^{Title: Pharmaceutical composition for topical use FIELD OF THE INVENTION The present invention relates to a composition comprising cannabidiol (CBD), probiotics and lipids, particularly cholesterol and ceramides. The present invention further relates to said composition for use in the treatment of skin inflammations, dermatitis, particularly atopic dermatitis, skin dryness, psoriasis, eczemas. BACKGROUND The skin is the largest organ in our body and is exposed to a wide variety of external stimuli, including microorganisms. One of the most important functions of the skin is to protect the host from harmful environmental stimuli including the invasion of pathogenic microorganisms (Segre JA, 2006). The protective function of the skin is implemented by the stratified squamous epithelium of the epidermis as a barrier function and by pro-inflammatory and antimicrobial molecules produced by keratinocytes and immune cells. Although most microorganisms living in human skin are harmless and even beneficial, some resident microorganisms are potentially harmful under certain conditions and are referred to as pathogens. Staphylococcus epidermidis colonizes human skin normally but can cause severe disease in some individuals (Blum RA, 1987). Similarly, the pathogen Staphylococcus aureus, which is a common cause of skin and systemic infections, may reside in the skin of 10-20% of healthy individuals as a harmless commensal. However, S. aureus colonizes the injured skin of approximately 90% of atopic dermatitis (AD) and its increased colonization is associated with disease flare-ups (Kong HH, 2012). Another characteristic of the skin of AD patients is the presence of a dysbiotic microbiota. The mechanism by which the normal skin microbiota switches from a neutral state to dysbiosis and the contribution of the abnormal microbiota to the pathogenesis of AD remain unclear. Recent advances in the analysis of the skin microbiota by high-throughput microbial gene sequencing have allowed partial identification and quantification of bacterial species, viruses, and fungi (Foulongne V, 2012; Grice EA, 2011). Cannabidiol (CBD) is one of at least 85 active cannabinoids identified from the cannabis plant. It is an important phytocannabinoid, accounting for up to 40% of the plant extract. CBD is believed to have} a wider range of medical applications than tetrahydrocannabinol. An orally administered liquid containing CBD has received drug status in the US and Europe for use as a treatment for Dravet syndrome, under the brand name Epidiolex.

The physiological action of endocannabinoids and psychoactive phytocannabinoids is driven by their interactions with the specific class of Gai protein-coupled receptors known as central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2), respectively. The highest abundance of CB1 is found in the central nervous system (basal ganglia, hippocampus, cerebellum, and cortex), where they mediate cannabinoid-related psychoactive effects. In addition, CB1 receptors are present in the nerve endings of the testis, uterus, vascular endothelium, eye, spleen, ileum, and adipocytes. CB2 receptors are distributed within the body's immune system, in the enriched areas of B lymphocytes (Schwitzer et al. 2015).

Atopic dermatitis (AD) is a common, clinically defined chronic inflammatory skin disease. The high variability of clinical phenotype and severity, genetic background and known pathological mechanisms strongly suggest a high degree of pathophysiological heterogeneity. The clinical pattern of eczematous skin lesions is relatively uniform and results from interactive alterations of the skin barrier and the innate and adaptive immune system. The pathogenesis of atopic dermatitis involves both epidermal barrier and immunological dysfunction. Some of these alterations are caused by mutations in genes coding for proteins with immune and barrier function, which may alter the regulation or structure of the gene product itself. Other alterations may be consequences of environmental factors such as stress, scratching behavior, exposure to allergens or washing habits (Thomsen 2014). The epidermal barrier consists of a thin layer of viable keratinocytes, which slowly differentiate into flat corneocytes as they move upwards in the epidermis. The thin layer of dead keratinocytes that makes up the stratum corneum covers the viable parts of the epidermis and protects it from water loss and microbial invasion. The complex process of epidermal differentiation is disturbed in AD lesions. The corneocytes are attached to each other by corneodesmosomes that can be degraded by the stratum corneum chymotryptic enzyme (SCCE). Decreased activity of the Kazal-type lymphoepithelial SCCE inhibitor (LEKTI) in AD skin results in increased SCCE activity, thus the amount of corneodesmosomes, lower cell cohesion and higher corneocyte dispersion (Vasilopoulos et al. 2004). The reduction of corneodesmosomes results in transepidermal water loss and less hydration of the stratum corneum. Epidermis protects internal organs from physical and chemical trauma, invasion by microorganisms and ultraviolet radiation. It also aids in the regulation of transepidermal movement of water and electrolytes, including the prevention of dehydration, which is essential for the sustenance of life. The main role of epidermis is considered to be in the stratum corneum and the lipid matrix, located in the intercellular space. The occurrence of dysfunction in the epidermal barrier is an important factor in the pathophysiology of skin diseases, particularly with reference to the damaged barrier caused, for example, by atopic dermatitis (Rio CD 2018).

In view of the above, it is evident the necessity of a pharmaceutical composition allowing to solve the above-mentioned cutaneous problems. BRIEF DESCRIPTION OF THE INVENTION

The Applicant has now found a pharmaceutical composition comprising probiotics, cannabidiol (CBD) and lipids.

Therefore, in a first aspect, the present invention relates to a pharmaceutical composition according to claim 1. Therefore, the present invention relates to a pharmaceutical composition comprising:

- cannabidiol (CBD);

- Lactobacillus ferment lysate;

- Bifida ferment lysate; and

- lipids selected from cholesterol, ceramide or a mixture thereof. Advantageously, the pharmaceutical composition according to the invention allows to effectively treat skin inflammations, dermatitis, particularly atopic dermatitis, skin dryness, psoriasis, eczemas. The pharmaceutical composition allows to restore the effectiveness of the epidermal barrier, usually damaged by the skin inflammations. The pharmaceutical composition according to the invention is capable of modifying the skin microbiota, reducing inflammation and related symptoms, for example itch, and also improving the barrier function of epidermis.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 refers to graphic representations of the effects of compound CH1030(4) ATOPIC CREAM on the release of IL-6, IL-8 and TSLP through RHE under stimulation conditions with (Poly(I:C) + IL-la).

Fig. 2 refers to a graphic summarizing the obtained results. Further advantages obtainable through the pharmaceutical composition of the invention will be described below.

DETAILED DESRIPTION OF THE INVENTION

For the purposes of the invention, the definitions of some terms used in the present description and in the attached claims are provided below.

The pharmaceutical composition according to the present invention can be alternatively indicated hereinafter simply as “composition”.

The term probiotics or para-probiotics means non-viable microbial cells (intact or fragments) which, when administered (orally or topically), provides advantage to the human consumer. According to a preferred aspect, the pharmaceutical composition of the invention comprises:

- cannabidiol from 0.001 to 10.000 mg;

- Lactobacillus ferment lysate from 0.01 mg to 10,000 mg;

- Bifida ferment lysate from 0.1 mg to 5,000 mg;

- lipids from 0.1 tol5,000 mg. Preferably, lipids comprise:

- ceramide from 0.1 to 10,000 mg;

- cholesterol from 0.01 to 5,000 mg.

According to a preferred aspect, the pharmaceutical composition of the invention comprises:

- cannabidiol from 0.001 to 10,000 mg; - Lactobacillus ferment lysate from 0.01 mg to 10,000 mg;

- Bifida ferment lysate from 0.1 mg to 5,000 mg;

- ceramide from 0.1 to 10,000 mg;

- cholesterol from 0.01 a 5,000 mg.

According to a preferred aspect, said lipids further comprise linolenic acid, linoleic acid or a mixture thereof. Preferably, the total amount of linoleic acid and linolenic acid is from 1 mg to 5.000 mg, more preferably from 1 mg to 1 ,000 mg.

According to a preferred aspect said lipids further comprise at least one from linoleic acid and linolenic acid. According to a preferred aspect, the pharmaceutical composition of the invention can comprise in addition pharmaceutically acceptable excipients.

The pharmaceutical composition of the invention can comprise urea.

With particular reference to lipids, they have a barrier function adjusting the differentiation of epidermal cells. Particularly, ceramides are the main lipid constituent of the lamellar sheets which are in the intracellular spaces of the stratum corneum (Corderch L. 2003).

According to a preferred aspect, the pharmaceutical composition of the invention comprises:

- cannabidiol from 0.1 to 500 mg;

- Lactobacillus ferment lysate from 0.1 mg to 3,000 mg; - Bifida ferment lysate from 0.1 mg to 1,000 mg;

- ceramide from 0.1 to 1 ,000 mg;

- cholesterol from 0.01 to 500 mg;

According to a preferred aspect, the pharmaceutical composition of the invention comprises:

- cannabidiol from 0.1 to 100 mg; - Lactobacillus ferment lysate from 0.1 mg to 1 ,000 mg;

- Bifida ferment lysate da 0.1 mg a 1,000 mg;

- ceramide from 0.1 to 500 mg;

- cholesterol from 0.01 to 500 mg.

According to a preferred aspect, the pharmaceutical composition is for topical use. Preferably, the composition for topical application is of 2-10 mg/cm ² , more preferably of 3-8 mg/cm ² , even more preferably

4-6 mg/cm ² . In a particularly preferred embodiment, the composition for topical application is of 5 mg/cm ² of composition.

Preferably, the composition is in a form selected from cream or gel.

In a second aspect, the present invention relates to a pharmaceutical composition as defined above, in the treatment and/or in the prevention of a disease or condition selected from skin inflammation, dermatitis, skin dryness, psoriasis, eczemas, skin inflammation related to a skin dysbiosis. Preferably such pharmaceutical composition is for topical use.

Preferably, dermatitis is atopic dermatitis (AD). With particular reference to the atopic dermatitis, it has been demonstrated that the pharmaceutical composition of the invention is particularly effective against AD symptoms, particularly itch, inflammation and clinical signs, such as integrity loss of skin barrier and hydration.

The combined action of the three elements (cbd+probiotics+lipids) interacts with the potential inflammatory effects represented in a RHE model (Reconstructed human epidermidis) stimulated with an inflammatory mix: (Poly (I: C) + IL-la). This stimulation results in a phenotype, in keratinocytes, which imitates the initial stages of the atopic dermatitis (AD), particularly through the induction of different types of cytokynes and especially TSLP (Thymic Stromal Lymphopoietin), a marker acting a key role in the allergic diseases such as AD (Kinoshita H, 2009).

The above is intended as illustrative, but not limiting, purpose. Furthermore, one skilled in the art will be able to understand that modifications can be made without departing from the scope of the present invention.

EXAMPLES

The following examples are aimed to further illustrate some embodiments of the present invention and therefore are not to be intended as limiting the same. In the following experimentation 1 a pharmaceutical composition was tested according to the invention (hereinafter, in experimentation 1, composition of the invention) as reported below.

Composition(s) of the invention:

- cannabidiol 2.000 mg;

- Lactobacillus ferment lysate 9.600 mg; - Bifida ferment lysate 3.750 mg;

- ceramide 1.750 mg;

- cholesterol 5.000 mg.

Experimentation 1

Using ELISA techniques by application 6 replicates according to pharmaceutical protocols of 5 mg/cm ² of composition(s) of the invention (not diluted) in emulsion in culture mean at 37°C with 5% CO ₂ for 10 days and then pre-incubated for 24 and reactivated for 6 hours. The results (see fig. 1) demonstrate an inhibition of timic stromal limphopoietin pro-inflammatory (TSLP) cytokines equal to 42% and of interleukin-6 (IL-6) equal to 71%. Variations on interleukin-8 (IL-8) was not observed, since the tissue was not injured (Amarbayasgalan T., 2013). Cytokines hyperexpressed in patients suffered from AD during the inflammatory phase activate mastocytes which in turn result in histamine release. The results show, on 6 replicates, statistically significant values with a p value <0.001.

The European Task Force on Atopic Dermatitis (ETFAD) developed the SCORAD index (SCORing AD). Patients involved (20+20) had a SCORAD index from 10 to 25.6 with a mean of 21.5 and a SEM of 0.1. The control group had an SCORAD index from 15 to 25 with a mean of 21.5 and a SEM of 0.7. The controlled and randomized double-blind study carried out, after 28 days, showed a reduction of 29.4% of the SCORAD index compared to placebo highlighting a reduction of 10.2%. The variation was statistically significant.

For itch evaluation, VAS scale was used. Patients involed (20+20) had a VAS index from 0.5 to 9 with a mean of 6.2 and a SEM of 0.6. The control group had a VAS index from 0.8 to 8.7 with a mean of 5.2 and a SEM of 0.5. The controlled and randomized double-blind study carried out, after 28 days, showed a reduction of 52.2% of VAS scale compared to placebo highlighting a reduction of 23.1%. The variation was statistically significant.

In order to evaluate the Transepidermal Water Loss a TEWAMETER T300 (Couraza - Khazaka) was used, involving 20 + 20 patients in a controlled randomized double blind study. Compared to the actives-free control group, results showed that at TO patients had an average data of 25.3 with a SEM of 2.3, while the control group of 21.4 with a SEM of 2.2. The treated group showed a reduction of 20.1% compared to placebo highlighting a reduction of 2%. The variation was statistically significant.

In order to evaluate skin hydration, a CORNEOMETER CM 825 (Couraza - Khazaka) was used, involving 20 + 20 patients in a randomized controlled double blind study. Compared to the actives-free control group, results showed that at TO patients had an average data of 21.0 with a SEM of 1.3, while the control group of 25.3 with a SEM of 2.2. The treated group showed a reduction of 28.7% compared to placebo highlighting a reduction of 2,1%. The variation was statistically significant.

Values are reported in figure 2.

Experimentation 2 fincluding examples 1 2 3. 4 51

Experimental tests were carried out in order to demonstrate the safety and therepeutic results obtainable by the composition of the invention. The composition used in the present sperimentation 2 is reported below, considering 1.54 g/die. Composition used in experimentation 2 (also indicated as “Composition exper. 2” or “ATOPIC

CREAM”)

- Cannabidiol 3,080 mg;

- Lactobacillus ferment lysate 30,184 mg;

- Bifida ferment lysate 5,775mg;

- colesterol 7,700 mg

- ceramide 3,311 mg

EXAMPLE 1 - Safety TEST/ASSAY Evaluation of skin irritation on 3D human reconstituted epidermis (EpiDermTM Model) through the investigation of: Cytotoxicity by MTT test

The purpose of this test is to evaluate if the tested material is or is not a skin irritant, according to the method described in OECD 439. This method uses human artificial skin model (EPISKIN™, EpiDerm™, SkinEthicTM RHE, epiCS®) to assess the skin irritation of chemical substances, mixture and to properly label them to this respect if applicable. The test is based on the evaluation of cell survival after the exposure to the substance through MTT assay and by comparison with epidermis treated with phosphate buffer only (negative control). The MTT method is a colorimetric assay that allows to determinate the percentage of cells alive within an in vitro cultured tissue. This assay is based on the ability of the mitochondrial succinate dehydrogenase enzyme to metabolise the nitro-blue tetrazolium salt, giving a coloured compound that can be measured by spectrophotometer reading.

The test has been carried out according to the method described in OECD 439 and in the MatTek validated protocol. In vitro test systems employed used tridimensional artificial system of human epidermis. A reconstructed artificial human skin model comprising normal human epidermal keratinocytes, growing as an integrated three-dimensional cell culture model, perfectly mimicking the human skin in vitro. The model exhibits normal barrier functions (presence of a well-differentiated stratum corneum). It was supplied by MatTek (Bratislava, Batch 34123).

3D epidermis model grown in vitro (MatTek®). Different layer of epidermis proliferation can be pointed out. At the surface, a well differentiated stratum corneum is evident, over which the product to be tested is placed. Below the epidermis there is a semipermeable membrane that communicate with the inferior well where the culture medium is placed (undernatant).

The sample is tested as that. The positive control SLS is dissolved at 5% in water. PBS alone has been used as negative control. 30m1 of the sample and controls have been applied on each epidermis unit in three replicates. The exposure has been carried out for 35’ at 37°C and 25’ at room temperature. At the end of the exposure period the product is removed with multiple washings with PBS and the tissues were further incubated at 37°C, 5% C02 for 42 h. At 24 h the medium has been discharged and renewed. At the end of the exposure, the viability assay is performed to evaluate the cell survival in the epidermis units.

MTT cell viability assay Epidermis units are treated with lmg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) for 3 h at 37°C. The solution is than removed and replaced with isopropanol, with further 2 h incubation at room temperature. 2 aliquots of every sample are transferred to a 96 well plate for the reading. The absorbance is read at the wavelength of 570 nm with a colorimeter (Tecan Infinite 200 PRO model) equipped with a microplate reader. The absorbance values are corrected by subtracting the reading of the blanks (diluent only).

Expression of results

The results are expressed in terms of viability:

% of cell viability = [mean OD(570 nm ) test product / mean OD(570 nm) negative control] x 100 Acceptance criteria of method For negative control (CN): the mean OD value of the 3 tissues has to be > 0.8 e < 2.8 the standard deviation has to be < 18%

For positive control (CP): the viability mean (expressed as % of the NC) has to be < 20% the standard deviation has to be < 18%

For the sample: the standard deviation has to be < 18% Results interpretation

Mean cell viability is linked of the irritant effect of the sample. The following criteria for the skin irritation assay interpretation apply:

Results and Conclusions Assay validity requirements

Acceptance criteria of the assay (see §) comply, hence the assay is valid. Results Conclusions: the composition exper. 2 tested is not a skin irritant EXAMPLE 2

Effects of the composition exper. 2 on IL-6, IL-8 and TSLP release in (Poly(LC) + IL-lα)-stimulated reconstructed human epidermis

The potential anti-inflammatory effects of Composition exper. 2 were evaluated in a reconstructed human epidermis (REIE) model stimulated with an inflammatory mix: (Poly(EC) + IL-lα). This stimulation results in keratinocytes in a phenotype mimicking the early phase of atopic dermatitis (AD), notably through the induction of different kind of cytokines and especially TSLP (Thymic Stromal Lymphopoietin), a marker which plays a key role in allergic diseases such as ADI .

The effects of Composition exper. 2 were assessed on IL-6, IL-8 and TSLP release by RHE under (Poly(LC) + IL-lα)-stimulated conditions, using specific ELISA kits.

Materials and Methods Biological Model

Test compound

Culture and treatment

At day 10, the RHE were placed in assay medium and incubated for 6 hours. The test compound was then topically applied or not (control) on the surface of the RHE and the tissues were pre- incubated for 24 hours. After pre-incubation, the treatments were renewed and assay medium containing the inflammatory mix (Poly(I:C) + IL-lα), tested at 1 pg/ml + 10 ng/ml, was added in all conditions except in the non-stimulated control. The RHE were then incubated for 48 hours.

All experimental conditions were performed in n=6. Enzyme-Linked Immunosorbent Assay < ELISA )

The quantity of cytokines, IL-6, IL-8 and TSLP released in culture subnatants was measured using specific ELISA kits according to the supplier’s instructions. Under non-stimulated control condition, a significant basal level of IL-8 and TSLP release was detected in RHE subnatants (106 and 11 pg/ml, respectively) whereas no IL-6 release could be detected (< 9 pg/ml). The treatment of RHE for 48 hours with the inflammatory mix (Poly(LC) + IL-la), resulted in a very strong stimulation of IL-8 release (5410 pg/ml), and also in a clear and significant increase of IL-6 release (219 pg/ml), and a weak stimulation of TSLP release (51 pg/ml). Altogether, these results were expected and validated the assay. Under the experimental conditions of this study, composition exper. 2, topically applied at 5 mg/cm ² , induced an inhibition of IL-6 and TSLP release by the (Poly(LC) + IL-la)-stimulated RHE (71% and 42% of relative inhibition, respectively). No effect was observed on IL-8 release. The results therefore indicate that compound composition exper. 2, displayed partial anti-inflammatory properties (no effect on IL-8) in a model mimicking early phase of atopic dermatitis.

In the following table 1 below, the effect of the composition exper. 2 on IL-6 release by RHE under (poly(LC) + IL-lα)-stimulated conditions.

TABLE 1

In the following table 2 below, the effect of the composition exper. 2 on IL-8 release by RHE under

(Poly(LC) + IL-lα)-stimulated conditions. TABLE 2

In the following table 3 below, the effect of the composition exper. 2 on TSLP release in (Poly(I:C) + IL- 1α)-stimulated conditions. TABLE 3

EXAMPLE 3

Skin sensitization assessment through the Reduced Local Lymph Node Assay ( rLLNA )

Test/Assay The assay measures cell proliferation in the lymph node proximal to the application site of the sample as a function of the sensitisation potential. The cell proliferation is evaluated through the titriated tymidine incorporation into DNA of dividing lymphocytes.

Purpose of the test is to evaluate the sensitising potential of a medical device using an in vivo test, the reduced Local Lymph Node Assay (rLLNA) according to the OECD 429 guideline [1] and to ESAC statement on the Reduced Local Lymph Node Assay of 27th April 2007. The LLNA assay is foreseen by the UNI EN ISO 10993-10 rule as alternative to the classic Magnusson & Kligman test (2). The test is performed according to the method described in OECD 429 (1) and in the ISO 10993-10:2010 standard (2). This test is an in vivo mouse assay, that is considered as an alternative method respect to the conventional guinea-pig methods because it reduces the number of animals and their stress. Furthermore, the reduced assay has been designed to further reduce the number of animals and it is advised to test substances or products supposed not to be sensitizers. In fact, differently from LLNA, the rLLNA is carried out testing the product at only one concentration, the highest compatible with the application of the sample. If the result will be different from what supposed suggesting a sensitising potential for the sample, it will be necessary to retest the substance at least with 3 scalar concentrations, to allow the determination of the potency. In the protocol, groups of mice (CBA/j strain) receive daily topical applications of the test chemical on the dorsum of both ears. Six days following the initiation of exposure, all mice receive an intravenous injection of 3H-labeled thymidine and then animals are sacrificed and draining (auricular) lymph nodes are excised, for measuring the incorporation of 3H-labeled thymidine. For each concentration of the test material a stimulation index (SI) is derived relative to the concurrent vehicle control, which classifies the test substance as a sensitising agent or not. This test is an in vivo mouse assay, that is considered as an alternative method respect to the conventional guinea-pig methods because it reduces the number of animals and their stress. Furthermore, the reduced assay has been designed to further reduce the number of animals and it is advised to test substances or products supposed not to be sensitizers. In fact, differently from LLNA, the rLLNA is carried out testing the product at only one concentration, the highest compatible with the application of the sample. If the result will be different from what supposed suggesting a sensitising potential for the sample, it will be necessary to retest the substance at least with 3 scalar concentrations, to allow the determination of the potency. In the protocol, groups of mice (CBA/j strain) receive daily topical applications of the test chemical on the dorsum of both ears. Six days following the initiation of exposure, all mice receive an intravenous injection of 3H-labeled thymidine and then animals are sacrificed and draining (auricular) lymph nodes are excised, for measuring the incorporation of 3H- labeled thymidine. For each concentration of the test material a stimulation index (SI) is derived relative to the concurrent vehicle control, which classifies the test substance as a sensitising agent or not. The test is performed according to the method described in OECD 429 (1) and in the ISO 10993-10:2010 standard (2). Eight- weeks old, CBA/j, female mice from Charles River Italia were used. Mice were divided into 3 groups of 5 animal each (Tab 2.4.1; 2.4.2; 2.4.3). Four mice per group were tested. Mice were housed in Standard Pathogen Free (SPF) conditions in a microbiologically controlled animal facility. Husbandry was at 20°C with 12 hours continuous artificial light within each 24-hrs period. Mice were housed at a density of 4 mice per cage since arrival to the animal facility. They were housed 7 days for acclimatization before testing. The Animal House Facility is a barrier facility and procedures include provision of sterilized bedding, autoclaved feed, and filtered drinking water. Standard laboratory rodent diet is used (see enclosure A). All caging equipment is washed in barrier processing facilities and autoclaved. All animals are housed in filter top cages (static microisolators). Individually ventilated cages and static microisolator cages are changed every week. Mice are transferred to clean cages using disinfected forceps. The cage, food, and bedding are autoclaved and the water is filtered (autoclaved only for immunodepressed mice). Procedures for barrier facilities include sanitation or sterilization of all supplies and equipment. Personnel working in barrier facilities wear sterilized clothing including bodysuit with hood, shoe covers, caps, masks, double gloves and passage via air shower prior to entry. Sentinels are exposed to dirty bedding from cages in their respective rooms. Sentinels are euthanized for serology, bacteriology, parassitology, and pathology screening every three months and/or annually as specified in the report (see enclosure B).

Mice of each group were weighted at time zero (TO) and after the 6-days time (T6d) of the protocol. Weights of individuals animals (in grams) and mean values were as follows:

Table 4

Group 1 : Negative control Acetone/olive oil (4/1 vol/vol) Table 5

Group 2: Positive control

DNFB 0.02%

Table 6

Group 3: treatment with the test sample Composition exper. 2; lab batch

Treatment and Exposure

The test substance was a cream, which appeared sufficiently persisting following its application on mouse ear. It was not expected to be directly toxic or systemically irritating. Thus, it was used for direct application (100%) to the mouse ear. As a negative control, acetone/olive oil (4/1 vol/vol) was used. As a positive control, DNFB 0.02 % was used. This concentration was chosen on the basis of the EC50 value of this substance, i.e. the concentration capable to elicit a 3x increase in 3H-thymidine incorporation in this test, without overt toxicity.

Test product and controls were applied daily for three days (25 mΐ per each ear pinnae) with a micropipette. Six days following the initiation of exposure all mice receive an intravenous injection of 3H-labeled thymidine and five hours later animals are sacrificed and draining (auricular) lymph nodes are excised and pooled for each experimental group. A single cell suspension of lymph nodes is prepared by gentle mechanical disaggregation and the cells washed and resuspended in trichloroacetic acid (TCA) for at least 12h at 4°C. Precipitates are resuspended in TCA and transferred to an appropriate scintillation fluid. The incorporation by draining lymph nodes of 3HTabeled thymidine is measured by scintillation counting and recorded as mean disintegrations per minute (dpm) for each experimental group. For each concentration of the test material a Stimulation Index (SI) is derived relative to the concurrent vehicle control.

Expression of results

Results for each treatment groups are expressed as the mean Stimulation Index (SI). The SI is the ratio of the mean dpm/mouse within each test product treatment group and the positive control treated group against the mean dpm/mouse for the solvent/vehicle treated control group.

Acceptance criteria of method For positive control S.I ≥ 3 When the SI is 3 or more, the test substance is regarded as a skin sensitizer.

Results and conclusions Assay validity requirements

Acceptance criteria of the assay (see § 2.3) comply, hence the assay is valid. Results No signs of general toxicity were observed. No animals had any weight loss. Locally, no signs of irritation were observed in any animal, including the DNFB treated mice. This last observation is likely due to the relatively low concentration (0.02%) of DNFB we used. DNFB showed an evident sensitising effect, as expected. In the following tables the obtained dpm and SI values are shown.

Group 1 : Negative control Acetone/olive oil (4/1 vol/vol) (SI= 1 .00)

Group 2: Positive control DNFB 0.02% (S.I. = 3.13)

Group 3: treatment with the tested sample Composition exper. 2; batch: lab batch (S.I. = 1.09)

Conclusions

In the above experimental conditions, the sample: of composition exper. 2 is not a skin sensitizer EXAMPLE 4 The study was aimed to assess the efficacy and the safety of the composition exper. 2 having activity on AD symptoms. In order to reach this goal a double-blind randomized placebo-controlled clinical study is carried out on 40 male and female subjects aged over 18 years old presenting a clinical feature characterized by atopic dermatitis on face/body (SCORAD - between 15 and 25). According to a predisposed randomization list included subjects are divided in 2 groups: - 20 subjects use the active ingredient

- 20 subjects use the placebo

The study foresaw 28 days of products use. Clinical/instrumental evaluations of the parameters under study were carried out at baseline (TO) and after 7 (T7), and 28 (T28) days of products use. The study was moreover integrated with a subjects self-assessment filled-in at the end of the study. A total of 40 female and male subjects have been enrolled according to the inclusion/non-inclusion criteria reported here below.

Inclusion criteria:

- Good general health;

- Female or male sex; - Caucasian subjects (phototype I to IV);

- Age more than 18 years old;

- SCORAD between 15 and 25;

- Subjects who have not been recently involved in any other similar study; - Willingness to stop topical or systemic treatments at least 2 weeks before the start of the study (wash-out period);

- Willingness to use during all the study period only the product to be tested;

- Willingness to not use products likely to interfere with the products to be tested; - Willingness to not vary the normal daily routine;

- Subject aware of the study procedures and having signed an informed consent form.

Non-inclusion criteria:

- Subjects who do not meet the inclusion criteria;

- Pregnant/breastfeeding female or who have planned a pregnancy during the study period; - Positive history for atopy or hypersensitive skin;

- Subjects under systemically pharmacological treatment;

- Subjects under pharmacological treatments that could interfere with the test;

- Subjects with known or suspected sensitization to one or more test formulation ingredients.

The study duration is 28 days (4 weeks). Clinical visits are planned at baseline (TO) and after 7 (T7) and 28 (T28) days of product use.

Screening - T-2 weeks

During the screening visit (T-2) the investigator evaluates if the subject is eligible to participate in the study. The following procedures are carried out:

- signature of the Informed Consent Form; - checking of the inclusion/non-inclusion criteria;

- start of the wash-out period;

- fixing the date of TO visit after 14 days.

Basal visit (TO)

The following procedures are carried out: - Checking of maintenance of subject eligibility;

- Objective evaluation - SCORAD;

- Subjective evaluation - YAS score;

- Instrumental evaluations - skin moisturization and TEWL;

- POEM questionnaire; - supplying the study active product / the placebo in accordance with the randomization list;

- fixing the date of the 1st check visit after 7 days.

T7 visit

The following procedures are carried out: - Checking of maintenance of subject eligibility;

- Clinical evaluation - product tolerability;

- Objective evaluation - SCORAD;

- Subjective evaluation - VAS score;

- Instrumental evaluations - skin moisturization and TEWL; - POEM questionnaire;

- fixing the date of the last check visit after 21 days.

Final check visit (T28)

The following procedures are carried out:

- Checking of maintenance of subject eligibility; - Clinical evaluation - product tolerability;

- Objective evaluation - SCORAD;

- Subjective evaluation - VAS score;

- Instrumental evaluations - skin moisturization and TEWL;

- POEM questionnaire; - filling of the self-assessment questionnaire.

Materials and methods Parameters below reported are assessed under controlled ambient conditions (T = 22±2°C and RH = 40- 60%).

Subjects are left to acclimatize to ambient condition for 15-20 minutes before the check visits.

CLINICAL, OBJECTIVE AND SUBJECTIVE EVALUATIONS Objective evaluation - SCORAD

The European Task Force on Atopic Dermatitis (ETFAD) has developed the SCORAD (SCORing AD) index to create a consensus on assessment methods for AD, so that study results of different trials can be compared. To measure the extent of AD, the rule of nines is applied on a front/back drawing of the patient's inflammatory lesions. The extent can be graded 0-100. The intensity part of the SCORAD index consists of six items: erythema, oedema/papulation, excoriations, lichenification, oozing/crusts and dryness. Each item can be graded on a scale 0-3. The subjective items include daily pruritus and sleeplessness. Both subjective items can be graded on a 10-cm visual analogue scale. The maximum subjective score is 20. All items should be filled out in the SCORAD evaluation form. The SCORAD index formula is: A/5 + 7B/2 + C. In this formula A is defined as the extent (0-100), B is defined as the intensity (0- 18) and C is defined as the subjective symptoms (0-20). The maximum SCORAD score is 103.

Subjective evaluation of itching (VAS score)

Itching feeling, as perceived by the volunteers, is recorded according to a Visual Analogue Scale (VAS 0- 10) where 0 is “no feeling” and 10 is “worst feeling”.

POEM questionnaire At each study time subjects will be asked to fill a Patient-Oriented Eczema Measure (POEM) questionnaire, a measurement tool for the self-measure of atopic dermatitis (AD) severity. The questionnaire is composed by 7 questions, each one carries equal weight and is scored from 0 to 4. Results are summed up and the obtained score can be helpful to classify the severity of the eczema as follow:

Clinical evaluation of product tolerability

Tolerability of the treatment was closely followed by the study principal investigator during the course of the study. Subjects had access to the investigators in case of intolerance reactions via a contact phone number provided with the informed consent form. If a subject reported an event, the principal investigator had to decide if it was pertinent or not. If yes, she reported it as a cosmetic or an adverse event. INSTRUMENTAL EVALUATIONS Skin moisturization

The measurement of skin moisturization is based on the Corneometer® method. Corneometer® method is based on the dielectric constant of water. The probe shows changes of capacitance according to the moisture content of the skin. An electric scatter field penetrates the very first layers of the skin (10-20 pm) and determines the dielectricity. The used device is the Corneometer® CM 825 (Courage+Khazaka, electronic GmbH).

Transepidermal Water Loss Transepidermal water loss is measured indirectly using a Tewameter® TM 300 (Courage+Khazaka, electronic GmbH). The measurement is based on the diffusion law, as described by the equation here below: where: A is the surface in m ² | m is the water transported (in g) 11 is the time (h) | D is the diffusion constant (0.0877 g/m(h(mm Hg)) | p is the vapor pressure of the atmosphere (mm Hg) | s is distance from skin surface to point of measurement (m).

The diffusion flow dm/dt indicates the mass of water, which is transported per cm ² in a specific period. It is proportional to the area A and the change of concentration per distance (dc/dx). D is the diffusion coefficient of water vapor in the air. The resulting density gradient is measured indirectly by two pairs of sensors (temperature and relative humidity) and is analyzed by a microprocessor. The measuring head of the probe is a narrow hollow cylinder (10 mm diameter and 20 mm height), in order to minimize influences of air turbulence inside the probe.

Digital macrophotography Digital pictures of the atopic dermatitis affected area are acquired at each experimental time using a reflex digital camera. The best 3 cases showing (active) product efficacy are reported in annex 1.

Self-assessment questionnaire

At the end of the study subjects are asked to express their personal opinion on the tested treatment by answering to a questionnaire about products acceptability and effects.

Results and Statistics Descriptive analysis

Demographic variables (age and sex) are reported. Data are summarized using frequency distributions (number and percentage) for categorical/ordinal variables. For continuous variables the following values are calculated:

- mean value;

- minimum value;

- maximum value;

- standard error of the mean (SEM); - individual variation/individual percentage variation;

- mean variation/mean ok the percentage variations.

All the calculations are done using a Microsoft® Excel 2013 (vers. 15.0.4885.1001; Microsoft, USA) worksheet running on Microsoft® Windows 8.1 Professional (Microsoft, USA).

The results of self-assessment questionnaire are calculated as percentage (%) of subjects who assigned a particular judgment (among those proposed). For each question, the number of subjects related to each judgment is counted → (number of subjects) and then divided by the total number of subjects → % of answers.

Statistical methods

The instrumental data are submitted to Bilateral Student’s Test t; clinical data are submitted to Wilcoxon signed test. Intra-group statistical analysis is made on the raw data vs. TO (T7vsT0 and T28vsT0) by means of Student’s/Wilcoxon Test for paired data; inter-group statistical analysis is made on the data variations versus TO (active vs. placebo), by means of Student’s/Wilcoxon Test for unpaired data. The statistical software used for statistical analysis is: NCSS 10 statistical software (NCSS, LLC. Kaysville, Utah, USA) running on Windows Server 2008 R2 Standard (Microsoft, USA). Interpretation of results

The study here above reported was designed to demonstrate the test product claim(s) in the current framework proposed by Commission Regulation (EU) No 655/2013. Endpoints are measured using techniques currently accepted in the cosmetic field while biases are minimized by procedure(s) standardization according to ISO 9001 Quality Management System. Data are analyzed and interpreted by skilled technician according to both descriptive and inferential statistical analysis procedures. Due to the lack of reference values in the cosmetic field, statistical significance (for instrumental analysis) and percentage of subjects showing an effect (for clinical/sensorial endpoints) are the primary criterion to evaluate the correspondence between the proposed claim(s) and the study output(s). In particular Intragroup (vs. TO) or intergroup (eg. active vs. placebo, treated vs not treated) statistical analysis criterion to reject the null hypothesis (no product effect) is set at p<0.05. For clinical evaluations, the positive effect of the product on the measured parameter is confirmed if more than 50% of the subjects register an improvement. Finally, for the self-assessment questionnaires, the performance and the pleasantness of the product must be perceived by at least 60% of the subjects. Whenever reference values or threshold values exists those values are used to validate product claim(s).

Conclusions

According to the obtained results we can conclude that the tested treatment determined a significant improvement of the clinical and instrumental monitored parameters, index of an efficacy in improving atopic dermatitis condition. EXAMPLE 5

In vitro efficacy study - evaluation of the skin barrier effect and protection of a cosmetic product The study described in this report concerns the in vitro evaluation of the capability of the tested product to preserve the integrity of the skin barrier by using a model of in vitro reconstructed epidermis treated with a solution of surfactant in order to alter the skin barrier. This evaluation was carried out by measurement of the TEER (Transepithelial/transendothelial Electrical Resistance) of in vitro reconstructed epidermis following the preceding pre-treatment and the contemporary treatment with the sample.

Experimental protocol provided:

- untreated epidermis (CTR-);

- epidermis treated with 50 pL of surfactant solution; - epidermis treated concomitantly with 50 mg of tested product and 50 pL of surfactant solution;

- epidermis pre-treated with 50 mg of tested product and exposed to 50 pL of surfactant.

MATERIALS AND METHODS

Experimental model The biological model used in the test consists of three-dimensional reconstructed human epidermis, built from primary cultures of keratinocytes (EpiDerm - MatTek, batch 34103). Particularly, this is 0.6 cm2 reconstructed epidermis by airlifted culture of keratinocytes for 17 days in chemically defined medium on inert polycarbonate filter at the air/liquid interface. The test system has an ultra-structure (tissue morphology and thickness) very similar to the human in vivo skin. All the tissue units were subjected to quality controls to ensure the suitability of the biological model and verify the absence of pathologies to ensure the operator safety.

Preparation of the test item and tissue exposure

For the test execution, reconstructed human epidermis was:

- treated with product under study and exposed to surfactant solution for 24h (contemporarery treatment); - Pre-treated with tested product for 24 h (pre-treatment) and then, after wash, exposed to a solution of surfactant for 24 hours. In particular, a solution of 2% Sodium dodecyl sulfate (SDS) was used. At the end of the experimental treatment the tissues were washed with PBS and TEER were measured. The results were compared to negative control (untreated epidermis, CTR-) and positive control (epidermis treated only with SDS, CTR+). In summary, the experimental protocol provided: - untreated epidermis (CTR-);

- epidermis treated with 2% SDS solution (CTR+) for 24 hours;

- epidermis pre-treated with product PURIMED CH1025 (1) for 24 hours, washed by the product, and then exposed to 2% SDS solution for 24 hours;

- epidermis treated with product PURIMED CH1025 (1) and exposed to 2% SDS solution for 24 hours. The test was conducted in triplicate.

TEER Measurement

The integrity of the skin barrier was determined by measuring the TEER performed using the MilliCell- ERS meter and electrode system (Millipore AG). Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in models of endothelial and epithelial cells. TEER measurement is a very sensitive and reliable method to confirm the integrity and permeability of the cell mono- and multilayer. For electrical measurements, two electrodes are used, with one electrode placed in the upper compartment and the other in the lower compartment and the electrodes are separated by the cellular monolayer. In theory, the ohmic resistance can be determined by applying a direct current (DC) voltage to the electrodes and measuring the resulting current. The ohmic resistance is calculated based on Ohm's law as the ratio of the voltage and current. An increase in TEER detected with the electronic circuit of the Millicell® ERS-2 meter and its electrode is an indication of cell monolayer health and confluence.

Statistical analysis Obtained results were subjected to statistical analysis by means of Student test. The variations versus negative control are considered not significant for °p>0.05 while the variations versus positive control are considered significant for *p<0.05.

Conclusion:

Under the experimental conditions described showed a potential skin barrier effect