PLECTRANTHUS AMBOINICUS extract for use in alleviating radiation-induced skin damage

Abstract

A method for alleviating radiation-induced skin damage (e.g., radiation dermatitis), comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of salvigenin and optionally silymarin, rosmarinic acid, carvacrol, or a combination thereof.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 355,995, filed on June 27, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Plectranthus amboinicus (also previously or alternatively known as Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus aromaticus auct., Plectranthus aromaticus Roxb., Plectranthus aromaticus Benth., and Plectranthus amboinicus (Lour.) Spreng.) is a perennial herb of the Lamiaceae (also known as Labiatae) family that inhabits southern and eastern Africa. Plectranthus amboinicus is also known as patchouli, Cuban oregano, Indian borage, Indian mint, Mexican mint, Mexican oregano, country borage, and Spanish thyme.

[0003] Centella asiatica (also previously or alternatively known as Centella asiatica Urban, Centella asiatica (L.) Urban, Hydrocotyle asiatica L., and Trisanthus cochinchinensis Lour.) is a perennial herb of the family Mackinlayaceae or subfamily Mackinlayoideae of the Apiaceae (also known as Umbelliferae) family, native to Asia, Africa, and South America. Centella asiatica is also known as European water-marvel, gotu kola, Kola, pennywort, Indian pennywort, marsh pennywort, pennyweed, Indian ginseng, Horse-hoof grass, Pegaga, Mandookaparni, Tiger herbal, Spadeleaf, or Tono. Extracts of Centella asiatica generally contain two major compounds: asiaticoside and madecassic acid.

Summary of the Invention

[0004] The present disclosure is at least partially based on the unexpected finding that compositions comprising salvigenin and optionally cirsimaritin, rosmarinic acid, carvacrol, or combinations thereof (e.g., Plectranthus amboinicus (PA) extract optionally combined with Centella asiatica (CA) extract) successfully reduced the disease score of radiation dermatitis and had a therapeutic effect superior to that of the corticosteroid control. Accordingly, the compositions disclosed herein are expected to exhibit excellent efficacy in alleviating radiation dermatitis.

[0005] In some embodiments, provided herein is a method for alleviating radiation-induced skin disorders (e.g., radiation dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin injury, or combinations thereof), the method comprising administering to a subject in need thereof a composition comprising an effective amount of salvigenin and a carrier. In some embodiments, the composition may be a pharmaceutical composition that may further comprise a pharmaceutically acceptable carrier. The compositions disclosed herein may further comprise silymarin, rosmarinic acid, carvacrol, or combinations thereof.

[0006] In some embodiments, the composition comprises a Plectranthus Amboinicus (PA) extract. In some examples, the PA extract can be prepared by a process comprising (i) mixing a portion of PA (e.g., the aerial part) with an extraction solution to produce a first PA extract, (ii) filtering and concentrating the first PA extract to produce a concentrated PA extract, (iii) contacting the concentrated PA extract with a hydrophobic interaction chromatography resin, and (iv) eluting the column with an elution solution to produce a PA extract. In some examples, the extraction solution comprises a solvent having a polarity index of about 2.9 - 6.6, and optionally, the extraction solution is acetone, butyl methyl ether, ethanol, ethyl acetate, isopropyl alcohol, methanol, or a mixture thereof. In some examples, the elution solution comprises a solvent having a polarity index of about 2.1 - 5.4, and optionally, the elution solution comprises a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, and hexane.

[0007] In some embodiments, the composition further comprises asiaticoside. For example, the composition further comprises a Centella asiatica (CA) extract comprising asiaticoside.

[0008] In some embodiments, the subject of treatment by any of the methods disclosed herein is a human patient having any of radiation-induced skin disorders (e.g., radiation dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin injury, or combinations thereof). In some embodiments, the composition is a topical formulation (e.g., in a suitable form such as a cream, gel, dressing, spray formulation, ointment, paste, patch, or lotion). In some embodiments, the composition is applied to the site where the skin disorder occurs. In some cases, the composition is administered to a subject undergoing radiation therapy (e.g., a human cancer patient).

[0009] In some embodiments, any of the compositions disclosed herein can be applied to the subject at least once a day (e.g., up to 6 times a day). Alternatively, the composition can be applied to the subject once every few days (e.g., up to 3 days).

[0010] Also within the scope of the present disclosure are compositions disclosed herein for use in alleviating radiation-induced skin disorders (e.g., those disclosed herein in a subject in need thereof), and compositions for use in the manufacture of a medicament for the intended medical use.

[0011] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and several modes for carrying out the invention, and will also become apparent from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present disclosure. Certain aspects of the present disclosure can be better understood by reference to the drawings in conjunction with the detailed description of specific modes for carrying out the invention presented herein.

[0013]

Figure 1

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Figures 5A - 5C

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Figures 7A - 7D

Mode for Carrying Out the Invention

[0014] Radiation can induce skin damage. Radiation dermatitis, also known as X-ray dermatitis, is a common side effect of radiation therapy. Radiation dermatitis is accompanied by damage to skin cells caused by radiation. Typical symptoms include redness, skin peeling, and ulcers. It usually begins to disappear after the end of treatment. In some cases, it may appear several years after the completion of radiation therapy. In patients with radiation dermatitis, skin cell damage can occur deep in the skin tissue without obvious open surface wounds. Instead of necrosis or apoptosis, it can modify the cell structure around the damaged area.

[0015] Unexpectedly, it is reported herein that PA extracts (e.g., containing salvigenin and optionally silimaritin, rosmarinic acid, carvacrol, or combinations thereof), alone or in combination with CA extracts (e.g., containing asiaticoside), as observed in animal models, have shown high therapeutic effects in the treatment of radiation dermatitis. Accordingly, provided herein is a pharmaceutical composition comprising a Plectranthus amboinicus extract or an active agent thereof, and optionally a Centella asiatica extract or an active agent thereof, for use in alleviating radiation dermatitis.

[0016] I. Composition The present disclosure herein provides compositions such as pharmaceutical compositions for alleviating radiation-induced skin disorders, such as radiation-induced dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin damage, or combinations thereof. The compositions disclosed herein may comprise a PA extract or one or more active agents contained therein (e.g., salvigenin, and optionally silimaritin, rosmarinic acid, carvacrol, or combinations thereof). In some embodiments, the pharmaceutical composition may further comprise a CA extract or one or more active agents contained therein (e.g., asiaticoside).

[0017] (i) Active agent The active agent in the compositions described herein includes salvigenin. In some embodiments, the active agent may further include silymarin, rosmarinic acid, carvacrol, or combinations thereof. In some cases, the composition includes a PA extract that includes salvigenin and optionally one or more of silymarin, rosmarinic acid, and carvacrol.

[0018] Plectranthus amboinicus extract The PA extract refers to an extract obtained from a PA plant using one or more suitable solvents. In some examples, the PA extract is prepared using the aerial parts of the PA plant. In some cases, at least one of the solvents used to prepare the extract has a polarity index of less than 7 (e.g., less than 5). See, for example, U.S. Patent No. 10,758,584, the relevant disclosures of which are incorporated by reference herein for the subject matter and purposes relevant hereto. As used herein, a solvent refers to a substance or mixture of substances that dissolves another to form a solution. The PA extract as described herein can be prepared using one solvent. The solvent used in each extraction step for preparing the extracts described herein (including both PA and CA extracts) can be a single solvent. Alternatively, it can be a mixture of two or more solvents.

[0019] The PA extract described herein can include terpenoids (e.g., monoterpenoids, diterpenoids, triterpenoids, and / or sesquiterpenoids), flavonoids, phenols, essential oils, or combinations thereof. The PA extract for making the pharmaceutical compositions disclosed herein can include salvigenin and optionally one or more of silymarin, rosmarinic acid, and carvacrol.

[0020] The PA extract described in this specification can be prepared by extracting the whole PA plant or a part thereof (e.g., the above-ground part) with one or more suitable solvents to form a solution, and then drying the solution to produce the PA extract. Since the PA extract contains flavonoids, terpenoids (e.g., monoterpenoids, diterpenoids, triterpenoids, and / or sesquiterpenoids), phenols, or essential oils, which are non-polar molecules, at least one of the extraction solvents can have a relatively low polarity (e.g., having a polarity index of less than 7) to facilitate the dissolution of non-polar molecules. "Extraction" can be carried out by bringing the PA material into direct contact with a suitable solvent or by eluting the active components of PA from the resin to which the active ingredients are attached.

[0021] In some examples, solvents having a polarity index of less than 7 can be used to extract the active components from PA and produce the PA extract. Such solvents can be ethyl acetate, methyl acetate, propanol, butanol, or chloroform. Alternatively, the solvent can be a mixture of one or more solvents having different polarity indices. Examples include, but are not limited to, mixtures of ethanol and ethyl acetate, ethyl acetate and butanol, ethanol and propanol, and methyl acetate and butanol.

[0022] In some embodiments, the PA extract can be prepared by a process involving the use of one solvent, such as a solvent having a polarity index of less than 7 (e.g., less than about 6.5, less than about 6.0, less than about 5.5, less than about 5.0, less than 4.9, less than 4.8, less than 4.7, less than 4.6, or less than 4.5). Examples include, but are not limited to, methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or combinations thereof.

[0023] The PA material can be the whole of the PA or a part thereof (e.g., the above-ground part such as leaves), and can be prepared by ordinary methodologies. The PA material can be a fresh plant or a part thereof. Alternatively, the PA material may be in a dried form. The PA can optionally be dried to form a powder, which can be used as the PA material for preparing the PA extract.

[0024] Any of the PA materials described herein can be extracted one or more times with a solvent suitable for producing a crude extract. The solvent for use in preparing the crude extract can be, for example, a highly polar solvent having a polarity index greater than 5 and preferably less than 7 (e.g., greater than 5.2, greater than 5.5, greater than 5.8, 6 or more, preferably less than 7). Examples include, but are not limited to, ethanol, acetone, methanol, water, or combinations thereof. Optionally, the crude extract can be concentrated by conventional methods to produce a concentrated crude extract.

[0025] The crude extract can then be contacted with a suitable resin (e.g., a nonionic absorbent resin) under suitable conditions that allow the active constituents in the crude extract to bind to the resin. Exemplary resins for use in preparing the Plectranthus amboinicus extract include, but are not limited to, DIAION® HP20, DIAION® HP20SS, Sepabeads® SP207, Amberlite™ XAD-2, or Amberlite™ XAD-4.

[0026] Thereafter, the resin can be washed one or more times and eluted with a suitable solvent, e.g., a solvent having a polarity index less than 7, to produce a PA extract, which can then be dried by conventional methods (e.g., freeze-drying, spray-drying, or concentration drying) to produce a dried PA extract that can be in a semi-solid or paste form.

[0027] In some examples, the resin absorption step can be performed by mixing the crude extract with the resin in a container. In other embodiments, the resin separation step can be performed by a chromatography column setup.

[0028] In one example, the extract of PA can be prepared as follows. The above-ground part of PA (about 1.5 g) containing leaves and / or stems can be collected and extracted with a solvent having a polarity less than 7 (e.g., methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or a combination thereof) at room temperature for 30 minutes to 6 hours. Alternatively, this extraction process can be carried out at a temperature of about 50 to 80 °C. The obtained crude extract can be directly packed into a non-ionic adsorptive resin column and eluted with a solvent having a polarity less than 6 (e.g., ethanol, ethyl acetate, butanol, dichloromethane, hexane, toluene, or a combination thereof). The eluted components can be collected and purified by extraction with a solvent having a polarity less than 6 (e.g., the solvents described herein). The obtained filtrate can be collected to produce a PA extract.

[0029] Centella asiatica extract In some embodiments, compositions such as the pharmaceutical compositions disclosed herein may further contain asiaticoside. In some cases, the composition may further contain a CA extract containing asiaticoside compounds. The CA extract described herein refers to an extract obtained from the whole CA plant or a part thereof. The CA extract may contain asiaticoside and optionally madecassic acid.

[0030] The CA extract can be prepared according to conventional methods, for example, the methods described in U.S. Patent Nos. 5,834,437, 6,417,349, 6,475,536, and 6,267,996, CN1313124, CN1089497, and CN1194154. The following is an example thereof.

[0031] CA materials can be prepared through daily practice. Such materials can be fresh CA plants or parts thereof, or dried CA. CA materials can be extracted with a suitable solvent such as water, ethanol, or a mixture thereof to produce a crude extract. The crude extract, which can optionally be concentrated, can be mixed with a suitable resin or filled into a column filled with resin. After washing one or more times, the resin can be eluted with a suitable solvent. The resulting eluate solution can be concentrated to form a paste, which can be dried by conventional methods, such as vacuum drying, to produce a powder of the CA extract. If necessary, the CA powder can be pulverized through a mesh (e.g., No. 100 mesh).

[0032] The extract of CA can be prepared by the same or similar process as described above for preparing the PA extract. The PA extract and / or the CA extract may be concentrated using a rotary evaporator under reduced pressure.

[0033] (ii) Pharmaceutical composition Any of the active compounds (e.g., salvigenin, and optionally one or more of silymaritin, rosmarinic acid, carvacrol, and / or asiaticoside), the PA extract can optionally be combined with the CA extract disclosed herein and mixed with a suitable carrier (e.g., a pharmaceutically acceptable carrier) to form a composition (e.g., a pharmaceutical composition) for use in alleviating radiation-induced skin disorders such as radiation-induced dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin damage, or combinations thereof. "Acceptable" means that the carrier must be compatible with the active ingredients of the composition (preferably, it can stabilize the active ingredients) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0034] Compositions such as the pharmaceutical compositions used in this method can be in the form of lyophilized preparations or aqueous solutions and can include pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, benzoate, sorbate, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, serine, alanine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0035] In some examples, the pharmaceutical compositions described herein may contain liposomes containing an active agent, prepared by methods known in the art or as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation times are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced from a lipid composition comprising phosphatidylcholine, cholesterol, and a PEG-derivatized phosphatidylethanolamine (PEG-PE) by the reverse phase evaporation method. The liposomes are extruded through a filter of a certain pore size to obtain liposomes having a desired diameter.

[0036] The active agent may also be encapsulated, for example, in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxyethylmethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, or may be encapsulated in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or may be encapsulated in a macroemulsion. Such techniques are known in the art; see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0037] In other examples, the pharmaceutical compositions described herein can be formulated in a sustained release format. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the active agent in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0038] Compositions such as pharmaceutical compositions for in vivo administration must be sterile. This can be readily accomplished, for example, by filtration through sterile filtration membranes. The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0039] When preparing solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, such as conventional tableting ingredients like corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum, and other pharmaceutical diluents such as water to form a solid preliminary preparation composition containing a homogeneous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preliminary preparation compositions being homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily divided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preliminary preparation composition is then divided into unit dosage forms of the above type containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition can be coated or otherwise formulated to provide a dosage form that offers the advantage of sustained action. For example, a tablet or pill may contain an inner dosage component and an outer dosage component, with the latter in a form that encapsulates the former. The two components may serve to resist disintegration in the stomach and may be separated by an enteric layer that allows the inner component to pass intact into the duodenum or delays its release. A variety of materials can be used for such an enteric layer or coating, and such materials include a number of polymeric acids, and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0040] Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85), and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions containing a surfactant will advantageously contain from 0.05 to 5% surfactant, and can be from 0.1 to 2.5%. It will be understood that other components, such as mannitol or other pharmaceutically acceptable vehicles, can be added if desired.

[0041] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient may be dissolved in a pre-mixed emulsion composition, or alternatively, the active ingredient may be dissolved in an oil (such as soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil), and may be dissolved in an emulsion formed upon mixing with a phospholipid (such as egg phospholipid, soybean phospholipid, or soybean lecithin) and water. It will be appreciated that other ingredients such as glycerol or glucose may be added to adjust the osmotic pressure of the emulsion. Suitable emulsions will typically contain up to 20% oil, such as 5 - 20% oil. The fat emulsion may contain fat droplets of 0.1 - 1.0 μm, particularly 0.1 - 0.5 μm, and can have a pH in the range of 5.5 - 8.0.

[0042] The emulsion composition can be one prepared by mixing the active agent with Intralipid™ or its components (soybean oil, egg phospholipid, glycerol, and water).

[0043] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions can contain the above-described suitable pharmaceutically acceptable excipients. In some embodiments, the composition is administered by the oral or nasal respiratory route for local or systemic effects.

[0044] The composition in a pharmaceutically acceptable solvent, preferably sterile, can be nebulized by the use of a gas. The nebulized solution may be breathed directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing apparatus. The solution, suspension, or powder composition can be administered preferably orally or nasally from a device that delivers the formulation in a suitable manner.

[0045] Topical preparation In some embodiments, the pharmaceutical compositions disclosed herein are topical preparations. Such topical preparations may include any of the PA extracts disclosed herein, or one or more of their active agents (e.g., salvigenin, and optionally, one or more combinations of silymaritin, rosmarinic acid, carvacrol). In some cases, the topical preparation may further include the CA extract disclosed herein, or one or more of its active agents (e.g., asiaticoside). In some examples, the topical preparations described herein may include salvigenin in an amount in the range of about 0.0001% to about 0.5% (w / w), and optionally, asiaticoside in an amount in the range of about 0.05% to about 5% (w / w). In some embodiments, the topical preparation includes Plectranthus amboinicus extract, Centella asiatica extract, or both in an amount of about 0.1 to 30% (w / w).

[0046] As used herein, the term "about" is not intended to denote a precise numerical boundary for the specified parameter (including both the upper and lower limits). One of ordinary skill in the art would have understood the meaning of "about" in the context of a particular application. In some cases, the term "about" refers to a particular value + / - 5% (e.g., + / - 3% or + / - 2%).

[0047] In some cases, the topical preparation may further include one or more carriers or excipients, including one or more of a thickening agent (e.g., about 1.0 to 10%), one or more ointment bases (e.g., one or more cream bases) that can be in the range of about 5 to 30%, one or more antimicrobial preservatives (e.g., about 0.005 to 0.2% by weight), one or more emulsifiers (about 0.5 to 10% by weight), or combinations thereof. These components can be dissolved or dispersed in a suitable solvent.

[0048] A "thickening agent" is a drug used to increase the viscosity of a formulation. Exemplary thickening agents include, for example, cetostearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or combinations thereof. The thickening agent may be present in the topical formulation at a concentration of about 1.0 to 10% (w / w). For example, the topical formulation may contain about 1 to 1.5%, 1.5 to 2.5%, 2 to 2.5%, 2.5 to 3%, 3 to 3.5%, 3.5 to 4%, 4 to 4.5%, 4.5 to 5%, 5 to 5.5%, 5.5 to 6%, 6 to 6.5%, 6.5 to 7%, 7 to 7.5%, 7.5 to 8%, 8 to 8.5%, 8.5 to 9%, 9 to 9.5%, or 9.5 to 10% (w / w) of the thickening agent. Alternatively, the topical formulation may contain about 1 to 5%, 2.5 to 7.5%, or 5 to 10% (w / w) of the thickening agent.

[0049] "Ointment base" can be any semi-solid preparation or vehicle into which an active agent can be incorporated. Exemplary ointment bases include, but are not limited to, oily ointment bases (e.g., white petrolatum or white ointment), absorbent ointment bases (e.g., hydrophilic petrolatum, anhydrous lanolin, Aquabase™, Aquaphor®, and Polysorb®), water / oil emulsion ointment bases (e.g., cold cream, hydrous lanolin, rose water ointment, Hydrocream™, Eucerin®, and Nivea®), oil / water emulsion ointment bases (e.g., hydrophilic ointment, Dermabase™, Velvachol®, and Unibase®), and water-miscible ointment bases (e.g., polyethylene glycol (PEG) ointment and Polybase™). The ointment base can be pharmacologically inert but can capture water to provide a skin-softening protective film. In certain embodiments, the ointment base can be any petrolatum compound (e.g., petrolatum, white petrolatum, white soft paraffin, liquid petrolatum, liquid paraffin). In a further particular embodiment, the ointment base is white petrolatum (CAS number 8009-03-8). The ointment base can be present in the topical formulation at a concentration of about 5-30% (w / w), for example, 10-30% (w / w). For example, the topical formulation can contain about 5-25%, 5-20%, 5-15%, 5-15%, 10-15%, 15-20%, 20-25%, or 25-30% (w / w) of the ointment base. Specifically, the topical formulation can contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 percent (w / w) of the ointment base.

[0050] In some embodiments, the "ointment base" described herein contains less than 20% water and volatile substances, and more than 50% hydrocarbons, waxes, or polyols as the vehicle.

[0051] In some embodiments, the "ointment base" described herein is a "cream base" that contains more than 20% water and volatile substances and / or typically contains less than 50% hydrocarbon, wax, or polyol as a vehicle for the active ingredient. The cream base may be a multiphase preparation containing a lipophilic phase and an aqueous phase. In some cases, the cream base is a lipophilic cream base having a lipophilic phase as the continuous phase. Such cream bases usually contain water-in-oil emulsifiers such as wool alcohol, sorbitan esters, and monoglycerides. In other cases, the cream base is a hydrophilic cream base having an aqueous phase as the continuous phase. Such cream bases typically contain oil-in-water emulsifiers such as sodium or trolamine soaps, sulfated fatty alcohols, polysorbates, and polyoxyl fatty acid and fatty alcohol esters, which may be combined with water-in-oil emulsifiers as necessary.

[0052] An "antimicrobial preservative" can be any compound that can destroy microorganisms, prevent the reproduction or growth of microorganisms, or prevent the pathogenic action of microorganisms. Exemplary antimicrobial preservatives include, but are not limited to, parabens (esters of para-hydroxybenzoic acid, such as paraben, methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, benzylparaben, isobutylparaben, isopropylparaben, benzylparaben, or their sodium salts), benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. The antimicrobial preservative can be present in topical formulations at a concentration of about 0.005 to 0.2%, for example, about 0.01 to 0.2% (w / w). For example, topical formulations can contain about 0.005 to 0.01%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.15%, or 0.15 to 0.2% (w / w) of the antimicrobial preservative. Specifically, topical formulations can contain about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 percent (w / w) of the antimicrobial preservative.

[0053] An "emulsifier" is a compound or substance that acts as a stabilizer for a mixture of two or more liquids that are normally immiscible (incapable of mixing or blending). Exemplary emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, propylene glycol monostearate, and polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span® 65], glyceryl monooleate, and sorbitan monooleate [Span® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and soltrol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g.,Polyoxyethylene lauryl ether (Brij®), and poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, and sodium doxsate, and / or combinations thereof may be mentioned. The emulsifier may be present in topical formulations at a concentration of about 0.5 to 10% (w / w), for example, 0.5 to 6% (w / w). For example, topical formulations may contain about 0.5 to 1%, 1 to 1.5%, 1.5 to 2%, 2 to 2.5%, 2.5 to 3%, 3 to 3.5%, 3.5 to 4%, 4 to 4.5%, 4.5 to 5%, 5 to 5.5%, 5.5 to 6%, 5 to 10%, 6 to 10%, or 8 to 10% (w / w) of the emulsifier. Specifically, topical formulations may contain about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 percent (w / w) of the emulsifier.,

[0054] The topical formulations of the present invention may further contain one or more solvents (e.g., non-aqueous solvents or water). Exemplary non-aqueous solvents include, but are not limited to, any known solvents including propylene glycol, glycols, and mixtures thereof. The non-aqueous solvent may be present in topical formulations at a concentration of about 2 to 65% (w / w). For example, topical formulations may contain about 2 to 15%, 15 to 30%, 30 to 45%, or 45 to 65% (weight / weight) of the solvent. In some embodiments, the topical formulations of the present invention may also contain water.

[0055] In some embodiments, the topical formulations of the present invention may further contain one or more skin softeners, fragrances, or pigments. The topical formulations may also be used in combination with wound dressings (e.g., bandages with adhesives, plasters, etc.).

[0056] (e.g., cyclohexane, n - hexane, n - decane, i - octane, octane, butyl ether, carbon tetrachloride, triethylamine, i - propyl ether, toluene, p - xylene, t - butyl methyl ether, benzene, benzyl ether, dichloromethane, methylene chloride, chloroform, dichloroethane, ethylene dichloride, 1 - butanol, i - butyl alcohol, tetrahydrofuran, ethyl acetate, 1 - propanol, 2 - propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzonitrile, 1,4 - dioxane, or p - dioxane).

[0057] Additional information regarding topical formulations can be found, for example, in U.S. Patent No. 10,758,584, and these related disclosures are incorporated by reference for the subject matter and purposes relevant herein.

[0058] The topical formulations disclosed herein can be in any suitable form. Examples include, but are not limited to, creams, coatings, ointments, lotions, pastes, and gels. In some cases, the topical formulation may be disposed on a device (e.g., a patch) for application to the site where the skin disorder occurs.

[0059] II. Method for Alleviating Radiation - Induced Skin Disorders To practice the methods disclosed herein, an effective amount of the composition described herein (e.g., a pharmaceutical composition) is administered to a subject (e.g., a human) in need of treatment via a suitable route, e.g., via intravenous administration, e.g., as a bolus, or via continuous infusion over a period of time by intramuscular, intraperitoneal, intrathecal, subcutaneous, intra - articular, intrasynovial, intratracheal, oral, inhalation, or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. The liquid formulation can be sprayed directly, and the lyophilized powder can be sprayed after reconstitution.

[0060] In some cases, the topical formations described herein can be used to alleviate radiation-induced skin diseases in a subject in need of treatment. Exemplary radiation-induced skin disorders include, but are not limited to, radiation dermatitis (e.g., acute or chronic), radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin injury, or combinations thereof.

[0061] The topical formulation can be applied to the wound site according to a suitable dosage and treatment plan. The dosage and dosing schedule of the methods described will depend on the nature and condition of the symptoms being treated, the age and condition of the patient, and any previous or concurrent treatments. In some cases, the topical formulation can be applied once a week, once every two days, once a day, twice a day, three times a day, or four times a day over a suitable period. Treatment can be terminated when the radiation-induced skin disorder has healed. If necessary, for example, if one or more symptoms of the skin disorder recur, treatment can be restarted.

[0062] The term "radiation-induced skin disorder" refers to any skin disorder associated with exposure to radiation. Examples include radiation dermatitis (e.g., acute or chronic), radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin injury, or combinations thereof.

[0063] The term "radiation dermatitis", also known as radiation burn, refers to dermatitis (e.g., skin irritation) associated with radiation. In some cases, radiation dermatitis is associated with radiation therapy received by a subject, e.g., a human cancer patient undergoing anti-cancer radiation therapy (e.g., X-ray therapy). Common symptoms associated with radiation dermatitis include redness, itching, peeling, flaking, pain, moisture, swelling, pigmentation changes, fibrosis, scarring of connective tissue, and the development of ulcers. The grades of radiation dermatitis are provided in Table 1 below according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v5.0 guidance.

Table 1

[0064] The subject to be treated by the compositions disclosed herein (e.g., any of the topical formulations disclosed herein) can be a human or non-human mammal. In some embodiments, the subject is a human patient having, suspected of having, or at risk of developing a skin disorder, such as a radiation-induced skin disorder (e.g., radiation dermatitis). In some cases, the subject can be a human patient who has received or is receiving radiation therapy (e.g., a human cancer patient). In some examples, treatment of a radiation-induced skin disorder and radiation therapy with any of the compositions disclosed herein can be performed simultaneously on a subject in need thereof.

[0065] Subjects having a radiation-induced skin disorder as disclosed herein can be identified by routine health examinations. In some cases, subjects having radiation dermatitis can exhibit one or more of the symptoms disclosed herein. In other cases, subjects having radiation dermatitis can suffer skin cell damage in deep skin layers with little or no surface wounds. Subjects suspected of having radiation dermatitis may exhibit one or more symptoms of the disease / disorder. Subjects at risk of radiation dermatitis can be subjects having one or more of the risk factors for that disease / disorder (e.g., a human patient who is undergoing or is at risk of being exposed to radiation therapy).

[0066] As used herein, "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. It will be apparent to those skilled in the art whether the amount of the composition has achieved a therapeutic effect. The effective amount will vary depending on parameters of the individual patient including the particular condition being treated, the severity of the condition, age, health status, size, gender, and weight, the duration of the treatment, the nature of concurrent therapy, if any, the particular route of administration, and like factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed by conventional experimental methods. Generally, the maximum dosage of the individual components or combinations thereof, i.e., the highest safe dosage by sound medical judgment, is preferably used.

[0067] Empirical considerations such as half-life will generally contribute to the determination of dosage. The dosing frequency can be determined and adjusted over the course of the therapy and generally depends on, but is not necessarily limited to, the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, a sustained continuous release formulation of the composition may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0068] In one example, the dosage of the composition described herein can be determined empirically in an individual who has received one or more administrations of the composition. The individual is given escalating doses of the agonist. To assess the efficacy of the agonist, indicators of the disease / disorder can be followed.

[0069] Generally, for the administration of any of the compositions described herein, an initial candidate dosage can be administered to a subject. For repeated administration over several days or more, depending on the condition, the treatment is continued until the desired suppression of symptoms occurs or until a sufficient treatment level is achieved to alleviate the target disease or disorder or its symptoms. Specific dosing regimens, i.e., dosage, timing, and repetition, will depend on the particular individual and the individual's medical history, as well as the characteristics of the individual agent (e.g., the half-life of the agent and other considerations well known in the art).

[0070] For the purposes of the present disclosure, the appropriate dosage of the compositions described herein will depend on the particular active agent contained therein, the type and severity of the disease / disorder, whether the composition is administered for preventive or therapeutic purposes, the patient's medical history and response to treatment, and the discretion of the attending physician. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in the severity of the disease (e.g., as represented by a disease score). The method of determining whether the dosage has resulted in the desired result will be apparent to those of skill in the art. Administration of any of the compositions disclosed herein can be continuous or intermittent, depending, for example, on the recipient's physiological state, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the composition can be essentially continuous over a preselected period of time or in a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0071] As used herein, the term "treating" refers to applying or administering to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease or disorder, a composition comprising one or more active agents for the purpose of curing, treating, alleviating, reducing, altering, healing, remitting, improving, or affecting the disorder, the symptoms of the disease, or the predisposition to the disease or disorder.

[0072] Relieving a target disease or disorder includes delaying the onset or progression of the disease, reducing the severity of the disease, or increasing the survival period. Relieving a disease or increasing the survival period does not necessarily require a cure outcome. As used herein, "delaying" the onset of a target disease or disorder means postponing, preventing, slowing, retarding, stabilizing, and / or extending the progression of the disease. This delay can be of various lengths depending on the disease history and / or the individual being treated. A method of "delaying" or relieving the onset of a disease or delaying the development of a disease is a method that reduces the probability of developing one or more symptoms of the disease and / or reduces the degree of the symptoms within a given time frame when compared to not using the method. Such a comparison is typically based on a clinical study using a sufficient number of subjects to obtain statistically significant results.

[0073] "Onset" or "progression" of a disease means the initial signs of the disease and / or its subsequent progression. The onset of a disease can be detectable and evaluable using standard clinical techniques well known in the art. However, onset also refers to progression that may not be detectable. For the purposes of this disclosure, onset or progression refers to the biological course of the symptoms. "Onset" includes occurrence, recurrence, and development. As used herein, "development" or "occurrence" of a target disease or disorder includes initial onset and / or recurrence.

[0074] Conventional methods known to those of ordinary skill in the medical field can be used to administer a pharmaceutical composition to a subject, depending on the type of disease being treated or the site of the disease. This composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, the composition can be administered to the subject via an injectable depot administration route, for example, using 1, 3, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intravitreally or intravitreally.

[0075] The specific dosing regimens used in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the medical history of that subject.

[0076] In some embodiments, any of the compositions disclosed herein can be co-administered with one or more additional therapeutic agents for treating the target disease. In some cases, the additional therapeutic agent can help enhance and / or complement the effectiveness of the compositions disclosed herein.

[0077] The therapeutic efficacy for the target disease / disorder can be evaluated by methods well known in the art.

[0078] III. Kit for Use in Alleviating Radiation-Induced Skin Disorders The present disclosure also provides a kit for use in alleviating radiation-induced skin disorders such as radiation dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin damage, or combinations thereof. Such a kit may include one or more containers containing any of the compositions (e.g., topical formulations) described herein that include one or more of their active agents such as Plectranthus amboinicus extract, Centella asiatica extract, a combination of Plectranthus amboinicus extract and Centella asiatica extract, or those disclosed herein.

[0079] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The instructions included can include instructions for administration of a composition (e.g., topical formulation) for alleviating a radiation-induced skin disorder such as those disclosed herein according to any of the methods described herein. The kit can further include instructions regarding selection of a suitable individual for treatment based on whether the individual has a skin disorder for which treatment is needed.

[0080] Instructions regarding use of the composition (e.g., topical formulation) generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The instructions provided within the kit of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included within the kit), but machine-readable instructions (e.g., instructions held on a magnetic or optical storage disk) are also acceptable.

[0081] The label or package insert indicates that the composition is used for alleviating a radiation-induced skin disorder as disclosed herein. Instructions for carrying out any of the methods described herein can be provided.

[0082] The kit of the present invention is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. At least one active agent in the composition is an active agent disclosed herein and may be included in Plectranthus amboinicus extract, Centella asiatica extract, or a combination of Plectranthus amboinicus extract and Centella asiatica extract.

[0083] The kit may optionally provide additional components such as interpretive information. Usually, the kit includes a container and a label or an accompanying document on or associated with the container. In some embodiments, the present invention provides a manufactured article comprising the contents of the above kit.

[0084] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the above description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way. All publications described herein are incorporated by reference for the purposes for which they are cited herein or for the subject matter.

[0085] Example 1: Alleviation of radiation dermatitis using Plectranthus Amboinicus (PA) extract, optionally in combination with Centella Asiatica (CA) extract This example explores the therapeutic efficacy of radiation dermatitis using (a) ON101 cream containing Plectranthus amboinicus (PA) extract and Centella asiatica extract, and (b) PA-F4 cream containing Plectranthus amboinicus (PA) extract. A topical corticosteroid cream (betamethasone) is used as a positive control.

[0086] Figure 1 shows the overall experimental design of a mouse radiation study to examine the therapeutic effects of ON101 cream and PA-F4 cream. Briefly, mice were shaved to remove dorsal hair, anesthetized, and then irradiated at the shaved site with a dose of 6.5 Gy once a day for 5 days (days 0 to 4). ON101 cream, PA-F4 cream, or a topical corticosteroid cream (control) was applied to the irradiated site. Radiation-induced dermatitis was evaluated twice a week from day 8 to day 28. 1 ml of cream was applied once a day for 5 days to each of the 5 cm 2 shaved areas, followed by a 2-day rest period. This cycle was repeated 4 times. See Figure 1. During this period, the level of radiation dermatitis was determined according to the definitions provided in Table 2 below. [Table 2]

[0087] For example, skin tissue samples were collected for the analysis of radiation exposure biomarkers via single-cell RNA sequencing (scRNA-seq).

[0088] As shown in Figure 2, both ON101 cream and PA-F4 cream showed a significant reduction in the radiation dermatitis score, and the therapeutic effect was better than that of the control cream.

[0089] Example 2: Therapeutic efficacy of PA extract on radiation-induced skin damage, optionally combined with CA extract This example evaluates the therapeutic effect of PA extract on radiation-induced skin damage, either alone or in combination with the CA extract disclosed herein. The treatment process is described in Example 1 above. Also, see Figure 1.

[0090] (i) Epidermal thickness The epidermal thickness in mice treated with ON101 cream, PA-F4 cream, corticosteroid (positive control), placebo cream (negative control), and untreated mice (blank control) was measured by hematoxylin and eosin (H&E) staining. Tissue sections obtained from the mice were immersed in distilled water, and alum hematoxylin was applied to stain the nuclei. Subsequently, the tissue sections were washed by rinsing under running tap water. 0.3% acid alcohol was used for differentiation. The tissue sections were rinsed again under running tap water, then immersed in Scott's tap water for further washing, and subsequently rinsed with tap water. The tissue sections were then stained with eosin solution for 2 minutes, dehydrated, cleared, and mounted.

[0091] The results indicate that both ON101 and PA-F4 reduce the epidermal thickness, with ON101 showing a better effect. Figure 3.

[0092] (ii) Fibrosis content The fibrosis content of the mice at the sites treated with radiation was analyzed by Masson staining. Masson's trichrome staining is a method widely used in histology for the identification and differentiation of collagen fibers and muscle fibers in tissue sections. This staining technique is particularly useful when studying various pathological conditions that affect organs. To perform Masson's trichrome staining, the following steps were carried out.

[0093] First, skin samples obtained from mice in various treatment groups were subjected to deparaffinization and rehydration by sequentially passing them through 100% alcohol, 95% alcohol, and 70% alcohol. This process ensures the removal of paraffin wax and enables the samples to be properly prepared for staining. Subsequently, the samples were thoroughly washed with distilled water to remove residual chemicals or debris and ensure a clean surface for staining.

[0094] Next, the processed skin samples were incubated in a Weigert's iron hematoxylin working solution for 10 minutes. This step facilitates the visualization of cell nuclei. Following the staining step, the samples were rinsed with warm running tap water for approximately 10 minutes. This washing process effectively removes excess dirt and enhances the transparency of the stained areas. After rinsing, the samples were washed again with distilled water to ensure the removal of any remaining traces of the staining solution.

[0095] Finally, the samples were stained with the Biebrich scarlet acid fuchsin solution for 10 - 15 minutes. This staining solution imparts distinct coloring to collagen fibers and enables differentiation from other tissue components.

[0096] As shown in Figure 4, skin samples from mice treated with PA - F4 cream and ON101 cream showed a reduction in fibrosis content compared to those treated with placebo cream or untreated mice.

[0097] (iii) Immunohistochemical analysis Immunohistochemical analysis was performed to examine the pathways related to oxidative stress, radiation damage, and inflammation in mice treated with various creams as described above. This experiment involved classifying mouse skin tissue samples obtained from mice treated with ON101, PA - F4, mock, or placebo cream. Immunostaining analysis was carried out to examine the levels of NLRP3, rH2AX, and IL1B via the immunohistochemistry (IHC) process as follows.

[0098] First, the slides were washed three times with PBS. Then, 600 μl of peroxidase block solution was added to the slides and incubated for 10 minutes. After washing with PBS once more, the slides were blocked in 2% skim milk for 15 minutes. Next, mouse anti-NLRP3, rH2AX, or IL1B (1:500 dilution) was applied to the slides and incubated at 4°C for 20 hours. After three washes, Dako HRP-labeled anti-mouse (1:5000) was added, and the slides were incubated for 30 minutes followed by another three washes. Then, substrate containing chromogen (one drop of chromogen per 1 ml of substrate) was added to the slides and incubated for 1 - 15 minutes. The slides were washed three times with PBS, and then counterstained with Mayer's hematoxylin for 12 seconds. After rinsing with tap water, the slides were dried.

[0099] The levels of NLRP3, rH2AX, and IL1B in mouse skin samples were summarized in Figures 5A - 5C, respectively. Mice treated with ON101 cream and PA-FA cream showed significantly lower levels of NLRP3, rH2AX, and IL1B in the skin tissue compared to untreated mice or mice treated with placebo cream.

[0100] (iv) Potential pathways for the protection of ON101 against radiation dermatitis In this study, RNA sequencing analysis was performed on mouse tissue samples affected by radiation dermatitis to investigate the molecular changes occurring in the skin tissue during radiation dermatitis and to investigate the pathways through which ON101 might exert its protective effect.

[0101] RNA transcripts collected from mouse skin samples were analyzed using high-throughput RNA sequencing technology. This technique enables a comprehensive view of gene expression changes that occur in the skin during radiation dermatitis, regardless of the presence or absence of ON101 treatment. The results of the RNA sequencing analysis can be used to identify differentially expressed genes and pathways associated with the onset and progression of radiation dermatitis. By comparing the gene expression profiles of samples obtained from mice treated with ON101 to those obtained from untreated mice, specific genes and pathways regulated by ON101 treatment can be deciphered.

[0102] Furthermore, RT-qPCR analysis was performed using the miDETECT A Track™ RNA qRT-PCR Starter Kit (RIBOBIO, Guangzhou, Taiwan) to investigate the levels of RNA transcripts. This process involved converting miRNAs into complementary DNA (cDNA) using the PrimeScript® First Strand cDNA Synthesis Kit (TaKaRa, Taipei, Taiwan). To quantify gene expression levels, qRT-PCR was performed using the SYBR Premix Ex Taq II Kit (Takara, Taipei, Taiwan). The qRT-PCR analysis was conducted on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). By utilizing these molecular biology assays and kits, miRNA and gene expression levels associated with the Nrf2-rH2AX axis were measured and analyzed.

[0103] The results of this study indicate that genes such as KEAP1, IL18, NK-κB, NLRP3, EGFR, VIM, and CTNNB1 were differentially expressed in samples from mice treated with ON101 compared to samples from untreated mice. More specifically, ON101 decreased the expression levels of KEAP1, IL18, NK-κB, NLRP3, and IL1B and increased the expression levels of EGFR, VIM, and CTNNB1. Figure 6. These results suggest that by targeting Keap1, a protein known to play a decisive role in the oxidative stress response and inflammation, ON101 can alleviate the onset and severity of radiation dermatitis.

[0104] (v) Activation of the p62-KEAP1-NRF2 pathway and promotion of Nrf2 nuclear translocation in mice with radiation dermatitis In this study, ON101 and PA-F4 were found to activate the p62-KEAP1-NRF2 pathway and promote Nrf2 nuclear translocation in mice with radiation dermatitis.

[0105] Protein lysates prepared from skin samples collected from mice treated with ON101, PA-F4, or placebo cream, and untreated mice, were heat-treated and prepared for analysis. To perform immunoblotting, a 5% non-fat milk solution in Tris-buffered saline with Tween® 20 (TBST) was used according to conventional immunoblotting practice. Briefly, the proteins in the protein lysate samples were transferred to a polyvinylidene fluoride (PVDF) membrane and then incubated overnight at 4 °C with primary antibodies specific for the target proteins (Keap1, p62, Nrf2, NFKB, r-H2AX, P53, GAPDH, Nucleus-Nrf2, histone H1, Cyto-Nrf2). The membrane was then washed several times with TBST and then incubated for 1 hour at room temperature with a secondary antibody labeled with horseradish peroxidase (HRP). After washing with TBST, the levels of the various target proteins on the membrane were examined by enhanced chemiluminescence (ECL). Finally, a BioSpectrum Imaging System (UVP, Upland, CA, USA) was used to capture and analyze the generated chemiluminescence signals.

[0106] The levels of the various target proteins examined are shown in FIGS. 7A and 7B. As shown in FIGS. 7C and 7D, samples from PA-F4-treated mice or mice treated with ON101 showed higher levels of nuclear Nrf2 and lower levels of cytoplasmic Nrf2 compared to samples from mice treated with placebo cream or untreated mice, indicating that PA-F4 and ON101 can enhance the nuclear-cytoplasmic translocation of Nrf2.

[0107] Other embodiments All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features that serve the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar functions.

[0108] From the above description, those skilled in the art can easily identify the essential features of the present invention and make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

[0109] Equivalents Although some embodiments of the invention have been described and illustrated herein, various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or advantages described herein will be readily apparent to those skilled in the art, and each such variation and / or modification is intended to be within the scope of the embodiments of the invention described herein. Generally, all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and it will be readily understood by those skilled in the art that actual parameters, dimensions, materials, and / or configurations may depend on the specific application or applications for which the inventive teachings are used. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced in a manner different from that specifically described and claimed. Embodiments of the invention disclosed herein are directed to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention disclosed herein if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0110] All definitions defined and used herein are to be understood as precedence over dictionary definitions, definitions in incorporated documents by reference, and / or ordinary meanings of defined terms.

[0111] All references, patents, and patent applications disclosed in this specification are incorporated by reference in their entirety for the subject matter to which each is cited, and in some cases may include the entire document.

[0112] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless clearly indicated to the contrary.

[0113] As used in this specification and the claims, the phrase "and / or" means "either or both" of the elements so joined in disjunction, i.e., the elements may be present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements are joined in disjunction. Other elements may optionally be present whether or not they are related to the specifically identified elements identified by the phrase "and / or". Thus, by way of non-limiting example, with reference to "A and / or B" when used in conjunction with non-limiting language such as "comprising", in one embodiment, only A (optionally including elements other than B), in another embodiment, only B (optionally including elements other than A), and in yet another embodiment, both A and B (optionally including other elements), etc. can be referred to.

[0114] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., includes at least one of several elements or a list of elements, but also includes two or more of them, and optionally, additional items not listed. Only terms explicitly indicated to the contrary, such as "only one of", or "exactly one of", or when used in the claims, "consisting of", will refer to including exactly one element of several elements or a list of elements. Generally, as used in this specification, the term "or" is interpreted as indicating exclusive alternatives (i.e., "either one or the other, but not both") only when preceded by an exclusive term such as "any one of", "one of", "only one of", or "exactly one of". When used within the claims, "consisting essentially of" has this ordinary meaning as used in the field of patent law.

[0115] As used in this specification and the claims, the phrase "at least one" with respect to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and is to be understood not to exclude any combinations of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements and referred to by the phrase "at least one" may optionally be present, whether or not they are related to the specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") can, in one embodiment, include at least one, optionally two or more, of A and no B (optionally including elements other than B), in another embodiment, include at least one, optionally two or more, of B and no A (optionally including elements other than A), and in yet another embodiment, include at least one, optionally two or more, of A and at least one, optionally two or more, of B (optionally including other elements), and so on.

[0116] Also, unless expressly indicated to the contrary, in any method claimed in this specification that includes two or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

1. A method for alleviating radiation-induced skin damage, comprising administering to a subject in need thereof a composition comprising an effective amount of salvigenin and a carrier, optionally, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein the composition further comprises cirsimaritin, rosmarinic acid, carvacrol, or a combination thereof.

3. The method according to claim 1 or 2, wherein the composition comprises a Plectranthus Amboinicus (PA) extract.

4. The PA extract is (i) mixing a portion of PA with an extraction solution to produce a first PA extract; (ii) filtering and concentrating the first PA extract to produce a concentrated PA extract; (iii) contacting the concentrated PA extract with a hydrophobic interaction chromatography resin; (iv) eluting the column with an elution solution to produce the PA extract, and is prepared by a process comprising the above steps. The method according to claim 3.

5. The method according to claim 4, wherein the portion of PA in step (i) is the aerial part.

6. The method according to claim 4 or 5, wherein the extraction solution comprises a solvent having a polarity index of about 2.9 to 6.

6.

7. The method according to claim 6, wherein the extraction solution is acetone, butyl methyl ether, ethanol, ethyl acetate, isopropyl alcohol, methanol, or a mixture thereof.

8. The method according to any one of claims 4 to 7, wherein the elution solution comprises a solvent having a polarity index of about 2.1 to 5.

4.

9. The method according to claim 8, wherein the elution solution comprises a mixture of at least two solvents selected from the group consisting of acetone, ethanol, ethyl acetate, and hexane.

10. The method according to any one of claims 1 to 9, wherein the composition further comprises asiaticoside.

11. The method according to claim 10, wherein the composition further comprises a pharmaceutical Centella asiatica (CA) extract containing the asiaticoside.

12. The method according to any one of claims 1 to 9, wherein the subject is a human patient having the radiation-induced skin damage.

13. The method according to any one of claims 1 to 12, wherein the composition is a topical preparation.

14. The method according to claim 13, wherein the topical formulation is in the form of a cream, gel, bandage, spray formulation, ointment, paste, patch, or lotion.

15. The method according to any one of claims 1 to 13, wherein the composition is applied to the site where the radiation-induced skin disorder occurs.

16. The method according to any one of claims 1 to 15, wherein the subject is undergoing radiotherapy.

17. The method according to any one of claims 1 to 16, wherein the subject is a cancer patient.

18. The method according to any one of claims 1 to 17, wherein the composition is administered to the subject once every three days to six times per day.

19. The method according to any one of claims 1 to 18, wherein the radiation-induced skin disorder includes radiation dermatitis, radiation-induced mucositis, radiation-induced lichen planus, radiation-induced vaginitis, radiation-induced skin inflammation, radiation-induced skin damage, or a combination thereof.

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