filter.

[0086] Preferably, the aforementioned topical composition comprises 4-t- butylcyclohexanol in a concentration between 0.1 % w / w and 7% w / w, more preferably between 0.2% w / w and 5% w / w, more preferably still between 0.35% w / w and 4% w / w.

[0087] Preferably, the aforementioned topical composition comprises niacinamide in a concentration between 0.1 % w / w and 9.5% w / w, more preferably between 0.5% w / w and 7% w / w, more preferably still between 0.75% w / w and 5% w / w.

[0088] Preferably, the aforementioned topical composition comprises silymarin at a concentration between 0.1 % w / w and 5.5% w / w, more preferably between 0.25% w / w and 4% w / w, more preferably still between 0.5% w / w and 3% w / w.

[0089] Preferably, the aforementioned topical composition comprises xylitol at a concentration between 0.1 % w / w and 15.5% w / w, more preferably between 0.25% w / w and 4% w / w, more preferably still between 0.5% w / w and 3% w / w.

[0090] Preferably, the aforementioned topical composition comprises acetylsalicylic acid or salicylic acid at a concentration between 0.1 % w / w and 35% w / w, more preferably between 0.25% w / w and 2% w / w. A concentration of acetylsalicylic acid or salicylic acid between 10% w / w and 30% w / w is particularly preferred for compositions intended for chemoexfoliation.

[0091] Preferably, in the aforementioned topical composition, the abovesaid at least one solar filter is chosen from the group comprising: benzalkonium camphor methosulphate; homosalate; benzophenone-3; phenylbenzimidazole sulfonic acid; terephthaiidenedicanephorsulfonic acid; butylmethoxydibenzoylmethane; benzylidene camphorsulfonic acid; octocrylene; polyacrylamidomethylbenzylidene camphor; ethylhexylmethoxycinnamate; peg-25 paba; isoamyl p-methoxycinnamate; ethylhexyl triazone; drometrizole trisiloxane; diethylhexyl butamido triazone; 4-methylbenzylidene camphor; ethylhexyl salicylate; ethylhexyl dimethyl paba; benzophenone-4, benzophenone-5; methylene bis- benzotriazolyl tetramethylbutylphenol; methylene bis-benzotriazolyl tetramethylbutylphenol (nano); disodium phenyl dibenzimidazole tetrasulfonate; bis-ethylhexyloxyphenol methoxyphenyl triazine; polysilicone-15; titanium dioxide; titanium dioxide (nano); diethylamino hydroxybenzoyl hexyl benzoate; tris-biphenyl triazine / tris-biphenyl triazine (nano); zinc oxide; zinc oxide (nano); phenylene bis-diphenyltriazine; methoxypropylamino cyclohexenylidenethoxyethylcyanoacetate.

[0092] Preferably, in the aforementioned topical composition, always expressed as a percentage by weight of the total weight of the topical composition, the maximum amount of solar filter is:

[0093] 6% for benzalkonium camphor methosulphate; 10% for homosalate;

[0094] 6% for benzophenone-3;

[0095] 8% as acid for phenylbenzimidazole sulfonic acid;

[0096] 10% as acid for terephthalidenedicanephorsulfonic acid;

[0097] 5% for butyl methoxydibenzoylmethane;

[0098] 6% as an acid for benzylidene camphorsulfonic acid;

[0099] 10% as acid for octocrylene;

[0100] 6% for polyacrylamidomethylbenzylidene camphor;

[0101] 10% for ethylhexylmethoxycinnamate;

[0102] 10% for peg-25 paba;

[0103] 10% for isoamyl p-methoxycinnamate;

[0104] 5% for ethylhexyl triazone; 15% for drometrizole trisiloxane;

[0105] 10% for diethylhexyl butamido triazone;

[0106] 4% for 4-methylbenzylidene camphor;

[0107] 5% for ethylhexyl salicylate;

[0108] 8% ethylhexyl dimethyl paba;

[0109] 5% as acid for benzophenone-4, benzophenone-5;

[0110] 10% for methylene bis-benzotriazolyl tetramethylbutylphenol;

[0111] 10% for methylene bis-benzotriazolyl tetramethylbutylphenol (nano);

[0112] 10% as acid for disodium phenyl dibenzimidazole tetrasulfonate;

[0113] 10% for bis-ethyihexyloxyphenol methoxyphenyl triazine;

[0114] 10% for polysillcone- 15;

[0115] 25% for titanium dioxide;

[0116] 25% for titanium dioxide (nano);

[0117] 10% diethylamino hydroxybenzoyl hexyl benzoate;

[0118] 10% for tris-biphenyl triazine / tris-biphenyl triazine (nano);

[0119] 25% for zinc oxide;

[0120] 25% for zinc oxide (nano);

[0121] 5% for phenylene bis-diphenyltriazine;

[0122] 3% for methoxypropylamino cyclohexenylidenethoxyethylcyanoacetate.

[0123] The amount of solar filters that can be used will depend on the maximum limits that can be used in accordance with the various regulations and the desired sun protection factor (SPF). The same applies to screens primarily intended to protect against UVA radiation.

[0124] In accordance with the foregoing and according to the present invention, the aforementioned topical composition may be in the form of an oil-in-water emulsion, water-in-oil emulsion, micellar emulsion, monophasic gel, aqueous or hydro glyceric solution, lotion, ointment, cream, paste, spray. As mentioned above, the topical composition according to the present invention has been shown to be particularly effective in treating an inflammatory condition of the skin, whether acute or chronic.

[0125] Therefore, in accordance with the foregoing, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of an inflammatory condition of the skin characterised by hyper-release of chemical mediators of inflammation.

[0126] In particular, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of rosacea.

[0127] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of atopic dermatitis.

[0128] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of contact dermatitis.

[0129] Furthermore, the present invention makes the aforementioned topical composition available for use as medicament in the treatment of seborrheic dermatitis.

[0130] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of sensitive skin syndrome.

[0131] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament to reduce the effect of ultraviolet radiation.

[0132] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of ultraviolet radiation-induced erythema.

[0133] Furthermore, the present invention makes available the aforementioned topical composition for use as a medicament for the maintenance, restoration or improvement of the barrier functions of the skin.

[0134] Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use for maintaining, restoring or improving the barrier functions of the skin, by exerting a clear soothing effect in all those skin conditions where such action may be useful.

[0135] Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use to reduce the effect of ultraviolet radiation.

[0136] Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use to reduce ultraviolet radiation-induced erythema.

[0137] Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use for the treatment of sensitive skin syndrome.

[0138] Essentially, the present topical composition has also been shown to be effective for cosmetic and non-therapeutic use in the treatment of skin conditions involving redness and / or swelling, particularly to improve its appearance.

[0139] In accordance with the above, the Applicant has surprisingly demonstrated that RO-peptide-23 and methyl sulfonyl methane together in the present topical composition result in a synergistic effect.

[0140] The aforementioned topical composition, for example, acts against ultraviolet radiation-induced erythema in a more pronounced manner when compared with individual components, i.e. when compared with related topical compositions each comprising only one of the aforementioned two active ingredients, as specified in more detail below, and this constitutes a significant effect in photoaggravated dermatoses.

[0141] Furthermore, the Applicant has shown that the components or active ingredients of the aforementioned topical composition, although acting with different mechanisms of action, contribute in synchrony to the modulation of the chemical mediators of inflammation, thus the topical composition according to the present invention is connoted by an overall action in the control of skin inflammation, either at a therapeutic level or at a non-therapeutic, cosmetic level.

[0142] The Applicant has also surprisingly demonstrated that the aforementioned topical composition has superior effects and toxicological advantages when compared to other therapeutic means used to control skin inflammation, such as corticosteroids, which disadvantageously delay or worsen the barrier functions of the skin, whereas the present topical composition has been shown to significantly improve the barrier functions of the skin. The epidermal barrier, when in good condition, is able to control the development of atopic dermatitis and nearly all inflammatory conditions affecting the skin, including rosacea.

[0143] The Applicant has further demonstrated that the aforementioned topical composition is able to effectively improve erythema in rosacea, decreasing its intensity, and simultaneously decreasing transderma! water loss (improvement of the barrier functions of the skin), in a more intense and long-lasting manner, especially when compared to drugs used in prior art in the control of erythema in rosacea, such as brimonidine tartrate.

[0144] The Applicant has also surprisingly ascertained that in erythematotelangiectatic rosacea, after twenty-eight days of treatment with a topical composition according to the present invention in emulsion form, ten weeks after the last treatment, the erythema was significantly less, compared to both time to (before treatment) and after twenty-eight days of use of said emulsion (tss), as illustrated more in detail below in the relevant clinical evaluation. This takes on absolute value when one considers that brimonidine tartrate, although considered the drug of choice in the treatment of erythema in rosacea, is disadvantageously characterised by a temporary effect and, an even more limiting factor, by an evident rebound effect, i.e. worsening of the erythema when therapy ceases.

[0145] In accordance with the above, the Applicant has ascertained that each of the components or active ingredients of the aforementioned topical composition exerts specific effects by modulating the release of the chemical mediators underlying an inflammatory process such as:

[0146] 1 . Controlling the inflammatory process induced by the cascade of reactions that, after TLR2 activation, leads to overexpression of KLK5 and KLK7 and, consequently, of antimicrobial peptide LL37.

[0147] 2. Controlling erythema as a neurogenic inflammation response after activation of TRPV1 receptors and subsequent release of substance P.

[0148] 3. Downstream control of VEGF angiokine release.

[0149] 4. Hindering the release of histamine as a consequence of basophil / mastocyte degradation.

[0150] 5. Exerting a marked antioxidant effect capable of counteracting the release of reactive oxygen species that may sustain and increase the inflammatory process.

[0151] 6. Exerting a specific protective effect on damage induced by ultraviolet radiation.

[0152] The topical composition in accordance with the present invention has thus been shown to be effective in controlling signs and symptoms of skin suffering, for example, from erythematotelangiectatic rosacea and general inflammatory states of the skin, whether present in an acute form or in a chronic form, for example, but not limited to, skin ailed by atopic dermatitis.

[0153] The use of this topical composition in accordance with the above has yielded clear results in controlling the erythema and discomfort such as tingling and burning sensations, as well as redness or swelling, that often attend irritative or allergic dermatitis, rosacea and other inflammatory states of the skin characterised by loss of its barrier functions.

[0154] The aforementioned topical composition has proved capable of acting on the evolution of the inflammatory picture, allowing clear and, moreover, sustainable results to be obtained over time, i.e. results consolidated over time, for example in the case of treatment of erythematotelangiectatic rosacea, and not having all the limitations associated with the use of corticosteroids in the case of treatment of general skin inflammation, probably due to the synchronous mechanism of action of the aforementioned two active ingredients of the topical composition.

[0155] Indeed, if on the one hand RO-peptide-23 is able to reduce the expression of serine proteases KLK5 and KLK7, peptide LL37, induction of the NFkB factor, Substance P, cytokines IL-1 p, IL-6, IL-8, IL17, on the other hand methyl sulfonyl methane has been shown to have a marked antioxidant effect, to decrease IL-1 a expression and, at the same time, to stimulate the release of receptor antagonist IL-1 oRA, to hinder the release of histamine caused by the degradation of basophils / mastocytes, to decrease the release of VEGF (not only causing neo-angiogenesls but also vasodilation), to have a vasoprotective effect and to protect both keratinocytes and fibroblasts from the toxic action of ultraviolet radiation.

[0156] These complementary mechanisms of action justify the use of the topical composition according to the present invention for the control of skin inflammation, acting synergistically in all situations where the receptors described above play a specific role in the evolution of this skin condition (e.g. in irritative and allergic dermatitis, rosacea, atopic dermatitis, photoaggravated inflammation).

[0157] As a demonstration of the above, the effect of the anti-erythema activity of the topical composition according to the present invention (composition 3) versus the individual components RO-peptide-23 and MSM - i.e. versus respective compositions each containing only one of the two aforementioned active ingredients (composition 1 and composition 2), in which skin erythema was induced by controlled exposure to ultraviolet radiation - was quantified.

[0158] The percentage reduction in erythema for each individual compound or active ingredient, i.e. for the aforementioned compositions 1 and 2, and for the topical composition according to the present invention comprising the two active ingredients (composition 3) was

[0159] - 1.12% for RO-peptide-23 - composition 1

[0160] - 3.12% for MSM - composition 2

[0161] - 4.91 % for topical composition 3 containing RO-peptide-23 and MSM The percentage reduction in erythema by the sum of the anti-erythema activity of each individual compound was

[0162] - 4.24% for single RO-peptide-23 (composition 1 ) + single MSM (composition 2)

[0163] The delta in percentage values of the topical composition comprising the two active ingredients versus the sum of the anti-erythema activity of each individual compound was 0.67%.

[0164] It was found that the combination of RO-peptide-23 and methyl sulfonyl methane, even when used at relatively low concentrations, is able to exert a synergistic effect in controlling ultraviolet radiation-induced erythema. Specifically against ultraviolet radiation-induced erythema, the anti-erythema activity of the topical composition according to the present invention comprising the two components is greater than the anti-erythema activity of the sum of percentage erythema inhibition of each individual component, i.e. the sum of the results achieved by compositions containing only one active ingredient: +0.67% in absolute value and +15.5% in activity-enhancing value - 4.24% vs -4.91 %. Ultimately, in the topical composition according to the present invention, each of the two components acts with its own mechanism that is different from the mechanism of action of the other component, wherein the different mechanisms of action are all intended to control skin inflammation.

[0165] Brief description of the figures

[0166] Further features and advantages of the present invention will be better evidenced by an examination of the following detailed description of certain preferred, but not exclusive, embodiments, illustrated for indicative and non-limiting purposes, with the support of the attached drawings, in which

[0167] - Figure 1 shows a graph of the % reduction in ultraviolet radiation- induced erythema evaluated by reflectance spectrophotometry at 60 hours, for three topical compositions in emulsion form comprising RO- peptide-23 (composition 1 ), MSM (composition 2) and RO-peptide-23 + MAS (composition 3);

[0168] - Figure 2 illustrates a graph of the % reduction in ultraviolet radiation-induced erythema, according to the data in the graph in Figure 1 , for topical composition 3 and for the sum of the anti-erythema activity of each individual compound (composition 1 + composition 2), as well as the delta in percentage values of the topical composition comprising the two compounds versus the sum of the anti-erythema activity of the compositions each comprising a single active ingredient;

[0169] - Figure 3 shows a graph of the photoprotective capacity exerted by MSM against UVB radiation on human keratinocytes;

[0170] - Figures 4 and 5 are fluorescence photomicrographs illustrating the photoprotective capacity exerted by MSM against UVB radiation on human keratinocytes, and show a negative control before irradiation (left) and after irradiation (right), and a sample protected with MSM before irradiation (left) and after irradiation (right), respectively;

[0171] - Figure 6 shows a graph of the photoprotective capacity exerted by MSM against UVB radiation on human fibroblasts;

[0172] - Figures 7 and 8 are fluorescence photomicrographs illustrating the photoprotective capacity exerted by MSM against UVB radiation on human fibroblasts, and show a negative control before irradiation (left) and after irradiation (right), and a sample protected with MSM before irradiation (left) and after irradiation (right), respectively;

[0173] - Figure 9 shows images acquired by means of the Antera 3D apparatus of some patients suffering from rosacea, each image illustrating the respective patient before (left) and after (right) treatment with a topical composition according to the present invention, in emulsion form, containing RO-peptide-23 and MSM, for a period of sixteen days with a single application every two days, for a total of eight applications;

[0174] - Figure 10 shows three photographs of a patient suffering from erythema treated with a topical composition according to the present invention in emulsion form comprising RO-peptide-23 and MSM, in which photograph 1 is at time to (start of treatment), photograph 2 is at time tss (28 days after the start of treatment) and photograph 3 is ten weeks after treatment was discontinued.

[0175] Detailed description of the invention

[0176] A topical composition according to the present invention comprises methyl sulfonyl methane and a peptide with sequence, in accordance with IUPAC base name:

[0177] H-DL-Phe-DL-Ala-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-DL- Leu-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-NH2, and in accordance with complete IUPAC name: DL-phenylalanyl-DL-alanyl-DL-lysyl-DL-alanyl-DL-leucyl-DL-lysyl-DL-alanyl- DL-leucyl-DL-leucyl-DL-lysyl-DL-alanyl-DL-leucyl-DL-lysyl-DL-alanyl-DL- leucinamide.

[0178] The aforementioned topical composition, intended for application to the skin, is also referred to as a composition for topical use. The aforementioned topical composition therefore includes methyl sulfonyl methane and the aforementioned peptide as active ingredients, also identified here as compounds or components.

[0179] In detail, methyl sulfonyl methane is also identified here as MSM, while the aforementioned peptide is also identified here as RO-peptide-23.

[0180] In accordance with the invention, the present topical composition was found to be particularly effective in the treatment of an inflammatory condition of the skin, presumably due to the different mechanisms of action of the two compounds, which contribute in synchrony to the modulation of the chemical mediators of inflammation, thus the present topical composition is connoted by an overall action in the control of skin inflammation, either at a therapeutic level or at a non-therapeutic, cosmetic level.

[0181] Indeed, the topical composition according to the present invention has been shown to be particularly effective in protecting the skin, in particular in maintaining in good state the condition of the skin or improving the condition of the skin by maintaining, restoring or improving the barrier functions of the skin, whether detectable in the presence of an inflammatory condition of the skin or prodromal thereto.

[0182] The present topical composition may therefore be conveyed in different chemical / physical forms for the alleviation, control and treatment of the symptoms of general skin inflammation, to be used as functional (non-therapeutic) or therapeutic aids, thus as cosmetics or medical devices based on the present topical composition, in particular in the form of oil-in-water emulsion, water-in-oil emulsion, micellar emulsion, monophasic gel, aqueous or hydro glyceric solution, lotion, ointment, cream, paste, spray.

[0183] In accordance with the foregoing, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of an inflammatory condition of the skin characterised by hyper-release of chemical mediators of inflammation. Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of rosacea.

[0184] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of atopic dermatitis.

[0185] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of contact dermatitis.

[0186] Furthermore, the present invention makes the aforementioned topical composition available for use as medicament in the treatment of seborrheic dermatitis.

[0187] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of sensitive skin syndrome.

[0188] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament to reduce the effect of ultraviolet radiation.

[0189] Furthermore, the present invention makes the aforementioned topical composition available for use as a medicament in the treatment of ultraviolet radiation-induced erythema.

[0190] Furthermore, the present invention makes available the aforementioned topical composition for use as a medicament for the maintenance, restoration or improvement of the barrier functions of the skin.

[0191] Furthermore, the present invention makes available the aforementioned topical composition for cosmetic and non-therapeutic use for the maintenance, restoration or improvement of the barrier functions of the skin.

[0192] Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use to reduce the harmful effect of ultraviolet radiation. Furthermore, the present invention makes the aforementioned topical composition available for cosmetic and non-therapeutic use for the treatment of sensitive skin syndrome.

[0193] Essentially, the present topical composition has been shown to be effective for cosmetic and non-therapeutic use in the treatment of skin conditions also involving redness and / or swelling, particularly to improve its appearance.

[0194] Preferably, the topical composition according to the present invention comprises RO-peptide-23 in a concentration between 0.000005% w / w and 1 % w / w, more preferably between 0.00005% w / w and 0.075% w / w, more preferably still between 0.0005% w / w and 0.005% w / w, where “% w / w" indicates the percentage by weight of the aforementioned peptide, and more in general of the compound considered, relative to the total weight of the topical composition.

[0195] Preferably, the topical composition according to the present invention comprises methyl sulfonyl methane in a concentration between 0.005% w / w and 25% w / w, more preferably between 0.05% w / w and 7.5% w / w, more preferably still between 0.075% w / w and 5% w / w.

[0196] Preferably, the topical composition according to the present invention comprises at least one of: 4-t-butylcyclohexanol, niacinamide (vitamin PP), silymarin, xylitol, acetylsalicylic acid, salicylic acid and at least one solar filter.

[0197] Preferably, the topical composition according to the present invention comprises 4-t-butylcyclohexanol in a concentration between 0.1 % w / w and 7% w / w, more preferably between 0.2% w / w and 5% w / w, more preferably still between 0.35% w / w and 4% w / w.

[0198] Preferably, the topical composition according to the present invention comprises niacinamide in a concentration between 0.1 % w / w and 9.5% w / w, more preferably between 0.5% w / w and 7% w / w, more preferably still between 0.75% w / w and 5% w / w.

[0199] Preferably, the topical composition according to the present invention comprises silymarin at a concentration between 0.1 % w / w and 5.5% w / w, more preferably between 0.25% w / w and 4% w / w, more preferably still between 0.5% w / w and 3% w / w.

[0200] Preferably, the topical composition according to the present invention comprises xylitol at a concentration between 0.1 % w / w and 15.5% w / w, more preferably between 0.25% w / w and 4% w / w, more preferably still between 0.5% w / w and 3% w / w.

[0201] Preferably, the topical composition according to the present invention comprises acetylsalicylic acid or salicylic acid at a concentration between 0.1 % w / w and 35% w / w, more preferably between 0.25% w / w and 2% w / w. A concentration of acetylsalicylic acid or salicylic acid between 10% w / w and 30% w / w is particularly preferred for topical compositions for cosmetic use or as a medical device, intended for chemoexfoliation.

[0202] Preferably, in the topical composition according to the present invention, the abovesaid at least one solar filter is chosen from the group comprising: benzalkonium camphor methosulphate; homosalate; benzophenone-3; phenylbenzimidazole sulfonic acid; terephthalidenedicanephorsulfonic acid; butylmethoxydibenzoylmethane; benzylidene camphorsulfonic acid; octocrylene; polyacrylamidomethylbenzylidene camphor; ethylhexylmethoxycinnamate; peg- 25 paba; isoamyl p-methoxycinnamate; ethylhexyl triazone; drometrizole trisiloxane; diethylhexyl butamido triazone; 4-methylbenzylidene camphor; ethylhexyl salicylate; ethylhexyl dimethyl paba; benzophenone-4, benzophenone-5; methylene bis-benzotriazolyl tetramethylbutylphenol; methylene bis-benzotriazolyl tetramethylbutylphenol (nano); disodium phenyl dibenzimidazole tetrasulfonate; bis-ethylhexyloxyphenol methoxyphenyl triazine; polysilicone-15; titanium dioxide; titanium dioxide (nano); diethylamino hydroxybenzoyl hexyl benzoate; tris-biphenyl triazine / tris-biphenyl triazine (nano); zinc oxide; zinc oxide (nano); phenylene bis-diphenyltriazine; methoxypropylamino cyclohexenylidenethoxyethylcyanoacetate.

[0203] In accordance with the invention, the Applicant has surprisingly demonstrated that RO-peptide-23 and methyl sulfonyl methane together in the present topical composition result in a synergistic effect. The aforementioned topical composition, for example, acts against ultraviolet radiation-induced erythema in a more pronounced manner when compared with individual components, i.e. relative compositions, each comprising only one active ingredient, as described in detail below, and this constitutes a significant effect in photoaggravated dermatoses.

[0204] Effect of the anti-erythema activity of the topical composition according to the invention versus compositions each containing one active ingredient only

[0205] A well-established and widely used method was used to quantify the synergistic anti-inflammatory effect of the two components or active ingredients of the topical composition according to the present invention in skin inflammation artificially caused by controlled exposure to ultraviolet radiation.

[0206] The topical composition according to the invention, as well as the compositions containing one active ingredient only were used in the form of emulsions at the following concentrations:

[0207] RO-peptide-23 used at a concentration of 0.000025% (w / w) ~ Composition 1 ;

[0208] MSM used at a concentration of 0.25% (w / w) - Composition 2;

[0209] Topical composition comprising RO-peptide-23 and MSM used at respective concentrations of 0.00025% and 0.25% (w / w) - composition 3 - was used on twelve healthy volunteers.

[0210] The skin erythema was induced using an ultraviolet lamp Mod. UVM- 57 (UVP, San Gabriel, CA) capable of emitting radiation in the 290-320 nm range with a peak at 302 nm. The minimal erythema dose (MED) was first determined for each subject, and then six skin sites of 1 cm2were identified and marked on the central part of each forearm, which were irradiated, to cause erythema, with exposure times equal to twice the subject’s MED.

[0211] After UVB irradiation, two sites were used as controls and thus untreated and the remaining sites were treated in duplicate with 100 mg of each of the above compositions 1 , 2, and 3. These compositions were applied to the skin sites using special chambers (Hill Top Chambers -Hill Top, Cincinnati, OH) for a period of three hours.

[0212] After the treatment period had elapsed (three hours), after removal of the Hill Top Chambers each site was washed with water to remove the residues of the compositions and left to rest for 30 minutes.

[0213] For each site, the erythema was evaluated at 60 hours with an X- Rite reflectance spectrophotometer mod.968. The instrument was calibrated to a blank standard in accordance with the National Bureau of Standards using a C light source and an observation angle of 2°.

[0214] The percentage reduction in erythema for each individual compound or active ingredient, i.e. for the aforementioned compositions 1 and 2, and for the topical composition according to the present invention comprising the two active ingredients (composition 3) was as shown in Figure 1 :

[0215] - 1 .12% for RO-peptide-23 - composition 1 .

[0216] - 3.12% for MSM -■ composition 2.

[0217] - 4.91 % for topical composition 3 containing RO-peptide-23 and MSM.

[0218] With reference to Figure 2, the percentage reduction in erythema by the sum of the anti-erythema activity of each individual compound was: - 4.24% for single RO-peptide-23 (composition 1 ) + single MSM (composition 2).

[0219] The delta in percentage values of the topical composition comprising the two active ingredients (composition 3) versus the sum of the antierythema activity of each individual compound was 0.67%.

[0220] According to the method performed, it was found that the combination of RO-peptide-23 and methyl sulfonyl methane, even when used at relatively low concentrations as described above, is able to exert a synergistic effect in controlling ultraviolet radiation-induced erythema in accordance with the method described above. Specifically against ultraviolet radiation-induced erythema, the anti-erythema activity of the topical composition according to the present invention comprising the two components is greater than the anti-erythema activity of the sum of percentage erythema inhibition of each individual component, i.e. the sum of the results of the compositions containing only one active ingredient: +0.67% in absolute value and +15.5% in activity-enhancing value - 4.24% vs -4.91 %.

[0221] Ultimately, it can be said that, most likely, in the topical composition according to the present invention, each of the two components acts with its own mechanism that is different from the mechanism of action of the other component, wherein the different mechanisms of action are all intended to control skin inflammation.

[0222] The Applicant has also surprisingly demonstrated that the topical composition according to the present invention has superior effects and toxicological advantages when compared to other therapeutic means used to control skin inflammation, such as corticosteroids, which disadvantageous^ delay or worsen the barrier functions of the skin, whereas the present topical composition has been shown to significantly improve the barrier functions of the skin. The epidermal barrier, when in good condition, is able to control the development of atopic dermatitis and nearly all inflammatory conditions affecting the skin, including rosacea.

[0223] The Applicant has further demonstrated that the topical composition according to the present invention is able to effectively improve erythema in rosacea, decreasing its intensity, and simultaneously decreasing transdermal water loss (improvement of the barrier functions of the skin), in a more intense and long-lasting manner, especially when compared to drugs used in prior art in the control of erythema in rosacea, such as brimonidine tartrate.

[0224] EFFECT OF RO-PEPTIDE-23 ON CHEMICAL MEDIATORS OVEREXPRESSED IN ROSACEA AND OTHER INFLAMMATORY DISEASES SUCH AS ATOPIC DERMATITIS

[0225] Surprisingly, RO-peptide-23 was shown to significantly affect the expression of KLK5, KLK7, LL37, NFkB, 11-1 p, IL-6, IL-8, IL-17, substance P.

[0226] - Expression of cathelicidins KLK5 and KLK7

[0227] The aim of the test is to highlight and quantify changes in gene expression of the levels of KLK5 and KLK7 (enzymes kallikrein 5 and 7 coding) in cell cultures exposed to treatment with vitamin D3 (1 a, 25- dihydroxy vitamin D3 - 'VD3') and RO-peptide-23 compared to controls of the same untreated cells. These peptides are expressed in keratinocytes and their levels have been found to be high in the skin of patients with rosacea.

[0228] In this test, the cells are pre-treated with the test substance for 24 hours and then with VD3 (vitamin D3) for a further 24 hours, to induce the mediators involved in the inflammatory processes of rosacea. Analyses are conducted by RT-qPCR, a highly sensitive method for detecting and quantifying the test protein-coding messenger RNA, hence the corresponding gene activation. RT-PCR measures the fluorescence emissions by the amplicons produced during each PGR cycle.

[0229] The cells were seeded in 24-well plates and left to grow for 24 hours. After that, they were treated for 24 h with fresh culture medium containing the sample diluted in water (RO-peptide-23) in order to achieve two different final dilutions: 0.005 and 0.001 mg / ml.

[0230] Each sample was tested in triplicate. After 48 hours of exposure (24h sample + 24h VD3), total RNA was extracted from the cells and resuspended in 50 pl sterile water, and its concentration was determined spectrophotometrically.

[0231] 150 ng of total RNA was reverse-transcribed into cDNA using random primers with the following protocol: 42cC for 10 minutes, 37°C for 2 hours, 85°C for 5 minutes.

[0232] Independent wells were used for viability analysis by MTT test.

[0233] At the end of the incubation, cell viability was evaluated by incubating the cells for 2 hours in an MTT solution. The solution was then removed and replaced with isopropanol or DMSO for formazan extraction. The plate was stirred to form a homogeneous solution and the absorbance at 570 nm was read with a spectrophotometer (Tecan Infinite F200) equipped with a plate reader, subtracting the background reading at 650 nm.

[0234] Changes in the gene expression profile were analysed by Real Time PCR using TaqMan assays. Commercially available, ad-hoc TaqMan primers and probe for KLK5 and KLK7 were purchased from Applied Biosystems (Thermo Scientific).

[0235] The TaqMan assay employs a sequence-specific, fluorescent oligonucleotide probe, as well as sequence-specific primers.

[0236] This assay exploits the 5’ -> 3’ exonuclease activity of Taq polymerase. The probe contains a fluorescent reporter at the 5' end and a quencher at the 3' end. When intact, the reporter fluorescence is switched off due to its proximity to the quencher. During the annealing / extension phase of the amplification reaction, the probe binds to the target sequence and the dsDNA-specific 5’ -> 3’ exonuclease activity of the polymerase cuts the reporter. As a result, the reporter is separated from the quencher and the resulting fluorescence signal is proportional to the amount of amplified product in the sample.

[0237] - Results The RO peptide-23 sample, at a concentration of 5pg and 1 pg / ml, was shown to inhibit the release of KLK5 and KLK7 proteases. The values below describe this ability of RO peptide-23 to modulate the release of said proteases. - Conclusions

[0238] RO-peptide-23 is able to reduce the expression of KLK5 and KLK7, prodromal to the ability of RO-peptide-23 to reduce LL37 peptide expression.

[0239] Expression of antimicrobial peptide LL37

[0240] The aim of the test is to highlight and quantify changes in gene expression of the levels of CAMP (peptide LL-37 coding) in cell cultures exposed to treatment with vitamin D3 (1 a, 25-dihydroxy vitamin D3 - “VD3”) and the test substance compared to controls of the same untreated cells. These peptides are expressed in keratinocytes and their levels have been found to be high in the skin of patients with rosacea.

[0241] In this test, the cells are pre-treated with the test substance for 24 hours and then with VD3 for a further 24 hours to induce the mediators involved in the inflammatory processes of rosacea. Analyses are conducted by RT-qPCR, a highly sensitive method for detecting and quantifying the test protein-coding messenger RNA, hence the corresponding gene activation. RT-PCR measures the fluorescence emissions by the amplicons produced during each PCR cycle.

[0242] The cells were seeded in 24-well plates and left to grow for 24 hours. After that, they were treated for 24 h with fresh culture medium containing the sample diluted in water (RO-peptide-23) in order to achieve a dilution of 0.005 mg / ml.

[0243] Each sample was tested in triplicate. After 48 hours of exposure (24h sample + 24h VD3), total RNA was extracted from the cells and resuspended in 50 pl sterile water, and its concentration was determined spectrophotometrically.

[0244] 150 ng of total RNA was reverse-transcribed into cDNA using random primers with the following protocol: 42°C for 10 minutes, 37°C for 2 hours, 85°C for 5 minutes.

[0245] Independent wells were used for viability analysis by MTT test. Changes in the gene expression profile were analysed by Real Time PCR using TaqMan assays. Commercially available, ad-hoc TaqMan primers and probe for CAMP were purchased from Applied Biosystems (Thermo Scientific).

[0246] The TaqMan assay employs a sequence-specific, fluorescent oligonucleotide probe, as well as sequence-specific primers.

[0247] This assay exploits the 5’ -> 3’ exonuclease activity of Taq polymerase. The probe contains a fluorescent reporter at the 5' end and a quencher at the 3' end. When intact, the reporter fluorescence is switched off due to its proximity to the quencher. During the annealing / extension phase of the amplification reaction, the probe binds to the target sequence and the dsDNA-specific 5’ -> 3’ exonuclease activity of the polymerase cuts the reporter. As a result, the reporter is separated from the quencher and the resulting fluorescence signal is proportional to the amount of amplified product in the sample.

[0248] - Results

[0249] RO-peptide-23, at a concentration of 5pg / ml, resulted in a significant reduction of LL37 peptide expression by approximately 18%. by reducing peptide LL37 expression, RO-peptide-23 hinders the inflammatory process induced by it. " RO-peptide-23: evaluation of NFkB factor expression

[0250] The aim of the test is to highlight and quantify changes in gene expression of NF-kB levels in human keratinocytes.

[0251] Under the impulse of toxic and stressful stimuli, such as exposure to bacterial lipopolysaccharide (LPS), keratinocytes respond by activating NF-kb, a transcription factor involved in the regulation of the immune response and in inflammation.

[0252] The increased transcription of this factor in cells after exposure to LPS leads to triggering a cascade of pro-inflammatory events, and can therefore be considered a marker of pro-inflammatory potential.

[0253] The analysis is conducted by RT-qPCR, a highly sensitive method for detecting and quantifying messenger RNA coding for the test protein, hence the corresponding gene activation. RT-PCR measures the fluorescence emissions by the amplicons produced during each PGR cycle.

[0254] The cell model used is stabilised human keratinocytes (HaCaT cells).

[0255] For the test, the sample was dissolved directly in DMEM low glucose culture medium and 10% FBS with added antibiotics at final concentrations of 0.0004 mg / ml and 0.0002 mg / ml.

[0256] The cells were seeded in 12-well plates at a concentration of 40000 cells / well in cell growth medium (Dulbecco’s Modified Eagle Medium, DMEM low glucose) and 10% FBS (Fetal Bovine Serum) and incubated for 24 hours at 37°C and 5% CO2. Then the culture medium was replaced with that containing the test sample at two different concentrations and the inflammatory stimulus (LPS 10pg / ml). Cells exposed to LPS alone were used as a positive control, while untreated cells were used as a blank. Unstressed, sample-treated cells were used as an internal control. The test was performed in duplicate. After 24 hours of exposure, total RNA was purified from the cells using TRIZOL reagent. Untreated cells were used as a negative control, while cells treated with the sample only were used as a cytotoxicity control.

[0257] Total RNA was extracted using a guanidine thiocyanate reagent (TRIZOL-INVITROGEN) following the instruction protocol. After precipitation and centrifugation (30’ at 12000 rpm at 4°C), the RNA was resuspended in 20 pl sterile water and its concentration was determined spectrophotometrically.

[0258] 300ng of total RNA was reverse-transcribed into cDNA using random primers at 37°C for 2 hours in a thermal cycler (Applied Byosistems, Foster City, CA).

[0259] Changes in the gene expression profile were analysed by real time PCR using SYBR green as fluorogenic molecule. Two sequences of primers for NF-kB were designed to pair in the intron exon boundary junction. The uniqueness of the gene pairing and thus amplification was verified thanks to the GENEBANK database. The signal emitted by SYBR Green increases in direct proportion with the amount of amplicon produced during the PCR reaction. By measuring the fluorescence emitted at each cycle, it is possible to monitor the PCR reaction during the exponential phase, i.e. where the first significant increases in the PCR product correlate with the initial amount of template.

[0260] At the beginning of PCR cycles, there is only a small change in the fluorescence signal; this period defines the baseline in the amplification curve. An increase in fluorescence above baseline indicates the detection of accumulating PCR products. Therefore, a fluorescence value called threshold is set, just above the baseline. The CT (cycle threshold) is defined as the number of cycles required to exceed the fluorescence threshold, (e.g. exceed the baseline level 10 times). The CT is inversely proportional to the amount of initial target nucleic acid present in the sample. High initial target levels mean less time in detecting fluorescence levels in the PCR, hence low CT. The CT value is very reproducible because the threshold value is taken during the exponential phase of PCR, where there is a linear relationship between the logarithm of fluorescence changes and the number of amplification cycles.

[0261] It can be observed that compared to the untreated control, in which protein expression levels are set arbitrarily equal to 1 , there is an increase in NF-kb expression in the presence of LPS.

[0262] The sample is able to reduce LPS-induced NF-kb transcription in a dose-response manner after 24 hours of exposure.

[0263] " RO-peptide-23: induction of neuropeptide Substance P

[0264] The study concerns the in vitro evaluation of the neuropeptide Substance P-coding TAC1 gene expression, which plays a role in vascularisation and inflammation processes.

[0265] In the test performed here, gene expression of TAC1 by skin cells is increased by exposure to capsaicin, and the ability of the tested substance to reduce this increase in gene expression is verified.

[0266] The study was carried out using an in-vitro reconstituted epidermis model in the presence of the test substance versus samples of untreated epidermis and epidermis treated with capsaicin alone.

[0267] The cell model used for the in-vitro tests is a three-dimensional system of in-vitro reconstituted human epidermis. This three-dimensional cell model consists of normal human primary keratinocytes, allowed to grow in vitro to simulate the multilayered architecture of the human epidermis, with a well-differentiated stratum corneum. The model was purchased from MatteK.

[0268] For the test, the initial induction of TAC1 gene expression was provoked by pre-treatment of the epidermis for 1 h30’ with 1 mM capsaicin.

[0269] The units were then washed and treated with the samples and exposure continued for 24 hours in a humidified incubator at 37cC, 5%CO2.

[0270] As a negative control the epidermis was treated with phosphate buffered saline (PBS), units were treated with capsaicin 1 mM only as a positive control. Units treated with the sample alone were used as an additional control. The test was conducted in duplicate.

[0271] At the end of the exposure, the samples were washed in PBS and RNA was extracted from the tissue.

[0272] After extraction, total RNA was quantified spectrophotometrically and was reverse-transcribed into cDNA before being analysed by real time PCR.

[0273] Changes in the gene expression profile were analysed by real time PCR using TaqMan assays. Commercially available, ad-hoc TaqMan primers and probe for TAC1 were purchased from Applied Biosystems (Thermo Scientific).

[0274] The TaqMan assay employs a sequence-specific, fluorescent oligonucleotide probe, as well as sequence-specific primers.

[0275] This assay exploits the 5’ --> 3’ exonuclease activity of Taq polymerase. The probe contains a fluorescent reporter at the 5' end and a quencher at the 3’ end. When intact, the reporter fluorescence is switched off due to its proximity to the quencher. During the annealing / extension phase of the amplification reaction, the probe binds to the target sequence and the dsDNA-specific 5’ --> 3’ exonuclease activity of the polymerase cuts the reporter. As a result, the reporter is separated from the quencher and the resulting fluorescence signal is proportional to the amount of amplified product in the sample.

[0276] Changes in the gene expression profile were measured using the comparative CT method (AACT method). Data were normalised against the expression levels of p- actin, as a control gene. For the quantitative analysis, the difference between the target CT value (TAC1 ) and that of the control gene (p-actin) was calculated, resulting in the ACT value.

[0277] ACT = CT (target) - CT (control)

[0278] This value was calculated for each sample to be quantified.

[0279] The untreated sample (calibrator) was used as a reference value for all the comparisons that were made.

[0280] AACT was calculated by the difference between the ACT of each sample and the ACT of the untreated control (calibrator)

[0281] AACT = ACT sample 1 - AACT untreated control (calibrator) AACT was then used to determine the Fold Change values:

[0282] Fold change = 2 '

[0283] - Results RO-peptide-23 was shown to be able to significantly reduce Substance P release under capsaicin-mediated stimulation (TRPV1 receptor agonist) by 49.71 % compared to the positive control (capsaicin alone), thus describing its direct action in controlling neurogenic inflammation.

[0284] ” RO-oeptide-23 - cytokine induction

[0285] The cell model used for the in-vitro tests is a three-dimensional system of in-vitro reconstituted human epidermis.

[0286] The sample was tested in 0.00025% water solution (w / v). Then 30pl of the sample was applied to each epidermis unit, in duplicate, and exposure was continued for 24 hours at 37°C, 5% CO2. At the end of the exposure, the product was removed by washing in phosphate buffered saline (PBS) and cell viability was evaluated using the MTT test.

[0287] Skin irritation characterised by the release of IL-1 a, IL-1 p, IL6, IL8, IL-17 was provoked by pre-treatment for 30 minutes with SLS 0.3%. A medium sample was taken after 24 hours for cytokine analysis.

[0288] The maintenance medium and the medium of two skin units treated with the non-irritated sample alone was used as negative control, while the culture medium of two skin units treated with a 0.3% SLS solution was used as positive control.

[0289] In this study IL-i p, IL6, IL8, IL-17 were assayed in the culture medium of treated and untreated epidermis units, using the direct ELISA (enzyme-linked immunosorbent assay) method, i.e. the colour change is directly proportional to the amount of cytokines present in the culture medium.

[0290] 50pl of sample are incubated for 1 .5 hours at room temperature in a plate loaded with the specific primary antibody for the cytokine under investigation. At the end of the incubation, the samples are washed 5 times with a washing solution. After that, 100pl of biotinylated secondary antibody is added and they are incubated for one hour at room temperature. The samples are washed 5 more times with the wash solution and incubated for 30 minutes with the peroxidase-conjugated (HP) streptavidin solution. After 5 washes, the colourimetric substrate is added for the detection of cytokine linked to the plate. The reading is taken at 450nm.

[0291] On the basis of the experimental data defined above, it appears that RO-peptide-23 is able to significantly reduce the expression of overexpressed chemical mediators of inflammation in patients with skin inflammation.

[0292] METHYL SULFONYL METHANE

[0293] - Antioxidant activity

[0294] - Antioxidant activity and purpose

[0295] The aim of the test is to assess whether the tested product possesses antioxidant activity in vitro, at different concentrations. Its ability to neutralise reactive oxygen species (ROS) and inhibit cell death is tested for this reason. This describes the ability to counteract ROS- induced skin inflammation.

[0296] - ROS assay

[0297] The test substance was diluted to the desired final concentrations in saline solution. Separately, dichlorofluorescein acetate (DCA) was dissolved in the appropriate buffer. DCA reacts with any free radicals present to produce a fluorescent derivative, and fluorimetry readings provide a quantitative indication of the presence of this substance in the cells analysed. An adequate number of cells (30,000 cells / pcs) is seeded in the wells of a 96-well plate. After an overnight pre-incubation period with the different sample concentrations, the growth medium is aspirated from the wells and replaced with 500 ml of DCA solution. The plates are incubated for 15’ in a CO2 thermostat at 37°C. At this point, the DCA solution is discarded. After a 5-minute UV exposure period, the oxidative stress is interrupted and the fluorimetry data are read. The lamp used in the experiments reproduces the solar spectrum with a constant UVA emission range between 315 and 400 nm. UVB emission is appropriately screened to avoid direct cytotoxic damage to the cell cultures. The plate containing the cells is irradiated at room temperature with an intensity of 1.7 mW / cm2UVA (5 J / cm2).

[0298] The fluorometer reading is taken at an excitation wavelength of 485 nm and an emission wavelength of 530 nm directly on the plates (Toxicol. Letters 1997 - 93 : 47-54).

[0299] - Protection from ROS

[0300] After 5 minutes of UV exposure.

[0301] The results are given in terms of fluorescence, which is directly proportional to the amount of ROS.

[0302] The percentage of antiradical protection offered by the substance is calculated using the following formula:

[0303] % protection = [(ROS (Positive Control) - ROS (Sample)) / (ROS (Positive Control) - ROS (Negative Control))]*’! 00

[0304] - Conclusions

[0305] Methyl sulfonyl methane exerts a marked antioxidant activity, evaluated by the method described above.

[0306] " Expression of cytokine IL-1 a and receptor antagonist IL-1 aRA

[0307] Methyl sulfonyl methane has surprisingly been shown to reduce the expression of IL-1 a (generic inflammation cytokine), whose increase is detectable in acute inflammation, and, at the same time, the increased expression of IL-1 aRA (receptor antagonist), which is normally overexpressed to protect the skin in chronic inflammation. This prerogative describes the high anti-inflammatory potential of methyl sulfonyl methane.

[0308] - Objective

[0309] Inhibition of the release of cellular mediators of inflammation (IL-1 a) after SLS-induced stress using a silicone emulsion containing 5% methyl sulfonyl methane.

[0310] Increased release of inflammation inhibitors (IL-1 a RA). The cell model used for the test is a three-dimensional system of in-vitro reconstituted human epidermis.

[0311] This three-dimensional cell model consists of normal human primary keratinocytes, allowed to grow in vitro to simulate the multilayered architecture of the human epidermis, with a well-differentiated stratum corneum. The model was purchased from Mattek (USA).

[0312] 1 mg of each sample was applied to each epithelium sample, in duplicate, and exposure continued for 24 hours at 37°C, 5% CO2 with or without pre-treatment for 30 minutes with 0.5% SLS. For the analysis of the cytokines produced, samples were taken at 6 and 24 hours.

[0313] The maintenance medium was used as a negative control, while a 0.5% SLS solution was used as a positive control.

[0314] At the end of the exposure, the products were removed, two PBS washes were performed and the cell viability was evaluated using the MTT test.

[0315] Under normal physiological conditions, unstimulated skin keratinocytes constitutively produce small doses of cytokines. Under the impulse of toxic and stressful stimuli, such as exposure to UV or somewhat aggressive biological and chemical substances, keratinocytes increase the production and release of some of these cytokines, such as lnterleukin-1 a (IL-1 a). This molecule is a soluble mediator involved in the genesis of the skin inflammatory process. The appearance or increase of this marker in the cell culture medium after exposure to certain substances, points to the triggering of a cascade of pro-inflammatory events and may therefore be considered a good marker of pro- inflammatory / pro-irritant potential.

[0316] On the contrary, the IL-1 aRA receptor antagonist is able to inhibit the pro-inflammatory effect triggered by IL-1 a by binding to the same receptor, thus its increase is linked to a protective effect.

[0317] In this study, cytokines were quantified in the culture medium using the ELISA method with commercial kits. A standard cytokine curve with relative absorbance, read in spectrophotometry at specific wavelengths, makes it possible to determine cytokine quantification in the analysed samples, after performing a colourimetry detection reaction using specific antibodies.

[0318] - Results

[0319] Description of the percentage of IL-1 alpha inhibition exerted by MSM.

[0320] Description of the percentage increase in IL-1 alpha RA exerted by MSM

[0321] - Conclusion The action of methyl sulfonyl methane has been shown to be similar to that of hydrocortisone in regulating the decrease in IL-1 alpha release and increasing the induction of IL-1 alpha Receptor Antagonist.

[0322] * Inhibition of basophil / mast cell degradation

[0323] - Objective The aim of the test is to assess whether methyl sulfonyl methane is able to inhibit the degranulation of basophils, the terminal effector cells of the in-vitro allergic response.

[0324] Indeed, basophil degranulation induced by specific allergens / aptens interacting with the human IgE receptor triggers in vivo the immediate hypersensitivity reaction, characterised by the release of specific inflammatory mediators (histamine, leukotrienes, basophilic granule enzymes, hexosaminidase).

[0325] - Results

[0326] The IC50 of the sample was 50mg / ml. On this basis, the highest concentration used in vitro was 10mg / ml. The following tables and graphs show the results obtained by analysing the release in the cell supernatant of p-hexosaminidase as an index of basophil degranulation. The numbers indicate optical density (405 nm).

[0327] - Conclusion

[0328] Methyl sulfonyl methane has a dose-dependent inhibitory effect on basophil degranulation.

[0329] - Methyl sulfonyl methane - VEGF expression

[0330] The cell model used for the in-vitro test is represented by: stabilised human keratinocytes (HaCaT cells).

[0331] - Treatment and exposure

[0332] The cells were seeded in 24-well plates in DMEM + 10% FCS culture medium containing methyl sulfonyl methane at the indicated final concentrations of 1 mg and 0.1 mg / ml and exposed for up to 72h with 5% CO2 The culture medium was renewed every day.

[0333] Each sample was tested in duplicate. Untreated cells were used as a negative control.

[0334] After 24 hours of incubation with sample concentrations between 1 and 0.1 mg / ml, the cytotoxicity test (MTT) was performed. The ELISA test to calculate the amount of VEGF produced was performed at 24, 48, and 72 hours.

[0335] - VEGF determination

[0336] 100 pL of culture medium was taken from each well, to be used for the determination of VEGF by ELISA, conducted at room temperature at each end-point (24, 48, and 72 hours).

[0337] The method involves calculating a standard curve over a range of appropriate concentrations.

[0338] The ELISA test uses a specific anti-human VEGF antibody immobilised on a solid substrate and a second anti-VEGF antibody marked with biotin.

[0339] - Evaluation of VEGF production

[0340] The amount of VEGF present in the culture medium was calculated after 24, 48, and 72 hours of exposure to methyl sulfonyl methane and in the negative control.

[0341] The sample containing methyl sulfonyl methane at a concentration of 0.1 mg / ml is appreciably able to inhibit VEGF production (more than 25%) already after 24 hours, while after 72 hours the inhibition percentage rises to 70%.

[0342] - In-vitro evaluation of the protective activity of MSM on human keratinocyte cell cultures after exposure to UVB radiation and on human fibroblast cell cultures after UVA exposure.

[0343] The photoprotective capacity of MSM was evaluated both by the MTT test (cell viability) and by morphological analysis (microscopy) of treated keratinocytes exposed to UVB radiation and treated human fibroblasts exposed to UVA radiation.

[0344] Cell morphology was evaluated by fluorescence microscopy after staining with acridine orange. An Olympus microscope with 20x magnification was used for the morphological analysis. Primary cultures of keratinocytes from human epidermis biopsies were used. The cultured cells were treated with 5% methyl sulfonyl methane, a concentration previously evaluated as non-cytotoxlc.

[0345] - Performing the test on keratinocytes

[0346] On the first day, 60,000 keratinocytes / well were seeded on plates, then incubated overnight. On the second day, the cells were transferred to microchambers and incubated for 60’ in PBS with 1 % (EM). After exposure to UVB for 30’, the cells were incubated overnight in CEC + medium in the presence of 5% foetal bovine serum (FCS). The cells not exposed to UVB radiation formed the negative control. On the third day, acridine orange staining was performed for observation under a fluorescence microscope.

[0347] - Performing the test on fibroblasts

[0348] On the first day, an adequate number of fibroblasts (80,000 cells / well) was seeded in plates and incubated overnight. On the second day, the cells were transferred to microchambers and incubated overnight in MEM (Minimal Essential Medium) - Sodium Pyruvate with added 5% fetal bovine serum (FCS). The negative control consisted of cells not exposed to UVA radiation. On the third day, acridine orange staining was performed for observation under a fluorescence microscope.

[0349] - Experimental irradiation conditions on keratinocytes

[0350] A lamp with constant UVB emission between 290 and 315 nm was used. The UVB radiation dose used was quantified in such a way as to induce cell death in the keratinocytes exposed to irradiation. The micro- chambers containing the ceils were irradiated for 30 minutes at room temperature with an intensity of 3.4 mW / cm2UVB (6 J / cm2). Lamp emission was measured using a PMA 2100 model radiometer by Solar Light Company Inc. (Philadelphia), calibrated with Beckmann 800 multimeter spectrophotometer.

[0351] - Experimental irradiation conditions on fibroblasts

[0352] A lamp with constant UVA emission between 320 and 400 nm was used. The UVA radiation dose used was quantified in such a way as to induce cell death in the keratinocytes exposed to irradiation. The microchambers containing the cells were irradiated for 30 minutes at room temperature with an intensity of 75 mW / cm2UVB (135 J / cm2). Lamp emission was measured using a PMA 2100 model radiometer by Solar Light Company Inc. (Philadelphia), calibrated with Beckmann 800 multimeter spectrophotometer.

[0353] - Cytoprotective activity on keratinocytes

[0354] Cell viability was assessed in the absence and after 30’ of UVB exposure Absorbance values (O.D.) at 540 nm were read. Depending on the results obtained, the percentage change in photoprotection provided by MSM was calculated according to the following formula: % protection = [MTT (UVB+ sample) - MTT (positive control) / MTT (positive control)] x 100.

[0355] Tab. 1 - Absorbance values (O.D.) at 540 nm: numerical values are directly proportional to the number of viable cells

[0356] The experimental data show the remarkable photoprotective capacity exerted by MSM against UVB radiation on human keratinocytes. Absorbance values, an indication of the concentration of viable cells, are increased by 198 %.

[0357] Indeed, in the negative control, after UVB irradiation, the O.D. value was 0.279 (± 0.027) compared to a value of 0.831 (± 0.090) for the keratinocytes protected with MSM, as illustrated in the graph in Figure 3, where:

[0358] C before = negative control before UVB exposure; c after = negative control after UVB exposure; MSM before = keratinocytes protected with MSM before UVB exposure; MSM after = keratinocytes protected with MSM after UVB exposure, and in Figures 4 and 5, showing the negative control before irradiation (left) and after irradiation (right), and the sample protected with MSM before irradiation (left) and after irradiation (right), respectively.

[0359] The morphological analysis showed that a greater number of keratinocytes protected by MSM survived compared to the negative control.

[0360] - Cytoprotective activity on fibroblasts

[0361] Cell viability was assessed in the absence and after 30’ of UVA exposure Absorbance values (O.D.) at 540 nm were read. Depending on the results obtained, the percentage change in photoprotection provided by MSM was calculated according to the following formula: % protection = [MTT (UVB+ sample) - MTT (positive control) / MTT (positive control)] x 100.

[0362] Tab. 2 ■ Absorbance values (O.D.) at 540 nm: numerical values are directly proportional to the number of viable cells.

[0363] The experimental data show the remarkable photoprotective capacity exerted by MSM against UVA radiation on human fibroblasts. Absorbance values, an indication of the concentration of viable cells, are increased by 86%.

[0364] Indeed, in the negative control, after UVA irradiation, the O.D. value was 0.378 (± 0.1 1 1 ) compared to a value of 0.637 (± 0.148) for the fibroblasts protected with MSM, as illustrated in the graph in Figure 6, where:

[0365] C before = negative control before UVB exposure; c after = negative control after UVB exposure; MSM before = keratinocytes protected with MSM before UVB exposure; MSM after = keratinocytes protected with MSM after UVB exposure, and in Figures 7 and 8, showing the negative control before irradiation (left) and after irradiation (right), and the sample protected with MSM before irradiation (left) and after irradiation (right), respectively.

[0366] The morphological analysis showed that a greater number of fibroblasts protected by MSM survived compared to the negative control.

[0367] The photo-protective action of MSM is considered significant in controlling directly or indirectly photo-induced inflammation.

[0368] - Conclusions regarding the synchronous action of RO-peotide23 and methyl sulfonyl methane on the expression of the chemical mediators of skin inflammation

[0369] As can be inferred from the above data, the combination of RO- peptide-23 and methyl sulfonyl methane exerts a synergistic action in mapping their respective activities that is able to reduce the expression of key skin inflammation markers. Furthermore, to complement the design described here, methyl sulfonyl methane is characterised by a marked antioxidant and protective activity against ultraviolet radiation, significantly contributing to the control of skin inflammation.

[0370] - Clinical evaluations

[0371] 20 subjects-patients with rosacea were selected in order to evaluate the efficacy of an emulsion containing RO-peptide-23 and methyl sulfonyl methane in concentrations as indicated above, in particular, 0.000025% w / w and 2% w / w respectively.

[0372] 2 ml of emulsion was applied to each subject.

[0373] The subjects were treated 8 times at 2-day intervals between one application and the next.

[0374] The product showed a specific and significant ability to control skin erythema, improve the barrier functions of the skin and hydration, as shown in figure 9 (reduction in erythema - red scale, decrease in telangiectasias - blue scale), and in the diagram below.

[0375] Indeed, a significant reduction in transepidermal water loss (TEWL) of 17.5% was observed after only 8 applications (16 days).

[0376] The product's ability to improve surface hydration (corneometry) amounted to a 15.2 per cent increase.

[0377] The intensity of the erythema (mexameter - reflectance spectrophotometry) after 16 days was reduced by 14.1 %.

[0378] Furthermore, in a further clinical evaluation carried out on 40 subjects, with application of a topical composition according to the present invention in emulsion form with concentrations of RO-peptide-23 and methyl sulfonyl methane in accordance with the foregoing, in particular 0.000025% w / w and 2% w / w respectively, carried out continuously for 28 days, it showed remarkable efficacy in controlling erythema, skin hydration and, very importantly, in improving the barrier functions of the skin, as shown in the diagram below.

[0379] The most surprising fact is defined by the product’s ability to improve results even after 10 weeks of using the topical composition, as shown in Figure 10 where:

[0380] 1 = baseline, 2= after 28 days, 3= 10 weeks after treatment was discontinued, and in the diagram below.

[0381] Indeed, the improvement in erythema is evident both at time to - 30.63% reduction in erythema intensity (start of treatment), and ten weeks after the last application with a result that consolidates and achieves an improvement of 1 1 .43%.

[0382] This surprisingly showed that the topical composition containing RO-peptide-23 and MSM, e.g. when compared to brimonidine tartrate, which is characterised by a less pronounced and short-lasting effect, exerts an action that persists and intensifies even after discontinuation of its use. EXAMPLES

[0383] - 1 ~ Oil-in-water emulsion RO-peptide-23 is used at a concentration of 10 pg / 100 g emulsion

[0384] PHASE PREPARATION:

[0385] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07 and raise the temperature to + °±3°C. PHASE B) a) Dissolve 08 in 1 1 and start heating to +75°±3°C. b) Disperse 12 in the solution of point a) by sprinkling and turbulate. c) Disperse 13 in the homogeneous dispersion obtained in step b), adding it by sprinkling, turbulate and continue heating to +75°±3°C. d) Mix 09 and 10 into the clear and fluid gel formed by step c) and continue heating to +75°±3°C.

[0386] PHASE C) a) Weigh separately components 14, 15, to be added at the end of preparation, in this precise order. EMULSION PREPARATION

[0387] METHOD

[0388] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both phases reach a temperature of +80°±3°C and +75°±3°C respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to + 40°±5°C.

[0389] PROCEDURE 02 a) addition of the components of PHASE C): Once the temperature has reached 4-40c±5°C, mix 14 and 15, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components continue cooling down to a temperature of +25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0390] 2 - Oil-in-water emulsion

[0391] RO-peptide-23 is used at a concentration of 100 pg / 100 g emulsion PHASE PREPARATION:

[0392] PHASE A) a) Mix 01 , 02, 03, 04, 05 and bring the temperature to +30°±5°C, mix with blades only until a homogeneous milky dispersion is obtained.

[0393] PHASE B) a) Dissolve 06 into 09 at a room temperature of +25°±3°C. b) Dissolve 07, 08 into the clear, colourless solution of point a), adding one component at a time, by sprinkling and stirring with blades only.

[0394] Check to ensure the complete solubilisation of the added ingredient before adding the next one. c) Mix 10 into the clear and colourless solution of step b), stir and check that the final solution remains clear and colourless.

[0395] PHASE C) a) Weigh component 1 1 , to be added at the end of preparation.

[0396] EMULSION PREPARATION METHOD PROCEDURE 01 a) Combining PHASE A) and PHASE B): Once the preparation of the two phases is complete, combine PHASE A) into PHASE B) at a room temperature of +25°±5°C by stirring and turbulate for 20 minutes.

[0397] PROCEDURE 02 a) Addition of the PHASE C) component: Mix 1 1 into the emulsion obtained in PROCEDURE 1 ) by turbulate for 10 minutes. b) While adding the component, maintain a temperature of + 25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0398] - 3 - Monophasic gel

[0399] RO-peptide-23 is used at a concentration of 75 pg / 100 g gel

[0400] GEL PREPARATION METHOD

[0401] PHASE A) a) Dissolve 01 into 04 at a room temperature of +25°±3°C.

[0402] Check to ensure the complete solubilisation of the added ingredient before adding the next one. b) Disperse 05 in the clear, colourless solution of point a), by sprinkling and turbulate. c) Disperse 06 into the clear, colourless gel formed in step b), by sprinkling and turbulate until a clear, colourless, viscous gel is obtained. d) Mix components 02, 03, 07, 08 into the gel mentioned under point c), adding one component at a time and turbulate for 10 minutes after every single addition.

[0403] Check to ensure the added ingredient has been mixed completely before adding the next one. e) Transfer the semi-finished product into the appropriate containers.

[0404] The following formula can be used to prepare a sprayable solution by removing the rheology modifier (No. 5 and No. 6).

[0405] - 4 - Oil-in-water emulsion with 4-t-butyicyclohexanol |

[0406] RO-peptide-23 is used at a concentration of 0.9 mg / 100 g emulsion.

[0407] PHASE PREPARATION:

[0408] PHASE A) a) Mix 01 , 02, 03, 04, 05 and bring the temperature to + 30°±5°C, mix with blades only until a homogeneous milky dispersion is obtained.

[0409] PHASE B) a) Dissolve 06 into 09 at a room temperature of +25°±3°C. b) Dissolve 07, 08 into the clear, colourless solution of point a), adding one component at a time by sprinkling and stirring with blades only.

[0410] Check to ensure the complete solubilisation of the added ingredient before adding the next one. c) Mix 10 into the clear and colourless solution of step b), stir and check that the final solution remains clear and colourless.

[0411] PHASE C) a) Weigh separately components 1 1 , 12, to be added at the end of preparation, in this precise order. EMULSION PREPARATION

[0412] METHOD

[0413] PROCEDURE 01 a) Combining PHASE A) and PHASE B): Once the preparation of the two phases is complete, combine PHASE A) into PHASE B) at a room temperature of +25O±5°C by stirring and turbulate for 20 minutes.

[0414] PROCEDURE 02 a) addition of the components of PHASE C): Mix 1 1 and 12 into the emulsion obtained in Procedure 1 ) by adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components, maintain a temperature of + 25±2°C. c) Transfer the semi-finished product into the appropriate containers - 5 - Oil-in-water emulsion with 4-t-butylcyclohexanol

[0415] RO-peptide-23 is used at a concentration of 5 mg / 100 g emulsion.

[0416] PHASE PREPARATION:

[0417] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07 and raise the temperature to +80°±3°C.

[0418] PHASE B) a) Dissolve 08. in 1 1 and start heating to +75°±3°C b) Disperse 12 in the solution of point a) by sprinkling and turbulate. c) Disperse 13 in the homogeneous dispersion obtained in step b), adding it by sprinkling, turbulate and continue heating to +75°±3°C. d) Mix 09 and 10 into the clear and fluid gel formed by step c) and continue heating to +75°±3°C.

[0419] PHASE C) a) Weigh separately components 14, 15, 16, to be added at the end of preparation, in this specific order.

[0420] EMULSION PREPARATION METHOD

[0421] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both PHASES reach a temperature of +80°±3°C and +75°±3°C, respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to + 40°±5°C.

[0422] PROCEDURE 0) a) addition of the components of PHASE C): Once the temperature has reached -t-40o±5°C, mix 14, 15 and 16, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components, continue the cooling operation until the temperature reaches +25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0423] - 6 - Emulsion with niacinamide

[0424] RO-peptide-23 is used at a concentration of 5 mg / 100 g emulsion.

[0425] PHASE PREPARATION:

[0426] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07 and raise the temperature to +80°±3°C.

[0427] PHASE B) a) Dissolve 08 and 09 in 12 and start heating to +75°±3°C. b) Disperse 13 in the solution of point a) by sprinkling and turbulate. c) Disperse 14 in the homogeneous dispersion obtained in step b), adding it by sprinkling, turbulate and continue heating to +75°±3°C. d) Mix 10 and 1 1 into the clear and fluid gel formed by step c) and continue heating to +75°±3°C.

[0428] PHASE C) a) Weigh separately components 15 and 16, to be added at the end of preparation, in this specific order.

[0429] EMULSION PREPARATION METHOD

[0430] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both PHASES reach a temperature of +80°±3°C and +75°±3°C, respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to +40°±5°C. PROCEDURE 02 a) addition of the components of PHASE C): Once the temperature has reached +40°±5°C, mix 15 and 16, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components continue cooling down to a temperature of +25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0431] 7 - Gel with niacinamide

[0432] RO-peptide-23 is used at a concentration of 5 mg / 100 g emulsion.

[0433] GEL PREPARATION METHOD

[0434] PHASE A) a) Dissolve 01 and 02 into 05 at a room temperature of +25c±3°C.

[0435] Check to ensure the complete solubilisation of the added ingredient before adding the next one. b) Disperse 06 in the clear, colourless solution of point a), by sprinkling and turbulate. c) Disperse 07 into the clear, colourless gel formed in step b), by sprinkling and turbulate until a clear, colourless, viscous gel is obtained. d) Mix components 03, 04, 07, 08, 09 into the gel under point c), adding one component at a time and turbulate for 10 minutes after every single addition.

[0436] Check to ensure the added ingredient has been mixed completely before adding the next one. e) Transfer the semi-finished product into the appropriate containers.

[0437] 8 - Emulsion with silymarin

[0438] RO-peptide-23 is used at a concentration of 5 mg / 100 g emulsion.

[0439] PHASE PREPARATION:

[0440] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07 and raise the temperature to + 80°±3°C.

[0441] PHASE B) a) Dissolve 08 in 1 1 and start heating to +75°±3°C. b) Disperse 12 in the solution of point a) by sprinkling and turbulate. c) Disperse 13 in the homogeneous dispersion obtained in step b), adding it by sprinkling, turbulate and continue heating to +75°±3°C. d) Mix 09 and 10 into the clear and fluid gel formed by step c) and continue heating to +75O±3°C.

[0442] PHASE C) a) Weigh separately components 14, 15, 16, to be added at the end of preparation, in this precise order.

[0443] EMULSION PREPARATION METHOD

[0444] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both PHASES reach a temperature of +80°±3°C and +75°±3°C, respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to + 40o±5°C.

[0445] PROCEDURE 02 a) addition of the components of PHASE C): Once the temperature has reached +40°±5°C, mix 14, 15 and 16, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components continue cooling down to a temperature of +25±2°C. c) Transfer the semi-finished product into the appropriate containers. 9 - Gel with xylitol

[0446] RO-peptide-23 is used at a concentration of 20 pg / 100 g gel.

[0447] GEL PREPARATION METHOD

[0448] PHASE A) a) Dissolve 01 and 02 into 05 at a room temperature of +25°±3°C.

[0449] Check to ensure the complete solubilisation of the added ingredient before adding the next one. b) Disperse 06 in the clear, colourless solution of point a), by sprinkling and turbulate. c) Disperse 07 into the clear, colourless gel formed in step b), by sprinkling and turbulate until a clear, colourless, viscous gel is obtained. d) Mix components 03, 04, 07, 08, 09 into the gel under point c), adding one component at a time and turbulate for 10 minutes after every single addition. Check to ensure the added ingredient has been mixed completely before adding the next one. e) Transfer the semi-finished product into the appropriate containers. 10 - Emulsion with xylitol

[0450] RO-peptide-23 is used at a concentration of 2.5 mg / 100 g emulsion.

[0451] PHASE PREPARATION:

[0452] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07 and raise the temperature to +80°±3°C.

[0453] PHASE B) a) Dissolve 08 and 09 in 12 and start heating to +75°±3°C. b) Disperse 13 in the solution of point a) by sprinkling and turbulate. c) Disperse 14 in the homogeneous dispersion obtained in step b), adding it by sprinkling, turbulate and continue heating to +75°±3°C. d) Mix 10 and 1 1 into the clear and fluid gel formed by step c) and continue heating to +75°±3°C.

[0454] PHASE C) a) Weigh separately components 15 and 16, to be added at the end of preparation, in this precise order.

[0455] EMULSION PREPARATION METHOD

[0456] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both PHASES reach a temperature of +80°±3°C and +75°±3°C, respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to + 40o±5°C.

[0457] PROCEDURE 02 a) addition of the components of PHASE C): Once the temperature has reached +40°±5°C, mix 15 and 16, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components continue cooling down to a temperature of +25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0458] 1 1 - Emulsion with solar filters (spf 50+)

[0459] RO-peptide-23 is used at a concentration of 125 pg / 100 g emulsion. PHASE PREPARATION:

[0460] PHASE A) a) Mix 01 , 02, 03, 04, 05, 06, 07, 08, 09, in this precise order, and start heating to +80°±3°C.

[0461] PHASE B) a) Dissolve 10 in 16, while stirring and start heating to +75O±3°C. b) Mix 1 1 and disperse 12 into the clear, transparent solution under point a), stirring and then turbulate, until the mass is completely gelled. Continue heating to +75°±3°C. c) After complete dispersion of 12, disperse 14, stirring and turbulate and continue heating to +75°±3°C. d) After obtaining a homogeneous gel, mix 13, continuing heating to +75°±3°C. e) Mix 15 into the gel under point e) and continue heating to +75°±3°C.

[0462] PHASE C) a) Weigh the components of the phase separately: 17, 18, 19, 20, to be added one at a time at the end of the preparation, in this precise order.

[0463] EMULSION PREPARATION METHOD

[0464] PROCEDURE 01 a) Combining PHASE A) and PHASE B): When both PHASES reach a temperature of +80°±3°C and +75°±3°C, respectively, combine them by stirring and turbulate for 20 minutes. b) Cool the emulsion formed to + 40o±5°C.

[0465] PROCEDURE 02 a) addition of the components of PHASE C): Once the temperature has reached +40°±5°C, mix 17, 18, 19, 20, adding one component at a time and stirring and turbulate for 10 minutes after every single addition. b) While adding the components continue cooling down to a temperature of +25±2°C. c) Transfer the semi-finished product into the appropriate containers.

[0466] The advantages of the present invention, which the above description makes apparent, can be summarised by pointing out that a topical composition is made available which can be conveyed in various chemical-physical forms, which is particularly effective in the control, mitigation or therapy of general skin inflammation, contact dermatitis, atopic dermatitis and rosacea, which is essentially free of side or otherwise undesirable effects and which can be used as a medicament in a therapeutic treatment or as a cosmetic in a non-therapeutic treatment, for the improvement of the barrier functions of the skin, therefore to maintain in good state the condition of the skin or improve the condition of the skin.

[0467] To the present invention, in the forms of embodiment illustrated and described, for the purpose of satisfying contingent and specific needs, a person skilled in the art may make numerous variations and modifications, all of which are within the scope of protection of the invention as defined by the following claims.

Claims

CLAMS1. Topical composition comprising methyl sulfonyl methane and a peptide with the following sequence:H-DL-Phe-DL-A!a-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu- DL-Leu-DL-Lys-DL-Ala-DL-Leu-DL-Lys-DL-Ala-DL-Leu-NH2.

2. Topical composition according to claim 1 , comprising said peptide at a concentration between 0.000005% w / w and 1 % w / w, preferably between 0.00005% w / w and 0.075% w / w, more preferably between 0.0005% w / w and 0.005% w / w.

3. Topical composition according to claim 1 or 2, comprising methyl sulfonyl methane at a concentration between 0.005% w / w and 25% w / w, preferably between 0.05% w / w and 7.5% w / w, more preferably between 0.075% w / w and 5% w / w.

4. Topical composition according to any one of the preceding claims, comprising at least one of the following: 4-t-butylcyclohexanol, niacinamide (vitamin PP), silymarin, xylitol, acetylsalicylic acid, salicylic acid and at least one solar filter.

5. Topical composition according to claim 4, comprising 4-t- butylcyclohexanol at a concentration between 0.1 % w / w and 7% w / w, preferably between 0.2% w / w and 5% w / w, more preferably between 0.35% w / w and 4% w / w.

6. Topical composition according to claim 4 or 5, comprising niacinamide at a concentration between 0.1 % w / w and 9.5% w / w, preferably between 0.5% w / w and 7% w / w, more preferably between 0.75% w / w and 5% w / w.

7. Topical composition according to claim 4, 5 or 6, comprising silymarin at a concentration between 0.1% w / w and 5.5% w / w, preferably between 0.25% w / w and 4% w / w, more preferably between 0.5% w / w and 3% w / w.

8. Topical composition according to any one of claims 4-7,comprising xylitol at a concentration between 0.1 % w / w and 15.5% w / w, preferably between 0.25% w / w and 4% w / w, more preferably between 0.5% w / w and 3% w / w.

9. Topical composition according to any one of claims 4-8, comprising acetylsalicylic acid or salicylic acid at a concentration between 0.1 % w / w and 35% w / w, preferably between 0.25% w / w and 2% w / w.

10. Topical composition according to any one of claims 4-9, wherein said at least one solar filters is selected from the group comprising: benzalkonium camphor methosulphate; homosalate; benzophenone-3; phenylbenzimidazole sulfonic acid; terephthaiidenedicanephorsulfonic acid; butylmethoxydibenzoylmethane; benzylidene camphorsulfonic acid; octocrylene; polyacrylamidomethylbenzylidene camphor; ethylhexylmethoxycinnamate; peg-25 paba; isoamyl p-methoxycinnamate; ethylhexyl triazone; drometrizole trisiloxane; diethylhexyl butamido triazone; 4-methylbenzylidene camphor; ethylhexyl salicylate; ethylhexyl dimethyl paba; benzophenone-4, benzophenone-5; methylene bis- benzotriazolyl tetramethylbutylphenol; methylene bis-benzotriazolyl tetramethylbutylphenol (nano); disodium phenyl dibenzimidazole tetrasulfonate; bis-ethylhexyloxyphenol methoxyphenyl triazine; polysilicone-15; titanium dioxide; titanium dioxide (nano); diethylamino hydroxybenzoyl hexyl benzoate; tris-biphenyl triazine / tris-biphenyl triazine (nano); zinc oxide; zinc oxide (nano); phenylene bis-diphenyltriazlne; methoxypropylamino cyclohexenylidenethoxyethylcyanoacetate.

11. Topical composition according to any one of the preceding claims, said topical composition being in the form of emulsion, lotion, aqueous solution, monophasic gel, ointment, cream, paste, spray.

12. Topical composition according to any one of the preceding claims, for use in the treatment of an inflammatory condition of the skin.

13. Topical composition according to any one of claims 1 -12, for use as a medicament in the treatment of an inflammatory condition of the skincharacterized by a hyper-release of chemical mediators of inflammation.

14. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of rosacea.

15. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of atopic dermatitis.

16. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of contact dermatitis.

17. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of seborrheic dermatitis.

18. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of sensitive skin syndrome.

19. Topical composition according to any one of claims 1 -13, for use as a medicament to reduce the effect of ultraviolet radiation.

20. Topical composition according to any one of claims 1 -13, for use as a medicament in the treatment of ultraviolet radiation-induced erythema.21 . Topical composition according to any one of claims 1 -13, for use as a medicament for maintaining, restoring or improving the barrier functions of the skin.

22. Topical composition according to any one of claims 1 -12, for cosmetic and non-therapeutic use for the maintenance, restoration or improvement of the barrier functions of the skin, to reduce the harmful effect of ultraviolet radiation, for the treatment of sensitive skin syndrome and / or to improve the appearance of skin characterized by redness.

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