Methods of screening for exfoliation capacity

Abstract

The present disclosure provides a method for measuring exfoliation capacity of an analyte on a skin sample. Also provided are exfoliating compositions comprising one or more alpha hydroxy acids (AHAs) and one or more polyhydroxy acids (PHAs). In some embodiments, it includes (a) contacting a skin sample with a test solution comprising an effective amount of an analyte, (b) incubating the skin sample with the test solution, (c) removing the test solution from contact with the skin sample and adding a detection reagent, and (d) detecting the presence or absence of cells and / or cellular debris from the test solution, wherein the presence of cells and / or cellular debris in the test solution is indicative of the exfoliation capacity of the analyte.

Description

Attorney Docket No.: ROF-018WOMETHODS OF SCREENING FOR EXFOLIATION CAPACITY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to United States Provisional Patent Application No. 63 / 773,829, filed on March 18, 2025, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides a quantifiable exfoliation assay to screen exfoliation capacity of chemical or biological exfoliants, (e.g., alpha hydroxy acid (AHA), beta hydroxy acid (BHA), polyhydroxy acid (PHA), enzymes, or amino acids or blends thereof) on human skin and exfoliating compositions comprising chemical and / or biological exfoliants.BACKGROUND

[0003] Skin exfoliation is a means to desquamate surface dead skin cells, remove dirt from pores and improve absorption of subsequent treatments. In terms of the clinical benefits, concentration of acid and pH of the chemical peel either used alone, or in combination with additional treatments, can reduce the appearance of mild dyschromic skin, photodamaged skin, enlarged pores and blemishes, revealing more even toned and smoother skin. While many compounds are used as cosmetic exfoliants, effective methods of measuring exfoliation capacity remain elusive. Prior methods of measuring exfoliation capacity suffer from inefficiencies and inaccuracies in quantifying cell debris removed during exfoliation.Accordingly, new methods are required to adequately measure exfoliation capacity.SUMMARY

[0004] Disclosed herein, in various embodiments, is a method of measuring exfoliation capacity of an analyte, the method comprising steps of: (a) contacting a skin sample with a test solution comprising an effective amount of the analyte; (b) incubating the skin sample with the test solution for a period of time to allow the analyte to exfoliate the skin sample; (c) removing the test solution from contact with the skin sample and adding a detection reagent to the test solution, wherein the detection reagent permits the detection of cells and cellular debris in the test solution; (d) detecting the presence or absence of cells and / or cellular debris from the test solution, wherein the presence of cells and / or cellular debris in the test solution is indicative of the exfoliation capacity of the analyte.Attorney Docket No.: ROF-018WO

[0005] In some embodiments, the method further comprises steps of: (a) after adding the detection reagent, isolating cells and / or cellular debris, if present, from the test solution; and (b) preparing a detection solution comprising the cells and / or cellular debris. In some embodiments, the method further comprises steps of: (a) after isolating the cells and cellular debris from the test solution, contacting the skin sample with a wash solution; (b) isolating cells and / or cellular debris, if present, from the wash solution; and adding the isolated cells and / or cellular debris to the detection solution. In some embodiments, the detection reagent is extracted from the cells and / or cellular debris in the detection solution. In some embodiments, the wash solution comprises phosphate-buffered saline (PBS). In some embodiments, the detection solution comprises PBS.

[0006] In some embodiments, the skin sample is a mammalian sample. In some embodiments, the skin sample is a human skin sample.

[0007] In some embodiments, the incubating is from about 30 minutes long to about 24 hours long. In some embodiments, the test solution comprises a buffering agent. In some embodiments, the buffering agent comprises citrate. In some embodiments, the test solution further comprises at least one humectant. In some embodiments, the at least one humectant is selected from the group consisting of glycerin, methylpropane diol, propane diol, and sodium hyaluronate. In some embodiments, the test solution further comprises a gelling agent. In some embodiments, the gelling agent is xanthan gum. In some embodiments, the test solution comprises trisodium citrate, citric acid, glycerin, methylpropane diol, xanthan gum, and sodium hyaluronate. In some embodiments, the test solution comprises trisodium citrate, citric acid, propane diol, and sodium hyaluronate. In some embodiments, the test solution has a pH ranging from about 3.5 to about 8. In some embodiments, the test solution has a pH of about 4. In some embodiments, the test solution has a pH of about 6.

[0008] In some embodiments, the detection reagent is a cell-staining dye. In some embodiments, the detection reagent is trypan blue. In some embodiments, the cells are skin cells. In some embodiments, the cells are comeocytes. In some embodiments, the detecting comprises measuring optical density of cells and / or cellular debris. In some embodiments, the detecting comprises measuring optical density of a detection reagent extracted from the cells and / or cellular debris. In some embodiments, the detecting is performed using a spectrophotometer. In some embodiments, the detecting comprises measuring the absolute count of cells and / or cellular debris. In some embodiments, the detecting is performed using aAttorney Docket No.: ROF-018WOcell counter. In some embodiments, the detecting is performed using a flow cytometer. In some embodiments, the method is performed ex vivo or in vitro.

[0009] Also disclosed herein, in various embodiments, is an exfoliating composition comprising one or more alpha hydroxy acids (AHAs) and one or more polyhydroxy acids (PHAs). In some embodiments, the one or more AHAs are independently selected from the group consisting of glycolic acid, lactic acid, citric acid, malic acid, mandelic acid, and tartaric acid. In some embodiments, the one or more PHAs are independently selected from the group consisting of glucono-D-lactone, lactobionic acid, and maltobionic acid.

[0010] In some embodiments, the exfoliating composition further comprises a beta hydroxy acid. In some embodiments, the BHA is salicylic acid. In some embodiments, the exfoliating composition further comprises a protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the exfoliating composition further comprises phytic acid.

[0011] In some embodiments, the composition comprises glycolic acid and glucono-D-lactone at a ratio of glycolic acid to glucono-D-lactone of about 1 : 1 by weight. In some embodiments, the composition comprises glycolic acid and maltobionic acid at a ratio of glycolic acid to maltobionic acid of about 3:1 by weight. In some embodiments, the composition comprises lactic acid and glucono-D-lactone at a ratio of lactic acid to glucono-D-lactone of about 1.2:1 by weight. In some embodiments, the composition comprises lactic acid and maltobionic acid at a ratio of lactic acid to maltobionic acid of about 2:1 by weight.

[0012] In some embodiments, the composition comprises glucono-D-lactone, salicylic acid, and glycolic acid. In some embodiments, the composition comprises glucono-D-lactone, maltobionic acid, and glycolic acid. In some embodiments, the composition comprises glucono-D-lactone, salicylic acid, lactic acid, and glycolic acid. In some embodiments, the composition comprises maltobionic acid, salicylic acid, lactic acid, and glycolic acid.

[0013] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, and about 3% glycolic acid by weight.

[0014] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 3% glucono-D-lactone by weight, about 1% maltobionic acid by weight, and about 3% glycolic acid by weight.

[0015] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 3% glucono-D-lactone by weight, about 0.2% salicylic acid by weight,Attorney Docket No.: ROF-018WOabout 3.7% lactic acid by weight, and about 3% glycolic acid by weight. In some embodiments, the exfoliating composition further comprises about 0.15% of a cysteine protease by weight.

[0016] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, about 3.7% lactic acid by weight, and about 3% glycolic acid by weight. In some embodiments, the exfoliating composition further comprises about 0.12% of a cysteine protease by weight.

[0017] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 1.2% glucono-D-lactone by weight, about 0.08% salicylic acid by weight, about 1.4% lactic acid by weight, and about 1.2% glycolic acid by weight. In some embodiments, the exfoliating composition further comprises about 0.065% phytic acid by weight.

[0018] Also disclosed herein, in various embodiments, is an exfoliating composition comprising about 1% maltobionic acid by weight, about 2% salicylic acid by weight, about 2% lactic acid by weight, and about 3% glycolic acid by weight.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGs. 1A-1F depict scatter plot representations of a trypan blue plate reader screening assay to quantify ex vivo exfoliation capacity after a 24 hour treatment with indicated component on human skin punch biopsies. The horizontal lines and error bars represent the mean and standard deviation, respectively. Solid datapoints indicate measurements taken at pH 4-5; hollow datapoints indicate measurements taken at pH 6. Significance: ****, p <0.0001; ***, p<0.0002; **, p < 0.0021; *P < 0.032. FIG. 1A depicts measurement of the exfoliation capacity of glycolic acid. FIG. IB depicts measurement of the exfoliation capacity of lactic acid. FIG. 1C depicts measurement of the exfoliation capacity of mandelic acid. FIG. ID depicts measurement of the exfoliation capacity of malic acid. FIG. IE depicts measurement of the exfoliation capacity of glucono-d-lactone. FIG. IF depicts measurement of the exfoliation capacity of carnitine tartrate.

[0020] FIGs. 2A-2C depict scatter plot representations of a trypan blue plate reader screening assay to quantify ex vivo exfoliation capacity after treatment of human skin punch biopsies with indicated compositions. The horizontal lines and error bars represent the mean and standard deviation, respectively. Solid datapoints indicate measurements taken at pH 4- 5; hollow datapoints indicate measurements taken at pH 6. Significance: ****, p <0.0001;Attorney Docket No.: ROF-018WO***, p<0.0002; **, p < 0.0021; *P < 0.032. FIG. 2A depicts measurement after 24 hrs. of treatment with Blend 1 and Blend 2. FIG. 2B depicts measurement after 30 min on human skin punch biopsies with indicated biological exfoliant compositions. FIG. 2C depicts measurement after 30 min on human skin punch biopsies with indicated biological exfoliant compositions.DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure provides methods of measuring exfoliation capacity of an analyte. In some embodiments, the method comprises steps of (a) contacting a skin sample with a test solution comprising the analyte; (b) incubating the skin sample with the test solution for a period of time to allow the analyte composition to exfoliate the skin sample; (c) removing the test solution from contact with the skin sample and adding a detection reagent, wherein the detection reagent permits the detection of cells in the test solution; and (d) detecting the presence or absence of cells from the test solution. In some embodiments, the presence of cells in the test solution is indicative of the exfoliation capacity of the analyte. In some embodiments, the method further comprises steps of after adding the detection reagent, isolating cells and / or cellular debris, if present, from the test solution; and preparing a detection solution comprising the cells and / or cellular debris. In some embodiments, the method further comprises steps of after isolating the cells and cellular debris from the test solution, contacting the skin sample with a wash solution; isolating cells and / or cellular debris, if present, from the wash solution; and adding the isolated cells and / or cellular debris to the detection solution.

[0022] The methods provided herein provide an improvement over a previously reported method that utilizes pig skin to screen exfoliation capacity by counting exfoliated corneocytes using a hemocytometer see Ahn, et al. (2019). Journal of cosmetic dermatology, 18(5), 1404-1409), which is neither efficient nor accurate in quantifying cell debris due to time- and labor-intensive manual cell counting and aggregation of cell debris. Modifying the previously reported method to count cells by flow cytometry is efficient and straight forward for intact corneocytes but difficult to accurately count intact cells and debris due to thresholding issues. In contrast, the presently disclosed method employs a detection reagent that permits the detection of cells and cellular debris and measures the optical density of the cells and / or debris after extraction by UV / vis spectroscopy. The presently disclosed method allows for rapid screening of analytes {e.g., chemical peeling agents), ranking of exfoliation efficiency, and evaluation of chemical and biological exfoliants. The presently disclosed method allowsAttorney Docket No.: ROF-018WOquantification, e.g., of corneocytes, released by analytes (e.g., chemical peeling agents) reproducibly and with limited variability, permitting screens for effective exfoliating analytes. Furthermore, the previously reported method may not be able to evaluate the exfoliation capacity of complete cosmetic formulas because other ingredients in the formula can interfere with the methods and generate inconsistent results.

[0023] The present disclosure also provides exfoliating compositions comprising one or more alpha hydroxy acids (AHAs) and one or more polyhydroxy acids (PHAs). The exfoliating compositions provided herein exhibit a surprising technical effect of having a comparable exfoliation capacity for the AHAs to compositions comprising approximately 1.5 to 3 times the concentration of AHA alone. The compositions provided herein are therefore advantageous as they can provide comparable exfoliating properties to compositions providing the AHAs alone while having reduced potential to cause skin irritation.Definitions

[0024] It must be noted that, as used herein, and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0025] As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45% 55%.

[0026] “Optional” or “optionally” may be taken to mean that the subsequently described structure, event or circumstance may or may not occur, and that the described includes instances where the event occurs and instances where it does not.

[0027] “Administering” when used in conjunction with a therapeutic means to administer a therapeutic directly or indirectly into or onto a target tissue to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted.“Administering” may include the act of self-administration or administration by another person such as a health care provider.

[0028] The term “improves” is used to convey that the present invention changes either the appearance, foim, characteristics, structure, function and / or physical attributes of the tissue to which it is being provided, applied or administered. “Improves" may also refer to the overall physical state of an individual to whom an active agent has been administered. For example, the overall physical state of an individual may “improve” if one or more symptoms of the disease, condition or disorder are alleviated by administration of an active agent.Attorney Docket No.: ROF-018WO

[0029] The term “cosmetic” means an agent utilized, and / or intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, altering the appearance of the skin or any combination thereof.

[0030] The terms “effective amount” or “effective dose” as used herein are interchangeable and may refer to the amount of an active agent or compound or composition that has the effect of moisturizing, cleansing, beautifying, promoting attractiveness, altering the appearance of the skin, or any combination thereof, that is being sought by the user. In some embodiments, “effective amount” or “effective dose” as used herein are interchangeable and may refer to the amount of an active agent or compound or composition that has the effect of promoting the exfoliation of dry skin, promoting the digestion of desmosomes, normalizing cell maturation, modulating keratinocyte function, normalizing keratinocyte differentiation, normalizing keratinocyte proliferation, modulating phosphatidylglycerol content of keratinocytes, modulating and / or improving the moisture content of the skin, promoting the retention of long lasting hydration in the skin, increasing skin moisture, improving skin water balance, increasing skin hydration, decreasing transepidermal water loss, reducing evaporation of water from the skin, treating and / or preventing dry and / or irritated skin, defending against, and reducing visible signs of aging for noticeably firmer, smoother, and flawless looking skin, erasing the appearance of premature aging, including brown spots, dullness and discoloration, visibly brightening the skin, reducing the appearance of fine lines and wrinkles, creating a radiant complexion, shielding the skin again biological and environmental aggressors associated with dry, irritated and sensitive skin, helping the skin retain moisture and remain comfortable when challenged by climate and other environmental aggressors, rehydrating the skin, to repairing, renewing, and / or enhancing the skin’s natural moisture barrier.

[0031] The term “inhibit,” “suppress,” “decrease,” “interfere,” and / or “reduce” (and like terms) generally refers to the act of reducing, either directly or indirectly, a function, activity, or behavior relative to the natural, expected, or average or relative to current conditions.

[0032] The term “increase,” “enhance,” “stimulate,” and / or “induce” (and like terms) generally refers to the act of improving or increasing, either directly or indirectly, a function or behavior relative to the natural, expected, or average or relative to current conditions.

[0033] The term “modulate,” “modify,” and / or “modulator” generally refers to the act of directly or indirectly promoting / stimulating or interfering with / inhibiting a specific functionAttorney Docket No.: ROF-018WOor behavior. In some instances a modulator may increase and / or decrease a certain activity or function relative to its natural state or relative to the average level of activity that would generally be expected or relative to a current level of activity.

[0034] As used herein, the term “normalize” refers to the act of establishing and / or maintaining a relative balance or equilibrium between two or more activities, functions or conditions.

[0035] The term “cosmetic composition” shall mean a composition including at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for detenuining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.

[0036] As used herein, the term “topically” and “topical” refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues.Methods of Measuring Exfoliation Capacity

[0037] In various embodiments, the methods provided herein measure the exfoliation capacity of an analyte. In some embodiments, the method is performed ex vivo or in vitro. “Exfoliation” refers to the removal of comeocytes, the dead cells composing the outer layer of the skin, or cellular debris, e.g., by chemical dissolution of the bonds that hold corneocytes together. Exfoliation is rooted in the skin’s natural desquamation process, where corneocytes are gradually shed as enzymes break down the corneodesmosomes that hold them together. As aging, environmental stress, or disrupted enzyme activity slows this natural shedding, dead cells accumulate, making the skin appear dull — so physical or chemical exfoliation helps accelerate the removal of these redundant corneocytes and supports smoother, more radiant skin. As used herein, the term “exfoliation capacity” refers to the relative efficiency with which a compound or composition removes corneocytes.

[0038] In various embodiments, the method provided herein comprises a step of incubating a skin sample with a test solution comprising an analyte for a period of time to allow the analyte to exfoliate the skin sample. As will be understood, the duration of the incubation can be any amount of time suitable to allow exfoliation and can be adjusted based on the analyte. In some embodiments, the incubation is for a duration from about 30 minutes to about 24 hours. In some embodiments, the incubation is for a duration of about 30 minutes. In someAttorney Docket No.: ROF-018WOembodiments, the incubation is for a duration of about 1 hour. In some embodiments, the incubation is for a duration of about 2 hours. In some embodiments, the incubation is for a duration of about 3 hours. In some embodiments, the incubation is for a duration of about 4 hours. In some embodiments, the incubation is for a duration of about 5 hours. In some embodiments, the incubation is for a duration of about 6 hours. In some embodiments, the incubation is for a duration of about 12 hours. In some embodiments, the incubation is for a duration of about 18 hours. In some embodiments, the incubation is for a duration of about 24 hours.Detection of Cells and Cellular Debris

[0039] In various embodiments, the method comprises a step of detecting the presence or absence of cells and / or cellular debris. As used herein “detecting the presence or absence of cells and / or cellular debris,” refers to detecting either or both of cells and cellular debris present in a test solution following incubation with a skin sample. The skilled person will understand that the method may be used to measure the exfoliation capacity of an analyte when the test solution is substantially free of either or both of cells and or cellular debris. In some embodiments, the cells are skin cells. In some embodiments, the cells are corneocytes. In some embodiments the cellular debris is debris from corneocytes. In some embodiments, a high detected level of cells and / or cellular debris indicates a high exfoliation capacity of the analyte.

[0040] As will be understood, the means of detecting cells can be selected based on the detection agent. In some embodiments, the detecting cells and / or cellular debris comprises measuring an optical density of cells and / or cellular debris. In some embodiments, the detecting cells and / or cellular debris comprises measuring an optical density of a detection reagent extracted from cells and / or cellular debris. In some embodiments, the detecting cells and / or cellular debris comprises an enzyme-linked immunosorbent assay (ELISA) against a surface marker present on the cells and / or cellular debris. In some embodiments, the detecting is performed using a spectrophotometer. In some embodiments, the detecting cells and / or cellular debris comprises measuring the absolute count of cells and / or cellular debris. In some embodiments the detecting is performed using a cell counter. In some embodiments the detecting is performed using a flow cytometer.ReagentsSkin SamplesAttorney Docket No.: ROF-018WO

[0041] In various embodiments, the methods provided herein employ skin samples to measure the exfoliation capacity of an analyte. In some embodiments, the skin sample is a mammalian skin sample. In some embodiments, the skin sample is a human skin sample, a porcine skin sample, or a rodent skin sample. In some embodiments, the skin sample is a porcine skin sample. In some embodiments, the skin sample is a human skin sample. In some embodiments the skin sample is a freshly isolated skin sample. In some embodiments, the skin sample is a cadaver skin sample. In some embodiments, the skin sample is an artificial skin sample. In some embodiments, the skin sample is a human skin equivalent. In some embodiments, the skin sample is reconstructed human skin.Analytes and Test Solutions

[0042] In some embodiments, the test solution further comprises a buffering agent. In some embodiments, the buffering agent is selected from the group consisting of citrate, phosphate, tris, HEPES, acetate, MES, borate, carbonate, TAPS, bicine, tricine, TAPSO, TES, MOPS, PIPES, and cacodylate. In some embodiments, the buffering agent comprises a citrate. In some embodiments, the buffering agent comprises trisodium citrate and citric acid.

[0043] In some embodiments, the test solution further comprises at least one humectant. In some embodiments, the at least one humectant is selected from the group consisting of glycerin, methylpropane diol, propane diol, sodium hyaluronate, diglycerin, betaine, diols, propylene glycol, butylene glycol, pentylene glycol, propanediol, 1,2-hexanediol, D-ribose, glucose, sorbitol, dextrose, urea, 2-Pyrrolidone-5-Carboxylic Acid and related salts, sea salt, inorganic salts of citric acid, inorganic salts of lactic acid, ectoin, lactic acid, betaine, glycolic acid, lactobionic acid, and any combination thereof.

[0044] In some embodiments, the test solution further comprises a gelling agent. In some embodiments, the gelling agent is a water phase gelling agent. In some embodiments, the water phase gelling agent is selected from the group consisting of xanthan gum, gellan gum, carrageenan, biosaccharide gum-I, sclerotium gum, pectin, pullulan, guar gum, gum arabic, chondroitin, sulfate, alginic acid, sodium hyaluronate, hydrolyzed hyaluronic acid sodium polyglutamate, chitin, chitosan, starch, and combinations thereof. In some embodiments, the gelling agent is xanthan gum.

[0045] In some embodiments, the test solution comprises trisodium citrate, citric acid, glycerin, methylpropane diol, xanthan gum, and sodium hyaluronate.Attorney Docket No.: ROF-018WO

[0046] In some embodiments, the test solution comprises trisodium citrate, citric acid, propane diol, and sodium hyaluronate.

[0047] In some embodiments, the test solution has a pH ranging from about 3.5 to about 8. In some embodiments, the test solution has a pH of about 3.5. In some embodiments, the test solution has a pH of about 4. In some embodiments, the test solution has a pH of about 4.5. In some embodiments, the test solution has a pH of about 5. In some embodiments, the test solution has a pH of about 5.5. In some embodiments, the test solution has a pH of about 6. In some embodiments, the test solution has a pH of about 6.5. In some embodiments, the test solution has a pH of about 7. In some embodiments, the test solution has a pH of about 7.5. In some embodiments, the test solution has a pH of about 8.

[0048] In some embodiments, the method comprises, after isolating the cells and cellular debris from the test solution, contacting the skin sample with a wash solution. In some embodiments, the method comprises isolating cells and / or cellular debris, if present, from the wash solution, and adding the isolated cells and / or cellular debris to the detection solution. In some embodiments, the wash solution comprises phosphate-buffered saline (PBS), trisbuffered saline (TBS), HEPES-buffered saline (HBS), or Hanks’ balanced salt solution (HBSS). In some embodiments, the wash solution comprises PBS.Detection Reagents and Solutions

[0049] In various embodiments, the method comprises contacting the cells with a detection reagent. In some embodiments, the detection reagent permits the detection of cells and cell debris in the test solution. In some embodiments, the detection agent is a dye capable of binding cells and cellular debris. In some embodiments, the detection agent is selected from the group consisting of trypan blue, erythrosin B, dansyl chloride, dihydroxy acetone, methylene blue, nile red, and propidium iodide. In some embodiments, the detection agent is trypan blue.

[0050] In various embodiments, the method comprises, prior to detecting the presence or absence of cells and / or cellular debris, isolating the cells and / or cellular debris, if present, from the test solution and preparing a detection solution comprising the cells and / or cellular debris. In some embodiments, the detection reagent is extracted from the cells and / or cellular debris into the detection solution. In some embodiments, the detection reagent is extracted into the detection solution by heating the cells and / or cellular debris in the detection solution. As will be understood by the skilled person, the detection solution can be selected based onAttorney Docket No.: ROF-018WOthe method of detection. For example, where the detection comprises measuring the optical density of the solution, the detection solution is preferably optically clear in the wavelength of detection. In some embodiments, the detection solution comprises phosphate-buffered saline (PBS), tris-buffered saline (TBS), HEPES -buffered saline (HBS), or Hanks’ balanced salt solution (HBSS). In some embodiments, the detection solution comprises PBS.Exfoliating Compositions

[0051] In various embodiments, the present disclosure provides exfoliation compositions comprising one or more alpha hydroxy acids (AHAs) and one or more polyhydroxy acids (PHAs). In some embodiments, the AHAs are selected from the group consisting of glycolic acid, lactic acid, citric acid, malic acid, mandelic acid, and tartaric acid. In some embodiments, the AHA is glycolic acid. In some embodiments, the AHAs are glycolic acid and lactic acid.

[0052] In some embodiments, the PHAs are selected from the group consisting of glucono-D-lactone, lactobionic acid, and maltobionic acid. In some embodiments, the PHA is glucono-D-lactone. In some embodiments, the PHA is maltobionic acid.

[0053] In some embodiments, the composition further comprises a beta hydroxy acid (BHA). In some embodiments, the BHA is salicylic acid.

[0054] In some embodiments, the composition comprises glucono-D-lactone, salicylic acid, and glycolic acid. In some embodiments, the composition comprises glucono-D-lactone, maltobionic acid, and glycolic acid. In some embodiments, the composition comprises glucono-D-lactone, salicylic acid, lactic acid, and glycolic acid. In some embodiments, the composition comprises maltobionic acid, salicylic acid, lactic acid, and glycolic acid.

[0055] In some embodiments, the concentration of AHA is about 2.6% by weight to about 6.7% by weight of the composition. In some embodiments, glycolic acid is present in the composition at a concentration of about 3% by weight. In some embodiments, glycolic acid is present in the composition at a concentration of about 1.2% by weight. In some embodiments, lactic acid is present in the composition at a concentration of about 3.7% by weight. In some embodiments, lactic acid is present in the composition at a concentration of about 1.4% by weight. In some embodiments, glycolic acid is present in the composition at a concentration of about 3% by weight and lactic acid is present in the composition at a concentration of about 3.7% by weight. In some embodiments, glycolic acid is present in theAttorney Docket No.: ROF-018WOcomposition at a concentration of about 1.2% by weight and lactic acid is present in the composition at a concentration of about 1.4% by weight.

[0056] In some embodiments, the concentration of PHA in the composition is about 3% by weight to about 4% by weight. In some embodiments, glucono-D-lactone is present in the composition at a concentration of about 3% by weight. In some embodiments, glucono-D-lactone is present in the composition at a concentration of about 1.2% by weight. In some embodiments maltobionic acid is present in the composition at a concentration of about 1%. In some embodiments, glucono-D-lactone is present in the composition at a concentration of about 3% by weight and maltobionic acid is present in the composition at a concentration of about 1% by weight.

[0057] In some embodiments, the concentration of BHA in the composition is about 0.2% by weight to about 2% by weight. In some embodiments, salicylic acid is present in the composition at a concentration of about 0.08% by weight. In some embodiments, salicylic acid is present in the composition at a concentration of about 0.2% by weight. In some embodiments, salicylic acid is present in the composition at a concentration of about 2% by weight.

[0058] In some embodiments, the composition further comprises a protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the protease is present in the composition at a concentration of about 0.5% by weight.

[0059] In some embodiments, the composition further comprises phytic acid. In some embodiments, the phytic acid is present in the composition at a concentration of about 2% by weight.

[0060] In some embodiments, the composition comprises glucono-D-lactone, salicylic acid, glycolic acid, and a cysteine protease. In some embodiments, the composition comprises maltobionic acid, glucono-D-lactone, phytic acid, glycolic acid, and a cysteine protease. In some embodiments, the composition comprises glucono-D-lactone, lactic acid, glycolic acid, salicylic acid, and a cysteine protease. In some embodiments, the composition comprises glucono-D-lactone, lactic acid, glycolic acid, and salicylic acid.

[0061] In some embodiments, the composition comprises a ratio of glycolic acid to glucono-D-lactone of about 1:1 by weight. In some embodiments, the composition comprises a ratio of glycolic acid to maltobionic acid of about 3:1 by weight. In some embodiments, the composition comprises a ratio of lactic acid to glucono-D-lactone of about 1.2:1 by weight. InAttorney Docket No.: ROF-018WOsome embodiments, the composition comprises a ratio of lactic acid to maltobionic acid of about 2: 1 by weight.

[0062] In some embodiments, the composition comprises about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, and about 3% glycolic acid by weight.

[0063] In some embodiments, the composition comprises about 3% glucono-D-lactone by weight, about 1% maltobionic acid by weight, and about 3% glycolic acid by weight.

[0064] In some embodiments, the composition comprises about 3% glucono-D-lactone by weight, about 0.2% salicylic acid by weight, about 3.7% lactic acid by weight, and about 3% glycolic acid by weight.

[0065] In some embodiments, the composition comprises about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, about 3.7% lactic acid by weight, and about 3% glycolic acid by weight.

[0066] In some embodiments, the composition comprises about 1.2% glucono-D-lactone by weight, about 0.08% salicylic acid by weight, about 1.4% lactic acid by weight, and about 1.2% glycolic acid by weight.

[0067] In some embodiments, the composition comprises about 1% maltobionic acid by weight, about 2% salicylic acid by weight, about 2% lactic acid by weight, and about 3% glycolic acid by weight.EXAMPLES

[0068] The subject matter is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the claims should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.Example 1: Design and Testing of an Exemplary Method of Measuring Exfoliation CapacityPreparation of Test Solutions

[0069] Acids were prepared to their corresponding % w / w by dissolution in minimal water and then neutralized to a pH of 4 or 6 with NaOH solution in water; QS (quantum satis to 100%) with water. Proteases were prepared by dissolution in water or Solution A (water, 10%Attorney Docket No.: ROF-018WObutylene glycol, and 1% xanthan gum) and their pH was adjusted with either a citric acid (aq.) or NaOH solution.Ethical Approval and Informed Consent

[0070] Sections of human skin used for screening were obtained from a consented adult volunteer donor during an elective surgical procedure in the United States. Each sample was derived from a competent volunteer adult donor who has signed an Institutional Review Board (IRB) validated donor consent form that specifically lists both the intended uses for the donation for non-clinical research and confirms the procedures for processing the samples are Standard Operating Procedure (SOP) managed protocols in compliance with ethical regulations. All samples were collected and processed in the United States.Preparation of Skin Samples

[0071] Due to the nature of the assay, tissue from multiple donors were needed for each study. Assay development studies were conducted on 12 mm punch biopsies obtained from a Caucasian, Female, age 45, BMI 21.4, abdomen (Donor 1), and screening studies were conducted on 6 mm punch biopsies obtained from female donors including Caucasian donors, age 31, BMI 29.8; thigh (Donor 2), the second age 54, BMI 25.2; abdomen (Donor 3), a third age 40, BMI 29.2; abdomen (Donor 4), a fourth age 34, BMI 32.9; abdomen (Donor 6) and an African American female donor age 47, BMI 30.6, abdomen (Donor 5).

[0072] Evaluation of the exfoliation efficiency of a series of chemical exfoliants (Table 1) was conducted on 6 mm punch biopsies obtained from two 100 cm2skin biopsies from a Caucasian, Female, age 31, BMI 29.8; Surgical depot: thigh (SKIN042921B, Donor 1). For the study of the exfoliation efficiency of a series of biological exfoliants, 6 mm punch biopsies were obtained from two 100 cm2skin biopsies from a Caucasian, Female, age 54, BMI 25.2; Surgical depot: Abdomen (SKIN021721B, Donor 2).Sample Treatment and Assay Methods

[0073] A trypan blue cellular debris assay (called “Trypan blue method”) was developed to quantify the media content of cell debris because of cell death. This method was modified for quantifying the corneocytes released by chemical peeling agents on skin. Assay conditions followed the general protocol outlined below, with samples being added on the surface of skin punches kept at a consistent size placed in microtiter plates. Trypan blue dye was added post treatment to corneocytes and removed after heating at 80°C.Trypan Blue MethodAttorney Docket No.: ROF-018WO1. Frozen skin tissues were thawed and rinsed 3 times with PBS to remove excess material from skin surface. 6 mm punches were collected and placed, epidermis side facing up, in 96-well plate.2. For solutions containing 0.1% TritonX-100, 1.5 pL of neat TritonX-100 was added to 1.5 mL of test solution (Table 1-2) and mixed.3. Explants were covered with 100 pl of test solution (infinite dose). Five (5) replicate explants were used for each test condition.4. Explants were then incubated in a 5% CO2 cell culture incubator for 24 or 8 hours or 30 minutes at 37° C, with relative humidity of 85-90%.5. At the end of treatment, the entire volume of samples containing the released comeocytes was removed and 100 pL PBS was added to the wells to rinse the explants. PBS was removed and combined with the corresponding samples.6. 200 pL 0.4% trypan blue solution was added to the samples and the released comeocytes were stained for 30 min at room temperature.7. The comeocytes were pelleted via centrifugation at 4,000-4,500 x g for 20 minutes. The supernatant was discarded and the comeocyte pellet was gently washed with 100-200 pL isopropanol for 2-5 minutes.8. Comeocytes were pelleted via centrifugation at 4,000-4,500 x g for 20 min at room temperature. Supernatant was removed and 50-100 pL of PBS was added to extract the stain from the corneocyte pellet for 15 minutes at 80° C.9. The PBS extract was separated from comeocytes via centrifugation at 4,000 x g for 15-20 minutes.10. The blue-colored extract was cooled to room temperature and 40 pl was transferred into a 384- well plate. The optical density of the PBS extract was measured using a spectrophotometer at a wavelength of 590 nm. Blanks without comeocytes were included.Pilot Exfoliation StudyA. Determine the number of comeocytes released from skin explants1. The surface of skin tissue was gently cleaned and blood and fluid were removed from the tissue.2. Skin explants were prepared from the skin tissue (diameter 6 mm, 200-300 mm thickness) and place them into a 96-well plate.3. 100 pL test solution containing 0.1% Triton X-100 was added to each well. The negative control was water. Each treatment was performed at least in triplicate.Attorney Docket No.: ROF-018WO4. Skin explants were incubated at 37° C with 50% humidity for 24 hrs.5. At the end of treatment, the supernatant was removed and then 100 pL PBS was added to rinse the explants in each well. Supernatant and PBS were combined in a microcentrifuge tube.6. 20 pL of supematant / PBS was placed on a hemocytometer to count the released comeocytes from the explants. Images of cell area on the hemocytometer were taken to check if the corneocytes clump together.7. The number of comeocytes was analyzed with cell flow cytometer and cell image analyzer to determine the best way to count corneocytes. If the corneocytes were not easily seen under microscope, they may have needed to be stained (e.g., with trypan blue) prior to the examination.B. Determine the intensity of trypan blue bound to comeocytes1. 2 sets of treatments were prepared. One was test solution + 0.1% Triton X-100 and the other was test solution only. Following the above steps 1-5, 200 pL 0.4% trypan blue solution were added to each micro-centrifuge tube containing supematant / PBS and tubes were briefly vortexed to mix the solution. Cell debris were allowed to stain on the bench top for 15 min at room temperature.2. Comeocytes were pelleted via centrifugation at 12,000 x g for 5 min at room temperature and the supernatant was discarded using a pipette, taking care not to disturb the pellet, if present.3. Pellets were washed by adding 500 pL of 100% isopropanol. The tubes were slowly inverted a couple of times, and then isopropanol was removed using a pipette while being careful not to disturb the trypan blue-stained comeocytes pellet.4. 100 pL of PBS was added to each of the tubes in order to extract the stain from the pellet. Pellets were agitated using a vortex and heat the tubes using a dry block heater for 15 min at 80° C.5. Samples were centrifuged at 12,000 x g for 5 min to remove corneocytes from the blue-colored PBS extract.6. Blue-colored PBS extract was loaded into a 96-well plate as technical duplicates of 45 pL and measure the optical density of the extract using a spectrophotometer at a wavelength of 590 nm. Make sure to include a blank without corneocytes.Statistical analysis:Attorney Docket No.: ROF-018WO

[0074] For each experimental condition, the mean of statistical replicates ± standard deviation (STDEV) and standard error of the mean (SEM) were calculated. Significance between two groups was determined using unpaired, two-tail Student's t test. Significance between means of one group and control samples was determined using one-way ANOVA with Dunnett’ s multiple comparisons and, significance between means of multiple groups was determined using one-way ANOVA with Tukey’s multiple comparisons. Statistical analysis was performed using GraphPad PRISM 9. For all statistical analysis, a p-value of <0.03 was considered significant. Designations were used from GraphPad PRISM software. Test solutions breakdown was used to validate the Trypan Blue human ex vivo exfoliation assay with mean OD590 reading.

[0075] The method was used to initially screen individual acids (alpha-hydroxy acid (AHAs), beta-hydroxy acid (BHAs), and poly-hydroxy acid (PHAs)), amino acids at two concentrations and two pHs (Tables 1-3) after treating tissues with test solution for 24 hours.

[0076] Chemical exfoliants were used to screen comeocyte removal after 24 hours of treatment on human skin explants and Trypan blue plate reader assay (96 wells, 6 mm punch biopsy, Donor 1) to quantify exfoliated corneocytes. Skin biopsies were treated with 100 pL of each test solution or control for 24 hours. Statistics were determined with a Students t-test with a p-value < 0.05 considered significant. Buffer 1 was a 0.1 M citric acid / sodium citrate buffer (pH 3.99). Buffer 2 was a 0.1 M citric acid / sodium citrate buffer at pH 5.9.Table 1. Human Skin Exfoliation Assay Screening of a-Hydroxy AcidsSolution FoldEx %w / w Acid pH Mean STDEV SEM p-value (QS) changewater n / a water - 1.22 1.0 0.14 0.06 0.936 TritonX- n / a water - 100 1.23 1.0 0.18 0.08 1.000Glycolicla Water 4acid 2.64 2.1 0.26 0.11 0.000 10 %Glycoliclb Water 6acid 2.60 2.1 0.08 0.03 0.000 Glycolic1c 3 % Water 4acid 2.66 2.2 0.16 0.07 0.000Attorney Docket No.: ROF-018WOGlycolicId Water 6acid 1.24 1.0 0.31 0.14 0.942 Lactic2a Water 4acid 2.74 2.2 0.02 0.01 0.000 10 %Lactic2b Water 6acid 2.30 1.9 0.27 0.12 0.000 Lactic2c Water 5acid 2.40 2.0 0.26 0.12 0.000 2.5 %Lactic2d Water 6acid 1.20 1.0 0.35 0.16 0.892 Mandelic3a Water 4acid 1.89 1.5 0.40 0.18 0.018 10 %Mandelic3b Water 6acid 1.32 1.1 0.16 0.07 0.463 Mandelic3c Water 4acid 1.29 1.0 0.24 0.11 0.686 3 %Mandelic3d Water 6acid 1.51 1.2 0.44 0.20 0.271 Malic4a Buffer 1 4acid 2.71 2.2 0.01 0.00 0.000 10 %Malic4b Buffer 2 6acid 1.96 1.6 0.40 0.18 0.012 Malic4c Buffer 1 4acid 2.47 2.0 0.25 0.11 0.000 3 %Malic4d Buffer 2 6acid 1.17 1.0 0.28 0.12 0.735

[0077] Chemical exfoliants were used to screen comeocyte removal after 24 hours of treatment on human skin explants and exfoliation capacity was assessed using the trypan blue plate reader assay (96 wells, 6 mm punch biopsy, Donor 1). Skin biopsies were treated with 100 pL of each test solution or control for 24 hours. Statistics were determined with a Students t-test with a p-value < 0.05 considered significant. Buffer 1 was a 0.1 M citric acid / sodium citrate buffer (pH 3.99). Buffer 2 was a 0.1 M citric acid / sodium citrate buffer at pH 5.9. Solution A contained 0.71 % w / w trisodium citrate, 0.31 %w / w citric acid, glycerin, methylpropane diol, xanthan gum, sodium hyaluronate (and) water. Solution B containedAttorney Docket No.: ROF-018WO1.21 %vilvi trisodium citrate, 1.13 %w7w citric acid, water, propane diol, sodium hyaluronate (and) water. PA = phytic acid, PHA = polyhydroxy acid.Table 2: Human Skin Exfoliation Assay Screening of p- and Poly-Hydroxy Acids Solution FoldEx %w / w Acid pH Mean STDEV SEM p- value (QS) changewater n / a water - 0.85 1.0 0.20 0.09 0.974 Tritonn / a water - 0.86 1.0 0.35 0.16 1.000 X-100Salicylic Buffer5a 4 1.22 1.4 0.38 0.17 0.183 acid 1 / Ethanol2 %Salicylic Buffer5b 6 1.14 1.3 0.20 0.09 0.168 acid 2 / EthanolSalicylic Buffer5c 4 1.10 1.3 0.18 0.08 0.222 acid 1 / Ethanol0.2 %Salicylic Buffer5d 6 1.06 1.2 0.33 0.15 0.417 acid 2 / EthanolGlucono-d- 6a Buffer 1 4 2.34 2.7 0.24 0.11 0.000 lactone10 %Glucono-d- 6b Buffer 2 6 1.22 1.4 0.29 0.13 0.140 lactoneGlucono-d- 6c Buffer 1 4 2.06 2.4 0.56 0.25 0.008 lactone3 %Glucono-d- 6d Buffer 2 6 1.00 1.2 0.38 0.17 0.588 lactoneAHA / BHABlend Solution / PHA 4 2.72 3.2 0.02 0.01 0.000 1 A(6:2:2)Blend PHA / PA Solution4 2.49 2.9 0.32 0.14 0.000 2 (2:2) B

[0078] Next, chemical exfoliants were used to screen corneocyte removal after 24 hours of treatment on human skin explants, and exfoliation potential was assessed using the trypan blue plate reader assay (96 wells, 6 mm punch biopsy, Donor 1) to quantify exfoliated corneocytes from skin biopsies treated with 100 pL of each test solution or control for 24Attorney Docket No.: ROF-018WOhours. Statistics determined with a Students t-test with a p-value < 0.05 considered significant. Buffer 1 was a 0.1 M citric acid / sodium citrate buffer (pH 3.99). Buffer 2 was a 0.1 M citric acid / sodium citrate buffer at pH 5.9.Table 3: Human Skin Exfoliation Assay Screening of Poly-Hydroxy Acids (PHA) and Amino Acids%w / Solution FoldEx Ingredient pH Mean STDEV SEM p-value w (QS) changewater n / a water - 1.15 0.7 0.31 0.14 0.222 Tritonn / a water - X-100 1.63 1.0 0.73 0.33 1.000Maltobionic7a Buffer 1 4acid 1.68 1.0 0.14 0.06 0.871 1 %Maltobionic7b Buffer 2 6acid 1.07 0.7 0.36 0.16 0.170 8a Phytic acid Buffer 1 5 1.64 1.0 0.40 0.18 0.976 2%8b Phytic acid Buffer 2 6 1.21 0.7 0.21 0.10 0.252 8c Phytic acid Buffer 1 4 1.68 1.0 0.60 0.27 0.913 1%8d Phytic acid Buffer 2 6 0.99 0.6 0.24 0.11 0.107L-Carnitine9a Buffer 1 4Tartrate 2.49 1.5 0.20 0.09 0.039 10 %L-Carnitine9b Buffer 2 6Tartrate 0.97 0.6 0.11 0.05 0.084 L-Carnitine9c Buffer 1 4Tartrate 2.19 1.3 0.29 0.13 0.157 3 %L-Camitine9d Buffer 2 6Tartrate 0.91 0.6 0.44 0.20 0.110 10a Carnosine Buffer 1 4 1.92 1.2 0.44 0.20 0.475 1 %10b Carnosine Buffer 2 6 1.18 0.7 0.51 0.23 0.313 Ila L-Serine Buffer 1 4 1.96 1.2 0.13 0.06 0.348 10 %lib L- Serine Buffer 2 6 1.35 0.8 0.35 0.16 0.472 11c L-Serine Buffer 1 4 1.81 1.1 0.63 0.28 0.697 3%lid L-Serine Buffer 2 6 1.48 0.9 0.22 0.10 0.669Attorney Docket No.: ROF-018WO

[0079] Based on the distribution of the data 3% GA (pH 4), 10% GA (pH 4 or 6), 10% LA (pH 4), and 10% MLA (pH 4) reached an apparent maximal level of corneocyte removal (> 2.1-fold) compared to control (FIGs. 1A-1D, Table 1). While only 2.5% LA (Sample 2c) or 3% MLA (Sample 4c) approached this level (2.0-fold) at a pH of 4-5 with a greater degree of variation (Samples 3a-d) also removed corneocytes, but it was overall less effective (1.5-fold) and required 10% at pH of 4 (Sample 3a, FIGs. 1B-1D). A solvent effect was observed for BHA, salicylic acid (SA), such that in water alone (no ethanol), 0.2% SA released more corneocytes (data not shown). Amino acids did not appear to release corneocytes using this modified Trypan blue protocol, as previously reported.(see Ahn, B., et al. (2019). Journal of cosmetic dermatology, 8(5), 1404-1409) While the carnitine tartrate at 3% or 10% CT was effective, the results suggest the tartaric acid was the active (Table 3). Glucono-d-lactone was also effective from 3% wt / wt as long as the pH was 4, albeit with a larger degree of variability compared to AHAs (FIGs. 1 A-1D).

[0080] In terms of blends, results from this screen indicated that blends of AHAs / BHA / PHAs (Blend 1, FIG. 2A) were better in terms of fold change and sample distribution than blends of polyacids such as PHA / PA (Blend 2, FIG. 2A). In terms of cosmetic grade biological exfoliants, such as proteases (cysteine / serine), the results revealed that, after 30 minutes of treatment, only Enzyme 1 removed corneocytes statistically significantly at more or less the same efficiency from 0.5-1 % (pH 4 or 6), when compared to water control using one-way ANOVA statistical analysis (FIG.2B and 2C), despite Student’s t tests suggesting 5% Enzyme 2 at pH 4 had slightly improved corneocyte removal when compared to control (Table 4).

[0081] Proteolytic enzymes’ exfoliation capacities were also screened in this assay after 30 min of treatment (Table 4). Biological exfoliants (proteases) were assessed using the trypan blue plate reader assay after 30 minutes of treatment on human skin biopsies (96 wells, 6 mm punch biopsy, Donor 5). Skin biopsies were treated with 100 LIL of each test solution or control for 30 minutes. Statistics were determined with a Students t-test, with a p-value < 0.05 considered significant. Solution A contained 0.71 %w / w trisodium citrate, 0.31citric acid, glycerin, methylpropane diol, xanthan gum, sodium hyaluronate (and) water. Solution B contained 1.21 %SN! N trisodium citrate, 1.13 %SN!SN citric acid, water, propane diol, sodium hyaluronate (and) water.Attorney Docket No.: ROF-018WOTable 4: Human Skin Exfoliation Assay Screening of Biological Exfoliants Solution Fold P- Ex %w / w Acid pH Mean STDEV SEM (QS) change value water n / a water - 0.86 1.0 0.12 0.05 1 la 10% Glycolic acid water 4 3.10 3.6 0.34 0.15 0.000Water / citric12a 1% Enzyme 1 4acid 1.72 2.0 0.53 0.24 0.008 Water / citric12b 1% Enzyme 1 6acid / NaOH 1.89 2.2 0.11 0.05 0.000 Water / citric12c 0.5% Enzyme 1 4acid 1.76 2.0 0.04 0.02 0.000 Water / citric12d 0.5% Enzyme 1 6acid / NaOH 1.66 1.9 0.12 0.05 0.000 13a 5% Enzyme 2 Solution A 4 1.09 1.3 0.17 0.08 0.039 13b 5% Enzyme 2 Solution B 6 0.82 1.0 0.12 0.06 0.678 Water / citric14a 4% Enzyme 3 5acid 0.75 0.9 0.04 0.02 0.099 Water / citric14b 4% Enzyme 3 6acid 0.85 1.0 0.10 0.05 0.895

[0082] Blends of acids and enzymes were next examined to distinguish faster exfoliation capacity after 30 min of treatment with blends of acids with proteolytic enzymes (Table 5).Formulated blends of AHAs, BHAs, PHAs were neutralized with 12.5% wt / wt sodium hydroxide solution and a Trypan blue plate reader assay was performed (96 wells, 6 mm punch biopsy, Donor 6) to quantify exfoliated corneocytes from skin biopsies treated with 100 pL of each test solution or control for 30 min. Abbreviations: CB, citrate buffer; LBA, lactobionic acid; GA, glycolic acid; LA, lactic acid; MA, mandelic acid; MLA, malic acid; SA, salicylic acid; MBA, Maltobionic Acid; GLU, glucono-d-lactone; PA, phytic acid; CT, carnitine tartrate; CA, Carnosine; SER, serine. Solution A contained glycerin, methylpropane diol, xanthan gum, sodium hyaluronate (and) water. Solution B contained water, propane diol, sodium hyaluronate (and) waterTable 5: Human Skin Exfoliation Assay Screening of Formulated blends of AHAs,Attomey Docket No.: ROF-018WOBHAs, PHAsAcid Pack SolutionExample MeanComposition STDEQ pH V p-value( S)water NA water 0.80823 0.174933 1Triton-X NA water 0.69251 0.153093 1le 10% GA water 4 2.80189 0.086135 3.98E-093% GLUBlend 3 1.9% SA water 4 3.09121 0.299463 2.39E-073% GA3% GLU1.9% SABlend 4 3% GA water 4 3.55521 0.018101 4.94E-100.5% CysteineProtease 11% MBA3% GLUBlend 5 water 4 1.72141 0.138577 3.77E-062% PA3% GA1% MBA3% GLU2% PABlend 6 water 4 3.40243 0.034799 8.71E-103% GA0.5% CysteineProtease 1Benchmarkemulsion 2.62905 0.286396 9.56E-07formula

[0083] Finally, the exfoliation capacities for acid packs at the same pH were analyzed to determine if it was affected by the format, such as in a toner versus and emulsion (Table 6).Formulated blends of AHAs, BHAs, PHAs and proteases were analyzed using the Trypan blue plate reader assay (30-minute and 8-hour plates combined, 96 wells, 6 mm punch biopsy, Donor 4) to quantify exfoliated comeocytes from skin biopsies treated with 100 pL of each test solution with the same acid pack in a toner versus emulsion format or control for 30 minutes and 8 hours. Full list of excipients in each emulsion are provided below. Solutions were neutralized with 45% wt / wt KOH solution. Statistics were determined with a Students t-test with a p-value < 0.05 considered significant. Solution A contained glycerin, methylpropane diol, xanthan gum, sodium hyaluronate (and) water. Solution B contained water, propane diol, sodium hyaluronate (and) waterEmulsion Formulations:

[0084] Emulsion 1: Water / Aqua / Eau, Dicaprylyl Carbonate, Caprylic / Capric Triglyceride, Lactic Acid, Gluconolactone, Glycolic Acid, Potassium Hydroxide, Glyceryl Stearate, PEG-Attorney Docket No.: ROF-018WO100 Stearate, Dimethicone, Cetearyl Alcohol, Butylene Glycol, Phenyl Trimethicone, Propanediol, Bakuchiol, Ceteareth-20, Cetyl Alcohol, Polyacrylate Crosspolymer-6, Phenoxyethanol, Arginine, Polymethylsilsesquioxane, Glucose, Fragrance / Parfum, Dipotassium Glycyrrhizate, Xanthan Gum, Salicylic Acid, Limonene, Caprylyl Glycol, Papain, Chlorphenesin, Sodium Phytate, Dextrin, Polydextrose, Amylopectin, Niacinamide, Linalool, Citral, Tocopherol

[0085] Emulsion 2: Water / Aqua / Eau, Dicaprylyl Carbonate, Caprylic / Capric Triglyceride, Lactic Acid, Gluconolactone, Glycolic Acid, Potassium Hydroxide, PEG-100 Stearate, Glyceryl Stearate, Dimethicone, Cetearyl Alcohol, Butylene Glycol, Phenyl Trimethicone, Propanediol, Bakuchiol, Ceteareth-20, Cetyl Alcohol, Polyacrylate Crosspolymer-6, Phenoxyethanol, Arginine, Polymethylsilsesquioxane, Fragrance / Parfum, Dipotassium Glycyrrhizate, Xanthan Gum, Salicylic Acid, Limonene, Caprylyl Glycol, Chlorphenesin, Sodium Phytate, Dextrin, Polydextrose, Amylopectin, Niacinamide, Linalool, Citral, Tocopherol.

[0086] Emulsion 3: Water / Aqua / Eau, Dicaprylyl Carbonate, Caprylic / Capric Triglyceride, Lactic Acid, Gluconolactone, Glycolic Acid, Potassium Hydroxide, Glyceryl Stearate, PEG-100 Stearate, Dimethicone, Cetearyl Alcohol, Butylene Glycol, Phenyl Trimethicone, Propanediol, Bakuchiol, Ceteareth-20, Cetyl Alcohol, Polyacrylate Crosspolymer-6, Phenoxyethanol, Arginine, Glucose, Citronellyl Methylcrotonate, Dipotassium Glycyrrhizate, Xanthan Gum, Salicylic Acid, Saccharomyces Ferment, Caprylyl Glycol, Papain, Chlorphenesin, Sodium Phytate, Dextrin, Polydextrose, Amylopectin, Niacinamide, Lauroyl Lysine, Tocopherol. Abbreviations: CB, citrate buffer; LB A, lactobionic acid; GA, glycolic acid; LA, lactic acid; MA, mandelic acid; MLA, malic acid: SA, salicylic acid; MBA, Maltobionic Acid; GLU, glucono-d-lactone; PA, phytic acid; CT, carnitine tartrate; CA, Carnosine; SER, serine.Table 6. Formulated Blends of AHAs, BHAs, PHAs and Proteases30 minutes treatment8-hour treatment plate Acid Pack plateExComposition Format pHMean STDEV P- Mean STDEV P- value value none water 0.71 0.09 1.000 0.79 0.336 1.000 0.1%none TritonX- 0.45 0.07 1.000 0.64 0.23 1.000100Attorney Docket No.: ROF-018WOArginine / le 10% GA 4 3.69 0.02 0.000 3.66 0.03 0.000 water3% GLU3.7% LABlend 3% GAArginine / 7 0.2% SA 4 3.74 0.01 0.000 3.73 1.65 0.000 water0.5%CysteineProtease 13% GLUBlend3.7% LA Arginine / 8 4 3.75 0.01 0.000 3.79 1.69 0.000 3% GA water0.2% SABlend3.7% LA Arginine / 9 4 3.61 0.12 0.000 3.80 1.68 0.000 3% GA water3% GLU3.7% LABlend 3% GA Arginine / 10 0.2% SA Emulsion 4 3.78 0.03 0.000 3.72 1.65 0.000 0.5% 1 and 3CysteineProtease 13% GLUBlend Arginine / 3.7% LA11 Emulsion 4 3.64 0.30 0.000 3.17 0.17 0.000 3% GA20.2% SA0087] The samples were read using Molecular Devices ID3 multimode reader. OD590 readings were collected and background was subtracted. The mean absorbance (n=5) is tabulated, with standard deviation and standard error of the mean listed. Each plate layout was analyzed with Student’ s t-test using unknown test solution and compared to 0.1 % TX-100 values as an in-plate reference. It was shown that the trypan blue human ex vivo exfoliation assay was useful in screening and distinguishing differences in these broad categories.

[0088] In conclusion, this optimized human skin exfoliation assay allowed rapidly comparison of exfoliation capacity of a series of AHAs, BHAs, PHA, amino acids and proteolytic enzymes cost effectively and reproducibly. Smaller AHAs at pH of 4 from 3-10% were most effective in removing comeocytes. Additionally, Enzyme 1 was identified as an effective biological exfoliant. Lastly, it was discovered that an optimized blend of AHAs and PHAs allowed for a lower level of AHAs to be included in a blend, which may help to reduce irritation potential whilst maintaining exfoliation capacity. The exfoliation effect of lower levels of AHA in the blends (e.g., % GA in Blends 3, 4 and 6 and 3% GA + 3.7% LA inAttorney Docket No.: ROF-018WOBlends 7, 8, 9, 10 and 11) was comparable to that of 10% GA (see Tables 5 and 6) after 30-min treatment. In addition, the exfoliation effect of blends was comparable to that of 8-hr and 24-hr treatments (Tables 1, 2, and 3). Exemplary compositions exhibiting this effect are listed in Table 7. In summary, the blends not only reduced the concentration of AHA but also decreased the duration of treatment to show similar exfoliation effect.Table 7: Exemplary Optimized AHA / BHA / PHA MixturesComposition Concentrations of AHAs, BHAs, and PHAs (w / w %)1 3% glucono-D-lactone2% salicylic acid3% glycolic acid2 3% glucono-D-lactone1 % maltobionic acid3% glycolic acid3 3% glucono-D-lactone0.2% salicylic acid3.7% lactic acid3% glycolic acid4 3% glucono-D-lactone2% salicylic acid3.7% lactic acid3% glycolic acid5 1.2% glucono-D-lactone0.08% salicylic acid1.4% lactic acid1.2% glycolic acid6 1 % maltobionic acid2% salicylic acid2% lactic acid3% glycolic acid

[0089] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by personsAttorney Docket No.: ROF-018WOskilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0090] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

Attorney Docket No.: ROF-018WOWHAT IS CLAIMED IS:

1. A method of measuring exfoliation capacity of an analyte, the method comprising steps of:(a) contacting a skin sample with a test solution comprising an effective amount of the analyte;(b) incubating the skin sample with the test solution for a period of time to allow the analyte to exfoliate the skin sample;(c) removing the test solution from contact with the skin sample and adding a detection reagent to the test solution, wherein the detection reagent permits the detection of cells and cellular debris in the test solution;(d) detecting the presence or absence of cells and / or cellular debris from the test solution,wherein the presence of cells and / or cellular debris in the test solution is indicative of the exfoliation capacity of the analyte.

2. The method of claim 1, further comprising steps of:(a) after adding the detection reagent, isolating cells and / or cellular debris, if present, from the test solution; and(b) preparing a detection solution comprising the cells and / or cellular debris.

3. The method of claim 2, further comprising steps of:(a) after isolating the cells and cellular debris from the test solution, contacting the skin sample with a wash solution;(b) isolating cells and / or cellular debris, if present, from the wash solution; and (c) adding the isolated cells and / or cellular debris to the detection solution.

4. The method of claim 2 or 3, wherein the detection reagent is extracted from the cells and / or cellular debris in the detection solution.

5. The method of claim 3, wherein the wash solution comprises phosphate-buffered saline (PBS).

6. The method of any one of claims 2-4, wherein the detection solution comprises PBS.

7. The method of any one of claims 1-6, wherein the skin sample is a mammaliansample.Attorney Docket No.: ROF-018WO8. The method of any one of claims 1-7, wherein the skin sample is a human skin sample.

9. The method of any one of claims 1-8, wherein the incubating is from about 30 minutes long to about 24 hours long.

10. The method of any one of claims 1-9, wherein the test solution comprises a buffering agent.

11. The method of claim 10, wherein the buffering agent comprises citrate.

12. The method of claim 10 or 11, wherein the test solution further comprises at least one humectant.

13. The method of claim 12, wherein the at least one humectant is selected from the group consisting of glycerin, methylpropane diol, propane diol, and sodium hyaluronate.

14. The method of any one of claims 10-13, wherein the test solution further comprises a gelling agent.

15. The method of claim 14, wherein the gelling agent is xanthan gum.

16. The method of any one of claims 1-9, wherein the test solution comprises trisodium citrate, citric acid, glycerin, methylpropane diol, xanthan gum, and sodium hyaluronate.

17. The method of any one of claims 1-9, wherein the test solution comprises trisodium citrate, citric acid, propane diol, and sodium hyaluronate.

18. The method of any one of claims 1-17, wherein the test solution has a pH ranging from about 3.5 to about 8.

19. The method of any one of claims 1-18, wherein the test solution has a pH of about 4.

20. The method of any one of claims 1-18, wherein the test solution has a pH of about 6.

21. The method of any one of claims 1-20, wherein the detection reagent is a cell-staining dye.Attorney Docket No.: ROF-018WO22. The method of any one of claims 1-21. wherein the detection reagent is trypan blue.

23. The method of any one of claims 1-22, wherein the cells are skin cells.

24. The method of any one of claims 1-23, wherein the cells are comeocytes.

25. The method of any one of claims 1-24, wherein the detecting comprises measuring optical density of cells and / or cellular debris.

26. The method of any one of claims 1-24, wherein the detecting comprises measuring optical density of a detection reagent extracted from the cells and / or cellular debris.

27. The method of any one of claims 1-26. wherein the detecting is performed using a spectrophotometer.

28. The method of any one of claims 1-24, wherein the detecting comprises measuring the absolute count of cells and / or cellular debris.

29. The method of any one of claims 1 -24 and 28, wherein the detecting is performed using a cell counter.

30. The method of any one of claims 1-24 and 28, wherein the detecting is performed using a flow cytometer.

31. The method of any one of claims 1-30, wherein the method is performed ex vivo or in vitro.

32. An exfoliating composition comprising one or more alpha hydroxy acids (AHAs) and one or more poly hydroxy acids (PH As).

33. The exfoliating composition of claim 32, wherein the one or more AHAs are independently selected from the group consisting of glycolic acid, lactic acid, citric acid, malic acid, mandelic acid, and tartaric acid.

34. The exfoliating composition of claim 32 or 33, wherein the one or more PHAs are independently selected from the group consisting of glucono-D-lactone, lactobionic acid, and maltobionic acid.Attorney Docket No.: ROF-018WO35. The exfoliating composition of any one of claims 32-34, further comprising a beta hydroxy acid.

36. The exfoliating composition of claim 35, wherein the BHA is salicylic acid.

37. The exfoliating composition of any one of claims 32-36, further comprising a protease.

38. The exfoliating composition of claims 37, wherein the protease is a cysteine protease.

39. The exfoliating composition of any one of claims 32-38, further comprising phytic acid.

40. The exfoliating composition of any one of claims 32-39, wherein the composition comprises glycolic acid and glucono-D-lactone at a ratio of glycolic acid to glucono- D-lactone of about 1 : 1 by weight.

41. The exfoliating composition of any one of claims 32-40, wherein the composition comprises glycolic acid and maltobionic acid at a ratio of glycolic acid to maltobionic acid of about 3: 1 by weight.

42. The exfoliating composition of any one of claims 32-41, wherein the composition comprises lactic acid and glucono-D-lactone at a ratio of lactic acid to glucono-D- lactone of about 1.2:1 by weight.

43. The exfoliating composition of any one of claims 32-42, wherein the composition comprises lactic acid and maltobionic acid at a ratio of lactic acid to maltobionic acid of about 2:1 by weight.

44. The exfoliating composition of any one of claims 32-43, wherein the composition comprises glucono-D-lactone, salicylic acid, and glycolic acid.

45. The exfoliating composition of any one of claims 32-43, wherein the composition comprises glucono-D-lactone, maltobionic acid, and glycolic acid.

46. The exfoliating composition of any one of claims 32-43, wherein the composition comprises glucono-D-lactone, salicylic acid, lactic acid, and glycolic acid.Attorney Docket No.: ROF-018WO47. The exfoliating composition of any one of claims 32-43, wherein the composition comprises maltobionic acid, salicylic acid, lactic acid, and glycolic acid.

48. An exfoliating composition comprising about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, and about 3% glycolic acid by weight.

49. An exfoliating composition comprising about 3% glucono-D-lactone by weight, about 1% maltobionic acid by weight, and about 3% glycolic acid by weight.

50. An exfoliating composition comprising about 3% glucono-D-lactone by weight, about 0.2% salicylic acid by weight, about 3.7% lactic acid by weight, and about 3% glycolic acid by weight.

51. The exfoliating composition of claim 50, further comprising about 0.15% of a cysteine protease by weight.

52. An exfoliating composition comprising about 3% glucono-D-lactone by weight, about 2% salicylic acid by weight, about 3.7% lactic acid by weight, and about 3% glycolic acid by weight.

53. The exfoliating composition of claim 52, further comprising about 0.12% of a cysteine protease by weight.

54. An exfoliating composition comprising about 1.2% glucono-D-lactone by weight, about 0.08% salicylic acid by weight, about 1.4% lactic acid by weight, and about 1.2% glycolic acid by weight.

55. The exfoliating composition of claim 54, further comprising about 0.065% phytic acid by weight.

56. An exfoliating composition comprising about 1% maltobionic acid by weight, about 2% salicylic acid by weight, about 2% lactic acid by weight, and about 3% glycolic acid by weight.

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