WO2022246529A2 - Compositions and methods to protect against oxidative hair dyes allergens - Google Patents

Abstract

Methods and kits of using Guayusa-containing compositions are disclosed. One method includes a) applying a composition to the scalp and the hair, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying an oxidative hair coloring formulation to the hair and the scalp. Another method includes a) mixing a composition with an oxidative hair coloring formulation to form a mixture, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying the mixture to the scalp and the hair. The kit of using the Guayusa-containing compositions includes a kit for protecting scalp and hair of a subject from an oxidative hair coloring process. The kit includes a) a composition comprising Ilex Guayusa leaf extract; and b) an oxidative hair coloring formulation.

Description

COMPOSITIONS AND METHODS TO PROTECT AGAINST OXIDATIVE HAIR DYES ALLERGENS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is related to, claims priority to, and incorporates herein by reference for all purposes U.S. Provisional Patent Application No. 63/193,462, filed May 26, 2021.

BACKGROUND

[0002]Many oxidative hair coloring formulations contain oxidants and allergens harmful to the hair and the scalp of the users, which often cause discomfort on the scalp of the users including burning and stinging feelings during application of these oxidative hair coloring formulations. These allergens include p-phenylenediamene (PPD), resorcinol (RCN), and hydrogen peroxide (H2O2).

[0003]A need exists for a formulation to be applied to the hair and the scalp prior to, during, or after an oxidative hair coloring process to prevent or reduce the discomfort and damage caused by the harmful oxidants and allergens in oxidative hair coloring formulations.

SUMMARY

[0004]In an aspect, the present disclosure provides a method of protecting scalp and hair of a subject from an oxidative hair coloring process. The method includes: a) applying a composition to the scalp and the hair, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying an oxidative hair coloring formulation to the hair and the scalp.

[0005]In another aspect, the present disclosure provides a method of protecting scalp and hair of a subject from an oxidative hair coloring process. The method includes: a) mixing a composition with an oxidative hair coloring formulation to form a mixture, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying the mixture to the scalp and the hair.

[0006]In yet another aspect, the present disclosure provides a kit for protecting scalp and hair of a subject from an oxidative hair coloring process. The kit includes a) a composition comprising Ilex Guayusa leaf extract; and b) an oxidative hair coloring formulation.

BRIEF DESCIPTION OF THE DRAWINGS

[0007]FIG. 1 is a plot illustrating quantification of radical proteins formation in HFDPCs culture treated with the investigational product SENSISHIELD SCALP and exposed to chemical inducers. HFDPCs were treated for 72 hours with the investigational product and, at the same time, exposed to chemical inducers, for aftermost quantification of the radical proteins.

[0008]FIG. 2 is a plot illustrating the semi-quantification of mitochondrial oxidative stress in HFDPCs culture treated with the investigational product SENSISHIELD SCALP and exposed to chemical inducers. HFDPCs were treated for 2 hours with the investigational product and, concomitantly, exposed to chemical inducers, for subsequent semi-quantification by image of the fluorescence emitted due to oxidative stress.

[0009]FIG. 3 is a plot illustrating the quantification of interleukin 8 in HFDPCs culture treated with the investigational product SENSISHIELD SCALP and exposed to chemical inducers. HFDPCs were treated for 72 hours with the investigational product and, at the same time, exposed to chemical inducers, for aftermost quantification of IL-8.

[0010]FIG. 4 is a plot illustrating the semi-quantification of filaggrin synthesis in human scalp ex vivo culture treated with the investigational product SENSISHIELD SCALP and exposed hair color. Fragments of human scalp ex vivo were treated for 40 minutes with the investigational product and exposed to hair color for further semi-quantification by image of the fluorescence emitted by the immunostaining of filaggrin. DETAILED DESCRIPTION

[0011]Before the present invention is described in further detail, it is to be understood that the invention is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0012]A Guayusa-containing composition and applications thereof are disclosed. It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.

[0013]As used herein, "oxidative hair coloring formulation" refers to a hair coloring formulation comprising at least one oxidizing agent. "Oxidative hair coloring process" refers to a process comprising the use of an oxidative hair coloring formulation described herein.

[0014]As used herein, "hair growth cycle" refers to a hair growth and loss process occurring in the hair follicles. The hair growth cycle consists of four phases—the anagen phase, the catagen phase, the telogen phase, and the exogen phase.

[0015]As used herein, "oxidative stress" refers to an imbalance between the production of reactive oxygen species in cells and tissues and the reduction of the reactive oxygen species by antioxidants. In particular, "oxidative stress" described herein is caused by the one or more oxidizing agents in an oxidative hair coloring formulation to the Human Follicle Dermal Papilla Cells (HFDPC) in the scalp, and is evaluated by the value of Radical Status Factor (RSF). A lower RSF indicates less oxidative stress in the scalp. [0016]As used herein, "Radical Status Factor" assesses the formation of radicalized proteins and determines whether a test system is protected from damage caused by oxidative stress. All products with the ability to protect against the generation of free radicals are rated with an RSF> 1. RSF is calculated according to the equation below:

RSF = Radical Proteins of stress group _

Radical proteins of investigacional product group

For RSF> 1 the product is protective against oxidative stress. For RSF <1 the product is a promoter of oxidative stress.

[0017]As used herein, "mitochondrial oxidative stress" refers to the overproduction of reactive oxygen species (e.g. superoxide) by mitochondria in the presence of free radicals.

[0018]The present disclosure provides a method of protecting the scalp and the hair from an oxidative hair coloring process. The method includes the application of a composition to the scalp and the hair prior to or during the application of an oxidative hair coloring formulation. The composition comprises Ilex Guayusa leaf extract. In some cases, the composition further comprises water and one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol. In some cases, the composition further comprises caffeine and chlorogenic acid.

[0019]In some cases, the oxidative hair coloring formulation contains one or more ingredient selected from the group consisting of p-phenylenediamine (PPD), ethanolamine, 2,4-diaminophenoxyethanol hydrochloride, p-aminophenol, 4- amino-2-hydroxytoluene, m-aminophenol, 6-hydroxyindoline, 2- methyl-5-hydroxyethylaminophenol, resorcinol, hydrogen peroxide (H2O2), l-hydroxyethyl-4,5-diamino pyrazole sulfate, 5-amino-6-chloro-o-cresol, and derivatives thereof.

[0020]In some cases, there is a reduced level of interleukin-8 (IL-8) synthesis in the scalp of the subject of from greater than 0% to 55% at the end of the oxidative hair coloring process, compared to the scalp of the same subject at the end of an oxidative hair coloring process in the absence of the composition. In some cases, the level of IL-8 synthesis is reduced by at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%. In some cases, the level of IL-8 synthesis is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0021]In some cases, the hair of a user has a maintained hair growth cycle starting from the end of the oxidative hair coloring process, compared to the hair of the same subject undergoing an oxidative hair coloring process in the absence of the composition.

[0022]In some cases, there is a maintained level of filaggrin protein synthesis in the scalp at the end of the oxidative hair coloring process.

[0023]In some cases, the composition is applied at least 10 minutes prior to the application of the oxidative coloring formulation. In some cases, the composition is applied at most 15 minutes prior to the application of the oxidative coloring formulation. [0024]In some cases, the composition is applied at least 10 minutes after the application of the oxidative coloring formulation. In some cases, the composition is applied at most 15 minutes after the application of the oxidative coloring formulation.

[0025]In some cases, the composition is applied substantially simultaneously as the oxidative coloring formulation.

[0026]In some cases, the composition is not a regular shampoo. In some cases, the composition is free of one or more surfactant selected from a group consisting of sodium lauryl sarcosinate, sodium lauryl sulfate (SLS), and sodium laureth sulfate (SLES).

[0027]In some cases, the composition is not a regular conditioner. In some cases, the composition is free of one or more cationic surfactant selected from a group consisting of cetrimonium chloride, behentrimonium methosulfate, distearyldimethylammonium chloride, and stearamidopropyl dimethylamine.

[0028]In some cases, the composition comprises from 0.001 wt% to 10.0 wt% of Ilex Guayusa leaf extract. In some cases, the composition comprises Ilex Guayusa leaf extract of at most 9.5 wt%, at most 8.5 wt%, at most 7.5 wt%, at most 6.5 wt%, at most 5.5 wt%, at most 4.5 wt%, at most 3.5 wt%, at most 2.5 wt%, at most 1.5 wt%, at most 0.5 wt%, or at most 0.1 wt%. In some cases, the composition comprises Ilex Guayusa leaf extract of at least 0.005 wt%, at least 0.2 wt%, at least 0.4 wt%, at least 0.6 wt%, at least 0.8 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, or at least 9.5 wt%.

[0029]In some cases, the composition comprises from 0.1 wt% to 1.0 wt% of Ilex Guayusa leaf extract. In some cases, the composition comprises Ilex Guayusa leaf extract of at most 0.9 wt%, at most 0.8 wt%, at most 0.7 wt%, at most 0.6 wt%, at most 0.5 wt%, at most 0.4 wt%, at most 0.3 wt%, or at most 0.2 wt%. In some cases, the composition comprises Ilex Guayusa leaf extract of at least 0.15 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, or at least 0.9 wt%.

[0030]In some cases, the composition comprises from 0.2 wt% to 0.7 wt% of Ilex Guayusa leaf extract.

[0031]In some cases, the composition comprises one or more humectants selected from glycerin, propylene glycol, and sorbitol. In some cases, the composition comprises from 0.1 wt% to 99.9 wt% of one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol. In some cases, the composition comprises from 0.1 wt% to 50.0 wt% of one or more humectants. In some cases, the composition comprises one or more humectants of at most 45 wt%, at most 40 wt%, at most 35 wt%, at most 30 wt%, at most 25 wt%, at most 20 wt%, at most 15 wt%, at most 10 wt%, at most 5 wt%, at most 3 wt%, at most 1 wt%, or at most 0.5 wt%. In some cases, the composition comprises one ore more humectants of at least 0.3 wt%, at least 0.6 wt%, at least 0.9 wt%, at least 1.5 wt%, at least 2 wt%, at least 4 wt%, at least 9 wt%, at least 13 wt%, at least 18 wt%, at least 23 wt%, at least 28 wt%, at least 33 wt%, at least 38 wt%, at least 43 wt%, or at least 48 wt%. [0032]In some cases, the composition comprises from 0.1 wt% to 30 wt% of one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol. In some cases, the composition comprises one or more humectants of at most 0.9 wt%, at most 0.8 wt%, at most 0.7 wt%, at most 28 wt%, at most 26 wt%, at most 24 wt%, at most 22 wt%, at most 20 wt%, at most 18 wt%, at most 16 wt%, at most 14 wt%, at most 12 wt%, at most 10 wt%, at most 8 wt%, at most 6 wt%, at most 4 wt%, at most 2 wt%, at most 1 wt%, at most 0.5 wt%, or at most 0.3 wt%. In some cases, the composition comprises one or more humectants of at least 0.2 wt%, at least 0.4 wt%, at least 0.8 wt%, at least 1.5 wt%, at least 3 wt%, at least 5 wt%, at least 7 wt%, at least 9 wt%, at least 11 wt%, at least 13 wt%, at least 15 wt%, at least 17 wt%, at least 19 wt%, at least 21 wt%, at least 23 wt%, at least 25 wt%, at least 27 wt%, or at least 29 wt%.

[0033]In some cases, the composition comprises from greater than 30 wt% to 99.9 wt% of one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol. In some cases, the composition comprises one or more humectants of at most 99.5 wt%, at 97 wt%, at most 95 wt%, at most 90 wt%, at most 85 wt%, at most 80 wt%, at most 75 wt%, at most 70 wt%, at most 65 wt%, at most 60 wt%, at most 55 wt%, at most 50 wt%, at most 45 wt%, at most 40 wt%, at most 35 wt%, or at most 33 wt%. In some cases, the composition comprises one or more humectants of at least 31 wt%, at least 34 wt%, at least 38 wt%, at least 43 wt%, at least 48 wt%, at least 53 wt%, at least 58 wt%, at least 63 wt%, at least 68 wt%, at least 73 wt%, at least 78 wt%, at least 83 wt%, at least 88 wt%, at least 93 wt%, at least 95 wt%, at least 97 wt%, at least 98 wt%, at least 99 wt%, or at least 99.3 wt%.

[0034]In some cases, the humectant is glycerin.

[0035]In some cases, the composition comprises water. In some cases, the composition comprises from 0.1 wt% to 99 wt% of water. In some cases, the composition comprises from 0.1 wt% to 50.0 wt% of water. In some cases, the composition comprises water of at most 45 wt%, at most 40 wt%, at most 35 wt%, at most 30 wt%, at most 25 wt%, at most 20 wt%, at most 15 wt%, at most 10 wt%, at most 5 wt%, at most 3 wt%, at most 1 wt%, or at most 0.5 wt%. In some cases, the composition comprises water of at least 0.3 wt%, at least 0.6 wt%, at least 0.9 wt%, at least 1.5 wt%, at least 2 wt%, at least 4 wt%, at least 9 wt%, at least 13 wt%, at least 18 wt%, at least 23 wt%, at least 28 wt%, at least 33 wt%, at least 38 wt%, at least 43 wt%, or at least 48 wt%.

[0036]In some cases, the composition comprises from 0.1 wt% to 30 wt% of water. In some cases, the composition comprises water of at most 0.9 wt%, at most 0.8 wt%, at most 0.7 wt%, at most 28 wt%, at most 26 wt%, at most 24 wt%, at most 22 wt%, at most 20 wt%, at most 18 wt%, at most 16 wt%, at most 14 wt%, at most 12 wt%, at most 10 wt%, at most 8 wt%, at most 6 wt%, at most 4 wt%, at most 2 wt%, at most 1 wt%, at most 0.5 wt%, or at most 0.3 wt%. In some cases, the composition comprises water of at least 0.2 wt%, at least 0.4 wt%, at least 0.8 wt%, at least 1.5 wt%, at least 3 wt%, at least 5 wt%, at least 7 wt%, at least 9 wt%, at least 11 wt%, at least 13 wt%, at least 15 wt%, at least 17 wt%, at least 19 wt%, at least 21 wt%, at least 23 wt%, at least 25 wt%, at least 27 wt%, or at least 29 wt%.

[0037]In some cases, the composition comprises from greater than 30 wt% to 99.9 wt% of water. In some cases, the composition comprises water of at most 99.5 wt%, at 97 wt%, at most 95 wt%, at most 90 wt%, at most 85 wt%, at most 80 wt%, at most 75 wt%, at most 70 wt%, at most 65 wt%, at most 60 wt%, at most 55 wt%, at most 50 wt%, at most 45 wt%, at most 40 wt%, at most 35 wt%, or at most 33 wt%. In some cases, the composition comprises water of at least 31 wt%, at least 34 wt%, at least 38 wt%, at least 43 wt%, at least 48 wt%, at least 53 wt%, at least 58 wt%, at least 63 wt%, at least 68 wt%, at least 73 wt%, at least 78 wt%, at least 83 wt%, at least 88 wt%, at least 93 wt%, at least 95 wt%, at least 97 wt%, at least 98 wt%, at least 99 wt%, or at least 99.3 wt%.

[0038]In some cases, the composition further comprises caffeine. In some cases, the composition comprises from 1 wt% to 20 wt% of caffeine. In some cases, the composition comprises caffeine of at most 19 wt%, at most 18 wt%, at most 17 wt%, at most 16 wt%, at most 15 wt%, at most 14 wt%, at most 13 wt%, at most 12 wt%, at most 11 wt%, at most 10 wt%, at most 9 wt%, at most 8 wt%, at most 7 wt%, at most 6 wt%, at most 5 wt%, at most 4 wt%, at most 3 wt%, at most 2 wt%, or at most 1.5 wt%. In some cases, the composition comprises caffeine of at least 1.2 wt%, at least 1.4 wt%, at least 1.8 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 11 wt%, at least 12 wt%, at least 13 wt%, at least 14 wt%, at least 15 wt%, at least 15 wt%, at least 16 wt%, at least 17 wt%, at least 18 wt%, at least 19 wt%, or at least 19.5 wt%.

[0039]In some cases, the composition further comprises chlorogenic acid. In some cases, the composition comprises from 1 wt% to 30 wt% of chlorogenic acid. In some cases, the composition comprises chlorogenic acid of at most 29.5 wt%, at most 29 wt%, at most 27 wt%, at most 25 wt%, at most 23 wt%, at most 21 wt%, at most 19 wt%, at most 17 wt%, at most 15 wt%, at most 13 wt%, at most 11 wt%, at most 9 wt%, at most 7 wt%, at most 5 wt%, or at most 3 wt%,. In some cases, the composition comprises chlorogenic acid of at least 2 wt%, at least 4 wt%, at least 6 wt%, at least 8 wt%, at least 10 wt%, at least 12 wt%, at least 14 wt%, at least 16 wt%, at least 18 wt%, at least 20 wt%, at least 22 wt%, at least 24 wt%, at least 26 wt%, at least 28 wt%, or at least 28.5 wt%.

[0040]In some cases, the scalp and the hair of a user are free of oxidative stress from the oxidative hair coloring process by virtue of using the methods or compositions disclosed herein.

[0041]In some cases, the composition reduces inflammation resulted from the oxidative hair coloring process by from greater than 0% to 80%. In some cases, inflammation is reduced by at most 70%, at most 60%, at most 50%, at most 40%, at most 30%, at most 20%, or at most 10. In some cases, the inflammation is reduced by at least 5%, at least 15%, at least 25%, at least 35%, at least 45%, at least 55%, at least 65%, or at least 75%.

[0042]The present disclosure provides a kit for protecting the scalp and the hair from an oxidative hair coloring process. The kit includes a composition and an oxidative hair coloring formulation. The composition is composed of Ilex Guayusa leaf extract, optionally glycerin, and optionally water.

[0043]The inventors surprisingly discovered that the application of a Guayusa-containing composition described herein prior to or during an oxidative hair coloring process has superior performance in protecting scalp and hair of a subject from the oxidative damage resulted from the oxidizing agents in the hair coloring formulations. The Guayusa- containing composition (i) contains antioxidants that guard hair and scalp from free radical damages, (ii) contains "antiirritation" or "anti-inflammatory" properties to keep the scalp healthy during hair coloring, (iii) preserves the natural protective barrier of the scalp, (iv) prevents oxidative stress on the scalp and hair during oxidative hair coloring process, (v) reduces the sensitiveness on scalp during oxidative hair coloring process, (vi) relieve the discomfort during oxidative hair coloring process, (vii) supports a healthy hair grow cycle. and (viii) reinforces the skin barrier resistance against oxidative hair dye damage.

[0044]Example 1.

[0045]In Vitro/Ex Vivo Tests

[0046]As used in this example, "Sensishield Scalp" refers to a composition as described above. Sensishield Scalp helps strengthen hair and promotes hair growth after an oxidative coloring process. The following experiments show that Sensishield Scalp contains antioxidants that reduce the amount of free radicals generated by the oxidizing agents in common hair coloring formulations, which are known to damage hair fiber and consequently causes protein loss in the hair. The following in vitro and ex vivo tests demonstrate that Sensishield Scalp act to protect scalp from oxidative hair dye damage on an external level at the scalp skin barrier and on an internal level at the mitochondria of the scalp cells.

[0047]Example 2

[0048]In Examples 2-5, citation numbers in the text correspond to the references identified at the end of the given Example. The first-listed reference corresponds to reference number 1, the second-listed to reference number 2, and so on.

[0049]Purpose

[0050]The purpose of the experiment described below is to evaluate the efficacy of Sensishield Scalp in the antioxidant action in culture of hair follicle dermal papilla cells (HFDPC) exposed to chemical inducers (PPD + RCN + H2O2) by quantifying the radical proteins and determining the radical status factor (RSF).

[0051]Introduction

[0052] "Permanent" hair dyes are among the most used cosmetic products by the population. Most formulations contain the dye precursor p-phenylenediamine (PPD) and a coupling agent such as Resorcinol (RCN) which, after successive reactions under oxidative conditions, result in hair color¹.

[0053]PPD, in addition to skin irritation, causes acute, subacute or chronic allergic contact dermatitis², with a prevalence of reported positive contact tests ranging from 4% to more than 6% of sensitive patients in Asia, Europe and North America³'⁴. People at risk cover a wide range of ages, with young and old people vulnerable to sensitization by contact after dermal exposure to PPD. [0054]Allergic contact dermatitis (ACD) is a skin inflammatory disease characterized by a late cell hypersensitivity response inducing an exacerbated inflammatory process with activation and migration of dendritic cells and production of CD4 + and CD8 + effector T cells residing in the skin. Subsequent exposure to the same contact allergen leads to the elicitation phase, characterized by the recruitment of several immune cells and the synthesis of pro-inflammatory cytokines and chemokines such as interleukin IL-8⁵ _' ⁶.

[0055]Another source that contributes significantly to skin allergy after exposure to these dyes is the generation of reactive oxygen species (ROS)⁷ _' ⁸. Topical exposure to irritants and sensitizers is known to increase ROS levels that can break through the epidermal barrier and activate several cellular pathways, including the increase in cytokines involved in allergic reactions⁹.

[0056]Reactive nitrogen species (RNS), such as peroxynitrite (ONOO-) and reactive oxygen species (ROS), such as peroxyl radicals (ROO ·), hydroxyl radicals (OH ·) or anion of superoxide radicals are associated with the formation of radical proteins (damaged by free radicals) by removing an electron or hydrogen atom compromising its function. One of the ways to classify products or ingredients that protect the skin or hair from the action of free radicals, preventing the formation of radical proteins is the use of the universal parameter known as Radical Status Factor (RSF). RSF is a quantitative tool that assesses the formation of radicalized proteins and determines whether the product can protect the test system from damage caused by oxidative stress. All products with the ability to protect against the generation of free radicals are rated with an RSF >1¹⁰.

[0057]Materials

[0058]Test-system

[0059]Human Follicle Papilla Dermal Cells (HFPDC) - (Cell Applications Inc.)

[0060]Positive Control

[0061]P-phenylenediamine dye (PPD), resorcinol dye (RCN), and hydrogen peroxide (H2O2). A solution in the proportion of 10 mM PPD and RCN was prepared with 3% H2O2 in culture medium to perform the cytotoxicity test, as described below. The results (in viable cells %) are as follows: 0.003 mg/mL = 165.61%; 0.01 mg/mL = 147.24%; 0.03 mg/mL = 93.15%; 0.10 mg/mL = 3.28%; 0.32 mg/mL = 1.30%; 1.00 mg/mL = 2.52%; 3.16 mg/mL = 2.11%; and 10.00 mg/mL = 5.99%. For the exposure of cultures as a positive control, a non-cytotoxic concentration of 0.01 mM PPD and RCN with 3% H2O2 was used.

[0062]Negative Control

[0063]Culture Medium: Human Follicle Papilla Dermal Cells (HFPDC) - (Cell Applications Inc.)

[0064]Equipment

•Laminar flow

•C02 incubator

•Water bath

•Vortex

•Pipetters

•Plate Reader - Infinite M200, Tecan

• Neubauer Chamber [0065]Supplies

•Culture plates (Corning)

•Culture Bottles (Corning)

•Tips (Axygen)

•Conical tubes (Corning)

•Microtubes (Axygen)

•Serological pipette (Corning)

•Papilla cell Growth Medium (Cell Applications Inc.) •Gentamicin (Thermo Fischer)

•Collagen Coating solution (Cell Applications Inc.) •Phosphate Buffer - PBS (Corning)

•Tryple Express (Thermo Fischer)

•Trypan blue (Sigma)

•DMPO (Sigma)

•Copper Sulfate (Sigma)

•H202 (Merck)

•Detection kit for radical proteins - RSF (STA-810_Cell Biolabs - San Diego, USA)

•Mycoplasma detection kit (LT07-710_Lonza - Basileia,

Suiga)

[0066]Methodology

[0067]HFDPCs were incubated with three non-cytotoxic concentrations of Sensishield Scalp for 72 hours with concomitant exposure to chemical inducers (PPD + RCN + H2O2). Most stabilization of proteins damaged by free radicals was achieved with the Spin Trap technique. Quantification of the radical proteins was achieved with the immuno-enzymatic assay (Elisa).

[0068]HFDPCs Culture

[0069]HFDPCs were seeded in culture bottles using the specific medium, supplemented with 1% gentamicin, and incubated at 37°C, with 5% CO2, until they reached confluence. These cells were used in the dose definition assay (Cytotoxicity). For this purpose, they were seeded in 96-well plates at a density of 5xl0³ cells per well and a final volume of 200 pL. For the effectiveness assay, cells were cultured in 6-well plates at a density of 2.5xl0⁵, with a final volume of 3 mL. Collagen coating was carried out on all plates and culture bottles to allow cell adherence.At the end of the experiments, a Mycoplasma detection test was performed by luminescence, proving the absence of Mycoplasma in the cultures.

[0070]Test to Define Treatment Concentrations - Cytotoxicity

[0071]To define the non-cytotoxic concentrations used in the treatment, the investigational product was solubilized in culture medium to assess the viability of cells exposed to concentrations of 100.00, 31.60, 10.00, 3.16, 1.00, 0.32, 0.10, and 0.03 mg/mL for 72 hours, using the MTT vital dye ((3- (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After the treatment, the optical density (OD) reading was performed at 570 nm. Results (in viable cells %) are as follows: 0.03 mg/mL = 118.03%; 0.10 mg/mL = 115.56%; 0.32 mg/mL = 122.50%; 1.00 mg/mL = 14.56%; 3.17 mg/mL = 13.83%; 10.00 mg/mL = 12.61%; 31.60 mg/mL = 8.71%; and 100.00 mg/mL = 0%. The same procedure was performed to define the non-cytotoxic dose of the chemical stress inducers (PPD + RCN + H2O2). Calculations to define the highest possible concentration that does not cause cell death were performed using the following equation for each concentration assessed:

% Viable Cells = DO mean treatment concentration ^* 100

DO Control group mean

[0072]Treatment protocol and chemical inducers

[0073]The cells were treated with concentrations of 0.32, 0.10, and 0.03 mg/mL, defined in the cytotoxicity assay, for 72 hours, concomitant with exposure to chemical inducers (PPD + RCN + H2O2) used as a positive stress control. At the end of this period, the cell lysate was collected for further quantification of the proposed mediator.

[0074]Quantification of mediator - RSF

[0075]The cell lysate was subjected to protein quantification using Bradford technique and then 10 μg/mL of protein from each sample was treated with 100 mM DMPO, 4.4 mM H2O2 and 50 mM CuS04, overnight at 37 ⁰ C. Subsequently, the formation of radicalized proteins in the samples was quantified by the immunoenzymatic assay technique (Elisa) using a commercially purchased kit, according to the manufacturer's instructions. RSF was determined according to the equation below:

RSF = Radical Proteins of stress group

Radical proteins of investigations! product group

[0076]For RSF> 1 the product is protective against oxidative stress. For RSF <1 the product is a promoter of oxidative stress. [0077]Statistical Analysis

[0078]In the statistical evaluation, ANOVA non-parametric analysis of variance was used, followed by the Bonferoni post hoc test, allowing the evaluation of comparative results and data between the groups. The significance level of 5% was used (GraphPad Prism v6).

[0079]RESULTS AND DISCUSSION

[0080]Cytotoxicity assessment

[0081]The cytotoxicity test of chemical inducers demonstrated that concentrations below 0.01 mM of PPD and RCN with 3% H202 do not induce the cell death. Thus, this was the dose chosen to induce stress as a positive control.

[0082]The cytotoxicity assay demonstrated that concentrations below 0.32mg/mL do not induce the cell death. Thus, concentrations of 0.32 were chosen for the tests; 0.10 and 0.03mg/mL.

[0083]Mediator quantification - RSF

[0084]The results obtained showed that the exposure of cultures to chemical inducers promoted an increase in the formation of radical proteins of 54% (P<0.001), when compared to the baseline control group.

[0085]The investigational product reduces the formation of radical proteins by 27%; 25% and 24% (P<0.001) at concentrations of 0.32; 0.10 and 0.03mg/mL, respectively, when compared to the group with exposure to chemical inducers, showing their ability to protect proteins from damage caused by free radicals. These results are shown in Fig. 1.

[0086]The RSF obtained was 1.37, 1.34 and 1.31 for concentrations of 0.32, 0.10 and 0.03mg/mL, respectively, indicating that the product is a protector against oxidative stress in all concentrations evaluated.

[0087]Additionally, we can observe that the product has a radical protection factor of 76%, 72% and 68%, in concentrations of 0.32; 0.10 and 0.03mg/mL, respectively.

[0088]Conclusion

[0089]Under the conditions in which the product described above was evaluated the data allow to conclude that: Sensishield Scalp has an antioxidant action reducing the damage to proteins caused by free radicals, with a radical protection factor of up to 76% and RSF of up to 1.37. These data demonstrate that the product can prevent oxidative stress caused by chemical inducers commonly present in hair dyes.

[0090]References for Example 2

[0091]Morel, 0. J. X., Christie, R.M. 2011. Current trends in the chemistry of permanent hair dyeing. Chem .Rev, 111, 4, 2537-2561.

[0092]Mukkanna, K.S., Stone, N.M., Ingram, J.R., 2017. Para-phenylenediamine allergy: current perspectives on diagnosis and management. J. Asthma Allergy 10, 9-15.

[0093]Krasteva, M., Bons, B., Ryan, C., Gerberick, G.F., 2009. Consumer allergy to oxidative hair coloring products: epidemiologic data in the literature. Dermatitis. 20, 123-141.

[0094]Thyssen, J.P., White, J.M.L., 2008. Epidemiological data on consumer allergy to pphenylenediamine. Contact Derm. 59, 327-343.

[0095]An, S., Kim, S., Huh, Y., Lee, T.R., Kim, H.K., Park, K.L., Eun, H.C., 2009. Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals. Contact Derm. 60, 185- 192.

[0096]Yoshida, Y., Sakaguchi, H., Ito, Y., Okuda, M., Suzuki, H., 2003. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol. In. Vitro. 17, 221-228.

[0097]Kidd P (2003) Thl/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8:223-46.

[0098]Bickers, R and Mohammad Oxidative Stress in the Pathogenesis of Skin Disease David Athar. Journal of Investigative Dermatology (2006) 126, 2565-2575.

[0099] Corsini, E., Galbiati, V., Nikitovic, D. 2013. Role of oxidative stress in chemical allergen induced skin cells activation. Food Chem Tox. 61:74-81.

[00100] Herrling T; Jung K. The Radical Status Factor (RSF): a novel metric to characterize skin products International Journal of Cosmetic Science, 2012, 34, 285-290

[00101] Example 3 [00102] INTRODUCTION

[00103] "Permanent" hair dyes are among the most used cosmetic products by the population. Most formulations contain the dye precursor p-phenylenediamine (PPD) and a coupling agent such as Resorcinol (RCN) which, after successive reactions under oxidative conditions, result in hair colorl. [00104] PPD, in addition to skin irritation, causes acute, subacute or chronic allergic contact dermatitis^, with a prevalence of reported positive contact tests ranging from 4% to more than 6% of sensitive patients in Asia, Europe and North America^{3, 4} . People at risk cover a wide range of ages, with young and old people vulnerable to sensitization by contact after dermal exposure to PPD.

[00105] Allergic contact dermatitis (ACD) is a skin inflammatory disease characterized by a late cell hypersensitivity response inducing an exacerbated inflammatory process with activation and migration of dendritic cells and production of CD4 + and CD8 + effector T cells residing in the skin. Subsequent exposure to the same contact allergen leads to the elicitation phase, characterized by the recruitment of several immune cells and the synthesis of pro-inflammatory cytokines and chemokines such as interleukin IL-8^'^.

[00106] Another source that contributes significantly to skin allergy after exposure to these dyes is the generation of reactive oxygen species (ROS)^'^. Topical exposure to irritants and sensitizers is known to increase ROS levels that can break through the epidermal barrier and activate several cellular pathways, including the increase in cytokines involved in allergic reactions^.

[00107] Reactive nitrogen species (RNS), such as peroxynitrite (ONOO-) and reactive oxygen species (ROS), such as peroxyl radicals (ROO hydroxyl radicals (OH ·) or anion of superoxide radicals are associated with the formation of radical proteins (damaged by free radicals) by removing an electron or hydrogen atom compromising its function. One of the ways to evaluate products or ingredients that protect the skin or hair from the action of free radicals is to evaluate the induction of mitochondrial oxidative stress. The production of superoxide by mitochondria can be visualized under fluorescence microscopy using the MitoSOX ™ Red reagent, which permeates living cells where the mitochondria are targeted. This reagent is rapidly oxidized by superoxide, but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxidized product is highly fluorescent when it binds to the nucleic acid, allowing semi-quantification by image of this mitochondrial oxidative stress.

[00108] STUDY PURPOSE

[00109] Evaluate the efficacy of the investigational product in the antioxidant action in culture of hair follicle dermal papilla cells (HFDPC) exposed to chemical inducers (PPD + RCN + H202) by the semi-quantification of mitochondrial oxidative stress.

[00110] QUALITY CONTROL

[00111] The study was conducted in accordance with the guidelines of Good Laboratory Practices. Quality control is performed at each step of the method.

[00112] MATERIALS

[00113] Test-System

[00114] Human Follicle Papilla Dermal Cells (HFPDC) - (Cell

Applications Inc.)

[00115] Positive Control

[00116] P-phenylenediamine dye (PPD); Resorcinol dye (RCN); Hydrogen peroxide (H202). A solution in the proportion of IOmM PPD and RCN was prepared with 3% H₂0₂ in culture medium to perform the cytotoxicity test, as described below, the results are discussed above.

[00117] For the exposure of cultures as a positive control, a non-cytotoxic concentration of O.OImM PPD and RCN with 3% H₂0₂ was used.

[00118] Negative Control

[00119] Culture Medium: Human Follicle Papilla Dermal Cells

(HFPDC) - (Cell Applications Inc.)

[00120] Equipment: Laminar flow; C02 incubator; Water bath;

Vortex; Pipetters; Plate Reader - Infinite M200, Tecan; Neubauer Chamber.

[00121] Supplies: Culture plates (Corning)Culture Bottles

(Corning); Tips (Axygen); Conical tubes (Corning); Microtubes (Axygen); Serological pipette (Corning); Papilla cell Growth Medium (Cell Applications Inc.); Gentamicin (Thermo Fischer); Collagen Coating solution (Cell Applications Inc.); Phosphate Buffer - PBS (Corning); Tryple Express (Thermo Fischer); Trypan blue (Sigma); DMPO (Sigma); Copper Sulfate (Sigma); H202 (Merck); MitoSOX™ Red (M36008_Thermo Fischer Scientific Waltham, Massachusetts, EUA); Mycoplasma detection kit (LT07- 710_Lonza - Basileia, Suiga)

[00122] METHODOLOGY

[00123] HFDPCs were incubated with 3 non-cytotoxic concentrations of the product for 72 hours concomitant exposure to chemical inducers (PPD + RCN + H202) for subsequent removal of treatments and an additional 18-hour incubation. Mitochondrial oxidative stress was semi-quantified by imaging the fluorescence emitted by the fluorogenic dye MitoSOX ™ Red. [00124] HFDPCs culture

[00125] HFDPCs were seed in culture bottles using the specific medium, supplemented with 1% gentamicin, and incubated at 37°C, with 5% C02, until they reached confluence. These cells were used in the dose definition assay (Cytotoxicity), for this purpose they were seeded in 96-well plates at a density of 5x10^ cells per well and a final volume of 200μL. For the effectiveness assay, cells were cultured in 24-well plates at a density of 1x10^, with a final volume of lmL. Collagen coating was carried out on all plates and culture bottles to allow cell adherence. At the end of the experiments, a Mycoplasma detection test was performed by luminescence, proving the ABSENCE of Mycoplasma in the cultures.

[00126] Test to define treatment concentrations - Cytotoxicity

[00127] To define the non-cytotoxic concentrations used in the treatment, the investigational product was solubilized in culture medium to assess the viability of cells exposed to concentrations of 100.00; 31.60; 10.00; 3.16; 1.00; 0.32; 0.01 and 0.03mg/mL for 72 hours, using the MTT vital dye ((3- (4,5- Dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium Bromide). After the treatment, the optical density (OD) reading was performed at 570nm.

[00128] The same procedure was performed to define the non- cytotoxic dose of the chemical stress inducers (PPD + RCN+ H202). [00129] Calculations to define the highest possible concentration that does not cause cell death were performed using the following equation for each concentration assessed:

¾ Viable Cells = DO mean treatment concentration ^* 100

DO Control group mean

[00130] Treatment protocol and chemical inducers

[00131] The cells were treated with concentrations of 0.32;

0.10 and 0.03mg/mL, defined in the cytotoxicity assay, for 72 hours, concomitant with exposure to chemical inducers (PPD + RCN + H202) used as a positive stress control. At the end of this period, the cell lysate was collected for further quantification of the proposed mediator.

[00132] Quantification of mediator - MitoSOX™ Red

[00133] The cells were incubated with the fluorogenic dye

MitoSOX™ Red at a concentration of ImM for 10 minutes at 37°C. Then the MitoSOXTM Red was removed and DAPI was added to label the cells nucleus (1: 500) for 5 minutes. The cells were washed with PBS and photographed using the Leica DMi8 S automated microscope. 12 images were collected per group and the analysis was performed using the ImageJ software.

[00134] Statistical Analysis

[00135] In the statistical evaluation, ANOVA non-parametric analysis of variance was used, followed by the Bonferoni post hoc test, allowing the evaluation of comparative results and data between the groups. The significance level of 5% was used (GraphPad Prism v6).

[00136] RESULTS AND DISCUSSION

[00137] For cytotoxicity assessment, see Example 2. [00138] Mediator quantification - MitoSOX™ Red

[00139] Images were acquired of each experimental group, which enable us to see the fluorescence emitted by the mitochondrial oxidative stress and the cell nucleus is also evident. These images do not reproduce adequately without the use of color images and their contents can be adequately described by the conclusions derived therefrom (see below), so we have elected to exclude these images from this application. If these images are desired by a patent examiner, then the Applicant for this application can provide them to that examiner upon request. If these images are desired by a member of the public, then they can be secured by accessing the provisional patent application to which this application claims priority from the United Stated Patent and Trademark Office. [00140] The results obtained showed that the exposure of cultures to chemical inducers promoted an increase in the mitochondrial oxidative stress of 73% (P <0.001), when compared to the baseline control group.

[00141] The investigational product reduces mitochondrial oxidative stress by 34%, 34% and 32% (P<0.001) at concentrations of 0.32; 0.10 and 0.03mg/mL, respectively, when compared to the group with exposure to chemical inducers, showing their ability to protect cells from damage caused by free radicals. These results are shown in Fig. 2.

[00142] Additionally, we can see that the product has the capacity to protect cells in relation to the maximum damage caused by chemical inducers of 81%, 81% and 76%, in concentrations of 0.32, 0.10 and 0.03mg/mL, respectively. [00143] CONCLUSION

[00144] Under the conditions in which the product described above was evaluated the data allow to conclude that: Sensishield Scalp has an antioxidant action protecting cells from mitochondrial oxidative stress caused by chemical inducers commonly present in hair dyes.

[00145] References for Example 3

[00146] Morel, 0. J. X., Christie, R.M. 2011. Current trends in the chemistry of permanent hair dyeing. Chem .Rev, 111, 4, 2537-2561.

[00147] Mukkanna, K.S., Stone, N.M., Ingram, J.R., 2017. Para-phenylenediamine allergy: current perspectives on diagnosis and management. J. Asthma Allergy 10, 9-15.

[00148] Krasteva, M., Bons, B., Ryan, C., Gerberick, G.F., 2009. Consumer allergy to oxidative hair coloring products: epidemiologic data in the literature. Dermatitis. 20, 123-141. [00149] Thyssen, J.P., White, J.M.L., 2008. Epidemiological data on consumer allergy to pphenylenediamine. Contact Derm. 59, 327-343.

[00150] An, S., Kim, S., Huh, Y., Lee, T.R., Kim, H.K., Park, K.L., Eun, H.C., 2009. Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals. Contact Derm. 60, 185- 192.

[00151] Yoshida, Y., Sakaguchi, H., Ito, Y., Okuda, M., Suzuki, H., 2003. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol. In. Vitro. 17, 221-228.

[00152] Kidd P (2003) Thl/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8:223-46.

[00153] Bickers, R and Mohammad Oxidative Stress in the Pathogenesis of Skin Disease David Athar. Journal of Investigative Dermatology (2006) 126, 2565-2575.

[00154] Corsini, E., Galbiati, V., Nikitovic, D. 2013. Role of oxidative stress in chemical allergen induced skin cells activation. Food Chem Tox. 61:74-81.

[00155] Example 4

[00156] "Permanent" hair dyes are among the most used cosmetic products by the population. Most formulations contain the dye precursor p-phenylenediamine (PPD) and a coupling agent such as Resorcinol (RCN) which, after successive reactions under oxidative conditions, result in hair colorl.

[00157] PPD, in addition to skin irritation, causes acute, subacute or chronic allergic contact dermatitis^, with a prevalence of reported positive contact tests ranging from 4% to more than 6% of sensitive patients in Asia, Europe and North America^{3, 4} _ People at risk cover a wide range of ages, with young and old people vulnerable to sensitization by contact after dermal exposure to PPD.

[00158] Allergic contact dermatitis (ACD) is a skin inflammatory disease characterized by a late cell hypersensitivity response inducing an exacerbated inflammatory process with activation and migration of dendritic cells and production of CD4 + and CD8 + effector T cells residing in the skin. Subsequent exposure to the same contact allergen leads to the elicitation phase, characterized by the recruitment of several immune cells and the synthesis of pro-inflammatory cytokines and chemokines such as interleukin IL-8^5,6.

[00159] Venkatesan et al. (2020)⁷ demonstrated that PPD induces an inflammatory response, with an allergenic potential associated with increased IL-8 synthesis.

[00160] Thus, including cosmetic ingredients in hair dyes that prevent an exacerbated inflammatory response is a way to protect the scalp, minimizing the occurrence of allergic reactions.

[00161] STUDY PURPOSE

[00162] Evaluate the efficacy of the investigational product in the anti-inflammatory action in culture of hair follicle dermal papilla cells (HFDPC) exposed to chemical inducers (PPD + RCN + H202) by quantifying interleukin 8 (IL- 8).

[00163] QUALITY CONTROL

[00164] The study was conducted in accordance with the guidelines of Good Laboratory Practices. Quality control is performed at each step of the method.

[00165] MATERIALS [00166] Test-System

[00167] Human Follicle Papilla Dermal Cells (HFPDC) - (Cell Applications Inc.)

[00168] Positive Control

[00169] P-phenylenediamine dye (PPD); Resorcinol dye (RCN); Hydrogen peroxide (H202). A solution in the proportion of 10mM PPD and RCN was prepared with 3% H202 in culture medium to perform the cytotoxicity test, as described herein.

[00170] Negative Control

[00171] Culture Medium: Human Follicle Papilla Dermal Cells

(HFPDC) - (Cell Applications Inc.)

[00172] Equipment

• Laminar flow

• C02 incubator

• Water bath

• Vortex

• Pipetters

• Plate Reader - Infinite M200, Tecan

• Neubauer Chamber

[00173] Supplies

• Conical tubes (Corning)

• Microtubes (Axygen)

• Serological pipette (Corning)

• Papilla cell Growth Medium (Cell Applications Inc.)

• Gentamicin (Thermo Fischer)

• Collagen Coating solution (Cell Applications Inc.)

• Phosphate Buffer - PBS (Corning)

• Tryple Express (Thermo Fischer)

• Trypan blue (Sigma)

• DMPO (Sigma)

• Copper Sulfate (Sigma)

• H202 (Merck)

• Human IL-8 detection kit (DY208-05_R&D Systems

Minneapolis, USA • Mycoplasma detection kit (LT07-710_Lonza - Basileia,

Suiga)

[00174] METHODOLOGY

[00175] HFDPCs were incubated with 3 non-cytotoxic concentrations of the product for 72 hours concomitant exposure to chemical inducers (PPD + RON + H202) for aftermost quantification of IL-8, using the immuno-enzymatic assay (Elisa).

[00176] HFDPCs culture

[00177] HFDPCs were seed in culture bottles using the specific medium, supplemented with 1% gentamicin, and incubated at 37°C, with 5% C02, until they reached confluence. These cells were used in the dose definition assay (Cytotoxicity), for this purpose they were seeded in 96-well plates at a density of 5x10^ cells per well and a final volume of 200μL. For the effectiveness assay, cells were cultured in 6-well plates at a density of 2.5x10^, with a final volume of 3mL. Collagen coating was carried out on all plates and culture bottles to allow cell adherence. At the end of the experiments, a Mycoplasma detection test was performed by luminescence, proving the ABSENCE of Mycoplasma in the cultures.

[00178] Test to define treatment concentrations -

Cytotoxicity

[00179] To define the non-cytotoxic concentrations used in the treatment, the investigational product was solubilized in culture medium to assess the viability of cells exposed to concentrations of 100.00; 31.60; 10.00; 3.16; 1.00; 0.32; 0.01 and 0.03mg/mL for 72 hours, using the MTT vital dye ((3- (4,5- Dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium Bromide). After the treatment, the optical density (OD) reading was performed at 570nm. The same procedure was performed to define the non-cytotoxic dose of the chemical stress inducers (PPD + RCN + H202).

[00180] Calculations to define the highest possible concentration that does not cause cell death were performed using the following equation for each concentration assessed:

% Viable Cells = DO mean treatment concentration ^* 100

DO Control group mean

[00181] Treatment protocol and chemical inducers

[00182] The cells were treated with concentrations of 0.32;

0.10 and 0.03mg/mL, defined in the cytotoxicity assay, for 72 hours, concomitant with exposure to chemical inducers (PPD + RCN + H202) used as a positive stress control. At the end of this period, the cell lysate was collected for further quantification of the proposed mediator.

[00183] Quantification of mediator - IL-8

[00184] The quantification of IL-8 in cell culture supernatants was performed by the immunoenzymatic assay technique (Elisa) using a commercially purchased kit, according to the manufacturer's instructions.

[00185] Statistical Analysis

[00186] In the statistical evaluation, ANOVA non-parametric analysis of variance was used, followed by the Bonferoni post hoc test, allowing the evaluation of comparative results and data between the groups. The significance level of 5% was used (GraphPad Prism v6). [00187] RESULTS AND DISCUSSION

[00188] Cytotoxicity assessment

[00189] See Example 2 for details.

[00190] Mediator quantification - IL-8

[00191] The results obtained demonstrated that the exposure of cultures to chemical inducers promoted an increase of 3.1- fold (P<0.001) in the IL-8 synthesis, when compared to the baseline control group.

[00192] The investigational product reduces the synthesis of

IL-8 by 54% (P<0.01) and 33% (P<0.05), at concentrations of

0.32 and 0.10 mg/mL, respectively, when compared to the group with exposure to chemical inducers, showing its ability to protect cells from inflammatory stress. These results are shown in Fig. 3.

[00193] The concentration of 0.03m/ mL had no significant effect in relation to the group with exposure to chemical inducers.

[00194] Additionally, we can observe that the product has a capacity of cell cultures protection against inflammatory stress caused by chemical inducers of 80% and 48%, in concentrations of 0.32 and O.lOmg/mL, respectively.

[00195] CONCLUSION

[00196] Under the conditions in which the product described above was evaluated the data allow to conclude that: SENSISHIELD SCALP has an anti-inflammatory action reducing the synthesis of IL-8 in cell cultures stimulated with chemical inducers (PPD + RCN + H202). These data demonstrate that the product can prevent the inflammatory stress caused by the dyes commonly present in hair dyes.

[00197] References for Example 4

[00198] Morel, 0. J. X., Christie, R.M. 2011. Current trends in the chemistry of permanent hair dyeing. Chem .Rev, 111, 4, 2537-2561.

[00199] Mukkanna, K.S., Stone, N.M., Ingram, J.R., 2017. Para-phenylenediamine allergy: current perspectives on diagnosis and management. J. Asthma Allergy 10, 9-15.

[00200] Krasteva, M., Bons, B., Ryan, C., Gerberick, G.F., 2009. Consumer allergy to oxidative hair coloring products: epidemiologic data in the literature. Dermatitis. 20, 123-141. [00201] Thyssen, J.P., White, J.M.L., 2008. Epidemiological data on consumer allergy to pphenylenediamine. Contact Derm. 59, 327-343.

[00202] An, S., Kim, S., Huh, Y., Lee, T.R., Kim, H.K., Park, K.L., Eun, H.C., 2009. Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals. Contact Derm. 60, 185- 192.

[00203] Yoshida, Y., Sakaguchi, H., Ito, Y., Okuda, M., Suzuki, H., 2003. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol. In. Vitro. 17, 221-228.

[00204] Kidd P (2003) Thl/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 8:223-46.

[00205] Bickers, R and Mohammad Oxidative Stress in the Pathogenesis of Skin Disease David Athar. Journal of Investigative Dermatology (2006) 126, 2565-2575.

[00206] Corsini, E., Galbiati, V., Nikitovic, D. 2013. Role of oxidative stress in chemical allergen induced skin cells activation. Food Chem Tox. 61:74-81.

[00207] Herling T; Jung K. The Radical Status Factor (RSF): a novel metric to characterize skin products International Journal of Cosmetic Science, 2012, 34, 285-290

[00208] Example 5

[00209] INTRODUCTION

[00210] "Permanent" hair dyes are among the most used cosmetic products by the population. Most formulations contain the dye precursor p-phenylenediamine (PPD) and a coupling agent such as Resorcinol (RON) which, after successive reactions under oxidative conditions, result in hair colorl.

[00211] PPD, in addition to skin irritation, causes acute, subacute or chronic allergic contact dermatitis^, with a prevalence of reported positive contact tests ranging from 4% to more than 6% of sensitive patients in Asia, Europe and North America^{3, 4} _ People at risk cover a wide range of ages, with young and old people vulnerable to sensitization by contact after dermal exposure to PPD.

[00212] Allergic contact dermatitis (ACD) is a skin inflammatory disease characterized by a late cell hypersensitivity response inducing an exacerbated inflammatory process with activation and migration of dendritic cells and production of CD4 + and CD8 + effector T cells residing in the skin. Subsequent exposure to the same contact allergen leads to the elicitation phase, characterized by the recruitment of several immune cells and the In addition to the intensification of the inflammatory response, ACD is associated with an impairment of the epidermal barrier, with filaggrin being the main biological marker for understanding the health of the epidermal barrier. This protein is responsible for adding keratin and other proteins in the most superficial layers of the epidermis to form the stratum corneum, which is the protective layer of our skin^.

[00213] STUDY PURPOSE

[00214] To evaluate the efficacy of investigational product in improving and protecting the epidermal barrier in human ex vivo scalp fragments culture submitted to hair color treatment. [00215] ETHICAL CONSIDERATIONS AND QUALITY CONTROL

[00216] The study was conducted in accordance with the guidelines of Good Laboratory Practices. Quality control is performed at each step of the method.

[00217] This study was conducted after the ethical approval by the Research Ethics Committee of University of San Francisco - SP, CAAE: 32317520.4.0000.55.

[00218] MATERIALS

[00219] Test-System

[00220] Ex vivo scalp fragments were obtained from elective plastic surgery (rhytidectomy) after approval by the Human Research Ethics Committee (CAAE: 32317520.4.0000.55). The fragments were obtained from a 48- year-old female research participant.

[00221] Positive Control [00222] Hair Color: Imedia Excellence cream; Brand: Loreal Paris,: Lot: 58T801; Validity: 06/2023. [00223] Negative Control [00224] Culture Medium (DMEM High Glucose). [00225] Equipment Laminar flow C02 incubator Water bath Vortex Pipetters

Cryostat (Leica, Germany)

Leica DMi8 S automated microscope

[00226] Supplies

Culture plates (Corning)

Culture Bottles (Corning)

Tips (Axygen)

Conical tubes (Corning)

Microtubes (Axygen)

Serological pipette (Corning)

Transwell inserts (Corning)

DMEM High Glucose (Corning)

Bovine Fetal Serum (Thermo Fischer)

Gentamicin (Thermo Fischer)

Phosphate Buffer - PBS (Corning)

Paraformolaldehyde (PFA) 4% (Synth)

Tissue-Tek

Silanized blades • Paraformaldehyde

• Sucrose

• Anti-Filaggrin antibody (NBPl-87528_Novus Biological -

Colorado, USA)

• Alexa Flour 488 secondary antibody - Goat anti Rabbit (L0319_Novus Biological - Colorado, USA)

• DAPI (4'-6-Diamidino-2-Phenylindole; DNA marker, Sigma-Aldrich, USA)

[00227] METHODOLOGY

[00228] Fragments of human scalp ex vivo were treated with a hair dye in the presence or absence of the investigational product with concentrations of 0.5% and 1%, for 40 minutes. Then the hair dye was removed, and the fragments were incubated for 24 hours, for subsequent fixation in 4% paraformaldehyde, cryopreservation, histological sections and filaggrin immunostaining, using the immunofluorescence technique.

[00229] Ex vivo human skin culture

[00230] The fragments of ex vivo human skin were transported to the laboratory in saline solution at 4°C for asepsis procedure using DMEM high glucose supplemented with gentamicin, for a period of 1 hour at 4°C. Then, the hypodermis was removed, and fragments were subjected to standard cuts with a diameter of 8 mm. The cuts were mounted on transwell inserts for 24- well plates and cultivated in an air-liquid interface.

[00231] Treatment protocol and exposure to hair dye [00232] The investigational product was prepared in a solution with 15.67% SENSISHIELD SCALP, 17.33% Glycerin and 67% distilled water. Subsequently, hair color was prepared according to the manufacturer's instructions in the presence of 0.5% and 1% of the SENSISHIELD SCALP solution previously prepared.

[00233] Then, the hair color was applied to the scalp fragments at a concentration of 25mg/cm2, according to the experimental groups: Baseline control (culture medium only); Hair Color; Hair color + SENSISHIELD SCALP 0.5%; Hair color + SENSISHIELD SCALP 1%.

[00234] The treatment was maintained for 40 minutes, then the hair color was removed, and the fragments were washed with lmL PBS for 3 times. The fragments were incubated for 24 hours in an air-liquid interface at 37°C with 5% C02. Then the fragments were collected for fixation, histological section, and immunostaining.

[00235] Semi-quantification of mediator - Filaggrin

[00236] For histological analysis, the ex vivo skin fragments were fixed in Paraformaldehyde 4% (pH 7.4) for 24 hours and cryoprotected in a 30% sucrose solution for 72 hours. Then, serial cuts of lOpm were collected directly on silanized slides using cryostat. For the evaluation by immunofluorescence the cuts were submitted to 3 washes of 5 minutes each with PBS and incubated overnight with the primary antibodies Anti-Filaggrin (1:200). Subsequently, the cuts were subjected to new washes with PBS and incubated for 1 hour with secondary antibody (1:2500). Immediately after the end of the above step, a new 5-minute incubation was performed with DAPI followed by 3 washes of 5 minutes with PBS. After the immunofluorescence procedures, the slides were mounted in a specific mounting medium, analyzed, and photographed under a fluorescence microscope.

[00237] The evaluated parameter was the fluorescence intensity emitted by the labeling with specific antibody.After obtaining the images, the fluorescence intensity was quantified with the aid of the ImageJ software (National Institutes of Health, USA) using arbitrary units (U.A.).

[00238] Statistical Analysis

[00239] Semi-quantification - Filaggrin

[00240] The results obtained demonstrated that the exposure of scalp ex vivo cultures to hair color promoted a reduction in the filaggrin synthesis of 37% (P<O.001), when compared to the baseline control group.

[00241] The investigational product increases the synthesis of filaggrin by 25% (P<0.01) and 35% (P<O.001), in concentrations of 0.5% and 1%, respectively.

[00242] In addition, we can observe that the product has the protection capacity of the baseline control group of 42% and 61% at concentrations of 0.5% and 1%, respectively.

[00243] The results are shown in Fig. 4.

[00244] CONCLUSION

[00245] Under the conditions in which the product described above was evaluated the data allow to conclude that: The investigational product induces the improvement of the epidermal barrier, protecting the scalp against the damage caused in the synthesis of filaggrin by hair color.

[00246] References for Example 5

[00247] Morel, 0. J. X., Christie, R.M. 2011. Current trends in the chemistry of permanent hair dyeing. Chem .Rev, 111, 4, 2537-2561.

[00248] Mukkanna, K.S., Stone, N.M., Ingram, J.R., 2017. Para-phenylenediamine allergy: current perspectives on diagnosis and management. J. Asthma Allergy 10, 9-15.

[00249] Krasteva, M., Bons, B., Ryan, C., Gerberick, G.F., 2009. Consumer allergy to oxidative hair coloring products: epidemiologic data in the literature. Dermatitis. 20, 123-141. [00250] Thyssen, J.P., White, J.M.L., 2008. Epidemiological data on consumer allergy to pphenylenediamine. Contact Derm. 59, 327-343.

[00251] An, S., Kim, S., Huh, Y., Lee, T.R., Kim, H.K., Park, K.L., Eun, H.C., 2009. Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals. Contact Derm. 60, 185- 192.

[00252] Yoshida, Y., Sakaguchi, H., Ito, Y., Okuda, M., Suzuki, H., 2003. Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol. In. Vitro. 17, 221-228.

[00253] M. Yamao, M. Inamatsu, Y. Ogawa, H. Toki, T. Okada, K. E. Toyoshima, and K. Yoshizato, Contact between dermal papilla cells and dermal sheath cells enhances the ability of DPCs to induce hair growth, J. Invest. Dermatol., 130, 2707- 2718 (2010).

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Claims

CLAIMS We claim:

1. A method of protecting scalp and hair of a subject from an oxidative hair coloring process, the method comprising: a) applying a composition to the scalp and the hair, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying an oxidative hair coloring formulation to the hair and the scalp.

2. The method of claim 1, wherein step b) occurs at least 10 minutes before the initiation of step a).

3. The method of claim 1, wherein step b) takes place after at least 10 minutes from the end of step a).

4. The method of claim 1, wherein step a) and step b) are performed at least partially simultaneously.

5. A method of protecting scalp and hair of a subject from an oxidative hair coloring process, the method comprising: a) mixing a composition with an oxidative hair coloring formulation to form a mixture, wherein the composition comprises Ilex Guayusa leaf extract; and b) applying the mixture to the scalp and the hair.

6. The method of any one of the preceding claims, wherein there is a reduced level of interleukin-8 (IL-8) synthesis in the scalp of the subject of from greater than 0% to 55% at the end of the oxidative hair coloring process, compared to the scalp of the same subject at the end of an oxidative hair coloring process in the absence of the composition.

7. The method of any one of the preceding claims, wherein the hair of the subject has a maintained growth cycle starting from the end of the oxidative hair coloring process, compared to the hair of the same subject undergoing an oxidative hair coloring process in the absence of the composition.

8. The method of any one of the preceding claims, wherein there is a maintained level of filaggrin protein synthesis in the scalp at the end of the oxidative hair coloring process.

9. The method of any one of the preceding claims, wherein the scalp and the hair of the subject are free of oxidative stress from the oxidative hair coloring process.

10. The method of any one of any one of the preceding claims, wherein the composition reduces inflammation resulted from the oxidative hair coloring process by from greater than 0% to 80%.

11. A kit for protecting scalp and hair of a subject from an oxidative hair coloring process, the kit comprising: a) a composition comprising Ilex Guayusa leaf extract; and b) an oxidative hair coloring formulation.

12. The method or kit of any one of the preceding claims, wherein the oxidative hair coloring formulation comprises one or more ingredients selected from the group consisting of p- phenylenediamine (PPD), ethanolamine, 2,4- diaminophenoxyethanol hydrochloride, p-aminophenol, 4-amino-2- hydroxytoluene, m-aminophenol, 6-hydroxyindoline, 2-methyl-5- hydroxyethylaminophenol, resorcinol, hydrogen peroxide (H2O2), 1-hydroxyethyl-4,5-diamino pyrazole sulfate, 5-amino-6-chloro- o-cresol, and derivatives thereof.

13. The method or kit of any one of the preceding claims, wherein the composition further comprises water and one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol.

14. The method or kit of any one of the preceding claims, wherein the composition consists essentially of the Ilex Guayusa leaf extract, the glycerin, and the water.

15. The method or kit of any one of the preceding claims, wherein the composition consists of the Ilex Guayusa leaf extract, the glycerin, and the water.

16. The composition or kit of any one of the preceding claims, wherein the composition is free of one or more surfactants selected from the group consisting of sodium lauryl sarcosinate, sodium lauryl sulfate (SLS), and sodium laureth sulfate (SLES).

17. The composition or kit of any one of the preceding claims, wherein the composition is free of one or more cationic surfactants selected from the group consisting of cetrimonium chloride, behentrimonium methosulfate, distearyldimethylammonium chloride, and stearamidopropyl dimethylamine.

18. The method or kit of any one of the preceding claims, wherein the composition comprises: a) from 0.001 to 10 wt% Ilex Guayusa leaf extract; b) from 0.1 to 99.9 wt% of one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol; and c) from 0.1 to 99.9 wt% water.

19. The method or kit of any one of the preceding claims, wherein the composition comprises: a) from 0.1 to 1.0 wt% Ilex Guayusa leaf extract; b) from 1.0 to 50.0 wt% of one or more humectants selected from the group consisting of glycerin, propylene glycol, and sorbitol; and c) from 1.0 to 50.0 wt% water.

20. The method or kit of any one of the preceding claims, wherein the composition further comprises caffeine and chlorogenic acid.

21. The method or kit of any one of the preceding claims, wherein the composition further comprises from 1 to 20 wt% of caffeine and from 1 to 30 wt% of chlorogenic acid.