STEM CELL STIMULATING COMPOSITIONS AND METHODS

Disclosed herein are compositions and methods of using a topical formulation on lips and nails. The topical formulation might comprise one for more defensins. The topical formulation might also comprise other cosmetically acceptable ingredients. The topical formulation might be used to alleviate a cosmetic concern of the lips or nails.

INCORPORATION BY REFERENCE

SUMMARY

The present disclosure provides compositions and methods for alleviating cosmetic concerns of an individual. Some aspects of the present disclosure provide compositions for topical formulations and methods of use for alleviating a cosmetic concern of an individual.

In one aspect, the present disclosure provides a composition comprising a topical formulation formulated for use on a lip or a nail that comprises: a) a first defensin, and b) a cosmetically acceptable ingredient.

In some embodiments, the topical formulation is formulated for use on the lip and the topical formulation is capable of alleviating a cosmetic concern of the lip. In some embodiments, the cosmetic concern of the lip comprises wrinkles on the lip, wrinkles around contour of the lip, an appearance of aging of the lip, discoloration of the lip, dryness of the lip, or any combinations thereof.

In some embodiments, the topical formulation is formulated for use on the nail and the topical formulation is capable of alleviating a cosmetic concern of the nail or stimulating a regeneration of the nail. In some embodiments, the nail comprises a nail portion selected from the group consisting of nail root, eponychium, proximal nail fold, lanula, cuticle, lateral nail fold, nail body/plate and hyponychium. In some embodiments, the cosmetic concern of the nail comprises brittle nail, thin nail, weakness of nail, nail discoloration, slow nail growth, nail surface irregularities, nail surface smoothness, nail vertical ridges, nail horizontal ridges, nail hydration, cuticle hydration, aesthetic appearance of the nail, aesthetic appearance of the cuticle, or any combinations thereof.

In some embodiments of any of the compositions of the present disclosure, the first defensin is selected from the group consisting of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3 and beta-defensin 4. In some embodiments of any of the compositions of the present disclosure, the first defensin is present in a concentration effective to recruit Lgr6+ stem cells. In some embodiments of any of the compositions of the present disclosure, the first defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml. In some embodiments of any of the compositions of the present disclosure, the first defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml. In some embodiments of any of the compositions of the present disclosure, the first defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

In some embodiments of any of the compositions of the present disclosure, the topical formulation further comprises a second defensin, wherein the second defensin is selected from the group consisting of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3 and beta-defensin 4. In some embodiments, the second defensin is present in a concentration effective to recruit Lgr6+ stem cells. In some embodiments, the second defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml. In some embodiments, the second defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml. In some embodiments, the second defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml. In some embodiments, the first defensin and the second defensin are present in a mass ratio of about 1:10 to about 10:1. In some embodiments, the first defensin and the second defensin are present in a mass ratio of about 1:5 to about 5:1.

In some embodiments of any of the compositions of the present disclosure, the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Caesalpinia spinosafruit extract,Helianthus annuussprout extract,Pistacia lentiscusgum, squalane, panthenol, biotin, niacinamide, d-l methionine and any combinations thereof. In some embodiments of any of the compositions of the present disclosure, the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Undaria pinnatifidaextract,Caesalpinia spinosafruit extract,Kappaphycus alvareziiextract,ceratonia siliquafruit extract,Brassica albasprout extract and any combinations thereof. In some embodiments of any of the compositions of the present disclosure, the cosmetically acceptable ingredient comprisesCaesalpinia spinosafruit extract.

In one aspect, the present disclosure provides a method of alleviating a cosmetic concern of a lip or a nail of an individual, the method comprises: providing a topical formulation comprising a first defensin and a cosmetically acceptable ingredient, applying the topical formulation to the lip or nail of the individual, and the topical formulation alleviates the cosmetic concern of the lip or nail of the individual.

In some embodiments, the topical formulation is applied to the lip or skin around the lip and the topical formulation alleviates the cosmetic concern of the lip. In some embodiments, the cosmetic concern of the lip comprises wrinkles on the lip, wrinkles around contour of the lip, an appearance of aging of the lip, discoloration of the lip, dryness of the lip, or any combinations thereof.

In some embodiments, the topical formulation is applied to the nail, and the topical formulation alleviates the cosmetic concern of the nail or stimulates the regeneration of the nail. In some embodiments, the nail comprises a nail portion selected from the group consisting of nail root, eponychium, proximal nail fold, lanula, cuticle, lateral nail fold, nail body/plate and hyponychium. In some embodiments, the cosmetic concern of the nail comprises brittle nail, thin nail, weakness of nail, nail discoloration, slow nail growth, nail surface irregularities, nail surface smoothness, nail vertical ridges, nail horizontal ridges, nail hydration, cuticle hydration, aesthetic appearance of the nail, aesthetic appearance of the cuticle, or any combinations thereof.

In some embodiments of any of the methods of the present disclosure, the first defensin is selected from the group consisting of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3 and beta-defensin 4. In some embodiments of any of the methods of the present disclosure, the first defensin is present in a concentration effective to recruit Lgr6+ stem cells. In some embodiments of any of the methods of the present disclosure, the first defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml. In some embodiments of any of the methods of the present disclosure, the first defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml. In some embodiments of any of the methods of the present disclosure, the first defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

In some embodiments of any of the methods of the present disclosure, the topical formulation further comprises a second defensin, wherein the second defensin is selected from the group consisting of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3 and beta-defensin 4. In some embodiments, the second defensin is present in a concentration effective to recruit Lgr6+ stem cells. In some embodiments, the second defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml. In some embodiments, the second defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml. In some embodiments, the second defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml. In some embodiments, the first defensin and the second defensin are present in a mass ratio of about 1:10 to about 10:1. In some embodiments, the first defensin and the second defensin are present in a mass ratio of about 1:5 to about 5:1.

In some embodiments of any of the methods of the present disclosure, the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Caesalpinia spinosafruit extract,Helianthus annuussprout extract,Pistacia lentiscusgum, squalane, panthenol, biotin, niacinamide, d-l methionine and any combinations thereof. In some embodiments of any of the methods of the present disclosure, the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Undaria pinnatifidaextract,Caesalpinia spinosafruit extract,Kappaphycus alvareziiextract,ceratonia siliquafruit extract,Brassica albasprout extract and any combinations thereof. In some embodiments of any of the methods of the present disclosure, the cosmetically acceptable ingredient comprisesCaesalpinia spinosafruit extract.

DETAILED DESCRIPTION

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

Although various features of the invention can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the invention can be described herein in the context of separate embodiments for clarity, the invention can also be implemented in a single embodiment.

Definitions

As used herein, the term “ready-to-use” indicates that the defensin-containing topical formulation is in a form that is presented for sale and application. It is contemplated that ready-to-use formulations can comprise a fully combined solution, cream, gel, serum, lotion, etc. Alternatively, the defensin can be packaged in a separate container (e.g., in a vial that pumps a defensin solution with a cream that the user blends before applying to unbroken skin) and combined with another topical formulation at the time of use/application.

As used herein, the phrase “sub-antimicrobially effective concentration” means concentration(s) of defensins which are characterized by an inability to inhibit the proliferation of microbes in an established infection.

As used herein, when the term “about” is used in conjunction with a numeral, “about” means a range of plus or minus ten percent of the numerical value given, including end points. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

As used herein, the term “non-injured” skin refers to skin in which dermis and hypodermis are substantially intact. Therefore, viewed from a different perspective, non-injured skin will appear intact to the unaided eye, with no breach sufficiently large or deep to result in bleeding. Thus, non-injured (or “healthy”) skin includes aged skin and skin with first degree sunburn, environmental exposure, bruising, or partially ablated stratum corneum. Non-injured (or healthy) skin also excludes skin displaying persistent infection with pathogens that result in visible symptoms and signs of infection.

As used herein, the term “Caesalpinia spinosa” or “tara” refers to a plant species native to Peru. Extracts of the tara plant can be made from the leaves, the fruit, the roots, or any combination thereof. Extracts from the tara plant can induce the formation of a protective form on the surface of human skin, reduce moisture loss, and skin peeling.

As used herein, the term “Leuconostoc/Radish Root Ferment Filtrate” refers to a chemical which is derived from the fermented root of the radish plant,Raphanus sativus. The root of the radish plant is fermented with the microorganismLeuconostoc. Leuconostoc/Radish Root Ferment Filtrate can enhance the stability and longevity of formulation. In addition,Leuconostoc/Radish Root Ferment Filtrate can have moisturizing properties.

As used herein, the term “Helianthus annuussprout extract” refers to extract made from the seeds of a sunflower. Sunflower seed extracts can have antioxidant, antimicrobial, antidiabetic, antihypertensive, anti-inflammatory, and wound-healing properties.

As used herein, the term “Pistacia lentiscusgum” refers to the bark of thePistacia lentiscustree. ThePistacia lentiscusgum can block the activity of5areductase type I, which has an important role in sebum production.

As used herein, the term “Undaria pinnatifidaextract” refers to extract made from the Wakame/sea mustard brown algae. Wakame extracts can prevent water loss and promote hydration.

As used herein, the term “Kappaphycus alvareziiextract” refers to extract made from theK. alvareziimacroalgae.K. alvareziiextract can help regulate skin barrier homeostasis.

As used herein, the term “Ceratonia siliquafruit extract” refers to extract made from the fruit of theC. siliquatree. TheC. siliquatree is also known as a carob tree, and the fruit of this tree is referred to as “Locust bean” or “carob tree fruit.” The Carob tree fruit extract can be used to thicken and stabilize formulations.

As used herein, the term “Brassica albasprout extract” refers to extract made from yellow mustard. The extract can be made with the yellow mustard seeds. The extract can be made with the yellow mustard leaves. The yellow mustard extract can increase hydration.

Topical Formulations

One aspect of the present disclosure provides a composition comprising a topical formulation. In some embodiments, the topical formulation can have a first defensin and a cosmetically acceptable ingredient. In some embodiments, the topical formulation can be formulated for use on a lip or nail. In some cases, the topical formulation can be formulated for use on a lip. In some examples, the topical formulation is applied to the surface of the lip. In some cases, the topical formulation can be formulated for use on a nail. In some examples, the topical formulation is applied to a surface of the nail, an end of the nail, a nail fold of the nail, and/or a nail root of the nail. In some embodiments, a composition can comprise at least one defensin present at a sub-antimicrobially effective concentration.

In some embodiments, the topical formulation can be formulated for use on a subject. In some cases, the subject is a mammal. In some cases, the mammal can be a non-human primate, a human, a dog, a cat, a rabbit, a rodent, or any combination thereof. In some cases, the subject is a human.

In some embodiments, the topical formulation can be applied yearly. In some embodiments, the topical formulation can be applied every six months. In some embodiments, the topical formulation can be applied every four months. In some embodiments, the topical formulation can be applied every three months. In some embodiments, the topical formulation can be applied every two months. In some embodiments, the topical formulation can be applied monthly. In some embodiments, the topical formulation can be applied biweekly. In some embodiments, the topical formulation can be applied weekly. In some embodiments, the topical formulation can be applied daily.

In some embodiments, the topical formulation may have one defensin, a combination of two defensins, or a combination of three or more defensins. The defensins used may be of the same or different types and subtypes. Non-limiting examples of defensins include one or more of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4. Especially preferred topical formulations contain alpha-defensin 5 and beta-defensin 3. When two more defensins are used in combination, each defensin may be present in equal quantities by mass or at mass ratios specified to achieve a desired result, such as 1:1.5, 1:2, 1:4, 1:5, etc. In some embodiments, the total concentrations of defensins used in the compositions are ineffective at inhibiting substantial proliferation of microbes in established skin infections in a therapeutically effective manner.

In some embodiments, the defensins can be from natural sources. In some embodiments, the defensins can be from synthetic sources. In some embodiments, defensins can be from both natural and synthetic sources. In some cases, defensins may be obtained from plants (e.g.,Arabidopsis, pea, tobacco, and spruce), mammals, or other animals. In some cases, defensins derived from natural sources may include human defensins, monkey defensins, mouse defensins, rat defensins, bovine defensins, sheep defensins, horse defensins, rabbit defensins, swine defensins, dog defensins, and/or cat defensins.

Synthetic defensins can include defensins produced by chemical synthesis (e.g., solid phase synthesis) or by recombinant technologies (e.g., produced by recombinant bacteria, yeast, tissue cultures, plants, or animals). In some embodiments, defensin analogues such as hapivirins and diprovirins may be used. In some embodiments, the defensins can be modified to increase their activity and specificity for cosmetic improvements to the appearance of lips and nails. In some cases, defensins may be unfolded and refolded under controlled conditions to ascertain proper disulfide bond formation (which can be monitored by MS analysis and/or CD spectroscopy).

Alternatively, in some embodiments, chemical modifications (e.g., using non-natural amino acids to increase half-life time, or derivatized proteinogenic amino acids to increase lipophilicity) can be used tailor the defensins to a particular need.

In some embodiments, synthetic defensins can use orthogonal protecting groups to protect selected cysteine residues, which can then be individually deprotected and bonded with the matching target cysteine residue, leading to coordinated non-random disulfide bond formation. Use of such protecting groups in the synthetic strategy can give rise to defensins with a specific activity that is comparable to the specific activity of the native defensin. Any suitable characterization and quality control measures may be employed. Typically, the specific activity of defensins incorporated into the inventive topical formulations can be measured by purity as determined by HPLC. In some embodiments, the defensin can be between 80% and 100% pure, the defensin can be at least 90% pure, the defensin can be at least 95% pure, the defensin can be at least 99% pure, or the defensin can be at least 99.9% pure. In some embodiments, proper amino acid sequence and disulfide bond formation can be confirmed by tandem MS/MS.

In some embodiments, the first defensin can be alpha-defensin 1. In some embodiments, the first defensin can be alpha-defensin 5. In some embodiments, the first defensin can be alpha-defensin 6. In some embodiments, the first defensin can be neutrophil defensin 1. In some embodiments, the first defensin can be neutrophil defensin 2. In some embodiments, the first defensin can be neutrophil defensin 3. In some embodiments, the first defensin can be neutrophil defensin 4. In some embodiments, the first defensin can be theta-defensin. In some embodiments, the first defensin can be beta-defensin 1. In some embodiments, the first defensin can be beta-defensin 2. In some embodiments, the first defensin can be beta-defensin 3. In some embodiments, the first defensin can be beta-defensin 4.

In some embodiments, the topical formulation can further have a second defensin. In some embodiments, the second defensin can be alpha-defensin 1. In some embodiments, the second defensin can be alpha-defensin 5. In some embodiments the second defensin can be alpha-defensin 6. In some embodiments, the second defensin neutrophil defensin 1. In some embodiments, the second defensin can be neutrophil defensin 2. In some embodiments, the second defensin can be neutrophil defensin 3. In some embodiments, the second defensin can be neutrophil defensin 4. In some embodiments, the second defensin can be theta-defensin. In some embodiments, the second defensin can be beta-defensin 1. In some embodiments, the second defensin can be beta-defensin 2. In some embodiments, the second defensin can be beta-defensin 3. In some embodiments, the second defensin can beta-defensin 4.

In some embodiments, the first defensin and the second defensin can be present in the composition at a mass ratio of about 1:100 to about 100:1. In some embodiments, the first defensin and the second defensin can be present in the composition at a mass ratio of about 1:100, about 1:90, about 1:80, about 1:70, about 1:60, about 1:50, about 1:40, about 1:30, 1:20, about 1:19, about 1:18, about 1:17, about 1:16, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, 100:1. In some cases, the first defensin and the second defensin can be present in a composition at a mass ratio of about 1:10 to about 10:1. In some cases, the first defensin and the second defensin can be present in a composition at a mass ratio of about 1:5 to about 5:1.

In some embodiments, the topical formulation of the present disclosure can increase the health and/or condition of a subject's lips. An increase in health and/or condition of a subject's lips can be evidenced by smoother lips, increased lip hydration, increased skin conductance, increased skin capacitance, decreased transepidermal water loss in the lips, decreased lip dryness, decreased dryness of lip skin or skin surrounding the lips, decreased wrinkles around the lips, decreased wrinkle depth or length around the lips, improved lip color, increased lip fullness, increased lip size, or any other measure of lip health or condition.

In some embodiments, the topical formulation of the present disclosure can increase hydration of a subject's lips. In some cases, the topical formulation can increase hydration in the subject's bottom lip. In some cases, the topical formulation can increase hydration in the subject's top lip. In some cases, the topical formulation can increase cutaneous hydration of a subject's lips. In some cases, the topical formulation can increase subcutaneous hydration of a subject's lips. In some cases, the topical formulation can increase cutaneous and subcutaneous hydration of a subject's lips. In some embodiments, the hydration of the subject's lips can be measured by a skin moisture meter. Non-limiting examples of skin moisture meters include cutometer, corneometer, novameter, or any combination thereof. In some embodiments, the hydration of the subject's lips can be measured by a physical test. In some cases, the physical test can be a pinch test. The pinch test can involve pinching the subject's lips and observing how quickly the skin returns to its original position. In some cases, the physical test can be a visual assessment. The visual assessment can involve observing the subject's lips to determine if there is any rough skin or dryness preset on the subject's lips. In some embodiments, the hydration of the subject's lips can be measured by an optical method. Non-limiting examples of optical sensors include near-infrared spectrometry, nuclear magnetic resonance, optical coherence tomography, raman spectroscopy, or any combination thereof. In some embodiments, the hydration of the subject's lips can be measured by calorimetry.

In some embodiments, the topical formulation of the present disclosure can decrease the transepidermal water loss from the subject's lips. In some cases, the topical formulation can decrease the transepidermal water loss from the subject's bottom lip. In some cases, the topical formulation can decrease the transepidermal water loss from the subject's top lip. In some cases, the transepidermal water loss from the subject's lips can be measured by a transepidermal water loss (TEWL) probe. Non-limiting examples of TEWL probes include AquaFlux™ AF200, DermaLab, Evaporimeter 2100, GPSkin Barrier Light, GPSkin Pro, H4300, H4500, MEECO, Noevir©-EVA, Evaporimeter EP1, Tewameter® TM210, Tewameter® TM300, Vapometer™ Vapometer™ SWL-2, Vapometer™ SWL-3, or any combination thereof.

In some embodiments, the topical formulation of the present disclosure can decrease dryness of the subject's lips. In some cases, the topical formulation can decrease dryness of the subject's bottom lip. In some cases, the topical formulation can decrease dryness of the subject's top lip. In some cases, the decrease of dryness of the subject's lips can be measured by visual observation. In some cases, the visual observation can comprise performing an evaluation of the subject's lips in a mirror. In some cases, the visual observation can comprise performing images of the subject's lips. In some examples, the images of the subject's lips are analyzed using image analysis software. In some cases, the visual observation can be performed by the subject and/or a separate individual. In some examples, the visual observation can comprise rating the subject's lips with a dryness score. The dryness score can include the following: a 0 dryness score corresponds to no dryness or chapping evident; a 1-2 dryness score corresponds to fine scaling; a 3-4 dryness score corresponds to coarse scaling and slight cracking; a 5-6 dryness score corresponds to coarse scaling and obvious cracking; and a 7-8 dryness score corresponds to course scaling and cracking progressing to fissuring. In some cases, the decrease of dryness of the subject's lips can be measured by physical evaluation. In some examples, the physical evaluation can comprise the subject and/or a separate individual touching the subject's lips. In some examples, the physical evaluation can comprise rating the subject's lips with a dryness score. The dryness score can include the following: a 0 dryness score correspond to smooth skin on the subject's lips; a 1-2 dryness score corresponds with slight, but definite roughness; a 3-4 dryness score corresponds with moderate rough skin on the subject's lips; a 5-6 dryness score corresponds with marked rough skin on the subject's lips; and a 7-8 dryness score corresponds with very marked rough skin on the subject's lips. In some embodiments, the decrease of dryness of the subject's lips can be measured by a visual observation and a physical evaluation.

In some embodiments, the topical formulation of the present disclosure can decrease wrinkles around contours of a subject's lips. In some cases, the topical formulation of the present disclosure can decrease wrinkles around the contour of the subject's bottom lip. In some cases, the topical formulation of the present disclosure can decrease wrinkles around the contour of the subject's top lip. In some cases, the topical formulation of the present disclosure can decrease wrinkles around the contour of the subject's bottom lip and around the contour of the subject's top lip. In some embodiments, the decrease of wrinkles around the contours of the subject's lips can be measured by a visual observation. The visual observation can comprise performing an evaluation of the subject's lips in a mirror. In some cases, the evaluation can include the subject rating the following wrinkle characteristics from 0 (e.g., not observed) to 10 (e.g., severe): overall number of wrinkles around the contours of the lips, depth of wrinkles around the contours of the lips, length of wrinkles around the contours of the lips, or any combination thereof. In some cases, the visual observation can comprise using the Daniell system of scoring the appearance of facial wrinkles. In some cases, the visual observation of the subject's lips can be performed by the subject and/or a separate observer. In some examples, the separate observer is a different individual from the subject. In some examples, the separate observer is a clinician. In some embodiments, the decrease of wrinkles around the contours of the subject's lips can be measured by an optical method. Non-limiting examples of optical methods to measure wrinkles around the contours of the subject's lips include image-analysis software, visual illusion-based image feature enhancement system, three-dimensional speckle tracking photogrammetry, digital fringe profilometry, three-dimensional acquisition, three-dimensional projecting grid technique, or any combination thereof.

In some embodiments, the topical formulation of the present disclosure can decrease wrinkles on the subject's lips. In some cases, the topical formulation of the present disclosure can decrease wrinkles on the subject's bottom lip. In some cases, the topical formulation of the present disclosure can decrease wrinkles on the subject's top lip. In some cases, the topical formulation of the present disclosure can decrease wrinkles on the subject's bottom lip and on the subject's top lip. In some embodiments, the decrease of wrinkles on the subject's lips can be measured by a visual observation. The visual observation can comprise performing an evaluation of the subject's lips in a mirror. In some cases, the self-evaluation can include rating the following wrinkle characteristics from 0 (e.g., not observed) to 10 (e.g., severe): overall number of wrinkles on the lips, depth of wrinkles on the lips, length of wrinkles on the lips, or any combination thereof. In some cases, the visual observation can comprise using the Daniell system of scoring the appearance of facial wrinkles. In some cases, the visual observation of the subject's lips can be performed by the subject and/or a separate observer. In some embodiments, the decrease of wrinkles on the subject's lips can be measured by an optical method. Non-limiting examples of optical methods to measure wrinkles on the subject's lips include image analysis software, visual illusion-based image feature enhancement system, three-dimensional speckle tracking photogrammetry, digital fringe profilometry, three-dimensional acquisition, three-dimensional projecting grid technique, or any combination thereof.

In some embodiments, the topical formulation can improve color of the subject's lips. In some embodiments, the topical formulation can improve color of the subject's bottom lip. In some embodiments, the topical formulation can improve color of the subject's top lip. In some cases, the topical formulation can result in a healthy lip color. Healthy lip colors can include pinks lips or light red lips. Non-healthy lip colors can include pale lips, purple lips, or blue lips. In some cases, improvement of the color of the subject's lips can be measured by a visual observation. The visual observation can comprise performing an evaluation of the subject's lips in a mirror and determining the current color of the subject's lips. In some cases, the visual observation can comprise taking images (e.g., pictures) of the subject's lips and determining the color of the subject's lips in the images. In some examples, the images of the subject's lips can be analyzed via an image analysis software to determine the color of the subject's lips. In some cases, the visual observation can be performed by the subject and/or a separate observer.

In some embodiments, the topical formulation of the present disclosure can increase fullness of the subject's lips. In some cases, the topical formulation of the present disclosure can increase fullness of the subject's bottom lip. In some cases, the topical formulation of the present disclosure can increase fullness of the subject's top lip. In some embodiments, the increase in the fullness of the subject's lips can be measured by visual observation. The visual observation can comprise performing an evaluation of the subject's lips in a mirror and/or taking images of the subject's lips. In some cases, the images of the subject's lips can be analyzed with an image analysis software. Non-limiting examples of image analysis software includes: PhotoGrammetrix® Image Analysis 3D Zephyr software, PhotoModeler software, Agisoft PhotoScan software, Metashape software, Pix4Dmapper software, or any combination thereof. In some cases, the visual observation of the subject's lips can be performed by the subject and/or a separate observer.

In some cases, the visual observation comprises grading the fullness of the subject's lips. The lip fullness grading scale can include grading lip size of the subject's lips, vermillion body (e.g., pink or red portion of the lips) of the subject's lips, and/or vermillion border (e.g., edge of the pink or red portion of the lips and the surrounding facial skin) of the subject's lips. In some examples, the lip fullness grading scale of the lip size of the subject's lips can include: a lip size score of −2 corresponds to very thin lips; a lip size score of −1 corresponds to thin lips; a lip size score of 0 corresponds to medium sized lips; a lip size score of 1 corresponds to full lips; and a lip size score of 2 corresponds to extremely full lips. In some examples, the lip fullness grading scale of the vermillion body can include: a vermillion body score of −1 corresponds with tight lips with almost no lines; a vermillion body score of 0 corresponds with rounded lips with natural lines; a vermillion body score of 1 corresponds with less rounded lips with fine lines; a vermillion body score of 2 corresponds with flattening lips with moderate wrinkles; and a vermillion body score of 3 corresponds with lips with flattening lips with severe wrinkles. In some examples, the lip fullness grading scale of the vermillion border can include: a vermillion border score of −1 corresponds with protruding vermillion borders and/or creating perioral shadow; a vermillion border score of 0 corresponds to distinct and intact vermillion borders with or without shadow from the mid-lower lip; a vermillion border scale of 1 corresponds to distinct vermillion borders but broken by fine lines with or without shadow from mid-lower lip; a vermillion border scale of 2 corresponds to indistinct vermillion borders broken by moderate lines with or without shadow from mid-lower lip; and a vermillion border scale of 3 corresponds with indistinct and severely lined vermillion borders without or without shadow from mid-lower lip.

In some embodiments, the lip health improvements of the topical formulation of the present disclosure can last for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, or at least about 10 months. In some embodiments, the lip health improvements of the topical formulation of the present disclosure can last for at most about 10 months, at most about 9 months, at most about 8 months, at most about 7 months, at most about 6 months, at most about 5 months, at most about 4 months, at most about 3 months, at most about 12 weeks, at most about 11 weeks, at most about 10 weeks, at most about 9 weeks, at most about 8 weeks, at most about 7 weeks, at most about 6 weeks, at most about 5 weeks, at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, or at most about 1 week. In some embodiments, the lip health improvements of the topical formulation of the present disclosure can last for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, or about 10 months.

In some embodiments, the topical formulation of the present disclosure can increase the health and/or condition of a subject's nails (e.g., fingernails, toenails). An increase in health and/or condition of a subject's nails can be evidenced by increased nail smoothness (e.g., nail surface smoothness), decreased number of nail ridges (e.g., horizontal ridges, vertical ridges), improved nail surface irregularities, decreased width and/or depth of nail ridges (e.g., horizontal ridges, vertical ridges), increased nail strength, increased nail thickness, increased nail hydration, increased nail nourishment, increased keratin production, increased antioxidant production, increased nail growth, increased nail plate hardness, increased nail fold hardness, increased nail free edge thickness, increased nail free edge length, increased nail root hydration, increased nail cuticle hydration, increased nail root conductance, increased nail root capacitance, or any other measure of nail health or condition.

In some embodiments, the topical formulation of the present disclosure can increase a thickness of a subject's nail. In some cases, the thickness of the subject's nail can be measured using a caliper. In some cases, the thickness of the subject's nail can be measured using topical coherence tomography. In some cases, the thickness of the subject's nail can be measured using ultrasonography. In some cases, the thickness of the subject's nail can be measuring using high frequency echography.

In some cases, the topical formulation can increase the thickness of a subject's nail compared to a baseline thickness of the subject's nail from a time point when the subject was not using the topical formulation by between about 0 micrometers (μm) to about 10 millimeters (mm). In some cases, the topical formulation can increase the thickness of a subject's nail by at least about 0 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, at least about 1 mm, at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 10 mm, or more. In some cases, the topical formulation can increase the thickness of the subject's nail by at most about 10 mm, at most about 9 mm, at most about 8 mm, at most about 7 mm, at most about 6 mm, at most about 5 mm, at most about 4 mm, at most about 3 mm, at most about 2 mm, at most about 1 mm, at most about 900 μm, at most about 800 μm, at most about 700 μm, at most about 600 μm, at most about 500 μm, at most about 400 μm, at most about 300 μm, at most about 200 μm, at most about 100 μm, at most about 90 μm, at most about 80 μm, at most about 70 μm, at most about 60 μm, at most about 50 μm, at most about 40 μm, at most about 30 μm, at most about 20 μm, at most about 10 μm, at most about 0 μm, or less. In some cases, the topical formulation can increase the thickness of the subject's nail by about 0 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm.

In some embodiments, the topical formulation of the present disclosure can decrease a depth of ridges on the subject's nail. In some embodiments, the depth of ridges on the subject's nail can be measured by a Visioscan imaging system®. In some embodiments, the depth of ridges on the subject's nail can be measured by a Visioline® imaging system.

In some embodiments, the topical formulation of the present disclosure can decrease a width of ridges on the subject's nail. In some embodiments, the width of ridges on the subject's nail can be measured by a Visioscan imaging system®. In some embodiments, the width of ridges on the subject's nail can be measured by a Visioline® imaging system.

In some embodiments, the topical formulation of the present disclosure can decrease an overall number of ridges (e.g., vertical ridge and horizontal ridges) on the subject's nail. In some embodiments, the overall number of ridges on the subject's nail can be measured by a Visioscan imaging system®. In some embodiments, the overall number of ridges on the subject's nail can be measured by a Visioline® imaging system.

In some embodiments, the topical formulation of the present disclosure can increase a smoothness of the subject's nail. In some embodiments, the smoothness of the subject's nail can be measured by a Visioscan imaging system®. In some embodiments, the smoothness of the subject's nail can be measured by a Visioline® imaging system.

In some embodiments, the topical formulation of the present disclosure can increase a nail plate hardness of the subject's nail. The nail plate can be a portion of the nail that is parallel to surface of the subject's finger or toe. In some cases, the nail plate hardness of the subject's nail can be measured by a durometer. In some cases, the nail plate hardness of the subject's nail can be measured by a Knoop indenter. In some cases, the nail plate hardness of the subject's nail can be measured by onychoscopy.

In some embodiments, the topical formulation of the present disclosure can increase a nail fold hardness of the subject's nail. The nail fold can be a portion of the nail that is perpendicular to surface of the subject's finger or toe. In some cases, the nail fold hardness of the subject's nail can be measured by a durometer. In some cases, the nail fold hardness of the subject's nail can be measured by a Knoop indenter. In some cases, the nail fold hardness of the subject's nail can be measured by onychoscopy.

In some embodiments, the topical formulation of the present disclosure can decrease the nail free edge of the subject's nail. The nail free edge thickness can be the thickness of an edge of the nail not directly connected to the subject's finger or toe. In some cases, the nail free edge thickness can be measured from a clipping taken from the nail free edge. In some cases, the nail free edge thickness can be measured from the nail free edge attached to the nail plate of the subject's nail. In some cases, the nail free edge can be white, translucent, yellow, or any combination thereof in appearance. In some cases, the nail free edge thickness can be measured by measuring the thickness of the nail clipping taken from the nail free edge.

In some embodiments, the topical formulation of the present disclosure can increase a length of the nail free edge of the subject's nail. The length of the nail free edge can be measured by measuring a length of the nail free edge starting wherein the nail free edge is connected to the nail plate to the end of the nail. In some cases, the length of the nail free edge can be measured via a measuring tape or ruler. In some cases, the length of the nail free edge can be measured via photographs of the subject's nail. In some examples, the photographs of the subject's nail can be analyzed to determine the length of the nail free edge of the subject's nail. In some examples, the photographs of the subject's nail can be analyzed via PhotoGrammetrix® Image Analysis 3D Zephyr software, PhotoModeler software, Agisoft PhotoScan software, Metashape software, Pix4Dmapper software, or any combination thereof.

In some embodiments, the topical formulation of the present disclosure can increase the hydration of a nail root of the subject's nail. The nail root can be the regions of the skin that are directly connected to the nail. In some embodiments, the hydration of the nail root can be measured by a nail blanch test. In some embodiments, the hydration of the nail root can be measured by spectral-resolved ellipsometry. In some embodiments, the hydration of the nail root can be measured by thermogravimetric analysis procedure. In some embodiments, the hydration of the nail root can be measured by a dermal phase meter. In some embodiments, the hydration of the nail root can be measured by a skin moisture meter.

In some embodiments, the nail health improvements of the topical formulation of the present disclosure can last for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, or at least about 10 months. In some embodiments, the nail health improvements of the topical formulation of the present disclosure can last for at most about 10 months, at most about 9 months, at most about 8 months, at most about 7 months, at most about 6 months, at most about 5 months, at most about 4 months, at most about 3 months, at most about 12 weeks, at most about 11 weeks, at most about 10 weeks, at most about 9 weeks, at most about 8 weeks, at most about 7 weeks, at most about 6 weeks, at most about 5 weeks, at most about 4 weeks, at most about 3 weeks, at most about 2 weeks, or at most about 1 week. In some embodiments, the nail health improvements of the topical formulation of the present disclosure can last for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, or about 10 months.

Other Ingredients

In some embodiments, the composition can have one or more cosmetically acceptable ingredients. In some embodiments, the one or more cosmetically acceptable ingredient can beLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Caesalpinia spinosafruit extract,Helianthus annuussprout extract,Pistacia lentiscusgum, squalane, panthenol, biotin, niacinamide, d-l methionine,Undaria Pinnatifidaextract,Kappaphycus alvareziiextract,Ceratonia Siliquafruit extract,Brassica albasprout extract, or any combinations thereof. In some embodiments, the one or more cosmetically acceptable ingredients can beLeuconostoc/radish root ferment filtrate. In some embodiments, the one or more cosmetically acceptable ingredients can be albumin. In some embodiments, the one or more cosmetically acceptable ingredients can be 1-alanyl-l-glutamine. In some embodiments, the one or more cosmetically acceptable ingredients can be phospholipids. In some embodiments, the one or more cosmetically acceptable ingredients can be tocopheryl acetate. In some embodiments, the one or more cosmetically acceptable ingredients can be ubiquinone. In some embodiments, the one or more cosmetically acceptable ingredients can beCaesalpinia spinosafruit extract. In some embodiments, the one or more cosmetically acceptable ingredients can beCaesalpinia spinosafruit pod extract. In some embodiments, the one or more cosmetically acceptable ingredients can beHelianthus annuussprout (Sunflower) extract. In some embodiments, the one or more cosmetically acceptable ingredients can bePistacia lentiscusgum. In some embodiments, the one or more cosmetically acceptable ingredients can be squalene. In some embodiments, the one or more cosmetically acceptable ingredients can be panthenol. In some embodiments, the one or more cosmetically acceptable ingredients can be biotin. In some embodiments, the one or more cosmetically acceptable ingredients can be niacinamide. In some embodiments, the one or more cosmetically acceptable ingredients can be d-l methionine. In some embodiments, the one or more cosmetically acceptable ingredients can beUndaria pinnatifidaextract. In some embodiments, the one or more cosmetically acceptable ingredients can beCaesalpinia spinosafruit extract. In some embodiments, the one or more cosmetically acceptable ingredients can beKappaphycus alvareziiextract. In some embodiments, the one or more cosmetically acceptable ingredients can beceratonia siliquafruit extract. In some embodiments, the one or more cosmetically acceptable ingredients can bebrassica albasprout extract. In some embodiments, the one or more cosmetically acceptable ingredients can be caprylic/capric triglycerides. In some embodiments, the one or more cosmetically acceptable ingredients can be water. In some embodiments, the one or more cosmetically acceptable ingredients can beRicinus Communis(Castor) seed oil. In some embodiments, the one or more cosmetically acceptable ingredients can be bis-diglyceryl polyaceyladipate-2. In some embodiments, the one or more cosmetically acceptable ingredients can be rapeseed oil. In some cases, the rapeseed oil can be hydrogenated rapeseed oil. In some cases, the rapeseed oil can be non-hydrogenated rapeseed oil. In some embodiments, the one or more cosmetically acceptable ingredients can be ethylhexyl palmitate. In some embodiments, the one or more cosmetically acceptable ingredients can beButyrospermum parkiishea) butter. In some embodiments, the one or more cosmetically acceptable ingredients can be Stearalkonium hectorite. In some embodiments, the one or more cosmetically acceptable ingredients can bePyrus malus(apple) fruit extract. In some embodiments, the one or more cosmetically acceptable ingredients can be glycerin. In some embodiments, the one or more cosmetically acceptable ingredients can beSambucus nigrafruit extract. In some embodiments, the one or more cosmetically acceptable ingredients can be lauroyl lysine. In some embodiments, the one or more cosmetically acceptable ingredients can beGlycine soja(soybean) seed extract. In some embodiments, the one or more cosmetically acceptable ingredients can be propylene carbonate. In some embodiments, the one or more cosmetically acceptable ingredients can be tocopheryl nicotinate. In some embodiments, the one or more cosmetically acceptable ingredients can be tetrahexyldecyl ascorbate. In some embodiments, the one or more cosmetically acceptable ingredients can be sodium hyaluronate. In some embodiments, the one or more cosmetically acceptable ingredients can beUndaria Pinnatifidaextract. In some embodiments, the one or more cosmetically acceptable ingredients can be palmitoyl tripeptide-1. In some embodiments, the one or more cosmetically acceptable ingredients can be Epigallocatechin Gallatyl glucoside. In some embodiments, the one or more cosmetically acceptable ingredients can beSpirodela polyrhizaextract. In some embodiments, the one or more cosmetically acceptable ingredients can be bisabolol. In some embodiments, the one or more cosmetically acceptable ingredients can be panthenol. In some embodiments, the one or more cosmetically acceptable ingredients can be tocopheryl acetate. In some embodiments, the one or more cosmetically acceptable ingredients can beGlycyrrhiza glabra(licorice) leaf extract. In some embodiments, the one or more cosmetically acceptable ingredients can beCitrus limon(lemon) peel extract. In some embodiments, the one or more cosmetically acceptable ingredients can be polyglyceryl-2 dipolyhydroxystearate. In some embodiments, the one or more cosmetically acceptable ingredients can be Glucomannan. In some embodiments, the one or more cosmetically acceptable ingredients can be trihydroxystearin. In some embodiments, the one or more cosmetically acceptable ingredients can be tribehenin. In some embodiments, the one or more cosmetically acceptable ingredients can be sorbitan isostearate. In some embodiments, the one or more cosmetically acceptable ingredients can be potassium sorbate. In some embodiments, the one or more cosmetically acceptable ingredients can be sodium benzoate. In some embodiments, the one or more cosmetically acceptable ingredients can be citric acid. In some embodiments, the one or more cosmetically acceptable ingredients can be sodium chloride. In some embodiments, the one or more cosmetically acceptable ingredients can be pentylene glycol. In some embodiments, the one or more cosmetically acceptable ingredients can be octyldodecanol. In some embodiments, the one or more cosmetically acceptable ingredients can be glyceryl stearate citrate. In some embodiments, the one or more cosmetically acceptable ingredients can be methionine. In some embodiments, the one or more cosmetically acceptable ingredients can be polyglyceryl-3 stearate. In some embodiments, the one or more cosmetically acceptable ingredients can be lecithin. In some cases, the lecithin can be hydrogenated lecithin. In some cases, the lecithin can be non-hydrogenated lecithin. In some embodiments, the one or more cosmetically acceptable ingredients can be phytic acid. In some embodiments, the one or more cosmetically acceptable ingredients can bePistacia lentiscusgum. In some embodiments, the one or more cosmetically acceptable ingredients can beRibes Nigrumseed oil. In some embodiments, the one or more cosmetically acceptable ingredients can beHelianthus annuusseed oil unsaponifiables. In some embodiments, the one or more cosmetically acceptable ingredients can beCardiospermum halicacabumflower extract. In some embodiments, the one or more cosmetically acceptable ingredients can beCardiospermum halicacabumleaf extract. In some embodiments, the one or more cosmetically acceptable ingredients can beCardiospermum halicacabumvine extract. In some embodiments, the one or more cosmetically acceptable ingredients can be tocopherol. In some embodiments, the one or more cosmetically acceptable ingredients can beHelianthus annuusseed oil. In some embodiments, the one or more cosmetically acceptable ingredients can beRosmarinus Officinalislead extract. In some embodiments, the one or more cosmetically acceptable ingredients can be epigallocatechin gallatyl glucoside. In some embodiments, the one or more cosmetically acceptable ingredients can be propylene glycol. In some embodiments, the one or more cosmetically acceptable ingredients can be polyacrylate crosspolymer-6. In some embodiments, the one or more cosmetically acceptable ingredients can be phenoxyethanol. In some embodiments, the topical formulate can comprise any combination of the one or more cosmetically acceptable ingredients disclosed herein.

In some embodiments, any formulation as described herein can comprise defensins, natural molecules that can assist regenerative cells. In some embodiments, any formulation as described herein can comprise methionine, an essential amino acid. In some embodiments, any formulation as described herein can comprise tara tree fruit extract, an antioxidant that can protect nails and keratin structures from damaging free radicals caused by environmental stress, UV light, and/or harsh chemicals. In some embodiments, any formulation as described herein can comprise a vitamin B complex comprising Vitamin B3 (niacinamide), Vitamin B5 (panthenol), and Vitamin B7 (biotin), which can repair natural moisturization of skin and keratin. In some embodiments, any formulation as described herein can comprise a blend of soothing and/or calming ingredients comprising flower extracts, epigallocatechin gallatyl glucoside, and black currant extract.

In some embodiments, the defensin can be associated with a cosmetically acceptable protein to increase stability and/or delivery characteristics. In some embodiments, the association can be non-covalent (e.g., electrostatic, ionic, hydrophobic, etc.). In some embodiments, the association can be covalent. Non-limiting examples of cosmetically acceptable proteins include lactoferrin, transferrin, and albumin (e.g., human serum albumin, bovine serum albumin, and egg albumin, recombinant albumin). The defensins and protein carriers may be in various ratios, including equimolar, sub-, and supramolar ratios. In some cases, combinations of two or more protein carriers may be used. In some examples, in a formulation in which two defensins are used, one defensin may be associated with one carrier, and the other defensin may be associated with a different carrier. In some embodiments, any combination of defensins and carriers are contemplated.

In some embodiments, lipid membrane delivery systems can comprise amphipathic or amphiphilic molecules such as phospholipids or combinations of phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphoinositides). In some embodiments, lipid membrane delivery systems can contain additive(s) such as sterols, polyethylene glycol, cholesterol, dicethylphosphate, stearyl amine, etc. In some embodiments, the lipid membrane delivery system content can be adjusted to achieve a sub-antimicrobial concentration of defensins. In some embodiments, unilamellar vesicles/liposomes can be produced using high shear techniques. In some cases, the unilamellar vesicles/liposome can have a greater Zeta Potential than the typical liposome and can allow for smaller/more uniform particle size with increased stability. Zeta Potential is an indicator of the electronic charge on the surface of any macroscopic material that is in contact with a liquid. In some embodiments, Zeta Potential can be used to predict and control the stability of suspensions. In some cases, a higher Zeta Potential can be associated with greater the stability of the molecule because the charged particles are able to repel and overcome their innate affinity to assemble.

In some embodiments, the defensins can have a profound effect on stem cell activity in dermal and hypodermal layers. In some embodiments, the liposomal formulations can have the ability to transport the defensins to invade the hair follicle and/or skin pores, and/or stratum corneum, and/or penetrate through nail plate to a depth and concentration sufficient to activate LGR6+ cells. In some embodiments, the use of lipid membrane delivery systems can aid in the delivery of defensins.

In one aspect, the composition can include additional ingredients that nourish and support the recruited stem cells in healthy nails and lips. Non-limiting examples of additional ingredients include albumin (e.g., human serum albumin, bovine serum albumin), egg albumin (albumen), recombinant albumin, plant hydrolysate, and P-cyclodextrin, glutamine, phospholipids (liposomes), fibronectin, hyaluronate, plant hydrolysate, L-alanyl-L-glutamine, gelatin, Vitamin E (tocopheryl nicotinate), ubiquinone (coenzyme Q10), gelatin, recombinant gelatin, hyaluronic acid, Epidermal Growth Factor, or any combinations thereof. In some embodiments, the additional ingredient can be albumin (e.g., human serum albumin, bovine serum albumin). In some embodiments, the additional ingredient can be egg albumin (albumen). In some embodiments, the additional ingredient can be recombinant albumin. In some embodiments, the additional ingredient can be plant hydrolysate. In some embodiments, the additional ingredient can be P-cyclodextrin. In some embodiments, the additional ingredient can be glutamine. In some embodiments, the additional ingredient can be phospholipids. In some embodiments, the additional ingredient can be fibronectin. In some embodiments, the additional ingredient can be hyaluronate. In some embodiments, the additional ingredient can be plant hydrolysate. In some embodiments, the additional ingredient can be L-alanyl-L-glutamine. In some embodiments, the additional ingredient can be gelatin. In some embodiments, the additional ingredient can be Vitamin E (tocopheryl nicotinate). In some embodiments, the additional ingredient can be ubiquinone (coenzyme Q10). In some embodiments, the additional ingredient can be gelatin. In some embodiments, the additional ingredient can be recombinant gelatin. In some embodiments, the additional ingredient can be hyaluronic acid. In some embodiments, the additional ingredient can be Epidermal Growth Factor.

Lip and Nail Formulations and Methods

One aspect of the present disclosure provides compositions and methods of use of a topical formulation as herein disclosed. In some embodiments, the topical formulation can be formulated for use on a lip or nail. In some embodiments, the topical formulation can be formulated for use on a lip or skin around the lip. In some embodiments, the topical formulation can be formulated for use on a nail. In some embodiments, the composition can be applied on broken, damaged or removed nail. In some embodiments, the composition can be applied on damaged or wounded lips.

In some embodiments, the method can comprise providing a topical formulation comprising a first defensin and a cosmetically acceptable ingredient as described herein. In some embodiments, the method can further comprise applying the topical formulation to the lip or nail of the individual. In some embodiments, the topical formulation can alleviate the cosmetic concern of the lip or nail of the individual.

In some embodiments, the topical formulation can be applied to the lip. In some cases, the topical formulation can alleviate the cosmetic concern of the lip. In some cases, the cosmetic concern of the lip can be wrinkles on the lip, wrinkles around contour of the lip, an appearance of aging of the lip, discoloration of the lip, dryness of the lip, or any combinations thereof. In some cases, the cosmetic concern of the lip can be wrinkles on the lip. In some cases, the cosmetic concern of the lip can be wrinkles around contour of the lip. In some cases, the cosmetic concern of the lips can the appearance of aging of the lip. In some cases, the cosmetic concern of the lip can be discoloration of the lip. In some cases, the cosmetic concern of the lip can be dryness of the lip.

In some embodiments, the topical formulation can be applied to the nail in some embodiments, the topical formulation can be applied to nail root, eponychium, proximal nail fold, lanula, cuticle, lateral nail fold, nail body/plate and hyponychium. In some cases, the topical formulation can alleviate the cosmetic concern of the nail of an individual. In some embodiments, the cosmetic concern of the nail can be brittle nail, thin nail, weakness of nail, nail discoloration, slow nail growth, or any combinations thereof. In some cases, the cosmetic concern of the nail can be brittle nail. In some cases, the cosmetic concern of the nail can be thin nail. In some cases, the cosmetic concern of the nail can be weakness of nail. In some cases, the cosmetic concern of the nail can be nail discoloration. In some cases, the cosmetic concern of the nail can be slow nail growth.

Any topical formulation as described herein can be packaged in a kit. A kit can comprise an applicator brush. An applicator brush can comprise a button on the bottom of the brush which, when pushed, dispenses serum onto the brush.

Embodiments

Embodiment 1. A composition comprising a topical formulation that comprises:a) a first defensin; andb) a cosmetically acceptable ingredient,
wherein the topical formulation is formulated for use on a lip or a nail.

Embodiment 2. The composition of embodiment 1, wherein the topical formulation is formulated for use on the lip, and wherein the topical formulation is capable of alleviating a cosmetic concern of the lip.

Embodiment 3. The composition of embodiment 2, wherein the cosmetic concern of the lip comprises wrinkles on the lip, wrinkles around contour of the lip, an appearance of aging of the lip, discoloration of the lip, dryness of the lip, or any combinations thereof.

Embodiment 4. The composition of embodiment 1, wherein the topical formulation is formulated for use on the nail, and wherein the topical formulation is capable of alleviating a cosmetic concern of the nail or stimulating a regeneration of the nail.

Embodiment 5. The composition of embodiment 4, wherein the nail comprises a nail portion selected from the group consisting of nail root, eponychium, proximal nail fold, lanula, cuticle, lateral nail fold, nail body/plate and hyponychium.

Embodiment 6. The composition of embodiment 4, wherein the cosmetic concern of the nail comprises brittle nail, thin nail, weakness of nail, nail discoloration, slow nail growth, nail surface irregularities, nail surface smoothness, nail vertical ridges, nail horizontal ridges, nail hydration, cuticle hydration, aesthetic appearance of the nail, aesthetic appearance of the cuticle, or any combinations thereof.

Embodiment 8. The composition of any of embodiments 1-7, wherein the first defensin is present in a concentration effective to recruit Lgr6+ stem cells.

Embodiment 9. The composition of any of embodiments 1-7, wherein the first defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml.

Embodiment 10. The composition of any of embodiments 1-7, wherein the first defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml.

Embodiment 11. The composition of any of embodiments 1-7, wherein the first defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

Embodiment 13. The composition of embodiment 11, wherein the second defensin is present in a concentration effective to recruit Lgr6+ stem cells.

Embodiment 14. The composition of embodiment 12, wherein the second defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml.

Embodiment 15. The composition of embodiment 12, wherein the second defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml.

Embodiment 16. The composition of embodiment 12, wherein the second defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

Embodiment 17. The composition of embodiment 12, wherein the first defensin and the second defensin are present in a mass ratio of about 1:10 to about 10:1.

Embodiment 18. The composition of embodiment 12, wherein the first defensin and the second defensin are present in a mass ratio of about 1:5 to about 5:1.

Embodiment 20. The composition of any of embodiments 1-18, wherein the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Undaria pinnatifidaextract,Caesalpinia spinosafruit extract,Kappaphycus alvareziiextract,ceratonia siliquafruit extract,brassica albasprout extract and any combinations thereof.

Embodiment 21. The composition of any of embodiments 1-18, wherein the cosmetically acceptable ingredient comprisesCaesalpinia spinosafruit extract.

Embodiment 22. A method for alleviating a cosmetic concern of a lip or a nail of an individual, comprising: providing a topical formulation comprising a first defensin and a cosmetically acceptable ingredient, applying the topical formulation to the lip or the nail of the individual, wherein the topical formulation alleviates the cosmetic concern of the lip or the nail of the individual.

Embodiment 23. The method of embodiment 22, wherein the topical formulation is applied to the lip or skin around the lip, and wherein the topical formulation alleviates the cosmetic concern of the lip.

Embodiment 24. The method of embodiment 23, wherein the cosmetic concern of the lip comprises wrinkles on the lip, wrinkles around contour of the lip, an appearance of aging of the lip, discoloration of the lip, dryness of the lip, or any combinations thereof.

Embodiment 25. The method of embodiment 22, wherein the topical formulation is applied to the nail, and wherein the topical formulation alleviates the cosmetic concern of the nail or stimulates a regeneration of the nail.

Embodiment 26. The method of embodiment 24, wherein the nail comprises a nail portion selected from the group consisting of nail root, eponychium, proximal nail fold, lanula, cuticle, lateral nail fold, nail body/plate and hyponychium.

Embodiment 27. The method of embodiment 24, wherein the cosmetic concern of the nail comprises brittle nail, thin nail, weakness of nail, nail discoloration, slow nail growth, nail surface irregularities, nail surface smoothness, nail vertical ridges, nail horizontal ridges, nail hydration, cuticle hydration, aesthetic appearance of the nail, aesthetic appearance of the cuticle, or any combinations thereof.

Embodiment 29. The method of any of embodiments 22-28, wherein the first defensin is present in a concentration effective to recruit Lgr6+ stem cells.

Embodiment 30. The method of any of embodiments 22-28, wherein the first defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml.

Embodiment 31. The method of any of embodiments 22-28, wherein the first defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml.

Embodiment 32. The method of any of embodiments 22-28, wherein the first defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

Embodiment 34. The method of embodiment 33, wherein the second defensin is present in a concentration effective to recruit Lgr6+ stem cells.

Embodiment 35. The method of embodiment 33, wherein the second defensin is present in a concentration of about 0.01 ng/ml to about 1000 ng/ml.

Embodiment 36. The method of embodiment 33, wherein the second defensin is present in a concentration of about 0.01 ng/ml to about 100 ng/ml.

Embodiment 37. The method of embodiment 33, wherein the second defensin is present in a concentration of about 1 ng/ml to about 100 ng/ml.

Embodiment 38. The method of embodiment 33, wherein the first defensin and the second defensin are present in a mass ratio of about 1:10 to about 10:1.

Embodiment 39. The method of embodiment 33, wherein the first defensin and the second defensin are present in a mass ratio of about 1:5 to about 5:1.

Embodiment 41. The method of any of embodiments 22-39, wherein the cosmetically acceptable ingredient is selected from the group consisting ofLeuconostoc/radish root ferment filtrate, albumin, l-alanyl-l-glutamine, phospholipids, tocopheryl acetate, ubiquinone,Undaria pinnatifidaextract,Caesalpinia spinosafruit extract,Kappaphycus alvareziiextract,ceratonia siliquafruit extract,brassica albasprout extract and any combinations thereof.

Embodiment 42. The method of any of embodiments 22-39, wherein the cosmetically acceptable ingredient comprisesCaesalpinia spinosafruit extract.

EXAMPLES

While the cosmetic formulations may be prepared using any number of ingredients and formulations known in the art, preferred topical formulations include those that are ready-to-use and can be applied by a user. Therefore, with respect to cosmetically acceptable carriers, all cosmetically acceptable carriers are contemplated and include creams, oil-in-water emulsions, water-in-oil emulsions, foams, mousses, ointments, lotions, suspensions, serum, gels, etc.

Example 1: Lip Formulation Testing

Exemplary Topical Cosmetic Lip Formulation Ingredient List 1:

Exemplary Topical Cosmetic Lip Formulation Ingredient List 2:

Lip Hydration

A number of volunteers with dry lips are included in the study to test the immediate hydration effect of the composition on lips. Before application of the composition, the cutaneous hydration of the lips of each volunteer is measured using a corneometer. A defined quantity of the composition is applied in a standardized way by a technician on the lips of the volunteers. Two hours after application of the composition on the lips, the cutaneous hydration of the lips is measured again to test for an increase in lip hydration after a single application

Wrinkles Around Contours of Lip

A number of volunteers with visible wrinkles around the contours of lips are included in the study. On day 0 of the study before application of the composition, 3D acquisitions of wrinkles around the contour of the lips are generated by fringe projection and volunteers fill out a self-evaluation of wrinkles using a mirror at the test center. Following initial acquisitions and evaluations, volunteers apply the composition to their lips. One hour after application, volunteers fill out a second self-evaluation of the wrinkles using a mirror and undergo a second 3D acquisition by fringe projection. The volunteers then return home with the composition and apply the composition once daily from day 1 to day 6 of the study design. On day 7, volunteers return to the test center to complete a third self-evaluation using a mirror and a third 3D acquisition by fringe projection. The three self-evaluations and three 3D acquisitions per volunteer are compared to see if wrinkles around the contours of the lips became less visible over the course of the study.

Example 2: Nail Formulation Testing

Example Topical Cosmetic Nail Formulation Ingredient List:

Nail Thickness

A number of volunteers with weak nails are included in this study. The volunteers are instructed to not wear nail polish during the duration of the study. On day 0 of the study, the nail thickness of the volunteer's nails is measured using a high frequency echography machine (e.g., DermaScan CR 2D) at the test center. The volunteers then apply the composition to their nails. The volunteers are sent home with the composition and instructed to apply the composition once a day on days 1-27 of the study. On day 28, the volunteers return to the test center to measure the thickness of their nails using the high frequency echography machine.

Example 3: Improvements in Nail Characteristics

Study Design

Four subjects (3 females and 1 male) were included in a study to determine the efficiency of a nail composition of the present disclosure. Prior to the start of the study, the baseline characteristics of the subject's nails were measured. The nail characteristics examined in this study included: horizontal and vertical ridges of the nail, surface smoothness of the nail, nail plate hardness, nail free edge thickness, nail free edge length measurement, and skin hydration.

The nail composition was applied to the subject's nails and the skin all around the nails on both hands twice a day. The subjects applied the nail composition twice a day every day for 91 days, with nail characteristics measured on day 0 (baseline characteristics), day 28, day 28, day 56, and day 91. The nail free edge was cut to control the length of the nail on day 14, day 42, and day 77 of the study. In addition, high resolution images of the subject's nail were taken on day 0, day 28, day 56, and day 91 of the study.

Horizontal and Vertical Ridges of Nails Nail Smoothness

The depth and depth of horizontal and vertical ridges of the subject's nails were measured using the Visioscan® imaging system. The Visioscan® imaging system has a measuring heading with a CCD-camera and two metal halogen lamps positioned opposite of each other in order to ensure even illumination of the measuring filed on the nail. The grey level distribution of the pixels in the image corresponded to different phenomena, such as dark pixels representing ridges on the nail. Following imaging on the subject's nails, the Visioscan® software automatically calculated the horizontal and vertical ridges (e.g., roughness) of the nail.

The nail composition was shown to reduce the horizontal and vertical ridges on the nail over the course of the study has shown inFIGS.1A-1B, with an average reduction in ridges of about 25% on day 56 and about 46% on day 91 of the study.

In addition, the Visioscan® calculations of the average depth and width of the ridges on the nail also showed that the nail composition improved the overall smoothness of the nail, as shown inFIGS.2A-2B. Overall, the nail composition resulted in an average reduction of ridges in the nail of about 11% on day 56 and about 38% on day 91 of the study.

Nail Plate Hardness

The Nail Plate hardness of the subject's nails were measured using a durometer. The durometer measured the resistance force of the penetration of a pin into the nail under a known spring load; the amount of penetration (e.g., 2.5 millimeter maximum) of the pin was converted into a hardness reading on a scale with 100 units maximum.

The nail composition was shown to increase the average nail plate hardness over the course of the study, with the nail plate being significantly harder on day 56 and day 91 of the study, as shown inFIGS.3A-3B.

Nail Free Edge Thickness

The nail free edge thickness was measured by cutting the nail free edge on the thumb (both right and left hand) and measuring the thickness of the cut nail free edge of optical micrometry on day 0, day 28, day 59, and day 91 of the study.

The nail composition was shown to reduce the average thickness of the nail free edge on the thumb nail by an average of about 9% on day 56 and day 91 of the study, as shown inFIGS.4A-Bbelow.

Nail Free Edge Length

The length of the nail free edge length was measured by high resolution matched scientific photography. Each nail of the subject on each hand was photographed at the following time points: day 0 (baseline), day 28, day 56, and day 91. The images taken at each time point were then analyzed by PhotoGrammetrix® Image Analysis to calculate the length of the nail free edge length.

The nail composition was shown to significantly increase the average nail free edge length at day 28, day 56, and day 91 of the study has shown inFIGS.5A-5B.

Skin Hydration

The hydration of the skin around the nail (e.g., nail root) was measured via a nova dermal phase meter. The nova dermal phase meter measured skin surface impedance to determine the electroconductivity of the skin around the nail. The measurements obtained were a relative measurement of the retained water content of the skin as a function of the skin's dielectric value. The skin impedance measurement was recorded automatically by the nova dermal phase meter once equilibrium was achieved.

The nail composition was shown to significantly increase the average level of skin moisture in the skin around the nail on day 56 and day 91 of the study, as shown inFIGS.6A-6B.

Conclusions

Overall, application of a nail composition of the present disclosure resulted in improvements in overall nail surface smoothness, nail plate hardness, nail free edge thickness and length, and hydration of skin around the nail.

Example 4: Analysis of Lip Characteristics Following Lip Composition Use

Subjects are included in a study to determine the efficiency of a lip composition of the present disclosure. Prior to the start of the study, baseline characteristics of the subject's lips are measured. This study examines the following lip characteristics: dry lips, trans-epidermal water loss of lips, lip plumping, lip color, and wrinkles on lip line.

The lip composition is applied to the subject's lip and skin around the lip twice a day. The subjects applied the nail composition twice a day every day for 91 days, with lips characteristics evaluated on day 0 (baseline characteristics), day 28, day 56, and day 91.

Dry Lips

The dryness of the subject's lips is determined by taking photographs of the subject's lips on day 0, day 28, day 56, and day 91 of the study. The images of the subject's lips are graded on the scale as shown in Table 1. At the conclusion of the study, the lip dryness grades from each timepoint are analyzed to determine if there is a decrease in lip dryness by using the lip composition.

Lip Hydration Trans-Epidermal Water Loss Assay

The trans-epidermal water loss (TEWL) of the subject's lips is measured using a vapometer. To measure TEWL, the vapometer is placed directly on the lips of the subject on day 0, day 28, day 56, and day 91 of the study. Once placed on the lips of the subject, the vapometer automatically calculates the TWEL of the subject's lips in units of grams per meter2per hour (g/m2/h). At the conclusion of the study, the TWEL values calculated by the vapometer at each timepoint are compared to determine if there is a decrease in the TWEL of the lips by using the lip composition.

Lip Plumping

The fullness (e.g., plumpness) of the subject's lips is determined by taking photographs of the subject's lips on day 0, day 28, day 56, and day 91 of the study. The images of the subject's lips are graded on the photometric scale as shown inFIG.7. At the conclusion of the study, the lip fullness grades from each timepoint are analyzed to determine if there is an increase lip fullness by using the lip composition.

Lip Color

The color of the subject's lips is determined by taking photographs of the subject's lips on day 0, day 28, day 56, and day 91 of the study. The photographs are analyzed for appearance of lip color (e.g., pink, red, pale, purple) and evenness of lip color (e.g., presence of spots on the lips, spotted lips). Healthy pinks are considered to be pink or light red lips. At the conclusion of the study, the images of the lips are compared to determine if there is an increase in the number of subject's with pink or light red lips.

Wrinkles at Lip Line

The evaluation of wrinkles at the lip line (e.g., lipstick wrinkles) of the subject's lips is evaluated by taking photographs of the subject's lips on day 0, day 28, day 56, and day 91 of the study. The photographs of the subject's lips are then analyzed by an image-analysis software to calculate a visual grading score of the wrinkles at the lip line of the subject. At the conclusion of the study, the scores of the wrinkles at the lip line at each timepoint are compared to determine if there is a decrease in the presence of wrinkles at the lip line by using the lip composition.

Example 4: Application of Nail Serum Composition

A subject applies a nail root serum of a formulation as described herein twice daily to nails. The application occurs once in the morning and once in the evening.

Application is done by using an applicator with a built-in brush. One to two pumps of the serum are applied to all nails and to the skin around the nails on both hands using the built-in applicator brush. A pump of the serum is released from the formulation packaging by pushing a button on the bottom of the brush and distributing the serum across all nails and the skin around the nails. The serum is brushed from the bed of the nail toward the end of the nail. The serum is applied in an even distribution to the skin surrounding all sides of the nail.

The serum is left on the nails and the skin around the nails for at least 30 minutes. In some experimental procedures, the serum is left on the nails and the skin around the nails overnight.