Natural formulations to reduce CPD levels

Abstract

Topical formulations have been developed which have been demonstrated to reduce ultraviolet ("UV")-induced cyclobutane pyrimidine dimer ("CPD") formation in a human clinical trial. The formulation includes all natural, non-toxic compounds which can be utilized in sunscreens, cosmetics and hair conditioners to protect from ultraviolet radiation. A preferred embodiment of the formulation contains 10% Ferulic Acid, 10% Diosmin, 5% Cytisine and 5% Resveratrol. A method of screening for compounds that are safe and effective to reduce UV-induced CPD formation also has been developed.

Description

[0001] NATURAL FORMULATIONS TO REDUCE CPD LEVELS

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under NCI P50CA121974 awarded by the National Cancer Institute. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] The invention is generally in the field of formulations to reduce cyclobutane pyrimidine dimer (CPD) formation by the sun and specifically in the area of natural compositions which lower risk of damaging the skin while preventing damage from ultraviolet radiation.

[0006] BACKGROUND OF THE INVENTION

[0007] Sunscreens are typically tested for protection from ultraviolet radiation and low toxicity (low to no systemic uptake, redness, burning, discoloration of skin, and other factors, many performed initially on animals). These are labeled based on how much ultraviolet light they block, known as sun protection factor (SPF). SPF is a measure of how much solar energy (ultraviolet (“UV”) radiation) is required to produce sunbum on protected skin (i.e., in the presence of sunscreen) relative to the amount of solar energy required to produce sunbum on unprotected skin. As the SPF value increases, sunburn protection increases.

[0008] At a molecular level, sun damage is harmful because it can lead to structural changes in DNA, including by creating cyclobutane pyrimidine dimers (CPDs). When these changes in DNA are not repaired, they may lead to mutations that drive skin cancer.

[0009] Currently available sunscreen agents are a cornerstone of skin cancer prevention strategies but have major limitations including ultraviolet light induced photodegradation, concerns about their environmental impact and safety concerns such as endocrine disrupting effects and paradoxical generation of reactive oxygen species (ROS) that may indirectly also induce CPDs. Sunscreen products must be tested for potential short-term and long- term health effects. This includes toxicity testing for irritation and skin allergies, as well as testing for skin absorption and the potential to cause cancer, disrupt the hormone system and cause harm during reproduction and development.

[0010] December 16, 2022, the Federal Food and Drug Administration (FDA) issued final sunscreen regulations. FDA regulates sunscreens, which are nonprescription drugs, to help make sure that consumers have access to safe and effective sun protection products. All sunscreens with an SPF 15 or higher must satisfy broad spectrum test requirements. This means that the ratio of UV-A1 to UV should be 0.7 or higher. All sunscreens must have a critical wavelength of 370 nm or higher. The maximum labelled SPF is 60 plus and the maximum marketed SPF is 80.

[0011] The FDA divided sunscreens into 3 categories. Category 1, GRASE, which is generally recognized as safe and effective, are those sunscreens that contain either zinc oxide or titanium dioxide. These are our physical blockers that reflect light. The second category is category 2 or non-GRASE and those are sunscreen ingredients that are no longer allowed, such as PABA [paraaminobenzoic acid] and trolamine salicylates. Category 3 is where the FDA says there is insufficient evidence of GRASE categorization meaning to say that these chemical sunscreen ingredients require additional evaluation prior to safety and efficacy determination.

[0012] Numerous studies have raised new concerns about endocrine- disrupting effects from homosalate, avobenzone and oxybenzone. The European Commission published preliminary opinions on the safety of three organic UV filters, oxybenzone, homosalate and octocrylene, and proposed a concentration limit of 2.2 percent for oxybenzone and 1.4 percent for homosalate. U.S. sunscreen manufacturers are legally allowed to use concentrations up to 6 and 15 percent, respectively, and hundreds of sunscreens manufactured in the U.S. use them at concentrations that far exceed the European Commission’s recommendations. Previous studies detected many sunscreen ingredients in breast milk and urine. There are a number of methods of screening for new sunscreen compounds. Most are tedious, requiring large amounts of labor and time. Even so-called automated robot screening (HelioScreen, Jasco, Inc.), only tests for a single parameter, efficacy in blocking ultraviolet radiation. Other systems, such as Waters Inc, use software to analyze multiple samples, but the data is still collected based on single sample testing.

[0013] There remains a need for natural topical formulations that provide effective photoprotection and are safe for use, and methods to rapidly and inexpensively screen for such compounds and formulations thereof.

[0014] It is therefore an object of the present invention to provide all natural photoprotection compositions that provide superior broad-spectrum photoprotection and safety relative to currently available sunscreen agents.

[0015] It is a further object of the present invention to provide methods and systems of screening candidate agents for photoprotection formulations.

[0016] It is yet another object of the present invention to provide methods of making and using the natural formulations to reduce cyclobutane pyrimidine dimers (CPD).

[0017] SUMMARY OF THE INVENTION

[0018] Photoprotective formulations have been developed that have been tested for safety and efficacy in reducing cyclobutane pyrimidine dimer (CPD) production, photostability, and safety. These are formulated for topical application to the skin as sunscreens or cosmetics, or to the hair as a spray or conditioner. In preferred embodiments, the photoprotective formulation contains the non-toxic natural products diosmin, ferulic acid, cytisine and resveratrol. A preferred embodiment of the photoprotective formulation for application to the skin contains 10% Ferulic Acid, 10% Diosmin, 5% Cytisine and 5% Resveratrol.

[0019] As described in the examples, a clinical trial was conducted with normal healthy humans in which 2 mg / cm2photoprotective formulation was applied 15 minutes before ultraviolet radiation (“UVR”) (2 MED) exposure. Skin biopsies were collected for quantification of CPD and formalin-fixed paraffin-embedded (FFPE) tissue prep for histological analysis, i.e. 3- nitrotyrosine immunofluorescence. Results show significantly lower levels of UVR induced DNA damaging CPDs with the natural photoprotective formulation. Photographs taken four hours after UVR shows bright red patch where there was no photoprotective formulation, and no change in coloration where the photoprotective formulation was applied.

[0020] A high throughput method of screening for compounds that are safe and effective as photoprotective agents has been developed. High- throughput screening of natural compounds is conducted in as few as four steps: (1) UV full-spectrum absorbance screening, (2) photostability screening, (3) cytotoxicity screening, and (4) reactive oxygen species generation and antioxidant capacity screening. FIG. 1 is a schematic of the process for high-throughput screening and testing of natural photoprotective agents. Following screening, candidate compounds that display effective UV absorption, photostability, effective safety properties and possible antioxidant properties are re-analyzed to confirm UV absorbance and photostability.

[0021] The examples demonstrate the method of screening and resulting formulations that were identified. In step 1, 915 compounds were placed into 384 micro well plates using acoustic non- touch robotic transfers. Compounds were then screened for absorbance in the UV-visible spectrum using a microplate reader. Compounds that do not absorb in the UV spectrum (peak absorbance < 0.5 or AUC sample 280-400 nm less than AUC blank 200-400 nm) were excluded. The plates are exposed to solar simulated UV light for 20 minutes to test photostability. The absorbance index is calculated at 300 nm, 340 nm, 360 nm: post UVR abs pre UVR abs and

[0022] UVA, UVB, and UV spectrum: post UVR AUC pre UVR AUC Compounds with all absorbance indices < 0.90 were excluded. Cytotoxicity screening was then performed. Products are then added to cells such as PDV, Pam 212, YUMMER1.7 in 384 well plates and incubated for 72 hours. The percent viability is assessed via a bioluminescent ATP-release assay. Toxicity is then classified as minimal cytotoxicity (<10% cell death), moderate cytotoxicity (10-30% cell death viability), and high cytotoxicity (>30% cell death). These compounds were then tested for efficacy as antioxidants by adding a fluorescent ROS probe such as dihydrorhodamine- 123 to the compounds and exposing them to UV.

[0023] The photoprotective compounds can be combined in various ratios to enhance efficacy. Studies were made of mixtures formed by combining one UVA agent with two UVB agents to test for in vitro SPF. Mixtures yielded an average SPF of about 47, with SPF values generally ranging between 30 and 50.

[0024] Some of the preferred compounds, and concentrations thereof that were tested, include 5% scopoletin, 5% methylxanthoxylin, 5% rutin, 5% ferulic acid, 5% diosmin, 5% cytisine, 5% gossypin, 5% isoliquiritigenin, and 5% 2,4-dihydroxychalcone (CAS 13323-66-5).

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a schematic of the process for high-throughput screening and testing of natural photoprotective agents.

[0027] FIG. 2A is a schematic of screening compounds for absorbance in the UV-visible spectrum using a microplate reader. FIG. 2B is a schematic showing results on a representative microwell plate.

[0028] FIG. 3 A is a schematic of testing for the compounds that do not absorb in the UV spectrum (peak absorbance < 0.5 or AUCsamPie 280-400 nm less than AUC blank 280-400 nm) in microwell plates exposed to solar simulated UV light for 20 minutes. FIG. 3B shows results on a representative microwell plate.

[0029] FIG. 4 is a schematic and results of the cytotoxicity screen.

[0030] FIG. 5 shows the results of an in vivo experiment to test the UV- blocking efficacy of tested photoprotective compositions in preventing cyclobutane pyrimidine dimer formation. FIG. 6. is a graph of absorbance demonstrating broad spectrum coverage.

[0031] FIG. 7A and 7B, comparing CPD levels with and without natural product photoprotective agents, immediately after UVR (7A) and four hours after UVR (7B).

[0032] FIG. 8A and 8B comparing levels of UVR induced DNA damaging CPUs immediately after UVR (8A) and four hours after UVR (8B).

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] I. Definitions

[0035] There are two basic types of ultraviolet (“UV”) rays: UVB (280 nm - 320 nm) and UVA (320 nm - 400 nm). UVB rays are responsible for producing sunburn, and play the greatest role in causing direct DNA damage, which is implicated in development of skin cancer. UVA rays also play a role in skin cancer formation, in part via the generation of reactive oxygen species, and can penetrate more deeply into the skin than UVB. UVA rays also play a role in premature skin aging changes including wrinkle formation (photoaging). There are approximately 500 times more UVA rays in sunlight than UVB rays.

[0036] “Sunblock” or “photoprotective formulation”, as used herein, refer to agents that block or filter ultraviolet light. Sunblock / photoprotective formulation agents provide protection against UVA, UVB, or both UVA and UVB light. “Sunblock formulation” or “Sunscreen formulation” are formulations containing one or more sunblock / sunscreen agent(s) applied to the skin; these may also be incorporated into hair conditioners and cosmetics.

[0037] The term “topical administration”, as used herein, refers to non- invasive administration to the skin or hair.

[0038] The terms “bioactive agent” and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0039] The term “pharmaceutically acceptable”, as used herein, refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration. A “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation which facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0040] The terms “effective amount”, as used herein, refers to an amount of agent effective to alleviate, delay onset of, or prevent one or more symptoms of a disease or disorder, to reduce penetration of UVB and UVA light into the skin, to improve hair growth or appearance, or to provide a desired look, color, complexion, or fragrance to an area of the skin or hair, as described herein, using criteria known and used by those skilled in the art.

[0041] “About" is intended to describe values either above or below the stated value in a range of approx. + / - 10%. The ranges are intended to be made clear by context, and no further limitation is implied.

[0042] Unless otherwise indicated, references to percentage of a component(s) in a composition generally or by weight, refers to “wt / wt%.” II. Photoprotective Agents and Additives

[0043] The formulations typically have a sun protection factor (SPF) of 15, 20, 25, 30, 35, 40, 45, 50, 55, or higher.

[0044] The compositions have a critical wavelength between 280 and 400 nm, more preferably between 370 and 400 nm to provide broad- spectrum protection. The compositions protect against DNA damage caused by UV exposure, and are non-irritating when applied topically to intact skin.

[0045] A. Photoprotective Agents

[0046] The compositions are formulated of a mixture of at least two or more photoprotective agents. In the preferred embodiment, there are at least four agents. Suitable agents include flavonoids, cinnamic acids (preferably trans- cinnamic acids), organic fused heterotricyclic compounds, inorganic compounds, coumarins, acetophenones (e.g., substituted acetophenones such as methylxanthoxylin), or combinations thereof. Preferably at least two of the active agents are from a different class of specified compounds.

[0047] Flavonoids include flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chaicones. Flavonoids are reviewed in Panche, et al., Journal of Nutritional Science 2016, 5, e47, ppl- 15.

[0048] Flavones include diosmins, gossypins, apiins, apigenins, tangeretins, baicaleins, rpoifolins, preferably diosmins, gossypins, apiins, and most preferably diosmins. Flavonols include rutins, quercetins, myricetins, morins, kaempferols, preferably rutins.

[0049] Chaicones include isoliquiritigenins, 2, 4-dihydroxy chaicones, phloretins, arbutins, phlioridzins, and chalconaringenins.

[0050] Coumarins include scopoletins, warfarins, acenocoumarols, phenprocoumons, umbelliferones, hydroxy coumarins, aesculetins, herniarins, psoralens, and imperatorins.

[0051] Inorganic compounds include inorganic oxides, such as transition metal oxides, preferably titanium dioxide and / or zinc oxide, more preferably titanium dioxide.

[0052] Representative combination formulations include flavonoids, trans- cinnamic acids, organic fused heterotricyclic compounds, and inorganic compounds, optionally containing coumarins and / or acetophenones.

[0053] In a representative embodiment, the composition contains diosmins, isoliquiritigenins, ferulic acids, cytisines, titanium dioxide, and optionally one or more of rutin, gossypins, scopoletins, methylxanthoxylins, apiins, and 2,4-dihydroxychalcones (CAS 13323-66-5). In another embodiment, the composition contains flavones, / ram-cinnamic acids, and organic fused heterotricyclic compounds such as diosmin; ferulic acids and sinapinic acids, preferably ferulic acids and cytisines, and a polyphenol such as the stilbenoid resveratrol (3,5,4'-trihydroxy-rran.y-stilbene. In another embodiment, the composition contains chaicones, / / rm-cinnamic acids, and organic fused heterotricyclic compounds, such as isoliquiritigenins, ferulic acids, and cytisines, respectively.

[0054] A preferred embodiment of the photoprotective formulation contains 10% Ferulic Acid, 10% Diosmin, 5% Cytisine and 5% Resveratrol. 2 mg / cm2photoprotective formulation applied 15 minutes before ultraviolet radiation (“UVR”) exposure.

[0055] B. Additives

[0056] Antioxidants

[0057] In some embodiments, the formulations may incorporate an antioxidant. Non-limiting examples of antioxidants include acetyl cysteine, ascorbic acid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanol pectinate, ascorbyl palmitate, ascorbyl stearate. BHA, BHT, t-butyl hydroquinone, cysteine, cysteine HC1, diamylhydroquinone, di-t- butylhydroquinone, dicetyl thiodipropionate, dioleyl tocopheryl methylsilanol, diosmin, disodium ascorbyl sulfate, distearyl thiodipropionate, ditridecylthiodipropionate, dodecyl gallate, erythorbic acid, esters of ascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters, hydroquinone, isooctylthioglycolate, kojic acid, magnesium ascorbate, magnesium ascorbyl phosphate, methylsilanol ascorbate, natural botanical anti-oxidants such as green tea or grape seed extracts, nordihydroguaiaretic acid, octyl gallate, phenyl thioglycolic acid, potassium ascorbyl tocopheryl phosphate, potassium sulfite, propyl gallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite, sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxide dismutase, sodium thioglycolate, sorbityl furfural, thiodiglycol, thiodiglycolamide, thiodiglycolic acid, thioglycolic acid, thiolactic acid, thio salicylic acid, tocophereth-5, tocophereth-10, tocophereth-12, tocophereth-18, tocophereth-50, tocopherol, tocophersolan, tocopheryl acetate, tocopheryllinoleate, tocopheryl nicotinate, tocopheryl Succinate, and tris(nonylphenyl)phosphite. A non-limiting list of preferred antioxidants include ferulic acid, diosmin, natural botanical anti-oxidants such as green tea or grape seed extracts, and other naturally-occurring antioxidant compounds.

[0058] Photostabilizers

[0059] In some embodiments, the formulations may contain a photostabilizer. Photostabilizers are primarily used to reduce the breakdown of sunscreens under prolonged exposure to UV light. In certain aspects, photostabilizers are organic compounds that help to prevent sunscreen agents from losing their effectiveness in sunlight. Some photostabilizers help to stabilize the molecules of sunscreen agents structurally and geometrically through electrostatic and van der Waals interactions, which makes them less likely to take part in chemical reactions. Another type of photostabilizer protects sunscreen agents by helping to dissipate the energy from UV more quickly, thus reducing or even eliminating the possibility of a chemical reaction. This process is called energy transfer, and it can take place when the UV filter and photostabilizer molecules exchange electrons, or even by action at a distance, as a radio transmitter sends a signal to a nearby receiver. In this way, the sunscreen agents are freed up to protect the skin by absorbing the harmful rays, while the photostabilizers dispose of the energy.

[0060] In some embodiments, the formulation may include avobenzone for the UV-A protection. Avobenzone has been shown to degrade significantly in light, resulting in less protection over time. The UV-A light in a day of sunlight in a temperate climate is sufficient to break down most of the compound. Data presented to the Food and Drug Administration by the Cosmetic, Toiletry and Fragrance Association indicates a -36% change in avobenzone’ s UV absorbance following one hour of exposure to sunlight. This degradation can be reduced by using a photo stabilizer, like octocrylene. Other photostabilizers include: 4-Methylbenzylidene camphor (USAN Enzacamene); Tinosorb S (USAN Bemotrizinol, INCI Bis- Ethylhexyloxyphenol Methoxyphenyl Triazine); Tinosorb M (USAN Bisoctrizole, INCI Methylene BisBenzotriazolyl Tetramethylbutylphenol); Butyloctyl Salicylate (Tradename HallBrite BHB); Hexadecyl Benzoate; Butyloctyl Benzoate; Mexoryl SX (USAN Ecamsule, INCI Terephthalylidene Dicamphor Sulfonic Acid); Corapan TQ (INCI Diethylhexyl 2,6-Naphthalate); Parsol SLX (INCI Polysilicone- 15). Oxynex ST (1 C1 Diethylhexyl Syringylidene Malonate) Poly cry lene (1 C1 Polyester-8); complexing avobenzone with cyclodextrins may also increase its photostability. Certain naturally-occurring antioxidants may also serve as photostabilizers; for example, the natural antioxidant quercetin has been shown to reduce photodegradation of organic sunscreen agents. In some forms, two or more of the above photostabilizers may be used in combination to provide the unexpected SPF enhancement effect. Photostabilizers may include but are not limited to Octocrylene, Corapan TQ (Diethylhexyl 2,6- Naphthalate), HallBrite BHB (Butyloctyl Salicylate), HallBrite PSF (Undecylcrylene Dimethicone), Mexoryl SX or Ecamsule (Terephthalylidene Dicamphor Sulfonic Acid), Neo Heliopan MBC or Enzacamene (4- Methylbenzylidene camphor), Oxynex ST (Diethylhexyl Syringylidene Malonate), Parsol SLX (Polysilicone- 15), Polycrylene (Polyester-8), Polycrene SI (Polyester-25), SolaStay SI (Ethylhexyl Methoxy cry lene), Tinosorb M or Bisoctrizole (Methylene Bis-Benzotriazolyl Tetramethylbutylphenol), Tinosorb S or Bemotrizinol (Bis- Ethylhexyloxyphenol Methoxyphenyl Triazine).

[0061] Avobenzone can degrade faster in light in combination with mineral UV absorbers like zinc oxide and titanium dioxide, thus currently not allowed in the United States. Consequently, in some forms, the formulation may comprise mineral UV absorbers in the absence of avobenzone, with SPF enhancement provided by a combination of photostabilizers such as octocrylene and polyester-8.

[0062] Although not preferred, one or more chemical sunscreen agents such as oxybenzone, dioxybenzone, meradimate, amiloxate, octinoxate, homosalate, octisalate, octocrylene, sulisobenzone, avobenzone, ecamsule, ensulizole, enzacamene, bisdisulizole disoidium, padimate O, p- aminobenzoic acid (PABA), phenylbenzimidazole sulfonic acid, cinoxate, trolamine salicylate, menthyl anthranilate, sulisobenzone, parsol compounds, tinosorb compounds, heliopan compounds, and combinations thereof could be added to the sunscreen formulations.

[0063] Additionally or alternatively, the formulation includes one or more natural anti-oxidant and / or other U V absorbing agents, including, but not limited to, polyphenols, indegoids, retinoids, melanin and melanin derivatives, ascorbic acid, ferulic acid, citric acid, oxalic acid, phytic acid, Polypodium leucotomos extract, carrageenan, milk thistle extract, coenzyme Q10, zinc gluconate, bioflavonoids, isoflavones, other flavones and flavonols, luteins, lycopenes, konjac, alpha lipoic acid, beta carotene, grape seed extract, alpha-tocopherol acetate, niacinamide, nicotinomide, green tea extract, copper gluconate, resveratrol and other stilbenoids, cholecalciferol, sodium selenite / selenium, bilberry fruit, folic acid, astaxanthin (red algae extract), canthaxanthin, zeaxanthin, anthocyanins and other cyanins, cinnamic acid and other cinnamates, salicylic acid, curcumin, other suitable agents discussed herein, and combination thereof. Resveratrol (3,5,4'- trihydroxy-trans-stilbene) is a stilbenoid, a type of natural phenol, and a phytoalexin produced by several plants in response to injury or when the plant is under attack by pathogens, such as bacteria or fungi.

[0064] III. Topical Formulations

[0065] The photoprotective agents can be formulated with appropriate pharmaceutically acceptable carriers for administration to an individual in need thereof. The formulations can be administered topically (e.g., to the skin via non-invasive topical application).

[0066] There are three categories of formulations that can provide photoprotection when applied topically: sunscreens, which protect the skin from UV radiation by reflecting, scattering, or absorbing UV radiation. cosmetics, which protect and improve the appearance of skin, including via counteracting the downstream effects of UV radiation and thus preventing photodamage and photoaging. hair conditioners, that can prevent drying and color bleaching by the sun.

[0067] Compositions containing a mixture of organic and inorganic active agents or of only organic active agents, in specific concentrations and ratios, are formulated in a vehicle suitable for use as a sunscreen, cosmetic or hair conditioners. These can be formulated as sprays, gels, lotions, powders, solutions, suspensions, shampoos or conditioners, and cosmetical formulations.

[0068] A. Sunscreen Formulations

[0069] Sunscreens are creams, lotions, sprays, gels, sticks, or other topical formulations that protect the skin from the sun; by absorbing or reflecting harmful UV radiation. Sunscreens can be formulated as sprays, solutions or suspensions, foams, creams or lotions, gels or spray powders, and may also include pigments or moisturizers. A representative vehicle is a FDA P2 vehicle, which includes water, sorbitol, lanolin, glyceryl stearate, theobroma cacao seed butter, stearic acid, triethanolamine, benzyl alcohol, methylparaben, and propylparaben.

[0070] The weight percentage of each active agent or the total weight percentage of the two or more active agents may range from about 1% to about 15% by weight. In some forms, the total weight percentage of the active agents (two or more, three or more, etc.), is between 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, .1% to 30%. The preferred weight percentage of each active agent is from about 2.5% to about 10%. The preferred total weight percentage of the active agents is from about 15% to about 25%.

[0071] 1. Emulsions

[0072] An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. In particular embodiments, the non-miscible components of the emulsion include a lipophilic component and an aqueous component. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil- in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0073] The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially nonionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0074] 2. Lotions

[0075] A lotion can contain finely powdered substances that are insoluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’ s surface.

[0076] 3. Creams

[0077] Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000- 50,000 centistokes. Creams are often time preferred over ointments, as they are generally easier to spread and easier to remove.

[0078] The difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations.

[0079] 4. Ointments

[0080] Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy than ointments prepared with the same components.

[0081] 5. Gels

[0082] Gels are semisolid systems containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alkylene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the compound. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited to, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.

[0083] 6. Foams and Sprays

[0084] Sprays may be aqueous and / or alcohol-based solutions or suspensions that are applied by aerosolization, pumping or wipes.

[0085] Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1, 1,2- tetrafluoroethane (FIFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (FIFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying. Furthermore, the formulations preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0086] B. Cosmetics

[0087] In another embodiment the formulations are incorporated into cosmetics and other skin care products which protect against wrinkling, pigmented spots, fine lines, atropic damage and sebaceous hyperplasia. These may include coloring, hydrating agents, or be what is referred to as an “anti-aging serum”

[0088] In the United States, the Food and Drug Administration (FDA), which regulates cosmetics, defines cosmetics as products "intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance without affecting the body's structure or functions". This broad definition includes any material intended for use as an ingredient of a cosmetic product, with the FDA specifically excluding pure soap from this category.

[0089] Cosmetics have various purposes. Those designed for personal care and skin care can be used to cleanse or protect the body or skin. Cosmetics designed to enhance or alter one's appearance (makeup) can be used to conceal blemishes, enhance one's natural features (such as the eyebrows and eyelashes), add color to a person's face, or change the appearance of the face. Moisturizers are creams or lotions that hydrate the skin and help it to retain moisture. Typical components are polyols such as glycerol and sorbitol as well as partially hydrolyzed proteins. Tinted moisturizers contain a small amount of foundation, which can provide light coverage for minor blemishes or to even out skin tones. They are usually applied with the fingertips or a cotton pad.

[0090] Eyes require a different kind of moisturizer compared with the rest of the face. The skin around the eyes is extremely thin and sensitive, and is often the first area to show signs of aging. Eye creams are typically very light lotions or gels, and are usually very gentle; some may contain ingredients such as caffeine or Vitamin K to reduce puffiness and dark circles under the eyes. Eye creams or gels should be applied over the entire eye area with a finger, using a patting motion. Combining a moisturizer with SPF is beneficial to prevent aging and wrinkles.

[0091] Serums are skin care products containing active ingredients that target the visible signs of aging, such as fine lines, wrinkles, and age spots, with the goal of improving the appearance of the skin. Serums are light and easily absorbed liquids that one spreads on their skin. The main purpose of the product is to apply before moisturizer and the serum provides a high concentration of any specific ingredient on the face. The benefits to serum are skin firmness, smoothness of face, and reducing the fine lines and wrinkles on one’s face.

[0092] Representative ingredients in serums that provide pro-aging support include retinoids, hyaluronic acid, peptides, ferulic acid, vitamin C and glycolic acid. Retinoids are derived from vitamin A. Retinoids work by increasing cell turnover and stimulating collagen production, which can improve the texture and appearance of the skin over time. Some common types of retinoids used in skin care include retinol and tretinoin.

[0093] Hyaluronic acid helps your skin maintain hydration and plumpness. When used in skin care products, hyaluronic acid can help improve skin texture and tone and minimize the appearance of fine lines and wrinkles.

[0094] Peptides are short amino acid chains that can penetrate the skin and signal cells to perform specific functions. When used in skin care products, peptides can help stimulate collagen production and reduce the appearance of fine lines and wrinkles.

[0095] Ferulic acid is a plant-based antioxidant commonly used in skin care products. When applied topically, ferulic acid can help neutralize free radicals and prevent oxidative stress, which is linked to collagen breakdown, hyperpigmentation, and other signs of aging. Ferulic acid may also enhance the effectiveness of other antioxidants, such as vitamin C and vitamin E.

[0096] Vitamin C is an antioxidant that helps protect the skin from damage caused by environmental stressors such as UV radiation and pollution. When applied topically in a serum, vitamin C can also help to stimulate collagen production, brighten the skin, and reduce the appearance of fine lines and wrinkles.

[0097] Glycolic acid is a type of alpha-hydroxy acid (AHA) that is commonly used in skin care products for its ability to exfoliate the skin and improve its texture and tone. When applied topically, glycolic acid can help to dissolve dead skin cells and other debris on the surface of the skin, which can clog pores and contribute to a dull complexion.

[0098] There has been a marketing trend towards the sale of cosmetics lacking controversial ingredients, especially those derived from petroleum, sodium lauryl sulfate (SLS), and parabens. Per- and polyfluoroalkyl substances (PFAS) are a class of about 9,000 synthetic organofluorine compounds that have multiple highly toxic fluorine atoms attached to an alkyl chain. PFAS are used by major cosmetics industry companies in a wide range of cosmetics.

[0099] C. Hair Conditioners

[0100] In another embodiment, the formulations are formulated as topical hair conditioners to protect against sun damage and fading of hair color. These can be shampoos or hair coloring formulations, conditioners, sprays, mousses, gels, or foams.

[0101] The addition of a photo protective formulation to hair products not only protects the hair from damage from exposure to sun, but can be used to avoid bleaching of hair color. This is particularly an issue for hair that is dyed to a dark shade of brown or black, especially when the underlying color is grey or white. Sun causes rapid loss of pigment, requiring more frequent dying. This may be aggravated by the use of products such as hydrogen peroxide which increases porosity of the hair fibers to allow pigment to penetrate, but also causes photoreactions that can degrade the pigment.

[0102] Hair spray or gel is a hairstyle product that is used to stiffen hair into a particular hairstyle. Hair gel can come in tubes, pots, small bags, or even in a spray form. A hair wax is typically provided as a small pot of hair pomade, a thick hair styling product containing wax, which helps hold hair in place. Many manufacturers are now releasing different versions of hair wax, such as pomade, putty, glue, glypto, whip, and styling paste.

[0103] Hair mousse is a product added to hair for extra volume and shine. It is most commonly produced as a foam, but can also be found as a spray. Hair mousse adds volume without causing clumps or buildup. It is a lighter alternative to hair gel. Mousse is generally applied to the roots of damp hair before blow drying or styling. Mousse may also be used to add definition to curls, or to add texture to hair for a beach blown effect.

[0104] A pomade is an oil-based or water-based product designed for slick and tight hairstyles. Unlike hair spray and hair gel, pomade does not dry and often takes several washes to remove. Most oil based pomades contain petroleum jelly and mineral oil, and many also contain some sort of wax. They may also contain perfume and coloring agents. Hair spray is an aqueous solution which is used to stiffen hair into a certain style. Weaker than hair gel or hair wax, it is sprayed directly onto the hair to hold styles for long periods of time. It sprays evenly over the hair using a pump or aerosol spray nozzle. The product may leave hair feeling 'crunchy' unless brushed out. Polymers, which keep the hair stiff and firm without snapping are dissolved in solvents such as alcohols and hydrocarbons. Representative solvents include polyvinylpyrrolidone, which is water-soluble. The non-water-soluble polymer polydimethylsiloxane is added to make the hold last a bit longer. Some less common polymers found in hair spray include copolymers with vinyl acetate and copolymers with maleic anhydride.

[0105] Some hair sprays use natural polymers and solvents like vegetable gums dissolved in alcohol. One popular ingredient in natural hair sprays is gum arabic or gum tragacanth.

[0106] Hair volumizers are used to temporarily add volume, body and shine to thin or flat hair. Hair volumizers are used by both men and women. Men turn to hair volumizers to make their hair look more dense. Volumizers come in many forms such as shampoos, conditioners, sprays, pomades and lotions. Hair volumizers contain humectants, which work by attracting moisture from the surrounding areas to the hair strand, thereby swelling the hair and making it look thicker. Various polymers present in the volumizer coat the hair strand, making it look thicker and shiny.

[0107] Shampoo and conditioner forms of the volumizers are used just like ordinary shampoo or conditioners. The spray and lotion form of volumizers are used on damp hair near the roots of the hair. In order to use a hair volumizer, the person using the product must flip their head downward and gradually blow dry the hair, with the air being blown along the shaft of the hair until the hair is dry. Drying the hair in this position will increase volume and achieve the desired effect.

[0108] Other types of products include serums, leave-in conditioner, clays, hair tonic, hair dry powder shampoo, and heat protection sprays are frequently used hairstyling products in salons and homes across the country. A hair care product that helps to improve the appearance, feel, volume, or shine of the hair is a hair conditioner. In general, conditioners make hair more manageable by reducing friction between hair strands and ease the process of combing the hair. It also strengthens the hair, reduces frizz, and makes them anti-static. At a neutral pH, the surface of hairs has a negative charge. Conditioners, just like shampoos, also contain surfactants that are positively charged. The positively charged molecules of the conditioners are attracted to the negatively charged surface of the hair. This attraction results in conditioner to deposit on the hair, especially on weathering parts. Conditioners help reduce the static charge, which develops due to interaction between the anionic surfactants of shampoo and the negatively charged surface of the hair. Friction is reduced by closing the cuticle scales. This results in a smooth feeling to the hair and makes combing hair easier both in wet and dry conditions. Conditioner also helps to improve color, seals the split ends of hair temporarily, and gives shine to the hair by increasing the light reflectance.

[0109] The primary ingredients of hair conditioners are water (typically about 80% of the formulation) and cationic surfactants, such as Stearamidopropyl dimethylamine, Behentrimonium or propyltrimonium, or cetyltrimethylammonium chloride. The positively charged molecules of conditioning agents "adsorb" on the hair's surface instead of being absorbed.

[0110] Cationic polymer, such as polypeptides derived from collagen, polyvinylpyrrolidone (PVP), or poly and monopeptides-like hydrolyzed proteins (amino acids), are added to achieve additional conditioning effects. These cations are attracted by the negatively charged surface of the hair shaft and make a film over the hair shaft making hair fibers smooth. Conditioning agents make 1 to 4% of the formulation. Humectants or Moisturizers attract and bind the water molecules from the surrounding environment to hair and provide a moisturizing effect to the hair. They are present in a quantity of 0 to 2% in the formulation. Emollients make the hair soft and lubricated and are usually from 0 to 2% of the formulation. They also replenish the lipids lost from the hair shaft. Oils, fats, and butter are the common types of emollients used in conditioners. Again, the type of hair for which the conditioner is being formulated needs to be considered while using the emollients as well, such as hair that are fine require lighter emollients; otherwise, heavy emollients make them weighed down and lank. Natural oils such as olive oil, jojoba oil, or grape seed oil, synthetic oils like silicones (cyclomethicone, amodimethicone, dimethiconol, and dimethicone) are used commonly in conditioners to give luster and shine to hair strands.

[0111] Conditioners also contain thickening agents, preservatives, fragrances, and pH stabilizers.

[0112] IV. Methods of Identifying Photoprotective Agents and Compositions A. Method for High throughput Screening of Sunscreen

[0113] Agents

[0114] A high throughput method of screening for compounds that are safe and effective as sunscreens has been developed. High-throughput screening of natural compounds is conducted in as few as four steps: (1) UV fullspectrum absorbance screening, (2) photostability screening, (3) cytotoxicity screening, and (4) reactive oxygen species generation and antioxidant capacity screening. FIG. 1 is a schematic of the process for high-throughput screening and testing of natural sunscreen agents.

[0115] Following screening, mixtures are made of the candidate compounds that display effective UV absorption, photostability, and favorable safety profiles, some of which also have strong antioxidant properties, and all of which have undergone confirmatory testing for UV absorbance and photostability.

[0116] In a preferred embodiment, compounds (25 pM - 0.5 mM) dissolved in dimethyl sulfoxide (DMSO) are added to microwell plates such as 384 well plates. The compounds can be obtained from libraries such as MicroSource Pure Natural Products and NCI Natural Products Set and plated into 384 well plates using acoustic non-touch robotic transfer systems. If desired, the concentration of compounds can be determined by plating libraries at different concentrations, obtaining UV absorbance spectra using a microplate reader, and selecting the concentration(s) at which significant UV absorbance is observable in at least some of the wells.

[0117] UV Absorbance Screening

[0118] In the preferred embodiment, the compounds in the microwell plate are screened for absorbance in the UV-visible spectrum using a microplate reader. Compounds that do not absorb in the UV spectrum are excluded. This determination is based on the peak absorbance at a wavelength between 280 and 400 nm being less than 0.5 or the AREA UNDER THE CURVE (AUC) between 280 and 400 nm being less than the average AUC of blank wells, according to the formula, where the background absorbance at the same wavelength is subtracted as the control:

[0119] AUCsampie (280-400nm) < AUCbiank (280-400nm)

[0120] Photostability Assay

[0121] The microwell plates containing the compounds are exposed to solar simulated UV light for a suitable time, such as 15 minutes to 2 hours, preferably 20 min. The duration of UV exposure for the photostability experiment is determined by the time it takes for avobenzone to degrade. Avobenzone is a currently available organic sunscreen agent known to photodegrade. The absorbance indices for the following were calculated: post UVR absorbance 300nm, 340nm, 360nm: pre UVR absorbance

[0122] . b. T U,TVTA. , T U,TVTrB,, and 1 T UTTVT spectrum:

[0123] Compounds that had absorbance indices less than 0.90 are excluded.

[0124] This exclusion threshold is determined by prior experiments assessing the absorbance indices of avobenzone.

[0125] Toxicity Screening

[0126] The compounds are then screened for toxicity according to three measures: cytotoxicity, photo-cytotoxicity and generation of reactive oxygen species (ROS).

[0127] Toxicity was classified according to the following scale:

[0128] Minimal toxicity = >90% viability

[0129] Moderate cytotoxicity = 70-90% viability High cytotoxicity = <70% viability

[0130] To screen for reactive oxygen species (ROS), fluorescent ROS / RNS probes can be added to candidate compounds. Examples of probes used include dihydrorodamine-123 and aminophenyl fluorescin. The candidate compounds can then be exposed to UV light.

[0131] Antioxidant Screening

[0132] Antioxidant capacity may be approximated by the reactive oxygen species screening protocol described above. Candidate compounds with post- UV exposure ROS probe fluorescence significantly less than the fluorescence of blank wells may be further investigated as possible antioxidants.

[0133] For definitive determination of antioxidant capacity, 25 pM - 0.5 rnM of candidate compounds are plated in 384-well plates. Free radical scavenger probes are added to candidate compounds to assess for antioxidant capacity. Examples of free radical scavenger probes include the colorimetric probe 2,2-Diphenyl-l-Picrylhydrazyl radical.

[0134] Confirmatory UV Absorbance and Photostability Testing

[0135] To confirm the UV-absorbing capability and photostability of promising compounds identified via high-throughput screening, 0.05 - 1 mg / ml of promising compounds are dissolved in DMSO and plated in triplicate in 96-well plates. The plate is then read using a microplate reader to assess for absorbance from 280 nm to 400 nm. The plate is exposed to solar simulated UV light for a suitable time, such as 15 minutes to 2 hours. The duration of exposure is determined by the time it takes for avobenzone and padimate O, two organic sunscreen agents, to degrade. The duration of exposure is also determined by the FDA’s recommendations for reapplication of sunscreen every two hours.

[0136] The compositions are formulated for topical application, with minimal to no transdermal penetration. The compositions can be applied to the skin, hair, etc., or other external surface of a subject in need thereof, using standard techniques. The present invention will be further understood from the following non-limiting Examples.

[0137] Example 1: High throughput Screening and Testing of Natural Compounds for Sunscreen Agents

[0138] The examples demonstrate the method of screening and resulting formulations that were identified.

[0139] Materials and Methods

[0140] Compounds from MicroSource Pure Natural Products (0.5 mM) and NCI Natural Products Set (25pM) were plated into 384-well plates using acoustic non-touch robotic transfer systems. This process yielded 915 candidate compounds.

[0141] Compounds were then screened for absorbance in the UV-visible spectrum using a microplate reader. See Fig. 2A.

[0142] Compounds that absorbed in the UV spectrum (peak absorbance > 0.5 and AUC sample 280-400 nm greater than AUC blank 280-400 nm) were added to microwell plates and the plates exposed to solar simulated UV light for 20 minutes. See Fig. 3A

[0143] The absorbance index was calculated at 300 nm, 340 nm, 360 nm: post UVR abs pre UVR abs and

[0144] UVA, UVB, and UV spectrum: post UVR AUC pre UVR AUC

[0145] Compounds with absorbance indices < 0.90 were excluded.

[0146] The compounds were then screened for toxicity according to 2 measures: cytotoxicity and generation of reactive oxygen species (ROS).

[0147] For cytotoxicity screening, 0.5 - 10[iM of each candidate compound was added to each of three cell lines (PDV, Pam 212, and YUMMER1.7) in 384-well plates. The samples were incubated for 72 hours, after which percent viability was assessed using a bioluminescent ATP-release assay. Toxicity was classified according to the following scale:

[0148] Minimal cytotoxicity = <10% cell death

[0149] Moderate cytotoxicity = 10-30% cell death

[0150] High cytotoxicity = >30% cell death

[0151] To screen for reactive oxygen species (ROS) and antioxidant capacity, fluorescent ROS / RNS probes were added to candidate compounds. Examples of probes used included dihydrorodamine-123 and aminophenyl fluorescin. The candidate compounds were then exposed to UV light and assessed for ROS probe fluorescence. The top 10% of photostable, UVA and UVB-absorbing candidates were selected for in depth assessment of cytotoxicity and potential to generate ROS.

[0152] To confirm the UV-absorbing capability and photostability of the most promising compounds identified via high-throughput screening, 0.125 mg / ml of compounds were dissolved in DMSO and plated in triplicate in 96- well plate. Absorbance spectra from 280 nm to 400 nm was collected. The plate was then exposed to solar simulated UV light, and absorbance spectra were collected at 20 minutes, 40 minutes, 60 minutes, and 120 minutes of UV exposure to assess for photostability.

[0153] Results

[0154] Of the 915 compounds, 322 (35.2%) passed the UV-VIS absorbance screen. See Fig. 2B.

[0155] 219 compounds (23.9% of the 915 compounds) passed the photostability screen. See Fig. 3B.

[0156] Of the 219 compounds that passed the absorbance testing and photostability screen, 118 (12.9% of the 915 compounds) had minimal cytotoxicity. See Fig. 4.

[0157] Table 1 demonstrates the percent of cells killed from 9 of these candidate compounds. Percent cell death was normalized to positive (20pM bortezomib, 100% toxicity) and negative (0.1% DMSO, 0% toxicity) controls according to the following formula: average test well luminescence

[0158] % cell death (toxicity ) = 1 - '■ - ; - — - ; - average negative control luminescence Table 1 : Percent of cells killed from candidate compounds

[0159] ROS screening of the candidate compounds was performed following 20-minute incubation periods in the UV- solar simulator. Table 2 demonstrates the relative fluorescence of the ROS probe dihydrorhodamine- 123 after incubation with 10 of the candidate compounds after exposure to UV light. Fluorescence in relative fluorescence units (RFU) was normalized to blank DMSO wells by subtracting the fluorescence of blank DMSO wells from the value of the test well. For reference, the average RFU of blank DMSO wells after normalization was 0.00, and the average RFU of wells containing padimate O, an organic sunscreen agent known to generate ROS on UV exposure, was +17093.32. Table 2: Post-UV exposure dihydrorhodamine-123 fluorescence after incubation with candidate compounds

[0160] The best performing and safe compounds identified via screening were then subjected to confirmatory UV absorbance and photostability screening.

[0161] From the original 915 compounds, 13 compounds were identified based on high UV absorbance, photostability, minimal cytotoxicity, and failing to generate ROS upon exposure to UV.

[0162] The best performing and commercially available compounds were further assessed using in vitro methods by mixing individual compounds into an FDA P2 vehicle without sunscreen actives and assessing the SPF values via the Diffey method.

[0163] Table 3 demonstrates results from in vitro SPF testing of formulations containing 5% of a single candidate compound compared to FDA P2 reference standard with a known SPF of approximately 16. All formulations containing only one candidate compound had SPF values ranging from 2 to 8.5. For example, some formulations had low in vitro SPF values. Formulations containing 5% diosmin (DIO) or 5% methylxanthoxylin (MX) had average SPF values of 2.6 and 2.2 respectively (Table 2). Other formulations had higher SPF values. For example, formulations containing 5% cytisine (CYT) from two separate suppliers had average SPF values of 7.2 and 8.1 respectively (Table 3). Other tested compounds included isoliquiritigenin (IQL), ferulic acid (FA), and 2,4- dihydroxychalcone (DHC; CAS 13323-66-5). These data demonstrate that compounds derived from high-throughput screening methods can be effectively used to block UV from the sun

[0164] Table 3’. Preliminary results from in vitro SPF testing of single candidate compounds

[0165] UVA- and UVB-absorbing compounds with the highest in vitro SPF values were combined at pre-determined ratios and further evaluated to determine which combinations provided broad-spectrum photoprotection. SPF values from in vitro tests of these combination formulations were calculated.

[0166] Example 2: Testing of Combinations of Compounds on Mice

[0167] Following screening, mixtures were made of the candidate compounds that displayed effective UV absorption, photostability, effective safety properties and strong antioxidant properties.

[0168] Materials and Methods

[0169] The identified compounds can be combined in various ratios to enhance efficacy. Studies were made of mixtures formed by combining one UVA agent with two UVB agents to test for in vitro SPF according to the Diffey method. These mixtures were then tested for in vivo protection against UV radiation by topical application to the shaved dorsal skin of C57B1 / 6J mice. Mice were exposed to UV radiation, after which DNA damage in the skin was quantified by performing enzyme-linked immunosorbent assay to detect cyclobutane pyrimidine dimers.

[0170] These mixtures were also tested for safety of topical application to live skin by applying the mixtures to shaved dorsal mouse skin every other day for 5 applications. Throughout the experiment, the skin was assessed for gross indication of toxicity or inflammation, and at the conclusion of the experiment, skin was examined histologically for evidence of toxicity or irritation.

[0171] Results

[0172] Some of the preferred compounds, and concentrations thereof that were tested, include 5% Scopoletin, 5% Methanylxanthoxylin, 5% Rutin, 5% Ferulic Acid, 5% Diosmin, 5% Cytisine, 5% Gossypin, 5% Isoliquiritigenin, and 5% 2,4-dihydroxychalcone (CAS 13323-66-5). Mixtures yielded an SPF of up to 57.1. For reference, the minimum recommended SPF recommended by the FDA is 15.

[0173] Combinations of natural compounds in specific concentrations and ratios likely to provide safe, effective photoprotection with the added benefit of antioxidant capacity are shown in Table 4.

[0174] Table 4 shows results from in vitro testing of a formulation containing 10% Diosmin (DIO), 10% Ferulic acid (FA) and 5% Cytisine (CYT). The formulation containing 10% DIO, 10% FA and 5% CYT exhibited a favorable average SPF value of between 48.6 and 57.1 (Tables 4- 6). Table 5 shows results from in vitro testing of selected formulations as well as the critical wavelength of these formulations. All tested formulations had critical wavelength greater than or equal to 370 nm, indicating broadspectrum photoprotection. Table 4: Preliminary results from in vitro SPF testing of a mixture containing 10% Diosmin (DO), 10% Ferulic acid (FA) and 5% Cytisine (CYT)

[0175] Table 5: Preliminary results from in vitro SPF testing of a mixture containing multiple compounds at varying percentages.

[0176] Table 6. Summary of selected results from in vitro SPF and critical wavelength testing of a mixture containing multiple compounds at varying percentages. Average % cytotoxicity: Compounds (10 uM) were tested for cytotoxic activity on keratinocyte (Pam 212, PDV) and melanocyte (YUMMER1.7) cell lines. Cytotoxicity data were normalized to positive and negative controls (bortezomib and 0.1 % DMSO, respectively; n=2 wells per group). The average % cytotoxicity of the 3 cell lines is presented. Final candidates were minimally cytotoxic (defined as <10% toxicity).

[0177] ROS probe fluorescence: dihydrorhodamine- 123 (reactive oxygen species probe) fluorescence after 20 minutes of exposure to UVR. ROS data were normalized to the fluorescence of blank DMSO and are presented in comparison to the ROS-inducing positive control, padimate O. All compounds demonstrated UV absorbance capacity on confirmatory absorbance testing. All compounds except sennoside B were relatively photostable, demonstrating minimal photodegradation. All compounds were plated at 0.125 mg / ral in DMSO (n=3 per group). Table 7: In vitro Photostability of Compounds

[0178] FIG. 5 shows results from in vivo testing of a mixture containing multiple compounds at varying percentages for protection against UV-induced DNA damage. These data demonstrate that all tested mixtures were effective in preventing UV from causing DNA damage in the form of cyclobutane pyrimidine dimers.

[0179] These mixtures were also tested for potential to cause skin irritation and toxicity after topical application. After 5 repeated applications, gross examination of mouse skin revealed no signs of toxicity, irritation, or inflammation. Histologic findings of skin were also benign. The epidermis, hair follicles, sebaceous glands, and dermis appeared normal without inflammatory infiltrate.

[0180] Example 3: In vivo testing for reduction of CPD production in Mice exposed to UV Radiation

[0181] Materials and Methods

[0182] Chronic UVR: A topical formulation consisting of 10% ferulic acid, 10% diosmin, 5% cytisine, 5% resveratrol was applied at 2mg / cm2on dorsal body wall skin of C57B1 / 6 mice (N=7 / group) 15 minutes before UVR exposure (1500 J / m2UVB + 2700 J / m2UVA) three times per week for 10 weeks.

[0183] Acute UVB: A topical formulation consisting of 10% ferulic acid, 10% diosmin, 5% cytisine, 5% resveratrol mixed into P2 vehicle was applied at 2mg / cm2on dorsal bodywall skin of C57B1 / 6 mice that were then exposed to 1000 J / m2UVB. Immediately after exposure skin samples were harvested and DNA prepared for cyclobutane pyrimidine dimer (CPD) quantification by ELISA. The topical formulation reduced CPD levels by 85%.

[0184] Results

[0185] Chronic UVR: Four days after the final exposure, skin was harvested for epidermal sheet immunofluorescent staining to quantify mutant p53 keratinocyte clones (called p53 islands).

[0186] Treatment with the topical formulation reduced the number of p53 islands by 92.54% and reduced the total size (area occupied by p53 islands) by 97.34%.

[0187] Acute UVB: In vivo protection of natural compound formulations applied at 2mg / cm2against UV-induced cyclobutane pyrimidine dimer (CPD) formation in C57 / BL6J mice (n=5 per group). Data were normalized to the +1000J / m2UVB, no sunscreen group. Natural compounds were mixed into P2 vehicle formulation. 10% DIO + 10% FA + 5% CYT was the best performer at 92.40% CPDs blocked (SD 3.51). P2 reference standard led to 84% CPDs blocked (SD 9.82), 10% DIO + 7.5% FA + 2.5% CYT led to 86.00% CPDs blocked (SD 5.75), 5% IQL + 10% FA + 5% CYT led to 86.40% CPDs blocked (SD 3.65), and 5% IQL + 7.5% FA -2.5% CYT led to 72.40% CPDs blocked (SD 13.05). Legend: CPDs, cyclobutane pyrimidine dimers; UV, ultraviolet; DIO, diosmin; FA, ferulic acid; CYT, cytisine; IQL, isoliquiritigenin.

[0188] Example 4: Clinical Trial Testing Sunscreen on Humans to assess effectiveness of Natural Product sunscreen in preventing DNA damage

[0189] Materials and Methods

[0190] Sunscreen:

[0191] 10% Ferulic Acid

[0192] 10% Diosmin

[0193] 5% Cytisine

[0194] 5% Resveratrol

[0195] In vivo efficacy in blocking UVR was tested.

[0196] 2 mg / cm2 Natural Product sunscreen applied 15 minutes before ultraviolet radiation (“UVR”) (2 MED) exposure

[0197] N = 7 subjects tested to date

[0198] Skin biopsy samples collected immediately post UVR and 4 hr post UVR

[0199] 1. No UVR, No Sunscreen

[0200] 2. UVR + Natural Product sunscreen; biopsy immediately post UVR

[0201] 3. UVR + No Sunscreen; biopsy immediately post UVR

[0202] 4. UVR + Natural Product sunscreen; biopsy 4 hr post UVR

[0203] 5. UVR + No Sunscreen; biopsy 4 hr post UVR

[0204] Each biopsy is bisected and stored for a) DNA purification for cyclobutane pyrimidine dimer (CPD) quantification by ELISA, and b) FFPE prep for histological analysis, i.e. 3-nitrotyrosine immunofluorescence

[0205] Results

[0206] FIG. 6 is a graph showing that the formulation provides broad spectrum coverage, with critical wavelength 371 nm (+ 0.57) and an in vitro SPF of 35.92 ± 9.53, when tested on PMMA HD6 at 0.75 mg / cm2. The USFDA P5 standard (SPF 30) tested at SPF 30.90 ± 4.01 under these same conditions.

[0207] Results shown in FIGs 7 A and 7B compare CPD levels with and without natural product sunscreen, immediately after UVR (7 A) and four hours after UVR (7B). The results show significantly lower levels of CPD with the natural sunscreen.

[0208] Results shown in FIGs 8A and 8B compare levels of UVR induced DNA damaging CPDs immediately after UVR (8A) and four hours after UVR (8B). The results show significantly lower levels of UVR induced DNA damaging CPDs with the natural sunscreen.

[0209] Photographs taken four hours after UVR shows bright red patch where there was no sunscreen, and no change in coloration where the sunscreen was applied.

[0210] Those skilled in the art will recognize, or be able to ascertain, modifications and variations of the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A formulation to reduce ultraviolet (“UV”)-induced cyclobutane pyrimidine dimer (“CPD”) formation comprising a mixture of compounds selected from the group consisting of flavonoids, cinnamic acids, organic fused heterotricyclic compounds, inorganic compounds, coumarins, and acetophenones, having the following properties: having an absorbance index: post UVR absorbance i. 300nm, 340nm, 360nm: pre UVR absorbanceT„r . TTITT-. 1 TTTT it. UVA, UVB, and UV spectrum:more than 0.90; being photostable; not being cytotoxic; and not generating reactive oxygen species.

2. The topical formulation of claim 1 , comprising compounds selected from the group consisting of flavones, chaicones, rrans-cinnamic acids, organic fused heterotricyclic compounds, and inorganic compounds.

3. The topical formulation of claim 1, wherein the mixture comprises flavonols, coumarins, acetophenones, or combinations thereof.

4. The topical formulation of any one of claims 1 to 3, wherein the compounds in the mixture are selected from diosmins, isoliquiritigenins, ferulic acids, cytisines, stilbenoids, and combinations thereof, and optionally from rutins, gossypins, scopoletins, methylxanthoxylin, apiins, 2,4- dihydroxychalcones, and combinations thereof.

5. The topical formulation of any one of claims 1 to 4, wherein the compounds in the mixture are selected from the group consisting of diosmins, isoliquiritigenins, ferulic acids, cytisines, and resveratrol.

6. The topical formulation of any one of claims 1 to 5, wherein the compounds are selected from diosmins, ferulic acids and sinapinic acids, preferably ferulic acids; and cytisines.

7. The topical formulation of claim 1 comprising Ferulic Acid, Diosmin, Cytisine and Resveratrol.

8. The topical formulation of claim 7 comprising 10% Ferulic Acid, 10% Diosmin, 5% Cytisine and 5% Resveratrol.

9. The topical formulation of any one of claims 1-8, wherein each compound has a percent weight from 1% wt / wt to 15% wt / wt, from 2.5% wt / wt to 10% wt / wt, or from 2.5% wt / wt to 15% wt / wt.

10. The topical formulation of claim 9, comprising from 1% wt / wt to 15% wt / wt diosmins, from 1% wt / wt to 15% wt / wt ferulic acids, and 1% wt / wt to 15% wt / wt cytisines, from 1% wt / wt to 15% wt / wt resveratrols, preferably about 10% wt / wt diosmins; about 7.5% wt / wt or about 10% wt / wt ferulic acids; about 5% wt / wt cytisines; and about 5% wt / wt resveratrols.

11. The topical formulation of any one of claims 1 to 10, having a sun protective factor value between 25 and 80, such as between 25 and 70, between 25 and 60, between 25 and 50, or between 40 and 50.

12. The topical formulation of any one of claims 1-11 formulated as a sunscreen.

13. The topical formulation of any one of claims 1-11 formulated as a hair conditioner.

14. The topical formulation of any one of claims 1-11 formulated as a cosmetic.

15. A method of reducing ultraviolet (“UV”)-induced cyclobutane pyrimidine dimer (“CPD”) formation comprising topically applying an effective amount to the skin and / or hair of the topical formulation of any of claims 1-14.

16. The method of claim 15 wherein the topical formulation is a sunscreen applied to the skin.

17. The method of claim 15 wherein the topical formulation is a hair conditioner, shampoo, styling formulation or coloring formulation applied to the hair to reduce sun damage or bleaching of the color of the hair.

18. The method of claim 17 where the hair has been artificially colored.

19. The method of claim 15 wherein the topical formulation is applied to the skin, eyes, or lips, to reduce wrinkles, fine lines, sebaceous hypertrophy, or pigmented spots, or other damage due to aging or sun exposure.

20. A high throughput assay of identifying sunscreen compounds, the assay comprising:(a) providing compounds to be tested in a multi- well tray, and(b) selecting the compounds with absorbance indices of at least 0.90.

21. The assay of claim 20, comprising:(c) measuring ultraviolet (UV) full-spectrum absorbance by measuring absorbance at wavelengths comprising 300, 340 and 360 nm before and after exposure to ultraviolet light, prior to step (b).

22. The assay of claim 20 or 21, comprising:(d) measuring the ultraviolet A (UVA), ultraviolet B (UVB) and UV spectrum prior to step (b).

23. The assay of any one of claims 20-22, wherein the absorbance indices are calculated from: r , post UVR absorbance i. 300nm, 340nm, 360nm: - , pre UVR absorbance ii. UVA,’ UVB,’ and UV sphectrum: - pre UVRAUC , or both (i) and (ii).

24. The assay of any one of claims 20 to 23, comprising screening the compounds for cytotoxicity, as determined by a percent cellular viability count in a bioluminescent adenosine triphosphate release assay, wherein compounds displaying a percent cellular toxicity of less than 10% are selected for further use.

25. The assay of any one of claims 20 to 24, comprising screening the compounds for photostability, as determined by an absorbance index of at least 0.90 and / or screening the compounds for reactive oxygen species generation, antioxidant capacity, or both, wherein: screening the compounds for (i) reactive oxygen species generation is performed via a fluorescent reactive oxygen species probe assay, and (ii) screening the compounds for antioxidant capacity is performed via the same fluorescent reactive oxygen species probe assay as well as colorimetric free radical scavenger assay.

26. The assay of any one of claims 20 to 25, the assay comprising:(a) providing compounds to be tested in a multi- well tray,(b) measuring ultraviolet (UV) full-spectrum absorbance by measuring absorbance at wavelengths comprising 300, 340 and 360 nm before and after exposure to ultraviolet light;(c) measure the ultraviolet A (UVA), ultraviolet B (UVB) and UV spectrum, and(d) calculating the absorbance indices: post UVR absorbance300nm, 340nm, 360nm: pre UVR absorbance, , , ,, , post UVRAUC ii. UVA, UVB, and UV spectrum: - pre UVR AUC(e) selecting the compounds with absorbance indices more than 0.90;(f) screening of these compounds for photostability;(g) screening of these compounds for cytotoxicity; and(h) screening of these compounds for reactive oxygen species generation and antioxidant capacity.

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