Pharmaceutical compositions for use in reducing hyperpigmentation

Abstract

A method for reducing pigmentation in a subject, comprising administering an effective amount of a pharmaceutical composition to a skin site of the subject in need, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a stilbene compound, for example, a compound of formula (I) (for example, formula (IA)), or a pharmaceutically acceptable salt thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit as of the filing date of U.S. Provisional Application No. 63 / 512,421, filed on July 7, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Pigmentation is the darkening of spots on the skin compared to the surrounding skin. It occurs when the skin produces too much melanin. Skin pigmentation is caused by the accumulation of melanin-containing melanosomes in the basal layer of the epidermis.

[0003] Melanin is a pleomorphic and multifunctional biopolymer represented by eumelanin, pheomelanin, and neuromelanin. A proper amount of melanin in the skin can absorb ultraviolet (UV) rays, maintain skin temperature, and thereby protect the skin from UV light. However, excessive melanin can make the skin appear dull and cause an undesirable appearance. Furthermore, excessive melanin can lead to skin melanosis or pigmented skin conditions such as age spots and freckles. Melanin production can also cause problems in other organs, such as the skin, hair, eyes, ears, or brain of animals.

[0004] Therefore, it is interesting to develop compositions that can regulate melanin levels and thereby reduce pigmentation. Thus, the ability to remove or reduce the appearance of pigmentation (e.g., melasma, seborrheic keratosis, chloasma, freckles, sunburn, or aged skin) may be of interest to individuals who desire an even skin tone and a more radiant complexion. A report published by Intelligence Research Group, "2017-2022 Monitoring and Prospect Forecast of Chinese Whitening Market," shows that over 80% of Chinese women aged 20-50 have a demand for skin whitening, and there is an annual market of nearly 66 billion for whitening products. Therefore, there is a significant market for safe and effective products to control skin pigmentation. [Overview of the project]

[0005] This application is at least in part based on the remarkable discovery that stilbene compounds (e.g., 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol) exhibited significant melanin inhibitory activity both in vitro and in vivo. Therefore, such compounds are expected to be highly effective in reducing hyperpigmentation.

[0006] Accordingly, a method for reducing pigmentation in a subject is provided herein, comprising administering an effective amount of a pharmaceutical composition to a skin site of the subject in need.

[0007] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a stilbene compound or a pharmaceutically acceptable salt thereof having the structure of formula (I): [ka] The formula includes, where each of R1, R2, R3, R4, and R8 is independently H, halogen, thiol, hydroxyl, C1-4 alkyl, C2-4 alkenyl, OR5, COOR6, or -OC(=O)R7. Each of R5, R6, and R7 is independently H or C1-4 alkyl.

[0008] In some examples, the stilbene compounds disclosed herein have the structure of formula (IA): [ka] The formula may have, where each of R1, R2, R3, and R4 is as defined herein.

[0009] In some cases, at least one of R1, R2, R3, and R4 is a hydroxyl group. For example, in the compounds of formula (I) or formula (IA) provided herein, three of R1, R2, R3, and R4 may be hydroxyls. In some cases, the other may be hydrogen. In other cases, all of R1, R2, R3, and R4 may be hydroxyls.

[0010] In some cases, at least one of R1, R2, R3, and R4 is methoxy. For example, in the compounds of formula (I) or formula (IA) provided herein, three of R1, R2, R3, and R4 may be methoxy. In some cases, the other may be hydrogen. In other cases, all of R1, R2, R3, and R4 may be methoxy.

[0011] In some cases, at least one of R1, R2, R3, and R4 is OC(=O)R7, where R7 is C 1~4 It is alkyl (e.g., methyl). For example, three of R1, R2, R3, and R4 in the compounds of formula (I) or formula (IA) provided herein may be OC(=O)R7, where R7 is C 1~4It is alkyl (e.g., methyl). In some cases, the other can be hydrogen. In other examples, all of R1, R2, R3, and R4 are OC(=O)R7, where R7 is C 1~4 It is alkyl (e.g., methyl).

[0012] Any of the compounds of formula (I) provided herein can be hydroxyl at the R8 position.

[0013] Exemplary stilbene compounds are provided below: [Chemical Structure] (4-[(E)-2-(3,5-Dihydroxyphenyl)ethenyl]benzene-1,3-diol), [Chemical Structure] (5-[(E)-2-(4-Hydroxyphenyl)ethenyl]benzene-1,3-diol), [Chemical Structure] (1,2,3-Trimethoxy-5-[(Z)-2-phenylethenyl]benzene), [Chemical Structure] (1-[(E)-2-(2,4-Dimethoxyphenyl)ethenyl]-3,5-dimethoxybenzene), [Chemical Structure] ([4-[(E)-2-(3,5-Diacetyloxyphenyl)ethenyl]phenyl]acetate), and [Chemical Structure] (5-[(E)-2-(3-Hydroxy-4-methoxyphenyl)ethenyl]benzene-1,3-diol).

[0014] In some cases, stilbene compounds are not resveratrol.

[0015] In some embodiments, pharmaceutically acceptable carriers used in any of the pharmaceutical compositions described herein may include a nonionic surfactant having a hydrophilic-lipophilic balance value (HLB value) greater than 5, a solvent (e.g., polyols or polymers thereof, e.g., ethylene glycol, polyoxyethylene, polyethylene glycol, or propylene glycol), acetone, dimethyl sulfoxide, glycerol, alcohol, or a combination thereof), a buffer, an oil phase (e.g., soybean oil and / or medium-chain triglycerides), or a combination thereof. In some specific examples, the nonionic surfactant includes polyoxyl castor oil (e.g., polyoxyl 35 castor oil) and / or polyoxyl hydrogenated castor oil (e.g., polyoxyl 40 hydrogenated castor oil).

[0016] In some embodiments, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier and a plurality of nanoparticles formed of a stilbene compound such as formula (A) or a pharmaceutically acceptable salt thereof. In some examples, the nanoparticles have an average particle size of up to 200 nm and a polydispersity index (PDI) value of up to 0.4. In some cases, the pharmaceutically acceptable carrier comprises one of the nonionic surfactants disclosed herein. Alternatively, the weight ratio of the compound of formula (A) or a pharmaceutically acceptable salt thereof to the nonionic surfactant is in the range of 1:1 to 1:1000. In certain examples, the weight ratio is in the range of 1:1 to 1:500.

[0017] In some embodiments, the pharmaceutical compositions described herein may further include additional activators for reducing pigmentation, other than the stilbene compound, such as the compound of formula (A) or a pharmaceutically acceptable salt thereof. In other embodiments, the stilbene compound, such as the compound of formula (A) or a pharmaceutically acceptable salt thereof, is the sole activator in the pharmaceutical composition for reducing pigmentation.

[0018] In some embodiments, the methods described herein may further include administering to the subject an additional activator for reducing pigmentation, which is different from the stilbene compound, such as the compound of formula (A) or a pharmaceutically acceptable salt thereof. In other embodiments, the method does not administer such an additional activator to the subject.

[0019] In some cases, the pharmaceutical composition may further contain antioxidants. Alternatively, the pharmaceutical composition may not contain any oxidizing agents.

[0020] In some embodiments, the pharmaceutical compositions provided herein do not contain water.

[0021] Any of the pharmaceutical compositions disclosed herein can be administered to a skin site by intradermal injection or topical application.

[0022] Any of the pharmaceutical compositions disclosed herein for use in reducing pigmentation in a subject, and the use of such pharmaceutical compositions for manufacturing a drug for use in reducing pigmentation in a subject are also provided herein.

[0023] Details of one or more embodiments of the present invention are described below. Other features or advantages of the present invention will become apparent from the following drawings and detailed descriptions of some embodiments, as well as from the appended claims. [Brief explanation of the drawing]

[0024] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by referring to the drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0025] [Figure 1]This figure shows the changes in melanin production in mouse melanoma B16-F10 cells treated with various 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol preparations. [Figure 2] This bar graph shows the inhibitory activity of various 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol preparations on melanin production in mouse melanoma B16-F10 cells. [Figure 3] These are photographs showing the darkening of the back skin of mice after UVB irradiation and treatment with the indicated compositions. N=3-4 / group. Data were analyzed by Student's t-test. Compared to control, #p<0.05, ##p<0.01; compared to UVB, *p<0.05, **p<0.01. [Figure 4A-4C]This includes graphs showing the reduction of melanin in mice treated with a 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition. Figure 4A is a graph showing the reduction in melanin content in mouse skin after UVB irradiation and treatment with the exemplary 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition shown. N=3-4 / group. Data were analyzed by Student's t-test. Compared to control, #p<0.05, ##p<0.01; compared to UVB, *p<0.05, **p<0.01. Figure 4B is a graph showing the reduction in melanin content in mouse skin after treatment with the exemplary 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition shown via UVB irradiation and intradermal injection. N=4-8 / group. Data were analyzed by Student's t-test. Compared to the control, #p<0.05, ##p<0.01, ###p<0.001; compared to UVB, *p<0.05, **p<0.01. Figure 4C is a graph showing the reduction in melanin content in mouse skin after treatment with an exemplary 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition shown via UVB irradiation and topical administration. N=4~8 / group. Data were analyzed by Student's t-test. Compared to the control, #p<0.05, ##p<0.01, ###p<0.001; compared to UVB, *p<0.05, **p<0.01. [Figure 5] This graph shows the changes in melanin production in mouse melanoma B16-F10 cells treated with 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol versus 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and other skin-whitening ingredients. [Figure 6A-6C]Includes figures showing the effect of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol on α-MSH-induced melanin production compared to other components such as arbutin and hydroquinone. Figure 6A: Graph showing that α-MSH induces melanin production. Figure 6B: Graph showing changes in melanin production in mouse melanoma B16-F10 cells treated with the indicated α-MSH and various 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol compositions and arbutin and hydroquinone compositions. Figure 6C: Graph showing changes in melanin production in mouse melanoma B16-F10 cells treated with the indicated α-MSH and various 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol compositions and arbutin compositions. [Figures 7A-7D] Figures showing the in vitro melanin-reducing activity and cytotoxicity of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, arbutin, and hydroquinone are included. Figure 7A: Melanin levels in cells induced by α-MSH and treated with different concentrations of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol. Figure 7B: Survival rates of cells treated with different concentrations of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol. Figure 7C: Melanin levels in cells induced by α-MSH and treated with 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, arbutin, or hydroquinone. Figure 7D: Survival rates of cells treated with 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, arbutin, or hydroquinone. N=3 / group, ND=not detected. Data were analyzed by Student's t-test. #p<0.05, ##p<0.01, ###p<0.001 compared to vehicle, *p<0.05, **p<0.01, ###p<0.01 compared to α-MSH. [Modes for carrying out the invention]

[0026] 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and its derivatives, such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, are polyphenol compounds that can be found in various natural sources, for example, in the fruit and skin of grapes. 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, also known as E-2,3',4,5'-tetrahydroxystilbene, [ka] It is a polyhydroxystilbene compound having the structure shown. Various biological activities have been reported for 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol derivatives, such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, including antitumor and neuroprotective activities.

[0027] Melanin is a group of natural pigments found in most living organisms. Melanin pigment is produced in a specialized group of cells known as melanocytes. There are five basic types of melanin: eumelanin, pheomelanin, neuromelanin, allomelanin, and piomelanin. The most common type is eumelanin, which has two subtypes: brown eumelanin and black eumelanin. Eumelanin is produced through a multi-step chemical process known as melanogenesis, in which polymerization follows the oxidation of tyrosine amino acid.

[0028] Skin pigmentation is caused by the accumulation of melanin-containing melanosomes in the basal layer of the epidermis. In mammalian skin, melanin formation is initiated by exposure to UV radiation, darkening the skin. Differences in skin pigmentation are due to both the relative ratio of eumelanin (brown / black) to pheomelanin (yellow / red) and the number of melanosomes within melanocytes.

[0029] This specification describes how 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and its derivatives, such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, successfully reduce melanin levels in cell cultures and animal models, and how 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol is reported to be more effective in reducing melanin levels compared to the known skin whitening agents arbutin and hydroquinone. Therefore, it is expected that 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and its derivatives, such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, may be effective in reducing skin pigmentation, potentially providing a variety of therapeutic and / or cosmetic effects. For example, 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol can reduce oxidative damage, premature skin aging, abnormal or excessive pigmentation, and other age-related disorders.

[0030] Accordingly, this disclosure provides a pharmaceutical composition comprising one or more stilbene compounds for use in reducing skin pigmentation in subjects requiring such treatment.

[0031] definition All definitions defined and used herein should be understood to control dictionary definitions, definitions in literature incorporated by reference, and / or the ordinary meanings of the terms defined.

[0032] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter they cite, and in some cases may encompass the entirety of the references.

[0033] When used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless otherwise explicitly stated.

[0034] When used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements thus combined, i.e., elements that exist constitutively in some cases and disjunctively in others. Multiple elements listed using "and / or" should be interpreted similarly, i.e., "one or more" of the elements thus combined. Other elements other than those specifically identified by the "and / or" clause may exist at their discretion, whether related to or unrelated to those specifically identified elements. Thus, as a non-restrictive example, a reference to "A and / or B," when used with open-ended language such as "comprising," may refer in one embodiment to A only (including elements other than B at their discretion), in another embodiment to B only (including elements other than A at their discretion), in yet another embodiment to both A and B (including other elements at their discretion), and so on.

[0035] Where used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when dividing items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of a number of elements or a list of elements, but also including two or more, and optionally including additional items not on the list. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of,” or, where used in claims, “consisting of,” should be interpreted as including exactly one element of a number of elements or a list of elements. In general, where used herein, the term “or” should be interpreted as indicating an exclusive substitution (i.e., “one or the other, but not both”) only when preceded by an exclusive term such as “either,” “one of,” “only one of,” or “exactly one of.” When used in patent claims, "consisting essentially of" shall have its ordinary meaning as it is used in the field of patent law.

[0036] As used herein in the 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 of the elements in the list of elements, but not necessarily including at least one of each element specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements to which the phrase “at least one” refers, whether related to or unrelated to those specifically identified elements, at the discretion of the definition. Therefore, as a non-restrictive 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”) may, in one embodiment, mean at least one A (including two or more A's of any choice) and no B (and optionally including elements other than B); in another embodiment, mean at least one B (including two or more B's of any choice) and no A (and optionally including elements other than A); and in yet another embodiment, mean at least one A (including two or more A's of any choice) and at least one B (including two or more B's of any choice) (and optionally including other elements), and so on.

[0037] Exemplary C 1~4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. 2~4 Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, alkynyl, aralkyl, alkyl halide, heteroalkyl, aryl, heterocyclyl, cycloalkyl, cycloalkenyl, and cycloalkynyl.

[0038] As used herein, the term "alkyl" refers to a linear, saturated, acyclic, monovalent hydrocarbon radical or a branched, saturated, acyclic, monovalent hydrocarbon radical having 1 to 3 carbon atoms bonded to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, or 1-methylethyl (isopropyl). An optionally substituted alkyl radical is an alkyl radical that is optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halo, cyano, nitro, oxo, hydroxyl, thio, or amino, as long as the valence allows.

[0039] As used herein, the term “alkenyl” refers to a linear, acyclic, monovalent hydrocarbon radical or a branched, acyclic, monovalent hydrocarbon radical having an intercarbon double bond and two or three carbon atoms bonded to the rest of the molecule by a single bond, such as ethenyl or propenyl. Optionally substituted alkenyl radicals are alkenyl radicals that are optionally substituted with one, two, or three substituents independently selected from the group consisting of halo, cyano, nitro, hydroxyl, thio, or amino, as far as the valence allows. As described herein, an alkene can be a Z or E alkene. Alkenes shown as Z alkenes include the E isomer of the alkene.

[0040] "Amino" refers to a radical of the formula -NRbRc (where Rb and Rc are each hydrogen atoms), or an alkyl radical containing 1 to 3 carbon atoms as defined above. The alkyl portion of an optionally substituted amino radical is optionally substituted with respect to the alkyl radical as defined above.

[0041] A "thiol" is a radical of the formula -SRd (wherein Rd is hydrogen), or an alkyl radical containing 1 to 3 carbon atoms, as defined above. The alkyl portion of an optionally substituted thiol radical is optionally substituted with respect to the alkyl radical, as defined above.

[0042] I. Stilbene compounds In some embodiments, stilbene compounds for use in reducing pigmentation in subjects are provided herein. As reported herein, an exemplary stilbene compound, 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, exhibited high bioactivity in reducing melanin with little to no cytotoxicity both in vivo and in vitro. The melanin-reducing activity achieved by 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol was significantly greater than that of arbutin and HQ, known compounds for use in skin whitening or reducing pigmentation.

[0043] When used herein, stilbene compounds have the following core structure: [ka] The term refers to a compound having and including a suitable substitution at one or more positions on one or both of the ring structure. Stilbene compounds for use in any of the methods disclosed herein may include 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and its derivatives, or pharmaceutically acceptable salts thereof. In some examples, the stilbene compound is a 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol derivative such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, or other derivatives provided herein.

[0044] In one embodiment, the stilbene compound is given by the following formula (I): [ka] Or it may have a pharmaceutically acceptable salt thereof. In formula (I), each of R1, R2, R3, R4, and R8 is independently H, halogen, thiol, hydroxyl, C 1~4 Alkyl, C 2~4is alkenyl, OR5, COOR6, or -OC(=O)R7, wherein each of R5, R6, and R7 is independently H or C 1~4 alkyl (e.g., methyl).

[0045] In some examples, the compound of formula (I) provided herein has the structure of formula (I-A): [Chemical formula] or a pharmaceutically acceptable salt thereof, wherein R1-R4 are independently or together H, halogen (e.g., Cl or F), thiol, hydroxyl, C 1~4 alkyl, C 2~4 is alkenyl, OR5, COOR6, or -OC(=O)R7, wherein each of R5, R6, and R7 is independently H or C 1~4 alkyl (e.g., methyl). Alternatively, each of R1-R4 can independently be hydroxyalkyl, thiol, alkoxy, or alkaryl.

[0046] In some cases, one or more of R1-R4 can be hydroxyl. Alternatively, one or more of R1-R4 can be methoxy. In other cases, one or more of R1-R4 can be OC(=O)R7, wherein R7 is H or C 1~4 alkyl (e.g., methyl).

[0047] Exemplary stilbene compounds are provided herein. In one particular example, the stilbene compound is a 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol derivative, e.g., 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol, or a pharmaceutically acceptable salt thereof.

[0048] The compounds described herein may contain one or more chiral centers and thus may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers, including a racemic mixture and a mixture concentrated in one or more stereoisomers. The isomers may be isolated from the mixture by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts, or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Wilen et al., Tetrahedron 33:2725 (1977), Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure also includes the compounds described herein as individual isomers substantially free from other isomers, and / or mixtures of various isomers.

[0049] II. Pharmaceutical Compositions Any of the stilbene compounds disclosed herein may be mixed with one or more pharmaceutically acceptable carriers to form a pharmaceutically acceptable composition that can be used to reduce pigmentation in subjects requiring treatment. Examples of pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. In particular, exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare such pharmaceutically acceptable salts and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from suitable inorganic bases (e.g., sodium hydroxide, barium hydroxide, iron(II) hydroxide, iron(III) hydroxide, magnesium hydroxide, calcium hydroxide, aluminum hydroxide, ammonium hydroxide, potassium hydroxide, cesium hydroxide, or lithium hydroxide) or suitable organic bases (e.g., pyridine, methylamine, imidazole, benzimidazole, histidine, phosphazene bases, or hydroxides of organic cations such as quaternary ammonium hydroxide and phosphonium hydroxide). Furthermore, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth salts such as lithium, sodium, potassium, or calcium salts.

[0050] The pharmaceutical compositions described herein 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 Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover. Such carriers, excipients, or stabilizers may enhance one or more properties of the active ingredients in the compositions described herein, such as bioactivity, stability, bioavailability, and other pharmacokinetics and / or bioactivity.

[0051] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; benzoates, sorbates, and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins. It may contain proteins such as robulin; 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® (polysorbate), PLURONICS® (nonionic surfactant), or polyethylene glycol (PEG).

[0052] In some embodiments, the pharmaceutically acceptable carrier may include a nonionic surfactant having a hydrophilic-lipophilic balance value (HLB value) greater than 5, a solvent, a buffer, an oil phase (e.g., medium-chain triglycerides or soybean oil), or a combination thereof. In some examples, the pharmaceutically acceptable carrier may include a nonionic surfactant that can form nanoparticles with the stilbene compound. Alternatively, the pharmaceutical composition containing the stilbene compound may not contain nanoparticles. In one embodiment, the pharmaceutical composition has a pH value in the range of 2.5 to 8.0 when dissolved in water.

[0053] In some embodiments, the pharmaceutically acceptable carrier may further include a co-surfactant, co-solvent, antioxidant, or any combination thereof. In some cases, the pharmaceutically acceptable carrier may include a lipophilic solvent, such as triacetin, medium-chain triglycerides, or oils.

[0054] In some cases, the pharmaceutical compositions provided herein may not contain water. Alternatively, the pharmaceutical compositions provided herein may not contain antioxidants.

[0055] Nonionic surfactants The pharmaceutical compositions disclosed herein may comprise one or more nonionic surfactants capable of forming nanoparticles with the activators and / or hydrophilic therapeutic agents disclosed herein. The nonionic surfactants used in this technology are beneficial at least in the formation of nanoparticles in the composition. In some embodiments, the nanoparticles have the effect of encapsulating the activators and / or hydrophilic therapeutic agents.

[0056] Nonionic surfactants used in any of the pharmaceutical compositions disclosed herein preferably have a hydrophilic-lipophilic balance (HLB) value greater than 5. Nonionic surfactants are used in this technology in the ratios described above. Examples of nonionic surfactants include, but are not limited to, polysorbate 80 (Tween® 80), polyoxyl 15 hydroxystearate (Solutol® HS-15), polyoxyethylene castor oil derivatives (e.g., polyoxyl 35 castor oil (Kolliphor® ELP), polyoxyl 40 hydrogenated castor oil (Kolliphor® RH40), and polyoxyl 60 hydrogenated castor oil (Cremophor® RH60)), polyoxyethylene (12) glyceryl laurate (UNIGLY, ML-212), polyoxyl 20 stearate (Myrj® S20), polyoxyl 40 stearate (Myrj® S40), polyoxyl cetostearyl ether 12 (Kolliphor® CS12), and polyoxyl 20 cetostearyl ether (Kolliphor® CS20).

[0057] In some cases, the nonionic surfactant may be a polyoxyethylene derivative, which is also known as a PEGylated excipient. In some cases, the polyoxyethylene derivative is a PEG castor oil derivative, which is obtained by reacting varying amounts of ethylene oxide with either castor oil or hydrogenated castor oil. Examples include PEG-35 castor oil and PEG-40 hydrogenated castor oil. In other cases, the polyoxyethylene derivative is a PEG ester, which can be produced by reacting polyethylene glycol with a fatty acid. Examples include PEG-40 stearate and PEG-15 hydroxystearate. In some cases, the esters are sorbitan fatty acid esters (e.g., PEG-20 sorbitan monooleate, PEG-40 sorbitan monooleate, PEG-60 sorbitan monooleate, PEG-80 sorbitan monooleate, PEG-20 sorbitan isostearate, PEG-30 sorbitan tetraoleate, PEG-40, PEG-60 sorbitan tetraoleate, PEG-40 sorbitan diisostearate, or PEG-60 sorbitan tetrastearate), alkyl glycerides (e.g., PEG-8 caprylic / capringlyceride, PEG-32 hydrogenated palm glyceride) The ester may be a ceride (or PEG-32 lauroyl glyceride), or an alkyl ether (e.g., PEG-6 cetostearyl ether, PEG-12 cetostearyl ether, PEG-20 cetostearyl ether, PEG-10 cetyl ether, PEG-20 cetyl ether, PEG-4 lauryl ether, PEG-23 lauryl ether, PEG-2 oleyl ether, PEG-10 oleyl ether, PEG-20 oleyl ether, PEG-2 stearyl ether, PEG-10 stearyl ether, PEG-21 stearyl ether, or PEG-100 stearyl ether). In other cases, the ester may be a laurate (e.g., PEG-2 laurate, PEG-4 laurate, PEG-6 laurate, PEG-8 laurate).PEG-9-14 laurate, PEG-20 laurate, PEG-32-150 laurate, or PEG-12 glyceryl laurate), dilaurate (e.g., PEG-2 dilaurate, PEG-4 dilaurate, or PEG-6-150 dilaurate), or stearate (e.g., PEG-2 stearate, PEG-3 stearate, PEG-4 stearate, PEG-4 isostearate, PEG-5-7 stearate, PEG-6-8 isostearate, PEG-8 stearate, PEG-9 stearate, PEG-10 stearate, PEG-10 isostearate, PEG-12 isostearate, PEG-12-18 stearate, PEG-20 stearate, PEG-23-45 stearate, P stearate It may be EG-40, PEG-50 stearate, PEG-100 stearate, PEG-75-150 stearate, or PEG-6 and PEG-32 palmitostearate), glyceryl stearate (e.g., glyceryl stearate / PEG-40 stearate, glyceryl stearate / PEG-100 stearate, PEG-120 glyceryl stearate, PEG-20 methyl glucose sesquistearate, or PEG-25 propylene glycol stearate), distearate (e.g., PEG-2 distearate, PEG-3-120 distearate, PEG-150 distearate, or PEG-175 distearate), or hydroxystearate (e.g., PEG-15 hydroxystearate).

[0058] In some cases, the nonionic surfactant may be a castor oil derivative (e.g., PEG-35 castor oil or PEG-40 castor oil) or hydrogenated castor oil (e.g., PEG-40 hydrogenated castor oil, PEG-54 hydrogenated castor oil, or PEG-60 hydrogenated castor oil).

[0059] Additional examples of nonionic surfactants include PEG-15 cocamine, polyethylene glycol succinate with vitamin E, PEG-75 lanolin, and PEG-120 methyl glucose dioleate.

[0060] Nanoparticles and preparations thereof In some embodiments, the pharmaceutical compositions disclosed herein may comprise nanoparticles formed from one or more nonionic surfactants disclosed herein and one or more stilbene compounds disclosed herein (e.g., 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol).

[0061] In some embodiments, the nanoparticles may contain a nonionic surfactant and a stilbene compound in a preferred weight ratio that yields a preferred particle size and / or a preferred polydispersity index (PDI) value. For example, a preferred weight ratio of stilbene compound to nonionic surfactant may range from about 1:1 to 1:1000, for example, about 1:1 to 1:800, about 1:1 to 1:600, about 1:1 to 1:500, about 1:1 to 1:200, or about 1:1 to 1:100. In other examples, suitable weight ratios of stilbene compounds to nonionic surfactants are 1:5 to 1:1000, for example, 1:5 to 1:150, 1:5 to 1:200, 1:5 to 1:300, 1:5 to 1:400, 1:5 to 1:500, approximately 1:8 to 1:10, approximately 1:8 to 1:20, approximately 1:8 to 1:40, approximately 1:8 to 1:100, 1:8 to 1:150, 1:8 to 1:200, 1:8 to 1:300, 1:8 to 1:400, 1:8 to 1:500, approximately 1:10 to 1:20, approximately 1:10 to 1:40, approximately 1:10 to 1:100, 1:10 to 1:150, 1:10 to 1:200, 1:10 to 1:300, 1:10~1:400, 1:10~1:500, approx. 1:20~1:40, approx. 1:20~1:100, 1:20~1:150, 1:20~1:200, 1:20~1:300, 1:20~1:400, 1:20~1:500, approx. 1:40~1:100, 1:40~1:1 The time ranges are 50, 1:40-1:200, 1:40-1:300, 1:40-1:400, 1:40-1:500, 1:100-1:150, 1:100-1:200, 1:100-1:300, 1:100-1:400, 1:100-1:500, or 1:150-1:500.

[0062] In some cases, the pharmaceutical compositions disclosed herein may include nanoparticles having an average size of up to 200 nm, for example, up to 150 nm, up to 100 nm, up to 50 nm, or up to 20 nm. In some cases, the average size of the nanoparticles may be in the range of 5 to 200 nm, for example, 5 to 150 nm, 5 to 100 nm, 5 to 50 nm, or 5 to 20 nm.

[0063] Alternatively, the nanoparticles in the pharmaceutical composition may have a suitable PDI value, for example, up to 0.4. In some examples, the PDI value may be in the range of 0.05 to 0.4, for example, 0.05 to 0.35, 0.05 to 0.3, 0.05 to 0.25, 0.05 to 0.2, 0.05 to 0.15, or 0.05 to 0.1.

[0064] Any of the nanoparticles disclosed herein can be prepared by conventional methods or as disclosed herein. An example is provided below. A suitable amount of an activator or hydrophilic therapeutic agent can be mixed with a suitable solvent (e.g., alcohol (e.g., methanol, ethanol, propanol), acetonitrile, chlorinated solvent (e.g., dichloromethane, chloroform), diethyl ether, and ethyl acetate), and the mixture can be stirred at a suitable temperature (e.g., 150-500 rpm) until the activator or hydrophilic therapeutic agent, as disclosed herein, is completely dissolved in the solvent and forms a solution. A suitable amount of a pharmaceutically acceptable nonionic surfactant (e.g., as disclosed herein) can be added to the solution. The resulting mixture can be stirred at a suitable temperature (e.g., 100-300 rpm) to evaporate the solvent. Once the solvent has completely evaporated, a suitable amount of a pharmaceutically acceptable aqueous solution (e.g., physiological saline) can be added to the mixture to form micelles containing the activator or hydrophilic agent. The resulting particles can be filtered using a suitable filter (e.g., a 0.2 μm filter), and the filtered solution containing drug-containing micelles can be stored in a dark place in a refrigerator for later use.

[0065] Further details relating to the preparation of micelles in this technology can be found in U.S. Patent No. 10,610,496, which incorporates relevant disclosures by reference with respect to the subject matter and objectives referenced herein.

[0066] Additional components of pharmaceutical compositions In embodiments, the pharmaceutical compositions disclosed herein may further include cosolvents (e.g., to increase the solubility of the drug), suspending agents (e.g., to reduce the sedimentation rate of the drug), oil phase excipients (e.g., to increase the stability of the pharmaceutical composition and the solubility of the drug), antimicrobial and antiseptic agents, or combinations thereof.

[0067] In some cases, the cosolvent may include polyethylene glycol, propylene glycol, ethanol, and other cosolvents, or combinations thereof. Alternatively, the suspending agent may include sodium alginate, glycerol, sodium carboxymethylcellulose, mannitol, and other suspending agents, or combinations thereof.

[0068] In some cases, the oil phase excipient may include unsaturated fatty acids, glycerol, triglycerides, and other oil phase excipients, or combinations thereof. For example, the oil phase excipient may include one or more unsaturated fatty acids, which may be oleic acid, castor oil, sesame oil, cottonseed oil, soybean oil, safflower oil, corn oil, and other unsaturated fatty acids, or combinations thereof. In some cases, the oil phase excipient may include triglycerides, which may be medium-chain triglycerides.

[0069] In some embodiments, the pharmaceutical compositions disclosed herein may include local anesthetics. Examples include, but are not limited to, amides, para-aminobenzoic acid esters, and amino ethers, or combinations thereof. In some examples, the local anesthetic may include amides such as dibucaine, lidocaine, mepivacaine HCl, bupivacin HCl, pirocaine HCl, prilocaine HCl, digammacaine, and oxethazaine, or combinations thereof. In other examples, the local anesthetic may include para-aminobenzoic acid esters such as butacaine, dimethocaine, and tutocaine, or combinations thereof. In yet another example, the local anesthetic may include amino ethers such as quinisocaine and pramocaine, or combinations thereof.

[0070] Alternatively, the pharmaceutical compositions disclosed herein may contain one or more antioxidants. Examples, but not limited to, include beta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C (ascorbic acid), vitamin E, citric acid, sodium thiosulfate, propyl gallate, uric acid, nitric oxide, nitrogen oxides, pyruvate, catalase, superoxide dismutase, glutathione peroxidase, N-acetylcysteine, and naringenin, or combinations thereof. Alternatively, the pharmaceutical compositions may not contain antioxidants.

[0071] Alternatively, or in addition, the pharmaceutical compositions provided herein may further comprise a second activator, which is not a stilbene compound disclosed herein. In some cases, the second activator may be encapsulated in nanoparticles.

[0072] In some embodiments, the second activator may include hydrophilic therapeutic agents, such as green tea extract, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine, L-carnitine, synephrine, chlorogenic acid, or a combination thereof.

[0073] In some embodiments, the pharmaceutical compositions described herein may be formulated as topical formulations, for example, as creams, lotions, or gels for topical application. Such creams, lotions, or gels may be formulated using ingredients known in the art to be suitable for topical administration. In some examples, the topical formulation may be in the form of a transdermal patch.

[0074] In other embodiments, the pharmaceutical compositions described herein may be formulated as injectable formulations (e.g., for intradermal injection). Sterile injectable compositions, such as sterile aqueous or oily suspensions for injection, can be formulated according to techniques known in the art using suitable dispersants or wetting agents (e.g., polysorbate 80) and suspending agents. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic salines. In addition, sterile fixative oils have conventionally been used as solvents or suspension media (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, as well as naturally pharmaceutically acceptable oils such as olive oil or castor oil, particularly their polyoxyethylated versions, are useful in the preparation of injectable formulations. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants. Other commonly used surfactants, such as Tweens or Spans or other similar emulsifiers or bioavailability enhancers (commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms), may also be used for formulation purposes.

[0075] In some embodiments, the pharmaceutical composition is in the form of a parenteral dosage form, and the target is humans. For example, a composition formulated for injection may be in the form of a powder (e.g., lyophilized powder), a sterile suspension, an injectable solution, an injectable emulsion, or an intravenous infusion. In some examples, the composition may be placed in a microneedle device for injection. Alternatively, the composition may be formulated for transdermal administration, for example, in the form of an ointment, lotion, topical agent, cream, gel, bandage, emulsion, film, patch, compress, poultices, or topical powder.

[0076] III. Methods to reduce hyperpigmentation Stilbene compounds (e.g., 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol derivatives, e.g., 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol) or any of the pharmaceutical compositions disclosed herein can be used to reduce hyperpigmentation in subjects such as human patients in need of treatment, for example, to treat, delay the onset of, or alleviate hyperpigmentation. Hyperpigmentation refers to any spots on the skin that appear darker than the natural skin tone of the same subject due to the overproduction of the brown pigment melanin, which can be caused by certain events or causes, such as sun exposure, inflammation, or hormonal effects. Hyperpigmentation can be seen in age spots or sunspots.

[0077] To carry out the methods disclosed herein, an effective amount of the pharmaceutically active compositions described herein may be administered to a subject in need of treatment (e.g., a human) via a preferred route, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0078] In some embodiments, the pharmaceutical composition may be administered to a subject, such as a human subject, by topical administration or intradermal injection. In some cases, a pharmaceutical composition containing 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol or similar may be applied to a skin site where reduction of pigmentation is required. As used herein, “effective dose” refers to the amount of each activator required to produce a therapeutic effect in a subject, either alone or in combination with one or more other activators. Determining whether the amount of antibody has achieved a therapeutic effect will be obvious to those skilled in the art. The effective dose will vary, as recognized by those skilled in the art, depending on the specific condition being treated, the severity of the condition, the individual patient's parameters including age, health status, size, sex, and weight, the duration of treatment, the characteristics of combination therapy (if any), the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed by routine experimentation alone. In general, it is preferable to use the maximum dose of the individual components or their combinations, i.e., the safest highest dose according to sufficient medical judgment.

[0079] Empirical considerations, such as half-life, generally contribute to dosage determination. For example, using antibodies adapted to the human immune system, such as humanized or fully human antibodies, can extend the antibody half-life and prevent the antibody from being attacked by the host immune system. The frequency of administration may be determined and adjusted throughout the course of therapy, and is generally not essential, but is based on the treatment and / or suppression and / or enhancement and / or delay of the target disease / impairment. Alternatively, a sustained-release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0080] For the purposes of this disclosure, the appropriate dosage of a pharmaceutical composition as described herein depends on the specific activator and optionally hydrophilic agent used, the type and severity of the disease / disorder, whether the composition is administered for preventive or therapeutic purposes, past therapies, the patient's medical history and response to the activator, and the judgment of the attending physician. Typically, the clinician administers the pharmaceutical compositions disclosed herein until a dosage is reached that achieves the desired outcome.

[0081] In some embodiments, the desired outcome is the reduction or elimination of pigmentation on a skin site, which can be achieved by reducing the level of melanin in skin cells. A method for determining whether a dosage has produced the desired outcome will be apparent to those skilled in the art. Specific dosage plans used in the methods described herein, i.e., dosage, timing, and repetitions, depend on the specific subject and their medical history. The therapeutic efficacy for the target disease / disorder can be determined by methods well known in the art.

[0082] In some cases, stilbene compounds such as 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol are administered in suitable doses, e.g., 0.01 to 100 mg / cm³ based on the therapeutic area. 2 Alternatively, it may be applied to skin areas where reduction of hyperpigmentation is needed (e.g., topically, via transdermal patch or intradermal injection) at a dose of 0.01 to 100 mg / kg based on the subject's body weight.

[0083] In some cases, subjects such as human subjects may have pigmentation caused by sun exposure. Such subjects may have sunspots, age spots, liver spots, or solar lentigines. A pharmaceutical composition containing an effective amount of a stilbene compound such as 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol derivative (e.g., 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol), or any of those disclosed herein, may be applied to sunspot, age spots, liver spots, or solar lentigines skin areas of subjects (e.g., human patients) to reduce pigmentation.

[0084] In some cases, subjects such as human patients for the treatments provided herein may have post-inflammatory hyperpigmentation (PIH), which may be caused by melanocytes that produce excessive melanin in response to inflammation, injury, or certain procedures.

[0085] In other cases, subjects such as human patients may have melasma, a skin condition that most commonly occurs on the face. In some cases, subjects are pregnant women with melasma.

[0086] As used herein, the term “to treat” means applying or administering a composition comprising one or more activators to a target disease or disorder, symptoms of a disease / disorder, or a subject predisposed to a disease / disorder, for the purpose of treating, curing, alleviating, reducing, modifying, correcting, improving, or acting upon the disorder, symptoms of a disease, or predisposition to a disease or disorder.

[0087] Alleviating a target disease / disorder includes delaying the development or progression of the disease, reducing the severity of the disease, or extending survival time. Alleviating a disease or extending survival time does not necessarily require a curative outcome. As used herein, “delaying” the development of a target disease or disorder means postponing, inhibiting, slowing, suppressing, stabilizing, and / or delaying the progression of the disease. This delay may be of varying lengths depending on the disease history and / or the individual being treated. A method for delaying or alleviating the development of a disease, or delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of those symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical trials using a sufficient number of subjects to give statistically significant results.

[0088] The “development” or “progression” of a disease means the initial symptoms of the disease and / or the subsequent progression. Disease development can be detected and determined using standard clinical techniques known in the art. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological course of symptoms. “Development” includes onset, recurrence, and onset. As used herein, “onset” or “onset” of a target disease or disorder includes the initial onset and / or recurrence.

[0089] Any of the pharmaceutical compositions disclosed herein may be administered to a subject 1 to 4 times daily for a suitable period, e.g., 1 month to 1 year. The duration of treatment may depend on the treatment outcome. In some cases, treatment may be terminated when the pigmentation in the skin area is reduced to a certain level, resulting in the skin spots having a natural skin tone. Treatment may be restarted when the pigmentation reappears.

[0090] IV. Kits for use in reducing hyperpigmentation This disclosure also provides kits for use in treating or alleviating hyperpigmentation in subjects requiring treatment. Such kits may include one or more containers containing the pharmaceutical compositions disclosed herein. In some cases, the pharmaceutical compositions may be used in conjunction with a second therapeutic agent.

[0091] In some embodiments, the kit may include instructions for use in accordance with any of the methods described herein. The included instructions may include instructions for administering a pharmaceutical composition for treating, delaying the onset of, or alleviating hyperpigmentation in a subject, and optionally a second therapeutic agent. The kit may further include instructions for selecting an individual suitable for treatment, for example, based on identifying whether the individual has hyperpigmentation by following a routine procedure.

[0092] Instructions for use relating to the use of the pharmaceutical compositions disclosed herein generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions for use accompanying a kit of the present invention are typically those printed on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0093] The label or accompanying information indicates that the composition is used to treat, delay, and / or alleviate hyperpigmentation. Instructions for use for carrying out any of the methods described herein may be provided.

[0094] The kit of the present invention is contained in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or infusion devices such as minipumps, are also intended. The kit may have a sterile access port (e.g., the container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous needle). The container may also have a sterile access port (e.g., the container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous needle).

[0095] The kit may optionally provide additional components such as buffers and interpretation information. Typically, the kit includes a container and labels or accompanying documents on or associated with the container. In some embodiments, the present invention provides a product comprising the contents of the kit described above.

[0096] General techniques Unless otherwise indicated, the implementation of this disclosure will involve conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, within the scope of the skills in the relevant art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991)、Short Protocols in Molecular Biology(Wiley and Sons,1999)、Immunobiology(CAJaneway and P.Travers,1997)、Antibodies(P.Finch,1997)、Antibodies: A Practice Approach(D.Catty.,ed.,IRL Press,1988-1989)、Monoclonal antibodies: a practical approach(P.Shepherd and C.Dean,eds.,Oxford University Press,2000)、Using antibodies:a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999). JDCapra,eds.Harwood Academic Publishers,1995)、DNA Cloning:A Practical Approach,Volumes I and II(DNGlover ed.1985)、Nucleic Acid Hybridization(BDHames&S.J.Higgins eds.(1985>>、Transcription and Translation(BDHames&S.J.Higgins,eds.(1984>>、Animal Cell Culture(RIFreshney,ed.(1986>>、Immobilized Cells and Enzymes(lRL Press,(1986>>) and B.Perbal,A Practical Guide To Molecular Cloning(1984);FMAusubel et al.(eds.) With a strong introduction to this trend.

[0097] Without further detail, those skilled in the art will be able to make the most of the present invention based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and in no way limit the remainder of this disclosure. All publications referenced herein are incorporated by reference with respect to the purposes or subjects referenced herein. [Examples]

[0098] The following description merely represents certain preferred embodiments of the present disclosure and is not intended to limit the scope of the patent application of the present disclosure. Accordingly, any modifications or alterations that do not deviate from the spirit of the disclosure herein should be included within the scope of the patent application of the present disclosure.

[0099] Example 1. Anhydrous 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol-ELP preparation preparation A desired amount of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol was taken and dissolved in a specific volume of ethyl acetate, and the mixture was stirred at room temperature until the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol was completely dissolved. Then, polyoxyl-35-castor oil (Kolliphor® ELP, or ELP) was added and the mixture was stirred into an anhydrous 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol-ELP formulation.

[0100] Table 1 provides the components of three 4-[(E)-2-(3,5-dihydroxyphenyl)benzene-1,3-diol-ELP formulations having different 4-[(E)-2-(3,5-dihydroxyphenyl)benzene-1,3-diol-ELP weight ratios, as shown. [Table 1]

[0101] Visual Analysis The 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol-ELP formulations listed in Table 1 were placed on a stand for one day, and then their appearance was observed visually. Group A (1:1.6 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol:ELP) presented a homogeneous composition without any precipitation, Group B showed a small amount of precipitation, and Group C was very turbid. Group A remained stable, clear, and precipitation-free after a period of up to 5 days.

[0102] Example 2. A 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol-ELP formulation containing water and / or ascorbic acid. preparation In this example, all samples were prepared as described in Example 1, except that water and / or 0.1% ascorbic acid were added to form aqueous solutions. Details of the formulations tested in this example are provided in Table 2 below. In all groups, the ELP concentration was approximately 20% (wt%). [Table 2]

[0103] Visual Analysis The appearance of the samples listed in Table 2 was observed with the naked eye. As the concentration of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol increased, the color of the solution became darker. The appearance of the prepared Group D to Group G samples was uniform as clear solutions.

[0104] Stability of pharmaceutical compositions by accelerated stability testing. The normal storage conditions for pharmaceutical compositions are 2-8°C. Accelerated stability tests were performed on the samples listed in Table 2 above. In short, the samples listed in Table 2 were placed in an environment with relatively high temperature and relatively high humidity (temperature 25°C ± 2°C, relative humidity 60% ± 5%) and the maintenance of nanoparticles in the samples was examined.

[0105] The content of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol in nanoparticles was determined by high-performance liquid chromatography (HPLC, e.g., HPLC-UV) and defined as the drug content after accelerated stability testing. The percentage of drug content was calculated by dividing the drug content after accelerated stability testing by the initial drug content. A drug content of 95% or more indicates good stability of the sample being tested.

[0106] Table 3 shows the results of the accelerated stability tests for samples D through G. Samples D, E, F, and G were stored at 25°C ± 2°C for 24 hours, 1 week, and 2 weeks. After 1 week, the percentage of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol drug content in each sample was greater than 95%, and there was no significant decrease compared to the initial drug content. This result indicates that the pharmaceutical composition possesses excellent stability. [Table 3-1] [Table 3-2]

[0107] The effect of antioxidants on the stability of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol formulations was investigated. The experimental designs are listed in Table 4. [Table 4]

[0108] The results of the accelerated stability tests for Group H and Group I are shown in Tables 5 and 6, respectively. [Table 5] [Table 6]

[0109] Particle size analysis The particle size distribution was determined using a particle size analyzer. After analysis by the particle analyzer, if the particle size of the pharmaceutical composition was smaller than 250 nm and the PDI value was less than 0.4, it corresponds to the fact that the solution of the pharmaceutical composition is considered clear and transparent when observed with the naked eye, and that light rays can be observed when the solution of the pharmaceutical composition is irradiated with a laser. The results showed that groups H and I were formulated to have similar particle sizes (Tables 7 and 8). [Table 7] [Table 8]

[0110] The results reported herein indicate that the tested 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol formulations are stable, with or without the addition of antioxidants.

[0111] Example 3.4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol inhibits melanin production in vitro. In vitro efficacy study B16-F10 cells are a melanoma cell line derived from C57BL / 6 mice that produces melanin and exhibits metastatic behavior. Therefore, B16-F10 cells are widely used to test melanin formation and depigmentation.

[0112] Alpha-melanocyte-stimulating hormone (α-MSH) is a peptide hormone that can induce melanin production. This study investigated the effect of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol samples (containing different concentrations of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol) on inhibiting melanin production. The experimental design is listed in Table 9. [Table 9]

[0113] Methods and materials A. Test conditions B16-F10 cells, 1 × 10 5 Cells were seeded in 96-well plates at a cell / well density and cultured for 24 hours. The cells were then incubated with 0.1 μM α-MSH for 24 hours to induce melanin production. Next, 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol samples of different concentrations were added to the cultured cells, and they were cultured for an additional 24 hours. α-MSH was not used in the vehicle control group.

[0114] B. Determination of melanin content in each group The cells were washed once with 1 mL of 1x PBS, then 300 μL of 1x trypsin-EDTA was added, and they were placed in a 37°C incubator for 5 minutes. Finally, 1 mL of DMEM was added to the cells, and the number of cells was counted by trypan blue staining.

[0115] The cell suspension was centrifuged at 1500g at room temperature for 5 minutes, followed by removal of the supernatant and air-drying of the pellet for 15 minutes. 1N NaOH (containing 10% DMSO) was added to decompose the pellet, followed by sonication for 30 minutes and heating in an 80°C water bath for 1.5 hours. After cooling, the suspension was collected and its absorbance at 405 nm was measured. The results were calculated by subtracting the reagent blank from the absorbance value at 405 nm and dividing by the average absorbance value of the α-MSH group.

[0116] Images and quantitative results obtained after extracting the melanin content of each group are shown in Figure 1 and Table 10 (wherein used herein, "TA" refers to the test article). EC of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol preparations 50 The concentration was found to be approximately 15.2 ppm (equivalent to 0.062 μM). The results showed that the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol samples tested herein significantly reduced melanin content in a dose-dependent manner compared to the control group (α-MSH only). The IM01-C4, IM01-C5, and IM01-C6 groups reduced melanin production to lower levels than the vehicle group (without α-MSH), indicating that the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol samples not only suppressed α-MSH-induced melanin production but also reduced baseline melanin production. [Table 10]

[0117] Within the concentrations tested, the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol sample was shown to reduce melanin levels in a dose-dependent manner. (Figure 2 and Table 11). [Table 11]

[0118] Example 4.4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol reduces melanin levels in vivo. This example investigates the therapeutic efficacy of a 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition for reducing melanin in vivo via different routes of administration or different administration schedules.

[0119] (A) In vivo efficacy of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition via different routes of administration Five-week-old male HRM-2 mice, which are melanin-bearing, hairless mice, were used in the study. A total of 20 mice were tested using a conventional diet during the adaptation, induction, and testing periods. After 7 days of adaptation, the mice were randomly divided into a control group and a UVB-c group based on their body weight and weight change; therefore, statistical differences in average body weight and weight change were not significant between the groups. The UVB-c group received 150 mJ / cm³ of UVB-c irradiation on the back of the mice three times a week. 2 The mice were subjected to daily UVB irradiation. After 4 weeks of irradiation (29th day of UV irradiation, the first day of the study period), the backs of the mice became significantly darker, and these were then divided into four groups according to the mean body weight and body weight change in the UVB induction group: UVB-c group (UVB control), IM01-ID group (intradermal injection), IM01-topic group (topical application), and color cream group (a "three-in-one" commercial cream containing 5% hydroquinone, 0.1% dexamethasone, and 0.1% tretinoin). (See below). The experimental design is shown in Table 12. [Table 12]

[0120] As shown in Table 12, the IM01-ID group was administered by intradermal injection. The injection frequency was once a week for a total of three times, with each injection being 6 cm long. 2 This was the average injection area of ​​the skin. IM01-topic group and color cream group were administered by smearing once daily for a total of 21 days. The color cream group consisted of a commercially available three-in-one whitening cream (containing 5% hydroquinone, 0.3% tretinoin, and 0.3% dexamethasone), which was used here to compare its efficacy with the formulations of this disclosure. During the treatment period, 150 mJ / cm² was applied to the backs of mice. 2 The animals were exposed to UVB radiation three times a week at a rate of [number] days until slaughter. Note that the timing of UVB exposure and drug administration was staggered.

[0121] In this experiment, to evaluate the whitening effectiveness of IM01 and compare it with a commercially available three-in-one whitening cream, HRM-2 mice were irradiated with UVB to induce melanin deposition and aging. As shown in Figure 3, the skin color of the UVB-c group was darker and showed melanin deposition compared to the control group. The skin of UVB-c mice was dry, wrinkled, and thickened. After treatment with the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition shown, via intradermal injection or smear, the skin color of mice in the IM01-ID group and IM01-topic group became whiter and lighter than that of the UVB-c group, and the amount of melanin deposition in both groups was lower than that of the UVB-c group. The color cream group showed less melanin than the UVB-c group, but skin dryness and peeling were observed in all treated areas. In some severe cases, wounds, redness, and even skin darkening occurred (consistent with the side effects listed in the package insert).

[0122] After sacrificing the mice on day 22, skin from the treatment area was collected and subjected to thermal extraction with 0.4 mL of NaOH for 1 hour. As shown in Figure 4A and Table 13, the melanin content in the UVB-c group was significantly higher than that of the control group, i.e., 31.4% (p<0.01), while the IM01-ID group and IM01-topic group showed significantly lower melanin levels than the UVB-c group, i.e., 33.6% (p<0.01) and 30.6% (p<0.05), respectively. Experimental data were expressed as mean ± standard deviation (n=3~5) using Student's t-test analysis, with #p<0.05, ##p<0.01 compared to the control group, *p<0.05, and **p<0.01 compared to the UVB-c group. In contrast, the skin melanin content in the color cream group appeared lower than that of the UVB-c group, but the difference was not statistically significant. [Table 13]

[0123] In summary, the results from this case demonstrate that the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition, administered either intradermally or topically, significantly reduced UVB-induced pigmentation in HRM-2 mice.

[0124] B. In vivo efficacy of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition by intradermal injection The in vivo efficacy of the IM01 formulation was investigated via intradermal injection at either twice-weekly (IM01-ID / L) or once daily (IM01-ID / H). The study design is provided in Table 14 below. [Table 14]

[0125] The results from this study indicate that intradermal injection of IM01 resulted in significant skin whitening, as demonstrated by a dose-dependent reduction in melanin levels at the injection site. IM01-ID / L and IM01-ID / H significantly reduced melanin production by 13.9% and 23.1%, respectively. See Table 15 and Figure 4B. [Table 15]

[0126] C. In vivo efficacy of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition by topical administration The in vivo efficacy of the IM01 formulation was investigated via topical administration (IM01-T) according to the study design provided in Table 16 below. [Table 16]

[0127] The results from this study indicate that IM01 administered topically resulted in significant skin whitening, as demonstrated by a reduction in melanin levels at the injection site. IM01-T significantly reduced melanin production by 21.4%. See Table 17 and Figure 4C. [Table 17]

[0128] Example 5.4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol showed better in vitro efficacy compared to other skin whitening compounds. In vitro efficacy comparison B16-F10 cells were seeded in 96-well plates. Except for the vehicle group, cells from the other groups were first cultured for 0.5 hours in the presence of α-MSH. The compositions for testing were then added to the culture medium, and the cells were cultured for a further 30 hours. The remaining experimental conditions and procedures were the same as those described in Example 3. Subsequently, cells were collected, and the effects of the 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition (IM01 group) and the arbutin and 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol composition (IM00 group) on reducing melanin levels were evaluated. The experimental design is summarized in Table 18, and the results are shown in both Table 18 and Figure 5. [Table 18]

[0129] The results indicate that 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol showed a better in vitro effect in reducing melanin compared to 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol and arbutin.

[0130] Example 6.4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol and known skin whitening agents in vitro efficacy This example aims to evaluate the in vitro effect of a 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition (dissolved in 0.2% DMSO at different concentrations shown) on melanin production in B16-F10 cells, compared to the known skin whitening agents arbutin and hydroquinone.

[0131] In this experiment, B16-F10 cells were seeded in 96-well plates and divided into several treatment groups as listed in Tables 19 and 20 below. Except for the vehicle group, cells from the other groups were first cultured in α-MSH for 24 hours. Then, the compositions for investigation (see Tables 19 and 20) were added to the culture medium, and the cells were cultured for a further 24 hours. The remaining experimental procedure was the same as that described in Example 3. Subsequently, the cells were collected, and the effects of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol (IM01 group), arbutin, and hydroquinone on melanin inhibition were evaluated. [Table 19] [Table 20]

[0132] After 24 hours of induction by α-MSH in melanocytes, melanin levels were significantly increased. Figure 6A shows that after 24 and 48 hours of induction by α-MSH, melanin content was significantly increased in treated cells compared to the vehicle control group.

[0133] Figures 6B-6C and Tables 21-22 show the melanin content of various treatment groups after culturing with compositions for 24-hour monitoring. The 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol composition (IM01 group) significantly reduced melanin content in a dose-dependent manner, for example, at both 60 μM and 120 μM. The melanin content of cells treated with 120 μM of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol was even lower than that of the vehicle group (without α-MSH induction), demonstrating that 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol can reduce baseline melanin levels (in the absence of α-MSH induction). In addition, the test results also showed that the effect of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol (e.g., 120 μM) in reducing melanin was better than that of the whitening agent arbutin (e.g., either 450 μM or 900 μM) and the whitening agent hydroquinone (e.g., 120 μM). [Table 21] [Table 22]

[0134] Example 7. In vitro whitening efficacy and toxicity test This example investigates the in vitro whitening efficacy and toxicity of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol compared to the compounds arbutin and hydroquinone.

[0135] A. In vitro efficacy and toxicity of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol Table 23 below lists test designs for evaluating the EC50 value of IM01 containing 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol. [Table 23]

[0136] IM01 exhibits whitening efficiency, demonstrated by the reduction of melanin levels by α-MSH, and the reduction activity is dose-dependent. Figure 7A. In this study, the EC of IM01 50 The value was determined to be approximately 41.2 μM. No significant toxicity was observed in cells treated with IM01. Figure 7B. Efficacy and toxicity results are also summarized in Table 24 below. [Table 24]

[0137] B. In vitro efficacy and toxicity of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol compared to other compounds Table 25 below lists test designs for evaluating the EC50 value of IM01 containing 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol. [Table 25]

[0138] Compared to other compounds investigated in this study, IM01 significantly inhibited melanin production and exhibited low cytotoxicity (Figures 7C and 7D). For example, the melanin inhibitory activity of IM01 was much greater than that of arbutin. Hydroquinone (HQ) showed high cytotoxicity. See also Table 26 below. [Table 26]

[0139] Example 8. In vitro reduction of pigmentation activity of additional derivatives. Design of in vitro efficacy trials B16-F10 cells are a melanoma cell line derived from C57BL / 6 mice that produces melanin and exhibits metastatic behavior. Therefore, B16-F10 cells can be used to test melanin formation and depigmentation.

[0140] Alpha-melanocyte-stimulating hormone (α-MSH) is a peptide hormone that can induce melanin production. This study investigated the effects of 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol and its derivative samples on inhibiting melanin production. The experimental design is listed in Table 27. [Table 27]

[0141] Methods and materials A. Test conditions B16-F10 cells, 1 × 10 5 Cells were seeded at a cell / well density in 96-well plates and cultured for 24 hours. The cells were then incubated with 0.1 μM α-MSH for 24 hours to induce melanin production. Next, 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol and derivative samples were added to the cultured cells and cultured for an additional 24 hours. α-MSH was not used in the vehicle control group.

[0142] B. Determination of melanin content in each group The cells were washed once with 1 mL of 1x PBS, then 300 μL of 1x trypsin-EDTA was added, and they were placed in a 37°C incubator for 5 minutes. Finally, 1 mL of DMEM was added to the cells, and the number of cells was counted by trypan blue staining.

[0143] The cell suspension was centrifuged at 1500g at room temperature for 5 minutes, followed by removal of the supernatant and air-drying of the pellet for 15 minutes. 1N NaOH (containing 10% DMSO) was added to decompose the pellet, followed by sonication for 30 minutes and heating in an 80°C water bath for 1.5 hours. After cooling, the suspension was collected and its absorbance at 405 nm was measured. The results were calculated by subtracting the reagent blank from the absorbance value at 405 nm and dividing by the average absorbance value of the α-MSH group.

[0144] C. Expected results The 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol and 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol derivative samples tested herein are expected to significantly reduce melanin content compared to the control group (α-MSH only). Groups IM01, IM02, IM03, IM04, and IM05 are also expected to reduce melanin production to lower levels than the vehicle group (without α-MSH).

[0145] 4-[(E)-2-(3,5-dihydroxyphenyl)ethenyl]benzene-1,3-diol and its derivatives are expected to suppress α-MSH-induced melanin production and reduce baseline melanin production.

[0146] Other Embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise specified, each disclosed feature is merely an example of a general set of equivalents or similar features.

[0147] From the above description, those skilled in the art will readily be able to identify the essential features of the present invention and make various changes and modifications to the invention to suit various uses and conditions without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

[0148] Equal parts While several embodiments of the present invention are described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or benefits, each of such variations and / or modifications will be considered within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will understand that all parameters, dimensions, materials and configurations described herein are illustrative, and that actual parameters, dimensions, materials and / or configurations will depend on the specific one or more applications in which the teachings of the present invention are used. Those skilled in the art will be able to verify many equivalents to specific embodiments of the present invention described herein using only routine experimentation. Accordingly, it should be understood that the embodiments described herein are presented only as examples, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be carried out in ways other than those specifically described and claimed. Embodiments of the present invention in this disclosure cover each individual feature, system, article, material, kit and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention as long as such features, systems, articles, materials, kits, and / or methods are not inconsistent with each other.

[0149] Furthermore, unless otherwise explicitly stated, in any method claimed herein that includes two or more steps or actions, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

Claims

1. A method for reducing pigmentation in a subject, comprising administering an effective amount of a pharmaceutical composition to a skin site of the subject in need, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a compound of formula (I) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 In the formula, R １ , R ２ , R ３ , R ４ , and R ８ Each of these independently consists of H, halogen, thiol, hydroxyl, C1-4 alkyl, C2-4 alkenyl, OR ５ COOR ６ , or -OC(=O)R ７ And, wherein R ５ , R ６ , and R ７ each independently is H or C1-4 alkyl, a method.

2. The compound of formula (I) has the following structure: 【Chemistry 2】 It has, in the formula, R １ , R ２ , R ３ , and R ４ Each of the following is the method according to claim 1.

3. R １ , R ２ , R ３ , and R ４ The method according to claim 1 or 2, wherein at least one of them is hydroxyl or methoxy.

4. R １ , R ２ , R ３ , and R ４ The method according to claim 3, wherein at least three of them are hydroxyl or methoxy.

5. R １ , R ２ , R ３ , and R ４ At least one of them is OC(=O)R ７ And in the formula, R ７ The method according to claim 1 or 2, wherein the molecule is C1-4 alkyl, and optionally methyl.

6. R １ , R ２ , R ３ , and R ４ At least three of them are OC(=O)R ７ And in the formula, R ７ The method according to claim 5, wherein the molecule is C1-4 alkyl, and optionally methyl.

7. R ８ The method according to any one of claims 1 and 3 to 6, wherein the compound is hydroxyl.

8. The compound has the following structure: 【Transformation 3】 The method according to claim 1, comprising one of the following.

9. The method according to any one of claims 1 to 8, wherein the pharmaceutically acceptable carrier comprises a nonionic surfactant, solvent, buffer, oil phase, or combination thereof having a hydrophilic-lipophilic balance value (HLB value) greater than 5.

10. The method according to claim 9, wherein the nonionic surfactant comprises polyoxyl castor oil and / or polyoxyl hydrogenated castor oil.

11. The method according to claim 9 or 10, wherein the pharmaceutically acceptable carrier comprises the solvent, the pharmaceutically acceptable carrier being a polyol or a polymer thereof, acetone, dimethyl sulfoxide, glycerol, an alcohol, or a combination thereof, and optionally the polyol or polymer thereof being ethylene glycol, polyoxyethylene, polyethylene glycol, or propylene glycol.

12. The method according to any one of claims 9 to 11, wherein the pharmaceutically acceptable carrier comprises the oil phase comprising soybean oil and / or medium-chain triglycerides.

13. The method according to any one of claims 1 to 12, wherein the pharmaceutical composition comprises a plurality of nanoparticles formed of the pharmaceutically acceptable carrier and the compound of formula (I) or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein the nanoparticles have an average particle size of up to 200 nm and a polydispersity index (PDI) value of up to 0.

4.

15. The method according to claim 13 or 14, wherein the pharmaceutically acceptable carrier comprises the nonionic surfactant, and the weight ratio of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the nonionic surfactant is in the range of 1:1 to 1:1000, optionally 1:1 to 1:

500.

16. The method according to any one of claims 1 to 15, wherein the pharmaceutical composition is administered to the skin site by intradermal injection or topical application.

17. The method according to any one of claims 1 to 16, wherein the pharmaceutical composition further comprises an activator for reducing pigmentation, the activator being different from the compound or a pharmaceutically acceptable salt thereof.

18. The method according to any one of claims 1 to 17, further comprising administering an activator to the subject for reducing pigmentation, wherein the activator is different from the compound or a pharmaceutically acceptable salt thereof.

19. The method according to any one of claims 1 to 18, wherein the pharmaceutical composition further comprises an antioxidant.

20. The method according to any one of claims 1 to 18, wherein the pharmaceutical composition does not contain water and / or an antioxidant.

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