Conditioned water containing amphibian bioactive molecules and uses thereof

Abstract

The present disclosure describes conditioned water containing amphibian bioactive molecules and purified bioactive molecules obtained from the conditioned water of amphibians. The present disclosure also describes methods of isolating bioactive molecules from the conditioned water. Moreover, the present disclosure describes using the purified bioactive molecules for treating skin conditions.

Description

Docket No. R217-0015PCTCONDITIONED WATER CONTAINING AMPHIBIAN BIOACTIVE MOLECULES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 744,632 filed January 13, 2025, and U.S. Provisional Application No. 63 / 939,332, filed December 12, 2025, which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The disclosure describes water used to grow amphibians and the use of the water for various purposes.BACKGROUND

[0003] Although all mammals including humans can spontaneously regenerate the tips of their fingers into adult life, mammals are not able to regenerate their limbs in contrast to amphibians. Most amphibians can regenerate missing body parts. However, their regenerative ability to regenerate varies extensively from species to species. Urodeles, for example, have exceptional regenerative capabilities.

[0004] Wound healing in amphibians and humans are similar. However, adult mammalian skin wound repair commonly results in scar tissue formation, while amphibians repair wounds by regeneration instead of scarring. It is desirable to develop methods for treating mammalian wounds that would not lead to scarring and in a shortened period.

[0005] A number of amphibian-derived wound healing molecules, including peptides have been reported. These molecules exist in skin secretions and contribute to skin repair in amphibians. Thus, there is an interest in developing ways to obtain the wound healing molecules of amphibians, in particular Urodeles, for treating mammalian skin.SUMMARY

[0006] This Summary is provided to introduce a selection of concepts in a simplified form that is further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0007] The present disclosure describes methods of preparing conditioned water and processing the conditioned water for use. In embodiments, processing the conditioned water includes isolating bioactive molecules from the conditioned water to obtain purified bioactive molecules (PBMs) of amphibians, such as Urodeles, or increasing the concentration of one or more bioactive molecules in the conditioned waterDocket No. R217-0015PCT

[0008] The present disclosure also describes using the PBMs and the conditioned water to treat and prevent skin conditions. The conditioned water can be processed conditioned water.BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 shows proteins present in conditioned water of baby axolotls. Silver staining is used to detect the proteins on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Control is deionized water (DI water). Ladder is the protein ladder showing the molecular weight of the proteins at the specific band. Sample 1 is conditioned water of the living quarter of a 5.1 -month old baby albino axolotl. Sample 2 is conditioned water from the living quarter of a 4.25-month old baby leucistic axolotl. Sample 3 is conditioned water from the living quarter of a baby axolotl of a 4-month old baby golden albino axolotl. The conditioned water was collected from after the baby axolotls have been in their living quarters for24hours.

[0010] FIGs. 2A and 2B show protein concentration of samples of conditioned water from FIG. 1 using Bradford assay (2A) and Sircoll assay (2B).

[0011] FIG. 3 shows the steps for processing the conditioned water.

[0012] FIGs. 4A, 4B, 4C, and 4D show examples of genes that are expressed after treatment with a mixture of purified bioactive molecules (PBMs), substantially free of endotoxin, and the mixture of bioactive molecules containing endotoxins (without endotoxin filtration). The bar graphs show linear fold change (LFC) in the expression of the genes. The top graph provides the combined total of the LFC of each of the genes on the x-axis. Sample 004 indicates treatment with a mixture of bioactive molecules containing endotoxins and Samples 005 and 006 are treatments with PBMs. Sample 005 was obtained using an ion exchange to remove the endotoxins, while sample 006 was obtained using an affinity column to remove the endotoxins.

[0013] FIG. 5 shows the locations of the punch biopsies performed on the subjects.

[0014] FIG. 6 shows the locations of the punch biopsies performed on the subjects.DETAILED DESCRIPTION

[0015] The present disclosure describes conditioned water from raising or growing amphibians for use in treating skin conditions.

[0016] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0017] The term “antigen” refers to a molecule that is a toxin or is foreign to the subject and induces an immune response in the form of the production of antibodies against the molecule. The compositions or activated skin described herein can have reduced antigenicity as compared to native skin, such that it can be used in a subject.Docket No. R217-0015PCT

[0018] The term “bioactivity” refers to biological effects. A substance is bioactive or has bioactivity includes a substance having a biological function. Bioactive molecules include proteins, peptides, polypeptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes, salts, and other functional molecules secreted by amphibians, such as axolotls. The conditioned water of one or more amphibians contains bioactive molecules. The bioactive molecules can be purified from the conditioned water of amphibians and are referred to as “purified bioactive molecules (PBMs)”.

[0019] The term "biocompatible" refers to a product and its normal degradation products in vitro, ex vivo, or in vivo that are substantially non-toxic and non-carcinogenic to a cell, tissue, organ, organism, or subject within useful, practical, and / or acceptable tolerances. The term "cytocompatible" refers to a product that can sustain the viability and growth of a population of cells.

[0020] The term “biomaterial" refers to a material suitable for in vitro, ex vivo, or in vivo use. As an example of in vivo use, the biomaterial is suitable for administering to a subject in need thereof. The material can be synthetic or natural. The compositions and activated skin described herein are examples of biomaterial.

[0021] The term "carrier" or “excipient” refers to a substance added to a composition that does not affect the active compound in the composition. The carrier can be a diluent. The excipient can be a substance added to the composition to facilitate the administration of the composition.

[0022] The term “cosmetics” refers to products (excluding pure soap) intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance. Examples of cosmetic benefits can include improving the appearance of skin such as improving the appearance of wrinkling and fine lines, removing oil and excess sebum, reducing the appearance of skin blemishes, cleansing, and conditioning the skin, toning, and tightening the skin, soothing irritation, and refreshing and cooling the skin.

[0023] The term “conditioned water” or “collected water” refers to the water that is used to raise one or more amphibians in a confined space or the water that the one or more amphibians have been living in a confined space. The confined space can be a tank of various sizes and is the living quarter of the one or more amphibians. Conditioned water includes purified water collected from the living quarter of the one or more amphibians 10 hours to 7 days, 12 hours to 5 days, 14 hours to 3 days, 16 hours to 2 days, 18 hours to 36 hours, 20 hours to 30 hours, 22, hours, 23 hours, 24 hours, 25 hours, or 26 hours, after the one or more amphibians have been placed in the living quarters. During the preparation of conditioned water for collection, fecal matters are removed on a daily basis and before collection of theDocket No. R217-0015PCTwater. The amphibians living the water are not fed any food and the water is not changed. The conditioned water includes bioactive molecules. After collecting the water, the conditioned water is further processed to remove nitrates, nitrites, endotoxins, and / or bacteria, such as E. coli.

[0024] The term “drugs,” “pharmaceuticals,” or “therapeutics” refers to articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease and articles (other than food) intended to affect the structure or any function of the body of man or other animals.

[0025] The term "derive", "derived," or "derives" refers to a product obtained from any stated source by any useful method. For example, an activated skin derived from an amphibian refers to an activated skin obtained from a member of the amphibian class.

[0026] The term “exogenous” refers to a product that originated outside of the organism, tissue, cell, organ, or subject. In contrast, the term “endogenous” refers to a product that originated from the organism, cell, tissue, organ, or subject.

[0027] The term “extracellular matrix” or “ECM” refers to a natural scaffolding having a three-dimensional structure including biomolecules and minerals that provide biochemical support to surrounding cells. The ECM includes structural and non-structural biomolecules, such as collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. The ECM can be obtained from various sources of tissues including the skin and non-cutaneous tissues.

[0028] The term "decellularized extracellular matrix" or "decellularized ECM" refers to ECM prepared by removing and / or devitalizing cells from ECM found in multicellular organisms, for example, amphibians or mammals. Decellularized ECM is substantially free of intact cells, lysed cells, and cellular components including cellular and nuclear debris such that the decellularized ECM exhibits reduced immunogenicity so that it can be administered to a subject, for example, a mammalian subject, as a non-toxic xenograft or biomaterial. A decellularized ECM that is “substantially free of immunogenic components” refers to an ECM in which immunogenic components are at a level that is not sufficient to induce an adverse immune response in a subject.

[0029] The term "isolated" refers to being separated or removed from its native surroundings, such that it is substantially free from components that accompany it in its naturally-occurring state. For example, a cell or a protein can be isolated from its naturally-occurring state.

[0030] The term “immunogenic” refers to relating to or producing an immune response. The term “immunogenicity” refers to the ability of a foreign substance, such as an antigen, to provoke an immune response in a subject. The compositions and activated skin describedDocket No. R217-0015PCTherein can have reduced immunogenicity as compared to the native skin, such that it can be used as a biomaterial in a subject.

[0031] The term "non-toxic" refers to a product that causes little or no adverse reaction or substantial harm to cells and tissues in vitro or ex vivo, and / or does not cause a substantial adverse or undesirable reaction or substantial harm to cells and tissues in the body (in vivo).

[0032] The term “prevent” or “prevention” refers to the prevention of the onset, recurrence, or spread of a condition or one or more symptoms of the condition. As an example, the condition could be a skin condition. The term includes the administration of a product described herein before the onset of symptoms in particular to subjects at risk of developing a condition, such as a skin condition. The term includes the inhibition or reduction of one or more symptoms associated with the skin condition. The term “prevention” can be used interchangeably with the term “prophylactic treatment”.

[0033] The term “Purified Protein Substrate (PPS)” refers to a mixture of proteins, peptides, amino acids, and / or polypeptides obtained from the conditioned water of animals that has been processed as described herein, for example, to remove endotoxins, water, and debris including fecal matters, loose tissues, cells and cellular debris, food, ammonia, nitrates, nitrites, negatively charged aliphatic lipids, salts, and microbes including bacteria. As an example, the PPS obtained from processed conditioned water of amphibians is “Purified Amphibian Protein Substrate (PAPS)”. The PPS can be obtained from the processed conditioned water of Urodeles, axolotls, axolotls of specific phenotype including albino, golden albino, leucistic, or wild type, or axolotls of a combination or a blend of phenotypes of axolotls.

[0034] The term “retain structural and functional integrity" used with reference to the ECM refers to retaining sufficient structure and function to permit and support the use of the matrix as a substrate for the growth of cells in vivo, ex vivo, or in vitro. For example, the decellularized ECM retains the structure and functional properties of a naturally occurring ECM enabling its use as a biomaterial.

[0035] The terms “scaffold” and “bioscaffold” are used interchangeably to refer to a substrate on which cells can grow in vitro, ex vivo, and / or in vivo. A scaffold or bioscaffold is an example of a biomaterial.

[0036] The terms “skin” and “skin sample” are used interchangeably to refer to the skin or a sample of skin from a subject.

[0037] The term “skin conditions” includes skin conditions that require therapeutic “drug” treatment including diseases, defects, and injuries including wounds, such as full-thickness wounds, deep dermal wounds, and burns.Docket No. R217-0015PCT

[0038] The term “cosmetic skin conditions” includes skin conditions that are related to tone, clarity, radiance, brightness, elasticity, thinning, firmness, pore size, inflammation, and / or hydration of the skin.

[0039] The term "subject" refers to an animal, for example, a mammal. Examples of mammals include humans, dogs, cats, horses, cows, goats, sheep, pigs, and non-human primates. A subject in need of treatment or a subject in need thereof includes a subject having a disease or condition that needs to be treated. A subject in need thereof also includes a subject that needs treatment and / or prevention of a skin condition.

[0040] The term "therapeutically effective amount" refers to an amount of a product or composition that provides a therapeutic benefit in the treatment, prevention, or management of a condition or disease, such as a skin disease, an injury to the skin, or a wound, for example, a drug product. The term “therapeutically effective amount” also includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.

[0041] The term "treatment" or "treating" in the context of administering a product, such as a biomaterial, to a subject refers to administering the product to achieve a desirable clinical / medical endpoint, including alleviating symptoms of a disease or condition. Examples of such desirable endpoints associated with skin disease or condition include wound healing, tissue closure, bulking tissue, preventing tissue adhesion, providing structural support to tissue, providing a protective barrier, and / or correcting a defect. Administering the product also includes applying the product to a subject.

[0042] The term “xenogenic” refers to a product derived or originated from a member of another species.

[0043] The term “amphibians” refers to cold-blooded vertebrate animals that include frogs, toads, newts, salamanders, and caecilians. They have an aquatic gill-breathing larval stage followed by a terrestrial lung-breathing adult stage. Amphibians include the class of amphibians and the orders of Anura (frogs or toads), Urodela (newts or salamanders), and Apoda (caecilians). In embodiments, the amphibians described herein are young or neotenic amphibians. A young amphibian includes a young frog, such as a froglet, tadpole, or larval stage young Apoda. The amphibian can be a young amphibian and the young amphibian can include a larval stage of any order of amphibian. The amphibian can be neotenic.

[0044] The term “salamanders” refers to a group of amphibians characterized by a lizardlike appearance and having a tail throughout life. The families of salamanders include the Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), Proteidae (mudpuppies and olms),Docket No. R217-0015PCTRhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The Ambystomatidae family includes Ambystoma altamirani, Ambystoma amblycephalum, Ambystoma andersoni, Ambystoma annulatum, Ambystoma barbourin, Ambystoma bishop, Ambystoma bombypellum, Ambystoma californiense, Ambystoma cingulated, Ambystoma dumerilii, Ambystoma flavipiperatum, Ambystoma gracile, Ambystoma granulosum, Ambystoma jeffersonianum, Ambystoma laterale, Ambystoma leorae, Ambystoma lermaense, Ambystoma mabeei, Ambystoma macrodactylum, Ambystoma maculatum, Ambystoma mavortium, Ambystoma mexicanum, Ambystoma opacum, Ambystoma ordinarium, Ambystoma rivulare, Ambystoma rosaceum, Ambystoma silvense, Ambystoma subsalsum, Ambystoma talpoideum, Ambystoma taylori, Ambystoma texanum, Ambystoma tigrinum, and Ambystoma velasci.

[0045] The families of salamanders are grouped under the order Urodela (or Caudata). The term “Urodele” refers to a salamander of the order Urodela, in the class Amphibia. The amphibians can be from the orders Urodela, Anura, and Apoda. Urodeles begin life as aquatic animals in a larval state, and some undergo metamorphosis from a juvenile form with gills to an adult, terrestrial, air-breathing form with lungs. During metamorphosis, a Urodele's physical features are altered in preparation for life on land. These alterations include caudal fin resorption, thickening of the skin, the development of dermal glands, and resorption of gills. Sexual maturity also occurs during this time in most Urodeles. However, some families of Urodeles are "neotenic," which means that individuals of such families, even after reaching sexual maturity, retain their juvenile aquatic form throughout their lives. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of neotenic salamanders. Instead of becoming a terrestrial amphibian, an adult axolotl remains aquatic and gilled. However, under certain circumstances, an axolotl will undergo metamorphosis and transform into a terrestrial form.

[0046] Axolotls possess pigment cells, called chromatophores, that are responsible for their colors. The chromatophores of axolotls include melanophores containing eumelanin, xanthophores containing pteridines, and iridophores containing crystallized purines. Eumelanin is a black-brown pigment; pteridine is a yellow and reddish pigment; and crystallized purine is an iridescent white pigment. These pigments which are encoded by their respective genes provide the different phenotypes of axolotls, such as wild-type, golden albino, leucistic, and melanistic.

[0047] Axolotls have the ability to fully regenerate lost or damaged body parts including organs, limbs, and parts of the central nervous system, throughout their entire life. Axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scar-less processes. The axolotl wound healing process resembles the scar-free healingDocket No. R217-0015PCTprocess of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than do the corresponding events in postnatal mammals.

[0048] Although the skin structure of amphibians is similar to mammals, Urodeles and anuran amphibians (frogs and toads) can regenerate their skin structures including the dermis and secretion glands without forming any scar after a deep skin injury. Moreover, the skin of amphibians contains ECM, which is rich in growth factors, which are favorable for wound healing. The ECM is a three-dimensional network of extracellular macromolecules and minerals including collagen, enzymes, glycoproteins, and hydroxyapatite which provide structural and biochemical support to surrounding cells. The ECM can include a combination of fibrous and network-type collagens. Examples of various types of collagens, such as one or more of type I, II, III, IV, V, and VI collagens. The ECM can also include elastin and / or elastic fibers. The ECM can also include laminin, fibronectin, hyaluronan, chondroitin sulfate, or both, and / or one or more proteoglycan, glycoprotein, glycosaminoglycan, or any combination thereof. The components and structure of the ECM play an important role in the healing process because the ECM components create scaffolding which provides the structural architecture of the matrix required for the healing process. Moreover, the ECM components are involved in stimulating the adhesion and migration of cells during the healing process as well as mediating the interactions among the cells and between the cells and the matrix, or between ECM proteins during the healing process. Further, the ECM components also serve as a reservoir and modulator of the action of the cytokines and growth factors to regulate wound repair activities.

[0049] Some amphibians start their life in water and transform into air-breathing adults with lungs and appendages while others remain aquatic their entire lives. They can secrete various bioactive molecules having therapeutic functions from their skin. Such molecules include peptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes.

[0050] The water used to raise amphibians, such as Urodeles, is purified water and is referred to as conditioned water and includes bioactive molecules from amphibians. The conditioned water is clean as compared to the water that wild amphibians live. The conditioned water is prepared by growing a specific one or more amphibians, for example, specific age, phenotype, or a combination thereof, in a confined amount of space for a specified amount of time and the amphibians are not fed any food. Additionally, the water is cleaned on a daily basis to remove fecal matter. After collection of the water from the living quarters of the amphibians, the collected water is further processed under specific conditions to remove mud,Docket No. R217-0015PCTsand, bacteria, such as E. coli, endotoxins, nitrites, and / or nitrates. Therefore, the collected water is conditioned water and is clean.

[0051] In embodiments, the amphibians are newborn to one year old, one-month to ten-month old, two-month to eight-month old, three-month to six-month old, three-month old, four-month old, five-month old, or six-month old. In embodiments, the amphibians are neotenic amphibians including neotenic Urodeles such as neotenic axolotls. The conditioned water is the water used to raise neotenic axolotls of various phenotypes including wild-type, albino, golden albino, leucistic, and melanistic, or a combination thereof. In embodiments, the conditioned water is obtained from axolotls of a certain age, a specific phenotype, a combination of phenotypes, a specific sex, a combination of sexes, or a combination thereof. As an example, the axolotls can be wild-type, golden albino, leucistic, melanistic, or a combination or blend of these phenotypes. In embodiments, the conditioned water from these animals is processed as described herein. The processed conditioned water of these animals contains specific bioactive molecules in an enhanced quantity as compared to a control. The control can be a sample of water from the wild where the amphibians are grown and bred. The control can be deionized water that is clean and has not been used for growing amphibians.

[0052] The conditioned water is collected from the living quarters of the one or more amphibians, such as Urodeles, for example, axolotls as described herein. The amphibians have been living in the collected water for 10 hours to 7 days, 12 hours to 5 days, 14 hours to 3 days, 16 hours to 2 days, 18 hours to 36 hours, 20 hours to 30 hours, 22, hours, 23 hours, 24 hours, 25 hours, or 26 hours, before being collected and before water is changed. Fecal matters in the living quarters are removed daily and before water collection. The amphibians are not fed any food until after the water has been changed. The amphibians are fed every 48 to 72 hours.

[0053] The conditioned water can be processed by concentration and / or filtering for ease of storage and / or use. Filtration removes undesirable amphibian matters and can also sterilize the conditioned water. Filtration can include membrane filtration. Examples of membrane filtration includes ultrafiltration, diafiltration, depth filtration, normal flow filtration and tangential flow filtration, which can reduce the volume of the collected water and increase the concentration of the bioactive molecules. The conditioned water can also be processed by concentration, for example, by chromatography, vacuum evaporation and freeze-drying (lyophilization). The collected conditioned water can also be frozen for storage before or after concentration and / or filtering. The frozen water can also be thawed for concentration and / or filtration for use.

[0054] In embodiments, about 10 to 20 liters of collected unprocessed water can be filtered by filtration, such as depth filtration (0.5 / 0.2 micron) for 2 hours (hrs). The filtered water isDocket No. R217-0015PCTfrozen for about 24 hrs and lyophilized for 24 hours to form a powder for ease of storage and / or transport. The powder can be reconstituted by acidification to a pH of about 4.5 to 6.5 followed by sonification to solubilize the bioactive molecules post lyophilization. Examples of acids used for acidification can include acetic acid and phosphoric acid. As an example, the powder can be reconstituted by dissolving 1 gram (g) of powder in 2 to 20 milliliters (mL), 5 to 15 mL, or 8 to 12 mL of acid. In embodiments, the powder can be reconstituted at a ratio of 1 gram (g) of powder to 10 mL of acid. As an example, 1.5 g of the powder can be reconstituted with 15 ml of acetic acid. The acidified mixture can be sonicated for about 2 to 10 seconds (secs) a few times with 30 secs in between to solubilize the bioactive molecules. The bioactive molecules can also be solubilized by microwaving the acidified mixture. The process of collecting and reconstituting the powder takes about 3 hrs (hrs).

[0055] The bioactive molecules are then diluted in water to a concentration of 1 pg / mL (1 microgram of protein per milliliter (mL) of water) to 30 pg / mL, 1 pg / mL to 20 pg / mL, 1 pg / mL to 15 pg / mL, 1 pg / mL to 10 pg / mL, 1 pg / mL to 5 pg / mL, 10 pg / mL to 200 pg / mL, 20 mg / mL to 30 pg / mL, 50 p / mL to 100 pg / mL, 100 pg / mL to 150 pg / mL, or 150 pg / mL to 200 pg / mL. . The water used is cell culture grade water (CCGW) or deionized water. The solubilized bioactive molecules can then be dialyzed for 24 hrs using tubing, such snakeskin tubing, or a cassette, to remove salts and small molecules such as ammonium, nitrates, and nitrites. The pore size of the tubing or cassette is about 10 kDa. After dialysis, the solubilized bioactive molecules undergo a 15 minutes (mins) pre-column sterilization using a 0.22 micron filter to obtain a sterilized solution containing bioactive molecules. However, the process can also be performed without dialysis and the solubilized bioactive molecules can proceed directly to precolumn sterilization.

[0056] Next, the sterilized solution undergoes column chromatography (endotoxin filtration) for about 7 hrs to remove endotoxins. The column can be an affinity column that contains a stable adsorbent for binding endotoxin with high efficiency to remove them from the bioactive molecules The type of affinity column used can include EtoxiClear® column which is a prepacked column. The driving force for the affinity column can be gravity or a peristaltic or syringe pump. Ion exchange and gravity columns can also be used to remove endotoxins. Elution buffers with appropriate salt concentrations can be used on the columns to elute the bioactive molecules. Examples of elution buffers can include 0.05 M sodium chloride to 0.1 M sodium chloride, phosphate buffered saline (PBS) pH 7.2, and PBS with 0.1 M sodium chloride. Other examples of buffers include citric acid, potassium phosphate buffer, bicine, boric acid, cacodolyic acid, sulphonic acid, glycine, glycylglycine, sodium phosphate, sulfonylethyl acid, ortricine or tris buffer. A pump, such as a peristatic pump or syringe pump, can also be used with the gravity column to push the bioactive molecules out of the column.Docket No. R217-0015PCTThe bioactive molecules in the solution can be eluted from the column with the application of the appropriate buffer(s) for about 1 to 7 times, 1 to 6 times, 1 to 5 times, 1 to 4 times, 1 to 3 times, or 1 to 2 times to obtain an eluted bioactive molecule solution. In embodiments, the endotoxins are separated from the bioactive molecules by binding the endotoxins to the column, and the bioactive molecules are comes off the column without the application of any elution buffer. The process of endotoxin filtration can take about a few minutes to 5 hours.

[0057] In embodiments, about 50% to 99%, 60%, 75%, 80% to 99%, 85% to 99%, 90% to 99%, or about 95% to 99% of the endotoxins are removed from the bioactive molecules in the solution. The solution of bioactive molecules after endotoxin removal is substantially free of endotoxins, such that about 75% to 99% of the endotoxins are removed. In embodiments, the solution of bioactive molecules contain less than 100 endotoxin units (EU) per mL, less than 80 EU / mL, less than 70 EU / mL, less than 50 EU / mL, less than 40 EU / mL, between 10 EU / mL to 100 EU / mL, between 10 EU / mL and 50 EU / mL, between 10 EU / mL and 40 EU / mL, and between 10 and 30 EU / mL, and between 10 EU / mL and 20 EU / mL Examples of endotoxins include lipopolysaccharides (LPS).

[0058] The solution of bioactive molecules, substantially free of endotoxins, need not be diluted but can also be diluted 10 times using a diluent such as CCGW, PBS, or acidified PBS. As an example, the solution can be diluted to a concentration of 20 to 30 ug / mL of CCGW at about pH 5.0. The solution then undergoes a final 15 mins sterilization process via a 0.22 micron filter to obtain the sterilized mixture of proteins. The sterilized mixture contains purified bioactive molecules (PBMs) from amphibians, such as Urodeles, for example axolotls. The sterilized mixture of PBMs includes proteins and other bioactive molecules for example, peptides, amino acids, glycoproteins, fatty acids, and steroids, and are substantially free of endotoxins. In embodiments, the purified PBMs include proteins, polypeptides, peptides, and / or amino acids, in which case the mixture can be referred to as “purified amphibian protein substrate (PAPS)”.

[0059] Sterile filtration is used at various stages of the process to remove bacteria and sediments. The sterile filters used can have a pore size of 0.2 microns to 0.5 microns. In embodiments, the sterile filters have a pore size of 0.22 microns.

[0060] The entire process takes about 2.5 days or about 60.5 hours.

[0061] The collected unprocessed water can also be filtered by tangential flow filtration (TFF) which would not require freezing and lyophilization of the bioactive molecules and saves about 26 hours. The retentate of the TFF contains bioactive molecules in a smaller volume of sterile conditioned water and can proceed to column chromatography for removal of endotoxin and sterile filtration.Docket No. R217-0015PCT

[0062] In embodiments, the removal of the endotoxin can also take place prior to filtration with TFF or after depth filtration and before freezing the filtered water. When endotoxin removal occurs before TFF, the retentate containing bioactive molecules are substantially free of endotoxins and can proceed to sterile filtration and / or lyophilization into a powder for storage. Similarly, when the endotoxin removal occurs immediately after filtration, such as depth filtration, the eluted solution is substantially free of endotoxin and can proceed to sterile filtration, freezing, and lyophilization.

[0063] The conditioned water contains bioactive molecules useful for treating skin conditions. The conditioned water can be minimally processed, for example, to remove the endotoxins and / or filtered to become more concentrated. The conditioned water can be processed as described herein to obtain compositions including an increased concentration of bioactive molecules that are substantially free of endotoxins. At least the concentration of the one or more bioactive molecules in the collected water are increased as compared to a control sample described herein. The processing of the conditioned water as described herein, which includes filtration, such as clarification filtration, concentration filtration, and / or sterile filtration, and column chromatography, removes nitrates, nitrites, endotoxins, and / or bacteria, such as E. coli.

[0064] Moreover, other methods of obtaining bioactive molecules from the conditioned water include precipitation or extraction from the collected water by well-known methods including the use of cold nonpolar solvents, such as petroleum ether, hexane, and / or acetone, to displace the water and force the bioactive molecules to precipitate into the insoluble nonpolar solvent. The solvent can then be evaporated off to obtain the bioactive molecules.

[0065] Additional methods for obtaining the bioactive molecules from the conditioned water include precipitation with chemicals like ammonium sulfate or trichloroacetic acid, filtration through membrane filters with specific pore sizes, ultracentrifugation, and chromatography such as affinity chromatography, ion exchange chromatography, and size exclusion chromatography.

[0066] The sterilized mixture of PBMs obtained after processing of the conditioned water can be used immediately or stored to be used at a later time. The sterilized mixture can be stored frozen or in the cold at 2 °C to 8 °C for later use. It can also be lyophilized to a powder and stored. The powder can be reconstituted and stored. In embodiments, the storage can be for a few weeks to more than 2 years depending on the means of storage. In embodiments, the sterilized mixture can be stored for 4 months to 8 months in the cold at 2 °C to 8 °C without deterioration.

[0067] The present disclosure describes compositions including the processed conditioned water and / or PBMs obtained from the conditioned water. The compositions described hereinDocket No. R217-0015PCTcan further include carriers or excipients. The compositions can be in the form of a pharmaceutical composition or cosmetic composition, and optionally including one or more pharmaceutically or cosmetically acceptable carriers, respectively. The compositions can be formulated in the form of a powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, a dispersion, an emulsion, an ointment, a gel, a hydrogel, or a gelatin.

[0068] Proteomic analyses of the conditioned water after minimal processing without the removal of endotoxins shows that it includes proteins that are essential for regenerative medicine such as wound healing, tissue regeneration, tissue repair, neuroprotection, treatment of neurodegenerative disease, immunomodulation, inflammation, cancer therapy, cell growth regulation, and metabolic regulation. Examples of such proteins include keratin 5 fragment, histones H3 and H4, thrombospondin-1 and -4, exotoses multiple-like 3 protein, and alpha-globin and beta-globin chains, that are essential for regeneration and repair of tissues. Other proteins found in the collected processed water include heat shock protein 70 (HSP70), activity dependent neuroprotector homeobox (ADPN), and arrestin-C, which are important for neuroprotection against neurodegenerative diseases. Additionally, proteins involved in immunomodulation and inflammation such as lysozyme g and Wnt-5a were present in the collected processed water. Further, examples of proteins, such as prominin-3 and E2F transcription factor 5, which are involved in cancer therapy and cell growth regulations, and proteins such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) involved in metabolic regulation were also present in the collected processed water without the removal of endotoxins. Minimal processing included one or more of concentration via lyophilization and reconstitution using acidified solvents, and diafiltration to remove some salts.

[0069] Gene marker studies were conducted to determine how the samples of the PBMs modulate gene expression in human skin. A 3D in vitro skin model (MatTek EFT-400) containing epidermal keratinocytes and dermal fibroblasts was used. Differential gene expression was assessed after 24 or 48 hours of exposure to the test material (TM or PBMs) using mRNA-Seq by comparing to control human skin which were not treated with PBMs to determine activation (upregulation) or suppression (downregulation) of genes.

[0070] Gene marker studies were used to compare treatment with PBMs, which is substantially free of endotoxins, with treatment with mixtures of bioactive molecules obtained from conditioned water containing endotoxins (without endotoxin removed). In FIGs. 4A-4D, Sample 004 is a mixture of bioactive molecules containing endotoxins, and Samples 005 and 006 are PBMs, which are substantially free of endotoxins. FIGs. 4A-4D show that Sample 004 activated: the epidermal barrier gene TGM1; the cell renewal / regeneration gene CASP3; the extracellular matrix integrity gene TNG; and the proinflammatory cytokine genes IL1B, and IL23. Sample 005 inhibited: the epidermal genes FLG, KRT1, LOR, PKP1, and ST14; the cellDocket No. R217-0015PCTrenewal / regeneration genes CALML5 and CASP14; and the extracellular matrix integrity genes DXC1, DSG1 , and DSG3. Sample 005 activated, the inflammation genes DEFB1 and IL23A. Sample 006 activated: the epidermal barrier genes CLDN1 , KRT1, and KRT10; the cell renewal / regeneration genes CASP3 and TP63; and the extracellular matrix integrity genes COL7A1 and DSG1. Sample 6 inhibited: the cell / renewal regeneration gene CASP14; the extracellular matrix integrity gene COL17A1; and the inflammation genes TLR2 and TNF.

[0071] FIGs. 4A-4D confirms that removal of endotoxins removes some of the bioactive molecules including proteins and changes the function of the mixtures of bioactive molecules. As an example, in FIG. 4D, the endotoxins present in sample (004) activated the proinflammatory cytokine genes, such as IL1B, IL23A. Both IL1B and IL23 are activated with 0.01 wt% of Sample 004 and only IL1B is activated at 1.0 wt% of Sample 004. Sample 005 (0.1 wt%) activated genes such as DEFB1 and IL23A. DEFB1 , beta-Defensin 1, is an antimicrobial peptide. Sample 006 (0.1 wt%) inhibited genes such as TLR2 and TNF. TLR2 (Toll-like Receptor 2) and TNF (Tumor Necrosis Factor) are important for innate immune responseTLR2 recognizes pathogens and triggers signaling pathways leading to the release of pro-inflammatory cytokines, including TNF. TNF can further increase TLR2. The inhibition of such genes indicates that the endotoxins are removed from the sample. These results indicate that the PBMs are functionally different from the mixture of amphibian proteins found in nature, as the removal of endotoxins makes the purified bioactive molecules more suitable for treating skin conditions.

[0072] Similar gene marker studies were conducted using the PBMs. The results of the gene marker studies show a trend towards an increased modulation of: anti-aging genes such as FOXO3, MFN2, PNK4, POLG1 / MD, and SIRT1 ; cell renewal / regeneration genes such as CASP3 and TP63 genes; epidermal barrier genes, such as CLDN1 , KRT1 , and KRT10; extracellular matrix integrity genes, such as COL7A1 and DSG3; and antioxidant / stress response genes, such as AHR, HMOX1 , MT1A, MT2A, and SIRT1 ; and decreased modulation of: inflammation / immune response genes, such as TNF, and TLR2; extracellular matrix breakdown genes, such as KLK5, KLK7, and SERPINB3 and modulation of tissue homeostasis such as PTGS1. Tables 1 and 2 below (in the Examples) show the linear fold change (LFC) as compared to the control of the representative genes.

[0073] The results of T ables 1 and 2 indicates that the PBMs treatment activated the genes involved in anti-aging, cell renewal / regeneration genes, epidermal barrier genes, extracellular matrix integrity genes, and antioxidant / stress response genes. The results also indicates that at the same time the PBMs treatment inhibited the inflammation / immune response genes and extracellular matrix breakdown genes. The results confirm that mixture of PBMs is effective in treating various skin conditions by activating the required genes associated with anti-aging,Docket No. R217-0015PCTcell renewal / regeneration, epidermal barrier, extracellular matrix breakdown and antioxidant / stress response, and at the same time by inhibiting inflammation / immune response and extracellular matrix breakdown genes.

[0074] The compositions described herein can be used to treat subjects with skin conditions. In embodiments, the compositions described herein can be used to treat and / or prevent various skin conditions including inflammatory skin conditions and injured skin having a wound or disrupted skin barrier. In embodiments, the compositions can be used to heal wounds.

[0075] One or more agents can be added to the compositions described herein for treating subjects with skin conditions. The one or more agents can be xenogenic to amphibians, for example a protein or a small molecule. In embodiments, the present disclosure provides a composition comprising the collected water and one or more agents that are xenogenic to amphibians. Examples of one or more agents xenogenic to amphibians include peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof. The proteins or peptides comprise growth factors, cytokines, or chemokines. The polymers comprise synthetic or natural polymers or copolymers. The drugs comprise retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics. The antibiotics comprise neomycin, polymyxin B, bacitracin, ora combination thereof. In embodiments, the one or more agents xenogenic to amphibians can be a mammalian molecule, for example, a mammalian peptide, protein, drug, or nutrient.

[0076] Examples of inflammatory skin conditions include psoriasis; dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. Other skin conditions needing treatment include fine lines and / or wrinkles, aging, redness, abrasion, burns, cuts, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The burns include acute thermal burns including first, second, or third-degree burns.

[0077] The compositions described herein can also be used in a skincare regimen for protecting the skin from damage including UV rays and environmental pollution and as an aesthetic agent for improving the appearance of the skin. The compositions described herein can be used to treat and improve photodamaged skin. Photodamaged skin is characterizedDocket No. R217-0015PCTby one or more skin changes including fine and coarse wrinkles, roughness, freckles, and pigmentation, such as brown spot, that occur as a result of prolonged exposure to the sun.

[0078] The compositions described herein can be used to treat or improve the appearance of uneven skin tones, visible hyperpigmentation, and dyschromia (changes in the color of the skin or nails). The compositions described herein can be used to treat and improve skin hydration, skin quality and texture, and reduce the appearance of fine lines and wrinkles.

[0079] The compositions described herein can be used to enhance treatment after nonablative fractional laser. Facial non-ablative fractional resurfacing refers to the use of lasers to reduce the signs of aging, to improve skin texture, laxity (loss of skin elasticity), and tone, and to reduce the appearance of scars and stretch marks. The process involves the microscopic injury of the epidermal, the thin outer layer of the skin, and the superficial dermal layers of the skin, the vascular tissue that plays a key role in skin function that includes the steady supply of oxygenated blood and nutrients, for reducing the signs of photodamaged skin. Photodamaged skin results from premature aging of the skin due to exposure to sun’s ultraviolet (UV) radiation. The compositions described herein can increase healing after a nonablative fractional laser procedure on the skin, including photodamaged skin and to decrease downtime (duration of discomfort).

[0080] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0081] Numbers expressing ranges or quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ± 20% of the stated value; ± 15% of the stated value; ± 10% of the stated value; ± 5% of the stated value; ± 4% of the stated value; ± 3% of the stated value; ± 2% of the stated value; ± 1% of the stated value; or ± any percentage between 1% and 20% of the stated value.

[0082] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” orDocket No. R217-0015PCT“comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. Lack of a material effect can include a lack of a statistically significant biological function of one or more PBMs in vitro or in vivo, such as the modulation of genes or a lack of a statistically significant improvement in using the PBMs in treating skin conditions.

[0083] 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. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.5, 2.7, 3, 4, 5, 5.1, 5.3, 5.8 and 6. Moreover, any ranges cited herein are inclusive.

[0084] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein.

[0085] The following exemplary embodiments and examples illustrate exemplary products, compositions, and methods described herein. These examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure.EXEMPLARY EMBODIMENTS

[0086] The following are exemplary embodiments:1. Purified bioactive molecules (PBMs), substantially free of endotoxins, including a mixture of one or more amphibian bioactive molecules present in the conditioned water of the living quarters of one or more amphibians; or conditioned water from the living quarters of one or more amphibians, wherein the conditioned water is substantially free of endotoxins and includes a mixture of one or more amphibian bioactive molecules.2. The PBMs or the conditioned water of embodiment 1, wherein the one or more amphibians are a frog, toad, newt, or salamander.Docket No. R217-0015PCT3. The PBMs or the conditioned water of embodiment 1 or 2, wherein the one or more amphibians are a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.4. The PBMs or the conditioned water of any one of embodiments 1 -3, wherein the one or more amphibians is a froglet, tadpole, Urodele, or larval stage young Apoda.5. The PBMs or the conditioned water of any one of embodiments 1 -4, wherein the one or more amphibians is a baby Urodele, and optionally the amphibian is a baby axolotl.6. The PBMs or conditioned water of any one of embodiments 1-5, wherein the mixture of bioactive molecules includes one of more proteins involved in wound healing, tissue regeneration and / or tissue repair, neuroprotection and / or treatment of neurodegenerative diseases, immunomodulation and / or inflammation, cancer therapy and / or cell growth regulation, metabolic regulation, and a combination thereof.7. The PBMs or conditioned water of any one of embodiments 1 -6, the PBMs or conditioned water including one or more proteins involved in skin wound healing, tissue regeneration, and / or tissue repair include keratin 5, histone H3, histone H4, thrombospondin -1 , thrombospondin-4, exostoses multiple-like 3 protein, alpha-globin chain, beta-globin chain, and a combination thereof;the PBMs or conditioned water including one or more proteins involved in neuroprotection and / or treatment of neurodegenerative diseases include heat shock protein 70 (HSP70), activity-dependent neuroprotector homeobox (ADNP), arrestin-C, and a combination thereof;the PBMs or conditioned water including one or more proteins involved in immunomodulation and inflammation include lysozyme g, wnt 5a, and a combination thereof;the PBMs or conditioned water including one or more proteins involved in cancer therapy and cell growth regulation include prominin 3; orthe PBMs or conditioned water including one or more proteins involved in metabolic regulation includes glyceraldehyde 3-phosphate dehydrogenase (GAPDH).8. The PBMs or conditioned water of any one of embodiments 1-7, wherein the PBMs include amphibian proteins, peptides, polypeptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes, salts, or a combination thereof9. A composition including the PBMs or the conditioned water of any one of embodiments 1-8, and optionally wherein the composition includes one or more carriers or excipients. 10. The composition of embodiment 9, wherein the composition is a pharmaceutical composition, and optionally including one or more pharmaceutically acceptable carriers orDocket No. R217-0015PCTexcipients; or wherein the composition is a cosmetic composition and optionally including one or more cosmetically acceptable carriers or excipients.11. The composition of embodiment 9 or 10, wherein the composition further includes one or more agents xenogenic to amphibians; and optionally, wherein the one or more agents include one or more peptides, proteins, drugs, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, and a combination thereof.12. A method of obtaining PBMs or conditioned water of any one of embodiments 1-8, wherein the method includesraising one or more amphibians in water for 10 hours to 7 days, 12 hours to 5 days, 14 hours to 3 days, 16 hours to 2 days, 18 hours to 36 hours, 20 hours to 30 hours, 22, hours, 23 hours, 24 hours, 25 hours, or 26 hours;removing fecal matters from the water on a daily basis;collecting the water;and processing the collected water to remove endotoxins to obtain PBMs and / or conditioned water substantially free of endotoxins.13. The method of embodiment 12, wherein the method further includes filtering the collected water to decrease volume and / or increasing the concentration of one or more bioactive molecules in the collected water.14. The method of embodiment 12 or 13, wherein the method further includes filtering the collected water before or after removal of the endotoxins by membrane filtration; and optionally wherein membrane filtration includes ultrafiltration, diafiltration, depth filtration, or tangential flow filtration.15. The method of any one of embodiments 12-14, wherein the method further includes filtering the collected water by depth filtration prior to removal of the endotoxin or filtering the collected water by tangential flow filtration after removal of the endotoxin.16. The method of any one of embodiments 12-15, wherein processing the collected water includes freezing the collected water before or after removal of the endotoxins to obtain frozen water; and optionally, storing the frozen water.17. The method of embodiment 16, wherein the method further includes lyophilizing the frozen water into a powder; and optionally storing the powder.18. The method of embodiment 17, wherein the method further includes reconstituting the powder into a solution and sterilizing the solution using a sterile filter to obtain a sterilized solution.19. The method of any one of embodiments 12-18, wherein removing the endotoxin includes using chromatography to obtain PBMs or conditioned water substantially free of toxins; andDocket No. R217-0015PCToptionally wherein the chromatography is affinity chromatography, ion exchange chromatography, gravity chromatography, or a combination thereof.20. The method of embodiment 19, wherein the method further includes sterilizing the PBMs or conditioned water by sterile filtration.21. The method of any one of embodiments 12-20, wherein the one or more amphibians are a frog, toad, newt, or salamander.22. The method of any one of embodiments 12-21, wherein the one or more amphibians are a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.23. The method of any one of embodiments 12-22, wherein the one or more amphibians is a froglet, tadpole, Urodele, or larval stage young Apoda.24. The method of any one of embodiments 12-23, wherein the one or more amphibians is a baby Urodele, and optionally the amphibian is a baby axolotl.25. A method of treating and / or preventing a skin condition including administering the PBMs or conditioned water of any one of embodiments 1-8 or the composition of any one of embodiments 9-11 on the skin of a subject in need of treatment.26. The method of embodiment 25, wherein the skin condition is an inflammatory skin condition; and optionally, wherein the inflammatory skin condition is psoriasis, dermatitis, acne, rosacea, or burn.27. The method of embodiment 25, wherein the method includes treating and / or preventing the skin from photodamage and environmental pollution.28. The method of embodiment 25, wherein the method includes treating and / or preventing uneven skin tones, visible hyperpigmentation, and dyschromia.29. The method of embodiment 25, wherein the method improves skin hydration, skin quality and texture, and reduce the appearance of fine lines and wrinkles, as compared to a control substance that do not contain the PBMs or conditioned water.30. The method of embodiment 25, wherein the method is used to treat subject after treatment with non-ablative fractional laser.31. The method of embodiment 25, wherein the PBMs or conditioned water activates expression of one or more anti-aging genes, cell renewal / regeneration genes, epidermal barrier genes, extracellular matrix integrity genes, or antioxidant / stress response genes; wherein the PBMs or conditioned water inhibits the expression of inflammation / immune response genes or the extracellular matrix breakdown genes; or wherein the PBMs or conditioned water activates and / or inhibits a combination of the genes.32. The method of embodiment 31 , wherein:the anti-aging genes include FOXO3, MFN2, PNK4, POLG1 / MD, and SIRT1;Docket No. R217-0015PCTthe cell renewal / regeneration genes include CASP3 and TP63;the epidermal barrier genes include CLDN1, KRT1, and KRT10;the extracellular matrix integrity genes include COL7A1, and DSG1;the antioxidant / stress response genes include AHR, HM0X1, MT1A, MT2A, and SIRT1; the extracellular matrix breakdown genes include KLK5, KLK7, and SERPINB3; and the inflammation / immune response genes include TNF.EXAMPLES

[0087] Example 1. Preparation and Analysis of the Conditioned Water Samples

[0088] One or more axolotls of ages between 3- to 6-month old were placed in a container with water. Fecal matter was removed daily from the water that the one or more axolotls were living in. Water was not changed, and the axolotls were not fed.

[0089] Water samples were collected from the axolotls’ living quarters for analysis of protein content and molecular weight distribution. Samples of water were taken at 24hours and frozen. The water was changed after collection at 24 hours, and the axolotls were fed (three pellets of food) every 72 hours. The water collected was cruelty free (CF) conditioned water that was unprocessed.

[0090] Analytical methods include SDS-PAGE for protein molecular weight assessment and the Bradford assay for total protein quantification.

[0091] Two types of samples were evaluated: unprocessed and minimally processed. The minimally processed samples underwent lyophilization to control protein concentration. Specifically, 1 mL of conditioned water was lyophilized in triplicates using six-well plates. Each lyophilized sample was then reconstituted with varying volumes of diluent (Cell Culture Grade Water (CCGW)): 0.5 mL, 0.25 mL, and 0.125 mL - yielding 1X, 2X, 4X, and 8X. These reconstituted samples were subsequently analyzed using SDS-PAGE and the Bradford and Sircoll assays were performed to assess protein concentration and molecular weight distribution.

[0092] The SDS-PAGE and Bradford assay together provide complementary information. The SDS-PAGE highlights protein diversity and molecular weight. FIG. 1 shows proteins present in the conditioned water of various baby axolotls. The Bradford and Sircoll assays validated the protein concentrations. FIGs. 2Aand 2B show protein concentrations of different samples of conditioned water. Proteomic analyses were performed on collected water samples to characterize the protein complex. Examples of proteins present in the samples include keratin 5 fragment, histones H3 and H4, thrombospondin-1 and -4, exotoses multiplelike 3 protein, and alpha-globin, beta-globin chains, heat shock protein 70 (HSP70), activity dependent neuroprotector homeobox (ADPN), arrestin-C, lysozyme g, Wnt-5a, prominin-3, E2F transcription factor 5, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Docket No. R217-0015PCT

[0093] Example 2. Processing of the CF Conditioned Water

[0094] Fifteen to 20 liters of CF conditioned water were collected from the living quarters of the baby axolotls as described above. The water was filtered by depth filtration (0.5 / 0.2 micron) for 2 hours (hrs) and then frozen for 24 hrs. The frozen water was lyophilized for 24 hrs and the resulting powder was subsequently reconstituted by acidification to a pH of about 4.5 to 6.5 followed by sonification to solubilize the bioactive molecules post lyophilization. The powder was reconstituted at a ratio of 1 gram (g) of powder to 10 mL of acid. The acidified mixture was then sonicated for about 2 to 10 seconds (secs) for a few times with 30 secs rest in between until the bioactive molecules were solubilized. The process of collecting and reconstituting the powder (solubilizing the bioactive molecules) took about 3 hours.

[0095] The solubilized bioative molecules were then diluted in water to a concentration of 10 militers (mL) of water per g of bioactive molecules to 100 mL per g of bioactive molecules. The water used was cell culture grade water (CCGW) or deionized water.

[0096] Next, the solubilized bioactive molecules were applied to a column chromatography (endotoxin filtration) to remove the endotoxins. The column used was the EtoxiClear® column, which is a pre-packed affinity column that contained a stable adsorbent for binding endotoxins with high efficiency to remove them from the bioactive molecules. Elution buffers of 0.05 M sodium chloride to 0.1 M sodium chloride, PBS pH 7.2, and PBS with 0.1 M sodium chloride were used to elute the sterilized bioactive molecules off the column and collected. The sterilized mixture of bioactive molecules were eluted from the column after applying the elution buffers for about 1 to 5 times. The process of endotoxin filtration using the affinity column took about 7 hours.

[0097] Some of the solubilized bioactive molecules underwent ion exchange chromatography using a Mustang™ E filter for the removal of endotoxins.

[0098] The bioactive molecules, substantially free of endotoxins, were then diluted to a concentration of 20 to 30 ug / mL of CCGW at about pH 5.0, and again, sterilized by filtration using a 0.22 micron filter for 15 minutes to obtain the final mixture of purified bioactive molecules (PBMs).

[0099] Fig. 3 shows the process steps in a flow chart.

[0100] Example 3. Gene Marker Studies

[0101] Gene marker studies were conducted to determine how the different samples of activated axolotl skin modulate gene expression in human skin. A 3D in vitro skin model (MatTek EFT-400) containing epidermal keratinocytes and dermal fibroblasts was used. Differential gene expression was assessed after 24 and 48 hours of exposure to the test material (TM) using mRNA-Seq. The TM was the mixture of PBMs obtained as described in Example 2. The gene expression of the test material was compared to control, which were notDocket No. R217-0015PCTtreated with the PBMs or with a mixture of bioactive molecules containing endotoxins (without endotoxin removal).

[0102] Gene marker studies were used to compare gene expression of human skin treated with PBMs, which is substantially free of endotoxins, and with mixtures of bioactive molecules obtained from conditioned water containing endotoxins (without endotoxin removed). In FIGs.4A-4D, Sample 004 is a mixture of bioactive molecules containing endotoxins, and Samples 005 and 006 are PBMs mixtures, which are substantially free of endotoxins. Sample 005 was obtained using ion exchange chromatography to remove the endotoxins, while sample 006 was obtained using an affinity column to remove the endotoxins. The bar graphs indicate that PBMs were functionally different from mixtures obtained from conditioned water containing endotoxins.

[0103] Gene marker studies were also performed to compare gene expression of human treated with PBMs as compared to the control (without treatment PBMs). Tables 1 and 2 summarize the results of the differential expression of representative relevant genes using PBMs as the test material for treating human skin. The numbers 0.01%, 0.10%, and 1.0% in the Tables is the concentration of PBMs (TM) in weight %.

[0104] Table 1. LFC after 24 hrs of exposure to PBMsDocket No. R217-0015PCT

[0105] Table 2. LFC after 24 hrs of exposure to PBMsDocket No. R217-0015PCT

[0106] FIGs. 4A-4D show examples of genes that are expressed after treatment with a mixture of PBMs.

[0107] Example 4. Efficacy of the Mixture of PBMs on Uneven Skin Tone

[0108] Introduction. Uneven skin tone, hyperpigmentation, and photodamage remain widespread dermatologic concerns affecting a significant portion of the global population. It contributes to epidermal thinning, degradation of collagen and elastin fibers, and disruption of the extracellular matrix (ECM) — a condition clinically recognized as solar elastosis. Numerous studies have confirmed that chronic UV exposure accelerates photoaging and is intricately associated with the onset of skin diseases and tumors, highlighting the critical need for targeted, evidence-based skincare interventions. This brightening and tone-correcting serumDocket No. R217-0015PCTis a comprehensive solution designed to address the visible signs of photodamage and pigmentation irregularities.

[0109] Formulation. The mixture of PBMs was formulated with depigmenting peptides, antioxidants, even-toners, and botanical extracts, to form a serum for topical application. The ingredients work synergistically to reduce melanin overproduction, redness, and inflammation, inhibit tyrosinase activity, visibly improve discoloration, and clarifies and brightens the skin. These active ingredients help fade existing pigmentation while preventing future discoloration, resulting in a clearer, more radiant complexion.

[0110] Objectives. The primary objective of this study was to investigate the efficacy and tolerability of the PBMs in the form of a topical skin care formulation in a full-face model on subjects with uneven skin tones, visible hyperpigmentation, dyschromia, and global improvement of photodamaged skin. The secondary objective was to show that the topical PBMs improved the appearance of photodamaged facial skin including uneven skin tones, visible hyperpigmentation, dyschromia, and global improvement of photodamaged skin as determined by:• Canfield Gen 5 VISIA CRP (Primos) camera system;• Physician visual assessment scale observation-visual assessment will be done at baseline, weeks 4, 8, and 12;• Subject questionnaires at weeks 4, 8, and 12;• Before and after digital photographs baseline, weeks 4, 8, and 12 with blinded observer;• Canfield Gen 5 VISIA CRP (Primos) camera system Photographs at baseline, weeks 4, 8, and 12; and• Biopsies for histology measurements-elastin, collagen deposition, epidermal thickness.

[0111] Study Design:

[0112] Female and male subjects, 30 to 70 years of age, were enrolled in this multi-site full face study.• (Site 1) Miami, Florida, 15 total subjects enrolled, 5 of which are control (placebo) • (Site 2) Birmingham, Michigan, 10 total subjects enrolled, 3 of which are control (placebo)• (Site 3) Pflugerville, Texas, 15 total subjects enrolled, 5 of which are control (placebo)Docket No. R217-0015PCT& & &Total of 9 biopsy subjects x 2 biopsies (One on Visit 1 and One on Visit 4)Total of 18 biopsy samplesBoth biopsies will be performed to left preauricular area (Left Superior preauricular on Visit 1 , Left Inferior preauricular on Visit 4)

[0113] Randomization and Blinding Procedure. Participants enrolled in the study were randomly assigned in a 2:1 ratio to receive either the serum or a control product. Randomization was conducted using an Artificial Intelligence (CHAT GPT) computergenerated randomization schedule to ensure allocation concealment and reduce selection bias. Neither the participants nor the study staff involved in the administration of the intervention or assessment of outcomes knew which product the participant was receiving (double-blind design).

[0114] The placebo was an inert substance that does not contain any active ingredients and was formulated to be indistinguishable in appearance and administration from the study serum. The use of a placebo Control Product allows for the evaluation of the safety and efficacy of the investigational product while minimizing bias.

[0115] Subjects who signed consent and met all inclusion criteria and none of the exclusion criteria were enrolled at the baseline visit. Subjects were asked to use the serum provided for the duration of the 12-week study. Subjects must be willing to replace their facial moisturizer, facial cleanser, and SPF with study supplied products and refrain from using any other skincare products on the face. Subjects may continue their current minimal make-up regimen if products were approved by the physician.

[0116] At baseline, subjects received the serum. Subjects were asked to apply the serum for 12 weeks to the full face twice daily (AM & PM). Subjects and the investigator completed the baseline subject skin type assessments including Fitzpatrick skin type (l-VI), GLOGAU Classification, and Expert Grader Photodamage Scale (see Appendix IV). Eligible participants had, for each of the parameters measured, mild to moderately severe to facial lines and wrinkles, erythema / redness, dryness / flaking, and dyschromia of photodamaged skin. DigitalDocket No. R217-0015PCTPhotographs were taken at baseline, 4 weeks, 8 weeks and 12 weeks. Canfield Gen 5 VISIA CRP (Primos) camera system Photographs were taken at baseline, 4 weeks, 8 weeks and 12 weeks.

[0117] To assess efficacy, subjects returned to the research center at 4 weeks, 8 weeks and 12 weeks for dermatologist investigator evaluations. Subjects were instructed to calendar their expected visit dates according to the schedule outlined hereunder; however, visit dates may vary + / - 3 days at each timepoint, actual visit dates will be recorded.

[0118] Photographs were taken of all subjects of the front, right, and left face with the using the Canfield Gen 5 VISIA CRP (Primos) camera system (see Photography for more specific information). Subjects completed a product questionnaire at each visit. Subjects were sent a compliance reminder text prior to each visit. Study product were checked for compliance, and accuracy of facial product application at each visit. Subjects were re-dispensed as needed. Study participation were completed at week 12.

[0119] To further enhance the comprehensiveness of this study, 9 subjects, 3 subjects from Florida, 3 subjects from Texas and 3 subjects from Michigan received two preauricular ex vivo 3mm punch biopsies taken for hematoxylin and eosin histology one at baseline and this procedure repeated with a second biopsy taken at week 12 from each of the selected subjects. Changes in histology (hyaluronic acid, collagen and elastin deposition and epidermal thickness) were measured to compare biopsies collected at baseline / day 0 (prior to application of the serum) face and biopsies collected at week 12 after topical application of the test product.

[0120] Biopsy Test Groups and Procedure.

[0121] A total of 9 subjects, 2 biopsies per subject (1 at baseline and 1 at 12 weeks from the left side) were collected for a total of 18 samples. At each site, one biopsy subject used the control product and 2 subjects used the serums.

[0122] Baseline, 3mm punch biopsy to left superior preauricular area

[0123] 12 Week, 3mm punch biopsy to left inferior preauricular area

[0124] Sterile Skin Prep and Lidocaine and Suture Closure, Store samples in formaldehyde. 7 days later the suture was removed.

[0125] Punch Biopsy site / locations for Visit 1 & Visit 4 are shown in FIG. 5.

[0126] Visit 1 (Screening Visit / Baseline <14 Days of Baseline)• Eligibility Criteria Assessment - Inclusion / Exclusion Criteria Review. Subjects were screened for suitability.• Review Product Ingredients - Subjects were prescreened for known irritations or allergies.• Medical History - Source Data Collection and Concomitant Medications, etc.Docket No. R217-0015PCT• Informed Consent Form - Subjects who signed consent and satisfied all inclusion criteria and none of the exclusion criteria were enrolled at the baseline visit.• Pregnancy Test (if applicable).• After subjects were enrolled based on satisfactory acceptable screening visit requirements, the following baseline clinical assessments were performed:o Digital Photographs.o Canfield Gen 5 VISIA CRP (Primos) camera system Photographs.o Baseline Clinical Evaluation: Fitzpatrick Skin Type Grader; GLOGAU Classification; and Expert Grader Skin Photodamage Scale Assessment:■ Erythema / Redness; Dryness / Flaking; Fine Lines / Wrinkles; and Dyschromiao Subject Education on Product Use Instructions:■ Subjects were asked to replace their facial moisturizer, facial cleanser and Broad-Spectrum SPF Sunscreen with study supplied products and refrain from using any other skincare products on the face. Subjects continued with minimal make-up regimen using product approved by the investigator.■ Subjects were asked to use the study facial moisturizer, facial cleanser and Broad-Spectrum SPF Sunscreen provided by the study sponsor for the duration of the 12-week study.■ Subjects received the serum for application twice a day (AM / PM) on entire face for 12 weeks.■ Subjects were asked to apply the serum for 12 weeks twice a day (AM / PM) to the face.o Distributed Subject Diary - informed that it will be checked for compliance and accuracy of facial product application at each visit.o Dispensed Study Products -Serum. Proper usage per 30-day period was approximately 1-30ml bottle.o Biopsies (if applicable) - Punch biopsy to the left superior pre-auricular area were performed before application of any study products.

[0127] Visit 2 (week 4, ± 3 days):• Pregnancy Test (if applicable)• Digital Photographs.• Clinical Evaluation: Expert Grader Skin Photodamage Scale Assessment:• Erythema / Redness; Dryness / Flaking; Fine Lines / Wrinkles; Dyschromia;Stinging / Burning; and Global Improvement.• Subject Self-Assessment Questionnaire was collected.Docket No. R217-0015PCT• Adverse Events, if any, were reviewed.• Reviewed Subject Diary and checked for compliance and accuracy of facial product application at each visit.• Dispensed / Collected Study Products and Instructions (as needed).• Visit 3 (week 8, ± 5 days):• Pregnancy Test (if applicable).• Digital Photographs.• Clinical Evaluation - Expert Grader Skin Photodamage Scale Assessment:o Erythema / Redness; Dryness / Flaking; Fine Lines / Wrinkles; Dyschromia;Stinging / Burning; and Global Improvement.• Subject Self-Assessment Questionnaire was collected.• Adverse Events, if any, were Review.• Reviewed Subject Diary and checked for compliance and accuracy of facial product application at each visit.• Dispensed / Collected Study Products and Instructions (as needed)- Serum.

[0128] Visit 4 (final visit, week 12, ± 3 days):• Pregnancy Test (if applicable).• Digital Photographs.• Canfield Gen 5 VISIA CRP (Primos) camera system Photographs.• Clinical Evaluation - GLOGAU Classification; GAIS Classification; and Expert Grader Skin Photodamage Scale Assessment:o Erythema / Redness; Dryness / Flaking; Fine Lines / Wrinkles; Dyschromia;Stinging / Burning; and Global Improvement.• Subject Self-Assessment Questionnaire was collected.• Adverse Events were reviewed.• Reviewed Subject Diary and checked for compliance and accuracy of facial product application at each visit.• Collected Study Products -Serum.• Biopsies (if applicable) - Punch biopsy were performed to the left inferior pre- auricular area.

[0129] Results / Conclusion. The results suggested that the topical serum trended towards an improvement in the appearance of photodamaged facial skin including uneven skin tones, visible hyperpigmentation, dyschromia, and global improvement of photodamaged skin.

[0130] Example 5: Efficacy of the Mixture of PBMs in Improving Skin Hydration

[0131] Objective. The main purpose of this study was to determine the effect of the mixture of PBMs on skin moisturization and hydration over the duration of the study. Specifically, theDocket No. R217-0015PCTstudy was performed to determine whether the PBMs improve the appearance of the skin including skin hydration, skin quality, skin texture, and reduce the appearance of fine lines and wrinkles. The study was also performed to evaluate the safety and effectiveness of the PBMs to decrease skin dryness.

[0132] Formulation. The PBMs was formulated with hyaluronic acid, glycolic acid, and vitamin C to form a serum for topical application to improve skin moisturization and hydration over the duration of the study.

[0133] Study Design

[0134] A total of 40 subjects were recruited for the study.• Site 1 : Miami Forida, 15 subjects enrolled, 5 of which are control (placebo).• Site 2: Birmingham, Michigan, 10 subjects enrolled, 3 of which are control (placebo).• Site 3: Pflugerville, Texas, 15 subjects enrolled, 5 of which are control (placebo).& & &Total of 9 biopsy subjects x 2 biopsies (One on Visit 1 and One on Visit 4)Total of 18 biopsy samplesBoth biopsies will be performed to left preauricular area (Left Superior preauricular on Visit 1 , Left Inferior preauricular on Visit 4)

[0135] Randomization and Blinding Procedure. Participants enrolled in the study were randomly assigned as discussed in Example 4.

[0136] The criteria and procedure for enrollment were as described in Example 4.

[0137] Punch Biopsies were collected on 9 subjects at the 3 study sites and analyzed. The procedure for the biopsies is as described above in Example 4.

[0138] Evaluation and assessment procedures were similar to those described above in Example 4. The subjects enrolled in the study at Visit 1 returned for evaluation and assessment at weeks 4 (Visit 2), 8 (Visit 3), and 12 (Visit 4). The procedures for Visits 1 , 2, 3, and 4 are described in detail in Example 4.Docket No. R217-0015PCT

[0139] Results / Conclusion. The results suggested that the topical serum trended towards improved hydration of the skin, increased water content to the skin to give radiance, improved skin quality and texture, and reduced appearance of fine lines and wrinkles.

[0140] Example 6. Efficacy of the Mixture of PBMs for Improving the Appearance of Skin

[0141] Objective. The main purpose of this study was to determine whether the mixture of PBMs would improve the appearance of the subject’s skin. Specifically, the study was performed to determine whether the PBMs improve erythema / redness, dryness / flaking, fine lines / wrinkles, dyschromia (change in color of the skin or nails), stinging / burning, and global (overall) improvement or photodamaged skin. Photodamaged skin results from skin changes such as fine and coarse wrinkles, roughness, freckles and pigmentation changes that occur as a result of prolonged exposure to the sun. The study was also performed to evaluate the safety and effectiveness of the PBMs in improving photodamaged skin.

[0142] Formulation. The PBMs was formulated with skin enhancing agents including oligopeptides, emollients, humectants, emulsifiers, and antioxidants to form a serum for topical application.

[0143] Study Design

[0144] A total of 40 subjects were recruited for the study.• Site 1: Miami Florida, 15 subjects enrolled, 5 of which are control (placebo).• Site 2: Birmingham, Michigan, 10 subjects enrolled, 3 of which are control (placebo).• Site 3: Pflugerville, Texas, 15 subjects enrolled, 5 ofwhich are control (placebo).& & &Total of 9 biopsy subjects x 2 biopsies (One on Visit 1 and One on Visit 4)Total of 18 biopsy samplesBoth biopsies will be performed to left preauricular area (Left Superior preauricular on Visit 1 , Left Inferior preauricular on Visit 4)

[0145] Randomization and Blinding Procedure. Participants enrolled in the study were randomly assigned as discussed in Example 4.

[0146] The criteria and procedure for enrollment were as described in Example 4.Docket No. R217-0015PCT

[0147] Punch Biopsies were collected on 9 subjects at the 3 study sites and analyzed. The procedure for the biopsies is as described above in Example 4.

[0148] Evaluation and assessment procedures were similar to those described above in Example 4. The subjects enrolled in the study at Visit 1 returned for evaluation and assessment at weeks 4 (Visit 2), 8 (Visit 3), and 12 (Visit 4). The procedures for Visits 1, 2, 3, and 4 are described in detail in Example 4.

[0149] Results / Conclusion. The results suggested that the topical serum reduced the appearance of fine line wrinkles and redness and inflammation and trended towards an improvement in the appearance of moderately to severely photodamaged facial skin.

[0150] Example 7. Efficacy of the Mixture of PBMs for Enhancing Treatment After Non-Ablative fractional laser

[0151] Objective. The main purpose of this study was to determine the safety and effectiveness of the mixture of PBMs to enhance treatment after non-ablative fractional laser. A gel containing the PBMs was used to treat the subject’s face after a non-ablative fractional laser procedure on photodamaged skin to determine whether the PBMs can decrease downtime (duration of discomfort) and increase healing. Facial non-ablative factional resurfacing is defined as a use of lasers to reduce the signs of gaining, improve skin texture, elasticity (loss of skin elasticity and tone, in addition to reducing the appearance of scars and stretch marks.

[0152] Formulation. The mixture of PBMs was formulated as a gel for topical application. The control product did not contain any active ingredients.

[0153] Study Design

[0154] A total of 40 subjects were recruited for the study.• Site 1: Florida, 15 subjects enrolled, 5 of which are control (placebo).• Site 2: Michigan, 10 subjects enrolled, 3 of which are control (placebo).• Site 3: Texas, 15 subjects enrolled, 5 of which are control (placebo).

[0155] Visit 1 (Screening Baseline Visit / biopsy / Laser T reatment)

[0156] Subjects received the cleanser and sunscreen (SPF 42+) provided by the study sponsor for a duration of the study which is 4 weeks. Subjects were required to replace their facial moisturizer with the study moisturizer provided by the study sponsor, and refrained from using any other moisturizing products on the face. The following were performed at Visit 1.• Eligibility Criteria Assessment / Inclusion / Exclusion Criteria Review. Subjects were screened for suitability.• Review Product Ingredients - Prescreened subjects for known irritations or allergies.• Medical History - Source Data Collection, Concomitant Medications, etc.Docket No. R217-0015PCT• Informed Consent Form - Subjects who signed consent and meet all inclusion criteria and none of the exclusion criteria were enrolled at the baseline visit.• In office urine Pregnancy Test (if applicable, a female of childbearing age). If pregnancy was a possibility, a small collection of urine was collected fortesting.

[0157] After the subjects were enrolled based on satisfactory acceptable screening visit requirements, baseline clinical assessments and photography were performed.• Digital Photographs (before and after laser).• Canfield Gen 5 VISIA CRP (Primos) camera system Photographs.• Baseline Clinical Evaluation:o GLOGAU Classification - a chart of photo-aging classification scale.o Baker-Wong Pain Scale (only post non-ablative laser therapy treatment.o Expert Grader Skin Photodamage Scale:■ Erythema / Redness■ Dryness / Flaking■ Fine Lines / Wrinkles■ Dyschromia (discoloration of the skin, usually red and brown coloration)• Subject Education on Product Use Instructionso Subjects were asked to use the study topical skincare products provided by the study sponsor for the duration of the 4-week study.o Subjects received the gel or control product for application twice a day (AM / PM) to entire face for 4 weeks.• Adverse Event Review• Distributed / Reviewed Subject Diary - to be checked for compliance and accuracy of facial product application at each visit.• Dispensed Study Products -Gel or Control Product. Proper usage per 30-day period is approximately 1 bottle.• Biopsies: Punch biopsy to the pre-auricular area were performed before application of the non-ablative laser. Punch biopsies were randomized prior to the study for 9 / 40 patients at the 3 clinical sites. Punch biopsy is a small removal of the skin equivalent to the size of a pencil tip. A numbing injection of lidocaine was used prior to removing the skin tissue. A 3 milimeter (mm) skin biopsy was performed.o Punch biopsy to the Left pre-auricular area (near the opening of the ear) was performed before treatment with non-ablative fractional laser.o Two punch biopsiesforhistology (the study of microscopic anatomy of tissue under the microscope) were performed one at Visit 1 / baseline and one at Week 4, Visit 5 (FIG. 6)Docket No. R217-0015PCTo After the biopsy procedure was completed, the physician placed 1-2 sutures (stitches) at the affected area of skin. Instructions were provided on post-biopsy care, and post-treatment care such as applying antibiotic ointment (Polysporin, Bactroban, Mupirocin, Gentamicin, orNeosporintothe affected area and cover with a bandage if needed). Aquaphor or Vaseline could also be applied to the affected area to create a protective barrier that adds moisture which aids the healing process. Antibiotic ointments and protective barrier for use were provided to the subjects. The sutures were removed 7 days post procedure.• Non-ablative laser treatment:o A 1550 nm non-ablative fractional laser (FRAXEL, Solta) for resurfacing the photodamaged skin of the subjects was used to reduce the signs of photoaging. o Laser Treatment - Duration was approximately 30 minutes.■ Preparation: The laser treatment preparation included cleansing the entire face using the provided cleanser, applying a topical numbing cream (benzocaine / tetracaine / lidocaine), and removing hair in the treatment area if necessary. Subjects were positioned lying down or reclined during treatment. Following laser, the gel or control product was applied to the skin.■ The laser procedure consisted of the following activities:- Skin Preparation of the skin areas in front of the ears.- Skin cleansing with mild cleanser given for study.Hair removal in treatment area by shaving if hair were present. - An FDA-approved 1550 nm wavelength Thulium laser (Solta Dual) was used to treat the entire face.- T opical application of gel or control product immediately after laser treatment.- Topical application of gel or control product twice daily (morning and evening) for 4 weeks. Subjects began this regimen the night of their first laser procedure.o Laser Device Preparation: The 1550 nm Thullium laser (Solta Dual) was prepared as per its instructions for use.o Subject Preparation: Prior to the laser treatment, the subject’s entire face was completely cleansed and then the topical numbing agent (Benzocaine USP 20% / Tetracaine USP 10% / Lidocaine USP 10%) was applied to the entire face. The numbing cream stayed on the subject’s face for at least 20 minutes. Afterwards, the numbing cream was wiped off of the face with a gauze and witch hazel (a plant-based botanical ingredient used to combat skin issues like irritation,Docket No. R217-0015PCTand redness. It also tightens and constricts small blood vessels). The subjects were given protective eyewear that was worn in the treatment room during the application of laser energy as required by the study guidelines.o Parameters (definition of operation) forthe laser optical pulses (flashes of light) was determined by the physician.o After the treatment, mild erythema (reddening of the skin, usually in patches, as a result of injury or irritation causing dilatation of the blood capillaries.) and mild edema (swelling caused by trapped fluid inside your tissues) were expected. o Subjects returned to the research center at Day 3, Day 5, and Day 7 for wound healing assessment. The physician conducted an evaluation for edema around the face and erythema using a quartile scale (a type of percentile).o Photographs were taken of the front, right, and left the subjects’ face with the digital camera. Subjects were required to complete a sponsor questionnaire. Compliance reminder text was sent prior to each of the visits.o A group of 9 patients (2 biopsies per patient) will have biopsies. Skin biopsy samples were collected from one pre-auricular side at baseline and other side at day 7 (before your laser treatment, and Day 7 post laser treatment.

[0158] Visit 2 (day 3 Post Laser Treatment)

[0159] The duration was 1 to 2 hours and the following evaluations were performed:• Digital Photographs• Canfield Gen 5 VISIA CRP (Primos) camera system Photographs• Clinical Evaluation: (1) Healing Assessment Scale; (2) Baker-Wong Pain Scale; and (3) Expert Grader Skin Photodamage Scale Assessment: (a) Erythema / Redness; (b) Dryness / Flaking; (c) Fine Lines / Wrinkles; (d) Dyschromia -discoloration of skin red and brown tones; (e) Stinging / Burning; and (f) Global Improvement.• Subject Self-Assessment Questionnaire were collected.• Adverse Events (AEs) were observed or reported. An Adverse Event is any unfavorable and / or unintended medical occurrence that presents during the course of the study• Reviewed Subject Diary - Checked for compliance and accuracy of facial product application at each visit.• Dispensed / Collected Study Products (gel and control product) and Instructions (as needed). Proper usage per 30-day period was approximately 1 bottle.

[0160] Visit 3 (day 5 Post Laser Treatment)

[0161] The duration was 1 to 2 hours and the same evaluations were performed as under Visit 2.Docket No. R217-0015PCT

[0162] Visit 4 (day 7 Post Laser Treatment)

[0163] The duration was 1 to 2 hours and the same evaluations were performed as under Visit 2, in addition to the following.• Healing Assessment Scale.• Baker-Wong Pain Scale.• Biopsies (if applicable)- Punch biopsy to the Right pre-auricular area will be performed before application of the gel or control product.o Punch biopsy to the right preauricular area (near the opening of your ear) o was performedo One punch biopsy for histology to the right preauricular area was performed: o Changes in histology (wound healing, collagen, blood vessels) were measured to compare biopsies collected at baseline (prior to non-ablative fractional laser surgery) on one side of face and with biopsies collected after 7 days of topical application of the gel or control product to opposite side of face.o A 3-mm skin punch biopsy was performed. Instructions were provided on postbiopsy care and post-treatment care such as applying antibiotic ointment (Polysporin, Bactroban, Mupirocin, Gentamicin, or Neosporin) to the affected area and covered with a bandage if needed. Alternatively, Aquaphor or Vaseline was applied to the affected area to create a protective barrier that adds moisture which aids, the healing process. The physician provided the antibiotic ointment and protective barrier use.o After the biopsy procedure was completed, the physician placed 1 -2 sutures at the affected area of skin.o Suture were removed 7 days post procedure. Instructions were provided for postbiopsy care, and post-treatment care such as applying antibiotic ointment (Polysporin, Bactroban, Mupirocin, Gentamicin, or Neosporin to the affected area and cover with a bandage if needed. Alternatively, Aquaphor or Vaseline was applied to the affected area to create a protective barrier that adds moisture which aids the healing process. The physician provided the antibiotic ointment and protective barrier for your use.

[0164] Visit 5 (Final Visit week 4, 30 days from baseline (± 5 days)

[0165] The duration was 1 to 2 hours and the following evaluations were performed.• Pregnancy Test (if applicable, meaning you are a female of childbearing age).• Digital Photographs.• Canfield Gen 5 VISIA CRP (Primos) camera system Photographs.Docket No. R217-0015PCT• Clinical Evaluations: (1) GLOGAU Classification; (2) GAIS Classification; (3) Healing Assessment Scale; (4) Baker-Wong Pain Faces Scale; (5) Expert Grader Skin Photodamage Scale Assessment: (a) Erythema / Redness; (b) Dryness / Flaking; (c) Fine Lines / Wrinkles; (d) Dyschromia (discoloration of the skin, usually red and brown coloration); (e) Stinging / Burning; and (f) Global Improvement.• Subject Self-Assessment Questionnaire.• Adverse Event Review Clinic staff or Investigator recorded any Adverse Events (AEs) that were observed or reported. An Adverse Event is any unfavorable and / or unintended medical occurrence that presents during the course of the study.• Reviewed Subject Diary and checked for compliance and accuracy of facial product application at each visit.• Collected Study Products -Gel or Control Product.

[0166] Results / Conclusions. The main reason for this study was to determine the effect of the gel to decrease post-operative recovery following facial non-ablative fractional resurfacing of photodamaged skin. The results suggested that the topical gel trended towards a decrease in post-operative recovery time following facial non-ablative fractional resurfacing of the photodamaged skin and decreased erythema and dyschromia at 4 weeks for the subjects that received the gel as compared to the subjects that received the control.

[0167] The subject matter described above is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.

[0168] All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entireties as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.

Claims

Docket No. R217-0015PCTCLAIMS1. Purified bioactive molecules (PBMs), substantially free of endotoxins, comprising one or more amphibian bioactive molecules present in the conditioned water of the living quarters of one or more amphibians; or conditioned water from the living quarters of one or more amphibians, wherein the conditioned water is substantially free of endotoxins and comprises a mixture of one or more amphibian bioactive molecules.

2. The PBMs or the conditioned water of claim 1 , wherein the one or more amphibians are a frog, toad, newt, or salamander.

3. The PBMs or the conditioned water of claim 1 , wherein the one or more amphibians are a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.

4. The PBMs or the conditioned water of claim 1 , wherein the one or more amphibians is a froglet, tadpole, Urodele, or larval stage young Apoda.

5. The PBMs or the conditioned water of claim 1 , wherein the one or more amphibians is a baby Urodele, and optionally the amphibian is a baby axolotl.

6. The PBMs or conditioned water of claim 1 , wherein the one or more bioactive molecules comprises one of more proteins involved in wound healing, tissue regeneration and / or tissue repair, neuroprotection and / or treatment of neurodegenerative diseases, immunomodulation and / or inflammation, cancer therapy and / or cell growth regulation, metabolic regulation, and a combination thereof.

7. The PBMs or conditioned water of claim 1 ,the PBMs or conditioned water including one or more proteins involved in skin wound healing, tissue regeneration, and / or tissue repair include keratin 5, histone H3, histone H4, thrombospondin -1, thrombospondin-4, exostoses multiple-like 3 protein, alpha-globin chain, beta-globin chain, and a combination thereof;the PBMs or conditioned water including one or more proteins involved in neuroprotection and / or treatment of neurodegenerative diseases include heat shock protein 70 (HSP70), activity-dependent neuroprotector homeobox (ADNP), arrestin-C, and a combination thereof;the PBMs or conditioned water comprising one or more proteins involved in immunomodulation and inflammation comprise lysozyme g, wnt 5a, and a combination thereof;the PBMs or conditioned water comprising one or more proteins involved in cancer therapy and cell growth regulation comprise prominin 3; orthe PBMs or conditioned water comprising one or more proteins involved in metabolic regulation comprises glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Docket No. R217-0015PCT8. The PBMs or conditioned water of claim 1 , wherein the PBMs comprise amphibian proteins, peptides, polypeptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes, salts, or a combination thereof9. A composition comprising the PBMs or the conditioned water of any one of claims 1-8, and optionally wherein the composition comprises one or more carriers or excipients.

10. The composition of claim 9, wherein the composition is a pharmaceutical composition, and optionally comprising one or more pharmaceutically acceptable carriers or excipients; or wherein the composition is a cosmetic composition and optionally comprising one or more cosmetically acceptable carriers.

11. The composition of claim 10, wherein the composition further comprises one or more agents xenogenic to amphibians; and optionally, wherein the one or more agents comprise one or more peptides, proteins, drugs, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, and a combination thereof.

12. A method of obtaining PBMs or conditioned water of any one of claims 1-8, wherein the method comprisesraising one or more amphibians in water for 10 hours to 7 days, 12 hours to 5 days, 14 hours to 3 days, 16 hours to 2 days, 18 hours to 36 hours, 20 hours to 30 hours, 22, hours, 23 hours, 24 hours, 25 hours, or 26 hours;removing fecal matters from the water on a daily basis;collecting the water;and processing the collected water to remove endotoxins to obtain PBMs and / or conditioned water substantially free of endotoxins.

13. The method of claim 12, wherein the method further comprises filtering the collected water to decrease volume and / or increasing concentration of one or more bioactive molecules in the collected water.

14. The method of claim 12, wherein the method further comprises filtering the collected water before or after removal of the endotoxins by membrane filtration; and optionally wherein membrane filtration comprises ultrafiltration, diafiltration, depth filtration, or tangential flow filtration.

15. The method of claim 12, wherein the method further comprises filtering the collected water by depth filtration prior to removal of the endotoxin or filtering the collected water by tangential flow filtration after removal of the endotoxin.

16. The method of claim 12, wherein processing the collected water comprises freezing the collected water before or after removal of the endotoxins to obtain frozen water; and optionally, storing the frozen water.Docket No. R217-0015PCT17. The method of claim 16, wherein the method further comprises lyophilizing the frozen water into a powder; and optionally storing the powder.

18. The method of claim 17, wherein the method further comprises reconstituting the powder into a solution and sterilizing the solution using a sterile filter to obtain a sterilized solution.

19. The method of claim 12, wherein removing the endotoxin comprises using chromatography to obtain PBMs or conditioned water substantially free of toxins; and optionally wherein the chromatography is affinity chromatography, ion exchange chromatography, gravity chromatography, or a combination thereof.

20. The method of claim 19, wherein the method further comprises sterilizing the PBMs or conditioned water by sterile filtration.

21. The method of claim 12, wherein the one or more amphibians are a frog, toad, newt, or salamander.

22. The method of claim 12, wherein the one or more amphibians are a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.

23. The method of claim 12, wherein the one or more amphibians is a froglet, tadpole, Urodele, or larval stage young Apoda.

24. The method of claim 12, wherein the one or more amphibians is a baby Urodele, and optionally the amphibian is a baby axolotl.

25. A method of treating and / or preventing a skin condition comprising administering composition of claim 10 on the skin of a subject in need of treatment.

26. The method of claim 25, wherein the skin condition is an inflammatory skin condition; and optionally, wherein the inflammatory skin condition is psoriasis, dermatitis, acne, rosacea, or burn.

27. The method of claim 25, wherein the method comprises treating and / or preventing the skin from photodamage and environmental pollution.

28. The method of claim 25, wherein the method comprises treating and / or preventing uneven skin tones, visible hyperpigmentation, and dyschromia.

29. The method of claim 25, wherein the method improves skin hydration, skin quality and texture, and reduce appearance of fine lines and wrinkles, as compared to a control substance that do not contain the PBMs or conditioned water.

30. The method of claim 25, wherein the method is used to treat subject after treatment with non-ablative fractional laser.

31. The method of claim 25, wherein the PBMs or conditioned water activates expression of one or more anti-aging genes, cell renewal / regeneration genes, epidermal barrier genes, extracellular matrix integrity genes, or antioxidant / stress response genes; wherein the PBMs or conditioned water inhibits expression of inflammation / immune response genes orDocket No. R217-0015PCTextracellular matrix breakdown genes; or wherein the PBMs or conditioned water activates and / or inhibits a combination of the genes.

32. The method of claim 31, wherein:the anti-aging genes comprise FOXO3, MFN2, PNK4, POLG1 / MD, and SIRT1; the cell renewal / regeneration genes comprise CASP3 and TP63;the epidermal barrier genes comprise CLDN1, KRT1, and KRT10;the extracellular matrix integrity genes comprise COL7A1, and DSG1;the antioxidant / stress response genes comprise AHR, HMOX1, MT1A, MT2A, and SIRT1;the extracellular matrix breakdown genes comprise KLK5, KLK7, and SERPINB3; and the inflammation / immune response genes comprise TNF.

Images