Extract of perfumed tomentosa for alleviating radiation-induced skin conditions

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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 incorporated herein by reference in its entirety. Background Art

[0003] Plectranthus amboinicus (formerly or alternatively 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 family (also known as Labiatae), native to 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.

[0004] Centella asiatica (formerly or alternatively also known as Centella asiatica Urban, Centella asiatica (L.) Urban, Hydrocotyle asiatica L. and Trisanthuscochinensis Lour.) is a perennial medicinal plant belonging to the Mackinlayoideae subfamily of the Mackinlayaceae or 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, marshpennywort, pennyweed, Indian ginseng, Horse-hoofgrass, Pegaga, Mandookaparni, Tiger herbal, Spadeleaf or Tono. Centella asiatica extracts generally contain two main compounds: asiaticoside and madecassic acid. Summary of the invention

[0005] The present disclosure is based at least in part on the unexpected discovery that a composition comprising salvigenin, and optionally cirsimaritin, rosmarinic acid, carvacrol, or a combination thereof (e.g., a Psoralea corylifolia (PA) extract, optionally in combination with a Centella asiatica (CA) extract) successfully reduces the disease score of radiation dermatitis, and the therapeutic effect is superior to a corticosteroid control. Therefore, it is expected that the compositions disclosed herein exhibit excellent efficacy in alleviating radiation dermatitis.

[0006] In some aspects, 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 damage, or a combination thereof), the method comprising administering to a subject in need thereof an effective amount of a composition comprising trilobate caryophyllein and a carrier. In some embodiments, the composition may be a pharmaceutical composition, which may further comprise a pharmaceutically acceptable carrier. The composition disclosed herein may further comprise cynarocephalin, rosmarinic acid, carvacrol, or a combination thereof.

[0007] In some embodiments, the composition comprises a Pennisetum odoratum (PA) extract. In some examples, the PA extract can be prepared by a method comprising: (i) mixing a portion of the PA (e.g., an aerial portion) 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 to a hydrophobic interaction chromatography resin; and (iv) eluting the column with an eluent solution to produce the PA extract. In some examples, the extraction solution comprises a solvent having a polarity index of about 2.9 to 6.6; optionally wherein the extraction solution is acetone, butyl methyl ether, ethanol, ethyl acetate, isopropanol, methanol, or a mixture thereof. In some examples, the eluent solution comprises a solvent having a polarity index of about 2.1-5.4; optionally wherein the eluent solution comprises a mixture of at least two solvents selected from the group consisting of: acetone, ethanol, ethyl acetate, and hexane.

[0008] In some embodiments, the composition further comprises asiatica saponin. For example, the composition further comprises a Centella asiatica (CA) extract, and the CA extract comprises the asiatica saponin.

[0009] In some embodiments, the subject treated by any of the methods disclosed herein is a human patient suffering from any of the 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 damage, or a combination 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 applied to a subject (e.g., a human cancer patient) who is receiving radiation therapy.

[0010] In some embodiments, any composition 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).

[0011] Also within the scope of the present disclosure are compositions as disclosed herein for ameliorating radiation-induced skin disorders in a subject in need thereof, compositions as disclosed herein for radiation-induced skin disorders, and compositions for the preparation of a medicament for the intended medical use.

[0012] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will become apparent from the following drawings and the following detailed description of several embodiments and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0014] Figure 1 Schematic representation of the experimental design of the mouse radiation study used to explore the efficacy of ON101 and PA-F4 cream in treating radiation dermatitis and to identify biomarkers of radiation exposure.

[0015] Figure 2 is a graph showing that ON101 and PA-F4 creams reduced disease scores compared to a corticosteroid cream control using a radiodermatitis mouse model. *P<0.05, **P<0.01, ***P<0.001.

[0016] Figure 3 is a graph showing the thickness of the epidermis of skin tissues from untreated mice or mice treated with placebo cream, PA-F4 or ON101. *P<0.05, **P<0.01, ***P<0.001.

[0017] Figure 4 is a graph showing the fibrotic content of skin tissues from untreated mice or mice treated with placebo cream, PA-F4 or ON101. *P<0.05, **P<0.01, ***P<0.001.

[0018] Figures 5A-5C Included are images showing NLRP3 ( Figure 5A )、IL1β( Figure 5B) and γ-H2AX( Figure 5C ) levels. Protein expression levels are represented by Q-score values. *P<0.05, **P<0.01, ***P<0.001.

[0019] Figure 6 is a graph showing the differential expression of various protein markers as indicated in skin tissues of untreated mice and mice treated by ON101. ***P<0.001.

[0020] Figures 7A-7D Included are graphs showing protein levels in skin tissue samples from untreated mice or mice treated with placebo cream, PA-F4, or ON101. Fig. 7A : Western blots to measure the levels of Keap1, p62, Nrf2, NF-κB, γ-H2AX, P53, and GAPDH. Figure 7B : Western blot used to measure the levels of nuclear and cytoplasmic Nrf2. Figure 7C : Nrf2 levels in the nucleus. Fig.7D : Levels of Nrf2 in the cytoplasm. DETAILED DESCRIPTION

[0021] Radiation can induce skin disorders. Radiation dermatitis, also known as X-ray dermatitis, is a common side effect of radiation therapy. Radiation dermatitis involves skin cell damage caused by radiation. Typical symptoms include redness, peeling, and ulcers. It usually starts to subside after treatment ends. In some cases, it may appear many years after radiation therapy ends. In patients with radiation dermatitis, skin cell damage may occur in the deep layers of the skin tissue without obvious open surface wounds. The cell structure around the damaged area may change instead of necrosis or apoptosis.

[0022] It is reported herein that, unexpectedly, PA extracts (e.g., comprising carnosine and optionally cyperine, rosmarinic acid, carvacrol or a combination thereof) alone or in combination with CA extracts (e.g., comprising asiatica saponin) show high therapeutic effects in treating radiation dermatitis, as observed in animal models. Therefore, provided herein is a pharmaceutical composition for alleviating radiation dermatitis, comprising a Fructus sylvestris extract or an active agent thereof, and optionally a Centella asiatica extract or an active agent thereof.

[0023] I. Composition

[0024] The present disclosure provides a composition such as a pharmaceutical composition for 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 a combination thereof. The composition disclosed herein may include a PA extract or one or more active agents contained therein (e.g., caryophyllin, and optionally cynaroside, rosmarinic acid, carvacrol, or a combination thereof). In some embodiments, the pharmaceutical composition may further include a CA extract or one or more active agents contained therein (e.g., Centella asiatica saponin).

[0025] (i) Active agent

[0026] The active agent in the compositions described herein comprises carnosine. In some embodiments, the active agent may further comprise cynagenin, rosmarinic acid, carvacrol, or a combination thereof. In some cases, the composition comprises a PA extract comprising carnosine and optionally one or more of: cynagenin, rosmarinic acid, and carvacrol.

[0027] Fragrance Extract

[0028] PA extract refers to an extract obtained from a PA plant using one or more suitable solvents. In some instances, the PA extract is prepared using the aerial parts of the PA plant. In some cases, the polarity index of at least one of the solvents used to prepare the extract is less than 7 (e.g., less than 5). See, for example, U.S. Patent No. 10,758,584, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes cited herein. As used herein, a solvent refers to a substance or mixture of substances that dissolves another substance to form a solution. The PA extract as described herein can be prepared using a 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.

[0029] The PA extracts described herein may comprise terpenes (e.g., monoterpenes, diterpenes, triterpenes and / or sesquiterpenes), flavonoids, phenols, essential oils or combinations thereof. The PA extracts used to prepare the pharmaceutical compositions disclosed herein may comprise trilobatin and optionally one or more of: cynarocephalin, rosmarinic acid and carvacrol.

[0030] The PA extracts described herein can be prepared by extracting the entire PA plant or a portion thereof (e.g., an aerial portion) with one or more suitable solvents to produce a solution, and then drying the solution to produce a PA extract. Since the PA extract contains flavonoids, terpenes (e.g., monoterpenes, diterpenes, triterpenes and / or sesquiterpenes), phenols or essential oils, which are non-polar molecules, the polarity of at least one extraction solvent can be relatively low (e.g., a polarity index of less than 7) to facilitate the dissolution of non-polar molecules. "Extraction" can be performed by directly contacting the PA material with a suitable solvent or by eluting the active components of the PA from a resin to which the active components are attached.

[0031] In some examples, solvents with a polarity index of less than 7 can be used to extract active components from PA to produce PA extracts. Such solvents can be ethyl acetate, methyl acetate, propanol, butanol, or chloroform. Alternatively, the solvent can be a mixture of one or more solvents with different polarity indices. Examples include, but are not limited to, mixtures of ethanol and ethyl acetate, ethyl acetate and butanol, ethanol and propanol, methyl acetate and butanol.

[0032] In some embodiments, the PA extract can be prepared by a method involving the use of a solvent, such as a solvent having a polarity index less than 7 (e.g., < about 6.5, < about 6.0; < about 5.5, < about 5.0, < 4.9, < 4.8, < 4.7, < 4.6, or < 4.5). Examples include, but are not limited to, methanol, ethanol, acetone, ethyl acetate, butanol, dichloromethane, or a combination thereof.

[0033] The PA material can be the whole PA or a part thereof (e.g., an aerial part such as a leaf), and can be prepared by conventional methods. The PA material can be a fresh plant or a part thereof. Alternatively, the PA material can be in a dried form. The PA can be optionally dried to form a powder, which can be used as a PA material for preparing a PA extract.

[0034] Any PA material as described herein can be extracted one or more times by a suitable solvent to produce a crude extract. The solvent used to prepare the crude extract can be a highly polar solvent, for example, with a polarity index greater than 5 and preferably less than 7 (e.g., >5.2; >5.5, >5.8, >6 or more, and preferably less than 7). Examples include, but are not limited to, ethanol, acetone, methanol, water, or a combination thereof. If desired, the crude extract can be concentrated by conventional methods to produce a concentrated crude extract.

[0035] The crude extract can then be contacted with a suitable resin (e.g., a nonionic absorbent resin) under suitable conditions that allow the active components in the crude extract to bind to the resin. Exemplary resins for preparing the hand fragrance extract include, but are not limited to HP20, HP20SS, SP207, Amberlite TM XAD-2 or Amberlite TM XAD-4.

[0036] The resin can then be washed one or more times and eluted with a suitable solvent (e.g., a solvent with a polarity index of less than 7) to produce a PA extract, which can then be dried by conventional methods (e.g., freeze drying, spray drying, or concentrated drying) to produce a dried PA extract, which can be in a semi-solid or paste form.

[0037] 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.

[0038] In one example, an extract of PA can be prepared as follows. The aerial parts of PA (about 1.5 g), including leaves and / or stems, can be collected and extracted with a solvent having a polarity of 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 method can be carried out at a temperature of about 50 to 80° C. The resulting crude extract can be directly loaded into a nonionic adsorption resin column and eluted with a solvent having a polarity of 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 of less than 6 (e.g., a solvent described herein). The resulting filtrate can be collected to produce a PA extract.

[0039] Centella Asiatica Extract

[0040] In some embodiments, the compositions disclosed herein (e.g., pharmaceutical compositions) may further comprise asiaticoside. In some cases, the composition further comprises a CA extract, which may comprise an asiaticoside compound. The CA extract as described herein refers to an extract obtained from the whole CA plant or a part thereof. The CA extract may comprise asiaticoside and optionally madecassic acid.

[0041] CA extracts can be prepared according to conventional methods, such as those described in US Pat. Nos. 5,834,437, 6,417,349, 6,475,536 and 6,267,996, CN 1313124, CN 1089497 and CN 1194154. The following are examples.

[0042] CA material can be prepared by conventional practice. Such material can be fresh CA plant or part thereof, or dried CA. CA material can be extracted with suitable solvents such as water, ethanol or mixtures thereof to produce a crude extract. The crude extract can be optionally concentrated, mixed with a suitable resin or loaded onto a column filled with resin. After washing once or multiple times, the resin can be eluted with a suitable solvent. The resulting eluate can be concentrated to form a paste, which can be dried by conventional methods, for example, vacuum drying to produce a powder of the CA extract. If necessary, the CA powder can be ground through a sieve (e.g., No. 100 sieve).

[0043] The extract of CA can be prepared by the same or similar method as described above for preparing the PA extract. The PA extract and / or the CA extract can be concentrated using a reduced pressure rotary evaporator.

[0044] (ii) Pharmaceutical Compositions

[0045] Any of the active compounds (e.g., carnosine, and optionally one or more of: cyperin, rosmarinic acid, carvacrol and / or asiaticoside), PA extracts optionally in combination with CA extracts as disclosed herein, can be mixed with a suitable carrier (e.g., a pharmaceutically acceptable carrier) to form a composition (e.g., a pharmaceutical composition) for 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 a combination thereof. "Acceptable" means that the carrier must be compatible with the active ingredients of the composition (and preferably, capable of stabilizing the active ingredients) and not harmful to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition (2000) Lippincott Williams and Wilkins, ed. KE Hoover.

[0046] Compositions used in the methods of the invention, such as pharmaceutical compositions, may include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy, 20th edition (2000), Lippincott Williams & Wilkins, ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; 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 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®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0047] In some examples, the pharmaceutical compositions described herein include liposomes containing an active agent, which can be prepared by methods known in the art or as described in the following literature: 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 time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to produce liposomes with a desired diameter.

[0048] Active agent can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules in coarse emulsions. These techniques are known in the art, see, for example, Remington: Pharmaceutical Science and Practice, 20th edition, Mack Publishing (2000).

[0049] In other examples, the pharmaceutical compositions described herein can be formulated into a sustained release format. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing active agents, which are in the form of molded articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly D-(-)-3-hydroxybutyric acid.

[0050] Compositions to be used for in vivo administration, such as pharmaceutical compositions, must be sterile. This is easily achieved, for example, by filtration through a sterile filtration membrane. The compositions described herein can be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration or by inhalation or insufflation.

[0051] In order to prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gum and other pharmaceutical diluents such as water to form a homogeneous mixture solid pre-formulated composition containing the compound of the present invention or its pharmaceutically acceptable non-toxic salt. When these pre-formulated compositions are called homogeneous, this means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily subdivided into equivalent unit dosage forms, such as tablets, pills and capsules. The solid pre-formulated composition is then subdivided into unit dosage forms of the type described above, and the unit dosage form contains 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the new composition can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, a tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of a coating on the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a variety of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0052] Suitable surfactants specifically include nonionic agents such as polyoxyethylene sorbitan (e.g., Tween TM 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span TM 20, 40, 60, 80 or 85). Compositions with surfactant will conveniently contain between 0.05 and 5% surfactant, and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added if desired, such as mannitol or other pharmaceutically acceptable vehicles.

[0053] You can use Intralipid TM , Liposyn TM Infonutrol TM , Lipofundin TM Lipiphysan TMSuitable emulsions can be prepared using commercially available fat emulsions such as glycerol, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, the active ingredient can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and in an emulsion formed by mixing with phospholipids (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the tension of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can contain fat droplets between 0.1 and 1.0 μm, specifically between 0.1 and 0.5 μm, and the pH can be in the range of 5.5 to 8.0.

[0054] The emulsion composition may be prepared by mixing the active agent with the Intralipid TM Or an emulsion composition prepared by mixing its components (soybean oil, lecithin, glycerin and water).

[0055] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as listed above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0056] Compositions in the form of preferably sterile pharmaceutically acceptable solvents can be aerosolized using gases. The aerosolized solution can be breathed directly from the aerosolizing device, or the aerosolizing device can be attached to a mask, oxygen tent, or intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions can be preferably administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0057] Topical preparations

[0058] In some embodiments, the pharmaceutical compositions disclosed herein are topical formulations. Such topical formulations may include any PA extract disclosed herein or one or more active agents thereof (e.g., salvinia trilobata, and optionally a combination of one or more styrax, rosmarinic acid, carvacrol). In some cases, the topical formulation may further include a CA extract as disclosed herein or one or more active agents thereof (e.g., asiaticoside). In some instances, the topical formulations described herein may include salvinia trilobata in an amount ranging from about 0.0001% to about 0.5% (w / w), and optionally asiaticoside, which may range in amount from about 0.05% to about 5% (w / w). In some embodiments, the topical formulation includes an amount of about 0.1-30% (w / w) of a safflower extract, a centella asiatica extract, or both.

[0059] As used herein, the term "about" is intended to set strict numerical boundaries for a specified parameter (including upper and lower limits). Those skilled in the art will understand the meaning of "about" associated with a specific context. In some cases, the term "about" refers to a specific value + / - 5% (e.g., + / - 3% or + / - 2%).

[0060] In some cases, the topical formulation may further comprise one or more carriers or excipients, including one or more viscosity enhancing agents (e.g., about 1.0-10%), one or more ointment bases (e.g., one or more cream bases) in the range of about 5-30%, one or more antimicrobial preservatives (e.g., about 0.005-0.2 wt %), one or more emulsifiers (about 0.5-10 wt %), or a combination thereof. These components may be dissolved or dispersed in a suitable solvent.

[0061] "Viscosifier" is an agent used to thicken a formulation. Exemplary viscosity enhancers can include, for example, cetearyl alcohol, cholesterol, stearyl alcohol, chlorocresol, white wax, stearic acid, cetyl alcohol, or a combination thereof. The viscosity enhancer can be present in a topical formulation at a concentration of about 1.0-10% (w / w). For example, a topical formulation can contain about 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, or 9.5-10% (w / w) viscosity enhancer. Alternatively, the topical formulation may comprise about 1-5%, 2.5-7.5%, or 5-10% (w / w) viscosity increasing agent.

[0062] An "ointment base" can be any semisolid formulation 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, TM , and ), water / oil emulsion ointment bases (e.g., cold cream, aqueous lanolin, rose water ointment, Hydrocream TM , and ), oil / water emulsion ointment bases (e.g., hydrophilic ointments, Dermabase TM , and ), and with water-miscible ointment bases (e.g., polyethylene glycol (PEG) ointments and Polybase TM ). The ointment base can be pharmacologically inert, but can entrap water to provide an emollient protective film. In a specific embodiment, the ointment base can be any petrolatum compound (e.g., petrolatum, white petrolatum, white soft paraffin, liquid petrolatum, liquid paraffin). In another specific embodiment, the ointment base is white petrolatum (CAS No. 8009-03-8). The ointment base can be present in the topical formulation at a concentration of about 5-30% (w / w), such as 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 may comprise 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 ointment base.

[0063] In some embodiments, an "ointment base" described herein contains less than 20% water and volatiles, and more than 50% hydrocarbons, waxes, or polyols as vehicles.

[0064] In some embodiments, the "ointment base" described herein is a "cream base" that contains more than 20% water and volatiles and / or generally contains less than 50% hydrocarbons, waxes or polyols as vehicles for drug substances. The cream base can 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 a continuous phase. Such cream bases generally contain water-in-oil emulsifiers, such as lanolin, sorbitol esters and monoglycerides. In other cases, the cream base is a hydrophilic cream base having an aqueous phase as a continuous phase. Such cream bases generally contain water-in-oil emulsifiers, such as sodium or triethanolamine soaps, sulfated fatty alcohols, polysorbates and polyoxygenated fatty acids and fatty alcohol esters, which can be combined with water-in-oil emulsifiers if necessary.

[0065] An "antimicrobial preservative" may be any compound capable of destroying microorganisms, preventing the reproduction or growth of microorganisms, or preventing the pathogenic effects of microorganisms. Exemplary antimicrobial preservatives include, but are not limited to, paraben compounds (esters of parabens; e.g., paraben, methylparaben, ethylparaben, propylparaben, butylparaben, heptylparaben, benzylparaben, isobutylparaben, isopropylparaben, benzylparaben, or their sodium salts), benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, bronopol, cetyltrimethylammonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. The antimicrobial preservative may be present in the topical formulation at a concentration of about 0.005-0.2%, such as about 0.01-0.2% (w / w). For example, the topical formulation may contain about 0.005-0.01%, 0.01-0.05%, 0.05-0.1%, 0.1-0.15% or 0.15-0.2% (w / w) of the antimicrobial preservative. Specifically, the topical formulation may 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) antimicrobial preservative.

[0066] An "emulsifier" is a compound or substance that acts as a stabilizer for a mixture of two or more normally immiscible (non-mixable or non-combinable) liquids. Exemplary emulsifiers can include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and magnesium aluminosilicate [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin, monostearate, ethylene glycol diols, glyceryl stearate ... stearic acid esters, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], glyceryl monooleate, sorbitan monooleate[ 80], polyoxyethylene esters (e.g., polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and thymic acid), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]) 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, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzalkonium chloride and docusate sodium and / or combinations thereof. The emulsifier may be present in the topical formulation at a concentration of about 0.5-10% (w / w), for example 0.5-6% (w / w). For example, the topical formulation can include about 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 5-10%, 6-10% or 8-10% (w / w) emulsifier. Specifically, the topical formulation can include 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) emulsifier.

[0067] 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 may include, but are not limited to, any known solvents, including propylene glycol, glycols, and mixtures thereof. The non-aqueous solvent may be present in the topical formulation at a concentration of about 2% to 65% (w / w). For example, the topical formulation may include about 2% to 15%, 15% to 30%, 30% to 45%, or 45% to 65% (w / w) of solvent. In some embodiments, the topical formulations of the present invention may also contain water.

[0068] In some embodiments, the topical formulations of the present invention may further comprise one or more emollients, fragrances or pigments. The topical formulations may also be combined with wound dressings (e.g., bandages with adhesives, plasters, etc.)

[0069] (e.g., cyclohexane, n-hexane, n-decane, isooctane, octane, butyl ether, carbon tetrachloride, triethylamine, isopropyl ether, toluene, p-xylene, tert-butyl methyl ether, benzene, benzyl ether, dichloromethane, methylene chloride, chloroform, dichloroethane, dichloroethylene, 1-butanol, isobutanol, tetrahydrofuran, ethyl acetate, 1-propanol, 2-propanol, methyl acetate, cyclohexanone, methyl ethyl ketone (MEK), nitrobenzene, benzonitrile, 1,4-dioxane or p-dioxane) can be used in combination.

[0070] Additional information regarding topical formulations may be found in U.S. Patent No. 10,758,584, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes cited herein.

[0071] The topical formulations disclosed herein can be in any suitable form. Examples include, but are not limited to, creams, dressings, ointments, lotions, pastes, and gels. In some cases, the topical formulation can be placed on a device (e.g., a patch) for application to the site where the skin condition occurs.

[0072] II. Methods for Alleviating Radiation-Induced Skin Disorders

[0073] In order to practice the methods disclosed herein, an effective amount of the compositions described herein (e.g., pharmaceutical compositions) can be administered to a subject (e.g., a person) in need of treatment by a suitable route, such as intravenous administration, for example, such as bolus injection or by continuous infusion over a certain period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhaled or topical routes. Commercially available nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers can be used for administration. Liquid formulations can be directly atomized and lyophilized powders can be atomized after reconstruction.

[0074] In some cases, topical formulations as described herein can be used to alleviate radiation-induced skin disorders 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 damage, or a combination thereof.

[0075] The topical formulation can be applied to the damaged area after a suitable dosage and treatment regimen. The dosage and application regimen of the described method will depend on the nature and condition of the symptoms being treated, the age and condition of the patient, and any previous or concurrent therapy. In some cases, the topical formulation can be applied once a week, once every other day, once a day, twice a day, three times a day, or four times a day for an appropriate period of time. When the radiation-induced skin disorder recovers, treatment can be terminated. If necessary, treatment can be resumed, for example, if the symptoms of one or more skin disorders recur.

[0076] 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 damage, or a combination thereof.

[0077] The term "radiation dermatitis", also known as radiation burns, refers to dermatitis (e.g., skin irritation) associated with radiation. In some cases, radiation dermatitis is associated with radiation therapy received by a subject, for example, a human cancer patient receiving anticancer radiation therapy (e.g., X-ray therapy). Common symptoms associated with radiation dermatitis include redness, itching, flaking, peeling, pain, moisture, blistering, pigmentation changes, fibrosis, connective tissue scarring, and ulcer development. According to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) v5.0 guidelines, the grading of radiation dermatitis is provided in Table 1 below:

[0078] Table 1. NCI CTCAE v5.0 radiation dermatitis grading

[0079]

[0080] The subject 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 suffering from a radiation-induced skin disorder (e.g., radiation dermatitis), suspected of having a skin disorder, or at risk of developing a skin disorder. In some cases, the subject can be a human patient (e.g., a human cancer patient) who has received or is receiving radiotherapy. In some embodiments, the subject in need of treatment can be treated with any of the compositions disclosed herein for radiation-induced skin disorders and radiotherapy simultaneously.

[0081] Subjects suffering from radiation-induced skin disorders disclosed herein can be identified by routine medical examinations. In some cases, subjects suffering from radiation dermatitis may show one or more symptoms of the symptoms disclosed herein. In other cases, subjects suffering from radiation dermatitis may suffer skin cell damage in the deep skin layer, with little or no surface wounds. Subjects suspected of having radiation dermatitis may show one or more symptoms of the disease / illness. Subjects at risk of radiation dermatitis can be subjects with one or more risk factors in the risk factors for the disease / illness (e.g., human patients who will receive radiotherapy or are at risk of being exposed to radiation).

[0082] As used herein, "effective amount" refers to the amount of each active agent required to give a subject a therapeutic effect, either alone or in combination with one or more other active agents. It will be apparent to those skilled in the art whether a certain amount of composition has achieved a therapeutic effect. As recognized by those skilled in the art, the variation in effective amount depends on the specific condition being treated, the severity of the condition, individual patient parameters, including age, physical condition, body shape, sex and weight, the duration of treatment, the nature of concurrent therapy (if present), specific routes of administration, and similar factors within the knowledge and expertise of health practitioners. These factors are well known to those of ordinary skill in the art and can be solved only by routine experiments. It is generally preferred to use the maximum dose of individual components or their combinations, i.e., the highest safe dose according to reasonable medical judgment.

[0083] Empirical considerations such as half-life will generally contribute to the determination of dosage. The frequency of administration can be determined and adjusted during the course of treatment, and is generally but not necessarily based on the treatment and / or inhibition and / or improvement 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.

[0084] In one example, the dosage of a composition as described herein can be determined empirically in an individual who has been given one or more administrations of the composition. Increasing doses of the agonist are given to the individual. In order to assess the efficacy of the agonist, an indicator of the disease / disorder can be followed.

[0085] In general, for administration of any of the compositions described herein, an initial candidate dose may be given to a subject. For repeated administration over several days or longer, treatment continues until desired symptom suppression occurs or until a therapeutic level sufficient to alleviate the target disease or disorder or its symptoms is reached, depending on the condition. The specific dosing regimen, i.e., dosage, timing, and repetition, will depend on the specific individual and the individual's medical history as well as the properties of the individual agents (such as the half-life of the agent and other considerations well known in the art).

[0086] For the purposes of this disclosure, the appropriate dosage of the composition as described herein will depend on the specific active agent contained therein, the type and severity of the disease / disorder, whether the composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and the response to treatment, and the judgment of the attending physician. Typically, the clinician will administer the composition until the dosage that achieves the desired result is reached. In some embodiments, the desired result is a reduction in the severity of the disease (e.g., represented by a disease score). The method for determining whether the dosage produces the desired result will be apparent to those skilled in the art. The administration of any composition in the composition disclosed herein can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to skilled practitioners. The composition administration can be substantially continuous within a preselected period, or a series of interval doses can be used, for example, before, during, or after the development of the target disease or disorder.

[0087] As used herein, the term "treat" refers to applying or administering a composition comprising one or more active agents to a subject suffering from a target disease or condition, symptoms of a disease / condition, or susceptibility to a disease / symptom, for the purpose of treating, curing, alleviating, alleviating, altering, remedying, ameliorating, improving, or affecting the condition, symptoms of a disease, or susceptibility to a disease or condition.

[0088] Relieve target disease / illness and comprise delaying the development or progress of disease or reducing disease severity or prolonging survival.Relieve disease or prolong survival and do not necessarily need to cure result.As used herein, "delaying" the development of target disease or illness means to postpone, hinder, slow down, slow down, stabilize and / or delay the progress of disease.This delay can have different time lengths, and this depends on the history of the disease and / or individual treated.The method of "delaying" or relieving the development of disease or delaying the onset of disease is to reduce the possibility of one or more symptoms of developing disease in a given time frame and / or the method of reducing the degree of symptom in a given time frame compared with not using described method.This type of comparison is usually based on clinical research, using many subjects that are enough to give statistically significant results.

[0089] "Development" or "progression" of a disease means the initial manifestation and / or subsequent progression of the disease. The progression of a disease may be detectable and can be assessed using standard clinical techniques as are well known in the art. However, progression also refers to progression that may not be detectable. For purposes of this disclosure, development or progression refers to the biological course of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein, an "onset" or "occurrence" of a target disease or condition includes initial onset and / or recurrence.

[0090] According to the type of disease to be treated or the position of disease, conventional methods known to those of ordinary skill in the medical field can be used to apply pharmaceutical composition to the subject.This composition can also be used by other conventional routes, for example, oral, parenteral, by inhalation spray, local, rectal, nasal cavity, cheek, vagina or by implantable drug reservoir.As used herein, term "parenteral" includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion technology.In addition, it can be applied to the subject by injectable reservoir administration route, as using 1 month, 3 months or 6 months reservoir injectable or biodegradable materials and methods.In some instances, pharmaceutical composition is applied intraocularly or intravitreally.

[0091] The specific dosing regimen, ie, dosage, timing, and repetition, used in the methods described herein will depend on the particular subject and the subject's medical history.

[0092] In some embodiments, any of the compositions disclosed herein can be used together with one or more additional therapeutic agents for treating a target disease. In some cases, additional therapeutic agents can be used to enhance and / or supplement the effectiveness of the compositions disclosed herein.

[0093] The efficacy of a treatment for a disease / disorder of interest can be assessed by methods well known in the art.

[0094] III. Kits for Alleviating Radiation-Induced Skin Disorders

[0095] The present disclosure also provides kits for 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 a combination thereof. Such kits may include one or more containers comprising any of the compositions described herein (e.g., topical formulations), the compositions comprising a scutellaria baicalensis extract, a Centella asiatica extract, a combination of a scutellaria baicalensis extract and a Centella asiatica extract, or one or more active agents thereof, such as those disclosed herein.

[0096] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of applying the composition (e.g., a topical formulation) according to any of the methods described herein to alleviate radiation-induced skin disorders, such as radiation-induced skin disorders disclosed herein. The kit may further include a description of selecting an individual suitable for treatment based on identifying whether the individual suffers from a skin disorder in need of treatment.

[0097] Instructions for use of the composition (e.g., topical formulation) typically include information about dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0098] The label or package insert indicates that the composition is used to alleviate a radiation-induced skin condition as disclosed herein.Instructions may be provided for practicing any of the methods described herein.

[0099] The kit of the present invention adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. At least one active agent in the composition is an active agent as disclosed herein, which can be contained in the extract of the scutellaria baicalensis, the extract of Centella asiatica, or the combination of the extract of the scutellaria baicalensis and the extract of Centella asiatica.

[0100] The kit may optionally provide additional components such as interpretative information. Typically, the kit comprises a container and a label or package insert located on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0101] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the greatest extent based on the above description. Therefore, the following specific examples should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. All disclosures cited herein are incorporated by reference for the purpose or subject matter cited herein.

[0102] Example 1: Relief of radiation dermatitis using a combination of a Palmatum odoratum (PA) extract, optionally with a Centella asiatica (CA) extract

[0103] This example explores the therapeutic efficacy of (a) ON101 cream containing Aristolochia odorata (PA) extract and Centella asiatica extract; and (b) PA-F4 cream containing Aristolochia odorata (PA) extract on radiation dermatitis. Topical corticosteroid cream (Betamethasone) was used as a positive control.

[0104] Figure 1The overall experimental design of a mouse radiation study for studying the therapeutic effects of ON101 cream and PA-F4 cream is described. Briefly, mice were shaved to remove dorsal hair, anesthetized, and then received a dose of 6.5 Gy once daily at the shaved site for 5 days (Day 0 to Day 4). ON101 cream, PA-F4 cream, or topical corticosteroid cream (control) was applied to the radiation site. Radiation-induced dermatitis was assessed every 2 days during Day 8 to Day 28. 1 ml of cream was applied to 5 cm 2 shaved area once a day for five days, followed by a two-day rest period. Repeat this cycle four times. Figure 1 During this time period, the level of radiation dermatitis was determined according to the definition provided in Table 2 below:

[0105] Table 2. Definition of radiation dermatitis scores

[0106] score symptom Corresponds to CTCAE 0 none 0.5 dry 1 Dry and flaky CTCAE Level 1 2 Early moist desquamation CTCAE Level 2 3 Moist desquamation in about 30% of the area CTCAE Level 3 4 Moist scaling over more than 50% of the area 5 Skin necrosis and ulcers

[0107] Skin tissue samples are collected for analysis of biomarkers of radiation exposure by, for example, single-cell RNA sequencing (scRNA-seq).

[0108] like Figure 2 As shown, both ON101 cream and PA-F4 cream showed a significant reduction in the radiodermatitis score, and the treatment effect was superior to that of the control cream.

[0109] Example 2: Therapeutic efficacy of PA extract, optionally in combination with CA extract, on radiation-induced skin damage

[0110] This example evaluates the therapeutic efficacy of PA extract alone or in combination with CA extract as disclosed herein on radiation-induced skin damage. The treatment process is described in Example 1 above. See also Figure 1 .

[0111] (i) Thickness of the epidermis

[0112] The thickness of the epidermis of mice treated with ON101 cream, PA-F4 cream, corticosteroids (positive control), placebo cream (negative control) and untreated mice (blank control) was measured by hematoxylin and eosin (H&E) staining. The tissue sections obtained from the mice were immersed in distilled water and the nuclei were stained with alum hematoxylin. The tissue sections were then cleaned by rinsing under running tap water. 0.3% acid ethanol was used for differentiation. The tissue sections were rinsed again under running tap water and then further rinsed by immersion in Scott's tap water, followed by tap water. The tissue sections were then stained in eosin solution for 2 minutes, and then dehydrated, cleaned and fixed.

[0113] The results showed that both ON101 and PA-F4 reduced the thickness of the epidermis, with ON101 showing a better effect. Figure 3 .

[0114] (ii) Fibrosis content

[0115] The fibrosis content of the mice at the irradiated sites was analyzed by Masson staining. Masson's trichrome stain is a widely used method in histology for identifying and distinguishing collagen fibers from muscle fibers in tissue sections. This staining technique is particularly valuable when studying various pathologies affecting organs. In order to perform Masson's trichrome staining, the following steps were performed.

[0116] Initially, skin samples obtained from mice in different treatment groups were dewaxed and rehydrated by sequentially passing through 100% alcohol, 95% alcohol, and 70% alcohol. This process ensured the removal of paraffin and properly prepared the samples for staining. Subsequently, the samples were thoroughly washed in distilled water to eliminate any residual chemicals or debris, ensuring a clean surface for staining.

[0117] The treated skin samples were then incubated in Weigert's iron hematoxylin working solution for 10 minutes. This step facilitates visualization of the cell nuclei. After the staining step, the samples were rinsed in running warm tap water for approximately 10 minutes. This rinsing process effectively removes excess stain and enhances the clarity of the stained sections. After rinsing, the samples were washed again in distilled water to ensure that any remaining traces of staining solution were removed.

[0118] Finally, the samples were stained with Biebrich scarlet acid fuchsin solution for a period of 10-15 minutes. This staining solution imparts a unique color to the collagen fibers, distinguishing them from other tissue components.

[0119] like Figure 4As shown, skin samples from mice treated with PA-F4 cream and ON101 cream showed reduced fibrosis content relative to mice treated with placebo cream or untreated mice.

[0120] (iii) Immunohistochemical analysis

[0121] Immunohistochemical analysis was performed to investigate pathways associated with oxidative stress, radiation damage, and inflammation in mice treated with various creams as described above. The experiment involved sorting mouse skin tissue samples obtained from mice treated with ON101, PA-F4, sham, or placebo creams. Immunostaining analysis was performed to detect the levels of NLRP3, rH2AX, and IL1B by the following immunohistochemical (IHC) procedure.

[0122] First, the slide was washed three times with PBS. Subsequently, 600 μl of peroxidase blocking solution was added to the slide, and the slide was then incubated for 10 minutes. After another round of PBS washing, the slide was blocked in 2% skim milk for 15 minutes. Then, mouse anti-NLRP3, rH2AX or IL1B (diluted at 1:500) was applied to the slide, and the slide was incubated at 4 ° C for 20 hours. After three washes, Dako (Dako) HRP-labeled X anti-mouse (1:5000) was added, and the slide was incubated for 30 minutes, and then washed three more times. Next, a substrate containing a chromogen (1 drop of chromogen to 1 ml substrate) was added to the slide and incubated for 1-15 minutes. The slide was washed three times with PBS, and then counterstained with Mayer hematoxylin for 12 seconds. After rinsing with tap water, the slide was dried.

[0123] Figures 5A-5C The levels of NLRP3, rH2AX, and IL1B in mouse skin samples were summarized separately. Mice treated with ON101 cream and PA-FA cream showed significantly lower levels of NLRP3, rH2AX, and IL1B in skin tissues compared to untreated mice or mice treated with placebo cream.

[0124] (iv) Potential pathways for ON101 to prevent radiation dermatitis

[0125] In this study, RNA sequencing analysis was performed on tissue samples from mice affected by radiation dermatitis to explore the molecular changes that occur in skin tissue during radiation dermatitis and investigate the potential pathways by which ON101 confers its protective effects.

[0126] RNA transcripts collected from mouse skin samples were analyzed using high-throughput RNA sequencing technology. This technology allows for a comprehensive look at the gene expression changes that occur in the skin during radiation dermatitis in 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 development and progression of radiation dermatitis. By comparing the gene expression profiles of samples obtained from ON101-treated mice with those obtained from untreated mice, specific genes and pathways regulated by ON101 treatment can be deciphered.

[0127] Further, using miDETECT A Track TM RT-qPCR analysis was performed using the RNA qRT-PCR Starter Kit (RIBOBIO, Guangzhou, Taiwan, China) to investigate the levels of RNA transcripts. The miRNA was converted into complementary DNA (cDNA) using a 1st strand cDNA synthesis kit (TaKaRa, Taipei, Taiwan, China). To quantify gene expression levels, qRT-PCR was performed using a SYBR Premix Ex TaqII kit (TaKaRa, Taipei, Taiwan, China). qRT-PCR analysis was performed in 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.

[0128] Results from this study showed that genes such as KEAP1, IL18, NK-κB, NLRP3, EGFR, VIM, and CTNNB1 were differentially expressed in samples from ON101-treated mice compared to samples from untreated mice. More specifically, ON101 reduced the expression levels of KEAP1, IL18, NK-κB, NLRP3, and IL1B, and enhanced the expression levels of EGFR, VIM, and CTNNB1. Figure 6 The results suggest that ON101 can mitigate the development and severity of radiation dermatitis by targeting Keap1, a protein known to play a key role in oxidative stress response and inflammation.

[0129] (v) Activation of the p62-KEAP1-NRF2 pathway and Nrf2 in radiation dermatitis mice

[0130] Promotion of nuclear translocation

[0131] In this study, ON101 and PA-F4 were found to activate the p62-KEAP1-NRF2 pathway and promote Nrf2 nuclear translocation in radiation dermatitis mice.

[0132] Protein lysates prepared from skin samples collected from mice treated with ON101, PA-F4, or placebo cream and from untreated mice were heat treated in preparation for analysis. For immunoblotting, a 50 μl ELISA kit containing 50 μl of 1% ELISA kit was used according to conventional immunoblotting practice. 20% skim milk solution in Tris buffered saline (TBST).In short, the protein in the protein lysate sample is transferred to polyvinylidene fluoride (PVDF) membrane, then the membrane and the primary antibody with specificity to target protein (Keap1, p62, Nrf2, NFKB, r-H2AX, P53, GAPDH, nucleus-Nrf2, histone H1, cytoplasm-Nrf2) are incubated overnight at 4 ℃.Then the membrane is washed several times with TBST, and then incubated at room temperature for 1 hour with the secondary antibody labeled with horseradish peroxidase (HRP).After being washed by TBST, the level of various target proteins on the film is detected by enhanced chemiluminescence (ECL).Finally, the chemiluminescent signal generated is captured and analyzed using a biospectral imaging system (UVP company (UVP, Upland, CA, USA) in Upland, California, USA).

[0133] Fig. 7A and 7B The levels of various target proteins detected are shown in FIG. Figure 7C and 7D As shown, samples from PA-F4-treated or ON101-treated mice showed higher levels of nuclease Nrf2 and reduced levels of cytoplasmic Nrf2 compared to samples from mice treated with placebo cream or untreated, indicating that PA-F4 and ON101 can enhance the nucleus-cytoplasm translocation of Nrf2.

[0134] Other embodiments

[0135] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a series of general equivalent or similar features.

[0136] Through the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to make it suitable for various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0137] Equivalent

[0138] Although various inventive embodiments have been described and illustrated herein, a person of ordinary skill in the art will readily conceive of various other devices and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each of such changes and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications used using the teachings of the present invention. Using only routine experiments, a person of ordinary skill in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, if such features, systems, articles, materials, kits, and / or methods are not mutually contradictory, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention disclosed herein.

[0139] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0140] All references, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter for which each is cited, in some cases in their entirety.

[0141] Unless clearly indicated to the contrary, the indefinite articles "a" and "an" as used in this specification and claims should be understood to mean "at least one".

[0142] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more elements" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "comprising", a reference to "A and / or B" may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0143] As used herein in this specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating the items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one element in many elements or lists of elements, but also including more than one element and optionally other unlisted items. Only explicitly indicating the opposite term (such as "only one" or "just one") or when used in the claims, "consisting of..." will refer to including exactly one element in many elements or lists of elements. Usually, as used herein, when it is an exclusive term (such as "any one of...", "one of...", "only one of..." or "just one of..."), the term "or" should only be interpreted as indicating exclusive alternatives (that is, "one or the other, but not both"). When used in the claims, "consisting essentially of..." should have the common meaning as used in the field of patent law.

[0144] As used herein in this specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one element of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that in addition to the elements specifically identified in the list of elements referred to by the term "at least one", elements may optionally be present, whether related to or unrelated to the specifically identified elements. Thus, as a non-limiting example, in one embodiment, "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") may refer to at least one that optionally includes more than one A, does not have B (and optionally includes elements other than B); in another embodiment, may refer to at least one that optionally includes more than one B, does not have A (and optionally includes elements other than A); in yet another embodiment, may refer to at least one that optionally includes more than one A, and optionally includes more than one B (and optionally includes other elements), etc.

[0145] It should also be understood that in any method claimed herein that includes more than one step or operation, the order of the steps or operations of the method is not necessarily limited to the method steps or order of steps recited unless clearly indicated to the contrary.

Claims

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

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

3. The method of claim 1 or claim 2, wherein the composition comprises an extract of Plectranthus Amboinicus (PA).

4. The method according to claim 3, wherein the PA extract is prepared by a method comprising: (i) mixing a portion of the 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, and (iv) eluting the column with an eluent solution to produce the PA extract.

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

6. The method of claim 4 or claim 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, isopropanol, methanol or a mixture thereof.

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

4.

9. The method of claim 8, wherein the eluent 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 medicinal Centella asiatica (CA) extract, wherein the CA extract comprises the Centella asiatica saponin.

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

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

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

15. The method of 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 of any one of claims 1 to 15, wherein the subject is receiving radiation therapy.

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

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

19. The method of any one of claims 1 to 18, wherein the radiation-induced skin disorder comprises 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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