Liposomal preparation encapsulating biotin

Abstract

A liposomal preparation of biotin comprising a lipid bilayer membrane encapsulating an aqueous solution of biotin, thereby constituting a biotin containing liposome. Compositions comprising these liposomes and their cosmetic and medical uses in hair or skin diseases.

Description

Case Ref. P274WO IPTector® Liposomal preparation of Biotin. Technical Field

[0001] The present invention relates to liposomal preparations of vitamin B, and in particular biotin and to non-therapeutic and therapeutic uses of biotin or liposomal biotin. Background

[0002] Biotin and biosynthetic production of biotin is known for example from prior patent applications WO2019 / 012058, WO2020 / 148351, WO2023 / 285585, and WO2021 / 254927.

[0003] Biotin, also known as vitamin B7 or vitamin H is a well-known molecule from the industry and from consumers. It is widely used as a nutritional complement. It has gained commercial popularity for its claimed benefits on healthy hair and nail growth. Despite its reputation, there is limited research to support the effectiveness of biotin in healthy individuals. Biotin is readily found in food, however, congenital or acquired biotin deficiency can happen. Typical signs of biotin deficiency include alopecia, eczematous skin rashes, seborrheic dermatitis, showing that biotin has a fundamental role in ensuring skin integrity and beauty. Biotin’s function in protein synthesis and more specifically, in keratin production, explains its contribution to healthy nail and hair growth.

[0004] Biotin is a required cofactor for carboxylase enzymes that become activated once they are joined together by holocarboxylase synthase. These enzyme complexes play an important role in multiple metabolic processes including gluconeogenesis, fatty acid synthesis, and amino acid catabolism.

[0005] Biotin serves as a cofactor for a number of carboxylases, such as 3- methylcrotonyl-CoA carboxylase in the leucine catabolic pathway, propionyl-CoA carboxylase in the valine catabolic pathway, and acetyl -CoA carboxylase, the rate-limiting enzyme for fatty acid synthesis. Additionally, biotin is a cofactor for the enzyme pyruvate carboxylase. Through its role in pyruvate carboxylase, biotin is essential for the replenishment of the citric acid (or Krebs) cycle metabolites which are essential for normal cellular functions.

[0006] While biotin it is required by all organisms, it is synthesized only by bacteria, yeast, moulds, algae, and some plant species. Biotin comes from a variety of sources, but very few would be considered “biotin rich”. So far, biotin has been industrially produced by chemical synthesis. While the effects of biotin have been studied on various types of cell cultures, little is known about the topical effects of biotin. There is a need to develop applications for biotin as well as formulations particularly suitable for such applications.Case Ref. P274WO IPTector® Summary

[0007] The present invention provides improvements offering solutions to certain drawbacks of the background art. One objective of the invention is to provide new formulation and delivery system for biotin and accordingly a liposomal preparation of biotin is provided comprising a lipid bilayer membrane encapsulating an aqueous solution of biotin, thereby constituting a biotin containing liposome.

[0008] Another objective of the invention is to provide new and improved compositions of biotin, and accordingly a composition is provided comprising the liposomal preparation of biotin described herein.

[0009] A further objective of the invention is to provide new and improved application of biotin and / or liposomal biotin or the composition comprising these, and accordingly provided herein is a cosmetic non-therapeutical use of the liposomal preparation for skin or hair care.

[0010] A further objective of the invention is to provide new and improved therapeutic applications of biotin or the liposomal preparation thereof. Accodingly a method is provided for treating or preventing skin or hair disease using biotin or the liposomal preparation described. Description of drawings and figures

[0011] The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out. Figure 1. Schematic representation of the signalling pathways regulating melanogenesis, with essential involvement of tyrosinase. Figure 2 shows a microscopic image of Normal Human Epidermal Melanocytes (NHEM) in culture as used in the example. Figure 3. Graphical representation of the results showing normalized cell viability after treatment of human melanocytes cells with Biotin, compared to the non-treated control. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as *** for p < 0.001 and **** for p < 0.0001. Figure 4. Graphical representation of the results showing cell viability after UVA exposure (left) and normalized melanin levels (right) of human melanocytes after treatment with Biotin. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. Figure 5. Graphical representation of the results showing normalized melanin levels after subtractingCase Ref. P274WO IPTector® basal values (C), of human melanocytes after treatment with Biotin. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. Figure 7. Graphical representation about the penetration capacity of different radiations (UVC, UVB, UVA and Visible) into the skin. Figure 8 shows a microscopic image of human keratinocytes in culture (HaCaT cell line) used. Figure 9 shows a graphical representation of the results showing normalized cell viability after treatment of human keratinocytes cells with Biotin, compared to the non-treated control. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as **** for p < 0.0001. Figure 10. Graphical representation of the results showing UVA-induced ROS levels. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as **** for p < 0.0001. Figure 11 shows a graphical representation of the results showing cell viability after UVA exposure and UVA-protection after treatment with Biotin after subtracting ROS basal levels. Data are presented as mean ± standard error of the median (SEM). Statistical significance is depicted as ** for p < 0.01. Figure 13 shows biotin quantification expressed as µg of biotin per mg of total protein in the skin. Figure 14 shows the total content of biotin expressed as µg of biotin per cm2of stratum corneum (assuming a stratum corneum thickness of 15 µm). Figure 15 shows the overlap of the quantification of the total content of biotin (expressed as µg of per cm2 of stratum corneum) after application of the products Biotin or LipoBiotin. Figure 16 shows the amount of biotin absorbed into the skin during the 120 min lapse for each test subject and for all of them (average). Figure 17 shows the amount of biotin absorbed into the skin during the 120 min lapse for each test subject and for all of them (average). Figure 18 shows the comparison of the average amount of biotin absorbed into the skin during the 120 min lapse for the products Biotin and LipoBiotin. The data are presented as µg of biotin absorbed per cm2of stratum corneum (assuming a depth of 15 µm). Figure 19 shows the device used in the Crema Facial study: Crème P.3041. “Crema Facial ref CAIV01- BS04-2403 lote:2403044 CORRESPONDIENTE AL ACTIVO” Figure 20 shows skin behavior against suction when assessed by Cutometer® MPA 580. Figure 21 shows graphical representation of the skin firmness and elasticity. The Mean and Standard Error of the Mean (SEM) are shown. ** Represents statistical significance with p-value < 0,005. Figure 22 shows representative image of a skin gloss measurement conducted with Skin-Glossymeter®Case Ref. P274WO IPTector® GL 200. Figure 23 shows representative image of brown spots measurement conducted with VISIA-CR. Figure 24 shows graphical representation of skin brightness (gloss). The Mean and Standard Error of the Mean (SEM) are shown. *Represents statistical significance with p-value < 0,05. Figure 25 shows graphical representation of brown spot area, number and contrast. The measure displayed are D28-D0. The Mean and Standard Error of the Mean (SEM) are shown. *Represents statistical significance with p-value < 0,05. Figure 26 shows graphical representation of the water content (^g) per cm2of SC before (Day 0) and 28 days after the treatment. The Mean and S.E.M. are shown. Asterisks represent statistical significance as ** for p < 0.01. Figure 27 shows increase in water content in the skin before (Day 0) and 28 days after the treatment for each volunteer individually (left panels) and the average of all of them (right panels). The increase in water content is expressed as µg water (top panels) or in percentage (bottom panels). The Mean and S.E.M. are shown. Figure 28 shows graphical representation of the SC thickness (^m) before (Day 0) and 28 days after the treatment. The Mean and S.E.M. are shown. Asterisks represent statistical significance as * for p < 0.05. Figure 29 shows iIncrease in the stratum corneum thickness before (Day 0) and 28 days after the treatment for each volunteer individually (left panels) and the average of all of them (right panels). The increase in stratum corneum thickness is expressed as µm (top panels) or in percentage (bottom panels). The Mean and S.E.M. are shown. Figure 30a shows the percentage of water as a function of depth is represented for Day 0 (baseline) and Day 28. Averaged values are represented in the top panel, while bottom panels show the individual values. Vertical lines in the top panel indicate the thickness of the SC for Day 0 (to the left) and Day 28 (to the right). Figure 30b shows the same data as in Figure 30a but with colors. Figure 31 shows graphical representation of the Ceramide and fatty acid content of the skin (arbitrary units) per cm2of SC before (Day 0) and 28 days after the treatment. The Mean and S.E.M. are shown. Asterisks represent statistical significance as ** for p < 0.01. Figure 32 shows percentage of the increase in Ceramide and fatty acid content of the skin in the SC before (Day 0) and 28 days after the treatment for each volunteer individually (left panel) and the average of all of them (right panel). The Mean and S.E.M. are shown. Figure 33a shows the averaged (top panel) or individual (bottom panels) amount of skin Ceramide and fatty acids as a function of depth is represented for Day 0 (baseline) and Day 28 (in arbitrary units).Case Ref. P274WO IPTector® Figure 33b shows the same data as Figure 33a but with colors. Figure 34 shows overlap representation of the graphs showing the amount of (i) water (Figure 30) and (ii) ceramides and fatty acids (Figure 33) as a function of depth. Circle legend indicates Day 28 measurements. Figure 35 shows graphical representation of results of self-assessment questionnaire. Figure 36 shows penetration of vectorised biotin versus free biotin. Figure 37 shows total amount of absorbed biotin for each volunteer. Figure 38 shows ceramides and fatty acids levels in the epidermis before and after application of a cream containing EraGlow beautin by Biosyntia™. Figure 39 shows effect of EraGlow beautin by Biosyntia™ on the synthesis of ceramides and fatty acids as a function of the epidermis depth. Incorporation by reference

[0012] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls. Detailed Description The features and advantages of the present invention is readily apparent to a person skilled in the art in view of the below detailed description of embodiments and examples of the invention with reference to the figures and drawings included herein. Definitions

[0013] Biotin as refered to herein refers to to compound of the general formula:

[0014] The term “liposome” as used herein refers to a a spherically shaped structure made up of a lipid bilayer membrane encapsulating an aqueous core. The lipid bilayer membrane can encapsulate a hydrophilic (water loving) ingredients in its core, or a lipophilic (oil loving) ingredients in the bilayerCase Ref. P274WO IPTector® membrane or both.

[0015] The term “brown spot” as used herein refers to freckles on sun-exposed skin which are ephelides (the plural of ephelis) and lentigines (the plural of lentigo). The difference between an ephelis and a lentigo is that an ephelis fades during the winter months while a lentigo persists in the absence of ultraviolet (UV) stimulation. Ephelides and lentigines can occur in the same individuals and the risk factors for both are generally the same. Ephelides are very common in fair-skinned people, especially in children with red hair, where the MC1R gene is thought to be the main gene involved. They are an inherited characteristic that also sometimes affects people with darker skin types. An ephelis is brown because of the pigment melanin. Melanin is made by melanocytes and diffused into keratinocytes. Melanin production by melanocytes decreases during the winter months and increases when the skin is exposed to the UV radiation in sunlight. The colour is due to the localised accumulation of melanin in keratinocytes. There is no increase in the number of melanocytes. Ephelides arise on an individual's mid face and sometimes more widely from early childhood onwards. As the person ages, this type of freckle generally become less noticeable. They are more prominent in summer but fade considerably or disappear in winter. An ephelis is usually less than 3 mm in diameter. Lentigines are brown flat lesions with a clearly defined edge. The most common type, solar lentigines, arise in middle age and result from sun damage. They are most often found on the face and hands, and they are larger and more defined than freckles. Other types of lentigo include ink spot lentigo and lentigo simplex. Lentigines are common in people with fair skin, but they also frequently arise in sun-exposed sites in people who tan easily or who have naturally dark skin. Lentigines are common after the age of 40 years, but they may also occur in younger people. Aspects and embodiments

[0016] In the first aspect a liposomal preparation of biotin is provided comprising a lipid bilayer membrane encapsulating an aqueous solution of biotin, thereby constituting a biotin containing liposome.

[0017] In some embodiments the liposomal preparation further comprises one or more ingredients selected from purified water, lecithin, vitamin E, glycerol, ethanol and / or potassium carbonate or combinations thereof. The liposomal preparation suitably comprises between 50% to 80 % by weight of water, such as between 60% to 70 % by weight, such as between 65% to 70 % by weight, such as between 67% to 69 % by weight. The liposomal preparation suitably comprises between 1% to 10 % by weight of lecithin, such as between 4% to 8 % by weight, such as between 6% to 7 % by weight, such as between 6,6% to 6,8% by weight. The liposomal preparation suitably comprises between 5% to 15% by weight wt of vitamin E, such as between 8% to 12 % by weight, such as between 9% to 11Case Ref. P274WO IPTector® % by weight, such as between 9,5% to 10,5% by weight. The liposomal preparation suitably comprises between 5% to 30% by weight of glycerol, such as between 10% to 25% by weight, such as 15% to 20% by weight, such as between 16% to 18% by weight. The liposomal preparation suitably comprises between 1% to 20% by weight of ethanol, such as between 2% to 15% by weight, such as 5% to 10% by weight, such as between 6% to 8% by weight. The liposomal preparation suitably comprises between 0,005% to 2% by weight of potassium carbonate, such as between 0,01% to 1% by weight, such as 0,05% to 0,09% by weight, such as between 0,06% to 0,08% by weight.

[0018] In some embodiments the the liposomal preparation comprises 0,01% to 10% by weight of biotin, such as between 0,05% to 1% by weight, such as 0,1% to 0,5% by weight, such as between 0,2% to 0,3% by weight of biotin.

[0019] In some embodiments the liposomal preparation comprises 0.06 %wt biotin.

[0020] In some embodiments the liposomal preparation comprises 0.02 %wt biotin.

[0021] In some embodiments the liposomal preparation comprises 2 %wt biotin.

[0022] In some embodiments the liposomal preparation comprises 25 to 50 %wt excipients selected from lechitin, vitamin E, glycerol, ethanol and / or potassium carbonate.

[0023] In some embodiments the liposomal preparation comprises 30 %wt excipients selected from lechetin, vitamin E, glycerol, ethanol and / or potassium carbonate.

[0024] In some embodiments the liposomal preparation comprises 30 %wt excipients selected from lechetin, vitamin E, glycerol, ethanol and potassium carbonate and 0.02 %wt biotin.

[0025] In some embodiments the liposomes have a diameter between 10 to 500 nm, such as 20 to 250 nm, such as 30 to 100 nm.

[0026] In a further aspect a new and improved composition is provided comprising the liposomal preparation of biotin described herein. The composistion is in some embodiments selected from bath preparations, body and hand preparations, skin or hair cleansing products, such as cold creams, cleansing lotions, liquids and pads), eye makeup, face and neck preparations, hair conditioners, hair preparations, hair sprays tanning preparations, manicuring preparations, moisturizing preparations, night skin care preparations, shampoos, skin fresheners, skin tonics, skin dressings, bath oils, bath soaps and detergents, eye lotions, eyeliners, foundations, hair dyes and colors, hair rinses, hair wave sets, makeup reparations, mascaras, nail polishes and enamels, paste masks, mud packs, skin care preparations, and sun tan preparations. In other embodiments the composition comprises between 0,001% to 10% by weight of the liposomal preparation, such as between 0,01% to 1% by weight, such as 0,1% to 0,3% by weight.

[0027] In a further aspect a cosmetic non-therapeutical use of the liposomal preparation for skin or hair care is provided. The cosmetic non-therapeutical use includes in some embodiments skin or hairCase Ref. P274WO IPTector® care providing photoaging reduction, hair conditioning, skin conditioning, skin lightening, skin brightening, reduction of senile lentigines, reduction of melanin, moisturizing of skin, oxygen elimination or a combination thereof. In other embodiments the skin or hair care comprises improvemenmts in one or more skin properies selected from pigmentation, UV protection, blemish spots, skin tone, hydration, ceramides and fatty acids contents, radiance, gloss, firmness and / or elasticity.

[0028] In a further aspect a therapeutic application of biotin or a liposomal preparation thereof is provided, including a method for treating or preventing skin or hair disease by administering biotin or a liposomal preparation thereof to a subject in need thereof in an effect amount to provide for said treatment or prevention. The skin or hair disease is preferably selected from skin inflammation, eczema, dermatosis, and / or psoriasis.

[0029] In a further aspect a method is provided for producing the biotin liposomal preparation or the composition described herein comprising mixing biotin with a lipid bilayer membrane at conditions allowing the formation of a spherical shaped nanostructure having an aqueous core comprising the biotin.

[0030] In some embodiments provided for herein is a therapeutic or non-therapeutic method for whitening melanocytes comprising contacting the melanocytes with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to whiten the melanocytes.

[0031] In some embodiments provided for herein is a therapeutic or non-therapeutic method for reducing melanin formation in melanocytes comprising contacting the melanocytes with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to reduce formation of melanin in the melanocytes.

[0032] In some embodiments provided for herein is a therapeutic or non-therapeutic method for depigmentation of the skin or hair of a human or animal comprising contacting the skin or hair with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to depigment the skin or hair.

[0033] In some embodiments provided for herein is a therapeutic or non-therapeutic method for protecting the skin or hair of a human or animal from ultraviolet light exposure, preferably UVA, comprising contacting the skin or hair with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to protecting the skin or hair from the ultraviolet light exposure.

[0034] In some embodiments provided for herein is a therapeutic or non-therapeutic method for increasing the firmness or elasticity of the skin or hair of a human or animal comprising contacting the skin or hair with biotin, preferably in the form of biotin liposomes in an amount and under conditionsCase Ref. P274WO IPTector® effective to increase the firmness or elasticity of the skin or hair.

[0035] In some embodiments provided for herein is a therapeutic or non-therapeutic method for increasing the brightness, tone, gloss and / or radiance of the skin or hair of a human or animal comprising contacting the skin or hair with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to increase the brightness, gloss or radiance of the skin or hair.

[0036] In some embodiments provided for herein is a therapeutic or non-therapeutic method for reducing or eliminating brown or blemish spots on the skin of a human or animal comprising contacting the brown or blemish with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to reduce or eliminate the brown or blemish spots on the skin.

[0037] In some embodiments provided for herein is a therapeutic or non-therapeutic method for increasing hydration of the skin of a human or animal comprising contacting the skin with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to increase hydration of the skin.

[0038] In some embodiments provided for herein is a therapeutic or non-therapeutic method for increasing the level of ceramide in the skin of a human or animal comprising contacting the skin with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to increase level of ceramide in the skin.

[0039] In some embodiments provided for herein is a therapeutic or non-therapeutic method for increasing the level of fatty acids in the skin of a human or animal comprising contacting the skin with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to increase level of fatty acids in the skin.

[0040] In some embodiments provided for herein is a therapeutic or non-therapeutic method for decrease aging of the skin of a human or animal comprising contacting the skin with biotin, preferably in the form of biotin liposomes in an amount and under conditions effective to decrease aging of the skin. Examples

[0041] Chemicals used in the examples herein e.g. for buffers and substrates are commercial products of at least reagent grade. Example 1 – preparation of biotin liposomes

[0042] Biotin was sources from a commercial vendor. For encapsulating biotin in a liposome the PlexoZome® liquid complex liposomal delivery technology was applied using the instruction of the commercial vendor (Phamako, Australia). PlexoZome® is a spherical shaped nanostructure made upCase Ref. P274WO IPTector® of a lipid bilayer membrane around an aqueous core. This structure encapsulates the hydrophilic biotin mainly in its core. The liposome preparation allowed for a faster and higher absorption in the skin, which significantly maximized the biotins functional benefits. The general characteristics of the biotin liposomes were as shown in table 1. Table 1 Test Specification Appearance Pale yellow to amber, clear to opaque, liquid Odour Characteristic Identification - FTIR Score ≥ 950 compared to in-house library Solubility 1 g freely soluble in 250 mL water Density (g / mL) 1.0 ± 0.1 pH 7.0 ± 0.5 Liposomal particle size analysis Z-Average (d.nm) < 100 Zeta Potential |negative charge|> 30 The biotin liposomes contained the ingredients as shown in table 2. Table 2 Raw Ingredient Name Label Overage Input Release Expiry - TGO101 Material Claim (LC) mg / g Amount (NLT LC) (90.0-120.0% Code mg / g mg / g mg / g LC) mg / g - Biotin 2.0 0.34 2.34 2.0 1.8 – 2.4 680.3 - - - RMO035 Purified water N / A (68.03) RMO055 Lecithin N / A - 67.2 (6.72) - - RMO043 Vitamin E TPGS N / A - 10.0 (1.0)- -169.5 - - - RMO004 Glycerol N / A (16.95) RMO044 Ethanol 70 - 70.0 (7.0)- -RMO051 Potassium carbonate N / A - 0.75 (0.075) - - The impurity and contamination profile of the probiotin liposomes was as shown in table 3. Table 3 Contaminants Test Specification Method / Monograph Elemental impurities TGO 101 Lead NMT 5 ppm Arsenic (inorganic) NMT 2 ppm NMI* Cadmium NMT 1 ppm Mercury NMT 0.2 ppm Pesticide Residues According to Australian New Zealand Food Standards Code – Schedule 20; Reg. (EC) 396 / 2005 NMI* and its amendments; and to 40 CFR Part 180Case Ref. P274WO IPTector® Residual Solvents Solvents class 1, class 2 and class 3 (except for ethanol) according to TGO101; USP <467>; Ph. Eur. USP <467> / SCU* 5.4; and ICH Q3C (R8) Microbiology TGO 100 Total aerobic microbial Less than or equal to 103 CFU per g count Total yeast and mould count Less than or equal to 102 CFU per g Bile-tolerant Gram-negative Less than or equal to 102 CFU per g ALS* bacteria Salmonella Absent in 10 g Escherichia coli Absent in 1 g Staphylococcus aureus Absent in 1 g TGO = Therapeutic Goods order; QBI = Quantified by input; EC = European Commission; NMI = National Measurement Institute, Australia; ALS = Australian Laboratory Services; CFR = The Code of Federal Regulations, USA; NLT = Not less than; NMT = Not more than;qsExample 2 – Analysis of the depigmentation effects of biotin in human melanocytes Introduction

[0043] Human skin is repeatedly exposed to ultraviolet radiation (UVR), which impacts the function and survival of many cell types and is considered one of the main factors underlying skin cancer. Skin pigmentation has traditionally been highlighted as the most important photoprotective factor in this organ, due to the UV absorption, antioxidant and scavenging properties of melanin [Brenner and Hearing, 2008]. Indeed, many epidemiological studies have shown a lower incidence of skin cancer in individuals with darker skin compared to those with fair skin. Besides, skin pigmentation is becoming an increasingly important cosmetic parameter, with new formulations that aim to boost or prevent, depending on the cultural preferences.

[0044] Melanin is one of the most common and widely distributed pigments in nature. It is a polymer of relatively variable composition and in all cases derivative of an essential amino acid, tyrosine. Visible pigmentation in mammals results from its synthesis and distribution in the skin, hair, bulbs and eyes [Gilchrest, 2011; Hearing et al., 1980]. Melanins are produced in a specific cell type, the melanocyte, and can be grouped into two basic types: eumelanins, which are brown or black, and pheomelanins, which are red or yellow [Urán et al., 2008; Quevedo et al., 1987]. In mammals, a mixture of bothCase Ref. P274WO IPTector® classes is typically found.

[0045] The biosynthesis of melanin strongly relies on tyrosinase, which is a critical rate-limiting enzyme for the production of this pigment [Wick et al., 1987]. Tyrosinase catalyses the two first steps in melanin biosynthesis: the hydroxylation of tyrosine to DOPA and its further oxidation to DOPAquinone. DOPAquinone undergoes several reactions that eventually give rise to eumelanins and pheomelanins [D’Orazio et al., 2011]. Melanin is found in specific compartments of melanocytes, called melanosomes. Several signalling pathways modulate melanogenesis, some of them through tyrosinase expression and activity. A summarized depiction of them is shown on Figure 1 [Goldsmith et al., 2014].

[0046] Due to the paramount relevance of tyrosinase in melanogenesis, it is not surprising that whitening treatments seek the inhibition of this enzymatic complex, while tanning treatments aim at its stimulation. However, mammalian melanogenesis is not solely regulated by tyrosinase at the enzymatic level, and there are additional melanogenic factors that can modulate pigmentation in either a positive or negative way.

[0047] The proportion of melanocytes in the skin is similar for most people. Nevertheless, melanocytes from individuals of different ethnic groups produce varying amounts of pheomelanin and eumelanin. This variation in the ratio of pheomelanin to eumelanin results in a wide variety of skin tones. Thus, different photo types are determined based on the skin's ability to respond to solar radiation. Their classification ranges from I to VI according to the Fitzpatrick scale, where I corresponds to people with very pale skin and VI to people with very dark or black skin [Kawada, 2000].

[0048] Ethnic differences in skin color are mostly determined by genetic background, which influences melanosome biogenesis. In addition, several non-genetic factors can influence the expression of melanin-related genes, thus contributing to changes in skin color. Hormonal changes, chronic inflammation and exposure to UV radiation are some examples of conditions in which altered melanogenesis causes hypopigmentation or hyperpigmentation [Boo, 2019].

[0049] Abnormal skin pigmentations are aesthetically significant conditions that can affect a person's quality of life. Several approaches are currently used in the field of dermatology and cosmetology to control hyper and hypopigmentation. However, consumer satisfaction to date has been low and therefore, a wider commercial availability of ingredients with effective and safe depigmentation effects is greatly needed [Boo, 2019].

[0050] For these reasons, the goal of this example was to assess the depigmentation effects of Biotin by quantifying the melanin levels in human melanocytes subjected to UVA irradiation to mimic sun exposure. Figure 2 shows a microscopic image of Normal Human Epidermal Melanocytes (NHEM) in culture as used in the example.Case Ref. P274WO IPTector® Material and Methods

[0051] Working concentrations of the biotin liposome product were determined using the MTT viability assay in human melanocytes. For melanin quantification, cells were cultured for 72 hours (h) with Biotin at 0.03 % and 0.003 %. During the incubation period, sequential UVA irradiation steps were applied to a final dose of 5 J / cm2 to stimulate melanin production. Human melanocytes were allowed to accumulate melanin after UVA exposure during a resting period, and the pigment was then extracted and quantified with a spectrophotometer. Data were statistically analysed. Analytical equipment:

[0052] Stereoscopic microscope, cell culture incubator (37°C, 5 % CO2, 90 % relative humidity (RH), statistical analysis software, laminar flow hood, micropipettes, pipettes, propipette, Bürker chamber, freezer -20ºC, freezer -80ºC, rack, heating block, plate reader spectrophotometer, Luzchem Irradiator LZC-420 with UVA lamps, UVA irradiance sensor, vortex and consumables. Reagents:

[0053] Normal Human Epidermal Melanocytes (NHEM, PromoCell), NHEM culture medium (PromoCell), Dulbecco’s Modified Essential Medium (DMEM, Gibco), Trypan Blue Solution (Bio- Rad), Phosphate buffered saline (Gibco), Trypsin / EDTA (Gibco), MTT reagent [3-(4,5- Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide] (Invitrogen), dimethyl sulfoxide (DMSO, Sigma) and sodium hydroxide (NaOH, Alfa Aesar). Procedure - cell viability (MTT assay):

[0054] Cell numbers and viability were determined using Trypan-Blue staining and counting in a Bürker chamber under the microscope. For the MTT viability assay, Normal Human Epidermal Melanocytes were cultured overnight at a 10.000 cells / well density in a 96 well plate, in supplemented growth medium.24 h later, the culture medium was replaced with fresh medium containing Biotin at 8 different concentrations (3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0.001 %). After 24 h of incubation, the medium was removed, and MTT solution was added to each well. Plates were incubated at 37ºC for 3 h. MTT reagent was removed and DMSO at 100 % was added to each well to solubilize formazan crystals, then the absorbance was measured at 550 nm and 620 nm as a reference on a scanning multi- well spectrophotometer. Melanin extraction and quantification:Case Ref. P274WO IPTector®

[0055] Cells were seeded in 24-well plates at a density of 2x104 cells / well and incubated overnight at 37 ºC in a CO2 incubator. Culture medium was then removed and replaced with fresh medium containing Biotin at 0.03 % and 0.003 % and incubated for 24 h in the presence of the product. To mimic sunlight exposure, cells were irradiated with UVA light in 5 cycles for a total dose of 5 J / cm2. After the last cycle, cells were maintained at 37 ºC in a CO2 incubator for a resting period of 24 h to allow melanin accumulation. Then, cells were collected, and melanin was extracted and quantified by measuring the absorbance at 405 nm, as reported elsewhere [Kollias and Bager, 1987; Dwyer et al., 1998; Ou-Yang et al., 2004; Kollias and Bager, 1985]. A MTT assay was performed in parallel under the same conditions to correct melanin quantification fluctuations due to changes in cell viability. Corrected absorbance data were normalized to non-treated, UVA-exposed cells. Statistical analysis:

[0056] The MTT assay was set with 8 replicates per condition and 16 for control, and for melanin quantification, 4 technical replicates per condition were set. Data outliers were identified with ROUT method (Q = 5 %) and excluded from the analysis if found. Both cell viability-corrected and normalized data were statistically analyzed through ordinary one-way ANOVA and Dunnet’s post hoc multiple comparisons test when applicable. Statistical significance was set at p < 0.05, 95% of confidence. Bars in the charts represent the mean value for each condition and error bars indicate the standard error of the mean (SEM) for each group of values.

[0057] To evaluate the depigmentation effects of Biotin by quantifying melanin levels in human melanocytes exposed to UVA radiation. Results and conclusion Cell viability (MTT assay):

[0058] Results showed that Biotin did not affect cell viability when used at concentrations below 0.1 %, in human melanocytes (Figure 8). Based on this, the selected concentration for Biotin were 0.03 % and 0.003 % (see figure 3). Melanin quantification:

[0059] For the melanin quantification assay, both melanin extraction and cell viability assessment were performed in parallel. Melanin levels were then normalized to cell viability for each condition, thus correcting possible interferences due to the potential cytotoxicity induced by the UVA irradiation protocol; the rationale for this correction is that UVA irradiation decreases the cell viability, thus compromising the melanin production. As Figure 4 shows, melanin levels were increased in responseCase Ref. P274WO IPTector® to UVA irradiation by 19.5 ± 5.1 %. On the other hand, treatment with Biotin reduced melanin levels by 16.0 ± 5.1 %, when used at 0.003 %, compared to UVA-treated control (Figure 4).

[0060] Additionally, when melanin basal values (control, C) were subtracted to all samples to assess the inhibiting effects of the treatment on UVA-induced melanin levels, results indicated that treatment with Biotin reduced the UVA-induced melanin levels by 82.1 ± 20.7 % (Figure 5). Statistical data obtained from the experiments: Table 4 Cell viability (MTT assay) Raw data (O.D.550 nm) C3 10.3 0.1 0.03 0.01 0.003 0.001 C 0.163 0.13 0.1320.134 0.152 0.159 0.164 0.152 0.153 0.151 0.176 0.148 0.151 0.138 0.14 0.167 0.167 0.167 0.186 0.159 0.171 0.154 0.160.129 0.139 0.157 0.172 0.168 0.172 0.176 0.185 0.157 0.149 0.152 0.152 0.142 0.172 0.172 0.177 0.192 0.181 0.149 0.143 0.151 0.142 0.163 0.181 0.164 0.186 0.17 0.182 0.177 0.151 0.167 0.147 0.166 0.169 0.174 0.173 0.162 0.164 0.141 0.153 0.147 0.146 0.174 0.159 0.173 0.176 0.155 0.152 0.128 0.128 0.159 0.13 0.164 0.153 0.145 0.144 0.159 Normalized data C 3 1 0.3 0.1 0.03 0.01 0.003 0.001 C 0.967 0.771 0.783 0.795 0.901 0.943 0.973 0.901 0.907 0.895 1.044 0.878 0.895 0.818 0.830 0.990 0.990 0.990 1.103 0.943 1.014 0.913 0.949 0.765 0.824 0.931 1.020 0.996 1.020 1.044 1.097 0.931 0.884 0.901 0.901 0.842 1.020 1.020 1.050 1.139 1.073 0.884 0.848 0.895 0.842 0.967 1.073 0.973 1.103 1.008 1.079 1.050 0.895 0.990 0.872 0.984 1.002 1.032 1.026 0.961 0.973 0.836 0.907 0.872 0.866 1.032 0.943 1.026 1.044 0.919 0.901 0.759 0.759 0.943 0.771 0.973 0.907 0.860 0.854 0.943 Table 5 ANOVA for Cell viability (MTT assay) ANOVA summary F 8.353 P value <0.0001 P value summary **** Significant diff. among means (P < 0.05) Yes R squared 0.4884 Brown-Forsythe test 0.9027 (8, F (DFn, DFd) 70) P value 0.5193 P value summary ns Are SDs significantly different (P < 0.05) No Bartlett's test Bartlett's statistic (corrected) 8.068 P value 0.4268 P value summary ns Are SDs significantly different (P < 0.05) NoCase Ref. P274WO IPTector® ANOVA table SS DF MS F (DFn, DFd) P value F (8, 70) = Treatment (between columns) 0.3183 8 0.03979 P<0.0001 8.353 Residual (within columns) 0.3334 70 0.004763 Total 0.6517 78 Data summary Number of treatments (columns) 9 Number of values (total) 79 Table 6 Multiple Comparison for Cell viability (MTT assay) Number of families 1 Number of comparisons per 8 family Alpha 0,05 Dunnett's multiple Mean 95,00% CI Adjusted P Significant Summary ?-A comparisons test Diff, of diff, Value -0.2051 to 3 vs. C -0.1223 Yes *** 0.0009 B 3 -0.03941 -0.2178 to 1 vs. C -0.1350 Yes *** 0.0002 C 1 -0.05216 -0.2105 to 0.3 vs. C -0.1276 Yes *** 0.0005 D 0.3 -0.04479 -0.2320 to 0.1 vs. C -0.1491 Yes **** <0.0001 E 0.1 -0.06629 -0.1251 to 0.03 vs. C -0.04225 No ns 0.6816 F 0.03 0.04059 - -0.09184 0.01 vs. C No ns 0.9996 G 0.01 0.009000 to 0.07384 - -0.09555 0.003 vs. C No ns 0.9997 H 0.003 0.008857 to 0.07784 -0.06946 0.001 vs. C 0.01337 No ns 0.9994 I 0.001 to 0.09621 Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF 3 vs. C 0.8778 1.000 -0.1223 0.02988 8 16 4.091 70 1 vs. C 0.8650 1.000 -0.1350 0.02988 8 16 4.517 70 0.3 vs. C 0.8724 1.000 -0.1276 0.02988 8 16 4.271 70 0.1 vs. C 0.8509 1.000 -0.1491 0.02988 8 16 4.990 70 0.03 vs. C 0.9578 1.000 -0.04225 0.02988 8 16 1.414 70 0.01 vs. C 0.9910 1.000 -0.009000 0.02988 8 16 0.3012 70 0.003 vs. C 0.9911 1.000 -0.008857 0.03128 7 16 0.2832 70 0.001 vs. C 1.013 1.000 0.01337 0.02988 8 16 0.4476 70 Table 7 Descriptive statistics for Cell viability (MTT assay) C 3 1 0.3 0.1 0.03 0.01 0.003 0.001 Number of 16 8 8 8 8 8 8 7 8 values Minimum 0.8950 0.7590 0.7590 0.7650 0.7710 0.8420 0.9070 0.9010 0.8540 25% 0.9430 0.7873 0.7993 0.8008 0.8255 0.9340 0.9505 0.9730 0.9353 PercentileCase Ref. P274WO IPTector® Median 0.9905 0.8810 0.8895 0.8835 0.8540 0.9700 0.9960 0.9960 1.035 75% 1.066 0.9265 0.9040 0.9325 0.8938 0.9885 1.020 1.026 1.090 Percentile Maximum 1.139 1.050 0.9490 0.9900 0.9010 1.032 1.073 1.032 1.103 Mean 1.000 0.8778 0.8650 0.8724 0.8509 0.9578 0.9910 0.9911 1.013 Std. Deviation 0.07378 0.09337 0.06462 0.07618 0.04362 0.05592 0.05101 0.04509 0.08885 Std. Error of 0.01845 0.03301 0.02285 0.02694 0.01542 0.01977 0.01803 0.01704 0.03141 Mean Lower 95% CI 0.9607 0.7997 0.8110 0.8087 0.8144 0.9110 0.9484 0.9494 0.9391 Upper 95% CI 1.039 0.9558 0.9190 0.9361 0.8873 1.005 1.034 1.033 1.088 Table 8 MTT after UVA exposure Raw data (O.D.550 nm) C UVA 0.03 0.003 0.184 0.172 0.148 0.173 0.188 0.159 0.152 0.166 0.191 0.168 0.126 0.172 0.178 0.182 0.131 0.164 Normalized data C UVA 0.03 0.003 1.081 1.010 0.869 1.016 1.104 0.934 0.893 0.975 1.122 0.987 0.740 1.010 1.046 1.069 0.769 0.963 Table 9 ANOVA for MTT after UVA exposure ANOVA summary F 19.62 P value <0.0001 P value summary **** Significant diff. among means (P < 0.05) Yes R squared 0.8306 Brown-Forsythe test 3.555 (3, F (DFn, DFd) 12) P value 0.0476 P value summary * Are SDs significantly different (P < 0.05) Yes Bartlett's test Bartlett's statistic (corrected) 3.433 P value 0.3296 P value summary ns Are SDs significantly different (P < 0.05) No ANOVA table SS DF MS F (DFn, DFd) P value F (3, 12) = Treatment (between columns) 0.1537 3 0.05124 P<0.0001 19.62 Residual (within columns) 0.03135 12 0.002612 Total 0.1851 15Case Ref. P274WO IPTector® Data summary Number of treatments (columns) 4 Number of values (total) 16 Table 10 Multiple Comparison for MTT after UVA exposure Number of families 1 Number of comparisons per 3 family Alpha 0,05 Dunnett's multiple comparisons Mean 95,00% Adjusted ?- Significant Summary test Diff. CI of diff P Value A - 0.008713 C vs. UVA 0.08825 No ns 0.0764 A C to 0.1852 -0.2792 0.03 vs. UVA -0.1823 to - Yes *** 0.0008 C 0.03 0.08529 -0.1060 - 0.003 vs. UVA to No ns 0.9889 D 0.003 0.009000 0.08796 Mean Test details Mean 1 Mean 2 SE of diff, n1 n2 q DF Diff, C vs. UVA 1.088 1.000 0.08825 0.03614 4 4 2.442 12 0.03 vs. UVA 0.8178 1.000 -0.1823 0.03614 4 4 5.043 12 0.003 vs. UVA 0.9910 1.000 -0.009000 0.03614 4 4 0.2490 12 Table 11 Descriptive statistics for MTT after UVA exposure C UVA 0.03 0.003 Number of values 4 4 4 4 Minimum 1.046 0.9340 0.7400 0.9630 25% Percentile 1.055 0.9473 0.7473 0.9660 Median 1.093 0.9985 0.8190 0.9925 75% Percentile 1.118 1.054 0.8870 1.015 Maximum 1.122 1.069 0.8930 1.016 Mean 1.088 1.000 0.8178 0.9910 Std. Deviation 0.03279 0.05593 0.07463 0.02599 Std. Error of 0.01639 0.02797 0.03732 0.01299 Mean Lower 95% CI 1.036 0.9110 0.6990 0.9496 Upper 95% CI 1.140 1.089 0.9365 1.032 Table 12 Melanin levels Raw data melanin (O.D.405 nm) C UVA 0.03 0.003 0.095 0.098 0.075 0.079 0.072 0.080 0.071 0.075Case Ref. P274WO IPTector® 0.078 0.093 0.073 0.076 0.079 0.099 0.073 0.078 Raw data MTT (O.D.550 nm) C UVA 0.03 0.003 0.184 0.172 0.148 0.173 0.188 0.159 0.152 0.166 0.191 0.168 0.126 0.172 0.178 0.182 0.131 0.164 Melanin / MTT C UVA 0.03 0.003 0.513 0.576 0.539 0.468 0.389 0.470 0.510 0.444 0.421 0.546 0.524 0.450 0.426 0.581 0.524 0.462 Normalized data C UVA 0.03 0.003 0.944 1.059 0.991 0.862 0.715 0.865 0.938 0.818 0.775 1.005 0.965 0.829 0.785 1.070 0.965 0.851 Table 13 ANOVA for Melanin levels ANOVA summary F 7.396 P value 0.0046 P value summary ** Significant diff. among means (P < 0.05) Yes R squared 0.6490 Brown-Forsythe test 1.141 (3, F (DFn, DFd) 12) P value 0.3722 P value summary ns Are SDs significantly different (P < 0.05) No Bartlett's test Bartlett's statistic (corrected) 9.283 P value 0.0258 P value summary * Are SDs significantly different (P < 0.05) Yes ANOVA table SS DF MS F (DFn, DFd) P value F (3, 12) = Treatment (between columns) 0.1072 3 0.03573 P=0.0046 7.396 Residual (within columns) 0.05797 12 0.004831 Total 0.1651 15 Data summary Number of treatments (columns) 4 Number of values (total) 16 Table 14 MultipleNumber of families 1Case Ref. P274WO IPTector® Number of comparisons per 3 family Alpha 0,05 Dunnett's multiple comparisons Mean 95,00% Adjusted ?- Significant Summary test Diff, CI of diff, P Value A -0.3269 C vs. UVA -0.1950 to - Yes ** 0.0050 A C 0.06315 -0.1669 0.03 vs. UVA -0.03500 to No ns 0.8188 C 0.03 0.09685 -0.2916 0.003 vs. UVA -0.1598 to - Yes * 0.0180 D 0.003 0.02790 Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF C vs. UVA 0.8048 0.9998 -0.1950 0.04915 4 4 3.968 12 0.03 vs. UVA 0.9648 0.9998 -0.03500 0.04915 4 4 0.7122 12 0.003 vs. UVA 0.8400 0.9998 -0.1598 0.04915 4 4 3.251 12 Table 15 Descriptive statistics for Melanin levels C UVA 0.03 0.003 Number of values 4 4 4 4 Minimum 0.7150 0.8650 0.9380 0.8180 25% Percentile 0.7300 0.9000 0.9448 0.8208 Median 0.7800 1.032 0.9650 0.8400 75% Percentile 0.9043 1.067 0.9845 0.8593 Maximum 0.9440 1.070 0.9910 0.8620 Mean 0.8048 0.9998 0.9648 0.8400 Std. Deviation 0.09784 0.09422 0.02164 0.02008 Std. Error of 0.04892 0.04711 0.01082 0.01004 Mean Lower 95% CI 0.6491 0.8498 0.9303 0.8080 Upper 95% CI 0.9604 1.150 0.9992 0.8720 Table 16 Melanin levels without basal values Raw data melanin (O.D.405 nm) C UVA 0.03 0.003 0.095 0.098 0.075 0.079 0.072 0.080 0.071 0.075 0.078 0.093 0.073 0.076 0.079 0.099 0.073 0.078 Raw data MTT (O.D.550 nm) C UVA 0.03 0.003 0.184 0.172 0.148 0.173 0.188 0.159 0.152 0.166 0.191 0.168 0.126 0.172 0.178 0.182 0.131 0.164 Melanin / MTT without basal values C UVA 0.03 0.003 0.138 0.101 0.031 0.033 0.073 0.007Case Ref. P274WO IPTector® 0.109 0.087 0.013 0.144 0.087 0.025 Normalized data C UVA 0.03 0.003 1.305 0.956 0.291 0.308 0.685 0.068 1.028 0.820 0.124 1.360 0.820 0.235 Table 17 ANOVA for Melanin levels without basal values ANOVA summary F 8.701 P value 0.0079 P value summary ** Significant diff. among means (P < 0.05) Yes R squared 0.6591 Brown-Forsythe test 2.018 (2, F (DFn, DFd) 9) P value 0.1888 P value summary ns Are SDs significantly different (P < 0.05) No Bartlett's test Bartlett's statistic (corrected) 7.972 P value 0.0186 P value summary * Are SDs significantly different (P < 0.05) Yes ANOVA table SS DF MS F (DFn, DFd) P value F (2, 9) = Treatment (between columns) 1.489 2 0.7444 P=0.0079 8.701 Residual (within columns) 0.7700 9 0.08556 Total 2.259 11 Data summary Number of treatments (columns) 3 Number of values (total) 12 Table 18 Multiple Comparison for Melanin levels without basal values Number of families 1 Number of comparisons per 2 family Alpha 0,05 Dunnett's multiple comparisons Mean 95,00% CI of Adjusted ?- Significant Summary test Diff, diff, P Value A -0.7207 to 0.03 vs. UVA -0.1800 No Ns 0.6072 B 0.03 0.3607 -1.361 to - 0.003 vs. UVA -0.8208 Yes ** 0.0060 C 0.003 0.2801 Mean Test details Mean 1 Mean 2 SE of diff, n1 n2 q DF Diff,Case Ref. P274WO IPTector® 0.03 vs. UVA 0.8203 1.000 -0.1800 0.2068 4 4 0.8703 9 0.003 vs. UVA 0.1795 1.000 -0.8208 0.2068 4 4 3.968 9 Table 19 Descriptive statistics for Melanin levels without basal values UVA 0.03 0.003 Number of values 4 4 4 Minimum 0.3080 0.6850 0.06800 25% Percentile 0.4880 0.7188 0.08200 Median 1.167 0.8200 0.1795 75% Percentile 1.346 0.9220 0.2770 Maximum 1.360 0.9560 0.2910 Mean 1.000 0.8203 0.1795 Std. Deviation 0.4838 0.1106 0.1017 Std. Error of Mean 0.2419 0.05532 0.05085 Lower 95% CI 0.2304 0.6442 0.01768 Upper 95% CI 1.770 0.9963 0.3413

[0061] These results showed that melanin levels were increased in response to UVA irradiation by 19.5 ± 5.1 %. On the other hand, treatment with Biotin reduced melanin levels by 16.0 ± 5.1 %, when used at 0.003 %, compared to UVA-treated control.

[0062] Additionally, when melanin basal values (control, C) were subtracted to all samples to assess the inhibiting effects of the treatment on UVA-induced melanin levels, results indicated that treatment with Biotin reduced the UVA-induced melanin levels by 82.1 ± 20.7 %.

[0063] In conclusion, our study shows that treatment with Biotin shows whitening effects by reducing the UVA-induced melanin levels in human melanocytes (see figure 6). References - Devasagayam T, Tilak JC, Boloor KK, Sane Ketaki S, Ghaskadbi Saroj, Lele RD. Free radicals and antioxidants in human health: Current status and future prospects. Journal of Association of Physicians of India.2004; 54:796. - Han D, Williams E, Cadenas E. Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. The Biochemical Journal. 2001; 353:411-6. - Ichihashi M, Ando H, Yoshida M et al. Photoaging of the skin. Anti-Aging Medicine 2009; 6: 46–59. - Kim MS, Kim YK, Cho KH, Chung JH. Regulation of type I procollagen and MMP-1 expressionCase Ref. P274WO IPTector® after single or repeated exposure to infrared radiation in human skin. Mech Ageing Dev 2006; 127: 875–882. - Muller F. The nature and mechanism of superoxide production by the electron transport chain: Its relevance to aging. Journal of the American Aging Association.2004; 23(4):227-53. - Murphy MP. How mitochondria produce reactive oxygen species. Biochem J.2009; 417:1-13. - Vayalil PK, Mittal A, Hara Y, Elmets CA, Katiyar SK. Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinases expression in mouse skin. J Invest Dermatol 2004; 122: 1480–1487. - Dykas, Carol (June 2004). How to protect patients from harmful sunlight. www.2020mag.com - Peak MJ and Peak JG. Photodermatol.1989; 6, 1–15. Examples 3 – Analysis of the protective effects of biotin against UVA radiation in human keratinocytes Introduction

[0064] The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus [Murphy et al., 2009]. Mitochondria constitute the main source of ROS. They convert energy produced from cellular metabolism into a usable form, the adenosine triphosphate (ATP). The process of ATP production involves the transport of hydrogen ions across the mitochondrial membrane, by means of an electron transport chain. In the electron transport chain, electrons are passed through a series of proteins via oxidation-reduction reactions. The last destination of an electron is an oxygen molecule. In normal conditions, the oxygen is reduced to produce water. Nevertheless, in a very low percentage of cases, the electrons passing through the chain can prematurely and incompletely reduce the oxygen molecule to generate the superoxide radical (O2-) and other ROS [Devasagayam et al., 2004].

[0065] The role of free radicals in tissue damage, due to oxygen deprivation, has been the subject of several studies. Superoxide can inactivate specific enzymes, thus activating the pathway of peroxidation [Muller, 2000]. Under specific stress conditions, this last reaction is predominant, and the cell undergoes through the process of apoptosis or programmed cell death. Heat shock is known to induce oxidative stress in a process linked to the dysfunction of the mitochondrial electron transport chain. The main source of endogenous radicals is represented from mitochondria, plasmatic membrane, endoplasmic reticulum and peroxisome [Han et al., 2001]. Nevertheless, several exogenous stimuli, as ionizing radiation, UV light, smoke, inflammatory processes and some human diseases, cause free radicals’ production, as well. Particularly, depending on the severity of theCase Ref. P274WO IPTector® damages provoked on mitochondria membranes, the response triggered is different. In the case of leak damages, the electronic chain in the mitochondria membranes would result disturbed, reducing ATP synthesis; thus, depriving the cell of energy. In the other cases of severe damage, the mitochondria membranes and the oxidizing enzymes will be affected, provoking the release of free radicals (ROS).

[0066] Approximately, 6 % of the solar spectrum is UV radiation (200 – 400 nm), 52 % is visible light (400–760 nm) and the remaining 42 % is infrared radiation (760 nm – 106 nm) [Ichihashi et al., 2009]. These proportions highlight the importance light-induced damage, rather than historical UVA and UVB associated pathological and dermatological damages. Figure 7 is a graphical representation illustrating the penetration capacity of different radiations (UVC, UVB, UVA and Visible) into the skin. Figure 8 shows a microscopic image of human keratinocytes in culture (HaCaT cell line), used in this example.

[0067] Although the mechanisms are not fully understood, it appears that UVA radiation is involved in human photoaging. For these reasons, in this study, we evaluate the protective effect against oxidative damage of Biotin in human keratinocytes. Material and Methods

[0068] Working concentrations of the products were determined using the MTT cell viability assay in human keratinocytes. Then, human keratinocytes were treated with Biotin at 0.3 % and 0.03 %. After 24 hours (h), cells were exposed to UVA radiation, and the accumulation of intracellular ROS was evaluated fluorometrically. In parallel, cell viability was quantified through MTT assay to normalize ROS levels to the number of live cells due to UVA-induced cytotoxicity. Data were statistically analysed. Analytical equipment:

[0069] Stereoscopic microscope, cell culture incubator (37°C, 5 % CO2, 90 % relative humidity (RH), statistical analysis software, laminar flow hood, micropipettes, pipettes, propipette, Bürker chamber, freezer -20ºC, freezer -80ºC, rack, heating block, plate reader spectrophotometer, Luzchem Irradiator LZC-420 with UVA lamps, Glomax Discovery Promega Multi-mode detection system (Promega), vortex and consumables. Reagents:

[0070] DMEM low glucose medium (Gibco), FBS (Gibco), Penicillin / Streptomycin (Life Technologies), L- Glutamine (Gibco), Phosphate buffered saline (Gibco), Trypan Blue Solution (Bio-Rad), Trypsin- EDTA (Gibco), MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (Invitrogen),Case Ref. P274WO IPTector® dimethyl sulfoxide (DMSO, Sigma-Aldrich) and Intracellular ROS detection kit (Sigma Aldrich). Procedure for cell viability (MTT assay)

[0071] Cell numbers and viability were determined using Trypan-Blue staining and counting in a Bürker chamber under the microscope. For the MTT viability assay, human keratinocytes were cultured overnight at a 10.000 cells / well density in a 96 well plate, in supplemented growth medium. 24 h later, the culture medium was replaced with fresh medium containing Biotin at 8 different concentrations. After 24 h of incubation, the medium was removed, and MTT solution was added to each well. Plates were incubated at 37ºC for 3 h. MTT reagent was removed and DMSO at 100 % was added to each well to solubilize formazan crystals, then the absorbance was measured at 550 nm and 620 nm as a reference on a scanning multi-well spectrophotometer. ROS quantification:

[0072] Human keratinocytes were cultured overnight at a 10.000 cells / well density in a black 96-well plate, in growth media. 24 h later, the culture media was removed and replaced by new culture medium supplied with Biotin at 0.3 % and 0.03 % concentrations. After additional 24 h of incubation, PBS and ROS master mix were added in all cultured wells and cells were exposed to UVA radiation. Non-irradiated controls were incubated at 37ºC during this time in the dark.2 hours after ROS master mix addition to cells, ROS levels were measured in all samples. The intracellular ROS react with a fluorogenic sensor localized in the cytoplasm, resulting in a fluorescent product whose appearance is proportional to ROS levels. Fluorescence quantification was measured at λex =490 / λem =525.

[0073] In parallel, cell viability was quantified through MTT assay to normalize ROS levels to the number of live cells due to UVA-induced cytotoxicity. 6.3.3 Statistical analysis

[0074] The MTT assay was set with 8 replicates per condition and 16 for control, and ROS quantification assay was set with 4 replicates per condition. Data outliers were identified with ROUT method (Q = 5 %) and excluded from the analysis if found. Raw data were statistically analyzed by Unpaired t-test or ordinary one-way ANOVA test and Dunnet’s post hoc multiple comparisons test. Statistical significance was set at p < 0.05, 95 % of confidence. Bars in the charts represent the mean value for each condition and error bars indicate the standard error of the mean (SEM) for each group of values.Case Ref. P274WO IPTector® Results and conclusion Cell viability (MTT assay):

[0075] Results showed that treatment with Biotin did not affect cell viability when used at concentrations below 1 %, in human keratinocytes (Figure 9). Based on this, the selected concentrations for Biotin were 0.3 % and 0.03 %. ROS quantification:

[0076] In the ROS quantification assay, results showed that UVA radiation significantly increased ROS levels in human keratinocytes by 101.4 ± 3.1 %, compared to the non-irradiated control, thus validating our experimental model to study the oxidative stress (Figure 10).

[0077] When ROS basal levels were subtracted to all samples to assess UVA-induced oxidative stress protection, results indicated that, treatment with Biotin at 0.3 % reduced UVA-induced ROS levels by 22.6 ± 2.8 % (Figure 11). Statistical data obtained from the experiments: Table 20 Cell viability (MTT assay) Raw data (O.D.550 nm) C 3 1 0.3 0.1 0.03 0.01 0.003 0.001 C 1488 0.923 0.990 0.998 1131 1125 1416 1251 1120 1290 1166 0.945 1100 1100 1218 1496 1500 1395 1372 1200 1155 0.914 0.948 1500 1380 1306 1318 1306 1403 1274 1568 0.920 0.990 1430 1600 1104 1263 1357 1349 1117 1516 0.919 0.951 1600 1177 1270 1129 1303 1500 1421 1502 0.876 0.931 1190 1300 1220 1319 1227 1690 1478 1512 0.882 0.889 1150 1260 1130 1153 1259 1460 1529 1326 0.837 0.900 1120 1800 1290 1256 1221 1323 1407 Normalized data C 3 1 0.3 0.1 0.03 0.01 0.003 0.001 C 1085 0.673 0.722 0.728 0.824 0.820 1032 0.912 0.816 0.940 0.850 0.689 0.802 0.802 0.888 1091 1093 1017 1000 0.875 0.842 0.666 0.691 1093 1006 0.952 0.961 0.952 1023 0.929 1143 0.671 0.722 1042 1166 0.805 0.921 0.989 0.983 0.814 1105 0.670 0.693 1166 0.858 0.926 0.823 0.950 1093 1036 1095 0.639 0.679 0.867 0.948 0.889 0.962 0.894 1232 1077 1102 0.643 0.648 0.838 0.918 0.824 0.840 0.918 1064 1115 0.967 0.610 0.656 0.816 1312 0.940 0.916 0.890 0.964 1026 Table 21 ANOVA for Cell viability (MTT assay)Case Ref. P274WO IPTector® ANOVA summary F 12.58 Pvalue<0.0001P value summary****Significant diff. among means (P < 0.05) Yes Rsquared0.5864 Brown-Forsythe test F(DFn, DFd)2.230 (8,71)Pvalue0.0349 P value summary*Are SDs significantly different (P < 0.05)YesBartlett's test Bartlett's statistic (corrected)31.37 Pvalue0.0001 P value summary***Are SDs significantly different (P < 0.05)YesANOVA tableSSDF MS F (DFn, DFd) P value Treatment (between columns)1155 8 0.1443 F (8,71) = 12.58 P<0.0001Residual (within columns)0.8144 71 0.01147 Total1969 79 Data summaryNumber of treatments (columns)9 Number of values (total)80 Table 22 Multiple Comparison for Cell viability (MTT assay) Number of families 1 Number of comparisons per 8 family Alpha 0,05 Dunnett's multiple 95,00% CI Adjusted P Mean Diff, Significant Summary A comparisons test of diff, Value -0.4709 to 3 vs. C -0.3424 Yes **** <0.0001 B 3 -0.2140 -0.4269 to 1 vs. C -0.2984 Yes **** <0.0001 C 1 -0.1700 -0.2095 to 0.3 vs. C -0.08106 No ns 0.4373 D 0.3 0.04740 -0.1385 to 0.1 vs. C -0.01006 No ns 0.9997 E 0.1 0.1184 -0.2226 to 0.03 vs. C -0.09419 No ns 0.2676 F 0.03 0.03427 -0.1850 to 0.01 vs. C -0.05656 No ns 0.8127 G 0.01 0.07190 -0.1883 to 0.003 vs. C -0.05981 No ns 0.7675 H 0.003 0.06865 -0.1066 to 0.001 vs. C 0.02181 No ns 0.9994 I 0.001 0.1503Case Ref. P274WO IPTector® Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF 3 vs. C 0.6576 1000 -0.3424 0.04638 8 16 7384 71 1 vs. C 0.7016 1000 -0.2984 0.04638 8 16 6435 71 0.3 vs. C 0.9190 1000 -0.08106 0.04638 8 16 1748 71 0.1 vs. C 0.9900 1000 -0.01006 0.04638 8 16 0.2170 71 0.03 vs. C 0.9059 1000 -0.09419 0.04638 8 16 2031 71 0.01 vs. C 0.9435 1000 -0.05656 0.04638 8 16 1220 71 0.003 vs. C 0.9403 1000 -0.05981 0.04638 8 16 1290 71 0.001 vs. C 1022 1000 0.02181 0.04638 8 16 0.4703 71 Table 23 Descriptive statistics for Cell viability (MTT assay) C 3 1 0.3 0.1 0.03 0.01 0.003 0.001 Number of values 16 8 8 8 8 8 8 8 8 Minimum 0.8140 0.6100 0.6480 0.7280 0.8240 0.8050 0.8230 0.8900 0.8160 25% Percentile 0.8885 0.6400 0.6618 0.8055 0.8655 0.8210 0.8590 0.8985 0.9688 Median 1031 0.6680 0.6920 0.8525 0.9330 0.9075 0.9410 0.9340 1012 75% Percentile 1100 0.6725 0.7220 1080 1126 0.9490 1015 0.9798 1086 Maximum 1143 0.6890 0.8020 1166 1312 1091 1093 1017 1232 Mean 1000 0.6576 0.7016 0.9190 0.9900 0.9059 0.9435 0.9403 1022 Std. Deviation 0.1116 0.02522 0.04864 0.1588 0.1679 0.09450 0.09050 0.04545 0.1187 Std. Error of Mean 0.02789 0.008916 0.01720 0.05613 0.05935 0.03341 0.03199 0.01607 0.04198 Lower 95% CI 0.9406 0.6365 0.6610 0.7863 0.8497 0.8269 0.8678 0.9023 0.9226 Upper 95% CI 1060 0.6787 0.7423 1052 1130 0.9849 1019 0.9782 1121 Table 24 UVA - induction RFU C UVA 1.878.453 3.533.600 1.735.543 3.761.460 1.829.834 3.638.629 1.803.315 3.659.991 Normalized data C UVA 1.037 1.950 0.958 2.076 1.010 2.008 0.995 2.020 Table 25 Unpaired t test for UVA - inductionCase Ref. P274WO IPTector® Unpaired t test P value <0.0001 P value summary **** Significantly different (P < 0.05)? Yes One- or two-tailed P value? Two-tailed t, df t=33.07, df=6 How big is the difference? Mean of column A 1.000 Mean of column B 2.014 Difference between means (B - A) ± 1.014 ± 0.03064 SEM 95% confidence interval 0.9385 to 1.088 R squared (eta squared) 0.9945 F test to compare variances F, DFn, Dfd 2.459, 3, 3 P value 0.4794 P value summary Ns Significantly different (P < 0.05)? No Data analyzed Sample size, column A 4 Sample size, column B 4 Table 26 Descriptive statistics for UVA - induction C UVA Number of values 4 4 Minimum 0.9580 1.950 25% Percentile 0.9673 1.965 Median 1.003 2.014 75% Percentile 1.030 2.062 Maximum 1.037 2.076 Mean 1.000 2.014 Std. Deviation 0.03295 0.05168 Std. Error of Mean 0.01648 0.02584 Lower 95% CI 0.9476 1.931 Upper 95% CI 1.052 2.096 Table 27 MTT after UVA exposure O.D.550 nm C UVA 0.3 0.03 0.625 0.607 0.76 0.784 0.709 0.612 0.714 0.683 0.664 0.582 0.61 0.456 0.717 0.446 0.503 0.464Case Ref. P274WO IPTector® Normalized data C UVA 0.3 0.03 1.113 1.081 1.353 1.396 1.262 1.089 1.271 1.216 1.182 1.036 1.086 0.812 1.276 0.794 0.895 0.826 Table 28 ANOVA for MTT after UVA exposure ANOVA summary F 0.9035 P value 0.4680 P value summary ns Significant diff. among means (P < 0.05)? No R squared 0.1843 Brown-Forsythe test 3.476 (3, F (DFn, DFd) 12) P value 0.0505 P value summary ns Are SDs significantly different (P < 0.05)? No Bartlett's test Bartlett's statistic (corrected) 4.350 P value 0.2260 P value summary ns Are SDs significantly different (P < 0.05)? No ANOVA table SS DF MS F (DFn, DFd) P value F (3, 12) = Treatment (between columns) 0.1025 3 0.03417 P=0.4680 0.9035 Residual (within columns) 0.4539 12 0.03782 Total 0.5564 15 Data summary Number of treatments (columns) 4 Number of values (total) 16 Table 29 Multiple Comparison for MTT after UVA exposure MULTIPLE COMPARISONS Number of families 1 Number of comparisons per family 3 Alpha 0,05 Dunnett's multiple comparisons test Mean Diff, 95,00% CI of diff, Significant? Summary Adjusted P Value ?-A UVA vs. C -0.2083 -0.5772 to 0.1607 No ns 0.3385 A C UVA vs.0.3 -0.1513 -0.5202 to 0.2177 No ns 0.5733 C 0.3 UVA vs.0.03 -0.06250 -0.4315 to 0.3065 No ns 0.9406 D 0.03Case Ref. P274WO IPTector® Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 Q DF UVA vs. C 1.000 1.208 -0.2083 0.1375 4 4 1.514 12 UVA vs.0.3 1.000 1.151 -0.1513 0.1375 4 4 1.100 12 UVA vs.0.03 1.000 1.063 -0.06250 0.1375 4 4 0.4545 12 Table 30 Descriptive statistics for MTT after UVA exposure DESCRIPTIVE STATISTICS C UVA 0.3 0.03 Number of values 4 4 4 4 Minimum 1.113 0.7940 0.8950 0.8120 25% Percentile 1.130 0.8545 0.9428 0.8155 Median 1.222 1.059 1.179 1.021 75% Percentile 1.273 1.087 1.333 1.351 Maximum 1.276 1.089 1.353 1.396 Mean 1.208 1.000 1.151 1.063 Std. Deviation 0.07581 0.1393 0.2041 0.2907 Std. Error of Mean 0.03790 0.06965 0.1020 0.1453 Lower 95% CI 1.088 0.7783 0.8265 0.6000 Upper 95% CI 1.329 1.222 1.476 1.525 Table 31 UVA – protection RFU UVA 0.03 0.003 1.721.814 1.421.302 1.648.624 1.949.673 1.415.117 1.645.273 1.826.843 1.446.041 1.775.981 1.848.205 1.402.748 1.809.496 Normalized data UVA 0.03 0.003 0.937 0.774 0.898 1.062 0.770 0.896 0.995 0.787 0.967 1.006 0.764 0.985 Table 32 ANOVA for UVA – protection ANOVA summary F 33.66 P value <0.0001 P value summary **** Significant diff. among means (P < 0.05) Yes R squared 0.8821 Brown-Forsythe test 3.081 (2, F (DFn, DFd) 9) P value 0.0957Case Ref. P274WO IPTector® P value summary ns Are SDs significantly different (P < 0.05) No Bartlett's test Bartlett's statistic (corrected) 5.422 P value 0.0665 P value summary ns Are SDs significantly different (P < 0.05) No ANOVA table SS DF MS F (DFn, DFd) P value F (2, 9) = Treatment (between columns) 0.1089 2 0.05447 P<0.0001 33.66 Residual (within columns) 0.01456 9 0.001618 Total 0.1235 11 Data summary Number of treatments (columns) 3 Number of values (total) 12 Table 33 Multiple Comparison for UVA – protection MULTIPLE COMPARISONS Number of families 1 Number of comparisons per family 2 Alpha 0,05 Dunnett's multiple comparisons test Mean Adjusted P ?- 95,00% CI of diff, Significant? Summary Diff, Value A UVA vs.0.3 0.2263 0.1519 to 0.3006 Yes **** <0.0001 B 0.3 UVA vs. 0.06350 -0.01086 to 0.1379 No ns 0.0918 C 0.03 0.03 Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF UVA vs.0.3 1.000 0.7738 0.2263 0.02844 4 4 7.954 9 UVA vs. 1.000 0.9365 0.06350 0.02844 4 4 2.232 9 0.03 Table 34 Descriptive statistics for UVA – protection DESCRIPTIVE STATISTICS UVA 0.3 0.03 Number of values 4 4 4 Minimum 0.9370 0.7640 0.8960 25% Percentile 0.9515 0.7655 0.8965 Median 1.001 0.7720 0.9325 75% Percentile 1.048 0.7838 0.9805 Maximum 1.062 0.7870 0.9850 Mean 1.000 0.7738 0.9365 Std. Deviation 0.05123 0.009743 0.04621 Std. Error of Mean 0.02562 0.004871 0.02310 Lower 95% CI 0.9185 0.7582 0.8630 Upper 95% CI 1.082 0.7893 1.010 These results showed that UVA radiation significantly increased ROS levels in human keratinocytes by 101.4 ± 3.1 %, compared to the non-irradiated control. When ROS basal levels were subtracted to allCase Ref. P274WO IPTector® samples to assess UVA-induced oxidative stress protection, results indicated that treatment with Biotin at 0.3 % reduced UVA-induced ROS levels by 22.6 ± 2.8 %. These examples shows that treatment with Biotin shows antioxidant effects by reducing UVA-induced oxidative damage (ROS) in human keratinocytes (see figure 12). References - Devasagayam T, Tilak JC, Boloor KK, Sane Ketaki S, Ghaskadbi Saroj, Lele RD. Free radicals and antioxidants in human health: Current status and future prospects. Journal of Association of Physicians of India.2004; 54:796. - Han D, Williams E, Cadenas E. Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. The Biochemical Journal. 2001; 353:411-6. - Ichihashi M, Ando H, Yoshida M et al. Photoaging of the skin. Anti-Aging Medicine 2009; 6: 46–59. - Kim MS, Kim YK, Cho KH, Chung JH. Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin. Mech Ageing Dev 2006; 127: 875–882. - Muller F. The nature and mechanism of superoxide production by the electron transport chain: Its relevance to aging. - Journal of the American Aging Association.2004; 23(4):227-53. - Murphy MP. How mitochondria produce reactive oxygen species. Biochem J.2009; 417:1-13. - Vayalil PK, Mittal A, Hara Y, Elmets CA, Katiyar SK. Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinases expression in mouse skin. J Invest Dermatol 2004; 122: 1480–1487. - Dykas, Carol (June 2004). How to protect patients from harmful sunlight. www.2020mag.com - Peak MJ and Peak JG. Photodermatol.1989; 6, 1–15. Example 4 – In vivo testing of penetration of Biotin and LipoBiotin into human skin using Confocal Raman Spectroscopy Introduction

[0078] Measurement of the in vivo biotin and biotin liposome penetration into the skin of volunteers was assessed with Confocal Raman Spectroscopy. Confocal Raman Spectroscopy provided in-vivo, non-invasive, quantitative analysis of dynamic processes such as skin penetration and permeation of topical formulations to optimize active delivery, molecular concentration profiles of the skin with highCase Ref. P274WO IPTector® spatial resolution, distribution of intrinsic skin constituents: amino acids, lipids, proteins, water, and biotin concentration measured directly through the detection of its Raman emission. Material and Methods Six volunteer subjects were selected as follows: VOLUNTEER VOLUNTEER ID AGE GENDER SKIN TYPE V1 3487 54 Female Combination V2 2950 46 Female Dry V3 2704 37 Female Combination V4 2615 53 Female Dry V5 3516 42 Female Combination V6 2374 53 Female Other Biologically produces biotin and biotin liposomes of example 1 was used: SAMPLE ABBREVIATION INTERNAL CODE PlexoZome Biotin (0.2%) LipoBiotin P.3007 Bio-B7 Batch number: BSY-22-026 Biotin P.3008

[0079] Measurement of the chemical composition of the skin (volar forearm) by confocal Raman spectroscopy was made before any treatment wioht biotin or biotin liposomes. Biotin or biotin liposomes were then applied on the skin of volunteers (volar forearm), whereafter the amount of biotin in the skin was measured and quantified at different time intervals at 1, 10, 20, 40, and 120 minutes. The biotin and LipoBiotin were applied independently on different areas of the volar forearm of volunteers. Results and conclusion

[0080] Figure 13 shows the biotin quantification in the skin resulting from the application of biotin on the skin of the test subjects, expressed as µg of biotin per mg of total protein in the skin. Figure 14 shows the total content of LipoBiotin is expressed as µg of biotin per cm2of stratum corneum (assuming a stratum corneum thickness of 15 µm). To analyze the behavior of both biotin and LipoBiotin in a more visual manner, figure 15 overlaps the quantification of the total content of biotin (expressed as µg of per cm2of stratum corneum) after application of Biotin or LipoBiotin.

[0081] After the application of biotin and LipoBiotin the amount of biotin measured at subsequent time points decreased gradually due (i) to the diffusion of molecule to deeper layers, or (ii) to the metabolism or degradation of the molecule.

[0082] When LipoBiotin was applied, the amount of biotin that entered into the skin was 284 % higher compared to when the product Biotin was applied, indicated by the product levels after 1 minute ofCase Ref. P274WO IPTector® the application.

[0083] Figures 16 and 17 shows the amount of biotin absorbed into the skin during the 120 min lapse for each test subject and for all of them (average). This information is shown for both Biotin and LipoBiotin. The data are presented as µg of biotin absorbed per cm2of stratum corneum (assuming a depth of 15 µm). Dashed line indicates the averaged value.

[0084] 81.2% of the biotin was absorbed into the skin after 120 min of the application of the product LipoBiotin, in contrast to the 38.9% of the biotin that was absorbed into the skin after application of the product Biotin.

[0085] Figure 18 shows the comparison of the average amount of biotin absorbed into the skin during the 120 min lapse for the products Biotin and LipoBiotin. The data are presented as µg of biotin absorbed per cm2of stratum corneum (assuming a depth of 15 µm). The absorption of biotin into the skin was higher (108.6%) when the product LipoBiotin was used, compared to when the product Biotin was used.

[0086] Theser results show that: - When the product LipoBiotin was applied, the amount of biotin that entered into the skin was 284 % higher compared to when the product Biotin was applied. - 81.2% of the biotin was absorbed into the skin after 120 min of the application of LipoBiotin, in contrast to the 38.9% of the biotin that was absorbed into the skin after application of the product Biotin. - The absorption of biotin into the skin was higher (108.6%) when LipoBiotin was used, compared to when Biotin was used. - The Raman signal of some components of the formulation could overlap with those of the natural composition of the skin and could have been underestimated. Therefore, the shown amounts of biotin correspond to minimum quantities biotin. - After the application of Bioting and LipoBiotin, the amount of biotin measured in subsequent time points decreased gradually due (i) to the diffusion of molecule to deeper layers, or (ii) to the metabolism or degradation of the molecule. References - Caspers et al. Method to quantify the in vivo skin penetration of topically applied materials based on confocal Raman spectroscopy. Translational Biophotonics (2019), doi.org / 10.1002 / tbio.201900004. - Akhtar et al. Penetration Enhancing Effect of Polysorbate 20 and 80 on the In Vitro Percutaneous Absorption of LAscorbic Acid. Tropical Journal of Pharmaceutical Research, Vol.Case Ref. P274WO IPTector® 10 No.3 (2011) doi: 10.4314 / tjpr.v10i3.1. - El-Leithy ES, Makky AM, Khattab AM, Hussein DG. Optimization of nutraceutical coenzyme Q10 nanoemulsion with improved skin permeability and anti-wrinkle efficiency. Drug Dev Ind Pharm. 2018 Feb;44(2):316-328. doi: 10.1080 / 03639045.2017.1391836. Epub 2017 Nov 2. PMID: 29096550. - Kage M, Tokudome Y, Hashimoto F. Permeation of hyaluronan tetrasaccharides through hairless mouse skin: an in vitro and in vivo study. Arch Dermatol Res.2013 Jan;305(1):69-77. doi: 10.1007 / s00403-012-1252-2. Epub 2012 Jun 28. PMID: 22740084. - Mateus R, Moore DJ, Hadgraft J, Lane ME. Percutaneous absorption of salicylic acid--in vitro and in vivo studies. Int J Pharm. 2014 Nov 20;475(1-2):471-4. doi:10.1016 / j.ijpharm.2014.08.061. Epub 2014 Aug 29. PMID: 25178827. - Naito C, Katsumi H, Yoneto K, Omura M, Nishidono M, Kamei S, Mizoguchi A, Tamba A, Tanaka A, Morishita M, Yamamoto A. Development of a Phosphoric Acid-Mediated Hyaluronic Acid Gel Sheet for Efficient Transdermal Delivery of Alendronate for Anti-Osteoporotic Therapy. Pharmaceutics. 2019 Dec 2;11(12):643. doi: 10.3390 / pharmaceutics11120643. PMID: 31810310; PMCID: PMC6956090. - Niu J, Yuan M, Zhang Z, Wang L, Fan Y, Liu X, Liu X, Ya H, Zhang Y, Xu Y. Hyaluronic Acid Micelles for Promoting the Skin Permeation and Deposition of Curcumin. Int J Nanomedicine.2022 Sep 7;17:4009-4022. doi: 10.2147 / IJN.S372711. PMID: 36105622; PMCID: PMC9464638. - Example 5 – In vivo testing of anti-aging efficacy of LipoBiotin on human skin Introduction

[0087] Assessment of the anti-aging efficacy in human skin of biotin delivered by biotin liposomes is made on a population of volunteer test subjects by measuring the following characteristics: - Anti-blemish spots (area and number) by multispectral analysis; - Even Skin tone contrast of the spots vs skin by multispectral analysis; - Deep hydration at different depths of the skin by Raman confocal spectroscopy; - Skin ceramides and fatty acids at different depths of the skin by Raman confocal spectroscopy; - Skin radiance and gloss of the skin by using a Glossymeter probe; - Firming and elasticity: by using a Cutometer probe. Material and Methods Biologically produced biotin and the biotin liposomes of example 1 is used: SAMPLE ABBREVIATION INTERNAL CODECase Ref. P274WO IPTector® PlexoZome Biotin (0.2%) LipoBiotin P.3007 Bio-B7 Batch number: BSY-22-026 Biotin P.3008

[0088] The above mentioned characteristics are measured at day 0 (before the treatment) and Day 28 (after the treatment), as well at a number times during the treatment. Biotin and LipoBiotin are applied independently on different areas of the volar forearm skin of the test subjects. Results and conclusion

[0089] Improvements are observed of one or more of the characteristics of blemish spots, even Skin tone, deep hydration, skin ceramides and fatty acids content, skin radiance and gloss, firming and elasticity. Crema Facial clinical study:

[0090] PRODUCT TESTED The tested samples were received at room temperature and labelled as indicated in Figure 19. Product formulation specification: The product was formulated with excipients as described in Table 1, 2 and 3. Further excipient ingredients comprise: Caprylic / Capric Triglyceride, Propanediol, Diheptyl Succinate, Quijalla Saponaria Wood Extract, Capryloyl Glycerin / Sebacic Acid Copolymer, Hydrogenated Ethylhexyl Olivate, Sclerotium Gum, Saponaria officinalis Leaf / Root Extract, Sucrose Laurate, Caesalpinia Spinosa Gum, Hydrogenated Olive Oil Unsaponifiables, Phenoxyethanol, Parfum, Ethylhexylglycerin, Tocophersolan and / or Tocopherol.

[0091] STUDY PROTOCOL OVERVIEW 23 volunteers were submitted to a 28-day face treatment with Crema Facial. The effect of treatment was assessed through quantification of skin firmness and elasticity using Cutometer. Skin brightness was measured using Glossymeter probe and brown spots were measured using Visia-CR. Skin hydration and ceramides / fatty acid levels were evaluated by Confocal Raman Spectroscopy using the Gen2 SCA Ultimate (RiverD) on 10 volunteers. The measurements were carried out before (D0) and after 28days (D28) from the beginning of the treatment. Furthermore, macroscopic images were taken before and immediately after applying the product using Visia-CR. Volunteers also completed a self-assessment questionnaire after 28 days of the treatment (use test). The study was performed under dermatological surveillance.

[0092] GOOD CLINICAL PRACTICE This study was carried out under the basic principles and spirit of Good Clinical Practices. Under the direct supervision of certified GCP researchers. This ICH E6 GCPCase Ref. P274WO IPTector® Investigator Site Training Certification meets the Minimum Criteria for ICH GCP Investigator Site Personnel Training identified by TransCelerate BioPharma as necessary to enable mutual recognition of GCP training among trial sponsors.

[0093] CHECKING OF THE ACCEPTABILITY The subjects were requested to note every day any reaction observed, and sensation of discomfort felt. An examination of the experimental area under standard daylight source was performed by the responsible technician, the same days of the technical measurements. Together with the clinical examinations performed during the treatment, each subject was questioned by the responsible technician about the possible sensations of discomfort they felt, at the end of the study.

[0094] CONSUMPTION CONTROL Consumption control was carried out to verify that volunteers follow the guidelines and apply the treatment. This value is used for evaluating the clinical adherence, as the percentage of volunteers who follow the protocol and consume the estimated product to be consumed according to the client's application protocol.

[0095] STATISTICAL ANALYSIS For the evaluation of skin brightness, firmness and elasticity five repeated measurements were performed on the skin of each volunteer. Data were analyzed using statistical software for the identification of outliers (ROUT method with coefficient Q=5%) and then averaged and an experimental value was obtained for each time point. For the evaluation of brown spots one experimental value was obtained for each timepoint. For statistical analysis, all data collected before (D0) and day (D28) were subjected to the normality test using the Shapiro-Wilk method (Shapiro, 1965). The following methods were then applied to evaluate the statistical significance by pairs 0 vs 28. All those variables whose normality test result was positive (parametric variable) were analyzed using the paired t-test method (David, 1997). All those variables whose normality test result was not positive (non-parametric) were analyzed using the paired Wilcoxon method (Wilcoxon, 1959). In the report, the difference in percentage of the normalized data is plotted with the error bars indicating the standard error of the mean (SEM) For normalization, the technical values obtained before (D0) and after treatment are relativized to the basal values before treatment (D0) and expressed as a percentage. For Ramnan analysis data were analyzed using statistical software for the identification of outliers (ROUT method with coefficient Q=5%). For statistical analysis, all data collected before (Day 0) and after treatment (Day 28) were analyzed using the paired t-test method. The data is represented as the mean ± the standard error of the mean (SEM). Statistical significance was declared at p < 0.05, 95 % of confidence. Example 6 – Firmness and elasticity in Crema Facial clinical study IntroductionCase Ref. P274WO IPTector®

[0096] Skin firmness and elasticity measurements are performed through Cutometer® MPA 580. The principle of measurement is based on the suction method. In brief, the probe creates a negative pressure and the skin is drawn to the opening of the probe. The penetration depth is measured by a non-contact optical measurement system consisting of a light source, a receiver, and two prisms, which project light from the emitter to the receiver. The results evaluate the skin’s resistance to suction (firmness) and its ability to return to its original position (elasticity). Units of measurement are μm, depth of penetration in the opening of the probe, expressed in form of curves (Figure 20). Measurement with Cutometer is used as a standard in dermatology and cosmetology. It is indispensable for the development of efficacy tests and supports the validation of all types of cosmetic products (especially anti-aging and firming products). In this assay, for the quantification of the skin firmness and elasticity levels on the cheek area, the ROI was defined by the researcher, and all the measurements were carried out on the same spot to ensure the reproducibility and significance of the measurements. Five repeated measurements were performed on the cheek of each volunteer at each of the time points. Results and conclusion

[0097] The results showed that the Crema Facial significantly increased elasticity values by 8.4 ± 2.9 % after 28 days of treatment compared with basal values (Figure 21 and Table 35). Table 35 Statistical analysis of the results shown in Figure 21. Example 7 – Brightness / gloss in Crema Facial clinical study Introduction

[0098] Measurement of gloss (brightness) is very important in the efficacy testing of skincare, hair care, and decorative cosmetics (lipsticks, make-up, etc.). Skin and hair are supposed to show a natural, luminous gloss without appearing oily. Also, for nail cosmetics, gloss is an important parameter. The measurement of gloss is based on light reflection. Parallel white light is created by LEDs in the probe head. To be able to emit light at 60º in a relatively small and uniquely designed measurement head, light is sent out at 0º and reflected by mirrors to 60º. Two separate measurement channels measure the direct reflected light (also guided by a mirror in the same angle into the reflection channel) andCase Ref. P274WO IPTector® the diffuse reflected (scattered) light. The scattered / diffuse reflected light is measured at 0º (completely vertically above the measured surface) under the assumption that light is scattered in the same way overall degrees (diffuse channel). Therefore, the Skin-Glossymeter® GL 200 (Figure 22) expresses both, the portion of directly reflected light (gloss) and the diffusely scattered portion from the skin surface. Skin varies in structure and brightness, and also in color. On the skin, some part of the light will penetrate the upper layers and will get scattered depending mainly on the skin color (dark skin scatters less than light skin). To obtain a gloss value mostly free from the influence of skin color, the diffuse light entering the gloss channel is deducted by a special formula. No other gloss measurement offers such a function. Five repeated measurements on the cheek of the volunteers in each of the time points were performed. Results and conclusion The results show that the Crema Facial significantly increased skin gloss values by 14.5 ± 5.4 % after 28 days of treatment compared with basal values (Figure 24 and Table 36). Table 36. Statistical analysis of the results shown in Figure 24. Example 8 – Brown spots in Crema Facial clinical study Introduction

[0099] Brown spots analysis were performed on 2D images taken with Visia-CRP-5 Primos technology. The captures obtained were analyzed by placing a mask and creating a ROI (region of interest). The baseline ROI was directly transferred into the D28 captures. The data (contrast) were obtained through a .csv file (Figure 23). Results and conclusion

[0100] Results showed that the Crema Facial significantly decreased brown spots area and number by 2.7 ± 1.4 % and 2.3 ± 1.3 % respectively, after 28 days of treatment compared with basal values (Figure 25, Table 37). Table 37. Statistical analysis of the results shown in Figure 25.Case Ref. P274WO IPTector® Brown spot area D0 vs D28 Mean Diff. -2.74 Std. Error of Mean 1.42 Adjusted to P Value 0.00134 Summary * Significant Yes Brown spot number D0 vs D28 Mean Diff. -2.25 Std. Error of Mean 1.30 Adjusted to P Value 0.0338 Summary * Significant Yes Brown spot contrast D0 vs D28 Mean Diff. 0.05 Std. Error of Mean 1.04 Adjusted to P Value 0.9254 Summary Ns Significant No Example 9 – Skin hydration in Crema Facial clinical study Introduction

[0101] For the evaluation of the skin hydration levels, ten volunteers were enrolled in a 28-day topical treatment. Each volunteer applied the products on the skin according to the client’s instructions. Skin hydration levels were evaluated by Confocal Raman Spectroscopy by using the Gen2 SCA Ultimate (RiverD International B.V., Rotterdam, Netherlands) before (baseline) and 28 days after applying the product. This instrument is a confocal Raman system of high sensitivity designed for in vivo skin analysis (Kourbaj et al., 2022) (Richters et al., 2017). The gen2-SCA Ultimate has two built-in lasers (wave class 3B lasers, 671 nm and 785 nm). For this measurement, we used the laser with a wavelength of 671 nm, recording the “Raman HWN region” with wavenumbers from 2500 to 4000 cm–1. The laser power complies with the maximum permissible levels for skin as defined by the international laser safety standard (IEC 60285-1:2007; < 0.05, 95 % of confidence. Data in the graphs are represented by mean ± standard error of the mean. Results and conclusion

[0102] With Confocal Raman Spectroscopy we can directly measure water molecules in the skin. HereCase Ref. P274WO IPTector® we represent the averaged water content (µg) per cm2 of stratum corneum (SC) before (Day 0) and 28 days after the treatment (Figure 26). The specific values for each volunteer are also shown, indicating the increase of water content in µg and in percentage (Table 38). Finally, the increase in the water content of the SC after 28 days of treatment is shown (Figure 27). Table 38. Values of the water content (^g) per cm2of SC before (Day 0) and 28 days after the treatment for each volunteer. The increase in water content (in ^g and in %) is shown. The results of the evaluation of skin hydration levels after 28 days of treatment with Crema Facial showed that: a) Crema Facial increased deep hydration (water content) of the skin by 16.4% (Figure 26 and 27, Table 38). b) The water content increases 110.5 µg per cm2of stratum corneum (Figure 26 and 27, Table 38). c) 80% of the volunteers presented an increase in the deep hydration of the skin (Figure 27, Table 38). d) Deep hydration levels increased up to 37.4% (Figure 27, Table 38).

[0103] Confocal Raman Spectroscopy allows to measure the thickness of the SC of the skin in vivo. Hydration of the skin increases the thickness and health of the stratum corneum. Figure 28 represents the averaged SC thickness before (Day 0) and 28 days after the treatment. The specific values for each volunteer are shown in Table 39, indicating the increase of the SC thickness in µm and in percentage. The increase in the SC thickness after 28 days of treatment is shown in Figure 29. The results of the evaluation of stratum corneum (SC) thickness after 28 days of treatment with Crema Facial showed that:Case Ref. P274WO IPTector® a) Crema Facial increased the SC thickness by 10.6% (Figure 28 and 29, Table 39). b) The SC thickness increased an average of 1.6 µm (Figure 28 and 29, Table 39). c) 80% of the volunteers presented an increase in the SC thickness (Figure 29, Table 39). d) SC thickness increased up to 24.8% (Figure 29, Table 39). Table 39 Values of the SC thickness (^m) before (Day 0) and 28 days after the treatment for each volunteer. The increase in the SC thickness (in ^m and in %) is shown.

[0104] Confocal Raman Spectroscopy allows to directly measure water molecules in the skin in different depth layers, from the stratum corneum to the dermis. Increase in the SC thickness is associated with an increase in the deep hydration of the skin. The averaged amount of water (in %) as a function of depth is represented for Day 0 and Day 28 in Figure 30, indicating the thickness of the SC for each time point. Example 10 – Ceramides and fatty acid in Crema Facial clinical study Introduction

[0105] For the evaluation of ceramides and fatty acid levels of the skin, ten volunteers were enrolled in a 28-day topical treatment. Each volunteer applied the products on the skin according to the client’s instructions. Skin ceramides / fatty acid levels were evaluated by Confocal Raman Spectroscopy by using the Gen2 SCA Ultimate (RiverD International B.V., Rotterdam, Netherlands) before (baseline)Case Ref. P274WO IPTector® and 28 days after applying the product. This instrument is a confocal Raman system of high sensitivity designed for in vivo skin analysis (Kourbaj et al., 2022) (Richters et al., 2017). The gen2-SCA Ultimate has two built-in lasers (wave class 3B lasers, 671 nm and 785 nm). For this measurement, we used the laser with a wavelength of 785 nm, recording the “Raman fingerprint region” with wavenumbers from 400 to 1800 cm–1. The laser power complies with the maximum permissible levels for skin as defined by the international laser safety standard (IEC 60285-1:2007). Results and conclusion

[0106] With Confocal Raman Spectroscopy we can directly measure the amount of ceramides / fatty acids of the skin. Here we represent the averaged ceramides / fatty acid content (arbitrary units, AU) per cm2of SC before (Day 0) and 28 days after the treatment (Figure 31). We also show the specific values for each volunteer, indicating the increase of the ceramides / fatty acid content in percentage (Table 40). Finally, the increase in the ceramides / fatty acid content of the SC after 28 days of treatment is shown (Figure 32). The results of the evaluation of ceramides and fatty acid levels after 28 days of treatment with Crema Facial showed that: a) Crema Facial increased ceramides and fatty acid levels by 20.3% (Figure 31 and 32, Table 40). b) 100% of the volunteers presented an increase in ceramides and fatty acid levels (Figure 32, Table 40). c) Ceramides and fatty acid levels increased up to 50.4% (Figure 32, Table 40). Table 40 Values of the Ceramide and fatty acid content of the skin (arbitrary units) per cm2 of SC before (Day 0) and 28 days after the treatment for each volunteer. The increase in Ceramide and fatty acid content (in %) is shown.Case Ref. P274WO IPTector®

[0107] Confocal Raman Spectroscopy allows to directly measure the Ceramide and fatty acid levels in the skin in different depth layers, from the stratum corneum to the dermis. Increase in the Ceramide and fatty acid levels is associated with an improved skin barrier effect, skin health and deep hydration of the skin. The averaged amount of Ceramide and fatty acid (AU) as a function of depth is represented for Day 0 and Day 28 in Figure 33. Table 41 shows the Ceramide and fatty acid levels (AU) for each specific skin depth, indicating the percentage of the increase in Ceramide and fatty acid in each analyzed skin depth. Our results shows that the product increases the ceramide and fatty acid levels up to a depth of 18 µm.

[0108] Figure 34 shows an overlap representation of the graphs showing the amount of (i) water (as shown in Figure 30) and (ii) ceramides and fatty acids (as shown in Figure 33) as a function of depth. The increase of the amount of hydrophobic molecules (ceramides / fatty acids) is mostly detected the upper layers of the skin, corresponding to the SC. This change in the chemical structure of the skin modifies the water distribution across the SC and its structure. Accumulation of ceramides and fatty acids in the upper layers of the SC decreases the trans-epidermal water loss, expands the thickness of the SC and increases the amount of water present in the SC, enhancing the skin barrier effect. Example 11- Self assesment in Crema Facial clinical study Introduction

[0109] The efficacy of the treatment was subjectively evaluated by a use test (self-assessment questionnaire), answered by each of the volunteers who completed the study. The self-assessmentCase Ref. P274WO IPTector® questionnaire after 28 days treatment contained 24 questions. Opinions are given according to parameters from 1 to 4, (1 = Strongly disagree; 2 = Disagree; 3 = Agree; 4 = Strongly agree). For positive impressions, satisfaction is considered when volunteers scored parameters from 3 to 4. The number and percentage of satisfied volunteers according to punctuation are shown for each parameter. For positive impressions, satisfaction was considered when 80% volunteers gave positive answer. Results and conclusion

[0110] The efficacy of the treatment was subjectively evaluated by a use test (self-assessment questionnaire), answered by each of the volunteers who completed the study. The results of the preliminary questions are shown below. Results from the self-assessment questionnaire after 28 days of treatment with Crema Facial are shown in Table 41. Table 41 Results from the self-assessment questionnaire after 28 days of treatment with Crema Facia.

[0111] A relevant percentage of volunteers (≥ 80 %) after 28 days treatment considered about Crema Facial that: a) The application of the product is easy. b) The product has a pleasant texture. c) The product absorption is fast. d) This product is gentle to the skin. e) The smell is pleasant.Case Ref. P274WO IPTector® f) The skin feels softer. g) The skin is more supple. h) The skin is more nourished. i) The treatment visually improves the quality of the skin. j) The skin looks younger. k) The skin feels hydrated. l) The skin is firmer. m) The skin is more elastic. n) The treatment increases the luminosity of the skin. o) The skin looks smoother. p) They were satisfied with the treatment received. q) They would use the treatment again. r) They would recommend the treatment to a friend.

[0112] Results after 28 days of treatment showed an overall acceptance average of 87%. Specifically, positive evaluations (overall acceptance ≥ 80 %) were obtained for 18 out of 24 evaluated parameters. A graphical representation of results of self-assement questionnare is shown in Figure 35. Example 12 EraGlow beautin by Biosyntia™

[0113] In order to improve its solubility and bioavailability, natural biotin is vectorised in highly stable liposomal structures. 0.2% of natural biotin is incorporated in sunflower lecithin in order to form bilayered vesicles that will ensure progressive natural biotin delivery to the epidermis. This product is commercially available under the tradename EraGlow beautin by Biosyntia™. A penetration study has been performed in vivo to assess the behaviour of biotin when applied on the skin either in a free from or in delivery enhancing complex. The same quantities (50 µl) of both products were applied by a trained technician on the arm of 5 female volunteers aged 37 to 54. The penetration of biotin in the stratum corneum up to a depth of 18 µm was directly monitored by confocal Raman spectroscopy using the Gen2 SCA Ultimate (RiverD International B.V., Rotterdam, Netherlands) after 1, 10, 20, 40 and 120 minutes in order to evaluate the speed and amount of biotin delivery.

[0114] One minute after application, the total amount of biotin that penetrated into the skin was 3 times (284%) higher with EraGlow beautin by Biosyntia™ than with free biotin (Fig.36). We could also notice an important difference in biotin amounts on the surface of the stratum corneum. Measurements revealed that the amount of biotin was 3.2 times higher in the case of EraGlow beautin by Biosyntia™, showing that the vectorisation protects the molecule from being either degraded or metabolised by the microbiome before it could penetrate the skin.Case Ref. P274WO IPTector®

[0115] After two hours, the total amount of biotin delivered into the skin is twice the amount (+109%, p<0.02) with the vectorisation compared to free biotin that is rapidly metabolised or degraded (Fig. 37).

[0116] The penetration flux of biotin was also determined within the first 10 minutes and the next 30 minutes (Table 42). These results express the quantity of biotin that diffused to deeper layers and indicate that EraGlow Beautin by Biosyntia™ increases the biotin penetration flux 8.7 times compared to free biotin within the first 10 minutes after the application and that EraGlow Beautin by Biosyntia™ increases the biotin penetration flux 3 times compared to free biotin between 10 and 40 minutes after the application. Table 42: Penetration flux of biotin in the stratum corneum. Penetration flux (µg / cm² / h) 0 to 10 minutes 10 to 40 minutes Free biotin 0.0002 0.0011 EraGlow beautin™ 0.0006 0.0096

[0117] This study shows the interest of vectorising natural biotin in a stable liposomal structure. EraGlow beautin by Biosyntia™ allows a gradual delivery of higher amounts of natural biotin into the skin and reduces immediate biotin degradation that may be due to microbiome metabolism to support its diffusion to deeper layers. Synthesis of endogen ceramides and lipids

[0118] The formation of a competent epidermal permeability barrier requires an approximately equal molar ratio of cholesterol, free fatty acids and ceramides, which are synthesised in the endoplasmic reticulum of the stratum spinosum keratinocytes within the epidermis. Deficiencies in any of these lipids can result in a defective epidermal permeability barrier. Studies have shown that the aged stratum corneum displays a >30% reduction in total lipid content in comparison to young stratum corneum, due to reduced epidermal lipid synthesis (Wang). The level of lipids in the stratum corneum is thus a relevant marker of ageing.

[0119] A clinical study was performed on 10 volunteers who applied on the face a cream containing 30% EraGlow beautin by Biosyntia™ for 28 days. The amount of ceramides / fatty acid levels were evaluated by confocal Raman spectroscopy using the Gen2 SCA Ultimate (RiverD International B.V., Rotterdam, Netherlands) at a wavelength of 785 nm.

[0120] The results of the measurements of ceramides and fatty acid levels after 28 days of treatment with a face cream containing EraGlow beautin by Biosyntia™ showed that endogen ceramides andCase Ref. P274WO IPTector® fatty acid levels were increased very significantly by 20.3%, up to 50.4% and that 100% of the volunteers presented an increase (Fig.38).

[0121] Lipids and ceramides are primarily produced in the spinous layer at about 30 µm depth and are externalised from the outermost stratum granulosum cells to form the epidermal brick and mortar impermeable structure (Feingold). Confocal Raman spectroscopy allows to directly measure the ceramide and fatty acid levels in the skin in different depth layers, from the stratum corneum to the dermis. It is them possible to visualise the effect of biotin in EraGlow beautin by Biosyntia™ on the increase in ceramide and fatty acids synthesis as a function of the epidermis depth on a graph (Fig. 39).

[0122] Results shows that the Eraglow beautin by Biosyntia™ increases the ceramide and fatty acid levels up to a depth of 18 μm. As expected, it is possible to see the settlement of these lipids in the layers of the stratum corneum where they are externalised from the outermost layers of keratinocytes. At the level of the most superficial corneocyte layers, the amount of ceramides and fatty acids has increased by more than 30%. Under 18 µm depth, lipids detected by CRS are mostly constitutive of the cellular membrane. Reinforcing the epidermis to lock in moisture:

[0123] The application of the facial cream containing EraGlow beautin by Biosyntia™ contributed to increase significantly the thickness of the stratum corneum by 10.6% (p<0.05).

[0124] Because it is well known that the application of a cream instantly brings a moisturising effect to the upper layers of the stratum corneum through emollience, i.e. impregnation, it is interesting to assess the effect of Eraglow beautin by Biosyntia™ on deep hydration after a 28-day treatment. References - Goldberg LJ, Lenzy Y: Nutrition and hair. Clin Dermatol 2010; 28: 412–419. - Mock DM (1999) Biotin. In: Modern Nutrition in Health and Disease, 9th edn. Shils ME, Olson JA, Shike M, Ross AC (eds.). Baltimore, MD: Lippincott Williams & Wilkins, pp.459–466. - Wang Z, Man MQ, Li T, Elias PM, Mauro TM. Aging-associated alterations in epidermal function and their clinical significance. Aging (Albany NY).2020 Mar 27;12(6):5551-5565 - Feingold KR. Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid Res.2007; 48:2531–2546. * * *

Claims

Case Ref. P274WO IPTector® Claims 1. A liposomal preparation of biotin comprising a lipid bilayer membrane encapsulating an aqueous solution of biotin, thereby constituting a biotin containing liposome.

2. The liposomal preparation of claim 1 further comprising one or more ingredients selected from purified water, lecithin, vitamin E, glycerol, ethanol and / or potassium carbonate.

3. The liposomal preparation of claim 2 comprising 50% to 80% by weight of purified water, 5% to 8% by weight of lecithin, 7,5% to 12,5% by weight of vitamin E, 15% to 20% by weight of glycerol, 5% to 10% by weight ethanol and 0,01% to 1% by weight of potassium carbonate.

4. The liposomal preparation of any preceding claim comprising 0,01% to 10% by weight of biotin.

5. The liposomal preparation of any preceding claim wherein the diameter of the liposome is between 10 to 500 nm, such as 20 to 250 nm, such as 30 to 100 nm.

6. A composition comprising the liposomal preparation of any preceding claim.

7. The composition of claim 6 wherein the composition is selected from bath preparations, body and hand preparations, skin or hair cleansing products, such as cold creams, cleansing lotions, liquids and pads), eye makeup, face and neck preparations, hair conditioners, hair preparations, hair sprays tanning preparations, manicuring preparations, moisturizing preparations, night skin care preparations, shampoos, skin fresheners, skin tonics, skin dressings, bath oils, bath soaps and detergents, eye lotions, eyeliners, foundations, hair dyes and colors, hair rinses, hair wave sets, makeup reparations, mascaras, nail polishes and enamels, paste masks, mud packs, skin care preparations, and sun tan preparations.

8. The composition of claim 7 comprising 25 to 50 %wt of the liposomal preparation of claims 1 to 6 and / or 0,0001 to 2 %wt of biotin.

9. The composition of claim 8 comprising 30 %wt of the liposomal preparation and / or 0.2 %wt biotin.

10. Cosmetic non-therapeutical use of the liposomal preparation of any preceding claim for skin or hair care.Case Ref. P274WO IPTector® 11. The use of claim 10, wherein the skin or hair care includes photoaging reduction, hair conditioning, skin conditioning, skin lightening, skin brightening, reduction of senile lentigines, reduction of melanin, moisturizing of skin, oxygen elimination or a combination thereof.

12. The use of claim 10, wherein the skin or hair care comprises improvements in one or more skin properies selected from pigmentation, UV protection, blemish spots, brown spots, skin tone, hydration, ceramides and fatty acids contents, radiance, gloss, firmness and / or elasticity.

13. The liposomal preparation of any preceding claim for use as a medicament for the treatment or prevention of a skin or hair disease.

14. The liposomal preparation of claim 12 wherein the treatment or prevention of a skin or hair disease is selected from skin inflammation, eczema, dermatosis, and psoriasis.

15. A method for producing the biotin liposomal preparation of claim 1 to 5 or the composition of claim 6 to 8 comprising mixing biotin with a lipid bilayer membrane at conditions allowing the formation of a spherical shaped nanostructure having an aqueous core comprising the biotin. * * *

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