Biomarkers for predicting atopic dermatitis to facilitate prevention and / or treatment of onset of atopic dermatitis

Abstract

A method of predicting the tendency of an infant to develop atopic dermatitis. The method comprises observing the expression of S100A8 / A9 on the skin area of the infant; comparing the expression to a determined criterion, wherein the determined criterion is ascertained by measuring the level of biomarkers in a subject or a repertoire of subjects for which the absence of atopic dermatitis has been demonstrated; and determining a tendency of the infant to develop atopic dermatitis, wherein an increase in expression as compared to the determined criteria indicates the tendency of the infant to develop atopic dermatitis.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 413,009, filed on October 4, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical field

[0003] The present invention relates to factors that can be used to predict the tendency of an infant to develop atopic dermatitis. The present invention also relates to methods for using these factors to evaluate the potential of skin treatment regimens, ingredients, and / or compositions to prevent and / or treat atopic dermatitis in infants. Background art

[0004] The skin is a group of cells and macromolecules assembled in the form of a resistant and flexible tissue that covers the entire body. It consists of two connected layers, the epidermis and the dermis, and the associated subcutaneous tissue.

[0005] The main function of the skin is to establish a protective barrier against environmental damage while allowing some exchange between the internal and external environments. The barrier function is particularly important in restricting epidermal water loss. This function is mainly provided by the stratum corneum, the outermost layer of the epidermis, which consists of flattened anucleate cells called corneocytes. The water - tightness of this "brick wall" is provided by an intercellular cement composed of specific lipids (cholesterol, cholesterol sulfate, free fatty acids, and ceramides). The regenerative capacity of the epidermis is conferred by adult stem cells, which allow for the regular replacement of differentiated cells eliminated during keratinization. This process is particularly crucial for the complete development and maintenance of the barrier function.

[0006] Adapting to life outside the womb is a process that begins at birth and continues throughout the first year of life. The first few months after birth are a period of restructuring of the skin's structure and function, allowing for physiological adaptation to the extra - uterine environment. For example, the underdevelopment of neonatal skin is prominently manifested in the differences in the structure and molecular composition of the stratum corneum compared to that of adults. These are incomplete and thus continue to develop for at least the first 12 months. In addition, the results of clinical studies have shown that infant skin exhibits a certain immaturity in its ability to capture water and regulate related mechanisms. Moreover, these studies have shown that the epidermal barrier is structurally organized from birth to 2 years and is thus not fully developed during this period. This helps to explain the vulnerability of infant and toddler skin and its sensitivity to chemical, physical, and microbial attacks.

[0007] In addition, the incomplete development of the skin can have significant clinical consequences. Therefore, it is important to allow the skin to build and develop properly and in a coordinated manner, otherwise its functional and structural organization may be impaired. In this regard, maintaining the barrier function and the renewal capacity of the epidermis is crucial.

[0008] Thus, the incomplete development of the barrier and the mechanisms regulating hydration in infant skin make infant skin even more vulnerable to pathological conditions such as atopic dermatitis.

[0009] Atopic dermatitis is one of the most common chronic diseases in the population. It is characterized by a set of clinical signs, the most important of which are pruritus and eczematous lesions, which can be acute, subacute or chronic. It almost always begins in infants or young children, and the barrier self-organizes structurally and functionally. Atopic dermatitis usually begins at around three months of age, but sometimes also in the first few weeks of life. It progresses in alternating relapsing and remitting phases. Depending on the severity of the child and the condition, it can last from months to years. Most will persist into adulthood.

[0010] Atopic dermatitis is primarily a chronic inflammatory skin disease that combines skin barrier damage and skin inflammation. In the first sensitization phase, skin barrier defects allow allergens to penetrate the skin. Allergens that penetrate the upper layers of the epidermis are processed (internalized) by epidermal Langerhans cells and dermal dendritic cells. Langerhans cells are antigen-presenting cells capable of capturing skin antigens, preparing them, and presenting them to T lymphocytes. This presentation, together with the direct activation of epidermal alarmins such as IL-33, IL-25, and TSLP, leads to the activation of the Th2 response, which results in the production of inflammatory cytokines such as IL-4, IL-5, and IL-13.

[0011] During all active periods of the disease, bacterial or viral skin superinfection is the most common complication. The skin of atopic dermatitis patients is highly vulnerable to secondary infections, which tend to become more extensive. For example, the bacterium Staphylococcus aureus is a major cause of skin infections. It usually colonizes the skin of atopic dermatitis patients, while it is only present transiently on healthy skin. The bacteria then secrete virulence factors that further reduce the barrier function, worsening the disease and contributing to its chronicity. In addition, S. aureus is usually found in the form of a homogeneous biofilm in atopic dermatitis patients, which is a form resistant to host defenses and treatment.

[0012] Various conventional solutions include methods for evaluating the effectiveness of C7 sugars or their derivatives in preventing and / or treating at least one defect of the skin barrier of a subject, methods for evaluating the in vitro efficacy of a formulation in preventing dehydration effects on children's skin, and methods for evaluating the effect of a product on epidermal lipogenesis, which include applying the product to the surface of a skin equivalent, measuring changes in markers of epidermal lipids, and then comparing the measurements with similar measurements of a control sample. Other methods include in vivo methods for measuring the binding of a compound or a mixture of compounds to skin components.

[0013] Some traditional examples of treating sensitive skin, irritable skin, reactive skin, atopic skin, itching, ichthyosis, acne, xerosis, atopic dermatitis, skin desquamation, skin subjected to actinic radiation or skin subjected to ultraviolet radiation include administering an effective amount of a composition comprising furan lipids including vegetable oil, thereby increasing the synthesis of skin lipids.

[0014] Exemplary methods of identifying a rinse-off personal care composition include: (a) generating one or more control skin profiles of two or more subjects; (b) contacting at least a portion of the skin of the subjects with a rinse-off test composition, rinsing the test composition from the skin portion, extracting one or more skin samples from each of the subjects, and generating one or more test profiles of the subjects based on the extracted samples; (c) comparing the one or more test profiles with the one or more control profiles and identifying the rinse-off test composition as effective in improving the stratum corneum barrier of a human subject who exhibits (i) a decrease in one or more inflammatory cytokines, (ii) an increase in one or more natural moisturizing factors, (iii) an increase in one or more lipids, and (iv) a decrease in total protein. Similarly, in infants at risk of developing atopic dermatitis, the use of some emollients is associated with increased abundance and a trend towards higher bacterial diversity compared to non-use of an emollient.

[0015] In some examples, the microbial abundance of baby bath products and lotions has been shown to be significantly greater than that of the bath product alone. For example, both the cleansing protocol alone and the cleansing and emollient protocol have shown good tolerance; throughout the study for each protocol, the skin pH remained slightly acidic; at any time point, there were no significant changes in the total scores for dryness, redness / erythema, rash / irritation, tactile roughness, or objective irritation or overall skin appearance compared to baseline for either group; an increase in microbial abundance was found after 2 weeks and 4 weeks of using the bath product and after 4 weeks of adding the lotion; after 4 weeks of use, the use of the lotion increased the abundance more than the bath product alone; the mild baby bath product + lotion routine best helped to improve microbial abundance, which can promote overall skin barrier health by providing a suitable environment for healthy skin microbes to thrive.

[0016] Additional studies have shown that thymus and activation-regulated chemokine (TARC), an immunobiomarker in infants, can help predict the onset and severity of childhood atopic eczema or neonatal atopic dermatitis (AD). For example, tape strips used on the back of a patient's hand or between their shoulder blades have been used to analyze immunobiomarkers of skin cells, showing that full-term and premature infants with elevated levels of TARC at 2 months of age are more than twice as likely to develop eczema by 2 years of age. A positive correlation has been identified between the level of TARC and the severity of eczema; the prevalence of the disease is 34.6% in full-term children and 21.2% in premature children. TARC levels are elevated in children with AD onset before 6 months of age and in children with AD onset between 6 and 24 months of age when compared to children who did not develop into AD. Researchers have reported that interleukin (IL)-8 and IL-18 are also associated with moderate to severe eczema.

[0017] To date, there is no cure for atopic dermatitis. Treatment is mainly topical and aims to improve symptoms and control disease progression. As mentioned above, the daily use of emollients is particularly necessary for restoring and protecting the damaged skin barrier. There are many different emollients on the market. However, the exact mechanism by which they exert their beneficial effects is not well understood. Therefore, there is still a need to further understand the mechanism and select effective and well-tolerated emollients to not only treat but also hopefully prevent atopic dermatitis. Summary of the Invention

[0018] The present invention relates to factors that can be used to predict the tendency of an infant to develop atopic dermatitis. According to the present invention, the method includes:

[0019] a) observing the expression of S100A8 / 9 alone or in combination with other biomarkers on the skin surface of an infant;

[0020] b) comparing the expression with a determined standard, wherein the determined standard is ascertained by measuring the level of the biomarker in a subject or a library of subjects who have been shown to be free of atopic dermatitis; and

[0021] c) determining the tendency of the infant to develop atopic dermatitis, wherein an increase in the expression compared to the determined standard indicates the tendency of the infant to develop atopic dermatitis.

[0022] The infant can be from about 1 week of age to about 12 months of age; from about 2 weeks of age to about 6 months of age; from about 1 month of age to about 3 months of age; from about 8 weeks of age to about 2 months of age; and about 8 weeks of age.

[0023] The skin can be selected from facial skin; skin of the extremities and / or body skin. The skin can be from the cheek. The skin can be from the elbow.

[0024] The method may further include:

[0025] A step of observing the mutational status of FLG using genetic material from an infant to determine the FLG genotype of the infant, wherein the FLG genotype is selected from the FLG wild-type genotype and the FLG loss-of-function genotype; and

[0026] If it is determined that the infant has the FLG wild-type genotype, observe the individual expression of S100A8 / 9 or the combined expression with other biomarkers on the skin surface of the infant.

[0027] Also according to the present invention, a method for evaluating the efficacy of a skin treatment regimen, ingredient, and / or composition in treating atopic dermatitis in infants includes:

[0028] Before applying the skin treatment regimen, ingredient, and / or composition, measure the individual level of S100A8 / 9 or the level in combination with another biomarker on the skin area of the infant;

[0029] Apply the skin treatment regimen, ingredient, and / or composition to the skin area for a period of time;

[0030] After applying the skin treatment regimen, ingredient, and / or composition to the skin area, measure the level of S100A8 / 9 in the skin area;

[0031] Wherein if the level of S100A8 / 9 is less than or equal to the level of the untreated control, the skin treatment regimen, ingredient, and / or composition is beneficial to the skin.

[0032] Skin swabs can be used for sample collection. This method provides benefits over, for example, tape stripping, which is invasive and damages the barrier.

[0033] These and any other methods, skin treatment regimens, ingredients, and / or compositions will be described in more detail below. Description of the Drawings

[0034] Figure 1 Is a box plot showing the measurement of S100A8 / 9 levels on the elbows using skin swabs in 8-week-old FLG wt infants and FLG LoF infants, and broken down by the development of AD at 6 months, 12 months, or 12-month cumulative.

[0035] Figure 2 Is a box plot showing the measurement of IL-36γ levels on the elbows using skin swabs in 8-week-old FLG wt infants and FLG LoF infants, and broken down by the development of AD at 6 months, 12 months, or 12-month cumulative.

[0036] Figure 3Is a box plot showing S100A8 / 9 levels on the elbow measured using skin swabs in 8-week-old FLG wt infants and FLG LoF infants, and broken down by no AD development, non-persistent AD, and persistent AD. Detailed Description

[0037] Definition

[0038] As used herein, the following terms shall have the meanings specified below:

[0039] As used herein, "biomarker" refers to any biomolecule (gene, protein, lipid, metabolite) that alone or in combination reflects the current state of a biological system or predicts the future state of a biological system. Thus, as used herein, various biomarkers are indicators of skin quality. The ability to prevent and / or treat skin disorders can also be evaluated by measuring one or more biomarkers.

[0040] As used herein, "consumer" refers to an individual who purchases and / or uses a skin treatment regimen, ingredient, and / or composition according to the present disclosure. Thus, in some cases, a consumer may alternatively be referred to herein as a "user".

[0041] As used herein, "control" means an area of epithelial tissue that has not been and / or is not in contact with a regimen, ingredient, and / or composition that has contacted an affected surface.

[0042] "Cumulative incidence" is the proportion of a risk group that develops an outcome of interest over a specified period of time.

[0043] As used herein, "effective amount" means an amount of a regimen, ingredient, and / or composition sufficient to significantly induce a positive skin benefit, including independently or in combination with other benefits disclosed herein. This means that the content and / or concentration of the active ingredient in the regimen, ingredient, and / or composition is sufficient such that when the regimen, ingredient, and / or composition is administered at a normal frequency and normal amount, the regimen, ingredient, and / or composition can result in treating one or more undesired skin conditions. For example, the amount can be an amount sufficient to inhibit or enhance some biochemical functions occurring in the skin. The amount of the active ingredient can vary depending on, among other factors, the type of the regimen, ingredient, and / or composition and the type of skin condition to be addressed.

[0044] As used herein, "emollient" refers to a chemical agent specifically designed to make the outer layer of the skin (epidermis) softer and more pliable.

[0045] As used herein, "epidermis" refers to the outer layer of the skin and is divided into five layers, which include: the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum contains many layers of dead, anucleate keratinocytes that are substantially filled with keratin. Even in healthy skin, the outermost layer of the stratum corneum is constantly sloughed off. The stratum lucidum contains two to three layers of anucleate cells. The stratum granulosum contains two to four layers of cells that are held together by desmosomes and contain keratohyalin granules. The stratum spinosum contains eight to ten layers of moderately active dividing cells that are also held together by desmosomes. The stratum basale contains a single layer of columnar cells that actively divide by mitosis and provide cells that are destined to migrate through the upper epidermal layers to the stratum corneum. The main cell type of the epidermis is the keratinocyte. These cells are formed in the stratum basale and are present through the epidermal layers up to the stratum granulosum, where they transform into cells called corneocytes or form the scales of the stratum corneum. During this transformation process, the nucleus is digested, the cytoplasm disappears, lipids are released into the intercellular spaces, keratin intermediate filaments aggregate to form microfibrils, and the cell membrane is replaced by a cell envelope made of cross-linked proteins, where lipids are covalently attached to its surface. Keratin is the main structural protein of the stratum corneum. Corneocytes are regularly sloughed off (a process known as desquamation) to complete the entire process in healthy human skin in about one month. In the stratum corneum that desquamates at its normal rate, corneocytes will persist in the stratum corneum for approximately 2 weeks before being sloughed off into the environment.

[0046] As used herein, "epithelial tissue" refers to all or any part of the epithelium, particularly the epidermis, and includes one or more parts of the epithelium that can be obtained from a subject by harvesting techniques known in the art, including those described herein. By way of example, and without limitation, epithelial tissue refers to cell debris and remnants, proteins, isolated cells from the epithelium, including harvested and cultured cells.

[0047] As used herein, "filaggrin" refers to a filament-associated protein that binds to keratin fibers in epithelial cells. Filaggrin is essential for the regulation of epidermal homeostasis. Within the stratum corneum, filaggrin monomers can bind into the lipid envelope that endows the skin with its barrier function. Alternatively, these proteins can interact with keratin intermediate filaments. Filaggrin undergoes further processing in the upper stratum corneum to release free amino acids that assist in water retention. Loss-of-function mutations in the filaggrin gene are among the most common and severe monogenic defects identified to date in the causation and modification of disease. Filaggrin encodes an important epidermal protein that is highly expressed in the outer layer of the epidermis. The key role of filaggrin in epidermal function underlies the pathogenic importance of this gene in common skin diseases and allergic disorders. The spectrum of such diseases includes monogenic disorders of keratinization with complex abnormalities in the epidermal transport of lipids and allergens. Loss-of-function FLG carriers have a significantly increased risk of common complex traits, including atopic dermatitis (which affects at least 42% of all mutation carriers), contact allergy, asthma, hay fever, and peanut allergy. These genetic variants also influence the severity of asthma and alopecia areata as well as susceptibility to herpes infections.

[0048] Human beta-defensin 1 (hβD1) is an antimicrobial peptide constitutively expressed by epithelial cells at mucosal surfaces and in the epidermis.

[0049] As used herein, "infant" refers to a human being in the age range from birth to approximately twelve months.

[0050] "Inflammatory cytokine" is a signaling molecule secreted by immune cells and certain other cell types that promote inflammation. Inflammatory cytokines are mainly produced by T helper cells (Ths) and macrophages and are involved in the upregulation of the inflammatory response.

[0051] The interleukin-1 family (IL-1 family) is a group of 11 cytokines that play important roles in the regulation of immune and inflammatory responses to infection or sterile injury.

[0052] IL-1RA (IL-1 receptor antagonist) is a natural antagonist of IL-1 family members.

[0053] Interleukin-36 gamma (IL-36gamma; IL-36γ) is a cytokine in the IL-1 family that has pro-inflammatory effects.

[0054] Linear regression is a linear method used to model the relationship between a scalar response and one or more explanatory variables (also known as dependent and independent variables). The relationship is modeled using a linear prediction function, and the unknown model parameters of the linear prediction function are estimated from the data. Such models are called linear models.

[0055] As used herein, "metabolite" refers to an intermediate end product of metabolism. The term metabolite is generally limited to small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulation and inhibition of enzymes, their own catalytic activity (usually as enzyme cofactors), defense, and interaction with other organisms (such as pigments, flavorants, and pheromones). Primary metabolites are directly involved in normal "growth", development, and reproduction. Secondary metabolites are not directly involved in those processes but generally have important ecological functions.

[0056] "Packaging" includes any suitable container for a personal care regimen, ingredient, and / or composition.

[0057] As used herein, "personal care composition" refers to a composition intended for topical application to the skin. Compositions used in accordance with the present disclosure include topically applied compositions, including leave-on formulations and rinse-off formulations, where the product is topically applied to the skin and then subsequently rinsed off with water from the skin within a few minutes, or otherwise wiped off using a substrate on which a portion of the composition has been deposited. Personal care compositions used in accordance with the present disclosure can generally be dispensed from a package. Thus, in some embodiments, dispensing can be by extrusion. In some embodiments, the packaging can be a single-chamber package or a multi-chamber package, or a set of discrete packages. Personal care compositions used in accordance with the present disclosure can be in the form of a liquid, semi-liquid, cream, lotion, or gel intended for topical application to the skin.

[0058] "p-value" is a measure of the probability of observing a difference that occurs only by random chance. The lower the p-value, the greater the statistical significance of the observed difference. The p-value can be used as an alternative or supplement to a preselected confidence level for hypothesis testing. The standard significance level for demonstrating a statistically significant effect is 0.05. The term statistical significance is synonymous with p ≤ 0.05.

[0059] A "rinse-off" ingredient or composition refers to an ingredient or composition that is topically applied to the skin and then subsequently and immediately (i.e., within a few minutes) rinsed off with water, or otherwise wiped off using a substrate or other suitable removal device.

[0060] The "skin" is divided into three main structural layers, namely the outer epidermis, the inner dermis, and the subcutaneous tissue.

[0061] As used herein, the "stratum corneum" refers to the outermost layer of the epithelium or epidermis and is the skin structure that provides a chemical and physical barrier between the body of an animal and the environment. The stratum corneum is a tightly packed structure that includes an intracellular fibrous matrix that is hydrophilic and capable of trapping and retaining moisture. The intercellular spaces are filled with lipids formed and secreted by keratinocytes, and these lipids provide a diffusion pathway for channel substances with low water solubility.

[0062] As used herein, "subject" refers to a human being on whom a test or use protocol, composition, and / or combination is tested according to the methods described herein.

[0063] Unless otherwise indicated, as used herein, "substantially free" means that a personal care protocol, composition, and / or combination includes less than about 2%, less than about 1%, less than about 0.5%, or even less than about 0.1% of the indicated composition. As used herein, the term "free" means that a personal care protocol, composition, and / or combination includes 0% of the indicated composition. However, these compositions may be incidentally formed as by-products or reaction products of other components of the personal care protocol, composition, and / or combination.

[0064] As used herein, "test composition and / or combination" includes and encompasses purified or substantially pure compositions and / or combinations, as well as formulations that include one or more compositions and / or combinations. Thus, non-limiting examples of test compositions and / or combinations include water, mixtures of pharmaceuticals or cosmeceuticals, products, compounds, or products, and other examples and their combinations and dilutions.

[0065] As used herein, "test surface" means an area of epithelial tissue that has been contacted and / or is contacted by a product (such as a consumer product and / or a test protocol, composition, and / or combination), whereby contact of the product and / or protocol, composition, and / or combination with the epithelial tissue has resulted in some positive or negative changes in and / or on the epithelial tissue, such as but not limited to physiological, biochemical, visible, and / or tactile changes. In some examples, positive effects caused by a protocol, composition, and / or combination may include but are not limited to reduction of one or more of erythema, transepidermal water loss (TEWL), skin discoloration, rash, dermatitis, inflammation, eczema, dandruff, edema, etc. The location of the affected surface will depend on the protocol, composition, and / or combination used or the location of some physiological, biochemical, visible, and / or tactile changes in and / or on the epithelial tissue.

[0066] As used herein, "topical application", "topically", and "topical" mean applying a protocol, composition, and / or combination used according to the present disclosure to the surface of the skin.

[0067] As used herein, "treatment" includes modulating and / or immediately improving the appearance and / or feel of the skin.

[0068] As used herein, "toddler" means a human being in the age range of about twelve months to about 3 years or about 5 years or about 7 years.

[0069] Example

[0070] The present inventors have identified inflammatory cytokines in the body that can be used to distinguish high-risk infants who are likely to develop AD within 12 months from low-risk infants who are least likely to develop AD.

[0071] STOP AD Trial

[0072] Implement STOP AD (Short-term Topical Application for Prevention of Atopic Dermatitis), a randomized, open-label, controlled study designed to investigate the effect of short-term neonatal skin barrier protection using defined skin treatment regimens and compositions on the prevention of atopic dermatitis in high-risk infants. (See co-pending U.S. Patent Application Serial No. 63 / 327,052; NiChaoimh et al., Parental compliance with an infant moisturization protocol in the first 2 months of life; Lad et al., Can more be done to implement translational weaning advise for new mothers; and Lad et al., Neonatal natural moisturizing factor concentrations in a high-risk cohort with parental history of atopy compared to a reference cohort, each presented at European Academy of Allergy and Clinical Immunology (EAACI) 2020).)

[0073] In this study, high-risk infants were identified using a parental history of atopic disease, and infants were recruited from the postnatal ward soon after delivery (about 4 days) until 8 weeks of age and randomized to a defined skin treatment regimen and composition or standard routine care. For the first 2 months, standardized routine bathing was recommended for both the intervention and standard care groups.

[0074] Skin treatment regimen and composition: Apply topically to the entire body twice daily Dermexa Fast & Long-Lasting Balm, on its packaging, contains the following ingredients: glycerin, water, cetearyl alcohol, isocetyl alcohol, dimethicone, cetyl alcohol, oat kernel flour, oat kernel extract, oat kernel oil, caprylic / capric triglyceride, ceramide 3, ethylhexylglycerin, p-methoxybenzoic acid, sodium cetearyl sulfate, palmitic acid, stearic acid, sodium sulfate, sodium chloride, citric acid, dipotassium phosphate, potassium phosphate, sodium hydroxide, tocopherol, benzyl alcohol, benzoic acid, and potassium sorbate.

[0075] Control: Standard routine care without moisturizer in the first two months. Both groups received: BabyDaily Care Gentle Wash, which contains the following ingredients on the package: water, glycerin, cocamidopropyl betaine, sodium laurylamphoacetate, coco-glucoside, sodium chloride, hydroxypropyl starch phosphate, oat kernel powder, aloe barbadensis leaf juice, olea europaea leaf extract, chamomile extract, helianthus annuus seed oil, sarcosine, magnesium aspartate, potassium aspartate, polyquaternium-7, polysorbate 20, sodium cocoyl amino acids, acrylates / C10-30 alkyl acrylate crosspolymer, propylene glycol, citric acid, sodium hydroxide, tocopherol, tocopheryl acetate, sodium benzoate, potassium sorbate, sodium sulfite, fragrance.

[0076] Infants with a positive history of at least one parent with an atopic disease (AD, allergic rhinitis, or asthma) were eligible for enrollment.

[0077] Within approximately 4 days of birth, infants were randomized to receive either treatment with the skin treatment regimen and composition or standard routine skin care without moisturizer until 2 months of age. 260 infants participated in the study, including 120 in the intervention group and 140 in the control group.

[0078] The study involved repeated measurements of body weight, transepidermal water loss (TEWL), and Raman-derived natural moisturizing factor (NMF) to assess skin barrier function and structure, in addition to monitoring skin health and feeding, during the first year of life, within four days of birth, and at six visits at two, four, and eight weeks, and at six and 12 months. Questionnaires were completed regarding infant health, bathing, feeding, and skin care, and skin swabs were taken for microbiome and immune biomarker analysis.

[0079] The primary outcome of the study was the effect of the intervention on the incidence of atopic dermatitis at 12 months. Secondary outcomes included the effect of the intervention on the incidence of atopic dermatitis at 6 months and the evolution of TEWL and NMF values ​​from 0 to 12 months.

[0080] Skin swabs were taken again at baseline, at 8 weeks, and at 12 months. Healthcare workers blinded to treatment allocation assessed the presence (yes / no), extent, and severity of atopic dermatitis at 6 months and 12 months. DNA samples were collected to test for loss-of-function mutations in filaggrin associated with the risk of atopic dermatitis.

[0081] I-SEAL (Insights towards understanding Skin Function in Early Life) involved collecting skin microbiome and immunobiomarkers within the larger intervention trial STOP AD in order to:

[0082] ● Examine the temporal shift in the skin microbiome between birth and 12 months and its impact on the development of atopic dermatitis;

[0083] ● Investigate the effect of using skin regimens and compositions during the first two months of life on the infant skin microbiota; and

[0084] ● Investigate the kinetics of immunobiomarkers collected from the surface of infant skin within the first 12 months and examine the association with atopic dermatitis.

[0085] 103 participants were diagnosed with atopic dermatitis. The prevalence of atopic dermatitis was 27.3% and 27.9% at 6 months and 12 months, respectively, and 17.0% of participants met the UK Working Party Diagnostic Criteria (Williams et al., Br J Dermatol. September 1994;131(3):406-16. doi:10.1111 / j.1365-2133.1994.tb08532.x.).

[0086] Interim Analysis at Six (6) Months

[0087] Among 262 participants, 260 (120 in the intervention group and 140 in the control group) were included in the midpoint analysis at 6 months. Two participants (one from the intervention group and one from the control group) were excluded from the analysis due to missing data on atopic dermatitis outcomes at 6 months. Baseline characteristics were evenly distributed across the study groups. In the total cohort, the point prevalence and cumulative incidence of atopic dermatitis at 6 months were 27.3% and 28.1%, respectively. The cumulative incidence of atopic dermatitis was 18.3% in the intervention group and 36.4% in the control group [relative risk (RR): 0.503, 95% CI: 0.325, 0.779], corresponding to a 50% reduction in the risk of atopic dermatitis at 6 months in the intervention group.

[0088] Collect DNA samples for analyzing the filaggrin gene status using oral swabs for filaggrin genotyping.

[0089] Collect skin swabs for microbiome and immunobiomarker analysis from study participants at baseline, at the end of the intervention period (8 weeks), and at 12 months. Swabs are collected from two skin sites: the cheek and the antecubital fossa (elbow pit). At the end of the 12-month evaluation, skin swabs from a subgroup of approximately 30 infants from each study group (control and intervention groups) are sent to CosmosID, Rockville, MD, USA for analysis.

[0090] The swabs collected for microbiome analysis at the end of sample collection at 12 months are analyzed as a single batch for common 16S rRNA gene sequencing.

[0091] In addition to the microbiome swabs, samples from the same subgroup identified for microbiome analysis are sent for analysis at the end of sample collection. Samples for immunobiomarker quantification are collected from skin sites using specialized swabs soaked in buffer (FibroTx, Tallinn, Estonia) at baseline, 8 weeks, and 12 months. The samples are stored on dry ice until further processing and stored at -80 °C until transported to FibroTx for biomarker analysis using a dot enzyme-linked immunosorbent assay.

[0092] The samples are analyzed as follows:

[0093] ● Measure 10 cytokines:

[0094] ○ CCL27, CXCL2, hBD1, IL18, IL8, IL1a, IL1RA, IL1b, S100A8 / 9, IL36g.

[0095] ● Samples are taken from 2 body sites, namely, the elbow and the cheek

[0096] ● There are 2 treatment groups, namely, A and B. The analysis is done in a blinded manner. Group A and Group B represent the intervention group or the control group.

[0097] ● Samples are taken at 3 time points, namely, at 0 weeks, 8 weeks, and 12 months.

[0098] ● There are 3 AD endpoints, namely, at 6 months, 12 months, and 12-month cumulative incidence.

[0099] ○ 43 subjects are in Group A, and 43 subjects are in Group B.

[0100] ■ Group A: 43 (0 weeks), 43 (8 weeks), 30 (12 weeks)

[0101] ■ Group B: 43 (0 weeks), 43 (8 weeks), 30 (12 weeks)

[0102] At 8 weeks of age, surface cytokine concentrations on the elbow were measured using skin swabs, and the development of AD was measured at 6 months and 12 months (Table 1). Table 1 shows the number of subjects who developed AD at each endpoint.

[0103] Table 1

[0104] 6 Months 12 Months 12-Month Cumulative Wt 12 22 28 LoF 10 11 13

[0105] Table 2 shows the biomarker p-values from the elbow at 8 weeks, which are from a linear model comparing wild-type FLG and loss-of-function FLG transformants to non-transformants.

[0106] Table 2

[0107]

[0108] Table 3 shows the biomarker p-values from the elbow at 8 weeks, which are from a linear model comparing wild-type FLG and loss-of-function FLG persistent or non-persistent AD transformants to non-transformants.

[0109] Table 3

[0110]

[0111]

[0112] Observations from the above data include:

[0113] ● FLG wt transformants had increased cytokine levels at week 8

[0114] ○ S100A8 / 9 was the most prominent cytokine associated with the future development of AD

[0115] ■ Therefore, by evaluating the level of S100A8 / 9, patients at higher risk of developing AD can be identified. Such patients may benefit from skin barrier repair therapy. FLG LoF transformants had the smallest cytokine differences at week 8

[0116] ○ Therefore, in this population, the conversion to AD can be driven by skin barrier function rather than skin inflammation.

[0117] Analyze the data to test whether cytokine levels can predict the future development of AD in the FLG wt group and the FLG LoF group separately. In the I-SEAL cohort, there were 65 wt FLG subjects and 20 subjects with FLG LoF mutations. Among the FLG wt subjects, for all 3 endpoints, S100A8 / 9 was significantly different between converters and non-converters ( Figure 1 , Table 2), while for the two 12-month endpoints, IL-36γ was significant ( Figure 2 , Table 2). Other cytokines with potential signals (p < 0.3 at any endpoint) in converters included human β-defensin (hBD)-1 and IL-1a. Subjects with FLG LoF mutations did not have any cytokines that could distinguish converters from non-converters (Table 2).

[0118] Compare cytokine levels between non-AD, non-persistent AD (AD only at 6 months or 12 months), and persistent AD (AD at 6-month and 12-month endpoints). S100A8 / 9 was significantly different between FLG wt converters and non-converters who developed persistent AD, but not significantly different in non-persistent AD. ( Figure 3 , Table 3). Subjects with FLG LoF mutations did not have any cytokines that could distinguish converters from non-converters who developed persistent AD or non-persistent AD (Table 3).

[0119] In summary, these data indicate that cytokine expression in FLG wt subjects measured at 8 weeks of age can predict the future onset of AD at 6 months and 12 months. Additionally, compared to non-persistent AD, S100A8 / A9 measured at 8 weeks of age has a strong prediction for persistent AD.

[0120] It should be understood that although various aspects of the present disclosure have been shown and described by way of example, the invention as claimed herein is not limited thereto, but may be otherwise differently implemented according to the scope of the claims set forth in this patent application and / or any derivative patent application.

Claims

1. A method for predicting the tendency of an infant to develop atopic dermatitis, the method comprising: observing the expression of S100A8 / A9 on an infant's skin area; comparing the expression with a determined standard, wherein the determined standard is ascertained by measuring the level of a biomarker in a subject or a pool of subjects who have been demonstrated to be free of atopic dermatitis; and determining the tendency of the infant to develop atopic dermatitis, wherein an increase in the expression compared to the determined standard indicates the tendency of the infant to develop atopic dermatitis.

2. The method according to claim 1, wherein the age of the infant is selected from about 1 week old to about 12 months old; about 2 weeks old to about 6 months old; about 1 month old to about 3 months old; about 8 weeks old to about 2 months old; and about 8 weeks old.

3. The method according to claim 1, wherein the skin is selected from facial skin, limb skin, and body skin.

4. The method according to claim 3, wherein the facial skin is from the cheek.

5. The method according to claim 3, wherein the limb skin is from the elbow.

6. The method according to claim 1, the method further comprising: observing the mutation status of FLG using genetic material from the infant to determine the FLG genotype of the infant, wherein the FLG genotype is selected from the FLG wild-type genotype and the FLG loss-of-function genotype; and if it is determined that the infant has the FLG wild-type genotype, observing the individual expression of S100A8 / 9 or the combined expression with other biomarkers on the skin surface of the infant.

7. A method for evaluating the efficacy of at least one of a skin treatment regimen, a component, or a composition for treating atopic dermatitis in an infant, the method comprising: measuring the level of S100A8 / A9 on an infant's skin area before administering the skin treatment regimen, component, and / or composition; administering the skin treatment regimen, component, and / or composition to the skin area for a period of time; and measuring the level of S100A8 / A9 after administering the skin treatment regimen, component, and / or composition to the skin area; wherein if the level of S100A8 / A9 is less than or equal to the level of the untreated control, the skin treatment regimen, component, and / or composition is beneficial to the skin.

8. The method according to claim 7, wherein the facial skin is from the cheek.

9. The method according to claim 7, wherein the limb skin is from the elbow.

10. The method according to claim 7, the method further comprising: observing the mutation status of FLG using genetic material from the infant to determine the FLG genotype of the infant, wherein the FLG genotype is selected from the FLG wild-type genotype and the FLG loss-of-function genotype; and if it is determined that the infant has the FLG wild-type genotype, observing the individual expression of S100A8 / 9 or the combined expression with other biomarkers on the skin surface of the infant.

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