RNA m6A regulator composition, preparation method and application thereof

Abstract

The application discloses a RNAm6A regulator composition and a preparation method and application thereof. The composition comprises a compound shown in a formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, oil phase substance 1, oil phase substance 2 and oil phase substance 3, and can further comprise a penetration enhancer and an antioxidant. The RNAm6A regulator composition can be used for preventing or treating skin diseases caused by RNAm6A methylation.

Description

A RNA m6A regulator composition and its preparation method and application

[0001] The present disclosure claims priority to Chinese patent application No. 2023118444059, filed with the Patent Office of China on December 28, 2023, entitled “A RNA m6A regulator composition, its preparation method and application”. The entire contents of the above application are incorporated into the present disclosure by reference. Technical Field

[0002] The present invention relates to the field of medicinal chemistry technology, and in particular to an RNA m6A regulator composition, a preparation method, and an application thereof. Background Art

[0003] Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR) are erythematous skin lesions on the palms and soles, primarily caused by cytotoxic chemotherapy and targeted tumor drugs. Severe cases can lead to loss of self-care ability. The main pathological features of HFS and HFSR are vacuolar degeneration of basal keratinocytes, perivascular lymphocytic infiltration of the skin, keratinocyte apoptosis, and skin edema. Microscopic examination also reveals inflammatory changes, vasodilation, edema, and leukocyte infiltration.

[0004] Drugs that can cause hand-foot syndrome (HFS) include chemotherapy drugs such as capecitabine, liposomal doxorubicin, cytarabine, docetaxel, vinorelbine, continuous infusion doxorubicin, and gemcitabine. Drugs that can cause HFS include targeted drugs such as sonitinib (Sutent), sorafenib (Nexavar), imatinib (Gleevec), and erlotinib (Tarceva). The World Health Organization (WHO) categorizes HFS into four grades: Grade 1: numbness, paresthesia, or tingling in the hands and feet; Grade 2: discomfort with holding objects and walking, painless swelling or erythema; Grade 3: painful erythema, edema of the palms and soles, periungual erythema and swelling; and Grade 4: peeling, ulceration, blistering, and severe pain. Currently, severe HFS and HFS are often treated with superficial skin care alone to alleviate symptoms, or even require discontinuation of medication. Existing treatments only address the symptoms, severely limiting the use of first-line cancer chemotherapy drugs. This represents a significant unmet medical need. There is a need to develop a drug with good efficacy, few side effects and low cost for the prevention and treatment of hand-foot syndrome and hand-foot skin reaction to meet the growing medical needs worldwide.

[0005] RNA m6A methylation is the most common RNA modification in mammals, regulating multiple signaling pathways and cellular processes (such as growth, development, and disease), thereby playing a key biological role (Frye M., et al. Science 2018, 361, 2073-2092; Yang C., et al. Cell Death & Disease 2020, 11, 960; Meyer KD & Jaffrey SR Nature Review Molecular Cell Biology 2014, 15, 313-326). m6A methylation of mRNA, miRNA, circRNA, and lncRNA is a dynamic and reversible process. Methyltransferases (such as METTL3, METTL14, and METTL16) attach methyl groups to RNA, while demethylases (FTO and ALKBH5) remove methyl groups from RNA, forming the regulatory basis of m6A. m6A affects RNA processing, translation, and degradation by recruiting specific binding proteins (such as YTHDF1, YTHDF2, YTHDC1, and IGF2BP), thereby leading to changes in downstream protein function and cell biological behavior (Hsu PJ, et al. Journal of Biological Chemistry 2019, 294, 19889-19895; Yao Y., et al. FASEB Journal 2019, 33, 7529-7544).

[0006] Currently, there are few known direct associations between RNA m6A and skin-related diseases. Most m6A-related methylases or demethylases play a role in skin diseases, especially skin tumors. For example, the mRNA expression levels of METTL3 and ALKBH5 in tumor tissues of patients with acral melanoma are significantly higher than those in adjacent tissues. Furthermore, the mRNA expression level of METTL3 is significantly higher in patients with advanced acral melanoma than in patients with early-stage acral melanoma (Le Zhanghui, Preliminary Study on the Pathogenesis of LncRNA and RNA m6A Methylation in Acral Melanoma, Dissertation, 2019). In the diagnosis of melanoma, the m6A-specific binding proteins YTHDF1 and HNRNPA2B1 can serve as novel biomarkers (Li TD, et al. Cancer Cell 2020, 20, 239). In keratinocytes, only long-term, low-level arsenic exposure has been reported to inhibit selective autophagy caused by m6A demethylase, thereby inducing the occurrence of skin tumors (Cui YH, et al. Nature Communications 2021, 12, 2183). In summary, there is currently no method to prevent and treat skin diseases by regulating RNA m6A methylation. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide an RNA m6A regulator and its preparation method and application, so as to significantly improve skin diseases caused by RNA m6A methylation.

[0009] Solutions for solving problems

[0010] The present invention provides an RNA m6A regulator composition, comprising: a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, and an oily phase substance.

[0011] Preferably, the pharmaceutically acceptable salts include hydrochloride, sulfate, phosphate, acetate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate;

[0012] Preferably, the cocrystal formers of the pharmaceutically acceptable cocrystal include nicotinamide, isonicotinamide, L-proline, and glycolic acid;

[0013] Preferably, the oil phase material includes oil phase material 1, oil phase material 2 and oil phase material 3;

[0014] Preferably, the oil phase substance 1 is selected from one or more of lanolin, beeswax, white beeswax, and paraffin, preferably white beeswax;

[0015] Preferably, the oil phase substance 2 is selected from one or more of vaseline and white vaseline, preferably white vaseline;

[0016] Preferably, the oil phase substance 3 is selected from one or more of light liquid paraffin and vegetable oil, preferably light liquid paraffin.

[0017] Preferably, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, and 15%-40% of oil phase substance 3;

[0018] Preferably, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 15%-25% of oil phase substance 1, 35%-64% of oil phase substance 2, and 20%-35% of oil phase substance 3;

[0019] Preferably, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of oil phase substance 1, 40%-58.7% of oil phase substance 2, and 22%-30% of oil phase substance 3;

[0020] Preferably, the composition comprises, by mass percentage, 0.3% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18% of oil phase substance 1, 58.7% of oil phase substance 2, and 23% of oil phase substance 3;

[0021] Preferably, the composition comprises, by mass percentage, 1% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18% of oil phase substance 1, 58% of oil phase substance 2, and 23% of oil phase substance 3;

[0022] Preferably, the composition comprises, by mass percentage, 3% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18% of oil phase substance 1, 57% of oil phase substance 2, and 22% of oil phase substance 3;

[0023] Preferably, the composition comprises, by mass percentage, 10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 20% of oil phase substance 1, 40% of oil phase substance 2, and 30% of oil phase substance 3.

[0024] Preferably, the composition comprises the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, white beeswax, white vaseline, and light liquid paraffin;

[0025] Preferably, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white vaseline, and 15%-40% of light liquid paraffin;

[0026] Preferably, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-64% of white vaseline, and 20%-35% of light liquid paraffin;

[0027] Preferably, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of white beeswax, 40%-58.7% of white vaseline, and 22%-30% of light liquid paraffin;

[0028] Preferably, the composition comprises, by mass percentage, 0.3% of the compound represented by formula (I), 18% of white beeswax, 58.7% of white vaseline, and 23% of light liquid paraffin;

[0029] Preferably, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white vaseline, and 23% of light liquid paraffin;

[0030] Preferably, the composition comprises, by mass percentage, 3% of the compound represented by formula (I), 18% of white beeswax, 57% of white vaseline, and 22% of light liquid paraffin;

[0031] Preferably, in terms of mass percentage, the composition comprises 10% of the compound represented by formula (I), 20% of white beeswax, 40% of white vaseline, and 30% of light liquid paraffin.

[0032] Preferably, the composition further comprises a penetration enhancer, more preferably, the penetration enhancer is selected from one or more of isopropyl myristate, isopropyl palmitate, propylene glycol dipelargonate, diethyl sebacate, azone, and propylene glycol, further preferably, the penetration enhancer is isopropyl myristate;

[0033] Preferably, the composition further comprises an antioxidant, more preferably, the antioxidant is selected from one or more of vitamin E, alkyl gallates, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), and further preferably, the antioxidant is butylated hydroxytoluene (BHT);

[0034] Preferably, the composition further comprises other pharmaceutically acceptable excipients.

[0035] Preferably, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, 15%-30% of oil phase substance 3, 0.1%-20% of a penetration enhancer, and 0%-2% of an antioxidant;

[0036] Preferably, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 15%-25% of oil phase substance 1, 35%-63% of oil phase substance 2, 20%-25% of oil phase substance 3, 1%-15% of a penetration enhancer, and 0%-1% of an antioxidant;

[0037] Preferably, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of oil phase substance 1, 40%-58% of oil phase substance 2, 20%-21% of oil phase substance 3, 2%-10% of a penetration enhancer, and 0%-0.2% of an antioxidant.

[0038] Preferably, the composition comprises the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, white beeswax, white vaseline, light liquid paraffin, and isopropyl myristate;

[0039] Preferably, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white vaseline, 15%-30% of light liquid paraffin, and 0.1%-20% of isopropyl myristate;

[0040] Preferably, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-63% of white vaseline, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate;

[0041] Preferably, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate;

[0042] Preferably, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white vaseline, 21% of light liquid paraffin, and 2% of isopropyl myristate;

[0043] Preferably, the composition comprises, by mass percentage, 10% of the compound represented by formula (I), 20% of white beeswax, 40% of white vaseline, 20% of light liquid paraffin, 10% of isopropyl myristate

[0044] Preferably, the composition comprises the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, isopropyl myristate, and butylated hydroxytoluene (BHT);

[0045] Preferably, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, 0.1%-20% of isopropyl myristate, and 0%-2% of butylated hydroxytoluene (BHT);

[0046] Preferably, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, 1%-15% of isopropyl myristate, and 0%-1% of butylated hydroxytoluene (BHT);

[0047] Preferably, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, 2%-10% of isopropyl myristate, and 0%-0.2% of butylated hydroxytoluene (BHT);

[0048] Further preferably, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 20.9% of light liquid paraffin, 2% of isopropyl myristate, and 0.1% of butylated hydroxytoluene (BHT).

[0049] The present invention also provides a method for preparing the composition, characterized in that:

[0050] When the composition does not contain a penetration enhancer and / or an antioxidant, the method comprises the following steps:

[0051] (1) Weighing oil phase material 1 and oil phase material 2, melting them in a water bath, and homogenizing to obtain a uniform mixture A;

[0052] (2) Weighing the oil phase substance 3, adding the weighed compound represented by formula (I) or its pharmaceutically acceptable salt or pharmaceutically acceptable cocrystal, heating in a water bath and stirring to obtain a uniform mixture B;

[0053] (3) After cooling the mixture A, the mixture B is added thereto, stirred and homogenized, and cooled to obtain the composition;

[0054] When the composition contains a penetration enhancer and / or an antioxidant, the method comprises the following steps:

[0055] (1) Weighing oil phase substance 1, oil phase substance 2, a penetration enhancer and / or an antioxidant, heating and melting in a water bath, and homogenizing to obtain a uniform mixture A;

[0056] (2) Weighing the oil phase substance 3, adding the weighed compound represented by formula (I) or its pharmaceutically acceptable salt or pharmaceutically acceptable cocrystal, heating in a water bath and stirring to obtain a uniform mixture B;

[0057] (3) After cooling the mixture A, the mixture B is added thereto, stirred and homogenized, and cooled to obtain the composition.

[0058] The present invention also provides an RNA m6A regulator composition, or use of the method described in preparing a preparation for preventing or treating skin diseases caused by RNA m6A methylation;

[0059] Preferably, the skin diseases include acanthosis, eczema, ichthyosis, psoriasis, keratosis, systemic lupus erythematosus, hand-foot syndrome, hand-foot skin reaction and dermatitis;

[0060] More preferably, the skin diseases include hand-foot syndrome and hand-foot skin reaction.

[0061] Preferably, the preparation comprises an ointment, a cream, a gel, a cream, an emulsion, a lotion, a solution, a paste, a film, an oil preparation or a patch;

[0062] Preferably, the preparation is an ointment;

[0063] More preferably, the preparation is a suspension ointment.

[0064] Effects of the Invention

[0065] In the RNA m6A regulator composition provided by the present invention, the compound represented by formula (I) has a high exposure to the skin and can effectively prevent or treat skin diseases by inhibiting RNA m6A methylation. The composition of the present invention has good stability and excellent appearance, viscosity, coating properties, physical stability, and pharmacokinetic properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 depicts exemplary results of cellular m6A levels and NAP1L2 m6A levels measured by nanopore sequencing 24 hours after administration of sorafenib, capecitabine, docetaxel, and osimertinib to keratinocytes HaCat.

[0067] FIG2 depicts exemplary results of cellular m6A levels and NAP1L2 m6A levels in mouse hind limb skin tissue in Example 3.

[0068] FIG3 depicts exemplary results of NAP1L2 mRNA expression measured by RT-PCR 24 hours after administration of human recombinant METTL3 protein, human recombinant FTO protein, or transfection of METTL3 siRNA and FTO siRNA to keratinocytes HaCat.

[0069] FIG4 depicts exemplary results of mRNA and protein expression of matrix metalloproteinases measured by RT-PCR and Western blotting 24 hours after administration of human recombinant NAP1L2 protein, sorafenib, capecitabine, docetaxel, and osimertinib, or transfection of NAP1L2 siRNA to keratinocytes HaCat.

[0070] FIG5 depicts exemplary results of mRNA and protein expression of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin measured by RT-PCR and Western blotting 24 hours after administration of human recombinant HBEGF protein, HBEGF antibody, or transfection of METTL3 and NAP1L2 siRNA to keratinocytes HaCat.

[0071] FIG6 depicts exemplary results of measuring the level of cellular m6A methylation inhibition by LC-MS 24 hours after administration of the compound of formula (I), nicotinamide, and UZH2 to THP-1 cells.

[0072] FIG7 depicts exemplary results of mRNA expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin measured by RT-PCR 24 hours after administration of human recombinant HBEGF protein and the compound of formula (I) to keratinocytes HaCaT.

[0073] FIG8 depicts exemplary results of the measurement of stratum corneum thickness of rat paw plantar skin in Example 10.

[0074] FIG9 depicts exemplary histopathological results of epithelial blisters, inflammatory cell infiltration, and skin tissue hyperemia in rat paw plantar skin after tissue staining in Example 10.

[0075] FIG10 depicts exemplary results of histopathological scoring of epithelial blisters, inflammatory cell infiltration, and skin tissue hyperemia in rat paw plantar skin after tissue staining in Example 10.

[0076] FIG. 11 depicts exemplary results of mouse hind limb skin in Example 11.

[0077] FIG. 12 depicts exemplary results of the degree of swelling of the mouse hind limb toes in Example 11.

[0078] FIG13 depicts exemplary histopathological results of mouse hind limb skin tissue in Example 11.

[0079] FIG14 depicts exemplary results of immunohistochemistry for IL-8 in the skin tissues of the hind limbs and toes of mice in the capecitabine modeling series in Example 11.

[0080] FIG15 depicts exemplary results of immunohistochemistry for IL-6 in the skin tissues of the hind limbs and toes of mice in the docetaxel modeling series in Example 11.

[0081] FIG. 16 depicts exemplary results of mouse hind limb skin in Example 12.

[0082] FIG. 17 depicts exemplary results of the degree of swelling of the mouse hind limb toes in Example 12.

[0083] FIG. 18 depicts exemplary histopathological results of mouse hind limb skin tissue in Example 12.

[0084] FIG19 depicts exemplary results of immunohistochemistry for IL-1β in the skin tissues of the hind limbs and toes of mice in the Sorafenib model series in Example 12.

[0085] FIG20 depicts exemplary results of immunohistochemistry for IL-1β in the skin tissues of the hind limbs and toes of mice in the osimertinib modeling series in Example 12.

[0086] FIG21 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide.

[0087] FIG22 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and isonicotinamide.

[0088] FIG23 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and L-proline.

[0089] FIG24 is an XRPD spectrum of the drug co-crystal formed by the compound represented by formula (I) and glycolic acid. DETAILED DESCRIPTION

[0090] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0091] The present invention provides an RNA m6A regulator composition, comprising: a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, and an oily phase substance.

[0092] In certain embodiments, the pharmaceutically acceptable salts include hydrochloride, sulfate, phosphate, acetate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0093] In certain embodiments, the cocrystal formers of the pharmaceutically acceptable cocrystal include nicotinamide, isonicotinamide, L-proline, and glycolic acid.

[0094] In certain embodiments, the compound represented by formula (I) or its pharmaceutically acceptable salt or pharmaceutically acceptable co-crystal is present in the form of solid microparticles.

[0095] In certain embodiments, the D90 of the solid particles is 0.01 to 50 μm.

[0096] In certain embodiments, the D90 of the solid particles is 0.01 to 20 μm.

[0097] In certain embodiments, the D90 of the solid particles is 0.01 to 10 μm.

[0098] In certain embodiments, the oil phase material includes oil phase material 1, oil phase material 2, and oil phase material 3;

[0099] In certain embodiments, the oil phase material 1 is selected from one or more of lanolin, beeswax, white beeswax, and paraffin.

[0100] In certain embodiments, the oil phase material 1 is white beeswax.

[0101] In certain embodiments, the oil phase substance 2 is selected from one or more of vaseline and white vaseline.

[0102] In certain embodiments, the oil phase substance 2 is white petrolatum.

[0103] In certain embodiments, the oil phase substance 3 is selected from one or more of light liquid paraffin and vegetable oil.

[0104] In certain embodiments, the oil phase substance 3 is light liquid paraffin.

[0105] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, and 15%-40% of oil phase substance 3.

[0106] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% of the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof.

[0107] In certain embodiments, the composition comprises, by mass percentage, 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% oil phase substance 1.

[0108] In certain embodiments, the composition comprises, by mass percentage, 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40%, or 41%, or 42%, or 43%, or 44%, or 45%, or 46%, or 47%, or 48%, or 49%, or 50%, or 51%, or 52%, or 53%, or 54%, or 55%, or 56%, or 57%, or 58%, or 59%, or 60%, or 61%, or 62%, or 63%, or 64%, or 65%, or 66%, or 67%, or 68%, or 69%, or 70% oil phase substance 2.

[0109] In certain embodiments, the composition comprises, by mass percentage, 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40% oil phase substance 3.

[0110] In certain embodiments, the composition comprises 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of oil phase substance 1, 35%-64% of oil phase substance 2, and 20%-35% of oil phase substance 3.

[0111] In certain embodiments, the composition comprises 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% of oil phase substance 1, 40%-58.7% of oil phase substance 2, and 22%-30% of oil phase substance 3.

[0112] In certain embodiments, the composition comprises 0.3% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18% of oil phase substance 1, 58.7% of oil phase substance 2, and 23% of oil phase substance 3.

[0113] In certain embodiments, the composition comprises 1% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18% of oil phase substance 1, 58% of oil phase substance 2, and 23% of oil phase substance 3.

[0114] In certain embodiments, the composition comprises 3% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18% of oil phase substance 1, 57% of oil phase substance 2, and 22% of oil phase substance 3.

[0115] In certain embodiments, the composition comprises 10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 20% of oil phase substance 1, 40% of oil phase substance 2, and 30% of oil phase substance 3.

[0116] In certain embodiments, the composition comprises the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, and light liquid paraffin.

[0117] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% white beeswax, 30%-70% white petrolatum, and 15%-40% light liquid paraffin.

[0118] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% of the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof.

[0119] In certain embodiments, the composition comprises, by mass percentage, 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% white beeswax.

[0120] In certain embodiments, the composition comprises, by weight percentage, 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40%, or 41%, or 42%, or 43%, or 44%, or 45%, or 46%, or 47%, or 48%, or 49%, or 50%, or 51%, or 52%, or 53%, or 54%, or 55%, or 56%, or 57%, or 58%, or 59%, or 60%, or 61%, or 62%, or 63%, or 64%, or 65%, or 66%, or 67%, or 68%, or 69%, or 70% white petrolatum.

[0121] In certain embodiments, the composition comprises, by weight percentage, 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% light liquid paraffin.

[0122] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-64% of white petrolatum, and 20%-35% of light liquid paraffin.

[0123] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58.7% of white petrolatum, and 22%-30% of light liquid paraffin.

[0124] In certain embodiments, the composition comprises the compound represented by formula (I), white beeswax, white petrolatum, and light liquid paraffin.

[0125] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% of white beeswax, 30%-70% of white petrolatum, and 15%-40% of light liquid paraffin.

[0126] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-64% of white petrolatum, and 20%-35% of light liquid paraffin.

[0127] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58.7% of white petrolatum, and 22%-30% of light liquid paraffin.

[0128] In certain embodiments, the composition comprises, by mass percentage, 0.3% of the compound represented by formula (I), 18% of white beeswax, 58.7% of white petrolatum, and 23% of light liquid paraffin.

[0129] In certain embodiments, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, and 23% of light liquid paraffin.

[0130] In certain embodiments, the composition comprises, by mass percentage, 3% of the compound represented by formula (I), 18% of white beeswax, 57% of white petrolatum, and 22% of light liquid paraffin.

[0131] In certain embodiments, the composition comprises, by mass percentage, 10% of the compound represented by formula (I), 20% of white beeswax, 40% of white petrolatum, and 30% of light liquid paraffin.

[0132] In certain embodiments, the composition is composed of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, and light liquid paraffin.

[0133] In certain embodiments, the composition consists, by mass percentage, of 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, and 15%-40% of light liquid paraffin.

[0134] In certain embodiments, the composition consists of, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 15%-25% white beeswax, 35%-64% white petrolatum, and 20%-35% light liquid paraffin.

[0135] In certain embodiments, the composition consists, by mass percentage, of 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58.7% of white petrolatum, and 22%-30% of light liquid paraffin.

[0136] In certain embodiments, the composition consists of the compound represented by formula (I), white beeswax, white petrolatum, and light liquid paraffin.

[0137] In certain embodiments, the composition consists of, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% of white beeswax, 30%-70% of white petrolatum, and 15%-40% of light liquid paraffin.

[0138] In certain embodiments, the composition consists of 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-64% of white petrolatum, and 20%-35% of light liquid paraffin, by mass percentage.

[0139] In certain embodiments, the composition consists of 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58.7% of white petrolatum, and 22%-30% of light liquid paraffin, by mass percentage.

[0140] In certain embodiments, the composition consists of 0.3% of the compound represented by formula (I), 18% of white beeswax, 58.7% of white petrolatum, and 23% of light liquid paraffin, by mass percentage.

[0141] In certain embodiments, the composition consists of 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, and 23% of light liquid paraffin, by mass percentage.

[0142] In certain embodiments, the composition consists of 3% of the compound represented by formula (I), 18% of white beeswax, 57% of white petrolatum, and 22% of light liquid paraffin, by mass percentage.

[0143] In certain embodiments, the composition consists of 10% of the compound represented by formula (I), 20% of white beeswax, 40% of white petrolatum, and 30% of light liquid paraffin, by mass percentage.

[0144] In certain embodiments, the composition further comprises a penetration enhancer.

[0145] In certain embodiments, the penetration enhancer is selected from one or more of isopropyl myristate, isopropyl palmitate, propylene glycol dipelargonate, diethyl sebacate, azone, and propylene glycol.

[0146] In certain embodiments, the penetration enhancer is isopropyl myristate.

[0147] In certain embodiments, the composition further comprises an antioxidant.

[0148] In certain embodiments, the antioxidant is selected from one or more of vitamin E, alkyl gallates, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT).

[0149] In certain embodiments, the antioxidant is butylated hydroxytoluene (BHT).

[0150] In certain embodiments, the composition further comprises other pharmaceutically acceptable excipients.

[0151] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, 15%-30% of oil phase substance 3, 0.1%-20% of a penetration enhancer, and 0%-2% of an antioxidant.

[0152] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% of the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof.

[0153] In certain embodiments, the composition comprises, by mass percentage, 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% oil phase substance 1.

[0154] In certain embodiments, the composition comprises, by mass percentage, 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40%, or 41%, or 42%, or 43%, or 44%, or 45%, or 46%, or 47%, or 48%, or 49%, or 50%, or 51%, or 52%, or 53%, or 54%, or 55%, or 56%, or 57%, or 58%, or 59%, or 60%, or 61%, or 62%, or 63%, or 64%, or 65%, or 66%, or 67%, or 68%, or 69%, or 70% oil phase substance 2.

[0155] In certain embodiments, the composition comprises, by mass percentage, 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% oil phase substance 3.

[0156] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% penetration enhancer.

[0157] In certain embodiments, the composition comprises, by weight percentage, 0%, or 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 1.1%, or 1.2%, or 1.3%, or 1.4%, or 1.5%, or 1.6%, or 1.7%, or 1.8%, or 1.9%, or 2% antioxidant.

[0158] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of oil phase substance 1, 35%-63% of oil phase substance 2, 20%-25% of oil phase substance 3, 1%-15% of a penetration enhancer, and 0%-1% of an antioxidant.

[0159] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of oil phase substance 1, 40%-58% of oil phase substance 2, 20%-21% of oil phase substance 3, 2%-10% of a penetration enhancer, and 0%-0.2% of an antioxidant.

[0160] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, 15%-30% of oil phase substance 3, 0.1%-20% of a penetration enhancer, and 0%-2% of an antioxidant.

[0161] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I), 15%-25% of oil phase substance 1, 35%-63% of oil phase substance 2, 20%-25% of oil phase substance 3, 1%-15% of a penetration enhancer, and 0%-1% of an antioxidant.

[0162] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I), 18%-20% of oil phase substance 1, 40%-58% of oil phase substance 2, 20%-21% of oil phase substance 3, 2%-10% of a penetration enhancer, and 0%-0.2% of an antioxidant.

[0163] In certain embodiments, the composition comprises the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, and isopropyl myristate.

[0164] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% white beeswax, 30%-70% white petrolatum, 15%-30% light liquid paraffin, and 0.1%-20% isopropyl myristate.

[0165] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% of the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof.

[0166] In certain embodiments, the composition comprises, by mass percentage, 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% white beeswax.

[0167] In certain embodiments, the composition comprises, by weight percentage, 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40%, or 41%, or 42%, or 43%, or 44%, or 45%, or 46%, or 47%, or 48%, or 49%, or 50%, or 51%, or 52%, or 53%, or 54%, or 55%, or 56%, or 57%, or 58%, or 59%, or 60%, or 61%, or 62%, or 63%, or 64%, or 65%, or 66%, or 67%, or 68%, or 69%, or 70% white petrolatum.

[0168] In certain embodiments, the composition comprises, by weight percentage, 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% light liquid paraffin.

[0169] In certain embodiments, the composition comprises, by mass percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% isopropyl myristate.

[0170] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate.

[0171] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate.

[0172] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, and 0.1%-20% of isopropyl myristate.

[0173] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate.

[0174] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate.

[0175] In certain embodiments, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 21% of light liquid paraffin, and 2% of isopropyl myristate.

[0176] In certain embodiments, the composition comprises, by mass percentage, 10% of the compound represented by formula (I), 20% white beeswax, 40% white petrolatum, 20% light liquid paraffin, and 10% isopropyl myristate.

[0177] In certain embodiments, the composition is composed of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, and isopropyl myristate.

[0178] In certain embodiments, the composition consists, by mass percentage, of 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, and 0.1%-20% of isopropyl myristate.

[0179] In certain embodiments, the composition consists, by mass percentage, of 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate.

[0180] In certain embodiments, the composition consists, by mass percentage, of 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate.

[0181] In certain embodiments, the composition consists of, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, and 0.1%-20% of isopropyl myristate.

[0182] In certain embodiments, the composition consists of, by mass percentage, 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate.

[0183] In certain embodiments, the composition consists of, by mass percentage, 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate.

[0184] In certain embodiments, the composition consists of 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 21% of light liquid paraffin, and 2% of isopropyl myristate, based on mass percentage.

[0185] In certain embodiments, the composition consists of 10% of the compound represented by formula (I), 20% white beeswax, 40% white petrolatum, 20% light liquid paraffin, and 10% isopropyl myristate, calculated by mass percentage.

[0186] In certain embodiments, the composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, isopropyl myristate, and butylated hydroxytoluene (BHT).

[0187] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 10%-30% white beeswax, 30%-70% white petrolatum, 15%-30% light liquid paraffin, 0.1%-20% isopropyl myristate, and 0%-2% butylated hydroxytoluene (BHT).

[0188] In certain embodiments, the composition comprises, by weight percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% of the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof.

[0189] In certain embodiments, the composition comprises, by mass percentage, 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% white beeswax.

[0190] In certain embodiments, the composition comprises, by weight percentage, 30%, or 31%, or 32%, or 33%, or 34%, or 35%, or 36%, or 37%, or 38%, or 39%, or 40%, or 41%, or 42%, or 43%, or 44%, or 45%, or 46%, or 47%, or 48%, or 49%, or 50%, or 51%, or 52%, or 53%, or 54%, or 55%, or 56%, or 57%, or 58%, or 59%, or 60%, or 61%, or 62%, or 63%, or 64%, or 65%, or 66%, or 67%, or 68%, or 69%, or 70% white petrolatum.

[0191] In certain embodiments, the composition comprises, by weight percentage, 15%, or 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26%, or 27%, or 28%, or 29%, or 30% light liquid paraffin.

[0192] In certain embodiments, the composition comprises, by mass percentage, 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 2%, or 3%, or 4%, or 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15%, or 16%, or 17%, or 18%, or 19%, or 20% isopropyl myristate.

[0193] In certain embodiments, the composition comprises, by weight percentage, 0%, or 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1%, or 1.1%, or 1.2%, or 1.3%, or 1.4%, or 1.5%, or 1.6%, or 1.7%, or 1.8%, or 1.9%, or 2% butylated hydroxytoluene (BHT).

[0194] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 15%-25% white beeswax, 35%-63% white petrolatum, 20%-25% light liquid paraffin, 1%-15% isopropyl myristate, and 0%-1% butylated hydroxytoluene (BHT).

[0195] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal thereof, 18%-20% white beeswax, 40%-58% white petrolatum, 20%-21% light liquid paraffin, 2%-10% isopropyl myristate, and 0%-0.2% butylated hydroxytoluene (BHT).

[0196] In certain embodiments, the composition comprises, by mass percentage, 0.1%-20% of the compound represented by formula (I), 10%-30% white beeswax, 30%-70% white petrolatum, 15%-30% light liquid paraffin, 0.1%-20% isopropyl myristate, and 0%-2% butylated hydroxytoluene (BHT).

[0197] In certain embodiments, the composition comprises, by mass percentage, 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, 1%-15% of isopropyl myristate, and 0%-1% of butylated hydroxytoluene (BHT).

[0198] In certain embodiments, the composition comprises, by mass percentage, 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, 2%-10% of isopropyl myristate, and 0%-0.2% of butylated hydroxytoluene (BHT).

[0199] In certain embodiments, the composition comprises, by mass percentage, 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 20.9% of light liquid paraffin, 2% of isopropyl myristate, and 0.1% of butylated hydroxytoluene (BHT).

[0200] In certain embodiments, the composition is composed of a compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, isopropyl myristate, and butylated hydroxytoluene (BHT).

[0201] In certain embodiments, the composition consists, by mass percentage, of 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, 0.1%-20% of isopropyl myristate, and 0%-2% of butylated hydroxytoluene (BHT).

[0202] In certain embodiments, the composition consists, by mass percentage, of 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, 1%-15% of isopropyl myristate, and 0%-1% of butylated hydroxytoluene (BHT).

[0203] In certain embodiments, the composition consists, by mass percentage, of 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable cocrystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, 2%-10% of isopropyl myristate, and 0%-0.2% of butylated hydroxytoluene (BHT).

[0204] In certain embodiments, the composition consists, by mass percentage, of 0.1%-20% of the compound represented by formula (I), 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, 0.1%-20% of isopropyl myristate, and 0%-2% of butylated hydroxytoluene (BHT).

[0205] In certain embodiments, the composition consists, by mass percentage, of 0.2%-15% of the compound represented by formula (I), 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, 1%-15% of isopropyl myristate, and 0%-1% of butylated hydroxytoluene (BHT).

[0206] In certain embodiments, the composition consists, by mass percentage, of 0.3%-10% of the compound represented by formula (I), 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, 2%-10% of isopropyl myristate, and 0%-0.2% of butylated hydroxytoluene (BHT).

[0207] In certain embodiments, the composition consists of 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 20.9% of light liquid paraffin, 2% of isopropyl myristate, and 0.1% of butylated hydroxytoluene (BHT), by mass percentage.

[0208] The present invention also provides a method for preparing the composition, characterized in that:

[0209] When the composition does not contain a penetration enhancer and / or an antioxidant, the method comprises the following steps:

[0210] (1) Weighing oil phase material 1 and oil phase material 2, melting them in a water bath, and homogenizing to obtain a uniform mixture A;

[0211] (2) Weighing the oil phase substance 3, adding the weighed compound represented by formula (I) or its pharmaceutically acceptable salt or pharmaceutically acceptable cocrystal, heating in a water bath and stirring to obtain a uniform mixture B;

[0212] (3) After cooling the mixture A, the mixture B is added thereto, stirred and homogenized, and cooled to obtain the composition;

[0213] When the composition contains a penetration enhancer and / or an antioxidant, the method comprises the following steps:

[0214] (1) Weighing oil phase substance 1, oil phase substance 2, a penetration enhancer and / or an antioxidant, heating and melting in a water bath, and homogenizing to obtain a uniform mixture A;

[0215] (2) Weighing the oil phase substance 3, adding the weighed compound represented by formula (I) or its pharmaceutically acceptable salt or pharmaceutically acceptable cocrystal, heating in a water bath and stirring to obtain a uniform mixture B;

[0216] (3) After cooling the mixture A, the mixture B is added thereto, stirred and homogenized, and cooled to obtain the composition.

[0217] In certain embodiments, the water bath heating in step (1) is heated to 65-70°C.

[0218] In certain embodiments, the homogenization in step (1) is performed at 350 rpm for 15-20 minutes.

[0219] In certain embodiments, the water bath heating in step (2) is heated to 40°C.

[0220] In certain embodiments, the stirring in step (2) is performed at 300-400 rpm for 10 minutes.

[0221] In certain embodiments, the cooling in step (3) is to 40°C.

[0222] In certain embodiments, the homogenizer speed in step (3) is 10,000 rpm and the homogenization time is 2 minutes.

[0223] In certain embodiments, the cooling in step (3) is cooling to room temperature.

[0224] The present invention also provides an RNA m6A regulator composition, or an application of the method in preparing a preparation for preventing or treating skin diseases caused by RNA m6A methylation.

[0225] In certain embodiments, the skin disease comprises acanthosis, eczema, ichthyosis, psoriasis, keratosis, systemic lupus erythematosus, hand-foot syndrome, hand-foot skin reaction, and dermatitis.

[0226] In certain embodiments, the skin disorder comprises hand-foot syndrome and hand-foot skin reaction.

[0227] In certain embodiments, the formulation comprises an ointment, cream, gel, cream, emulsion, lotion, solution, paste, film, oil, or patch.

[0228] In certain embodiments, the formulation is an ointment.

[0229] In certain embodiments, the formulation is a suspension ointment.

[0230] Example 1: Preparation of the compound represented by formula (I)

[0231] Ethyl 3-bromo-2-oxocyclohexane-1-carboxylate (20 mg, 0.08 mmol) and 4-chloroaniline (25 mg, 0.2 mmol) were mixed and heated to 150°C. After reacting for 3 hours, the reaction solution was cooled to room temperature, diluted with 100 mL of dichloromethane, washed three times with 100 mL of 1N HCl and once with 100 mL of saturated NaHCO3. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The mixture was separated and purified by silica gel column chromatography to obtain ethyl 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylate (16 mg). 10 mg of ethyl 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxylate (0.036 mmol) was dissolved in 10 mL of ethanol, and 2 mL of 2M LiOH solution was added. The mixture was stirred at room temperature for 1 hour. After rotary distillation, 20 mL of water was added to dilute the mixture and the pH was adjusted to 2. The mixture was then extracted three times with 50 mL of dichloromethane. The organic phases were combined, dried, concentrated, and then ammonia was added. The mixture was heated at 60°C for 24 hours. The mixture was purified by silica gel column chromatography to obtain 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (compound 1, 7.6 mg) as a white solid. (S)-6-chloro-2,3,4,9-tetrahydro-1H-carbazolecarboxamide (compound represented by formula (I)) was obtained by separation using a Chiralpak AD chiral column. LCMS of compound represented by formula (I) [M+H] + :249. 1 H-NMR (400MHz, DMSO-d6): δ10.79 (s, 1H), 7.37-7.39 (m, 2H), 7.28 (d, J = 8.0Hz, 1H), 7. 08(s,1H),6.98-7.01(m,1H),3.64-3.67(m,1H),2.58-2.61(m,2H),1.66-2.04(m,4H).

[0232] Example 2: Drug-induced changes in keratinocyte mRNA methylation levels

[0233] 1*10 8HaCat cells were plated in culture dishes and treated with 50 nM of each drug (sorafenib, capecitabine, docetaxel, and osimertinib). The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was removed, the cells were washed with PBS, centrifuged, and the supernatant discarded. 400 μL of RLT cell lysis buffer (QIAGEN, Cat. #79216) was added to the cells. A portion of the sample was added with 400 μL of 70% ethanol, stirred thoroughly, and transferred to an RNeasy mini column. RNA was extracted using the RNeasy mini kit (QIAGEN, Cat. #74104). 2 μL of RNA sample was dissolved in 98 μL of 10 mM Tris-HCl buffer, and the absorbance at 260 nm was measured using a Nanodrop (Thermo Fisher Scientific) to determine RNA concentration.

[0234] 40 μg of RNA sample was taken and mRNA (PolyA+RNA) was enriched using the NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490). The 3' end of the enriched mRNA (PolyA+RNA) molecule was then connected to the reverse transcription adapter RTA using the Direct RNA Sequencing Kit (Oxfod Nanopore Technologies, Cat.#SQK-RNA002). The mRNA (PolyA+RNA) molecule was used as a template for reverse transcription reaction to synthesize its complementary chain. The reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the library was sequenced using Qubit TM dsDNA HS assay kit (Invitrogen, Cat.# LOT2133187) was used for quantitative detection.

[0235] After library quality control, the prepared sequencing library was loaded onto a PromethION Flow Cell chip (Oxford Nanopore Technologies, Cat.# FLO-MIN106D) and sequenced using a PromethION sequencer (Oxford Nanopore Technologies) in the matching sequencing mode. Sequencing data were base-called using guppy, and m6A methylation analysis was performed using the original Fast5 data.

[0236] The results, as shown in Figure 1, show that compared to the blank control group, the m6A methylation levels of HaCat cells increased by 5.05-fold, 6.01-fold, 3.97-fold, and 5.13-fold after addition of sorafenib, capecitabine, docetaxel, and osimertinib, respectively. This suggests that anti-cancer drugs (sorafenib, capecitabine, docetaxel, and osimertinib) can induce aberrant m6A methylation in human keratinocyte mRNA. Further bioinformatics analysis revealed significant differences in the m6A methylation levels of NAP1L2, primarily manifested in m6A methylation of the UGAGGACUCA fragment.

[0237] Example 3: Changes in mRNA methylation levels in a drug-induced mouse skin adverse reaction model

[0238] ICR male mice (5-6 weeks old, weighing approximately 35 grams) were acclimated for 7 days and then randomly divided into 6 groups: blank control, sorafenib, capecitabine, docetaxel, and osimertinib. Each group was gavaged with the corresponding modeling drug (sorafenib 100 mg / kg, capecitabine 200 mg / kg, docetaxel 25 mg / kg, osimertinib 10 mg / kg) once daily for 30 consecutive days before being sacrificed. Skin tissue from the hind limbs and toes of the mice was collected. A small amount of tissue sample was ground into a powder in a mortar filled with liquid nitrogen. The single-phase lysis buffer was added and the sample was allowed to stand at room temperature for 5 minutes before centrifugation (12,000 rpm) for 5 minutes. 1 mL of the supernatant was removed, 200 μL of chloroform was added, and the mixture was shaken and allowed to stand at room temperature for 15 minutes. The sample was then centrifuged (12,000 rpm, 4°C) for 15 minutes. The upper aqueous phase was aspirated and an equal volume of isopropanol was added. The sample was allowed to stand at -20°C for 1 hour and then centrifuged (12,000 rpm, 4°C). The supernatant was discarded. 400 μL of 70% ethanol was added to the pellet, mixed thoroughly, and transferred to an RNeasy mini column. RNA was extracted using the RNeasy mini kit (QIAGEN, Cat. #74104). 2 μL of RNA sample was dissolved in 98 μL of 10 mM Tris-HCl buffer, and the absorbance at 260 nm was measured using a Nanodrop (Thermo Fisher Scientific) to determine RNA concentration.

[0239] 40 μg of RNA sample was taken and mRNA (PolyA+RNA) was enriched using the NEBNext Poly(A) mRNA Magnetic Separation Module (NEB, Cat.#E7490). The 3' end of the enriched mRNA (PolyA+RNA) molecule was then connected to the reverse transcription adapter RTA using the Direct RNA Sequencing Kit (Oxfod Nanopore Technologies, Cat.#SQK-RNA002). The mRNA (PolyA+RNA) molecule was used as a template for reverse transcription reaction to synthesize its complementary chain. The reverse transcription adapter RTA end was connected to the sequencing adapter (RNA adapter) to form the final sequencing library, and the library was sequenced using Qubit TM dsDNA HS assay kit (Invitrogen, Cat.# LOT2133187) was used for quantitative detection.

[0240] After library quality control, the prepared sequencing library was loaded onto a PromethION Flow Cell chip (Oxford Nanopore Technologies, Cat.# FLO-MIN106D) and sequenced using a PromethION sequencer (Oxford Nanopore Technologies) in the matching sequencing mode. Sequencing data were base-called using guppy, and m6A methylation analysis was performed using the original Fast5 data.

[0241] The results are shown in Figure 2. Compared with the positive control group, the m6A methylation level in the skin tissue of the hind limbs and toes of mice in the drug modeling group was significantly increased. The m6A methylation level of NAP1L2 in the drug-treated group was significantly different from that in the blank control group.

[0242] Example 4: m6A regulates NAP1L2 mRNA expression levels

[0243] 1*10 8HaCat cells were plated in culture dishes and treated with recombinant m6A methylase METTL3 protein (Abcam, Cat.#ab271611) or m6A demethylase FTO protein (Abcam, Cat.#ab271525), or transfected with siRNA for METTL3 (sh-METTL3, GeneChip) or siRNA for FTO (sh-FTO, GeneChip). The cells were cultured at 37°C in an incubator containing 5% carbon dioxide for 24 hours, washed with PBS, and collected by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (Beyotime, Cat. #QH18721S), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction mixture. After completion of the reaction, the reaction mixture was subjected to agarose gel electrophoresis to determine the expression level of NAP1L2 mRNA.

[0244] The results are shown in Figure 3. When the m6A methylase METTL3 or the m6A methylase FTO function was inhibited (sh-FTO) in keratinocytes HaCat, the mRNA expression of NAP1L2 increased significantly; when the m6A methylase FTO or the m6A methylase METTL3 function was inhibited (sh-METTL3) in keratinocytes HaCat, the mRNA expression of NAP1L2 decreased significantly. This indicates that m6A methylation can regulate the mRNA expression of NAP1L2.

[0245] Example 5: NAP1L2 regulates the expression of matrix metal proteins in human keratinocytes HaCat

[0246] 1*10 8 HaCat cells were plated in culture dishes and treated with recombinant human NAP1L2 protein (Abcam, Cat.#ab117213), sorafenib, capecitabine, docetaxel, and osimertinib, or transfected with NAP1L2 siRNA (sh-NAP1L2, GeneChip). The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After washing with PBS, the cells were harvested by high-speed centrifugation. RLT lysis buffer was added to the harvested cells, shaken at 4°C for half an hour, and the supernatant was collected after centrifugation for measurement of matrix metal protein expression by Western blotting.

[0247] Total RNA was extracted from the collected cells using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (SinoBiological, Cat. #HP100168 & HP100367), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction solution. Following completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression level of the metal matrix protein.

[0248] The results are shown in Figure 4. When NAP1L2 recombinant protein was added to keratinocytes HaCat, the expression levels of the mRNA and protein of the metalloproteinases MMP2 and MMP9 increased significantly. When NAP1L2 siRNA was added to keratinocytes HaCat to inhibit the expression of NAP1L2, the expression levels of the mRNA and protein of the metalloproteinases MMP2 and MMP9 decreased significantly. Many studies have confirmed that abnormal expression of metalloproteinases is closely related to various skin-related diseases (Kumper M. et al., Am. J. Physiol. Cell. Physiol. 2022, 323(4): 1290-1303).

[0249] Example 6: m6A regulates the expression of human keratinocyte differentiation markers

[0250] 1*10 8 HaCat cells were plated in culture dishes and incubated with recombinant HBEGF protein (Thermo Fisher Scientific, Cat.#100-47-1mg) at 37°C in an incubator containing 5% CO2 for 24 hours. HBEGF antibody (Invitrogen, Cat.#406316) or transfection with siRNA for METTL3 (sh-METTL3, GeneChip) or NAP1L2 (sh-NAP1L2, GeneChip) were then added and incubated at 37°C in an incubator containing 5% CO2 for 24 hours. After washing with PBS, the cells were harvested by high-speed centrifugation. RLT lysis buffer was added to the harvested cells, shaken at 4°C for half an hour, and the supernatant was collected after centrifugation. Protein expression of the keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin was measured by Western blotting.

[0251] Total RNA was extracted using Trizol reagent (Sigma Aldrich), and 1 μg of RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, Cat. #AT311-03). RT-PCR was performed by adding 1.25 μL of primers (primer sequences are shown in Table 2), 10 μL of iTag Universal SYBR Green supermix (Bio-Rad, Cat. #172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction solution. After completion of the reaction, the reaction solution was subjected to agarose gel electrophoresis to measure the mRNA expression levels of the keratinocyte differentiation markers KRT1, KRT10, loricrin, and involucrin.

[0252] The results are shown in Figure 5. When HBEGF recombinant protein was added to keratinocytes HaCat, the mRNA and protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly increased, indicating that the keratinocytes HaCat were in a highly differentiated state. However, when HBEGF antibodies were added or siRNAs targeting METTL3 and NAP1L2 were used to inhibit intracellular m6A methylation, the mRNA and protein expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly decreased. This suggests that m6A can regulate the expression of keratinocyte differentiation markers, thereby regulating keratinocyte differentiation.

[0253] Example 7: Effects of compounds on m6A methylation levels in THP-1 cells

[0254] 1*10 8THP-1 cells were plated in a culture dish and treated with 4 nM and 400 nM of the compound of formula (I), 4 nM and 4 μM nicotinamide, and 5 μM of the m6A methylase inhibitor UZH2. The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The medium was discarded, the cells were washed with PBS, and the supernatant was discarded by centrifugation. 400 μL of RLT cell lysis buffer was added and the cells were stored at -80°C. A portion of the sample was taken, 400 μL of 70% ethanol was added and mixed thoroughly. 700 μL of the sample was transferred to an RNeasy spin column and centrifuged for 15 seconds (8000 g, 25°C). 700 μL of RW1 buffer was added and centrifuged for 15 seconds (8000 g, 25°C). Then, 500 μL of RPE buffer was added and centrifuged for 16 seconds (8000 g, 25°C). This step was repeated twice, followed by centrifugation for 2 minutes to completely remove the eluate. Add 30 μL of nuclease-free water to the column, incubate for 5 minutes, and centrifuge for 2 minutes (12,000 g, 25°C). Repeat this step three times. Dissolve 2 μL of RNA sample in 98 μL of 10 mM Tris buffer and measure the absorbance at 260 nm using a Nanodrop to determine the RNA concentration.

[0255] Dissolve 50 μg of total RNA in 100 μL of nuclease-free water. Resuspend the Oligo(dT) magnetic beads, transfer 50 μL of beads to a 1.5 mL tube, add 500 μL of binding buffer, let stand, and remove the supernatant. Add 100 μL of RNA sample to 200 μL of magnetic bead suspension, mix, and incubate in a Theromixer at 25°C for 5 minutes. Discard the supernatant and add 200 μL of wash buffer. Repeat twice, then centrifuge for 10 seconds (2000 g, 25°C). Add 50 μL of elution buffer and mix thoroughly. Heat to 75°C, transfer the supernatant to a new 1.5 mL tube, and purify using the RNA Clean & Concentrator kit to determine the RNA concentration.

[0256] Add 20 μL (about 200 ng) of mRNA sample and 20 μL of Nuclease P1 digestion premix (0.5 μL of 2 unit / μL Nuclease P1, 0.4 μL of 5 M NaCl, 2 μL of 0.1 M ZnCl2 and 17.1 μL of PCR-grade water) to each test tube, stir and incubate at 37°C in a Theromixer for 2 hours, add 2 μL of 2M NH4HCO3 solution and 1 unit of alkaline phosphatase, stir and incubate at 37°C in a Theromixer for 2 hours, add 1 μL of 1.2 M HCl neutralization solution, centrifuge for 30 minutes (16000g, 4°C), take 20 μL of supernatant and analyze m6A methylation fragments by LC-MS, and calculate the m6A inhibition rate.

[0257] m6A inhibition rate (%) = (measured ion peak area - blank control) / (positive ion peak area - blank control) * 100

[0258] The results, as shown in Figure 6, show that the compound of formula (I) can significantly inhibit the m6A methylation level of cellular mRNA in a concentration-dependent manner. At a concentration of 400 nM, the compound of formula (I) has comparable m6A inhibitory activity to the m6A methylase inhibitor UZH2 at a concentration of 5 μM. However, nicotinamide had no inhibitory effect on the m6A methylation level of cellular mRNA at either low (4 nM) or high (4 μM) concentrations.

[0259] Example 8: Effects of compounds on m6A methylation levels in human keratinocytes

[0260] HaCat cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture dishes were placed in an incubator containing 5% CO2 and cultured at 37°C for 24 hours. An equal volume of DMSO was added to control group 1, and 50 nM sorafenib, capecitabine, docetaxel, and osimertinib were added to control groups A, B, C, and D, respectively. After adding 50 nM of the corresponding compound, 400 nM of the test compound or nicotinamide was added to sample groups A1, A2, B1, B2, C1, C2, D1, and D2. The cells were cultured for 24 hours, washed with PBS, and collected by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich), and m6A methylation in the sample RNA was quantitatively detected using the EpiQuik m6A RNA Methylation Quantification Kit. The standard curve used positive control samples at concentrations of 0.01 ng / μl, 0.02 ng / μl, 0.05 ng / μl, 0.1 ng / μl, 0.2 ng / μl, and 0.5 ng / μl. The absorbance at 450 nm was read using a Tecan GENios microplate reader. The m6A quantitative calculation formula is as follows:

[0261] m6A (ng) = (absorbance of sample well – absorbance of background well) / slope of standard curve

[0262] m6A (%) = m6A (ng) / sample RNA amount (ng) * 100%

[0263] The results, as shown in Table 1, show that treatment with sorafenib, capecitabine, docetaxel, and osimertinib significantly increased m6A methylation levels in human keratinocytes (HaCat) (calculated as follows: fold change = measured m6A amount / m6A amount measured in control 1). The compound of formula (I) effectively inhibited the abnormal increase in RNA m6A levels in human keratinocytes caused by chemotherapy drugs and multikinase inhibitors.

[0264] Table 1: Fold changes in m6A methylation levels relative to control group 1.

[0265] Example 9: Inhibitory effect of compounds on keratinocyte HaCaT differentiation

[0266] Human keratinocytes HaCaT were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. 6 Cells were plated in 96-well plates at a density of 10 cells per square centimeter and incubated at 37°C in a 5% CO2 incubator for 24 hours. An equal volume of DMSO was added to control well 1, and 2.5 ng / mL human recombinant HBEGF protein (Abcam, ab205523) was added to control well 2. Sample wells were then incubated for 24 hours. After washing with PBS, cells were harvested by high-speed centrifugation. Total RNA was extracted using Trizol reagent (Sigma Aldrich). 1 μg of RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (Transgene Biotech, AT311-03). RT-PCR reactions were performed by adding 1.25 μL of primers (primer sequences are shown in Table 2), 10 μL of iTag Universal SYBR Green Supermix (Bio-Rad, 172-5125), and an appropriate amount of DEPC ultrapure water to a 20 μL reaction mixture. After the reaction, the reaction solution was subjected to agarose gel electrophoresis to determine the mRNA expression levels of keratinocyte differentiation markers KRT1, KRT10, Loricrin, and Involucrin.

[0267] Table 2: Some primer sequence information

[0268] As shown in Figure 7, after induction with the addition of human recombinant HBEGF protein, the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin significantly increased. However, after addition of the compound of formula (I), the mRNA expression levels of HaCaT differentiation markers KRT1, KRT10, Loricrin, and Involucrin were significantly suppressed.

[0269] Example 10: Inhibitory effect of the compound on keratinocyte differentiation in a rat hand-foot skin reaction model

[0270] After SD rats (weighing approximately 200 g) were bred and adapted for one week, they were divided into groups according to their weight, with 12 rats in each group. The modeling drugs (sorafenib, erlotinib, afatinib and osimertinib) were dissolved in solutions containing 5% DMSO, 45% PEG400 and 50% H2O, respectively, and the modeling drugs were diluted to the required concentrations and administered orally once a day according to the doses shown in Table 3. One hour after oral administration, 0.05 g of ointment containing different mass ratios of the test compound was evenly applied to the left paw of the rat, while the right paw was applied with a blank matrix ointment as a self-control. The rats in the blank control group were not applied with the drug. After applying the drug, the limbs were fixed for 2 hours, and then the residual drug was wiped off with clean water, the fixation was released and free movement was restored. The modeling drug, blank matrix ointment, and test compound ointment were all administered once daily for 30 consecutive days. The rats were euthanized, and paw plantar skin tissue was obtained, fixed in 10% neutral formaldehyde, cut into 5 μm sections, dehydrated, and embedded in paraffin. The test compound ointment was prepared by mixing the compound, white beeswax, white petrolatum, and light liquid paraffin in specific ratios (weight ratios of the three ointments were: 1:18:58:23, 3:18:57:22, and 10:20:40:30, respectively). The blank matrix ointment was prepared by mixing white beeswax, white petrolatum, and light liquid paraffin in specific ratios (weight ratio: 18:60:22).

[0271] Tissue staining: After dewaxing and rehydrating, the paw and plantar skin tissue sections of the above rats were stained in a hematoxylin solution for several minutes. After washing, the sections were immersed in 1% acidic alcohol until the sections faded to a light blue-red color. After washing with running water for 5 minutes, the sections were stained with eosin for 2-3 minutes. After washing to remove excess dye, the sections were dehydrated and transparentized in xylene for several minutes. The sections were then sealed with a neutral resin. The stratum corneum was observed under an optical microscope, and the thickness of the epidermal stratum corneum was measured using Dmetrix software.

[0272] Immunohistochemical staining: After dewaxing and hydration, the paw plantar skin tissue sections of the above rats were incubated with 3% H2O2 at room temperature for 30 minutes. After antigen retrieval, they were blocked with 10% goat serum for 30 minutes. KRT1 antibodies (Abcam, ab93652) and Loricrin antibodies (Abcam, ab183646) were added dropwise and incubated overnight at 4°C. HRP secondary antibodies (ZSGB-BIO, PV-6001) and DAB kits (ZSG-BIO, ZLI9017) were added for color development. The sections were counterstained with hematoxylin, washed, and mounted with central resin. The expression levels of KRT1 and Loricrin were observed under a light microscope.

[0273] The criteria for successful rat model establishment as described above are: (i) the appearance of symptoms such as erythema, swelling, desquamation, ulceration, or blisters on the paw; and / or (ii) a significantly higher stratum corneum thickness than normal rats in tissue staining; and / or (iii) a significant increase in markers such as KRT1, KRT5, and Loricrin. The incidence rate was calculated as the proportion of animals in each group meeting the above criteria for successful model establishment: incidence rate = (number of rats with successful model establishment / total number of rats in the group) * 100%.

[0274] The rat tissue staining pathology scoring criteria are as follows: no blisters, 0 points; 1-3 blisters, 1 point; 4-6 blisters, 2 points; 7-9 blisters, 3 points; and more than 10 blisters, 4 points. Inflammation occupying less than 10% of the total section area is scored as 0 points; inflammation occupying 10-25% of the total section area is scored as 1 point; inflammation occupying 25-50% of the total section area is scored as 2 points; inflammation occupying 50-75% of the total section area is scored as 3 points; and inflammation occupying more than 75% of the total section area is scored as 4 points. A score of 0 was assigned if the hyperemia area accounted for less than 10% of the total slice area; a score of 1 was assigned if the hyperemia area accounted for 10-25% of the total slice area; a score of 2 was assigned if the hyperemia area accounted for 25-50% of the total slice area; a score of 3 was assigned if the hyperemia area accounted for 50-75% of the total slice area; and a score of 4 was assigned if the hyperemia area accounted for more than 75% of the total slice area. Scoring was performed using a double-blind scoring system, and data from each group were presented as mean ± SEM (N = 12).

[0275] Table 3: Drug dosage and experimental results in rat models

[0276] As shown in Table 3, once-daily oral administration of sorafenib, erlotinib, afatinib, and osimertinib to SD rats at doses of 100 mg / kg, 70 mg / kg, 50 mg / kg, and 60 mg / kg, respectively, resulted in 75%, 83.3%, 66.7%, and 75% success rates for establishing hand-foot skin reaction models, respectively. The compound of formula (I) significantly reduced the incidence of hand-foot skin reaction at the application site at low doses (e.g., 1% by weight of the compound). The compound of formula (I) significantly reduced the incidence of hand-foot skin reaction at all paws of rats at high concentrations (e.g., 3% and 10% by weight of the compound).

[0277] As shown in Figure 8, the stratum corneum thickness measurements of rats successfully treated with sorafenib, erlotinib, afatinib, and osimertinib showed significantly higher stratum corneum thickness in the paw and plantar skin than in normal rats. The compound of formula (I) can effectively reduce the stratum corneum thickness of the paw and plantar skin in the model rats.

[0278] As shown in Figure 9, the tissue staining results show that the skin toxicity caused by the modeling drug can form blisters in the subcutaneous and epidermal regions of the rat paws and feet, inflammatory cell infiltration in the dermis, and congestion in the skin tissue. The compound of formula (I) can effectively ameliorate the formation of subcutaneous and epidermal blisters caused by the modeling drug, inhibit inflammatory cell infiltration in the dermis, and significantly improve skin congestion.

[0279] As shown in the pathological scoring results in Figure 10, the modeling drug can cause the formation of blister subcutaneously and intraepidermally in the plantar region of the rat paw, inflammatory cell infiltration in the dermis, and congestion in the skin tissue. The compound of formula (I) can effectively ameliorate the formation of blister subcutaneously and intraepidermally induced by the modeling drug, inhibit inflammatory cell infiltration in the dermis, and significantly improve skin congestion.

[0280] Immunohistochemical staining results also showed that KRT1 and Loricrin levels increased significantly in tissues of rats successfully treated with sorafenib, erlotinib, afatinib, and osimertinib. However, with the addition of the compound, KRT1 and Loricrin levels decreased significantly in tissues. In summary, the compound of formula (I) can effectively inhibit keratinocyte differentiation in vitro and in vivo, and has the potential to treat hyperkeratosis-related diseases.

[0281] Example 11: Effect of the compound of formula (I) in a mouse hand-foot syndrome model induced by chemotherapy drugs

[0282] After 7 days of adaptive feeding, SPF ICR mice (male, 5-6 weeks old, weighing approximately 35 grams) were randomly divided into the following groups: blank control group (6 mice), capecitabine model group (6 mice), docetaxel model group (6 mice), capecitabine + compound of formula (I) group (6 mice), docetaxel + compound of formula (I) group (6 mice), capecitabine + nicotinamide group (6 mice), and docetaxel + nicotinamide group (6 mice). Each group was orally administered with the corresponding modeling drug (capecitabine 200 mg / kg, docetaxel 25 mg / kg) by gavage once daily; the blank control group was given an equal volume of normal saline. After 2 weeks of chemotherapy drug administration, each group was topically applied with the corresponding ointment of the compound of formula (I) used in Example 10 (compound content 3%) or orally administered with nicotinamide (100 mg / kg) once daily for 16 consecutive days.

[0283] Measurement of toe swelling: The degree of toe swelling of the mouse hind limbs was measured using a toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) before administration of the modeling drug, 2 weeks after administration of the modeling drug, and before sacrifice. The same position was marked on the joint of the mouse hind foot before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was exactly on the same horizontal line as the marked position, and the value was recorded after it stabilized.

[0284] Skin histopathological staining: Skin tissue samples from the hind limbs of mice were fixed in 4% paraformaldehyde at room temperature for 4 hours. The tissues were then removed and rinsed with running water for several hours. After dehydration with 70%, 80%, and 90% ethanol solutions, they were treated with a mixture of equal parts pure alcohol and xylene for 15 minutes. Permeabilization was performed twice with xylene for 15 minutes each, until the sample became transparent. The samples were then immersed in a mixture of 50% xylene and 50% paraffin for 15 minutes, followed by permeabilization with Paraffin I and Paraffin II for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed, and hydrated. The hydrated sections were then stained in hematoxylin solution (Zhongshan Jinqiao, Catalog No. 23041001) for 3 minutes, differentiated in hydrochloric acid ethanol solution for 15 seconds, washed with water, and blued in Scott blue solution (Servicebio, Catalog No. 20230801) for 15 seconds. After rinsing with running water, the sections were stained with eosin stain (Solarbio, Catalog No. 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparentized, mounted, and examined under a microscope.

[0285] Immunohistochemical staining: Paraffin sections of mouse hind limb skin tissue were baked, dewaxed, and hydrated, followed by antigen retrieval using 0.2 M citric acid buffer (1.534 g citric acid, 3.1 g sodium citrate, 100 mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed with 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., Catalog No. 20290902). After incubation at room temperature for 10 minutes, sections were rinsed thoroughly with PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 1 M HCl, pH 7.4). Sections were permeabilized with 0.5% Triron-X-100 (Aimejie Technology, Catalog No. A-CSH436-100 mL). Mouse anti-IL-1β and rabbit anti-IL-8 were permeabilized for 10 minutes each, while rabbit anti-IL-6 was not permeabilized. After blocking with 5% BSA antigen, a 1 / 150 dilution of mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), a 1 / 200 dilution of rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998), or a 1 / 150 dilution of rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified chamber at 4°C overnight. Remove the wet chamber after overnight incubation and let it stand at room temperature for 45 minutes. Wash the slides three times with PBS buffer for 15 minutes each. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, Cat. No. 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 markers. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, Cat. No. 226700804) to mouse anti-IL-1β markers. Incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, Cat. No. 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, Cat. No. 23041001) for 3 minutes, differentiate with hydrochloric acid and alcohol to turn blue, and wash with water. The sections were sequentially placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II for rapid dehydration, and then transparentized with xylene I and xylene II. The sections were then sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

[0286] Figure 11 shows the skin appearance of mice. Fourteen days after the capecitabine and docetaxel treatment, significant lesions appeared on the skin of the hind limbs and toes of the mice. Compared with the normal control group, the capecitabine and docetaxel groups showed significant redness, swelling, cracking, and blisters on the skin of the hind limbs and toes of the mice.

[0287] Figure 12 shows the extent of hind paw swelling in mice. The modeling drugs capecitabine and docetaxel caused hind paw swelling in mice after 14 consecutive days of oral administration. Topical application of the compound of formula (I) ointment to the hind paws of mice significantly improved drug-induced paw swelling (p < 0.05). However, oral administration of 100 mg / kg nicotinamide did not significantly improve hind paw swelling in mice.

[0288] The results of pathological staining of the toe skin tissue are shown in Figure 13. Compared with the normal control group, the modeling drugs capecitabine and docetaxel can cause significant thickening of the epidermis and stratum corneum in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days, an increase in basal layer granular cells, and visible inflammatory cell infiltration. Local application of the ointment of the compound of formula (I) on the hind limbs and toes of mice can effectively improve the thickening of the epidermis and stratum corneum of the skin tissue, and the morphology of the basal layer cells returns to normal, and no inflammatory cell infiltration is seen. However, oral administration of 100 mg / kg nicotinamide has a slight improvement in the thickening of the epidermis and stratum corneum of the skin tissue of the hind limbs and toes of mice, with a slightly increased number of basal layer granular cells and still visible inflammatory cell infiltration.

[0289] The immunohistochemical results of the capecitabine modeling series are shown in Figure 14. Compared with the normal control group, the modeling drug capecitabine caused a significant increase in the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p < 0.05). This shows that oral administration of capecitabine can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes (p < 0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-8 in the skin tissue of the mouse hind limbs and toes.

[0290] The immunohistochemical results of the docetaxel modeling series are shown in Figure 15. Compared with the normal control group, the modeling drug docetaxel caused a significant increase in the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p < 0.05). This shows that oral administration of docetaxel can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes (p < 0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-6 in the skin tissue of the mouse hind limbs and toes.

[0291] Example 12: Effects of the compound of formula (I) in a kinase inhibitor-induced mouse hand-foot skin reaction model

[0292] After 7 days of adaptive feeding, SPF ICR mice (male, 5-6 weeks old, weighing approximately 35 grams) were randomly divided into the following groups: blank control group (6 mice), sorafenib modeling group (6 mice), osimertinib modeling group (6 mice), sorafenib + compound of formula (I) group (6 mice), osimertinib + compound of formula (I) group (6 mice), sorafenib + nicotinamide group (6 mice), and osimertinib + nicotinamide group (6 mice). Each group was orally gavaged with the corresponding modeling drug (sorafenib 100 mg / kg, osimertinib 10 mg / kg) once daily; the blank control group was given an equal volume of normal saline. After 2 weeks of chemotherapy, each group was topically applied with the corresponding ointment of the compound of formula (I) used in Example 10 (compound content 3%) or orally administered with nicotinamide (100 mg / kg) once daily for 16 consecutive days.

[0293] Measurement of toe swelling: The degree of toe swelling of the mouse hind limbs was measured using a toe swelling tester (KW-7C, Nanjing Calvin Biotechnology Co., Ltd.) before administration of the modeling drug, 2 weeks after administration of the modeling drug, and before sacrifice. The same position was marked on the joint of the mouse hind foot before administration, 2 weeks after administration, and before sacrifice. During measurement, the water was exactly on the same horizontal line as the marked position, and the value was recorded after it stabilized.

[0294] Skin histopathological staining: Skin tissue samples from the hind limbs of mice were fixed in 4% paraformaldehyde at room temperature for 4 hours. The tissues were then removed and rinsed with running water for several hours. After dehydration with 70%, 80%, and 90% ethanol solutions, they were treated with a mixture of equal parts pure alcohol and xylene for 15 minutes. Permeabilization was performed twice with xylene for 15 minutes each, until the sample became transparent. The samples were then immersed in a mixture of 50% xylene and 50% paraffin for 15 minutes, followed by permeabilization with Paraffin I and Paraffin II for one hour each. After paraffin embedding, the samples were sliced, baked, dewaxed, and hydrated. The hydrated sections were then stained in hematoxylin solution (Zhongshan Jinqiao, Catalog No. 23041001) for 3 minutes, differentiated in hydrochloric acid ethanol solution for 15 seconds, washed with water, and blued in Scott blue solution (Servicebio, Catalog No. 20230801) for 15 seconds. After rinsing with running water, the sections were stained with eosin stain (Solarbio, Catalog No. 33535) for 3 minutes. After rinsing with running water, the sections were dehydrated, transparentized, mounted, and examined under a microscope.

[0295] Immunohistochemical staining: Paraffin sections of mouse hind limb skin tissue were baked, dewaxed, and hydrated, followed by antigen retrieval using 0.2 M citric acid buffer (1.534 g citric acid, 3.1 g sodium citrate, 100 mL distilled water, pH 4.7). Endogenous hydrogen peroxide was removed with 3% hydrogen peroxide (Shandong Anjie High-Tech Disinfection Technology Co., Ltd., Catalog No. 20290902). After incubation at room temperature for 10 minutes, sections were rinsed thoroughly with PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 1 M HCl, pH 7.4). Sections were permeabilized with 0.5% Triron-X-100 (Aimejie Technology, Catalog No. A-CSH436-100 mL). Mouse anti-IL-1β and rabbit anti-IL-8 were permeabilized for 10 minutes each, while rabbit anti-IL-6 was not permeabilized. After blocking with 5% BSA antigen, a 1 / 150 dilution of mouse anti-IL-1β (Affinity Biosciences, Catalog No. BF8021), a 1 / 200 dilution of rabbit anti-IL-8 (Affinity Biosciences, Catalog No. DF6998), or a 1 / 150 dilution of rabbit anti-IL-6 (Affinity Biosciences, Catalog No. DF6087) was added to each slide and incubated in a humidified chamber at 4°C overnight. Remove the wet chamber after overnight incubation and let it stand at room temperature for 45 minutes. Wash the slides three times with PBS buffer for 15 minutes each. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (Zhongshan Jinqiao, Cat. No. 234750811) to rabbit anti-IL-8 and rabbit anti-IL-6 markers. Add a 1 / 100 dilution of horseradish enzyme-conjugated goat anti-mouse IgG (H+L) (Zhongshan Jinqiao, Cat. No. 226700804) to mouse anti-IL-1β markers. Incubate at 37°C for 30 minutes and then rinse thoroughly with PBS buffer. Add DAB (CWBIO, Cat. No. 13723) for color development for 3-5 minutes, wash with PBS buffer for 1 minute, counterstain with hematoxylin (Zhongshan Jinqiao, Cat. No. 23041001) for 3 minutes, differentiate with hydrochloric acid and alcohol to turn blue, and wash with water. The sections were sequentially placed in 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II for rapid dehydration, and then transparentized with xylene I and xylene II. The sections were then sealed with neutral resin glue (Solarbio, product number 20230725) and examined under a microscope.

[0296] The results, as shown in Figure 16, showed that 14 days after the sorafenib and osimertinib treatments, the skin of the hind limbs and toes of the mice developed significant lesions. Compared with the normal control group, the skin of the hind limbs and toes of the mice in the sorafenib and osimertinib groups showed significant redness, swelling, and cracking.

[0297] The degree of swelling in the mouse hind limbs and toes is shown in Figure 17. The modeling drugs sorafenib and osimertinib caused significant swelling in the mouse hind limbs and toes after 14 consecutive days of oral administration. Topical application of the compound of formula (I) ointment to the mouse hind limbs and toes effectively alleviated the swelling caused by the modeling drugs. However, oral administration of 100 mg / kg of nicotinamide did not significantly improve the swelling in the mouse hind limbs and toes.

[0298] The results of pathological staining of the toe skin tissue are shown in Figure 18. Compared with the normal control group, the modeling drugs sorafenib and osimertinib can cause significant thickening of the epidermis and stratum corneum in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days, an increase in basal layer granular cells, and visible inflammatory cell infiltration. Local application of the ointment of the compound of formula (I) on the hind limbs and toes of mice can effectively improve the thickening of the epidermis and stratum corneum of the skin tissue, and the morphology of the basal layer cells returns to normal, and no inflammatory cell infiltration is seen. However, oral administration of 100 mg / kg nicotinamide has no significant improvement in the thickening of the epidermis and stratum corneum of the skin tissue of the hind limbs and toes of mice, and the morphology of the basal layer cells recovers slightly, and inflammatory cell infiltration is still seen.

[0299] The immunohistochemical results of the Sorafenib modeling series are shown in Figure 19. Compared with the normal control group, the modeling drug Sorafenib caused a significant increase in the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice after continuous oral administration for 14 days (p<0.05). This shows that oral administration of Sorafenib can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice (p<0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-1β in the skin tissue of the hind limbs and toes of mice.

[0300] The immunohistochemical results of the osimertinib modeling series are shown in Figure 20. Compared with the normal control group, the modeling drug osimertinib caused a significant increase in the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes after 14 consecutive days of oral administration (p<0.05). This shows that oral administration of osimertinib can cause skin inflammation in mice, which is consistent with the results of clinical trial observations. Topical application of the ointment of the compound of formula (I) to the hind limbs and toes of mice can significantly reduce the expression level of the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes (p<0.05). However, oral administration of 100 mg / kg nicotinamide had no significant effect on the expression level of the cytokine IL-1β in the skin tissue of the mouse hind limbs and toes.

[0301] Example 13: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and nicotinamide

[0302] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 10.3 mg of nicotinamide, and acetonitrile (0.25 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and nicotinamide. The XRPD spectrum is shown in Figure 21, and the characteristic peak positions are shown in Table 4. The DSC spectrum showed that the melting point T onset @156.75℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0303] Table 4: XRPD diffraction peak data of the drug co-crystal formed by the compound represented by formula (I) and nicotinamide

[0304] Example 14: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and isonicotinamide

[0305] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 10.2 mg of isonicotinamide, and ethanol (0.2 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and isonicotinamide. The XRPD spectrum is shown in Figure 22, and the characteristic peak positions are shown in Table 5. The DSC spectrum showed that the melting point T onset 160.45°C. The chemical dosage ratio of the obtained drug cocrystal is 1:1.

[0306] Table 5: XRPD diffraction peak data of the drug co-crystal formed by the compound represented by formula (I) and isonicotinamide

[0307] Example 15: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and L-proline

[0308] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 9.7 mg of L-proline, and ethanol (0.25 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and L-proline. The XRPD spectrum is shown in Figure 23, and the characteristic peak positions are shown in Table 6. The DSC spectrum shows that the melting point T onset @183.55℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0309] Table 6: XRPD diffraction peak data of the drug co-crystal formed by the compound represented by formula (I) and L-proline

[0310] Example 16: Preparation of pharmaceutical co-crystals formed by the compound represented by formula (I) and glycolic acid

[0311] About 20 mg of the compound represented by formula (I) was weighed and placed in a 2 mL glass bottle with 6.3 mg of glycolic acid, and isopropyl acetate (0.2 mL) was added to obtain a suspension. The obtained sample was suspended and stirred at 5°C for 3 days. The obtained suspension was centrifuged at 14,000 rpm through a 0.45 μm nylon filter membrane, and the obtained solid was vacuum dried at 25°C for 4 hours to obtain the product. X-ray powder diffraction analysis showed that the product was a drug co-crystal formed by the compound represented by formula (I) and glycolic acid. The XRPD spectrum is shown in Figure 24, and the characteristic peak positions are shown in Table 7. The DSC spectrum shows that the melting point T onset @135.68℃. The obtained drug cocrystal has a chemical dosage ratio of 1:1.

[0312] Table 7: XRPD diffraction peak data of the drug co-crystal formed by the compound represented by formula (I) and glycolic acid

[0313] Example 17: Screening of drug-containing prescriptions

[0314] Based on the properties of the compound represented by formula (I), the formulation screening was evaluated based on appearance, viscosity, coating properties, physical stability, etc. The evaluation results are shown in Table 8:

[0315] Table 8: Screening of prescriptions containing drugs

[0316] According to the above results, the ratios in prescription A and prescription B showed better ointment properties in terms of appearance, consistency, hardness, and spreadability.

[0317] Example 18: Ointment formulation containing diethylene glycol monomethyl ether and PEG400

[0318] Based on the properties of the compound represented by formula (I), drug-containing prescriptions 1 to 4 are designed as shown in Table 9 below:

[0319] Table 9: Prescriptions containing medication 1 to 4

[0320] The stability test results of medicated prescriptions 1 to 4 are shown in Tables 10 and 11:

[0321] Table 10: Stability test of drug-containing prescription 1 and drug-containing prescription 2 1 RRT:Relative Retention Time, relative retention time; 2 RH:Relative Humidity, relative humidity; 3 ND: not detected

[0322] Table 11: Stability test of drug-containing prescription 3 and drug-containing prescription 4 1 RRT:Relative Retention Time, relative retention time; 2 RH:Relative Humidity, relative humidity; 3 ND: not detected

[0323] From the above test results, it can be seen that in the medicated ointment in the medicated formula 1 containing the PEG matrix, the impurities RRT=0.86, RRT=1.06, and RRT=1.18 all increased significantly under the accelerated stability test conditions. After the addition of the BHT antioxidant, the impurities at RRT=0.86 and RRT=1.06 of the medicated formula 2 were slightly improved, but the impurity growth trend at RRT=1.18 was still significant.

[0324] In the medicated ointment in medicated formulation 3 containing diethylene glycol monomethyl ether as a matrix, the impurities RRT = 0.86 and RRT = 1.37 showed a significant increase under the conditions of the accelerated stability test. After the addition of BHT antioxidant in medicated formulation 4, the impurity change trend at RRT = 1.37 was still significant.

[0325] Example 19: Suspension ointment preparation containing the compound represented by formula (I) (1%)

[0326] A suspension ointment preparation containing the compound represented by formula (I) was prepared. The specific formulation is shown in Table 12:

[0327] Table 12: Prescription information of suspension ointment formulation (1%) containing the compound represented by formula (I)

[0328] Preparation method:

[0329] (1) Weigh white beeswax and white vaseline, heat to 65-70°C in a water bath to melt, and homogenize at 350 rpm for 20 minutes to obtain a uniform mixture A;

[0330] (2) Weigh light liquid paraffin, add the weighed compound of formula (I) thereto, heat in a water bath to about 40°C and stir for 10 minutes until the active ingredient is completely dispersed, stirring at 400 rpm to obtain a uniform mixture B;

[0331] (3) Mixture A was cooled to 40°C, and mixture B was added thereto. The mixture was stirred continuously and homogenized at 10,000 rpm for 2 minutes. The mixture was then cooled to room temperature to obtain a suspension ointment preparation containing the compound of formula (I).

[0332] Example 20: Suspension ointment preparation containing the compound represented by formula (I) (3%)

[0333] A suspension ointment preparation containing the compound represented by formula (I) was prepared using the same preparation method as in Example 19. The specific formulation is shown in Table 13.

[0334] Table 13: Prescription information of suspension ointment formulation (3%) containing the compound represented by formula (I)

[0335] Example 21: Suspension ointment preparation containing the compound represented by formula (I) (10%)

[0336] A suspension ointment preparation containing the compound represented by formula (I) was prepared using the same preparation method as in Example 19. The specific formulation is shown in Table 14.

[0337] Table 14: Prescription information of suspension ointment formulation (10%) containing the compound represented by formula (I)

[0338] The ointment prepared in this example has suitable consistency and hardness and is easy to apply.

[0339] Example 22: Stability test of binary mixture of compound of formula (I) and penetration enhancer

[0340] Different types of penetration enhancers and the compound of formula (I) were mixed at a weight ratio of 10:1, and stability tests were performed under accelerated conditions of 40°C / 75% RH. The test results are shown in Table 15.

[0341] Table 15: Stability test of binary mixture of compound of formula (I) and penetration enhancer 1 RRT:Relative Retention Time, relative retention time; 2 ND: not detected

[0342] The results showed that the compound of formula (I) was unstable in both propylene carbonate and diisopropyl oxalate.

[0343] Example 23: Suspension ointment preparation containing a compound of formula (I) and a penetration enhancer

[0344] Design drug-containing prescriptions 5 to 6, as shown in Table 16:

[0345] Table 16: Ratios of drug-containing prescriptions 5 and 6

[0346] The stability test results of drug-containing prescriptions 5 to 6 are shown in Table 17:

[0347] Table 17: Stability test of drug-containing prescriptions 5 and 6 1 RRT:Relative Retention Time, relative retention time; 2 RH:Relative Humidity, relative humidity; 3 ND: not detected

[0348] The above test results show that in the drug-containing formulation 5 containing the penetration enhancer propylene glycol, the impurities RRT = 0.86, RRT = 0.93, RRT = 1.05, RRT = 1.21, and RRT = 1.36 all showed significant increases under the accelerated stability test conditions. In the drug-containing formulation 6 containing the penetration enhancer isopropyl myristate, the overall stability was good.

[0349] Example 24: Suspension ointment preparation containing the compound represented by formula (I) and a penetration enhancer (1%)

[0350] A suspension ointment preparation containing the compound represented by formula (I) and a penetration enhancer was prepared. The specific formulation is shown in Table 18:

[0351] Table 18: Prescription information of suspension ointment formulation (1%) containing the compound represented by formula (I) and a penetration enhancer

[0352] Preparation method:

[0353] (1) Weigh white beeswax, white petrolatum, and isopropyl myristate, heat to 65-70°C in a water bath to melt, and homogenize at 350 rpm for 15 minutes to obtain a uniform mixture A;

[0354] (2) Weigh light liquid paraffin, add the weighed compound of formula (I) thereto, heat in a water bath to about 40°C and stir for 10 minutes until the active ingredient is completely dispersed, stirring at a speed of 300 rpm to obtain a uniform mixture B;

[0355] (3) Mixture A was cooled to 40°C, and mixture B was added thereto. The mixture was stirred continuously and homogenized at 10,000 rpm for 2 minutes. The mixture was then cooled to room temperature to obtain a suspension ointment preparation containing the compound of formula (I) and a penetration enhancer.

[0356] Example 25: Suspension ointment preparation containing the compound represented by formula (I) and a penetration enhancer (10%)

[0357] A suspension ointment preparation containing the compound represented by formula (I) and a penetration enhancer was prepared. The preparation method was the same as that of Example 24. The specific formulation is shown in Table 19.

[0358] Table 19: Prescription information of a suspension ointment formulation (10%) containing a compound represented by formula (I) and a penetration enhancer

[0359] Example 26: Suspension ointment preparation (1%) containing a compound represented by formula (I), a penetration enhancer and an antioxidant

[0360] A suspension ointment preparation containing a compound represented by formula (I), a penetration enhancer and an antioxidant was prepared. The specific formulation is shown in Table 20:

[0361] Table 20: Prescription information of a suspension ointment formulation (1%) containing a compound represented by formula (I), a penetration enhancer, and an antioxidant

[0362] Preparation method:

[0363] (1) Weigh white beeswax, white petrolatum, isopropyl myristate, and butylated hydroxytoluene (BHT), heat to 65-70°C in a water bath to melt, and homogenize at 350 rpm for 15 minutes to obtain a uniform mixture A;

[0364] (2) Weigh light liquid paraffin, add the weighed compound of formula (I) thereto, heat in a water bath to about 40°C and stir for 10 minutes until the active ingredient is completely dispersed, stirring at a speed of 300 rpm to obtain a uniform mixture B;

[0365] (3) Mixture A was cooled to 40°C, and mixture B was added thereto. The mixture was continuously stirred and homogenized at 10,000 rpm for 2 minutes. The mixture was then cooled to room temperature to obtain a suspension ointment preparation containing the compound of formula (I), a penetration enhancer, and an antioxidant.

[0366] The 1% ointment containing isopropyl myristate and the antioxidant BHT was placed under 40°C / 75% accelerated conditions for one month and had good overall stability. The results are shown in Table 21.

[0367] Table 21: Stability test results of ointments containing penetration enhancers and antioxidants 1 RRT:Relative Retention Time, relative retention time; 2 RH:Relative Humidity, relative humidity; 3 ND: not detected

[0368] Example 27: Suspension ointment preparation containing the compound represented by formula (I) (0.3%)

[0369] A suspension ointment preparation containing the compound represented by formula (I) was prepared using the same preparation method as in Example 19. The specific formulation is shown in Table 22.

[0370] Table 22: Prescription information of suspension ointment formulation (0.3%) containing the compound represented by formula (I)

[0371] Example 28: Pharmacokinetic study of CD-1 mice after single skin application of an ointment containing the compound of formula (I)

[0372] Six CD-1 mice were divided into two groups, with three mice in each group, and received 1 mg / cm 2 The ointment containing the compound represented by formula (I) (3% suspension ointment) was applied to the back once, with the application area of ​​1 cm 2 The plasma concentration and skin tissue homogenate concentration of the compound represented by formula (I) were determined by LC-MS / MS.

[0373] The blood concentration of the compound represented by formula (I) in the first group of three CD-1 mice at 4 hours was 6.15 ng·h / mL. At the same time, the skin tissue homogenate of the back of CD-1 mice was taken at 4 hours, and the average drug concentration of the compound represented by formula (I) in the skin was 33033 ng / g.

[0374] The area under the plasma concentration-time curve (AUC) of the compound represented by formula (I) from time 0 to the last quantifiable concentration time (24 h) in the second group of 3 CD-1 mice was0-last The average value of C of the compound represented by formula (I) in plasma was 40.7 ng·h / mL. max The mean value was 14.0 ng / mL, and the peak time (T max ) appeared 1.67 h after administration, and the elimination half-life (T 1 / 2 ) with an average of 5.15 h. The dorsal skin tissue homogenates from the three CD-1 mice were sampled 24 hours after the endpoint. The average concentration of the compound of formula (I) in the skin was 7907 ng / g. The concentration of the compound of formula (I) in the plasma samples of the CD-1 mice at 24 hours was below the detection limit.

[0375] The concentration of the compound represented by formula (I) in skin tissue is much higher than that in plasma.

[0376] All animals tolerated the dose and no abnormalities were observed during the entire study.

[0377] The average PK parameters of the compound represented by formula (I) after a single dorsal skin application in CD-1 mice are summarized in Table 23.

[0378] Table 23: Pharmacokinetic parameters of the compound represented by formula (I) after single transdermal administration in CD-1 mice

[0379] Example 29: Pharmacokinetic study of Bama miniature pigs after single skin application of an ointment containing the compound of formula (I)

[0380] Two Bama miniature pigs were given 20 mg / cm 2 An ointment (3% suspension ointment) containing the compound of formula (I) was administered by single application to the back and abdomen, with the total application area covering 6.8% of the body surface area, with the dorsal and abdominal application areas approximately in a 1:1 ratio. The plasma concentration and skin tissue homogenate concentration of the compound of formula (I) were determined by LC-MS / MS.

[0381] The area under the plasma concentration-time curve (AUC) of the compound represented by formula (I) from time 0 to the last quantifiable concentration time (24 h) in two Bama miniature pigs 0-last The average value of C of the compound represented by formula (I) in plasma was 304 ng·h / mL. max The mean value was 21.6 ng / mL, and the peak time (T max ) appeared 16 hours after administration. The mean plasma drug concentration at the end point 24 hours was 17.7 ng / mL.

[0382] Skin tissue homogenates from two Bama minipigs were sampled at the 24-hour endpoint. The average drug concentration of the compound represented by formula (I) in the dorsal dermis was 88,336 ng / g at 24 hours. The average drug concentration of the compound represented by formula (I) in the dorsal epidermis was 1,109,245 ng / g at 24 hours, and the average drug concentration of the compound represented by formula (I) in the abdominal skin was 110,952 ng / g at 24 hours. The concentrations of the compound represented by formula (I) in each skin tissue were significantly higher than those in plasma.

[0383] All animals tolerated the dose and no abnormalities were observed during the entire study.

[0384] The average PK parameters of the compound represented by formula (I) after a single dorsal skin application to Bama miniature pigs are summarized in Table 24.

[0385] Table 24: Pharmacokinetic parameters of the compound represented by formula (I) after single transdermal administration in Bama minipigs

[0386] Example 30: Pharmacokinetic study of SD rats after single skin application of an ointment containing the compound of formula (I)

[0387] Six SD rats were divided into two groups, 3 rats in each group, one of which received 20 mg / cm 2 The ointment containing the compound represented by formula (I) (3% suspension ointment) was applied to the back skin once, with the application area of ​​20 cm 2 , and the other group received 20 mg / cm 2 The ointment containing the compound represented by formula (I) (10% suspension ointment) was applied to the back skin once, with the application area of ​​20 cm 2 The plasma concentration and dorsal skin tissue homogenate concentration of the compound represented by formula (I) were determined by LC-MS / MS.

[0388] The mean plasma drug concentration at 24 hours in three SD rats that received the 3% suspension ointment was 3.21 ng / mL, and the dorsal skin homogenate drug concentration at 24 hours was 161,167 ng / g. The mean plasma drug concentration at 24 hours in three SD rats that received the 10% suspension ointment was 17.3 ng / mL, and the dorsal skin homogenate drug concentration at 24 hours was 799,500 ng / g. The concentration of the compound represented by formula (I) in skin tissue is much higher than the drug concentration in plasma.

[0389] All animals tolerated the dose and no abnormalities were observed during the entire study.

[0390] The average PK parameters of the compound represented by formula (I) after a single dosing of SD rats by dorsal skin application are summarized in Table 25.

[0391] Table 25: Pharmacokinetic parameters of the compound represented by formula (I) after single transdermal administration in SD rats

[0392] The PK results above demonstrate that, following transdermal administration of a suspension ointment of the compound represented by Formula (I) to mice, rats, and pigs, plasma exposure was significantly lower than skin exposure. This ointment offers low systemic exposure and high safety in the treatment of skin-related diseases.

[0393] Example 31: Stability Study of Suspension Ointment Formulation

[0394] 1% suspension ointment formulations, 3% suspension ointment formulations, and 10% suspension ointment formulations prepared according to the methods of Examples 19, 20, and 21 were placed under two different conditions, 25°C / 60% RH and 40°C / 75% RH, for 3 months and 1 month, respectively. The formulation stability was observed by HPLC analysis of changes in the content of related substances. The results are shown in Table 26. The related substances in the formulations did not change significantly, and the formulations were generally stable.

[0395] Table 26: Stability study of suspension ointment formulations 1 RRT:Relative Retention Time, relative retention time; 2 RH:Relative Humidity, relative humidity; 3 ND: not detected

[0396] The three drug-containing formulations of Examples 19, 20, and 21 performed well in the stability test. The comprehensive ointment performance in terms of appearance, consistency, hardness, and coating properties was also very suitable. The feasibility of large-scale production was also comprehensively considered. The excipient ingredients were simple and commercially available. These formulations of the compound of formula (I) are very suitable for clinical development.

[0397] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. An RNA m6A regulator composition, characterized in that, The composition comprises: a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable co-crystal, and an oil phase substance, 2. The composition according to claim 1, wherein The pharmaceutically acceptable salts include hydrochloride, sulfate, phosphate, acetate, mesylate, benzenesulfonate, p-toluenesulfonate; Preferably, the eutectic formers of the pharmaceutically acceptable eutectics include nicotinamide, isonicotinamide, L-proline, glycolic acid; Preferably, the oil phase substances include oil phase substance 1, oil phase substance 2 and oil phase substance 3; Preferably, the oil phase substance 1 is selected from one or more of lanolin, beeswax, white beeswax, paraffin wax, preferably white beeswax; Preferably, the oil phase substance 2 is selected from one or more of petrolatum, white petrolatum, preferably white petrolatum; Preferably, the oil phase substance 3 is selected from one or more of light liquid paraffin, vegetable oil, preferably light liquid paraffin.

3. The composition according to claim 2, characterized in that, By mass percentage, the composition comprises 0.1%-20% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 10%-30% of oil phase substance 1, 30%-70% of oil phase substance 2, 15%-40% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 0.2%-15% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 15%-25% of oil phase substance 1, 35%-64% of oil phase substance 2, 20%-35% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 0.3%-10% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 18%-20% of oil phase substance 1, 40%-58.7% of oil phase substance 2, 22%-30% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 0.3% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 18% of oil phase substance 1, 58.7% of oil phase substance 2, 23% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 1% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 18% of oil phase substance 1, 58% of oil phase substance 2, 23% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 3% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 18% of oil phase substance 1, 57% of oil phase substance 2, 22% of oil phase substance 3; Preferably, by mass percentage, the composition comprises 10% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 20% of oil phase substance 1, 40% of oil phase substance 2, 30% of oil phase substance 3.

4. The composition according to claim 3, characterized in that, The composition comprises the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, white beeswax, white petrolatum, light liquid paraffin; Preferably, by mass percentage, the composition comprises 0.1%-20% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable eutectic, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-40% of light liquid paraffin; Preferably, by mass percentage, the composition comprises 0.2%-15% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal, 15%-25% white beeswax, 35%-64% white petrolatum, and 20%-35% light liquid paraffin; Preferably, by mass percentage, the composition comprises 0.3%-10% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal, 18%-20% white beeswax, 40%-58.7% white petrolatum, and 22%-30% light liquid paraffin; Preferably, by mass percentage, the composition comprises 0.3% of the compound shown by formula (I), 18% white beeswax, 58.7% white petrolatum, and 23% light liquid paraffin; Preferably, by mass percentage, the composition comprises 1% of the compound shown by formula (I), 18% white beeswax, 58% white petrolatum, and 23% light liquid paraffin; Preferably, by mass percentage, the composition comprises 3% of the compound shown by formula (I), 18% white beeswax, 57% white petrolatum, and 22% light liquid paraffin; Preferably, by mass percentage, the composition comprises 10% of the compound shown by formula (I), 20% white beeswax, 40% white petrolatum, and 30% light liquid paraffin.

5. The composition according to any one of claims 1-4, wherein Preferably, the composition further comprises a penetration enhancer. More preferably, the penetration enhancer is selected from one or more of isopropyl myristate, isopropyl palmitate, propylene glycol dinonanoate, diethyl sebacate, azone, and propylene glycol. Even more preferably, the penetration enhancer is isopropyl myristate; Preferably, the composition further comprises an antioxidant. More preferably, the antioxidant is selected from one or more of vitamin E, alkyl gallate, butylated hydroxyanisole (BHA), and dibutylhydroxytoluene (BHT). Even more preferably, the antioxidant is dibutylhydroxytoluene (BHT); Preferably, the composition further comprises other pharmaceutically acceptable excipients.

6. The composition according to claim 5, characterized in that, By mass percentage, the composition comprises 0.1%-20% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal, 10%-30% oil phase substance 1, 30%-70% oil phase substance 2, 15%-30% oil phase substance 3, 0.1%-20% penetration enhancer, and 0%-2% antioxidant; Preferably, by mass percentage, the composition comprises 0.2%-15% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal, 15%-25% oil phase substance 1, 35%-63% oil phase substance 2, 20%-25% oil phase substance 3, 1%-15% penetration enhancer, and 0%-1% antioxidant; Preferably, by mass percentage, the composition comprises 0.3%-10% of the compound shown by formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal, 18%-20% oil phase substance 1, 40%-58% oil phase substance 2, 20%-21% oil phase substance 3, 2%-10% penetration enhancer, and 0%-0.2% antioxidant.

7. The composition according to claim 6, wherein The composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, and isopropyl myristate; Preferably, by mass percentage, the composition comprises 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, and 0.1%-20% of isopropyl myristate; Preferably, by mass percentage, the composition comprises 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, and 1%-15% of isopropyl myristate; Preferably, by mass percentage, the composition comprises 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, and 2%-10% of isopropyl myristate; Preferably, by mass percentage, the composition comprises 1% of the compound represented by formula (I), 18% of white beeswax, 58% of white petrolatum, 21% of light liquid paraffin, and 2% of isopropyl myristate; Preferably, by mass percentage, the composition comprises 10% of the compound represented by formula (I), 20% of white beeswax, 40% of white petrolatum, 20% of light liquid paraffin, and 10% of isopropyl myristate; Preferably, the composition comprises a compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, white beeswax, white petrolatum, light liquid paraffin, isopropyl myristate, and butylated hydroxytoluene (BHT); Preferably, by mass percentage, the composition comprises 0.1%-20% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 10%-30% of white beeswax, 30%-70% of white petrolatum, 15%-30% of light liquid paraffin, 0.1%-20% of isopropyl myristate, and 0%-2% of butylated hydroxytoluene (BHT); Preferably, by mass percentage, the composition comprises 0.2%-15% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 15%-25% of white beeswax, 35%-63% of white petrolatum, 20%-25% of light liquid paraffin, 1%-15% of isopropyl myristate, and 0%-1% of butylated hydroxytoluene (BHT); Preferably, by mass percentage, the composition comprises 0.3%-10% of the compound represented by formula (I) or a pharmaceutically acceptable salt or a pharmaceutically acceptable co-crystal thereof, 18%-20% of white beeswax, 40%-58% of white petrolatum, 20%-21% of light liquid paraffin, 2%-10% of isopropyl myristate, and 0%-0.2% of butylated hydroxytoluene (BHT); Preferably, by mass percentage, the composition comprises 1% of the compound shown in formula (I), 18% white beeswax, 58% white petrolatum, 20.9% light liquid paraffin, 2% isopropyl myristate, and 0.1% butylated hydroxytoluene (BHT).

8. A method for preparing the composition according to any one of claims 1-7, characterized in that when the composition does not contain a penetration enhancer and / or an antioxidant, the method comprises the following steps: (1) Weigh oil phase substance 1 and oil phase substance 2, melt them by water bath heating, and obtain a uniform mixture A after homogenization; (2) Weigh oil phase substance 3, add the compound shown in formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal that has been weighed, heat it by water bath and stir to obtain a uniform mixture B; (3) After cooling the mixture A, add the mixture B thereto, stir and then homogenize, and obtain the composition after cooling; when the composition contains a penetration enhancer and / or an antioxidant, the method comprises the following steps: (1) Weigh oil phase substance 1, oil phase substance 2, a penetration enhancer and / or an antioxidant, melt them by water bath heating, and obtain a uniform mixture A after homogenization; (2) Weigh oil phase substance 3, add the compound shown in formula (I) or its pharmaceutically acceptable salt or its pharmaceutically acceptable co-crystal that has been weighed, heat it by water bath and stir to obtain a uniform mixture B; (3) After cooling the mixture A, add the mixture B thereto, stir and then homogenize, and obtain the composition after cooling.

9. Use of an RNA m6A regulator composition according to any one of claims 1-7, or a method according to claim 8, in the preparation of a preparation for preventing or treating skin diseases caused by RNA m6A methylation; Preferably, the skin diseases include acanthosis, eczema, ichthyosis, psoriasis, keratosis, systemic lupus erythematosus, hand-foot syndrome, hand-foot skin reaction, and dermatitis; More preferably, the skin diseases include hand-foot syndrome and hand-foot skin reaction.

10. The application according to claim 9, wherein The preparation includes ointment, cream, gel, cream, lotion, lotion, solution, paste, film, oil preparation or patch; Preferably, the preparation is an ointment; More preferably, the preparation is a suspension ointment.

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