WO2024079664A1

WO2024079664A1 - Compositions and methods for skincare

Info

Publication number: WO2024079664A1
Application number: PCT/IB2023/060251
Authority: WO
Inventors: Kevin Dietzel; João C. FERNANDES; Helena Rocha MOREIRA; Adelia MENDES; Morgan TARDY; Daan THORN LEESON; Annie TSONG; Mariana VEIGA; Raaj P. KHUSIAL
Original assignee: Amyris Bio Products Portugal, Unipessoal, Ltda.; Universidade Católica Portuguesa
Priority date: 2022-10-11
Filing date: 2023-10-11
Publication date: 2024-04-18

Abstract

The disclosure provides compositions including a human milk oligosaccharide (HMO), as well as methods of using the same for reducing inflammation of the skin, treating or preventing psoriasis, acne, rosacea, eczema, erythema, UV radiation-induced inflammation, environmental pollution-induced inflammation, and/or dehydration-induced inflammation, increasing the hydration of the skin cells, improving skin barrier function, reducing fine lines or wrinkles, and/or increasing the firmness or elasticity of the skin.

Description

COMPOSITIONS AND METHODS FOR SKINCARE

BACKGROUND OF THE INVENTION

Inflammatory and irritated skin conditions are known to affect at least 5 million Americans, causing pain, itching, discomfort, dryness, and, potentially, a lack of confidence in the subject. Inflammatory skin conditions, while common, are challenging to diagnose and manage. In some instances, cosmetics may be used to improve a person’s appearance or treat a disease or condition of the skin. With respect to cosmetics, a person may apply lotions to promote the skin’s condition and hydration, and may also apply short-term cosmetic formulations, such as concealer or foundation, to hide imperfections, such as redness, dryness, and dullness. However, given the challenge of managing inflammatory skin conditions and to address the underlying causes of physical appearance to the skin, there remains a need for effective options for improving the outward appearance of skin and for treating the underlying inflammation or irritation in the skin.

SUMMARY OF THE INVENTION

The present disclosure provides compositions (e.g., pharmaceutical compositions and cosmetic compositions) that contain a human milk oligosaccharide (HMO). The pharmaceutical compositions of the disclosure may be used to treat inflammation in a subject, particularly in the subject’s skin. The skin may become inflamed due to an underlying disease or condition. For example, the inflammation of the skin may be caused by psoriasis. Additionally or alternatively, the skin may become inflamed due to pollution or other environmental conditions. The cosmetic compositions of the disclosure can be used to engender a series of beneficial results for the end user. Particularly, the cosmetic compositions described herein can be administered to a subject so as to improve the quality and/or the outward appearance of the subject’s skin by reducing the appearance of fine lines and wrinkles or increasing the firmness and elasticity of the skin. Cosmetic compositions containing an HMO may also reduce redness in a subject’s skin, such as redness due to inflammation caused by one or more of a variety of underlying conditions. Additionally, cosmetic compositions containing an HMO may hide one or more imperfections in a subject’s skin, such as a blemish caused by lack of hydration or chronic or acute inflammation. Cosmetic compositions containing an HMO may also be used for their anti-aging effects.

The foregoing examples illustrate some of the ways in which the compositions (e.g., pharmaceutical compositions and cosmetic compositions) of the disclosure may be used. Further examples are provided herein.

In a first aspect, the disclosure provides a method of reducing inflammation in the skin of a subject including topically applying to the skin of the subject a composition including an HMO. In some embodiments, the inflammation is caused by psoriasis (e.g., the subject having been diagnosed as having psoriasis). In some embodiments, the inflammation is caused by acne (e.g., the subject having been diagnosed as having acne). In some embodiments, the inflammation is caused by rosacea (e.g., the subject having been diagnosed as having rosacea). In some embodiments, the inflammation is caused by eczema (e.g., the subject having been diagnosed as having eczema). In some embodiments, the inflammation is caused by erythema (e.g., the subject having been diagnosed as having erythema). In some embodiments, the inflammation is caused by UV radiation. In some embodiments, the inflammation is caused by environmental pollution. In some embodiments, the environmental pollution is air pollution. In some embodiments, the inflammation is caused by exposure to a chemical compound. In some embodiments, the inflammation in the skin of the subject is reduced by at least 10% in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied. In some embodiments, the inflammation in the skin of the subject reduced by between 10% and 50% (e.g., between 10% and 45%, 10% and 40%, 10% and 35%, 10% and 30%, 10% and 25%, 10% and 20%, 10% and 15%, 12% and 50%, 15% and 50%, 20% and 50%, 25% and 50%, 30% and 50%, 35% and 50%, 40% and 50%, or 45% and 50%) in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied.

In another aspect, the disclosure provides a method of reducing irritation in the skin of a subject, including topically applying to the skin of the subject a composition comprising an HMO. In some embodiments, the irritation is caused by psoriasis (e.g., the subject having been diagnosed as having psoriasis). In some embodiments, the irritation is caused by acne (e.g., the subject having been diagnosed as having acne). In some embodiments, the irritation is caused by rosacea (e.g., the subject having been diagnosed as having rosacea). In some embodiments, the irritation is caused by eczema (e.g., the subject having been diagnosed as having eczema). In some embodiments, the irritation is caused by erythema (e.g., the subject having been diagnosed as having erythema). In some embodiments, the irritation is caused by UV radiation. In some embodiments, wherein the irritation is caused by environmental pollution. In some embodiments, the environmental pollution is air pollution. In some embodiments, the irritation is caused by exposure to a chemical compound. In some embodiments, the irritation of the skin of the subject is reduced by at least 10% in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied. In some embodiments, the irritation of the skin of the subject reduced by between 10% and 50% (e.g., between 10% and 45%, 10% and 40%, 10% and 35%, 10% and 30%, 10% and 25%, 10% and 20%, 10% and 15%, 12% and 50%, 15% and 50%, 20% and 50%, 25% and 50%, 30% and 50%, 35% and 50%, 40% and 50%, or 45% and 50%) in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied.

In another aspect, the disclosure provides a method of treating or preventing psoriasis (e.g., the subject having been diagnosed as having psoriasis), acne (e.g., the subject having been diagnosed as having acne), rosacea (e.g., the subject having been diagnosed as having rosacea), eczema (e.g., the subject having been diagnosed as having eczema), erythema (e.g., the subject having been diagnosed as having erythema), UV radiation-induced inflammation or irritation, environmental pollution-induced inflammation or irritation, dehydration-induced inflammation, or irritation, and/or chemical exposure induced inflammation or irritation in a subject. The method may include topically applying to the skin of the subject a composition comprising an HMO.

In another aspect, the disclosure provides a method of achieving an anti-aging effect in the skin of a subject including topically applying to the skin of the subject a composition including an HMO.

In another aspect, the disclosure provides a method of reducing the appearance of fine lines or wrinkles in the skin of a subject including topically applying to the skin of the subject a composition including an HMO.

In another aspect, the disclosure provides a method of increasing skin firmness or elasticity in the skin of a subject including topically applying to the skin of the subject a composition including an HMO.

In another aspect, the disclosure provides a method of reducing oxidative stress in skin cells of a subject including topically applying to the skin of the subject a composition including an HMO.

In another aspect, the disclosure provides a method of increasing hydration in skin cells of a subject and/or improving skin barrier function in a subject. The method may include topically applying to the skin of the subject a composition including an HMO.

In some embodiments, the HMO is selected from L-fucose, 2’-fucosyllactose (2’-FL), lacto-N- neotetraose (LNnT), 3-fucosyllactose (3’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N- fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N-difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto-N-hexaose (F-LNH) I, F-LNH II, difucosyllacto-N-hexaose (DFLNH) I, DFLNH II, difucosyllacto-N-neohexaose (DFLNnH), difucosyl-para- lacto-N-hexaose (DF-para-LNH), difucosyl-para-lacto-N-neohexaose (DF-para-LNnH), trifucosyllacto-N- hexaose (TF-LNH), 3’-siallylactose (3’-SL), 6’-siallylactose (6’-SL), sialyllacto-N-tetraose (LST) a, LST b, LST c, disialyllacto-N-tetraose (DS-LNT), fucosyl-sialyllacto-N-tetraose (F-LST) a, F-LST b, fucosyl- sialyllacto-N-hexaose (FS-LNH), fucosyl-sialyllacto-N-neohexaose (FS-LNnH) I, f ucosyl-disialy llacto-N- hexaose (FDS-LNH) II, or a combination thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the HMO is L-fucose.

In some embodiments, the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL (e.g., between 1 mg/mL and 120 mg/mL, 1 mg/mL and 100 mg/mL, 1 mg/mL and 80 mg/mL, 1 mg/mL and 60 mg/mL, 1 mg/mL and 40 mg/mL, 1 mg/mL and 20 mg/mL, 20 mg/mL and 150 mg/mL, 40 mg/mL and 150 mg/mL, 60 mg/mL and 150 mg/mL, 80 mg/mL and 150 mg/mL, 100 mg/mL and 150 mg/mL, or 125 mg/mL and 150 mg/mL). In some embodiments, the composition has a concentration of the HMO of between about 10 mg/mL and about 50 mg/mL (e.g., between 10 mg/mL and 40 mg/mL, 10 mg/mL and 30 mg/mL, 10 mg/mL and 20 mg/mL, 20 mg/mL and 50 mg/mL, 30 mg/mL and 50 mg/mL, or 40 mg/mL and 50 mg/mL). In some embodiments, the composition has a concentration of the HMO of about 10 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 25 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 50 mg/mL. In some embodiments, the composition further includes squalane. In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 60% (w/w) (e.g., about 1% (w/w) and 50% (w/w), 1 % (w/w) and 40% (w/w), 1 % (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 20% (w/w) and 60% (w/w), 30% (w/w) and 60% (w/w), 40% (w/w) and 60% (w/w), or 50% (w/w) and 60% (w/w)). In some embodiments, the composition has a concentration of squalane of between about 20% (w/w) and about 50% (w/w) (e.g., between 20% (w/w) and 45% (w/w), 20% (w/w) and 40% (w/w), 20% (w/w) and 35% (w/w), 20% (w/w) and 30% (w/w), 20% (w/w) and 25% (w/w), 25% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), 35% (w/w) and 50% (w/w), 40% (w/w) and 50% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the composition has a concentration of squalane of about 42% (w/w). In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 20% (w/w) (e.g., 1 % (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 1 1% (w/w), 12% (w/w), 13% (w/w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w), and 20% (w/w)). In some embodiments, the composition has a concentration of squalane of about 10% (w/w).

In some embodiments, the composition further includes carboxymethyl cellulose (CMC). In some embodiments, the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w) (e.g., between 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1% (w/w), 1% (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), or 4% (w/w) and 5% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 1 % (w/w) and about 3% (w/w) (e.g., between 1 % (w/w) and 2.5% (w/w), 1% (w/w) and 2% (w/w), 1% (w/w) and 1 .5% (w/w), 1 .5% (w/w) and 3% (w/w), 2% (w/w) and 3% (w/w), or 2.5% (w/w) and 3% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 2% (w/w).

In some embodiments, the composition further includes lecithin. In some embodiments, the composition has a concentration of lecithin of between about 0.01% (w/w) and about 1% (w/w) (e.g., between 0.01 % (w/w) and 0.8% (w/w), 0.01% (w/w) and 0.6% (w/w), 0.01% (w/w) and 0.4% (w/w), 0.01 % (w/w) and 0.2% (w/w), 0.01 % (w/w) and 0.05% (w/w), 0.05% (w/w) and 1% (w/w), 0.2% (w/w) and 1% (w/w), 0.4% (w/w) and 1% (w/w), 0.6% (w/w) and 1 % (w/w), or 0.8% (w/w) and 1 % (w/w)). In some embodiments, the composition has a concentration of lecithin of between about 0.05% (w/w) and about 0.5% (w/w) (e.g., between 0.05% (w/w) and 0.4% (w/w), 0.05% (w/w) and 0.3% (w/w), 0.05% (w/w) and 0.2% (w/w), 0.05% (w/w) and 0.1% (w/w), 0.1 % (w/w) and 0.5% (w/w), 0.2% (w/w) and 0.5% (w/w), 0.3% (w/w) and 0.5% (w/w), or 0.4% (w/w) and 0.5% (w/w)). In some embodiments, the composition has a concentration of lecithin of about 0.1% (w/w).

In some embodiments, the composition further comprises phenoxyethanol. In some embodiments, the composition has a concentration of phenoxyethanol of about between about 0.01% (w/w) and 5% (w/w) (e.g., between about 0.05% (w/w) and 5% (w/w), 0.1 % (w/w) and 5% (w/w), 0.5% (w/w) and 5% (w/w), 1 % (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), 4% (w/w) and 5% (w/w), 0.01 % (w/w) and 4% (w/w), 0.01% (w/w) and 3% (w/w), 0.01% (w/w) and 2% (w/w), 0.01% (w/w) and 1 % (w/w), 0.01% (w/w) and 0.5% (w/w), 0.01% (w/w) and 0.1% (w/w), 0.1% (w/w) and 1 % (w/w), or 0.3% (w/w) and 0.7% (w/w)). In some embodiments, the composition has a concentration of phenoxyethanol of about 0.5% (w/w).

In some embodiments, the composition further comprises cetearyl glucoside. In some embodiments, the composition further comprises cetearyl alcohol and cetearyl glucoside. In some embodiments, the composition comprises Montanov®. In some embodiments, the composition has a concentration of cetearyl alcohol and cetearyl glucoside of between about 0.5% (w/w) and about 10% (w/w) (e.g., between about 0.5% (w/w) and 9% (w/w), 0.5% (w/w) and 8% (w/w), 0.5% (w/w) and 7% (w/w), 0.5% (w/w) and 6% (w/w), 0.5% (w/w) and 5% (w/w), 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1 % (w/w), 1% (w/w) and 10% (w/w), 2% (w/w) and

10% (w/w), 3% (w/w) and 10% (w/w), 4% (w/w) and 10% (w/w), 5% (w/w) and 10% (w/w), 6% (w/w) and

10% (w/w), 7% (w/w) and 10% (w/w), 8% (w/w) and 10% (w/w), 9% (w/w) and 10% (w/w), 1% (w/w) and

5% (w/w), or 2% (w/w) and 7% (w/w)). In some embodiments, the composition has a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w).

In some embodiments, the composition further comprises oil. In some embodiments, the oil is soy oil. In some embodiments, the composition has a concentration of soy oil of between about 1% (w/w) and about 25% (w/w) (e.g., between about 1 % (w/w) and 20% (w/w), 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 5% (w/w) and 25% (w/w), 10% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 20% (w/w) and 25% (w/w), 5% (w/w) and 15% (w/w), or 5% (w/w) and 20% (w/w)). In some embodiments, the composition has a concentration of soy oil of about 10% (w/w).

In some embodiments, the composition further includes glyceride. In some embodiments, the composition includes Gelucire®. In some embodiments, the composition has a concentration of glyceride of between about 1 % (w/w) and about 20% (w/w) (e.g., between 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 0.5% (w/w) and 20% (w/w), 10% (w/w) and 20% (w/w), or 15% (w/w) and 20% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w) (e.g., 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), or 10% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 8% (w/w).

In some embodiments, the composition is a patch, liquid, gel, lotion, paste, cream, foam, serum, ointment, or stick. In some embodiments, the composition is applied to the skin of the subject one or more times daily, optionally wherein the composition is applied to the subject once daily, twice daily, or three times daily. In some embodiments, the composition is applied to the skin of the subject once daily. In some embodiments, the composition is applied to the skin of the subject twice daily, optionally wherein the composition is administered to the subject once in the morning and once in the evening. In some embodiments, the composition is applied to the skin of the subject three times daily. In some embodiments, the composition is applied to the skin of the subject for at least 1 day. In some embodiments, the composition is applied to the skin of the subject for at least 2 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 7 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 14 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 28 continuous days.

In another aspect, the disclosure provides a pharmaceutical composition including an HMO and one or more excipients selected from squalane, carboxymethyl cellulose, lecithin, and glyceride. In some embodiments, the HMO is selected from any one of L-fucose, LNnT, 2’-FL, 3’-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, 6’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, FDS-LNH II, or a combination thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the HMO is L-fucose. In some embodiments, the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL (e.g., between 1 mg/mL and 120 mg/mL, 1 mg/mL and 100 mg/mL, 1 mg/mL and 80 mg/mL, 1 mg/mL and 60 mg/mL, 1 mg/mL and 40 mg/mL, 1 mg/mL and 20 mg/mL, 20 mg/mL and 150 mg/mL, 40 mg/mL and 150 mg/mL, 60 mg/mL and 150 mg/mL, 80 mg/mL and 150 mg/mL, 100 mg/mL and 150 mg/mL, or 125 mg/mL and 150 mg/mL). In some embodiments, the composition has a concentration of the HMO of between about 10 mg/mL and about 50 mg/mL (e.g., between 10 mg/mL and 40 mg/mL, 10 mg/mL and 30 mg/mL, 10 mg/mL and 20 mg/mL, 20 mg/mL and 50 mg/mL, 30 mg/mL and 50 mg/mL, or 40 mg/mL and 50 mg/mL). In some embodiments, the composition has a concentration of the HMO of about 10 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 25 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 50 mg/mL.

In some embodiments, the composition includes squalane. In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 60% (e.g., about 1% (w/w) and 50% (w/w)_a 1 % (w/w) and 40% (w/w), 1 % (w/w) and 30% (w/w), 1 % (w/w) and 20% (w/w), 20% (w/w) and 60% (w/w), 30% (w/w) and 60% (w/w), 40% (w/w) and 60% (w/w), or 50% (w/w) and 60% (w/w)). In some embodiments, the composition has a concentration of squalane of between about 20% (w/w) and about 50% (w/w) (e.g., between 20% (w/w) and 45% (w/w), 20% (w/w) and 40% (w/w), 20% (w/w) and 35% (w/w), 20% (w/w) and 30% (w/w), 20% (w/w) and 25% (w/w), 25% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), 35% (w/w) and 50% (w/w), 40% (w/w) and 50% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the composition has a concentration of squalane of about 42% (w/w). In some embodiments, the composition has a concentration of squalane of between about 1 % (w/w) and about 20% (w/w) (e.g., 1% (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 1 1% (w/w), 12% (w/w), 13% (w/w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w), and 20% (w/w)). In some embodiments, the composition has a concentration of squalane of about 10% (w/w). In some embodiments, the composition includes CMC. In some embodiments, the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w) (e.g., between 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1% (w/w), 1 % (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), or 4% (w/w) and 5% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 1% (w/w) and about 3% (w/w) (e.g., between 1 % (w/w) and 2.5% (w/w), 1% (w/w) and 2% (w/w), 1% (w/w) and 1.5% (w/w), 1.5% (w/w) and 3% (w/w), 2% (w/w) and 3% (w/w), or 2.5% (w/w) and 3% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 2% (w/w).

In some embodiments, the composition includes lecithin. In some embodiments, the composition has a concentration of lecithin of between about 0.01% (w/w) and about 1% (w/w) (e.g., between 0.01% (w/w) and 0.8% (w/w), 0.01 % (w/w) and 0.6% (w/w), 0.01 % (w/w) and 0.4% (w/w), 0.01% (w/w) and 0.2% (w/w), 0.01% (w/w) and 0.05% (w/w), 0.05% (w/w) and 1 % (w/w), 0.2% (w/w) and 1% (w/w), 0.4% (w/w) and 1 % (w/w), 0.6% (w/w) and 1% (w/w), or 0.8% (w/w) and 1% (w/w)). In some embodiments, the composition has a concentration of lecithin of between about 0.05% (w/w) and about 0.5% (w/w) (e.g., between 0.05% (w/w) and 0.4% (w/w), 0.05% (w/w) and 0.3% (w/w), 0.05% (w/w) and 0.2% (w/w), 0.05% (w/w) and 0.1% (w/w), 0.1 % (w/w) and 0.5% (w/w), 0.2% (w/w) and 0.5% (w/w), 0.3% (w/w) and 0.5% (w/w), or 0.4% (w/w) and 0.5% (w/w)). In some embodiments, the composition has a concentration of lecithin of about 0.1 % (w/w).

5% (w/w), or 2% (w/w) and 7% (w/w)). In some embodiments, the composition has a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w). In some embodiments, the composition further comprises oil. In some embodiments, the oil is soy oil. In some embodiments, the composition has a concentration of soy oil of between about 1% (w/w) and about 25% (w/w) (e.g., between about 1 % (w/w) and 20% (w/w), 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 5% (w/w) and 25% (w/w), 10% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 20% (w/w) and 25% (w/w), 5% (w/w) and 15% (w/w), or 5% (w/w) and 20% (w/w)). In some embodiments, the composition has a concentration of soy oil of about 10% (w/w).

In some embodiments, the composition includes glyceride. In some embodiments, the composition includes Gelucire®. In some embodiments the composition has a concentration of glyceride of between about 1 % (w/w) and about 20% (w/w) (e.g., between 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 0.5% (w/w) and 20% (w/w), 10% (w/w) and 20% (w/w), or 15% (w/w) and 20% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w) (e.g., 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), or 10% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 8% (w/w).

In another aspect, the disclosure provides a cosmetic composition including an HMO and one or more excipients selected from squalane, carboxymethyl cellulose, lecithin, and glyceride. In some embodiments, the HMO is selected from any one of L-fucose, LNnT, 2’-FL, 3’-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, 6’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, FDS-LNH II, or a combination thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the HMO is L-fucose. In some embodiments, the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL (e.g., between 1 mg/mL and 120 mg/mL, 1 mg/mL and 100 mg/mL, 1 mg/mL and 80 mg/mL, 1 mg/mL and 60 mg/mL, 1 mg/mL and 40 mg/mL, 1 mg/mL and 20 mg/mL, 20 mg/mL and 150 mg/mL, 40 mg/mL and 150 mg/mL, 60 mg/mL and 150 mg/mL, 80 mg/mL and 150 mg/mL, 100 mg/mL and 150 mg/mL, or 125 mg/mL and 150 mg/mL). In some embodiments, the composition has a concentration of the HMO of between about 10 mg/mL and about 50 mg/mL (e.g., between 10 mg/mL and 40 mg/mL, 10 mg/mL and 30 mg/mL, 10 mg/mL and 20 mg/mL, 20 mg/mL and 50 mg/mL, 30 mg/mL and 50 mg/mL, or 40 mg/mL and 50 mg/mL). In some embodiments, the composition has a concentration of the HMO of about 10 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 25 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 50 mg/mL.

In some embodiments, the composition includes squalane. In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 60% (w/w) (e.g., about 1% (w/w) and 50% (w/w), 1 % (w/w) and 40% (w/w), 1 % (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 20% (w/w) and 60% (w/w), 30% (w/w) and 60% (w/w), 40% (w/w) and 60% (w/w), or 50% (w/w) and 60% (w/w)). In some embodiments, the composition has a concentration of squalane of between about 20% (w/w) and about 50% (w/w) (e.g., between 20% (w/w) and 45% (w/w), 20% (w/w) and 40% (w/w), 20% (w/w) and 35% (w/w), 20% (w/w) and 30% (w/w), 20% (w/w) and 25% (w/w), 25% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), 35% (w/w) and 50% (w/w), 40% (w/w) and 50% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the composition has a concentration of squalane of about 42% (w/w). In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 20% (w/w) (e.g., 1 % (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 1 1% (w/w), 12% (w/w), 13% (w/w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w)_a and 20% (w/w)). In some embodiments, the composition has a concentration of squalane of about 10% (w/w).

In some embodiments, the composition includes CMC. In some embodiments, the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w) (e.g., between 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1% (w/w), 1 % (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), or 4% (w/w) and 5% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 1% (w/w) and about 3% (w/w) (e.g., between 1 % (w/w) and 2.5% (w/w), 1% (w/w) and 2% (w/w), 1% (w/w) and 1.5% (w/w), 1.5% (w/w) and 3% (w/w), 2% (w/w) and 3% (w/w), or 2.5% (w/w) and 3% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 2% (w/w).

In some embodiments, the composition includes glyceride. In some embodiments, the composition includes Gelucire®. In some embodiments, the composition has a concentration of glyceride of between about 1 % (w/w) and about 20% (w/w) e.g., between 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 0.5% (w/w) and 20% (w/w), 10% (w/w) and 20% (w/w), or 15% (w/w) and 20% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w) (e.g., 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), or 10% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 8% (w/w).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of images and graphs which show the immunofluorescence of the capacity of 2’-FL at 5% (w/v) in comparison to PAL at 0.5pM to stimulate extracellular matrix proteins associated with skin firmness and elasticity, using human primary dermal fibroblasts.

FIG. 2 is a series of images and graphs which show the immunofluorescence of the capacity of 2’-FL at 5% (w/w) in comparison to Matrixyl 3000 at 3% (v/w) to stimulate extracellular matrix proteins associated with skin firmness and elasticity, using a skin ex vivo model.

FIG. 3A and FIG. 3B are graphs showing the anti-inflammatory effect of 2-FL in epidermal keratinocytes from psoriasis donors by inhibition of pro-inflammatory cytokine IL-1 a release (FIG. 3A) and psoriatic human dermal fibroblasts by inhibition of pro-inflammatory cytokine IL-6 release (FIG. 3B). FIG. 4 is a series of images and graphs which show the immunofluorescence of 2’-FL at 5% (w/w) and betamethasone 0.05% (w/w) capacity to reduce proteins associated with keratinocyte hyperproliferation, using an inflammatory psoriatic-like skin (IPS) ex vivo model.

FIG. 5 is a graph showing the analysis via qPCR of 2’-FL at 5% (w/w) and betamethasone 0.05% (w/w) impact upon genes associated with inflammatory and keratinocyte hyperproliferation processes characteristic from psoriasis, using an inflammatory psoriatic-like skin ex vivo model

FIG. 6A and FIG. 6B are graphs which show the capacity of 2’-FL to reduce the release of pro- inflammatory cytokine IL-1 a (FIG. 6A) and ROS production (FIG. 6B) by HaCaT cells exposed to urban air pollution (left side).

FIG. 7A and FIG 7B are graphs illustrating the mean transepidermal water loss (TEWL) (FIG. 7A) and the mean percent change in TEWL (FIG. 7B) after administration of a cosmetic composition containing 2’-FL or L-fucose on skin that was irritated with sodium lauryl sulfate.

FIG. 8A and FIG. 8B are graphs illustrating the mean TEWL (FIG. 8A) and the mean percent change in TEWL (FIG. 8B) after administration of a cosmetic composition containing 2’-FL or L-fucose on skin that was irritated with UV radiation.

FIG. 9A and FIG. 9B are graphs illustrating the mean visual irritancy (FIG. 9A) and the mean percent change in visual irritancy (FIG. 9B) after administration of a cosmetic composition containing 2’- FL or L-fucose on skin that was irritated with sodium lauryl sulfate.

FIG. 10A and FIG. 10B are graphs illustrating the mean visual irritancy (FIG. 10A) and the mean percent change in visual irritancy (FIG. 10B) after administration of a cosmetic composition containing 2’- FL or L-fucose on skin that was irritated with UV radiation.

FIG. 11 shows the quantification of collagen I in skin explants treated with 2-FL, retinol or niacinamide by immunofluorescence microscopy where the results are expressed in fold-change relative to control (no treatment). Statistical significance: * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 .

FIG. 12 shows the quantification of elastin in skin explants treated with 2-FL, retinol or niacinamide by immunofluorescence microscopy where the results are expressed in fold-change relative to control (no treatment). Statistical significance: * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 .

FIG. 13 shows the quantification of fibronectin in skin explants treated with 2-FL, retinol or niacinamide by immunofluorescence microscopy where the results are expressed in fold-change relative to control (no treatment). Statistical significance: * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 .

FIG. 14A and FIG. 14B are graphs showing the quantification of IL-1 a (FIG. 14A) and IL-6 (FIG. 14B) levels in skin explants aggressed with SDS and treated with 2-FL, retinol or niacinamide by immunohistochemistry where the results are expressed in fold-change relative to skin aggressed with SDS. Statistical significance: * P < 0.05; ** P < 0.01 ; *** P < 0.001 ; **** P < 0.0001 .

FIG. 15 is a graph which shows fold change in filaggrin measured in skin samples after treatment with 0.1 % (w/w) retinol, 0.2 %(w/w) retinol, 2.5 % (w/w) niacinamide, 5 % (w/w) niacinamide, 2.5 % (w/w) 2’-FL, or 5 % (w/w) 2’-FL. FIG. 16 is a series of images showing the filaggrin area and the total stratum corneum area after samples were treated with 0.1 % (w/w) retinol, 0.2 %(w/w) retinol, 2.5 % (w/w) niacinamide, 5 % (w/w) niacinamide, 2.5 % (w/w) 2’-FL, or 5 % (w/w) 2’-FL.

DEFINITIONS

As used herein the singular forms “a,” “an,” and, “the” include plural reference unless the context clearly dictates otherwise.

The term “about” when modifying a numerical value or range herein includes normal variation encountered in the field, and specifically includes plus or minus 1 -10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%) of the numerical value or end points of the numerical range. Thus, a value of 10 includes all numerical values from 9 to 1 1 . All numerical ranges described herein include the endpoints of the range unless otherwise noted, and all numerical values in-between the end points, to the first significant digit.

As used herein, the terms “administering” and “administration” refer to the administration of a composition (e.g., a compound or a preparation that includes a compound as described herein) to a subject. Administration to a subject may be by any appropriate route; for example, the administration may be topical administration, for instance, in the form of a patch, liquid, gel, lotion, paste, cream, foam, serum, ointment, or stick.

As used herein in the context of improving the outward appearance of a subject’s skin, the term “brighten” refers to a process of increasing a subject’s skin tone uniformity, skin complexion uniformity, skin luminosity, and/or skin firmness. The term “brighten” may also refer to a process of decreasing discoloration and/or dullness in a subject’s skin, which may be caused by an accumulation of dead cells on the skin surface. Each of the foregoing parameters - skin tone uniformity, skin complexion uniformity, skin luminosity, skin firmness, skin discoloration, and skin dullness - can be evaluated by a visual inspection of a subject’s skin. For example, an increase in skin tone uniformity can be readily detected by visually monitoring a subject’s skin and assessing whether the subject’s skin tone at a location of interest has become more evenly distributed, with fewer areas of dissimilar color. Similarly, skin complexion uniformity can be evaluated by visually monitoring a subject’s skin and assessing whether the texture of the subject’s skin has become increasingly even, with fewer fluctuations in texture from one area to another. Skin luminosity can also be assessed by visual evaluation of the intensity of light that is reflected from the surface of a subject’s skin. Additionally or alternatively, skin luminosity can be evaluated using photodetection methods known in the art for assessing the intensity of reflected light, for example, using methods described in Jeudy et al. (2015) Measurement of Skin Radiance. In: Humbert et al., Agache’s Measuring the Skin. ISBN: 978-3-319-26594-0, the disclosure of which is incorporated herein by reference.

As used herein, the term “capric/caprylic triglyceride” and its abbreviation, “OCT,” refers to a mixture of esters including caprylic and capric fatty acids covalently bound to a glycerin backbone. Capric/caprylic triglyceride may include from about 50% to about 70% caprylic acid and from about 30% to about 50% caprid acid. Using International Union of Pure and Applied Chemistry (IUPAC) nomenclature, capric/caprylic triglyceride is also referred to as decanoic acid esterified with 1 ,2,3- propanetriol octanoate.

As used herein, the term “cosmetic composition” refers to a composition that is intended to be applied to a user’s skin (e.g., the skin of a male or female human subject) so as to regulate a condition of the skin and/or to improve the outward appearance of the skin. Cosmetic compositions of the disclosure may further include one or more carriers, diluents, or excipients, such as a carrier, diluent, or excipient described herein.

As used herein, the terms “decrease” and “reduce” refer to reduction in the level of a property of interest by a statistically significant or visually apparent amount as compared to a reference level of the property. The reference level may be, for example, a level observed in the absence of using a cosmetic composition of the disclosure. In some embodiments of the disclosure, the “decrease” or “reduction” observed in connection with a particular property (such as inflammation, redness, or dehydration, among other epidermal properties described herein) is, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. A “decrease” or “reduction” may also refer to a reduction in a particular property that is readily apparent from a visual inspection of a subject. For example, a “decrease” or “reduction” in skin redness refers to a reduction in skin redness that can be readily observed by the user of a cosmetic composition of the disclosure upon visual inspection of the affected area of the skin. A “decrease” or “reduction” in visually assessed properties, such as skin redness, may be observed by examining photographic images of a subject before and after administration of a cosmetic composition of the disclosure to the affected area of the skin.

As used herein, the term “erythema” refers to any abnormal redness of the skin. Erythema may be caused by dilation and irritation of the superficial capillaries wherein the augmented flow of blood through them imparts a reddish hue to the skin. The redness of the skin may be caused, for example, by acne, rosacea, sunburn, atopic dermatitis, contact dermatitis, an allergic reaction to a medication, or as a result of an underlying infection, In some embodiments, the methods described herein are used to treat erythema multiforme, erythema ab igne, erythema chronicum migrans, erythema induratum, erythema infectiosum, erythema marginatum, erythema migrans, erythema nodosum, erythema toxicum, erythema elevatum diutinum, erythema gyratum repens, keratolytic winter erythema, or palmar erythema.

As used herein, the terms “increase,” “enhance,” and “improve” refer to an increase in a property of interest by a statistically significant or visually apparent amount as compared to a reference level of the property. The reference level may be, for example, a level observed in the absence of using a cosmetic composition of the disclosure. In some embodiments of the disclosure, the “increase,” “improvement,” or “enhancement” observed in connection with a particular property (such as skin barrier function or skin hydration, among other beneficial epidermal properties described herein) is, for example, an increase by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. An “increase,” “improvement,” or “enhancement” may also refer to an improvement in a particular property that is readily apparent from a visual inspection of a subject. For example, an “increase,” “improvement,” or “enhancement” in skin hydration refers to an increase in skin hydration that can be readily observed by the user of a cosmetic composition of the disclosure upon visual inspection of the affected area of the skin. An “increase,” “improvement,” or “enhancement” in visually assessable properties, such as skin hydration, may be observed by examining photographic images of a subject before and after administration of a cosmetic composition of the disclosure to the affected area of the skin.

As used herein, the term “inflammation” refers to an immunological response involving increased blood flow and/or blood vessel permeability to the site of inflammation, leukocyte migration and/or exudation at the site of inflammation, and/or release of chemical mediators, including cytokines (TNF-a, interleukins, etc.). Signs of inflammation may include redness, heat, swelling, pain, and/or stiffness.

As used herein, the term “irritation” refers to a physical condition in which part of the body becomes itchy, red, dry, scaley, swollen, or painful, or develops a rash as a result, e.g., of exposure to an injury, infection, or irritant.

As used herein, the term “Montanov® 68” refers to a non-ionic, liquid crystal emulsifier comprising cetearyl alcohol and cetearyl glucoside which has a melting point of between 61 .0 °C and 65.0 °C and a pH of between 5.5 and 7.5.

As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic compound or prophylactic compound to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the mammal.

As used herein, the term “squalane” refers to linear hydrocarbon obtainable from squalene by way of a hydrogenation reaction. Squalane has the following chemical structural formula:

As used herein, the term “skin barrier function” refers to the ability of the skin of a subject (e.g., a male or female human subject) to retain hydration and prevent excessive transepidermal water loss. Skin barrier function can be assessed, for example, by monitoring transepidermal water loss in a subject using a transepidermal water loss assay described herein, such as the transepidermal water loss assay outlined in Example 1 , below.

As used herein, the term “subject” refers to an animal, such as a mammal (e.g., a male or female human), to which a cosmetic composition described herein may be administered.

As used herein in the context of administration of a composition to a subject, the term “topical” refers to administration of the cosmetic composition to any skin or exposed mucosal surface. “Skin” includes any exposed epidermal region of the subject’s body, including, without limitation, the skin of the subject’s scalp, face (including lips), hands, nails, legs, neck, abdominal area, eyes, nose, and chest.

As used herein, the term “transepidermal water loss” refers to the amount of water that is lost to evaporation over the skin barrier provided by the epidermis. A variety of methods may be used to measure transepidermal water loss. Examples of such methods are provided in, e.g., Antonov et al., Curr. Probl. Dermatol. 49:61 -70 (2016), the disclosure of which is incorporated herein by reference.

As used herein, “treatment” and “treating” refer to an approach for obtaining beneficial or desired results, e.g., therapeutic results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. Those in need of treatment include those already with the disease or condition, those prone to or at risk of developing the disease or condition, and those in which the disease or condition is to be prevented.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions (e.g., pharmaceutical compositions and cosmetic compositions) that contain a human milk oligosaccharide (HMO). The compositions of the disclosure may further include squalane. In some embodiments, the compositions of the disclosure may include carboxymethyl cellulose (CMC). The compositions may also have one or more additional components, such as lecithin and/or glyceride. Additionally, the disclosure provides methods of using the compositions described herein to reduce inflammation in the skin of a subject (e.g., a male or female human subject), as well as methods of using the compositions to treat or prevent a disease or condition in a subject. Furthermore, the disclosure provides methods of using the compositions described herein for achieving one or more improvements to the skin of the subject. For example, the method may be used to achieve an anti-aging effect, reduce fine lines or wrinkles, improve skin barrier function, and/or increase the firmness or elasticity of the skin.

The present disclosure is based, in part, on the discovery that compositions containing an HMO can reduce inflammation or redness of the skin, reduce hyperproliferation of keratinocytes, and reduce transepidermal water loss, thereby improving hydration of a subject’s skin. Exemplary compositions of the disclosure, as well as methods of using the same to achieve these beneficial outcomes, are described in further detail below.

Human Milk Oligosaccharides

The compositions described herein in an HMO. The HMO may be without limitation L-fucose, 2’- fucosyllactose (2’-FL), lacto-N-neotetraose (LNnT), 3-fucosyllactose (3’-FL), difucosyllactose (DFL), lacto- N-tetraose (LNT), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N- difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto- N-hexaose (F-LNH) I, F-LNH II, difucosyllacto-N-hexaose (DFLNH) I, DFLNH II, difucosyllacto-N- neohexaose (DFLNnH), difucosyl-para-lacto-N-hexaose (DF-para-LNH), difucosyl-para-lacto-N- neohexaose (DF-para-LNnH), trifucosyllacto-N-hexaose (TF-LNH), 3’-siallylactose (3’-SL), 6’-siallylactose (6’-SL), sialyllacto-N-tetraose (LST) a, LST b, LST c, disialyllacto-N-tetraose (DS-LNT), fucosy l-sialyllacto- N-tetraose (F-LST) a, F-LST b, fucosyl-sialyllacto-N-hexaose (FS-LNH), fucosyl-sialyllacto-N-neohexaose (FS-LNnH) I, fucosyl-disialyllacto-N-hexaose (FDS-LNH) II, or a combination thereof. In some embodiments, the HMO is 2’-FL. In some embodiments, the HMO is L-fucose.

In some embodiments, the composition includes one or more (e.g., 2, 3, 4, 5, or more) HMOs. For example, the composition may include both 2’-FL and LNnT.

The concentration of the HMO may be between about 1 mg/mL and about 100 mg/mL (e.g., between 1 mg/mL and 80 mg/mL, 1 mg/mL and 60 mg/mL, 1 mg/mL and 40 mg/mL, 1 mg/mL and 20 mg/mL, 20 mg/mL and 100 mg/mL, 40 mg/mL and 100 mg/mL, 60 mg/mL and 100 mg/mL, or 80 mg/mL and 100 mg/mL). For example, the composition may have an HMO concentration of between about 10 mg/mL and about 50 mg/mL (e.g., between 10 mg/mL and 40 mg/mL, 10 mg/mL and 30 mg/mL, 10 mg/mL and 20 mg/mL, 20 mg/mL and 50 mg/mL, 30 mg/mL and 50 mg/mL, or 40 mg/mL and 50 mg/mL). In some embodiments, the composition has a concentration of the HMO of about 10 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 25 mg/mL. In some embodiments, the composition has a concentration of the HMO of about 50 mg/mL.

Heterologous expression of HMOs from host cells

In some embodiments, the HMO may be produced using a host cell (e.g., a yeast cell) that is modified to express one or more enzymes of the HMO biosynthetic pathway and is thus capable of producing an HMO or a precursor of an HMO. In some embodiments, for example, cells of the disclosure (e.g., yeast cells) may naturally express some of the enzymes of the biosynthetic pathway for a given HMO. Such cells may be modified to express the remaining enzymes of the biosynthetic pathway. In some embodiments, for instance, a cell (e.g., a yeast cell) may naturally express many of the enzymes of the biosynthetic pathway of a desired HMO, and the cells may be modified so as to express the remaining enzymes of the biosynthetic pathway for the desired HMO by providing the cells with one or more heterologous nucleic acid molecules that, together, encode the remaining enzymes of the biosynthetic pathway.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing LNnT, including a p-1 ,3-N- acetylglucosaminyltransferase (LgtA), a p-1 ,4-galactosyltransferase (LgtB), and a UDP-N- acetylglucosamine diphosphorylase. Exemplary LgtA and LgtB enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 2’-FL, including a lactose permease, a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an a-1 ,2-fucosyltransferase, and a fucosidase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3-fucosyllactose, including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an a-1 ,3-fucosyltransferase, and a fucosidase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing lacto-N-tetraose, including a p-1 ,3-N-acetylglucosaminyltransferase, a p-1 ,3-galactosyltransferase, and a UDP-N-acetylglucosamine diphosphorylase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 3’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP-N- acetylglucosamine diphosphorylase, and a CMP-N-acetylneuraminate-p-galactosamide-a-2,3- sialyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing 6’-sialyllactose, including a CMP-Neu5Ac synthetase, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a UDP-N- acetylglucosamine diphosphorylase, and a p-galactoside-a-2,6-sialyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, cells of the disclosure are provided with heterologous nucleic acid molecules that encode one or more enzymes of a pathway for synthesizing difucosyllactose, including a GDP-mannose 4,6-dehydratase, a GDP-L-fucose synthase, an a-1 ,2-fucosyltransferase, and an a-1 ,3- fucosyltransferase. Exemplary enzymes useful in conjunction with the compositions and methods of the disclosure are described in the sections that follow.

In some embodiments, the cells of the disclosure express an LgtA polypeptide. The LgtA polypeptides of the disclosure can be used to produce one or more of a variety of HMOs, including, without limitation, L-fucose, LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, and FDS-LNH II.

In some embodiments, the cells of the disclosure express a LgtB polypeptide. In some embodiments, the cells of the disclosure express a protein that transports lactose into the cell. In some embodiments, the cells of the disclosure express a GDP-mannose 4,6-dehydratase. In some embodiments, the cells of the disclosure express a GDP-L-fucose synthase. In some embodiments, the cells of the disclosure express an a-1 ,2-fucosyltransferase polypeptide.

Host cells capable of producing exemplary HMOs and their precursors

In some embodiments, the host cells of the disclosure are capable of producing one or more HMOs (e.g., LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, 6’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS- LNH II) and their precursors. The sections that follow describe host cells that are capable of producing exemplary HMOs, as well as the biosynthetic pathways that are involved in the production of each exemplary HMO.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the UDP-glucose HMO precursor. The activated sugar UDP-glucose is composed of a pyrophosphate group, the pentose sugar ribose, glucose, and the nucleobase uracil. UDP-glucose is natively produced by yeast cells, and its production levels can be increased with overexpression of, for example, phosphoglucomutase-2 (PGM2) or UTP glucose-1 -phosphate uridylyltransferase (UGP1 ).

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the UDP-galactose HMO precursor. The activated sugar UDP-galactose is composed of a pyrophosphate group, the pentose sugar ribose, galactose, and the nucleobase uracil. UDP-galactose is natively produced by yeast cells, and its production levels can be increased with overexpression of, for example, UDP-glucose-4-epimerase (GAL10).

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the UDP-N-acetylglucosamine HMO precursor. The activated sugar UDP-N-acetylglucosamine consists of a pyrophosphate group, the pentose sugar ribose, N-acetylglucosamine, and the nucleobase uracil. UDP-N-acetylglucosamine is natively produced by yeast cells, and its production levels can be increased with expression of, for example, UDP-N-acetylglucosamine-diphosphorylase, or overexpression of, for example, glucosamine 6-phosphate N-acetyltransferase (GNA1 ) or phosphoacetylglucosamine mutase (PCM1 ).

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the GDP-fucose HMO precursor. The activated sugar GDP-fucose consists of a pyrophosphate group, the pentose sugar ribose, fucose, and the nucleobase guanine. GDP-fucose is not natively produced by yeast cells, and its production can be enabled with the introduction of, for example, GDP-mannose 4,6- dehydratase, e.g., from Escherichia coli, and GDP-L-fucose synthase, e.g., from Arabidopsis thaliana.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the CMP-sialic acid HMO precursor. The activated sugar CMP-sialic acid consists of a pyrophosphate group, the pentose sugar ribose, sialic acid, and the nucleobase cytosine. CMP-sialic acid is not natively produced by yeast cells, and its production can be enabled with the introduction of, for example, CMP- Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, and UDP- N-acetylglucosamine 2-epimerase, e.g., from C. jejuni.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing 2’-FL. In addition to one or more heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include one or more heterologous nucleic acids encoding one or more of GDP-mannose 4,6-dehydratase, e.g., from Escherichia coli, GDP-L-fucose synthase, e.g., from Arabidopsis thaliana, a-1 ,2-fucosyltransferase, e.g., from Helicobacter pylori, and a fucosidase, e.g., an a- 1 ,3-fucosidase. In some embodiments, the fucosyltransferase is from Candidata moranbacterium or Pseudoalteromonas haloplanktis.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose, e.g., a GDP-mannose 4,6-dehydratase. In some embodiments, the GDP-mannose 4,6-dehydratase is from Escherichia coli. Other suitable GDP-mannose 4,6-dehydratase sources include, for example and without limitation, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Mus musculus, Drosophila melanogaster, Sinorhizobium fredii HH103, Sinorhizobium frec//7 NGR234, Planctomycetes bacterium RBG_13_63_9, Silicibacter sp. TrichCH4B, Pandoraea vervacti, Bradyrhizobium sp. YR681 , Epulopiscium sp. SCG-B11 WGA-EpuloA1 , Caenorhabditis briggsae, Candidates Curtissbacteria bacterium RIFCSPLOWO2_12_FULL_38_9, Pseudomonas sp. EpS/L25, Clostridium sp. KLE 1755, Nitrospira sp. SG-bin2, Cricetulus griseus, Arthrobacter siccitolerans, and Paraburkholderia piptadeniae. In some embodiments, the GDP-mannose dehydratase is from Caenorhabditis briggsae or Escherichia coli.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-4-dehydro-6- deoxy-D-mannose to GDP-L-fucose, e.g., a GDP-L-fucose synthase. In some embodiments, the GDP-L- fucose synthase is from Arabidopsis thaliana. Other suitable GDP-L-fucose synthase sources include, for example and without limitation, Mus musculus, Escherichia coli K-12, Homo sapiens, Marinobacter salaries, Sinorhizobium fredii NGR234, Oryza sativa Japonica Group, Micavibrio aeruginosavorus ARL- 13, Citrobacter sp. 86, Pongo abelii, Caenorhabditis elegans, Candidates Staskawiczbacteria bacteriem RIFCSPHIGHO2_01_FULL_41_41 , Drosophila melanogaster, Azorhizobiem caelinodans ORS 571 , Candidates Nitrospira nitrif leans, Mycobacteriem elephantis, Elesimicrobia bacteriem RBG_16_66_12, Vibrio sp. JCM 19231 , Planktothrix serta PCC 8927, Thermodeselfovibrio sp.

RBG_19FT_COMBO_42_12, Anaerovibrio sp. JC8, Dictyosteliem discoideem, and Criceteles grisees.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-L-fucose and lactose to 2’-FL, e.g., an a-1 ,2-fucosyltransferase. In some embodiments, the a-1 ,2-fucosyltransferase is from Helicobacter pylori. In some embodiments, the fucosyltransferase is from Candidata moranbacteriem or Pseedoalteromonas haloplanktis ANT/505. Other suitable a-1 ,2-fucosyltransferase sources include, for example and without limitation, Escherichia coli, Ses scrota, Homo sapiens, Chlorocebes sabaees, Pan troglodytes, Macaca melatta, Oryctolages ceniceles, Pongo pygmaees, Mes mesceles, Rattes norvegices, Caenorhabditis elegans, Hylobates lar, Bos taeres, Hylobates agilis, Eelemer felves, and Helicobacter hepatices ATCC 51449. In some embodiments, the source of the a- 1 ,2-fucosyltransferase is Pseedoalteromonas haloplanktis ANT/505, Candidates moranbacteria bacterium, Acetobacter sp. CAG:267, Bacteroides velgates, Selferovem lithotrophicem, Thermosynechococces elongates BP-1 , Geobacter eraniiredecens Rf4, Bacteroides fragilis str. S23L17, Chromobacteriem vaccinii, Herbaspirillem sp. YR522, or Helicobacter bills ATCC 43879.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of difucosyllactose to 2’- FL and fucose, e.g., an a1 -3,4-fucosidase. Suitable a1 -3,4-fucosidase sources include, for example and without limitation, Bacteroides thetaiotaomicron, Bifidobacteriem bifidem, Bifidobacteriem longem, Bifidobacteriem longem sebsp. infantis, Clostridiem perfringens, Lactobacilles casei, Paenibacilles thiaminolytices, Pseedomonas petida, Thermotoga maritima, Arabidopsis thaliana, and Rattes norvegices.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing 3-fucosyllactose. In addition to one or more heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include one or more heterologous nucleic acids encoding one or more of GDP-mannose 4,6-dehydratase, e.g., from Escherichia coli, GDP-L-fucose synthase, e.g., from Arabidopsis thaliana, a-1 ,3-fucosyltransferase, e.g., from Helicobacter pylori, and a fucosidase, e.g., an a-1 ,2-fucosidase.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-L-fucose and lactose to 3-fucosyllactose, e.g., an a-1 ,3-fucosyltransferase. In some embodiments, the a-1 ,3- fucosyltransferase is from Helicobacter pylori. Other suitable a-1 ,3-fucosyltransferase sources include, for example and without limitation, Homo sapiens, Escherichia coli, Sus scrota, Chlorocebus sabaeus, Pan troglodytes, Macaca mulatta, Oryctolagus cuniculus, Pongo pygmaeus, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Hylobates lar, Bos taurus, Hylobates agilis, Eulemur fulvus, Helicobacter hepaticus ATCC 51449, Akkermansia muciniphila, Bacteroides fragilis, and Zea mays.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing lacto-N-tetraose. In addition to one or more heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include one or more heterologous nucleic acids encoding one or more of p-1 ,3-/V-acetylglucosaminyltransferase, e.g., from Neisseria meningitidis, p-1 , 3- galactosyltransferase, e.g., from Escherichia coli, and UDP-N-acetylglucosamine-diphosphorylase, e.g., from E. coli.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-N-acetyl-alpha- D-glucosamine and lactose to lacto-N-triose II and UDP, e.g., a p-1 ,3-/V-acetylglucosaminyltransferase. In some embodiments, the p-1 ,3-/V-acetylglucosaminyltransferase is from Neisseria meningitidis. Other suitable p-1 ,3-/V-acetylglucosaminyltransferase sources include, for example and without limitation, Arabidopsis thaliana, Streptococcus dysgalactiae subsp. equisimilis, Escherichia coli, e.g., Escherichia coli K-12, Pseudomonas aeruginosa PAO1 , Homo sapiens, Mus musculus, Mycobacterium smegmatis str. MC2 155, Dictyostelium discoideum, Komagataeibacter hansenii, Aspergillus nidulans FGSC A4, Schizosaccharomyces pombe 972h-, Neurospora crassa OR74A, Aspergillus fumigatus Af293, Ustilago maydis 521 , Bacillus subtilis subsp. subtilis str. 168, Rattus norvegicus, Listeria monocytogenes EGD-e, Bradyrhizobium japonicum, Nostoc sp. PCC 7120, Haloferax volcanii DS2, Caulobacter crescentus CB15, Mycobacterium avium subsp. silvaticum, Oenococcus oeni, Neisseria gonorrhoeae, Propionibacterium freudenreichii subsp. shermanii, Escherichia coli 0157:H7, Aggregatibacter actinomycetemcomitans, Bradyrhizobium diazoefficiens USDA 1 10, Francisella tularensis subsp. novicida U112, Komagataeibacter xylinus, Haemophilus influenzae Rd KW20, Fusobacterium nucleate m subsp. nucleatum ATCC 25586, Bacillus phage SPbeta, Coccidioides posadasii, Populus tremula x Populus alba, Rhizopus microsporus var. oligosporus, Streptococcus parasanguinis, Shigella flexneri, Caenorhabditis elegans, Hordeum vulgare, Synechocystis sp. PCC 6803 substr. Kazusa, Streptococcus agalactiae, Plasmopara viticola, Staphylococcus epidermidis RP62A, Shigella phage Sf 11, Plasmid pWQ799, Fusarium graminearum, Sinorhizobium meliloti 1021 , Physcomitrella patens, Sphingomonas sp. S88, Streptomyces hygroscopicus subsp. jinggangensis 5008, Drosophila melanogaster, Phytophthora infestans, Staphylococcus aureus subsp. aureus Mu50, Penicillium chrysogenum, and Tribolium castaneum.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-galactose and lacto-N-triose II to lacto-N-tetraose and UDP, e.g., a -1 ,3-galactosyltransferase. In some embodiments, the p-1 ,3-galactosyltransferase is from Escherichia coli. Other suitable p-1 ,3-galactosyltransferase sources include, for example and without limitation, Arabidopsis thaliana, Streptococcus dysgalactiae subsp. equisimilis, Pseudomonas aeruginosa PAO1 , Homo sapiens, Mus muse ulus, Mycobacterium smegmatis str. MC2 155, Dictyostelium discoideum, Komagataeibacter hansenii, Aspergillus nidulans FGSC A4, Schizosaccharomyces pombe 972h-, Neurospora crassa OR74A, Aspergillus fumigatus Af293, Ustilago maydis 521 , Bacillus subtilis subsp. subtilis str. 168, Rattus norvegicus, Neisseria meningitidis, Listeria monocytogenes EGD-e, Bradyrhizobium japonicum, Nostoc sp. PCC 7120, Haloferax volcanii DS2, Caulobacter crescentus CB15, Mycobacterium avium subsp. silvaticum, Oenococcus oeni, Neisseria gonorrhoeae, Propionibacterium freudenreichii subsp. shermanii, Aggregatibacter actinomycetemcomitans, Bradyrhizobium diazoefficiens USDA 110, Francisella tularensis subsp. novicida U112, Komagataeibacter xylinus, Haemophilus influenzae Rd KW20, Fusobacterium nucleatum subsp. nucleatum ATCC 25586, Bacillus phage SPbeta, Coccidioides posadasii, Populus tremula x Populus alba, Rhizopus microsporus var. oligosporus, Streptococcus parasanguinis, Shigella flexneri, Caenorhabditis elegans, Hordeum vulgare, Synechocystis sp. PCC 6803 substr. Kazusa, Streptococcus agalactiae, Plasmopara viticola, Staphylococcus epidermidis RP62A, Shigella phage Sf II, Plasmid pWQ799, Fusarium graminearum, Sinorhizobium meliloti 1021 , Physcomitrella patens, Sphingomonas sp. S88, Streptomyces hygroscopicus subsp. jinggangensis 5008, Drosophila melanogaster, Phytophthora infestans, Staphylococcus aureus subsp. aureus Mu50, Penicillium chrysogenum, and Tribolium castaneum.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N-acetyl-a-D- glucosamine 1 -phosphate to UDP-N-acetyl-a-D-glucosamine, e.g., a UDP-N-acetylglucosamine- diphosphorylase. In some embodiments, the UDP-N-acetylglucosamine-diphosphorylase is from Escherichia coli.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing lacto-N-neotetraose. In addition to one or more heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include one or more heterologous nucleic acids encoding one or more of p-1 ,3-/V-acetylglucosaminyltransferase, e.g., from Neisseria meningitidis, p-1 , 4- galactosyltransferase, e.g., from N. meningitidis, and UDP-N-acetylglucosamine-diphosphorylase, e.g., from E. coli.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-galactose and lacto-N-triose II to lacto N-neotetraose and UDP, e.g., a p-1 ,4-galactosyltransferase. In some embodiments, the p-1 ,4-galactosyltransferase is from Neisseria meningitidis. Other suitable p-1 ,4- galactosyltransferase sources include, for example and without limitation, Homo sapiens, Neisseria gonorrhoeae, Haemophilus influenzae, Acanthamoeba polyphaga mimivirus, Haemophilus influenzae Rd KW20, Haemophilus ducreyi 35000HP, Moraxella catarrhalis, [Haemophilus] ducreyi, Aeromonas salmonicida subsp. salmonicida A449, and Helicobacter pylori 26695.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing 3’-sialyllactose. In addition to heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cells may further include heterologous nucleic acids encoding CMP-Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, UDP-N- acetylglucosamine 2-epimerase, e.g., from C. jejuni, UDP-N-acetylglucosamine-diphosphorylase, e.g., from E. coli, and CMP-N-acetylneuraminate-p-galactosamide-a-2,3-sialyltransferase, e.g., from N. meningitides MC58.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-N-acetyl-a-D- glucosamine to N-acetyl-mannosamine and UDP, e.g., a UDP-N-acetylglucosamine 2-epimerase. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase is from Campylobacter jejuni. Other suitable UDP-N-acetylglucosamine 2-epimerase sources include, for example and without limitation, Homo sapiens, Rattus norvegicus, Mus musculus, Dictyostelium discoideum, Plesiomonas shigelloides, Bacillus subtilis subsp. subtilis str. 168, Bacteroides fragilis, Geobacillus kaustophilus HTA426, Synechococcus sp. CC931 1 , Sphingopyxis alaskensis RB2256, Synechococcus sp. RS9916, Moorella thermoacetica ATCC 39073, Psychrobacter sp. 1501 (2011 ), Zunongwangia profunda SM-A87, Thiomicrospira crunogena XCL-2, Polaribacter sp. MED152, Vibrio campbellii ATCC BAA-1 1 16, Thiomonas arsenitoxydans, Nitrobacter winogradskyi Nb-255, Raphidiopsis brookii D9, Thermoanaerobacter italicus Ab9, Roseobacter litoralis Och 149, Halothiobacillus neapolitanus c2, Halothiobacillus neapolitanus c2, Bacteroides vulgatus ATCC 8482, Zunongwangia profunda SM-A87, Moorella thermoacetica ATCC 39073, Paenibacillus polymyxa E681 , Desulfatibacillum alkenivorans AK- 01 , Magnetospirillum magneticum AMB-1 , Thermoanaerobacter italicus Ab9, Paenibacillus polymyxa E681 , Prochlorococcus marinus str. MIT 921 1 , Subdoligranulum variabile DSM 15176, Kordia algicida OT-1 , Bizionia argentinensis JUB59, Tannerella forsythia 92 A2, Thiomonas arsenitoxydans, Synechococcus sp. BL107, Escherichia coli, Vibrio campbellii ATCC BAA-1116, Rhodopseudomonas palustris HaA2, Roseobacter litoralis Och 149, Synechococcus sp. CC931 1 , Subdoligranulum variabile DSM 15176, Bizionia argentinensis JU B59, Selenomonas sp. oral taxon 149 str. 67H29BP, Bacteroides vulgatus ATCC 8482, Kordia algicida OT-1 , Desulfatibacillum alkenivorans AK-01 , Thermodesulfovibrio yellowstonii DSM 11347, Desulfovibrio aespoeensis Aspo-2, Synechococcus sp. BL107, and Desulfovibrio aespoeensis Aspo-2.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N-acetyl- mannosamine and phosphoenolpyruvate to N-acetylneuraminate, e.g., a sialic acid synthase. In some embodiments, the sialic acid synthase is from Campylobacter jejuni. Other suitable sialic acid synthase sources include, for example and without limitation, Homo sapiens, groundwater metagenome, Prochlorococcus marinus str. MIT 9211 , Rhodospirillum centenum SW, Rhodobacter capsulatus SB 1003, Aminomonas paucivorans DSM 12260, Ictalurus punctatus, Octadecabacter antarcticus 307, Octadecabacter arcticus 238, Butyrivibrio proteoclasticus B316, Neisseria meningitidis serogroup B., Idiomarina loihiensis L2TR, Butyrivibrio proteoclasticus B316, and Campylobacter jejuni.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N-acetylneuraminate and CTP to CMP-N-acetylneuraminate, e.g., a CMP-Neu5Ac synthetase. In some embodiments, the CMP-Neu5Ac synthetase is from Campylobacter jejuni. Other suitable CMP-Neu5Ac synthetase sources include, for example and without limitation, Neisseria meningitidis, Streptococcus agalactiae NEM316, Homo sapiens, Mus musculus, Bacteroides thetaiotaomicron, Pongo abelii, Danio rerio, Oncorhynchus mykiss, Bos taurus, Drosophila melanogaster, and Streptococcus suis BM407.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of CMP-N- acetylneuraminate and lactose to 3’-sially llactose and CMP, e.g., a CMP-N-acetylneuraminate-p- galactosamide-a-2,3-sialyltransferase. In some embodiments, the CMP-N-acetylneuraminate-p- galactosamide-a-2,3-sialyltransferase is from N. meningitides MC58. Other suitable CMP-N- acetylneuraminate-p-galactosamide-a-2,3-sialyltransferase sources include, for example and without limitation, Homo sapiens, Neisseria meningitidis alpha14, Pasteurella multocida subsp. multocida str. Pm70, Pasteurella multocida, and Rattus norvegicus.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing 6’-sialyllactose. In addition to one or more heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include one or more heterologous nucleic acids encoding one or more of CMP-Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, UDP-N-acetylglucosamine 2-epimerase, e.g., from C. jejuni, UDP-N- acetylglucosamine-diphosphorylase, e.g., from E. coll, and p-galactoside a-2,6-sialyltransferase, e.g., from Photobacterium sp. JT-ISH-224.

In some embodiments, the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of CMP-N- acetylneuraminate and lactose to 3’-sialyllactose and CMP, e.g., a p-galactoside-a-2,6-sialyltransferase. In some embodiments, the p-galactoside-a-2,6-sialyltransferase is from Photobacterium sp. JT-ISH-224. Other suitable p-galactoside-a-2,6-sialyltransferase sources include, for example and without limitation, Homo sapiens, Photobacterium damselae, Photobacterium leiognathi, and Photobacterium phosphoreum ANT-2200.

Exemplary host cell strains

In some embodiments, the host cell used to produce an HMO is a yeast cell, such as Saccharomyces cerevisiae. Saccharomyces cerevisiae strains suitable for genetic modification and cultivation to produce HMOs as disclosed herein include, but are not limited to, Baker's yeast, CBS 7959, CBS 7960, CBS 7961 , CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1 , CR-1 , SA-1 , M-26, Y-904, PE-2, PE-5, VR-1 , BR-1 , BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1 , CB-1 , NR-1 , BT-1 , CEN.PK, CEN.PK2, and AL-1 . In some embodiments, the host cell is a strain of Saccharomyces cerevisiae selected from the group consisting of PE-2, CAT-1 , VR-1 , BG-1 , CR-1 , and SA-1 . In certain aspects, the strain of Saccharomyces cerevisiae is PE-2. In certain embodiments, the strain of Saccharomyces cerevisiae is CAT-1 . In some aspects, the strain of Saccharomyces cerevisiae is BG-1 .

In some embodiments, the host cell is Saccharomyces cerevisiae, and in addition to heterologous nucleic acids encoding one or more of the aforementioned enzymes, the host cell may further include a heterologous nucleic acid encoding a lactose transporter. In some embodiments, the lactose transporter is a lactose permease, e.g., LAC12 from Kluyveromyces lactis (SEQ ID NO: 14). In some embodiments, the lactose permease is from Neurospora crassa, e.g., Cdt2. In some embodiments, the lactose permease is from Neofusicoccum parvum, e.g., Neofusicoccum parvum UCRNP2 (1287680). Other suitable lactose permease sources include, for example and without limitation, Scheffersomyces stipitis, Aspergillus lentulus, Emericella nidulans, Dacryopinax primogenitus, Microdochium bolleyi, Beauveria bassiana, Metarhizium robertsii, Phialocephala, Botryosphaeria parva, Moniliophthora roreri, Cordyceps fumosorosea, Diplodia seriata, Hypocrea jecorina, and Kluyveromyces marxianus.

Methods of producing HMOs

In some embodiments, the host cells of the disclosure are cultured under conditions suitable for the production of a desired HMO. The culturing can be performed in a suitable culture medium in a suitable container, such as a cell culture plate, a flask, or a fermentor. Any suitable fermentor may be used, including, but not limited to, a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof. In particular embodiments utilizing Saccharomyces cerevisiae as the host cell, strains can be grown in a fermentor as described in detail by Kosaric et al., in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co. KDaA, Weinheim, Germany. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Materials and methods for the maintenance and growth of cell cultures are well known to those skilled in the art of microbiology or fermentation science (see, for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration should be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process.

In some embodiments, the culturing is carried out for a period of time sufficient for the transformed population to undergo a plurality of doublings until a desired cell density is reached. In some embodiments, the culturing is carried out for a period of time sufficient for the host cell population to reach a cell density (GD600) of between 0.01 and 400 in the fermentation vessel or container in which the culturing is being carried out. The culturing can be carried out until the cell density is, for example, between 0.1 and 14, between 0.22 and 33, between 0.53 and 76, between 1.2 and 170, or between 2.8 and 400. In terms of upper limits, the culturing can be carried until the cell density is no more than 400, e.g., no more than 170, no more than 76, no more than 33, no more than 14, no more than 6.3, no more than 2.8, no more than 1 .2, no more than 0.53, or no more than 0.23. In terms of lower limits, the culturing can be carried out until the cell density is greater than 0.1 , e.g., greater than 0.23, greater than 0.53, greater than 1 .2, greater than 2.8, greater than 6.3, greater than 14, greater than 33, greater than 76, or greater than 170. Higher cell densities, e.g., greater than 400, and lower cell densities, e.g., less than 0.1 , are also contemplated.

In other embodiments, the culturing is carried for a period of time, for example, between 12 hours and 92 hours, e.g., between 12 hours and 60 hours, between 20 hours and 68 hours, between 28 hours and 76 hours, between 36 hours and 84 hours, or between 44 hours and 92 hours. In some embodiments, the culturing is carried out for a period of time, for example, between 5 days and 20 days, e.g., between 5 days and 14 days, between 6.5 days and 15.5 days, between 8 days and 17 days, between 9.5 days and 18.5 days, or between 1 1 days and 20 days. In terms of upper limits, the culturing can be carried out for less than 20 days, e.g., less than 18.5 days, less than 17 days, less than 15.5 days, less than 14 days, less than 12.5 day, less than 11 days, less than 9.5 days, less than 8 days, less than 6.5 days, less than 5 day, less than 92 hours, less than 84 hours, less than 76 hours, less than 68 hours, less than 60 hours, less than 52 hours, less than 44 hours, less than 36 hours, less than 28 hours, or less than 20 hours. In terms of lower limits, the culturing can be carries out for greater than 12 hours, e.g., greater than 20 hours, greater than 28 hours, greater than 36 hours, greater than 44 hours, greater than 52 hours, greater than 60 hours, greater than 68 hours, greater than 76 hours, greater than 84 hours, greater than 92 hours, greater than 5 days, greater than 6.5 days, greater than 8 days, greater than 9.5 days, greater than 1 1 days, greater than 12.5 days, greater than 14 days, greater than 15.5 days, greater than 17 days, or greater than 18.5 days. Longer culturing times, e.g., greater than 20 days, and shorter culturing times, e.g., less than 5 hours, are also contemplated.

In certain embodiments, the production of the one or more HMOs by the population of host cells is inducible by an inducing compound. Such host cells can be manipulated with ease in the absence of the inducing compound. The inducing compound is then added to induce the production of one or more HMOs by the host cells. In other embodiments, production of the one or more HMOs by the host cells is inducible by changing culture conditions, such as, for example, the growth temperature, media constituents, and the like.

In certain embodiments, an inducing agent is added during a production stage to activate a promoter or to relieve repression of a transcriptional regulator associated with a biosynthetic pathway to promote production of one or more HMOs. In certain embodiments, an inducing agent is added during a build stage to repress a promoter or to activate a transcriptional regulator associated with a biosynthetic pathway to repress the production of one or more HMOs, and an inducing agent is removed during the production stage to activate a promoter or to relieve repression of a transcriptional regulator to promote the production of one or more HMOs.

As discussed above, in some embodiments, the host cells may include a promoter that regulates the expression and/or stability of a heterologous nucleic acid described herein. Thus, in certain embodiments, the promoter can be used to control the timing of gene expression and/or stability of proteins.

In some embodiments, when fermentation of a host cell capable of producing a desired HMO is carried out in the presence of a small molecule, e.g., at least about 0.1% maltose or lysine, HMO production is substantially reduced or eliminated. When the small molecule is removed from the fermentation culture medium, HMO production is stimulated. Such a system enables the use of the presence or concentration of a selected small molecule in a fermentation medium as a switch for the production of an HMO. Controlling the timing of non-catabolic compound production so as to occur only when production is desired redirects the carbon flux during the non-production phase into cell maintenance and biomass. This more efficient use of carbon can greatly reduce the metabolic burden on the host cells, improve cell growth, increase the stability of the heterologous genes, reduce strain degeneration, and/or contribute to better overall health and viability of the cells.

In some embodiments, the fermentation method includes a two-step process that utilizes a small molecule as a switch to affect the “off” and “on” stages. In the first step, i.e., the “build” stage, wherein production of the compound is not desired, the host cells are grown in a growth or “build” medium including the small molecule in an amount sufficient to induce the expression of genes under the control of a responsive promoter, and the induced gene products act to negatively regulate production of the non- catabolic compound. In the second step, i.e., the “production” stage, the fermentation is carried out in a culture medium including a carbon source wherein the small molecule is absent or present in sufficiently low amounts such that the activity of a responsive promoter is reduced or inactive. As a result, the production of the desired non-catabolic compound by the host cells is stimulated.

In some embodiments, the culture medium is any culture medium in which a host cell (e.g., yeast cell) can subsist, i.e., maintain growth and viability. In some embodiments, the culture medium is an aqueous medium including assimilable carbon, nitrogen, and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients. In some embodiments, the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation media, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells, which convert the carbon source to a biomass.

In some embodiments, the method of producing one or more HMOs includes culturing host cells in separate build and production culture media. For example, the method can include culturing the host cells in a build stage, wherein the cells are cultured under non-producing conditions, e.g., non-inducing conditions, thereby producing an inoculum. The inoculum may then be transferred into a second fermentation medium under conditions suitable to induce production of one or more HMOs, e.g., inducing conditions. Steady state conditions may then be maintained in the second fermentation stage so as to produce a cell culture containing one or more desired HMOs.

In some embodiments, the culture medium includes sucrose and lactose. In some embodiments, the carbon sources in the culture medium consist essentially of sucrose and lactose. In some embodiments, the carbon sources in the culture medium consist of sucrose and lactose. In some embodiments, the mass ratio of the sucrose to the lactose is selected to influence, adjust, or control the relative production rates of HMO(s) produced by the yeast cells. Controlling the composition of the produced HMO(s) in this way can advantageously permit the increasing of desired products, the decreasing of undesired products, the targeting of a desired product ratio, and the simplification of downstream product separation processes.

The mass ratio of the sucrose to the lactose in the culture medium can be, for example, between 3 and 40, e.g., between 3 and 25.6, between 7.6 and 29.2, between 1 1.2 and 32.8, between 14.8 and 36.4, between 18.4 and 40, between 3 and 10, between 3 and 5, or between 3 and 4. In terms of upper limits, the mass ratio of the sucrose to the lactose can be less than 40, e.g., less than 36.4, less than 32.8, less than 29.2, less than 25.6, less than 22, less than 18.4, less than 14.8, less than 1 1 .2, less than 7.6, or less than 5. In terms of lower limits, the mass ratio of the sucrose to the lactose can be greater than 3, e.g., greater than 7.6, greater than 11.2, greater than 14.8, greater than 18.4, greater than 22, greater than 25.6, greater than 29.2, greater than 32.8, or greater than 36.4. Higher ratios, e.g., greater than 40, and lower ratios, e.g., less than 3, are also contemplated.

Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1 .0 g/L. In some embodiments, the addition of a nitrogen source to the culture medium beyond a certain concentration is not advantageous for the growth of the yeast. As a result, the concentration of the nitrogen sources in the culture medium can be less than about 20 g/L, e.g., less than about 10 g/L or less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culturing.

The effective culture medium can contain other compounds, such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in carbon, nitrogen or mineral sources in the effective medium or can be added specifically to the medium.

The culture medium can also contain a suitable phosphate source. Such phosphate sources include both inorganic and organic phosphate sources. Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate and mixtures thereof. Typically, the concentration of phosphate in the culture medium is greater than about 1 .0 g/L, e.g., greater than about 2.0 g/L or greater than about 5.0 g/L. In some embodiments, the addition of phosphate to the culture medium beyond certain concentrations is not advantageous for the growth of the yeast. Accordingly, the concentration of phosphate in the culture medium can be less than about 20 g/L, e.g., less than about 15 g/L or less than about 10 g/L.

A suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used. Typically, the concentration of magnesium in the culture medium is greater than about 0.5 g/L, e.g., greater than about 1 .0 g/L or greater than about 2.0 g/L. In some embodiments, the addition of magnesium to the culture medium beyond certain concetrations is not advantageous for the growth of the yeast. Accordingly, the concentration of magnesium in the culture medium can be less than about 10 g/L, e.g, less than about 5 g/L or less than about 3 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of a magnesium source during culturing.

In some embodiments, the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate. In such instance, the concentration of a chelating agent in the culture medium can be greater than about 0.2 g/L, e.g., greater than about 0.5 g/L or greater than about 1 g/L. In some embodiments, the addition of a chelating agent to the culture medium beyond certain concentrations is not advantageous for the growth of the yeast. Accordingly, the concentration of a chelating agent in the culture medium can be less than about 10 g/L, e.g., less than about 5 g/L or less than about 2 g/L.

The culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.

The culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride. Typically, the concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 mg/L, e.g., within the range of from about 20 mg/L to about 1000 mg/L or in the range of from about 50 mg/L to about 500 mg/L.

The culture medium can also include sodium chloride. Typically, the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, e.g., within the range of from about 1 g/L to about 4 g/L or in the range of from about 2 g/L to about 4 g/L.

In some embodiments, the culture medium can also include trace metals. Such trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Typically, the volume of such a trace metal solution added to the culture medium is greater than about 1 mL/L, e.g., greater than about 5 mL/L, and more preferably greater than about 10 mL/L. In some embodiments, the addition of a trace metals to the culture medium beyond certain concentrations is not advantageous for the growth of the host cells (e.g., yeast cells). Accordingly, the amount of such a trace metals solution added to the culture medium may desirably be less than about 100 mL/L, e.g., less than about 50 mL/L or less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.

The culture media can include other vitamins, such as pantothenate, biotin, calcium, inositol, pyridoxine-HCI, thiamine-HCI, and combinations thereof. Such vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. In some embodiments, the addition of vitamins to the culture medium beyond certain concentrations is not advantageous for the growth of the host cells (e.g., yeast cells).

The fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous, and semi-continuous. In some embodiments, the fermentation is carried out in fed-batch mode. In such a case, some of the components of the medium are depleted during culture, e.g., during the production stage of the fermentation. In some embodiments, the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and/or HMO production (e.g., HMO production) is supported for a period of time before additions are required. The preferred ranges of these components can be maintained throughout the culture by making additions as levels are depleted by culture. Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations. Alternatively, once a standard culture procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the culture. As will be recognized by those of ordinary skill in the art, the rate of consumption of nutrient increases during culture as the cell density of the medium increases. Moreover, to avoid introduction of foreign microorganisms into the culture medium, addition can be performed using aseptic addition methods, as are known in the art. In addition, a small amount of anti-foaming agent may be added during the culture.

The temperature of the culture medium can be any temperature suitable for growth of the host cells (e.g., yeast cells). For example, prior to inoculation of the culture medium with an inoculum, the culture medium can be brought to and maintained at a temperature in the range of from about 20 °C to about 45 °C, e.g., to a temperature in the range of from about 25 °C to about 40 °C, such as from about 28 °C to about 32 °C. For example, the culture medium can be brought to and maintained at a temperature of 25 °C, 25.5 °C, 26 °C, 26.5 °C, 27 °C, 27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C.

The pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases, when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium. In some embodiments, the pH is maintained at from about 3.0 to about 8.0, e.g., at from about 3.5 to about 7.0 or from about 4.0 to about 6.5.

Formulations and Routes of Administration

The compositions described herein may include one more excipients or carriers. The composition may include squalane, CMC, lecithin, and/ or glyceride.

The composition may include squalane. In some embodiments, the composition has a concentration of squalane of between about 1% (w/w) and about 60% (w/w) (e.g., about 1 % (w/w) and 50% (w/w)_a 1% (w/w) and 40% (w/w), 1% (w/w) and 30% (w/w), 1% (w/w) and 20% (w/w), 20% (w/w) and 60% (w/w), 30% (w/w) and 60% (w/w), 40% (w/w) and 60% (w/w), or 50% (w/w) and 60% (w/w)). For example, the squalane may be present in an amount of between about 20% (w/w) and about 50% (w/w) (e.g., between 20% (w/w) and 45% (w/w), 20% (w/w) and 40% (w/w), 20% (w/w) and 35% (w/w), 20% (w/w) and 30% (w/w), 20% (w/w) and 25% (w/w), 25% (w/w) and 50% (w/w), 30% (w/w) and 50% (w/w), 35% (w/w) and 50% (w/w), 40% (w/w) and 50% (w/w), or 45% (w/w) and 50% (w/w)). In some embodiments, the composition has a concentration of squalane of about 42% (w/w). In some embodiments, the composition has a concentration of squalane of between about 1 % (w/w) and about 20% (w/w) (e.g., 1 % (w/w), 2% (w/w), 3% (w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 1 1% (w/w), 12% (w/w), 13% (w/w), 14% (w/w), 15% (w/w), 16% (w/w), 17% (w/w), 18% (w/w), 19% (w/w), and 20% (w/w)). In some embodiments, the composition has a concentration of squalane of about 10% (w/w).

The composition may include CMC. The CMC may be present in a concentration of between about 0.5% (w/w) and about 5% (w/w) (e.g., between 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1 % (w/w), 1 % (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), or 4% (w/w) and 5% (w/w)). For example, the composition may have a concentration of CMC of between about 1 % (w/w) and about 3% (w/w) (e.g., between 1 % (w/w) and 2.5% (w/w), 1% (w/w) and 2% (w/w), 1 % (w/w) and 1.5% (w/w), 1.5% (w/w) and 3% (w/w), 2% (w/w) and 3% (w/w), or 2.5% (w/w) and 3% (w/w)). In some embodiments, the composition has a concentration of CMC of between about 2% (w/w).

The composition may include lecithin. In some embodiments, the composition has a concentration of lecithin of between about 0.01 % (w/w) and about 1% (w/w) (e.g., between 0.01 % (w/w) and 0.8% (w/w), 0.01 % (w/w) and 0.6% (w/w), 0.01% (w/w) and 0.4% (w/w), 0.01% (w/w) and 0.2% (w/w), 0.01% (w/w) and 0.05% (w/w), 0.05% (w/w) and 1 % (w/w), 0.2% (w/w) and 1% (w/w), 0.4% (w/w) and 1 % (w/w), 0.6% (w/w) and 1% (w/w), or 0.8% (w/w) and 1% (w/w)). For example, the lecithin may be present in the composition may be present in the amount of between about 0.05% (w/w) and about 0.5% (w/w) (e.g., between 0.05% (w/w) and 0.4% (w/w), 0.05% (w/w) and 0.3% (w/w), 0.05% (w/w) and 0.2% (w/w), 0.05% (w/w) and 0.1 % (w/w), 0.1 % (w/w) and 0.5% (w/w), 0.2% (w/w) and 0.5% (w/w), 0.3% (w/w) and 0.5% (w/w), or 0.4% (w/w) and 0.5% (w/w)). The composition may have a concentration of lecithin of about 0.1% (w/w).

The composition may include phenoxyethanol. In some embodiments, the composition has a concentration of phenoxyethanol of about between about 0.01% (w/w) and 5% (w/w) (e.g., between about 0.05% (w/w) and 5% (w/w), 0.1 % (w/w) and 5% (w/w), 0.5% (w/w) and 5% (w/w), 1% (w/w) and 5% (w/w), 2% (w/w) and 5% (w/w), 3% (w/w) and 5% (w/w), 4% (w/w) and 5% (w/w), 0.01% (w/w) and 4% (w/w), 0.01% (w/w) and 3% (w/w), 0.01% (w/w) and 2% (w/w), 0.01 % (w/w) and 1% (w/w), 0.01% (w/w) and 0.5% (w/w), 0.01% (w/w) and 0.1% (w/w), 0.1 % (w/w) and 1% (w/w), or 0.3% (w/w) and 0.7% (w/w)). For example, the composition may have a concentration of phenoxyethanol of about 0.5% (w/w).

The composition may include cetearyl glucoside. In some embodiments, the composition further comprises cetearyl alcohol and cetearyl glucoside. For example, the composition may include Montanov®. In some embodiments, the composition has a concentration of cetearyl alcohol and cetearyl glucoside of between about 0.5% (w/w) and about 10% (w/w) (e.g., between about 0.5% (w/w) and 9% (w/w), 0.5% (w/w) and 8% (w/w), 0.5% (w/w) and 7% (w/w), 0.5% (w/w) and 6% (w/w), 0.5% (w/w) and 5% (w/w), 0.5% (w/w) and 4% (w/w), 0.5% (w/w) and 3% (w/w), 0.5% (w/w) and 2% (w/w), 0.5% (w/w) and 1 % (w/w), 1 % (w/w) and 10% (w/w), 2% (w/w) and 10% (w/w), 3% (w/w) and 10% (w/w), 4% (w/w) and 10% (w/w), 5% (w/w) and 10% (w/w), 6% (w/w) and 10% (w/w), 7% (w/w) and 10% (w/w), 8% (w/w) and 10% (w/w), 9% (w/w) and 10% (w/w), 1 % (w/w) and 5% (w/w), or 2% (w/w) and 7% (w/w)). For example, the composition may have a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w).

The composition may include an oil, such as soy oil. In some embodiments, the composition has a concentration of soy oil of between about 1 % (w/w) and about 25% (w/w) (e.g., between about 1% (w/w) and 20% (w/w), 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 5% (w/w) and 25% (w/w), 10% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 15% (w/w) and 25% (w/w), 20% (w/w) and 25% (w/w), 5% (w/w) and 15% (w/w), or 5% (w/w) and 20% (w/w)). For example, the composition may have a concentration of soy oil of about 10% (w/w).

The composition includes glyceride. In some embodiments, the composition includes Gelucire®. The glyceride may be present in an amount of between about 1% (w/w) and about 20% (w/w) e.g., between 1% (w/w) and 15% (w/w), 1% (w/w) and 10% (w/w), 1% (w/w) and 5% (w/w), 0.5% (w/w) and 20% (w/w), 10% (w/w) and 20% (w/w), or 15% (w/w) and 20% (w/w)). For example, the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w) (e.g., 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), or 10% (w/w)). In some embodiments, the composition has a concentration of glyceride of between about 8% (w/w). In some embodiments, the composition is formulated for topical administration to the skin of a human subject. In some embodiments, the composition may be administered to any skin or exposed mucosal surface. Skin surfaces includes any part of the body, including but not limited to face, hands, legs, neck, abdominal area, eyes, nose, and chest. In some embodiments, the composition can be in any form suitable for topical use such as, for example, an aerosol, dusting powder, jelly, patch, liquid, gel, lotion, paste, cream, foam, serum, ointment, or stick.

Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the “internal” phase, may include, for example, petrolatum and a fatty alcohol, such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic, or amphoteric surfactant.

Lotions are preparations to be applied to the skin surface without friction and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably, for the present purpose, include a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations herein for treating large body areas, because of the ease with which a more fluid composition can cover large surfaces. It is generally desirable that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin.

Solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution may contain other acceptable chemicals to buffer, stabilize or preserve the solute. Common examples of solvents used in preparing solutions are ethanol, water, propylene glycol or any other acceptable vehicles.

Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol, and, optionally, an oil. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for a number of desirable characteristics, such as emollience or the like. As with other carriers or vehicles, an ointment base may desirably be inert, stable, nonirritating, and nonsensitizing. As explained in Flemington: The Science and Practice of Pharmacy, 19^th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid.

Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from singlephase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

In some embodiments, the composition further includes an additive. Examples of additives include, but are not limited to, diluents, buffers, binders, surface-active agents, lubricants, humectants, pH adjusting agents, preservatives (including antioxidants), emulsifiers, occlusive agents, opacifiers, antioxidants, colorants, flavoring agents, gelling agents, thickening agents, stabilizers, and surfactants, among others.

Methods for Using Pharmaceutical Compositions

The pharmaceutical compositions described herein may be used to treat a disease or condition in a subject. For example, the pharmaceutical compositions may be used to reduce inflammation or redness in a subject. Exemplary sources of inflammation include contact dermatitis, seborrheic dermatitis, nummular dermatitis, stasis dermatitis, atopic dermatitis, dermatitis herpetiformis, psoriasis, eczema, acne, or rosacea among others. The inflammation may be caused by an environmental factor. For example, in some embodiments, the inflammation is due to exposure to ultraviolet (UV) radiation. The inflammation may be caused by aridness, salt concentration present in the air, or air pollution, among other sources.

In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing psoriasis in a subject (e.g., the subject having been diagnosed as having psoriasis). In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing acne in a subject (e.g., the subject having been diagnosed as having acne). In some embodiments, an HMO- containing composition of the disclosure is used for treating or preventing rosacea in a subject (e.g., the subject having been diagnosed as having rosacea). In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing erythema in a subject (e.g., the subject having been diagnosed as having erythema). In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing eczema in a subject (e.g., the subject having been diagnosed as having eczema). In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing UV radiation-induced inflammation in the skin of a subject. In some embodiments, an HMO-containing composition of the disclosure is used for treating or preventing environmental pollution-induced inflammation in the skin of a subject. In some embodiments, an HMO- containing composition of the disclosure is used for treating or preventing dehydration-induced inflammation in the skin of a subject.

In some embodiments, the composition is applied to the skin of the subject one or more times daily, optionally wherein the composition is applied to the subject once daily, twice daily, or three times daily. In some embodiments, the composition is applied to the skin of the subject once daily. In some embodiments, the composition is applied to the skin of the subject twice daily, optionally wherein the composition is administered to the subject once in the morning and once in the evening. In some embodiments, the composition is applied to the skin of the subject three times daily. In some embodiments, the composition is applied to the skin of the subject for at least 1 day. In some embodiments, the composition is applied to the skin of the subject for at least 2 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 7 continuous days.

In some embodiments, the composition is applied to the skin of the subject for at least 14 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 28 continuous days.

Methods for Using Cosmetic Compositions

The cosmetic compositions described herein may be used to improve the outward appearance of the skin of a human subject (e.g., a male or female human subject). For example, the compositions described herein may be used to achieve an anti-aging effect in the skin of a subject. In some instances, this may mean that composition may be used to reduce the appearance of fine lines or wrinkles. In some instances, the composition may be used to increase the firmness or elasticity in the skin of the subject. Furthermore, the compositions described herein may be used to increase the hydration of the skin cells of a subject and improve skin barrier functionality.

In some embodiments, the composition is a patch, liquid, gel, lotion, paste, cream, foam, serum, ointment, or stick. In some embodiments, the composition is applied to the skin of the subject one or more times daily, optionally wherein the composition is applied to the subject once daily, twice daily, or three times daily. In some embodiments, the composition is applied to the skin of the subject once daily. In some embodiments, the composition is applied to the skin of the subject twice daily, optionally wherein the composition is administered to the subject once in the morning and once in the evening. In some embodiments, the composition is applied to the skin of the subject three times daily. In some embodiments, the composition is applied to the skin of the subject for at least 1 day. In some embodiments, the composition is applied to the skin of the subject for at least 2 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 7 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 14 continuous days. In some embodiments, the composition is applied to the skin of the subject for at least 28 continuous days. EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regards as their invention.

Example 1 . Effects of 2’-fucosyllactose on the skin

The results reported in this study showed that 2’-FL achieved anti-aging effects when applied topically. Using in vitro and ex vivo models it was shown that 2’-FL can regulate extracellular matrix proteins turnover, namely collagen type 1 , elastin, and fibronectin, thus improving skin firmness and elasticity, and leading to a decrease of fine and age-induced wrinkles. It was also shown that 2’-FL had anti-psoriatic and anti-inflammatory capacity in an inflammatory psoriatic-like skin model when applied topically. Additionally, using in vitro and ex vivo models, it was shown that 2’FL can reduce keratinocytes hyperproliferation markers (keratin 16, keratin 17, psoriasin) and inflammation mediators (pro- inflammatory cytokines TNF-a, IL-17, IL-1a), hallmark features of psoriasis. Furthermore, it was shown that 2’-FL can ameliorate the impact of urban air pollution in the skin; using in vitro models and was shown that 2’FL can reduce air-pollution induced inflammation and oxidative stress in skin cells.

Methods:

Anti-aging

Study 1

In vitro tests:

Cells’ isolation and growth conditions: Human cells were isolated from skin specimens of healthy donors (IMG 20.8 - 26.8) undergoing abdominoplasties after written informed consent and under the protocol established and approved between the Ethical Committee of Hospital Lusiadas (Porto, Portugal) and Universidade Catolica Portuguesa. Skin pieces were cut into small fragments (1 - 2 cm²) and incubated with 2.5 U/mL of dispase (BD Biosciences, USA) overnight at 4 °C. The epidermis was peeled off and the dermis (without epidermis) was digested overnight at 37 °C using the whole skin dissociation kit (Miltenyi Biotec, USA) as per the manufacturer’s instructions. Briefly, tissue fragments were incubated overnight at 37 °C under agitation (160 rpm) with 435 pL buffer L, 12.5 pL enzyme P, 50 pL enzyme D and 2.5 pL enzyme A. Then, cell suspension was applied to a 70 pm separation filter place on a 50 mL falcon. The filter was washed with 4 mL cold a-MEM and cell suspension was centrifuged at 300 g for 10 min at 4 °C. Afterwards, the suspension of freshly isolated human dermal fibroblasts was cultured in a- MEM, supplemented with 10% fetal bovine serum (FBS, Gibco, Portugal) and 1 % penstrep (Gibco, Portugal) at 37 °C in a humidified atmosphere with 5% CO2. Palmitoyl Tripeptide- 1 was used as benchmark (PAL, 0.5 pM; Cayman, #27059). Collagen type I production: Collagen I production was evaluated by immunofluorescence (FIG. 1 ). For that, cells were seeded in a 6-well plate at a cell density of 2.5x10⁵ cells/ml for 24 h. After, cells were exposed to 2’-FL at a final concentration of 10, 25, and 50 mg/ml, over 24 h. Cells were collected and further incubated for 24 h with primary antibody anti-collagen (1 :100; Abeam, ab34710). For detection, cells were incubated for 1 h at room temperature (RT) with the secondary antibody - Alexa Fluor 488 donkey anti-rabbit (Life Technologies, USA) - at a concentration of 1 :500. Nuclei were counterstained with DAPL All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers were automatically quantified with the Cell Profiler software.

Fibronectin production: Fibronectin production was evaluated via immunofluorescence (FIG. 1 ). For that, cells were seeded in a 6-well plate at a cell density of 2.5x10⁵ cells/ml for 24 h. After, cells were exposed to 2’-FL at a final concentration of 10, 25, and 50 mg/ml, over 24 h. Cells were collected and further incubated for 24h with primary antibody anti-fibronectin (1 :100; Abeam, ab2413). For detection, cells were incubated for 1 h at room temperature (RT) with the secondary antibody - Alexa Fluor 488 donkey anti-rabbit (Life Technologies, USA) - at a concentration of 1 :500. Nuclei were counterstained with DAPL All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers were automatically quantified with the Cell Profiler software.

Elastin production: Cells were seeded in a 6-well plate at a cell density of 2.5x10⁵ cells/ml, in complete cell culture medium, for 24 h. After that, cells were exposed to 2’-FL at a final concentration of 10, 25, and 50 mg/mL, and incubated for 48 h. The culture medium was then replaced by fresh cell culture medium supplemented with 2’-FL at the same concentration, and cells were incubated for an additional 24 h. Next, cells were harvested by trypsin digestion, counted by Trypan Blue Stain (0.4%, Invitrogen), and elastin protein content was obtained by oxalic acid digestion. Elastin quantification was performed by colorimetric assay, using the Elastin Assay - Fastin™ Elastin Economy kit (Biocolor), according to the manufacturer instructions.

Ex vivo tests:

Skin explants: Samples were obtained from healthy donors undergoing abdominoplasties, after written informed consent and under the protocol established and approved between the Ethical Committee of Hospital Lusiadas (Porto, Portugal) and Universidade Catolica Portuguesa. Before any experiment, the fat adipose tissue was carefully removed with scissors, taking care to preserve the dermis from damage. 12-mm-diameter punch biopsies were excised.

Anti-aging model: Skin biopsies were transferred to transwell plates with the epidermal surface left in contact with the air (air-liquid interface) and the dermal compartment was fed with high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Denmark) supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS; Gibco) and 1 % (v/v) Pen-Strep (Lonza, Switzerland). Models were allowed to stabilize for at least 18 h prior studies at 37 °C in a humidified atmosphere with 5% CO2. For treatment studies, 25 pL of each formulation was topically applied once daily for 2 days. The formulation used for topical application of 2’-FL was the following: 5% (w/w) of 2’FL + 41 .7% (w/w) Squalane + 2% (w/w) CMC + 8.2% (w/w) Gelucire® 43/01 + 43% (w/w) water + 0.1 % (w/w) Lecithin. Matrixyl 3000 from Sederma was used as benchmark (3% of Matrixyl + 41.7% (w/w) Squalane + 2% (w/w) CMC + 8.2% (w/w) Gelucire® 43/01 + 45% (w/w) water + 0.1% (w/w) Lecithin). A blank formulation (same formulation without 2’-FL) was also used as control.

Histology: Skin models were fixed in 4% (m/v) paraformaldehyde (Fisher Scientific, UK), dehydrated, embedded in paraffin (Thermo Scientific, USA) and cut into 5 pm sections. Tissue sections were stained with Masson’s Trichrome kit (collagen deposition; Bio-Optica, Italy), Picrosirius red (collagen fibers; Bio-Optica), and Orcein (elastin fibers; Bio-Optica, Italy) according to manufacturer’s instructions. For immunohistochemistry, paraffin tissue sections were deparaffinized in xylene, re-hydrated and antigen retrieval was performed for 20 min at 37 °C in a Proteinase K buffer. Afterwards, unspecific staining was blocked with 2.5 % Horse Serum (Vector Labs, USA). Primary antibodies - Collagen type I (1 :100; Abeam, UK, ab34710) and Fibronectin (1 :100; Abeam, ab2413) - were incubated overnight at 4 °C. For detection, sections were incubated for 1 h at room temperature (RT) with the secondary antibodies - Alexa Fluor 488 donkey anti-rabbit (Life Technologies, USA) - at a concentration of 1 :500. Nuclei were counterstained with DAPL All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers were automatically quantified with the Cell Profiler software (FIG. 2). qPCR: Total RNA was extracted by using Tri-reagent (Sigma, Portugal) and an ultra-turrax for the mechanical lysis of the tissue. Chloroform was added and samples were incubated for 15 min at RT and centrifuged. The aqueous phase was collected, and isopropanol was added. Samples were further incubated for 10 min at RT and centrifuged. The supernatants were discarded, and the pellets were washed twice with 100 % cold ethanol. Extracted RNA was kept in RNAse/DNAse free water. RNA was converted to cDNA by NZY First-Strand cDNA Synthesis Kit (Portugal) according to the manufacturer’s instruction. Real-time PCR was used to detect the expression of ELN, FN, COL1 A1 , COL3A1. Actin, Tubulin and B2M were used as reference genes. The values were calculated following the mathematical model 2-AACt.

Study 2

Ex vivo tests:

Skin explants: Samples were harvested from routine abdominoplasties of healthy female anonymous donors. It was also established that donors' body mass index (BMI) could not be superior to 29.9. Written informed consent from all participants was obtained under the protocol established and approved by the Ethical Committee of Hospital Lusiadas (Porto, Portugal) and Universidade Catolica Portuguesa (UCP, Porto, Portugal). Before any experiment, the fat adipose tissue was carefully removed with scissors, taking care to preserve the dermis from damage, and 12-mm-diameter punch biopsies were excised.

Anti-aging model: Skin biopsies were transferred to transwell plates with the epidermal surface left in contact with the air (air-liquid interface) and the dermal compartment was fed with high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Denmark) supplemented with 10 % (v/v) heat inactivated Fetal Bovine Serum (FBS; Gibco) and 1 % (v/v) Pen-Strep (Lonza, Switzerland). Models were allowed to stabilize for at least 18 h prior studies at 37 °C in a humidified atmosphere with 5% CO2. For treatment studies with 2’-FL, retinol (Acofarma, Spain) and niacinamide (Lotioncrafter, USA), 25 pL of each formulation was topically applied once daily for 2 days. The formulation employed for the topical application of 2’-FL, retinol, and niacinamide consisted of the following components: 5%, 2.5%, or 1% of 2’-FL (or niacinamide) + 10% (w/w) Squalane + 10% (w/w) Soy oil + 3% (w/w) Montanov 68 + 0.5% (w/w) phenoxyethanol + the remaining percentage of water. The formulations containing retinol were identical, except retinol was utilized at concentrations of 0.1% and 0.2%, and the remaining proportion comprised water. As a control, a vehicle formulation (the same formulation without the biomolecules under investigation) was also included. Matrixyl 3000 from Sederma was used as benchmark (3% of Matrixyl + 10% (w/w) Squalane + 10% (w/w) Soy oil + 3% (w/w) Montanov 68 + 0.5% (w/w) phenoxyethanol + 73.5% (w/w) water).

Histology: Skin models were fixed in 4 % (m/v) paraformaldehyde, dehydrated, embedded in paraffin (Thermo Scientific, USA) and cut into 4 pm sections. Tissue sections were stained with Orcein (elastin fibers; Bio-Optica) according to manufacturer’s instructions. For immunohistochemistry, paraffin tissue sections were deparaffinized in xylene, re-hydrated and antigen retrieval was performed for 20 min at 37 °C in a Proteinase K buffer. Afterwards, unspecific staining was blocked with 2.5 % Horse Serum (Vector Labs, USA). Primary antibodies - Collagen type I (1 :100; Abeam, UK) and Fibronectin (1 :100; Abeam) - were incubated overnight at 4 °C. For detection, sections were incubated for 1 h at room temperature (RT) with the secondary antibodies - Alexa Fluor 488 donkey anti-rabbit (Life Technologies, USA) - at a concentration of 1 :500. Nuclei were counterstained with DAPL All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers were automatically quantified with the Cell Profiler software (FIGS. 12 and 13). qPCR: Total RNA was extracted by using Tri-reagent (Sigma, Portugal) and an ultra-turrax for the mechanical lysis of the tissue. Chloroform was added and samples were incubated for 15 min at RT and centrifuged. The aqueous phase was collected, and isopropanol was added. Samples were further incubated for 10 min at RT and centrifuged. The supernatants were discarded, and the pellets were washed twice with 100 % cold ethanol. Extracted RNA was kept in RNAse/DNAse free water. RNA was converted to cDNA by NZY First-Strand cDNA Synthesis Kit (Portugal) according to the manufacturer’s instruction. Real-time PCR was used to detect the expression of ELN, FN, COL1 A1 , COL3A1. Actin, Tubulin and B2M were used as reference genes. The values were calculated following the mathematical model 2-AACt.

Anti-Psoriasis:

In vitro tests:

Cells’ growth conditions: Psoriatic Human Dermal Fibroblasts (P-HDFa) derived from lesional (Catalog #219736 and Lot #6F5008) and non-lesional (Catalog #219737 and Lot #6F5006) site were attained from Lonza (Switzerland). Cells were grown using high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Scientific, Denmark) supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS; Gibco, Thermo Scientific) and 1% (v/v) Pen-Strep (Lonza, Basel, Switzerland). All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Epidermal Keratinocytes from Psoriasis Donor (Non-Lesion Site Derived, P-HEK) were obtained from Lonza (Catalog #219735 and Lot #6F5005, Switzerland). Cells were grown at 37 °C in a humidified atmosphere with 5% CO2, using KBM- Gold™ Basal Growth Medium (Lonza, Switzerland) supplemented with KGM-Gold™ SingleQuots™ (Lonza, Switzerland) as follows: 0.1% (v/v) hydrocortisone; 0.1% (v/v) transferrin; 0.05% (v/v) epinephrine; 0.1 % (v/v) gentamicin sulfate-amphotericin (GA-1000); 0.4% (v/v) bovine pituitary extract (BPE); 0.1 % (v/v) human epidermal growth factor (hEGF) and 0.1% (v/v) insulin. Corticosteroid betamethasone dipropionate (20 pM; Sigma, B1152) was used as anti-inflammatory control.

Cytokine analysis: Cells were seeded into 12 well-plates at 2 x 10⁵ cells/mL. After 24 hours, cells were exposed to 2'-FL at 50, 25 and 10 mg/mL. Betamethasone at 20 pM was used as an anti-inflammatory control, whereas culture media was used as a negative control. Interleukin (IL)-1 a and IL-6 levels were determined from the cell supernatant after a 24-hour exposure period using an enzyme-linked immunosorbent assay (ELISA) (Abeam, United Kingdom), according to the manufacturer's instructions (FIG. 3A and FIG. 3B). Protein was extracted and quantified through Pierce™ BCA Protein Assay Kit (ThermoFisher, United Kingdom) and used to normalize ELISA’s results.

Ex vivo tests:

Inflammatory psoriatic-like skin (IPS) model: Skin biopsies were intradermally injected with an activation cocktail containing 0.2 ng recombinant human (rh) IL-2 (Peprotech, USA, 200-02), 1000 ng anti-CD3 antibody (Sigma, 16-0037-85) and 1000 ng anti-CD28 antibody (Sigma, 16-0289-85) in order to activate skin-resident T cells. Samples were transferred to transwell plates and left in air-liquid interface. The skin models were cultured up to 7 days in 12-well plates in a serum-free DMEM with 1 % Pen-Strep and supplemented with a Th17 polarization cocktail containing 10 ng/mL rhlL-1 protein (Peprotech, 200- 01 B), 50 ng/ mL rhlL-23 protein (Peprotech, 200-23) and 10 ng/mL rhTGF- protein (Peprotech, 100-21 ) in a humidified atmosphere of 5% CO2 at 37 °C. For treatment studies, 25 pL of each formulation was topically applied once daily after inflammation was induced (from day 4 - day 6 of culture). Corticosteroid betamethasone dipropionate (20 pM; Sigma, B1152) was used as anti-inflammatory control. The formulation used for topical application of 2’-FL was the following: 5% (w/w) of 2’FL + 41.7% (w/w) Squalane + 2% (w/w) CMC + 8.2% (w/w) Gelucire® 43/01 + 43% (w/w) water + 0.1% (w/w) Lecithin. Corticosteroid betamethasone dipropionate (Sigma, B1 152) was used as an anti-inflammatory control (0.05% (w/w) of betamethasone + 41 .7% (w/w) Squalane + 2% (w/w) CMC + 8.2% (w/w) Gelucire® 43/01 + 47.95% (w/w) water + 0.1 % (w/w) Lecithin). A blank formulation (same formulation without 2’-FL) was also used as control.

Histology: Skin models were fixed in 4 % (m/v) paraformaldehyde, dehydrated, embedded in paraffin and cut into 5 pm sections. Tissue sections were stained with Haematoxylin & Eosin (Sigma, USA) following a routine protocol. For immunohistochemistry, paraffin tissue sections were deparaffinized in xylene, re-hydrated and antigen retrieval was performed for 1 h at 55 °C in Tris-EDTA pH 9 buffer. Afterwards, unspecific staining was blocked with 2.5 % Horse Serum (Vector Labs, USA). Primary antibodies - K16 (1 :40; Abeam, ab76416), K17 (1 :400; Abeam, ab51056) and Psoriasin (1 :100; Abeam, ab13680) were incubated overnight at 4 °C. For detection, sections were incubated for 1 h at RT with the secondary antibodies - Alexa Fluor 488 donkey anti-rabbit or Alexa Fluor 594 donkey anti-mouse (Life Technologies) - at a concentration of 1 :500. Nuclei were counterstained with DAPI. All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers were automatically quantified with the Cell Profiler software (FIG. 4). qPCR: Total RNA was extracted by using Tri-reagent (Sigma, Portugal) and an ultra-turrax for the mechanical lysis of the tissue. Chloroform was added and samples were incubated for 15 min at RT and centrifuged. The aqueous phase was collected, and isopropanol was added. Samples were further incubated for 10 min at RT and centrifuged. The supernatants were discarded, and the pellets were washed twice with 100 % cold ethanol. Extracted RNA was kept in RNAse/DNAse free water. RNA was converted to cDNA by NZY First-Strand cDNA Synthesis Kit (Portugal) according to the manufacturer’s instruction. Real-time PCR was used to detect the expression of K16, K17, IL-1-a, TNF-a, and PI3. Actin, Tubulin and B2M were used as reference genes. The values were calculated following the mathematical model 2-AACt (FIG. 5).

Anti-pollution

In vitro response to 2-FL in cells exposed Urban Particulate Matter:

Cells’ growth conditions: Immortalized human keratinocytes (HaCaT) were attained from Cell Lines Service (#300493, Germany). Cells were cultured in a humidified atmosphere at 37 °C, 5% CO2. Unless otherwise specified, cells were grown in complete cell culture medium: Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and 1 % Antibiotic- Antimycotic (Penicillin/Streptomycin/Amphotericin B).

Urban Particulate Matter: Urban Particulate Matter (UPM) standard reference material SRM1648a was purchased from the National Institute of Standards & Technology (NIST). SRM1648a is composed of a mixture of atmospheric particulate matter collected in an urban area and is used to evaluate cellular responses to selected polycyclic aromatic hydrocarbons (PAHs), nitro-substituted PAHs (nitro-PAHs), polychlorinated biphenyl (PCB) congeners, and chlorinated pesticides. Unless otherwise specified, SRM1648a particles are resuspended in phosphate buffer saline (PBS) solution at a concentration of 20 mg/ml. The suspension is then sonicated for 15 minutes and stored at -20 °C until further use. At the time of use, SRM1648a 20mg/ml is thawed at room temperature and sonicated for 30 s, to avoid particle aggregation. Then, SRM1648a is diluted to final exposure concentration in complete culture medium.

Anti-inflammatory: The inflammatory response to SRM1648a was evaluated by the cellular secretion of pro-inflammatory cytokines IL-6 and IL-1 a (FIG. 6A). For that, HaCaT cells were seeded in a 12-well plate, at a cell density of 2.5x10⁵ cells/ml in complete cell culture medium for 24 h. After that, cells were exposed to 2’-FL at a final concentration of 10, 25 and 50 mg/mL, in the presence of SRM1648a (500 pg/ml) over 24 h, after which cell culture supernatants were collected for further analysis by ELISA. Cells were then harvested for protein extraction, using cell lysis buffer (50 mM Tris-HCI, pH 7.8, 150 mM NaCI, 1 mM EGTA, 1.5 mM MgCI2, 0.4% sodium dodecyl sulfate (SDS), 1 pl/ml benzonaze [25 U/ml], 1% Nonidet-P40, and protease inhibitor cocktail tablets (Roche, Basel, Switzerland). IL-6 and IL-1 a secretion was measured in the cell culture supernatant by ELISA (ELISA MAX™ Set Human IL-6, and ELISA MAX™ Deluxe Set Human IL-1 a, respectively, BioLegend), following the manufacturer instructions. Intracellular protein content was quantified by biuret reaction (reduction of Cu to Cu ) using bicinchoninic acid (BCA, Pierce BCA™ Protein Assay Kit, ThermoScientific). The inflammatory response to SRM1648a is expressed as a fold change of cytokine secreted. SRM1648a exposure potentiates the production of the pro-inflammatory cytokines IL-6 and IL-1 a. To evaluate the protective effect of 2-FL in response to SRM1648a exposure, the corticosteroid betamethasone dipropionate (20 pM; Sigma, B1 152) was used as anti-inflammatory control.

Oxidative Response (generation of intracellular reactive oxygen species, ROS): The production of intracellular ROS was evaluated with the fluorescent probe dichlorofluorescein diacetate (H2DCFDA, Sigma Aldrich) (FIG. 6B). Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). The SRM1648a reference material is composed by a mixture of particles that range between 0.1 - 100 pm in diameter, and presence of particles with high diameter interferes with the fluorescence detection. To overcome this problem, in this assay, cells were exposed to the ultrafine and soluble components of SRM1648a (sUPM). For that, SRM1648a was resuspended in the experimental culture medium (composition detailed below) at a concentration of 500 pg/ml. SRM1648a suspension was then incubated overnight with medium agitation. After, the suspension was centrifuged (5000 rpm, 5 min) and filtered using a 0.22 gm membrane filter. HaCaT cells were seeded in a 96-well black, clear bottom plate at a cell density of 1 x10⁵ cells/ml in DMEM without phenol red (GIBCO™), supplemented with 1% Glutamax (GIBCO™), 10% FBS, and 1% antibiotic-antimycotic (GIBCO™) and incubated for 24 h. Then, cells were exposed to 2’-FL at final concentration of 10, 25 and 50 mg/mL, in the presence of sUPM at the equivalent concentration of 500 pg/ml, over 24 h. The H2DCFDA probe was then added to cell culture at a final concentration of 250 pM and incubated for 2-4 h. Fluorescence was then measured using fluorescence excitation and emission wavelengths of 495 nm and 529 nm, respectively.

Example 2: Investigation of the skin protectant and anti-inflammatory properties of human milk oligosaccharides on damaged skin

Summary of the clinical study

This example describes a double blinded single centre study in twenty-two healthy male and female subjects to investigate the skin protectant and anti-inflammatory properties of two test articles including an HMO and a placebo versus a control site on damaged skin. The objective of this study was to evaluate the relative degree of improvement to skin barrier functionality and determine the antiinflammatory efficacy of the test articles and a placebo against a control site following open application and TEWL assessments. The test articles that were studied included Test Article #1 including 2’-FL and Test Article #2 including L-Fucose.

The subjects underwent transepidermal water loss (TEWL) assessments at baseline (Day 0) after which Sodium Lauryl Sulfate (SLS) was applied to the test sites (Volar forearm) on a patch for 24 hours of exposure to cause skin irritation. Irritation assessment and TEWL readings were taken post SLS removal on day 1 , and the test articles were applied to four sites, the other site remained untreated. The test sites on the back were exposed to UV Irradiation (4 x MED) to cause skin irritation. Irritation assessment, and TEWL readings were taken 24 hours post exposure on day 1 , and the test articles were applied to four sites, the other site remained untreated.

On day 1 , the test articles were applied to the test sites and allowed to absorb for 10 minutes before each assessment was performed. On days 1 , 4, 9 and 15, TEWL readings were taken prior to test article reapplication. The test articles were applied to the same sites on each study day for a total of 9 applications. Clinical grading of the test sites was performed prior to the application of the test articles at baseline, post SLS removal and on study days 1 , 2, 3, 4, 7, 8, 9, 10, 11 and 14 posttest article removal. The study was performed for 15 Days. Including nine, open applications of the test articles to the 22 healthy male and female subjects, aged 18 years and older.

Introduction and Objective

The objective of this study was to evaluate the relative degree of improvement to skin barrier functionality and determine the anti-inflammatory efficacy of the test articles and a placebo against a control site following open application and TEWL assessments. Material and Methods

Study Design: The study was conducted single blind, within subject comparison skin barrier functionality and anti-inflammatory study. A total of 22 subjects received four open-applications.

Selection of Subjects

Screening: 22 subjects were recruited into the study. Subjects satisfied the following inclusion and exclusion criteria, and were prepared to accept the prohibitions and restrictions and had to have given written informed consent. The suitability of each potential subject was confirmed before his or her acceptance by review of a study specific pre-treatment questionnaire.

Inclusion criteria

• Subject is a healthy male or female, aged 18 years or older.

• Subject has signed a written Informed Consent. Exclusion criteria

• Pregnancy or lactation.

• Inadequate precaution or procedure to prevent pregnancy (women of childbearing potential only).

• A current skin disease of any type at the test site (e.g., eczema, psoriasis)

• Heavy alcohol consumption in the opinion of the investigator.

• A fever in the last 12 hours, prior to the initial patch application.

• Significant past medical history of hepatic, renal, cardiac, pulmonary, digestive, haematological, neurological, locomotor or psychiatric disease, which in the opinion of the Investigator would compromise the safety of the subject.

• History of malignant disease.

• Insulin dependent or non-insulin dependent diabetes.

• Concurrent medication likely to affect the response to the test articles or confuse the results of the study, i.e., routine high dosage use of anti-inflammatory drugs (aspirin, ibuprofen, corticosteroids).

• Known sensitivity to the treatment solutions or their constituents including patch materials.

• Sensitization or questionable sensitization in a Repeat Insult Patch Test.

• Use of self-tanning lotion on the test area, one week previous to the start of the study. Prohibitions and restrictions for the duration of the study

• No use of aspirin or non-steroidal anti-inflammatory drugs for the duration of the study.

• No swimming during the study.

• No deliberate exposure of the test sites to natural sunlight or to other sources of UV light during the study. • No immunizations during the study.

• No use of self-tanning lotion on the test area during the study.

Method

Test articles: The following test articles were used and labelled as follows:

1 . 2’-FL - at a concentration of 1 % (w/w), 2.5 % (w/w), or 5 % (w/w)

2. L-Fucose

3. Vehicle

4. Untreated site (Volar forearm and Back sites) wherein the test articles were formulated with 10 % (w/w) squalane, 10 % (w/w) soy oil, 3 % (w/w) Montanov® 68, 0.5 % (w/w) phenoxyethanol, and the remaining portion was water.

Additionally, the induction of inflammation control for the study, was labelled as follows:

• 0.75% W/V Sodium Lauryl Sulfate (SLS) in water

Regime for Inflammation Induction

On Day 0 - The SLS solution was applied to occlusive patches that consisted of a breathable tape with non-breathable adhesive and a centre portion of 2cm by 2cm Webril patch pads on the volar forearm. The seven sites on the volar forearm were marked with skin markers. These SLS patches stayed in place for approximately 24 hours for the induction of an inflammatory skin response.

On Day 0 - Each subject was treated with a series of five light exposures in order to determine the MED for unprotected skin. Each exposure time was 1 .25 times greater than the previous one. This procedure continued until each subject produced a reaction 4 x their MED.

The MED study area was outlined on the lower back between waist and scapula and lateral to the midline. Subjects were exposed upright in a backless chair and the test sites were outlined while the subject was in this position.

A template of the study area contained seven subsites, each 3 cm x 2.5 cm, marked on the back. The template was located ensuring that no moles or skin lesions are present in any of these subsites. Only the subsites were exposed. A record of individual exposure times and subsites were kept. After test exposure any immediate skin responses were noted and subjects were instructed to keep the test area covered from sunlight or other sources of UV light for the next 24 hours before they returned to the study centre.

Dose-regime - Open Application

Day 1 - Subjects returned to the testing facility and had the SLS patches removed. The seven sites had TEWL readings and grading conducted for each site. Subjects had one control site (untreated) and the other site received an open application of the two test articles including an HMO and 1 placebo. Enough of the test articles were applied to cover the marked area. The application area was covered with a thin gauze pad that was taped on all sides so that the test article remained in place.

Approximately 10 minutes (±2 min) post application, the gauze and test articles were removed and each of the test sites was graded for irritation and TEWL readings were taken. After TEWL readings were taken, the test articles were re-applied in the same manner and again covered with the gauze pad and all sides taped to cover the application.

Subjects were instructed to keep the open application sites covered and the bandaging dry and given an appointment to return to the testing facility the next day.

Days 4, 9 and 15, - Subjects returned to the testing facility approximately 24 hours after each application. The subjects had the test article applications removed and the sites were gently wiped to remove any residual test article. TEWL measurements were taken, and the test articles were applied and covered as indicated above.

Applications occurred on Days 1 , 2, 3, 4, 7, 8, 9, 10, 1 1 and 14 with a re-application after the first application (after clinical grading and TEWL readings). Application of the test articles were made to the same sites on each day. The control site remained untreated. Prior to the next application, the test sites were gently wiped with a damp tissue to remove any remaining product. Clinical grading of the test sites was performed prior to the application of the test articles at baseline, post SLS removal and on study days 1 , 2, 3, 4, 7, 8, 9, 10, 1 1 and 14 posttest article removal.

Day 15 - Subjects returned to the testing facility approximately 24 hours after the final test article application and the same procedures were conducted. After all assessments were completed, subjects were considered complete and compensated for their participation.

Tewameter® Assessments of TEWL (Transepidermal Water Loss)

TEWL measurements were performed using the Tewameter® TM300 (Courage and Khazaka, Germany). The measurement of the water evaporation, and therefore TEWL, was based on the diffusion principle in an open chamber. The density gradient was measured indirectly by two pairs of sensors in the probe attachment, one for temperature and the other for relative humidity. This density gradient was then analyzed by a microprocessor in the instrument. A 15-minute warm-up period were allowed before using the Tewameter®.

TEWL readings were taken at all test sites marked on the volar forearm on Day 0, before application of the SLS patches. These readings acted as the baseline reading.

TEWL readings were taken at the treated and untreated sites after removal of the SLS patches and 24 hours post-irradiation (MED) on Day 1 , and again on all sites approximately 10 minutes (±2 min.) after application of the test product to three of the test sites and the placebo to one test site. The test products were allowed to sit on the surface of the treatment site for approximately 10 minutes (±2 min.) covered with a loose gauze pad adhered on 4 sides with tape. After which time it was removed from the test sites and gently wiped off with a clean tissue. TEWL readings of all test sites were performed. TEWL readings were taken for each test site on Days 1 , 4 and 9 after removal of the test articles and prior to reapplication. Final TEWL readings were taken on Day 15 of all test sites.

Grading of Test Sites

Clinical grading of the test sites was performed prior to the application of SLS at on Day 0, post removal of the SLS patches and 24 hours post-irradiation (MED) on study day 1 and 10 minutes +/-2 minutes) after the first application of the products on day 1 (test articles will be removed immediately prior to grading). After which the test articles were reapplied to the treated sites. Clinical grading also occurred prior to product application on days 2, 3, 4, 7, 8, 9, 10 and 1 1 and a final grading on Day 14 of the study.

As far as possible, the same person was assessed for all test sites for the duration of the study according to the scoring scale shown below. All assessors were named in the final report. Illumination of the test sites was by a 60-watt pearl bulb, approximately 30 cm from the site.

Irritancy Grading Scale and Key to Symbols Used

0.0 No apparent cutaneous involvement.

0.5 Faint, barely perceptible erythema, or slight dryness (glazed appearance).

1 .0 Faint but definite erythema, no eruptions or broken skin or No erythema but definite dryness; may have epidermal fissuring.

1 .5 Well-defined erythema or faint erythema with definite dryness, may have epidermal fissuring.

2.0 Moderate erythema, may have a very few papules or Deep fissures, moderate-to-severe erythema in the cracks.

2.5 Moderate erythema with barely perceptible oedema or severe erythema not involving a significant portion of the patch (halo effect around the edges), may have a few papules or moderate-to-severe erythema.

3.0 Severe erythema (beet redness), may have generalized papules or moderate-to-severe erythema with slight oedema (edges well defined by raising).

3.5 Moderate-to-severe erythema with moderate oedema (confined to patch area) or moderate- to-severe erythema with isolated eschar formations or vesicles.

4.0 Generalized vesicles or eschar formations or moderate-to-severe erythema and/or oedema extending beyond the area of the patch.

A Tape reaction.

E Oedema

P Papules.

S Reaction spread. Results

22 subjects of both sexes were recruited into, and completed, the study having the demographics as described in Table 1. Table 1 . Demographics of participants

No adverse events or reactions were reported. All subjects completed the study.

Method of Statistical Analysis • Demographics: 1 ) Descriptive statistics

• TEWL/Visual Irritancy: 1 ) Descriptive Statistics (mean and standard deviation) 2) Change from Baseline (Paired T-Test, mean percent improvement from Baseline, percent of subject improving) 3) Change from baseline between treatment analysis, analysis of covariance

• All statistical tests of hypothesis employed a level of significance of 0.05 and no adjustments were made for the number of tests performed.

A summary of the TEWL results from samples irritated with SLS are summarized in Table 2 and Table 3 and in FIG. 7A and FIG. 7B. Table 2. Results of Statistical Analysis - TEWL - SLS

* Significant change from baseline

** Significant difference between treatments - multiple comparisons are indicated on the following page [1] Percent change is calculated from the mean changes from baseline [2] Percent change is calculated individually by subject and averaged (Note if 0 at baseline, % not calculated for that subject) Table 3. Dunnett’s Multiple Comparison Test Results - TEWL -SLS

(Compares 4-Untreated and Treatments 1 ,2, and 3)

** Significant difference between Treatmentl and Treatment 2 Multiple Comparison Summary:

Dunnett’s test comparing Vehicle and All other treatments are shown in the data tables. Tukey’s test comparing All Treatments to All Treatments are shown in the data tables. A summary of the TEWL results from samples irritated with UV are summarized in Table 4 and

Table 5 and in FIG. 8A and FIG. 8B.

Table 4. Results of Statistical Analysis - TEWL -UV

* Significant change from baseline ** Significant difference between treatments - multiple comparisons are indicated on the following page

[1] Percent change is calculated from the mean changes from baseline [2] Percent change is calculated individually by subject and averaged (Note if 0 at baseline, % not calculated for that subject)

Table 5. Dunnett’s Multiple Comparison Test Results - TEWL - UV (Compares Untreated and Treatments 1 ,2, and 3)

** Significant difference between Treatment 1 and Treatment 2

Multiple Comparison Summary:

Dunnett’s test comparing Vehicle and the other treatments are shown in the data tables. Tukey’s test comparing All Treatments to All Treatments are shown in the data tables.

A summary of the visual irritancy results from samples irritated with SLS are summarized in Table 6 and Table 7 and in FIG. 9A and FIG. 9B. Table 6. Results of Statistical Analysis - Visual Irritancy - SLS

* Significant change from baseline

** Significant difference between treatments - multiple comparisons are indicated on the following page

[1] Percent change is calculated from the mean changes from baseline

[2] Percent change is calculated individually by subject and averaged (Note if 0 at baseline, % not calculated for that subject)

Table 7. Dunnett’s Multiple Comparison Test Results - Visual Irritancy - SLS

(Compares Untreated to Treatments 1 ,2, and 3)

** Significant difference between Treatment 1 and Treatment 2

Multiple Comparison Summary:

A summary of the visual irritancy results from samples irritated with UV are summarized in Table 8 and Table 9 and in FIG. 10A and FIG. 10B. Table 8. Results of Statistical Analysis - Visual Irritancy - UV

* Significant change from baseline

[1] Percent change is calculated from the mean changes from baseline

Table 9. Results of Statistical Analysis - Visual Irritancy and UV

Dunnett’s Multiple Comparison Test Results

(Compares Untreated to Treatments 1 ,2, and 3)

** Significant difference between Treatment 1 and Treatment 2

Conclusions

Based on the statistical analysis, it can be concluded, test articles 1 and 2 show anti-inflammatory efficacy and improvements to skin barrier functionality when compared to the untreated site and vehicle after pre-treatment SLS irritation under the conditions of the study.

Furthermore, based on the statistical analysis, it can be concluded, test articles 1 and 2 show antiinflammatory efficacy and improvements to skin barrier functionality when compared to the untreated and vehicle sites after pre-treatment irritation from UV (MED) exposure under the conditions of the study.

Example 3. Investigation of the efficacy of various moisturizer formulations on skin firmness, barrier function and fine lines and wrinkles over a 28-day use period

Summary of the Clinical Study

The investigation was a split-face, subject-blind, single center, home use designed study performed over a 28-Day use period. The Test Articles studied included: Test Article #1 : 2’-FL, Test Article #2: L-Fucose, Test Article #3: Matrixyl+ Vehicle, Test Article #4: Glycerin (3%), and Test Article #5: Vehicle. An adequate number of female subjects were enrolled so that at least 105 subjects completed the study with 15 subjects per treatment group. The subjects were healthy female subjects, aged 18 - 65 years, representing all skin types. The objective of the study is to evaluate the efficacy of various moisturizer formulations on skin firmness, barrier function and fine lines and wrinkles over a 28-Day use period using bioinstrumentation and imaging. Consumer perception information was also collected. Skin firmness was measured with the Cutometer® at baseline and on Days 14 and 28 on each side of the face. TEWL was measured with a Tewameter® at Baseline, and on Days 14 and 28 on each side of the face. Fine lines and wrinkles were measured using VISIA-CR® Photographic images taken at Baseline, and on Days 14 and 28. A Self-Perception Questionnaire (SPQ) assessing skin attributes, product efficacy and perceived improvement completed by subjects on Days 14 and 28.

Objective The objective of this study was to evaluate the efficacy of various moisturizer formulations on skin firmness, barrier function and fine lines and wrinkles over a 28-Day use period using bioinstrumentation, imaging, and collection of subject perception.

Study Design The study was a split-face, subject-blind, single center, home use design. Potential female subjects participated in a 28-Day treatment period. Subjects were issued the test articles to use twice a day, over the 28-Day study duration. Study assessments were conducted as shown in the table below.

Table 10. Summary of Study Timeline

I Adverse Event Review |

Selection of Subjects

Female subjects were enrolled into the study to allow for at least 15 subjects per treatment group (seven groups in total) to complete the study. Subjects satisfied the following inclusion and exclusion criteria. The suitability of each subject to participate was confirmed prior to their acceptance onto the study by completion and review of a study specific eligibility questionnaire.

Inclusion Criteria

• Healthy female volunteers aged 18 through 65 years (inclusive).

• All skin types i.e., normal, dry, oily and combination.

• Score > 2.0 for facial lines/wrinkles as determined by the evaluator at the

• baseline visit.

• Willing to follow study instructions and available to attend all study visits.

• Provide written Informed Consent and Photo Release Form.

• Completed a three-day wash-out period before the first (Baseline) visit having not used any moisturizing products (i.e., moisturizers, creams, lotions, eye creams, serums, treatments) on the face.

Exclusion Criteria

• Subject had a known allergy or hypersensitivity to moisturizing products or similar materials or their ingredients.

• Self-reported pregnant or breast feeding or planning to become pregnant during the course of the study.

• Any skin disease on the face (e.g., rosacea, eczema, psoriasis, etc.).

• Moderate to severe acne on the face.

• Any conditions on the face that would interfere with evaluations (i.e., tattoos, scars, open cuts, sunburn, piercings, excessive hair, etc.).

• Use of facial chemical peel in the last 14 days.

• Use of topical treatments such as OTC (over the counter) acne medication, hydroquinone, or hydrocortisone on the test site in the last month.

• Insulin-dependent diabetes.

• Medical condition which, in the opinion of the Investigator, would compromise the safety of the subject or confound study results.

• Concurrent medication that might affect the response to the test articles including routine use of anti-inflammatory medications, antihistamines, and steroids.

• Use of Isotretinoin/Accutane, Retin-A, Retinol, in the last three months. • Cosmetic medical procedures in the test area such as injectable antiwrinkle products, facial cosmetic surgery, etc. in the last year.

Prohibitions/Reauirements

• Subject agreed to use the test articles twice a day as instructed during the 28-Day treatment period.

• During the 28-Day treatment period, subject agreed not to apply any moisturizers or treatments to the face other than the provided test articles.

• Subjects could continue to use their usual cleanser, sunscreen, lip products and make-up.

• Subject agreed to attend all study visits with a clean face, no make-up or other facial products, including test articles, applied.

• Subject agreed not to introduce any new products (cleanser, sunscreen, cosmetics) to their face while participating in the study.

• Subject agreed to protect their face from excessive sun exposure or any form of tanning for the duration of the study. Subjects could wear their usual sunscreen to aid in this effort.

Method

Test Articles

The Sponsor supplied a sufficient quantity of the following test articles for each subject enrolled onto the study, which included the components described in Table 1 1. Test articles were labelled to maintain a subject-blind study design and were formulated with 10 % (w/w) squalane, 10 % (w/w) soy oil, 3 % (w/w) Montanov® 68, 0.5 % (w/w) phenoxyethanol, and the remaining portion was water.

Table 11 . Components of Test Articles

The test articles were used as supplied by the Sponsor. Subjects who qualified to participate in the study were placed in one of seven treatment groups. Subjects were issued two test articles according to the randomization, one for one side of the face selected from the test articles 1 to 5 listed above and the vehicle (test article 5) for the other side of the face. Subjects were instructed to apply the test articles to the appropriate side of the face, as determined by the randomization, twice a day during the 28-Day treatment period. Use Instructions:

Morning

1 . Wash face and pat dry.

2. Take the moisturizer marked Right and dispense a pea-sized amount. Massage onto the Right side of the face until absorbed.

3. Take the moisturizer marked Left and dispense a pea-sized amount. Massage onto the Left side of the face until absorbed.

Evening

1 . Wash face and pat dry.

Subjects will be given the instructions verbally and in writing.

Study Procedure

During screening, potential study participants were contacted via telephone or e-mail to inform them not to apply any moisturizing products (i.e., moisturizers, creams, lotions, eye creams, serums, treatments) on their face during the three days prior to the Baseline visit. Use of usual cleansers and/or cosmetics was permitted. Potential participants were also be informed to attend the first study visit with a clean face (no make-up or other facial products).

Visit 1 ; Day 1 : Participants attended the test center at Baseline where they will be provided with the Informed Consent Form to read and given the opportunity to ask questions about the study during the consent discussion. Once written Informed Consent and Photo Release form had been provided, a brief medical history was collected, concomitant medications were recorded, and eligibility criteria were reviewed. The subject agreed to follow all study instructions and the prohibitions/requirements. Subjects eligible to participate in the study were evaluated by the clinical grader for facial fine lines/wrinkles according to the scoring scale. Subjects who qualified with a score of > 2.0 for fine lines/wrinkles subjects were assigned a final subject number. Following a 20-minute acclimation period to indoor ambient conditions, subjects underwent remaining Baseline assessments. Photographic images were taken with VISIA-CR®, firmness measurements will be taken with Cutometer®, and trans-epidermal water loss were measured with Tewameter® Subjects were given two test articles according to the randomization along with instructions (written and verbal) to apply each test article twice a day, morning and evening by massaging a pea-sized amount onto the designated half of their clean face until absorbed. Subjects were instructed to replace their usual facial moisturizer with the test articles provided and not to use any other skin care products (only usual cleanser, make-up, lip product, and sunscreen permitted) on the face during the 28-Day study period. New facial skin care or cosmetic products were not to be introduced. Subjects also received a diary to record the twice daily application of the test articles during the 28-Day treatment period.

Visit 2; Day 14 (± 1 day): Subjects returned to the testing facility after 14 days of test articles use. Subjects were instructed to return with the test articles and usage diary for review. Study staff asked and document any changes to subjects’ health or medication or adverse event to the test articles. The diary and test articles were assessed for compliance with test articles usage instructions. Following a 20- minute acclimation period to indoor ambient conditions, subjects had firmness measurements with Cutometer®, TEWL measurements with Tewameter® and photographic images taken with VISIA-CR®. Subjects completed an SPQ assessing the test articles’ acceptability and attributes.

Visit 3; Day 28 (± 1 day): Subjects returned to the testing facility after 28 days of test articles use. Subjects were instructed to return with the test articles and usage diary for review. Study staff asked and documented any changes to subjects’ health or medication or adverse event to the test articles. The diary and test articles were assessed for compliance with test articles usage instructions. Following a 20- minute acclimation period to indoor ambient conditions, subjects had firmness measurements with Cutometer®, TEWL measurements with Tewameter® and photographic images taken with VISIA-CR®. Subjects completed an SPQ assessing the test articles’ acceptability and attributes. At the conclusion of the visit, the subject’s participation was considered complete.

Assessments

Assessments included in this study consisted of bioinstrumentation and self-perception questionnaires. Prior to having bio-instrument measurements, subjects acclimated to indoor ambient temperatures for at least 20 minutes.

Clinical Grader Assessments

The same trained clinical grader conducted assessments of fine lines and wrinkles according to the following scoring scale in Table 12. At the first visit, the clinical grader evaluated prospective subjects to determine eligibility to participate in the study. Subjects meeting qualification criteria score of > 2.0 for fine lines and wrinkles on the face were assigned a final number and continued on the study. Table 12. Clinical Assessment Scoring Scale

VISIA-CR® Image Capture

High resolution digital facial images were taken of the front and both sides of the face using the VISIA-CR® (Canfield Scientific; Parsippany, NJ) under standard 1 , standard 2, parallel polarized, crosspolarized, and UV lighting modes. Images were taken of the subject’s clean face i.e., no make-up (including mascara, lipstick and eyeliner) or jewelry at Baseline, Day 14 and Day 28. Subjects were required to wear a black headband and a black cape to make the images as similar as possible. Subjects were instructed to remain still and in a relaxed state while photos are captured using the VISIA-CR® equipment. VISIA-CR® images were captured first, prior to any other bioinstrumentation assessments. Facial images were taken at Baseline, Day 14, and Day 28.

Firmness via Cutometer®

The Cutometer® (Courage + Khazaka; Koln, Germany) was used to measure firmness. One measurement was taken from both the right and left cheek. Measuring mode 1 was used with constant suction of 400 mbar for five seconds followed by relaxation time of 3 seconds with three repetitions. The parameter measured was RO. The Cutometer® measurements were taken at Baseline, Day 14 and at Day 28.

Trans-epidermal Water Loss (TEWL) via Tewameter®

Trans-epidermal water loss was measured with the Tewameter® TM150 (Courage and Khazaka; Koln, Germany). The measurement of the water evaporation, and therefore TEWL, was based on the diffusion principle in an open chamber. The density gradient was measured indirectly by two pairs of sensors in the probe attachment, one for temperature and the other for relative humidity. This density gradient was then analyzed by a microprocessor in the instrument. One reading was taken from each side of the face. The Tewameter® measurements were taken at Baseline, Day 14 and at Day 28.

Self-Perception Questionnaire

Subjects completed a Self-Perception Questionnaire (SPQ) to gauge the subject’s perception of the test article’s properties, efficacy, and perceived improvements on Day 14 and at the final study visit, Day 28. Subjects determined their level of agreement to statements about the test articles utilizing a four-point Likert scale and questionnaire in Table 13.

Table 13. Self-Perception Questionnaire

Statistical Analysis

The data for statistical analysis included the following:

• Cutometer® measurements of firmness (RO) collected at Baseline, Day 14, and Day 28.

• Tewameter® measurements of trans-epidermal water loss collected at Baseline, Day 14, and Day 28.

• VISIA®-CR fine lines and wrinkles parameters from images collected at

• Baseline, Day 14, and Day 28.

• Subjective responses on the SPQ collected on Day 14 and Day 28.

Descriptive statistics (mean, standard deviation, N) were provided for all data. Within treatment Bioinstrumentation analysis utilized t-tests on the changes from Baseline. Between treatment analysis of bioinstrumentation data consisted of analysis of covariance utilizing Tukey’s multiple comparison test when appropriate. Self-Perception Questionnaire responses were summarized specifying percent agreement for each SPQ statement (with Strongly Agree and Agree as the top box scores). Between treatment analysis of Self-Perception Questionnaire data consisted of comparing the top box percentages using chi-squared statistics. All statistical tests of hypothesis employed a level of significance of 0.05 and no adjustments will be made for the number of tests performed. A summary of the results for Test Article #1 , Test Article #2, Test Article #3, Test Article #4, and Test Article #5 are found in Tables 14-17 respectively. A summary of the results achieved by each test article is provided in Table 18.

Table 14. Results of Statistical Analysis - Test Article 1 vs Vehicle

* Significant change from baseline ** Significant difference between treated and Vehicle

[1] Percent change is calculated from the mean changes from baseline

[2] Percent change is calculated individually by subject and averaged

5

Table 15. Results of Statistical Analysis - Test Article 2 vs Vehicle

[1] Percent change is calculated from the mean changes from baseline

[2] Percent change is calculated individually by subject and averaged

5

Table 16. Results of Statistical Analysis - Test Article 3 vs Vehicle

[1] Percent change is calculated from the mean changes from baseline

[2] Percent change is calculated individually by subject and averaged

Table 17. Results of Statistical Analysis - Test Article 4 vs Vehicle

[1] Percent change is calculated from the mean changes from baseline

[2] Percent change is calculated individually by subject and averaged

Table 18. Comparison of Test Articles

Evaluation

** Significant overall difference between treatments

Example 4. Investigation of the efficacy of 2’-FL on reducing inflammation and treating erythema

An erythema like ex vivo model was used where skin biopsies were exposed to 2% (w/v) sodium dodecyl sulphate (SDS, Merck, Germany) for 10 min at 37 °C. After incubating, culture media was removed, and each explant was abundantly washed with phosphate buffer saline (PBS). For treatment studies, 25 pL of each formulation was topically applied once daily after inflammation was induced, for three days. The formulation employed for the topical application of 2’-FL, retinol, and niacinamide consisted of the following components: 5% (w/w), 2.5% (w/w), or 1 % (w/w) of 2’-FL (or niacinamide) + 10% (w/w) Squalane + 10% (w/w) Soy oil + 3% (w/w) Montanov® 68 + 0.5% (w/w) phenoxyethanol + the remaining percentage of water. The formulations containing retinol were identical, except retinol was utilized at concentrations of 0.1 % and 0.2%, and the remaining proportion comprised water. As a control, a vehicle formulation (the same formulation without the biomolecules under investigation) was also included.

Histology: Skin models were fixed in 4 % (m/v) paraformaldehyde, dehydrated, embedded in paraffin, and cut into 4 pm sections. Tissue sections were stained with haematoxylin & eosin (Sigma, USA) following a routine protocol. For immunohistochemistry, paraffin tissue sections were deparaffinized in xylene, re-hydrated and antigen retrieval was performed for 1 h at 55 °C in Tris-EDTA pH 9 buffer. Afterwards, unspecific staining was blocked with 2.5 % Horse Serum (Vector Labs, USA). Primary antibodies - IL-6 (1 :50; R&D Systems, MAB2061 ) and IL-1 a (1 :40; Abeam, ab7632) were incubated overnight at 4 °C. For detection, sections were incubated for 1 hour at room temperature with the secondary antibodies - Alexa Fluor 488 donkey anti-rabbit or Alexa Fluor 594 donkey anti-mouse (Life Technologies) - at a concentration of 1 :500. Nuclei were counterstained with DAPL All samples were examined under an upright Imager.MI Microscope (Zeiss, Germany). Image analyses of several markers was automatically quantified with the Cell Profiler software (FIGS. 14A and 14B).

Example 5. Effects of 2’-fucosyllactose on filaggrin in the skin Materials and Methods

Contact Irritation ex vivo model: Samples were harvested from routine abdominoplasties of healthy female anonymous donors. It was also established that donors' BMI could not be superior to 29.9. Written informed consent from all participants was obtained under the protocol established and approved by the Ethical Committee of Hospital Lusiadas (Porto, Portugal) and Universidade Catolica Portuguesa (UCP, Porto, Portugal). Before any experiment, the fat adipose tissue was carefully removed with scissors, taking care to preserve the dermis from damage, and 12-mm-diameter punch biopsies were excised. Skin biopsies were exposed to 2 % (v/v) sodium dodecyl sulfate (SDS, Merck, Germany) for 10 min at 37 °C. After incubating, culture media was removed, and each explant was abundantly washed with phosphate buffer saline (PBS, Sigma-Aldrich). Then, 25 pL of 0.1 % (w/w) retinol, 0.2 %(w/w) retinol, 2.5 % (w/w) niacinamide, 5 % (w/w) niacinamide, 2.5 % (w/w) 2’-FL, or 5 % (w/w) 2’-FL was topically applied once daily for two days.

Immunohistochemistry: At day 3, skin samples were fixed in 10% (v/v) neutral buffered formalin (Bio Optica, Italy), dehydrated, embedded in paraffin using a tissue processor (Leica Biosystems, USA) and cut into 5 pm sections (Microtome CUT 5062, SLEE medical GmbH, Germany). Tissue sections were deparaffinized in xylene, re-hydrated, and incubated for 1 h in Tris-EDTA buffer (0 mM Tris Base, 1 mM EDTA solution and 0.05% (v/v) Tween 20, pH 9) in a water bath at 55 °C for antigen retrieval. Afterwards, unspecific staining was blocked with 2.5% (v/v) Horse Serum (Vector Labs, USA). Unconjugated primary anti-filaggrin antibody (1 :500, Abeam, USA) was incubated overnight in a humidified chamber at 4 °C. For detection, sections were then incubated with the universal antibody from the R.T.U. VECTASTAIN Elite ABC Kit (Vector Labs, USA). The peroxidase substrate kit (DAB, Vector Labs, USA) was used according to the manufacturer’s instructions. Nuclei were stained with Gill’s hematoxylin (Bio-Optica, Italy). All samples were examined under an upright Imager.MI Microscope and images were processed using the Zen Software 3.2.

Image analysis: Five images of random fields were acquired for each condition and independent experiment and used to infer about the filaggrin area. For each image, the filaggrin area and the total stratum corneum area were quantified with the FIJI for Imaged software (version 2.0.0-rc42). Results are presented as filaggrin area per total stratum corneum area.

Statistical analysis: GraphPad Prism software (v 8.2.1 , La Jolla, USA) was used to perform the statistical analysis. Data was analyzed using the Shapiro-Wilk normality test. A one-way analysis of variance (ANOVA) with a Tukey multiple comparison post-test was used. Significance was set to * p < 0.05.

Results

After exposure of the skin to the irritant, SDS, the negative and the positive controls showed almost the complete absence of filaggrin. However, when samples were treated with either concentrations of 2’-FL or niacinamide, there was an increase in filaggrin deposition, re-establishing the skin’s stratum corneum that was damaged after SDS exposure (FIGS. 15 and 16).

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing inflammation in the skin of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising a human milk oligosaccharide (HMO).

2. A method of reducing irritation in the skin of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

3. The method of claim 1 or 2, wherein the inflammation or irritation is caused by psoriasis.

4. The method of claim 1 or 2, wherein the inflammation or irritation is caused by acne.

5. The method of claim 1 or 2, wherein the inflammation or irritation is caused by rosacea.

6. The method of claim 1 or 2, wherein the inflammation or irritation is caused by eczema.

7. The method of claim 1 or 2, wherein the inflammation or irritation is caused by erythema.

8. The method of claim 1 or 2, wherein the inflammation or irritation is caused by ultraviolet (UV) radiation

9. The method of claim 1 or 2, wherein the inflammation or irritation is caused by environmental pollution, optionally wherein the environmental pollution is air pollution.

10. The method of claim 1 or 2, wherein the inflammation or irritation is caused by dehydration.

11 . The method of claim 1 or 2, wherein the inflammation or irritation is caused by exposure to a chemical compound.

12. The method of any one of claims 1 -1 1 , wherein the inflammation or irritation in the skin of the subject is reduced by at least 10% in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied, optionally wherein the inflammation or irritation in the skin of the subject reduced by between 10% and 50% in comparison to a reference skin of a subject to which the composition comprising an HMO was not applied.

13. A method of treating or preventing psoriasis, acne, rosacea, eczema, erythema, UV radiation- induced inflammation or irritation, environmental pollution-induced inflammation or irritation, dehydration- induced inflammation or irritation, and/or chemical exposure induced inflammation or irritation in a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

14. A method of achieving an anti-aging effect in the skin of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

15. A method of reducing the appearance of fine lines or wrinkles in the skin of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

16. A method of increasing skin firmness or elasticity in the skin of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

17. A method of reducing oxidative stress in skin cells of a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

18. A method of increasing hydration in skin cells of a subject and/or improving skin barrier function in a subject, wherein the method comprises topically applying to the skin of the subject a composition comprising an HMO.

19. The method of any one of claims 1 -18, wherein the HMO is selected from L-fucose, 2’-fucosyllactose (2’-FL), lacto-N-neotetraose (LNnT), 3-fucosyllactose (3’-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N-difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto-N-hexaose (F- LNH) I, F-LNH II, difucosyllacto-N-hexaose (DFLNH) I, DFLNH II, difucosyllacto-N-neohexaose (DFLNnH), difucosyl-para-lacto-N-hexaose (DF-para-LNH), difucosyl-para-lacto-N-neohexaose (DF-para- LNnH), trifucosyllacto-N-hexaose (TF-LNH), 3’-siallylactose (3’-SL), 6’-siallylactose (6’-SL), sialyl lacto-N- tetraose (LST) a, LST b, LST c, disialyllacto-N-tetraose (DS-LNT), fucosyl-sialyllacto-N-tetraose (F-LST) a, F-LST b, fucosyl-sialyllacto-N-hexaose (FS-LNH), fucosyl-sialyllacto-N-neohexaose (FS-LNnH) I, fucosyl-disialyllacto-N-hexaose (FDS-LNH) II, or a combination thereof.

20. The method of claim 19, wherein the HMO is 2’-FL.

21. The method of claim 19, wherein the HMO is L-fucose.

22. The method of any one of claims 1 -21 , wherein the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL.

23. The method of claim 22, wherein the composition has a concentration of the HMO of between about 10 mg/mL and about 50 mg/mL.

24. The method of claim 22, wherein the composition has a concentration of the HMO of about 10 mg/mL.

25. The method of claim 22, wherein the composition has a concentration of the HMO of about 25 mg/mL.

26. The method of claim 22, wherein the composition has a concentration of the HMO of about 50 mg/mL.

27. The method of any one of claims 1 -26, wherein the composition further comprises squalane.

28. The method of claim 27, wherein the composition has a concentration of squalane of between about 1% (w/w) and about 60% (w/w).

29. The method of claim 28, wherein the composition has a concentration of squalane of between about 10% (w/w) and about 50% (w/w).

30. The method of claim 29, wherein the composition has a concentration of squalane of about 42% (w/w).

31 . The method of claim 28, wherein the composition has a concentration of squalane of between about 1% (w/w) and about 20% (w/w).

32. The method of claim 31 , wherein the composition has a concentration of squalane of about 10% (w/w).

33. The method of any one of claims 1 -32, wherein the composition further comprises carboxymethyl cellulose (CMC).

34. The method of claim 33, wherein the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w).

35. The method of claim 34, wherein the composition has a concentration of CMC of between about 1% (w/w) and about 3% (w/w).

36. The method of claim 35, wherein the composition has a concentration of CMC of between about 2% (w/w).

37. The method of any one of claims 1 -36, wherein the composition further comprises lecithin.

38. The method of claim 37, wherein the composition has a concentration of lecithin of between about 0.01% (w/w) and about 1% (w/w).

39. The method of claim 38, wherein the composition has a concentration of lecithin of between about 0.05% (w/w) and about 0.5% (w/w).

40. The method of claim 39, wherein the composition has a concentration of lecithin of about 0.1% (w/w).

41 . The method of any one of claims 1 -40, wherein the composition further comprises phenoxyethanol.

42. The method of claim 41 , wherein the composition has a concentration of phenoxyethanol of about between about 0.01% (w/w) and 5% (w/w).

43. The method of claim 42, wherein the composition has a concentration of phenoxyethanol of about 0.5% (w/w).

44. The method of any one of claims 1 -43, wherein the composition further comprises cetearyl glucoside.

45. The method of claim 44, wherein the composition further comprises cetearyl alcohol and cetearyl glucoside.

46. The method of claim 45, wherein the composition comprises Montanov®.

47. The method of claim 45 or 46, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of between about 0.5% (w/w) and about 10% (w/w).

48. The method of claim 47, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w).

49. The method of any one of claims 1 -48, wherein the composition further comprises oil.

50. The method of claim 49, wherein the oil is soy oil.

51. The method of claim 50, wherein the composition has a concentration of soy oil of between about 1 % (w/w) and about 25% (w/w).

52. The method of claim 51 , wherein the composition has a concentration of soy oil of about 10% (w/w).

53. The method of any one of claims 1 -52, wherein the composition further comprises glyceride.

54. The method of claim 53, wherein the composition comprises Gelucire®.

55. The method of claim 53 or 54, wherein the composition has a concentration of glyceride of between about 1% (w/w) and about 20% (w/w).

56. The method of claim 55, wherein the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w).

57. The method of claim 56, wherein the composition has a concentration of glyceride of between about 8% (w/w).

58. The method of any one of claims 1 -57, wherein the composition is a patch, liquid, gel, lotion, paste, cream, foam, serum, ointment, or stick.

59. The method of any one of claims 1 -58, wherein the composition is applied to the skin of the subject one or more times daily, optionally wherein the composition is applied to the subject once daily, twice daily, or three times daily.

60. The method of claim 59, wherein the composition is applied to the skin of the subject once daily.

61 . The method of claim 59, wherein the composition is applied to the skin of the subject twice daily, optionally wherein the composition is administered to the subject once in the morning and once in the evening.

62. The method of claim 59, wherein the composition is applied to the skin of the subject three times daily.

63. The method of any one of claims 1 -62, wherein the composition is applied to the skin of the subject for at least 1 day.

64. The method of any one of claims 1 -62, wherein the composition is applied to the skin of the subject for at least 2 continuous days.

65. The method of any one of claims 1 -62, wherein the composition is applied to the skin of the subject for at least 7 continuous days.

66. The method of any one of claims 1 -62, wherein the composition is applied to the skin of the subject for at least 14 continuous days.

67. The method of any one of claims 1 -62, wherein the composition is applied to the skin of the subject for at least 28 continuous days.

68. A pharmaceutical composition comprising an HMO and one or more excipients selected from squalane, carboxymethyl cellulose, lecithin, cetearyl alcohol and cetearyl glucoside, oil, phenoxyethanol, and glyceride.

69. The pharmaceutical composition of claim 68, wherein the HMO is selected from any one of L-fucose, LNnT, 2’-FL, 3’-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, 6’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, FDS-LNH II, or a combination thereof.

70. The pharmaceutical composition of claim 69, wherein the HMO is 2’-FL.

71 . The pharmaceutical composition of claim 69, wherein the HMO is L-fucose.

72. The pharmaceutical composition of any one of claims 68-71 , wherein the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL.

73. The pharmaceutical composition of claim 72, wherein the composition has a concentration of the HMO of between about 10 mg/mL and about 50 mg/mL.

74. The pharmaceutical composition of claim 72, wherein the composition has a concentration of the HMO of about 10 mg/mL.

75. The pharmaceutical composition of claim 72, wherein the composition has a concentration of the HMO of about 25 mg/mL.

76. The pharmaceutical composition of claim 72, wherein the composition has a concentration of the HMO of about 50 mg/mL.

77. The pharmaceutical composition of any one of claims 68-76, wherein the composition comprises squalane.

78. The pharmaceutical composition of claim 77, wherein the composition has a concentration of squalane of between about 1 % (w/w) and about 60% (w/w).

79. The pharmaceutical composition of claim 78, wherein the composition has a concentration of squalane of between about 10% (w/w) and about 50% (w/w).

80. The pharmaceutical composition of claim 79, wherein the composition has a concentration of squalane of about 42% (w/w).

81 . The pharmaceutical composition of claim 28, wherein the composition has a concentration of squalane of between about 1 % (w/w) and about 20% (w/w).

82. The pharmaceutical of claim 31 , wherein the composition has a concentration of squalane of about 10% (w/w).

83. The pharmaceutical composition of any one of claims 62-82, wherein the composition comprises CMC.

84. The pharmaceutical composition of claim 83, wherein the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w).

85. The pharmaceutical composition of claim 84, wherein the composition has a concentration of CMC of between about 1 % (w/w) and about 3% (w/w).

86. The pharmaceutical composition of claim 85, wherein the composition has a concentration of CMC of between about 2% (w/w).

87. The pharmaceutical composition of any one of claims 68-86, wherein the composition comprises lecithin.

88. The pharmaceutical composition of claim 87, wherein the composition has a concentration of lecithin of between about 0.01% (w/w) and about 1 % (w/w).

89. The pharmaceutical composition of claim 88, wherein the composition has a concentration of lecithin of between about 0.05% (w/w) and about 0.5% (w/w).

90. The pharmaceutical composition of claim 89, wherein the composition has a concentration of lecithin of about 0.1% (w/w).

91 . The pharmaceutical composition of any one of claims 68-90, wherein the composition further comprises phenoxyethanol.

92. The pharmaceutical composition of claim 91 , wherein the composition has a concentration of phenoxyethanol of about between about 0.01% (w/w) and 5% (w/w).

93. The pharmaceutical composition of claim 92, wherein the composition has a concentration of phenoxyethanol of about 0.5%.

94. The pharmaceutical composition of any one of claims 68-93, wherein the composition further comprises cetearyl glucoside.

95. The pharmaceutical composition of claim 94, wherein the composition further comprises cetearyl alcohol and cetearyl glucoside.

96. The pharmaceutical composition of claim 95, wherein the composition comprises Montanov®.

97. The pharmaceutical composition of claim 95 or 96, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of between about 0.5% (w/w) and about 10% (w/w).

98. The pharmaceutical composition of claim 97, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w).

99. The pharmaceutical composition of any one of claims 68-98, wherein the composition further comprises oil.

100. The pharmaceutical composition of claim 99, wherein the oil is soy oil.

101 . The pharmaceutical composition of claim 100, wherein the composition has a concentration of soy oil of between about 1 % (w/w) and about 25% (w/w).

102. The pharmaceutical composition of claim 91 , wherein the composition has a concentration of soy oil of about 10% (w/w).

103. The pharmaceutical composition of any one of claims 68-102, wherein the composition comprises glyceride.

104. The pharmaceutical composition of claim 103, wherein the composition comprises Gelucire®.

105. The pharmaceutical composition of claim 103 or 104, wherein the composition has a concentration of glyceride of between about 1% (w/w) and about 20% (w/w).

106. The pharmaceutical composition of claim 105, wherein the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w).

107. The pharmaceutical composition of claim 106, wherein the composition has a concentration of glyceride of between about 8% (w/w).

108. A cosmetic composition comprising an HMO and one or more excipients selected from squalane, carboxymethyl cellulose, lecithin, cetearyl alcohol and cetearyl glucoside, oil, phenoxyethanol, and glyceride.

109. The cosmetic composition of claim 108, wherein the HMO is selected from any one of L-fucose, LNnT, 2’-FL, 3’-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, 6’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, FDS-LNH II, or a combination thereof.

110. The cosmetic composition of claim 109, wherein the HMO is 2’-FL.

111. The cosmetic composition of claim 109, wherein the HMO is L-fucose.

112. The cosmetic composition of any one of claims 108-11 1 , wherein the composition has a concentration of the HMO of between about 1 mg/mL and about 150 mg/mL.

113. The cosmetic composition of claim 112, wherein the composition has a concentration of the HMO of between about 1 mg/mL and about 50 mg/mL.

114. The cosmetic composition of claim 112, wherein the composition has a concentration of the HMO of about 10 mg/mL.

115. The cosmetic composition of claim 1 12, wherein the composition has a concentration of the HMO of about 25 mg/mL.

116. The cosmetic composition of claim 1 12, wherein the composition has a concentration of the HMO of about 50 mg/mL.

117. The cosmetic composition of any one of claims 108-116, wherein the composition comprises squalane.

118. The cosmetic composition of claim 1 17, wherein the composition has a concentration of squalane of between about 1 % (w/w) and about 60% (w/w).

119. The cosmetic composition of claim 1 18, wherein the composition has a concentration of squalane of between about 10% (w/w) and about 50% (w/w).

120. The cosmetic composition of claim 1 19, wherein the composition has a concentration of squalane of about 42% (w/w).

121 . The pharmaceutical composition of claim 118, wherein the composition has a concentration of squalane of between about 1 % (w/w) and about 20% (w/w).

122. The pharmaceutical of claim 121 , wherein the composition has a concentration of squalane of about 10% (w/w).

123. The cosmetic composition of any one of claims 108-122, wherein the composition comprises CMC.

124. The cosmetic composition of claim 123, wherein the composition has a concentration of CMC of between about 0.5% (w/w) and about 5% (w/w).

125. The cosmetic composition of claim 124, wherein the composition has a concentration of CMC of between about 1 % (w/w) and about 3% (w/w).

126. The cosmetic composition of claim 125, wherein the composition has a concentration of CMC of between about 2% (w/w).

127. The cosmetic composition of any one of claims 108-126, wherein the composition further comprises phenoxyethanol.

128. The cosmetic composition of claim 127, wherein the composition has a concentration of phenoxyethanol of about between about 0.01% (w/w) and 5% (w/w).

129. The cosmetic composition of claim 128, wherein the composition has a concentration of phenoxyethanol of about 0.5%.

130. The cosmetic composition of any one of claims 108-129, wherein the composition further comprises cetearyl glucoside.

131 . The cosmetic composition of claim 130, wherein the composition further comprises cetearyl alcohol and cetearyl glucoside.

132. The cosmetic composition of claim 131 , wherein the composition comprises Montanov®.

133. The cosmetic composition of claim 131 or 132, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of between about 0.5% (w/w) and about 10% (w/w).

134. The cosmetic composition of claim 133, wherein the composition has a concentration of cetearyl alcohol and cetearyl glucoside of about 3% (w/w).

135. The cosmetic composition of any one of claims 108-134, wherein the composition further comprises oil.

136. The cosmetic composition of claim 135, wherein the oil is soy oil.

137. The cosmetic composition of claim 136, wherein the composition has a concentration of soy oil of between about 1 % (w/w) and about 25% (w/w).

138. The cosmetic composition of claim 137, wherein the composition has a concentration of soy oil of about 10% (w/w).

139. The cosmetic composition of any one of claims 108-139, wherein the composition comprises lecithin.

140. The cosmetic composition of claim 139, wherein the composition has a concentration of lecithin of between about 0.01% (w/w) and about 1% (w/w).

141 . The cosmetic composition of claim 140, wherein the composition has a concentration of lecithin of between about 0.05% (w/w) and about 0.5% (w/w).

142. The cosmetic composition of claim 141 , wherein the composition has a concentration of lecithin of about 0.1% (w/w).

143. The cosmetic composition of any one of claims 78-98, wherein the composition comprises glyceride.

144. The cosmetic composition of claim 143, wherein the composition comprises Gelucire®.

145. The cosmetic composition of claim 143 or 144, wherein the composition has a concentration of glyceride of between about 1% (w/w) and about 20% (w/w).

146. The cosmetic composition of claim 145, wherein the composition has a concentration of glyceride of between about 5% (w/w) and about 10% (w/w).

147. The cosmetic composition of claim 146, wherein the composition has a concentration of glyceride of between about 8% (w/w).