Novel medicine for treating psoriasis by recovering keratinocyte Pp6 and application of novel medicine

Abstract

The invention provides a novel medicine for treating psoriasis by recovering keratinocyte Pp6 and application of the novel medicine. The novel medicine has the structural formula shown in the formula (I), has the remarkable effects of inhibiting epidermal hyperplasia, reducing the infiltration number of dermal inflammatory cells and the like on a psoriasis focus area, and has the very excellent relieving or treating effect on psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine; more specifically, this invention relates to a novel drug for treating psoriasis by restoring keratinocyte Pp6 and its application. Background Technology

[0002] Psoriasis, commonly known as "cowhide rash," is a chronic inflammatory skin disease that is difficult to cure completely, affecting the quality of life of a certain percentage of the global population. Histopathological changes in psoriasis include epidermal hyperkeratosis, parakeratosis, loss of the granular layer, significant thickening of the acanthosis, accelerated proliferation of basal cells with visible mitosis; tortuous, dilated, and congested dermal papillary vessels; and infiltration of T lymphocytes and dendritic cells around capillaries, with some lymphocytes migrating into the epidermis.

[0003] The pathogenesis of psoriasis is influenced by the interaction of environmental factors, genetics, and immune responses, and it is often accompanied by systemic diseases such as diabetes, coronary heart disease, hypertension, depression, and metabolic syndrome. Psoriasis seriously harms the physical and mental health and quality of life of patients, and is an important research topic in the field of dermatology worldwide.

[0004] Treatment options for psoriasis include topical therapies (topical corticosteroids, vitamin D derivatives, calcineurin inhibitors, etc.), phototherapy (narrowband UVB radiation, psoralen combined with UVA, etc.), systemic therapies (methotrexate, cyclosporine, etc.), and biologics (anti-tumor necrosis factor drugs, IL-17 inhibitors, etc.). These treatments are often used in combination clinically to achieve better therapeutic effects. Nevertheless, psoriasis treatment remains symptom-controlled, and patients typically require lifelong treatment to achieve disease remission. To date, there is no cure for psoriasis. Furthermore, the side effects of some treatments warrant attention, including increased risk of infection, liver fibrosis, and cancer. Therefore, further exploration of the pathogenesis of psoriasis, identification of new therapeutic targets, and screening of safe and effective drugs are of great significance, and bioactive natural small molecule compounds are one of the important tools for finding drug targets.

[0005] Protein phosphatase 6 (Pp6) is an evolutionarily conserved and universally expressed serine / threonine protein phosphatase closely related to skin inflammation and homeostasis. Pp6 is significantly reduced in psoriatic keratinocytes, and downregulation of Pp6 leads to the accumulation of ARG1 and increased polyamine production from the urea cycle. Polyamines can bind to autoRNA generated during keratinocyte lysis, activating myeloid dendritic cells and promoting IL-6 production, thereby promoting the development of inflammation.

[0006] Therefore, the reduction of Pp6 is closely related to the abnormality of keratinocytes in psoriatic diseases, and it is necessary to explore new treatment strategies and drugs that upregulate Pp6 in keratinocytes to treat psoriasis. Summary of the Invention

[0007] The purpose of this invention is to provide a novel drug for treating psoriasis by restoring keratinocyte Pp6 and its application.

[0008] In a first aspect of the invention, the use of the compound of formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for the preparation of pharmaceutical compositions for relieving or treating psoriasis is provided.

[0009]

[0010] R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or halogen.

[0011] In one or more embodiments, in the compound represented by formula (I), R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl.

[0012] In one or more embodiments, R is hydrogen in the compound represented by formula (I).

[0013] In one or more embodiments, the compound of formula (I), when mixed in a composition, can serve as the sole active component in the composition for alleviating or treating psoriasis.

[0014] In one or more embodiments, the pharmaceutical composition is also used to upregulate the expression of Pp6 (serine threonine protein phosphatase-6) under inflammatory conditions.

[0015] In one or more embodiments, the pharmaceutical composition is also used to: downregulate the expression of ARG1 (arginase 1) under inflammatory conditions.

[0016] In one or more embodiments, the pharmaceutical composition is further used in a signaling pathway in which Pp6 and ARG1 are contained, wherein ARG1 is located downstream of Pp6.

[0017] In one or more embodiments, the Pp6 or ARG1 is Pp6 or ARG1 in (human) keratinocytes.

[0018] In one or more embodiments, the pharmaceutical composition is also used to: reduce dermal inflammatory cell infiltration in the psoriatic lesion area.

[0019] In one or more embodiments, the pharmaceutical composition is also used to: reduce the epidermal thickness of psoriatic lesions or inhibit epidermal proliferation in psoriatic lesions.

[0020] In one or more embodiments, the pharmaceutical composition is also used to reduce skin scaling in psoriatic lesion areas.

[0021] In one or more embodiments, the pharmaceutical composition is also used to reduce skin erythema in areas of psoriatic lesions.

[0022] In another aspect of the invention, compounds of formula (I) or isomers thereof, solvates or precursors thereof, or pharmaceutically acceptable salts thereof are provided;

[0023]

[0024] R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or halogen.

[0025] In one or more embodiments, the pharmaceutical composition is further wherein R1 or R2 is each independently selected from: hydrogen, hydroxyl, C1-C2 alkyl; preferably, R1 or R2 is each independently hydrogen.

[0026] In one or more embodiments, the pharmaceutical composition is also used in compounds of formula (I) with the following structural formula:

[0027]

[0028] In another aspect of the invention, a method for preparing the compound of formula (I) is provided, wherein R1 or R2 is each independently hydrogen;

[0029]

[0030] The method includes performing the following reaction:

[0031]

[0032] In one or more embodiments, the method includes: mixing L-menthol, triethylamine and 4-dimethylaminopyridine, cooling the mixture to 0°C and mixing it thoroughly, adding cyclohexyl chloride dropwise, and then allowing the mixture to naturally warm to room temperature to react after the addition is complete, and terminating the reaction when it is complete.

[0033] In one or more embodiments, the method further includes: extracting the reaction solution after the aforementioned reaction is completed with an organic solvent (such as dichloromethane), washing, drying, concentrating, purifying by chromatography, and separating to obtain a white solid.

[0034] In another aspect of the invention, a method for preparing a pharmaceutical composition for relieving or treating psoriasis is provided, comprising mixing a compound of formula (I) as claimed in claim 4 or 5, or an isomer thereof, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier.

[0035] In another aspect of the invention, a pharmaceutical composition is provided comprising: the compound of formula (I) or an isomer thereof, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0036] In one or more embodiments, the pharmaceutical composition is used to relieve or treat psoriasis, or to prepare a pharmaceutical composition for relieving or treating psoriasis.

[0037] In one or more embodiments, the compound of formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, are present in an effective amount in the pharmaceutical composition; preferably, the effective amount is 0.01-50% by weight, for example, but not limited to, 0.01-5%, 0.03-3%, 0.05-1%, 20-30%, 40-50%, etc.; more preferably 0.03-30%; and even more preferably 0.05-10%.

[0038] In another aspect of the invention, the pharmaceutical composition is provided as a topical dosage form.

[0039] In another aspect of the invention, the dosage forms provided for the pharmaceutical compositions include: solutions, suspensions, ointments (such as creams, pastes), gels, foams, sprays, powders, or creams.

[0040] In another aspect of the invention, a kit is provided comprising: the compound of formula (I) or an isomer thereof, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof.

[0041] In another aspect of the invention, a medicine box is provided, comprising: the aforementioned pharmaceutical composition.

[0042] In another aspect of the invention, a method for preventing, alleviating or treating psoriasis is provided, the method comprising: administering to a subject in need of treatment an effective amount of the compound of formula (I) or an isomer thereof, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof.

[0043] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0044] Figures 1-3Mass spectrum of compound L1 (02-005-34-1-H, 02-005-34-1-H-1, 02-005-34-1-C).

[0045] Figure 4 Protein level of Pp6 in HaCaT cells: Protein level of Pp6 in HaCaT cells after treatment with 10 μM L-menthol or L1 for 12 hours following treatment with 1 μg / mL R848-stimulated HaCaT cells.

[0046] Figure 5 ARG1 mRNA level in HaCaT cells: ARG1 mRNA level in HaCaT cells after 12 hours of treatment with 10 μM L-menthol or L1 on HaCaT cells stimulated with 1 μg / mL R848.

[0047] Figure 6 Protein levels of Pp6 and ARG1 in NHEK cells: Protein levels of Pp6 and ARG1 in NHEK cells after 12 hours of treatment with 10 μM L-menthol or L1 on NHEK cells stimulated with 200 ng / mL IL-17A.

[0048] Figure 7 Compared with HaCaT cells transfected with the control plasmid, the protein level of Pp6 in HaCaT cells transfected with the Pp6 overexpression plasmid was significantly increased.

[0049] Figure 8 The proliferation capacity of HaCaT cells transfected with Pp6 overexpression plasmid was significantly reduced.

[0050] Figure 9 Compared with normal individuals, the expression of PP6 in the skin of psoriasis patients is significantly reduced.

[0051] Figure 10 L1 effectively reduced scaling and erythema in an IMQ-induced psoriasis model, and its effect was significantly better than that of L-menthol (top image). HE staining of mouse ear skin (bottom image): HE staining of ear skin of IMQ-induced mice after treatment with solvent control (anhydrous ethanol), or L-menthol, or L1.

[0052] Figure 11 1. Statistical analysis of the epidermal thickness of mouse ear skin: The epidermal thickness of the ear skin of IMQ-induced mice after treatment with solvent control (anhydrous ethanol), or L-menthol, or L1.

[0053] Figure 12 1. Count of dermal infiltration in mouse ear skin: The number of inflammatory cells infiltrating the dermis of mice after treatment with solvent control (anhydrous ethanol), L-menthol, or L1 induced by IMQ.

[0054] Figure 13 Pp6 in mouse ear skin: IMQ-induced protein levels of Pp6 in mouse ear skin after treatment with solvent control (anhydrous ethanol), or L-menthol, or L1. Detailed Implementation

[0055] Through in-depth research, the inventors have for the first time revealed a new compound with the structural formula shown in formula (I), which can restore serine threonine protein phosphatase-6 (Pp6) in keratinocytes under inflammatory conditions and has excellent alleviating or therapeutic effects on psoriasis.

[0056] the term

[0057] As used in this invention, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group containing 1-4 carbon atoms (preferably 1-2 carbon atoms). For example, alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0058] As used in this invention, the term "alkenyl" includes straight-chain and branched hydrocarbon groups containing at least one carbon-carbon double bond and 2-4 carbon atoms (preferably 2-3 carbon atoms).

[0059] The term "chain alkynyl" as used in this invention includes straight-chain and branched hydrocarbon groups containing at least one carbon-carbon triple bond and 2-4 carbon atoms (preferably 2-3 carbon atoms).

[0060] The term "halogen" as used in this invention refers to F, Cl, Br, or I.

[0061] The term "isomer" as used in this invention includes: geometric isomers, enantiomers, and diastereomers (such as cis-trans isomers and conformational isomers).

[0062] The present invention uses The representation method is well known to those skilled in the art, and it indicates that the group R can substitute for any one or more substituted positions on the ring. Furthermore, the choice of R can be different for different substitution positions. In this invention, R can be R1 or R2.

[0063] As used in this invention, the term "solvent" refers to a compound carrying solvent molecules; for example, the solvate may be a hydrate.

[0064] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing".

[0065] In this invention, a "pharmaceuticalally acceptable" ingredient is a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio.

[0066] In this invention, the pharmaceutical composition may further include a "pharmaceutically acceptable carrier," which is a pharmaceutically or food-grade solvent, suspending agent, or excipient used to deliver the compound of formula (I), isomer, solvate, precursor, or their pharmaceutically acceptable salt to an animal or human. The carrier may be liquid or solid.

[0067] compound

[0068] In this invention, a compound as shown in structural formula (I) is first provided:

[0069]

[0070] R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or halogen.

[0071] This invention also includes isomers, solvates, precursors, or pharmaceutically acceptable salts of compounds of formula (I) above, provided they also have the same or substantially the same function as compounds of formula (I). A "pharmaceutically acceptable salt" is a salt formed by the reaction of a compound with an inorganic acid, an organic acid, an alkali metal, or an alkaline earth metal. These salts include (but are not limited to): (1) salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; and (2) salts formed with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium) in the form of esters, carbamates, or other conventional "prodrugs." The compound has one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, or cis or trans isomers.

[0072] The term "precursor of the compound" refers to a compound that, when taken by an appropriate method, undergoes metabolism or chemical reaction in the patient's body to transform into a compound of structural formula (I), or a salt or solution of a compound of chemical structural formula (I).

[0073] In a preferred embodiment of the present invention, the compound has the following structure:

[0074]

[0075] The novel drug has a molecular weight of 266.425 and the chemical formula C17H30O2, which the inventors have named L1. It is a solid and soluble in organic solvents such as ethanol.

[0076] It should be understood that, after learning the structure of the compounds of the present invention, the compounds of the present invention can be obtained by a variety of methods well known in the art and using known raw materials, such as chemical synthesis or extraction from biological sources (e.g., animals or plants), all of which are included in the present invention.

[0077] As a preferred embodiment of the present invention, a method for preparing the compound represented by formula (I) of the present invention is provided, comprising: mixing L-menthol, triethylamine and 4-dimethylaminopyridine, cooling the mixture to 0°C and mixing it, adding cyclohexyl chloride dropwise, and then naturally heating the mixture to room temperature to react after the addition is complete, and terminating the reaction after it is complete.

[0078] The synthesized compound can be further purified by column chromatography, high-performance liquid chromatography, etc. Alternatively, it can be further purified by crystallization. It should be understood that various purification methods can be used in this invention. Preferably, the purification includes: extracting the reaction solution after the aforementioned reaction with an organic solvent (such as dichloromethane), washing, drying, concentrating, chromatographically purifying, and separating to obtain a white solid.

[0079] Applications to relieve or treat psoriasis

[0080] Psoriasis is a special disease with significant differences from other types of skin diseases in its pathogenesis and symptoms, making the development of its treatment drugs quite difficult.

[0081] In this field, L-menthol is considered a useful drug for treating psoriasis. L-menthol is a natural cyclic terpene small molecule compound with a molecular weight of approximately 156 Da. It can be extracted from peppermint or obtained through chemical synthesis, and its structural formula is as follows:

[0082]

[0083] L-Menthol possesses antipyretic, analgesic, and antipruritic properties. Furthermore, L-menthol inhibits psoriatic skin inflammation by restoring protein phosphatase 6 (Pp6) in keratinocytes under inflammatory conditions. However, research and practice have revealed that the therapeutic efficacy of L-menthol needs improvement; and high concentrations of L-menthol have been found to be irritating to the skin and may affect its therapeutic effect on psoriasis, indicating a need for the development of more effective psoriasis medications.

[0084] The compound of the present invention, as shown in structural formula (I), has the function of relieving or treating psoriasis, and its technical effect is superior to that of L-menthol.

[0085] The compound or its analogues represented by formula (I) of this invention are formulated into a topical dosage form using conventional pharmaceutical methods and pharmaceutically acceptable excipients. In a preferred embodiment, the weight content of the compound or its derivatives represented by formula (I) can be 0.01%-1.5%. It should be understood that physicians may also make appropriate dosage variations based on clinical practice.

[0086] The present invention also provides a method for relieving or treating psoriasis, the method comprising: administering to a subject in need of treatment an effective amount of the compound of formula (I) or an isomer thereof, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof.

[0087] The experimental results of embodiments of the present invention show that the novel drug L1 effectively upregulates Pp6 in keratinocytes under inflammatory conditions, and the upregulated Pp6 level is significantly higher than that of L-menthol previously reported. Furthermore, the compound of formula (I) of the present invention exhibits significant therapeutic effects in psoriatic animals, including inhibition of epidermal proliferation and reduction of dermal inflammatory cell infiltration. In addition, L1's ability to upregulate IMQ-induced Pp6 in mouse skin is significantly higher than that of L-menthol previously reported. In summary, the novel drug L1 can be used in psoriasis drugs and pharmaceutical compositions.

[0088] Composition

[0089] The dosage form of the pharmaceutical composition described in this invention can be diverse, as long as it enables the active ingredient to effectively reach the mammalian body. Examples include: gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, or suspensions. Those skilled in the art can choose a convenient dosage form to treat the type of disease treated by the compounds according to this invention.

[0090] From the perspective of ease of preparation and administration, preferred pharmaceutical compositions are solid or semi-solid compositions. The compounds of the present invention or their pharmaceutical compositions may also be stored in sterile instruments suitable for injection or infusion.

[0091] When used to relieve or treat psoriasis, topical administration of the pharmaceutical composition is preferred. Various pharmaceutical carriers suitable for formulating topical dosage forms can be used in this invention. As a preferred embodiment of this invention, the pharmaceutical composition containing the compound shown in formula (I) is a topical dosage form, including (but not limited to) those selected from: ointments (such as creams, pastes), gels, foams, sprays, powders, and creams.

[0092] As a preferred embodiment of the present invention, a pharmaceutical composition for treating psoriasis is provided, comprising: (a) an effective amount of the compound of formula (I), or an isomer thereof, solvate, precursor, or a pharmaceutically acceptable salt thereof; and (b) an excipient. The excipient is suitable to synergistically enhance the therapeutic effect of the compound of formula (I). The excipient comprises: excipient 1, selected from one or more of white petrolatum, paraffin, lanolin, microcrystalline wax, beeswax, cetyl wax, ceresin, glyceryl stearate, stearic acid, or cholesterol; excipient 2, selected from one or more of isopropyl myristate, azone, or N-methylpyrrolidone; and excipient 3, selected from one or more of vitamin E, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, propyl gallate, or tert-butylhydroquinone.

[0093] The effective dosage of the compound of formula (I) as the active ingredient can vary depending on the administration method and the severity of the disease to be treated. For example, satisfactory effects can be obtained when the compound of the present invention is administered daily at a dose of about 0.0001-500 mg / kg, preferably 0.001-300 mg / kg, more preferably 0.01-200 mg / kg of animal body weight, preferably in 1-3 separate doses daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0095] Example 1: Preparation and Identification of Compound L1

[0096] Compound L1 has the chemical formula C17H30O2; its structural formula is as follows:

[0097]

[0098] The preparation method is as follows:

[0099] L-menthol (156 mg, 1 mmol), triethylamine (303 mg, 3 mmol), and 4-dimethylaminopyridine (24.4 mg, 0.2 mmol) were weighed and placed in 10 mL of dichloromethane solution. The mixture was cooled to 0 °C, and cyclohexyl chloride (190 mg, 1.3 mmol) was slowly added dropwise while stirring. After the addition was complete, the mixture was allowed to rise naturally to room temperature and reacted for 2 hours. After the reaction was complete, 5 mL of water was added to terminate the reaction. The reaction solution was extracted with dichloromethane (10 mL * 3), washed once with saturated NaCl (10 mL), and finally dried over anhydrous Na2SO4. The organic layer was concentrated and separated by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1 to 6:1) to obtain a white solid (185 mg, 69.5%).

[0100] The mass spectra for L1 compound identification are as follows: Figure 1 , Figure 2 and Figure 3 .

[0101] The reaction formula for preparing compound L1 is as follows:

[0102]

[0103] Example 2: Effect of compound L1 on Pp6 expression in keratinocytes

[0104] L-menthol exerts its function of inhibiting psoriatic skin inflammation by restoring protein phosphatase 6 (Pp6) in keratinocytes under inflammatory conditions. However, experimental studies have shown that high concentrations of L-menthol are irritating to the skin and can affect its therapeutic effect on psoriasis. Therefore, the inventors conducted extensive research and analysis to find a substance that is superior to L-menthol in inhibiting psoriasis. Based on large-scale screening, preparation of new compounds, and experimental analysis, compound L1 was obtained.

[0105] R848 stimulates immortalized human keratinocytes (HaCaT cells) to induce an inflammatory environment and reduce PP6 expression in keratinocytes, thereby mimicking the reduced PP6 expression in keratinocytes of psoriasis patients.

[0106] HaCaT cells treated with R848 (Resiquimod) (HaCaT cells themselves express PP6, but PP6 expression decreases after the addition of R848) were then treated with L-menthol or L1, and the effects of L-menthol or L1 on Pp6 expression were analyzed. HaCaT cells stimulated with 1 μg / mL R848 were treated with 10 μM L-menthol or L1, and Pp6 expression was observed. The steps are as follows:

[0107] (1) Dissolve L-menthol, L1 and R848 in DMSO to obtain a stock solution.

[0108] (2) HaCaT cells were seeded into 24-well plates at a density of 100,000 cells / well and 1 mL of culture medium was added.

[0109] (3) After 24 hours, HaCaT cells were stimulated with 1 μg / mL R848 while the cells adhered to the wall, and were treated with 10 μM L-menthol or L1.

[0110] (4) After 12 hours, extract the protein from the cells.

[0111] (5) The expression of Pp6 in cells was detected by Western blot.

[0112] Immunoblotting analysis showed that, compared with L-menthol, compound L1 more significantly upregulated Pp6 expression. Figure 4 ).

[0113] Higher Pp6 expression indicates that keratinocyte proliferation is inhibited, suggesting that compound L1 has a more significant function in inhibiting keratinocyte proliferation and can be used to inhibit the excessive proliferation of keratinocytes in psoriatic diseases.

[0114] Example 3: Effect of compound L1 on ARG1 expression in keratinocytes

[0115] In this embodiment, HaCaT cells stimulated with 1 μg / mL R848 (HaCaT cells endogenously express ARG1) were treated with 10 μM L-menthol or L1, and the expression of ARG1 in the cells was analyzed. The steps are as follows:

[0116] (1) Dissolve L-menthol, L1 and R848 in DMSO to obtain a stock solution.

[0117] (2) HaCaT cells were seeded into 24-well plates at a density of 100,000 cells / well and 1 mL of culture medium was added.

[0118] (3) After 24 hours, HaCaT cells were stimulated with 1 μg / mL R848 while the cells adhered to the wall, and were treated with 10 μM L-menthol or L1.

[0119] (4) After 12 hours, mRNA was extracted from the cells and reverse transcribed to obtain cDNA.

[0120] (5) Use qPCR to detect the expression of ARG1 in cells.

[0121] The results showed that, compared with L-menthol, L1 was able to more significantly downregulate ARG1 expression. Figure 5 ).

[0122] The lower the ARG1 expression, the more likely it is that PP6 in keratinocytes can be upregulated by L1 and further inhibit the expression of downstream ARG1, suggesting that compound L1 has a more significant function in inhibiting the expression of downstream ARG1 of PP6.

[0123] Example 4: Effects of compound L1 on Pp6 protein levels and ARG1 expression in downstream Pp6 signaling pathways in keratinocytes under inflammatory conditions.

[0124] In this embodiment, human epidermal keratinocytes (NHEK cells) stimulated with 200 ng / mL IL-17A were treated with 10 μM L-menthol or L1.

[0125] (1) Dissolve L-menthol and L1 in DMSO to obtain a stock solution; dilute IL-17A with PBS to obtain a stock solution.

[0126] (2) Seed NHEK cells into 24-well plates at a density of 100,000 cells / well and add 1 mL of culture medium.

[0127] (3) After 24 hours, NHEK cells were stimulated with 200 ng / mL IL-17A while the cells were adherent, and were also treated with 10 μM L-menthol or L1.

[0128] (4) After 12 hours, extract the protein from the cells.

[0129] (5) The expression of Pp6 and ARG1 in cells was detected by Western blotting.

[0130] The results showed that, compared with L-menthol, L1 more significantly upregulated Pp6 expression and more significantly inhibited ARG1 expression. Figure 6 ).

[0131] The results above indicate that, compared with L-menthol, L1 can more significantly upregulate the Pp6 protein level in keratinocytes under inflammatory conditions and significantly inhibit the expression of ARG1 in the downstream signaling pathway of Pp6.

[0132] Example 5: Effect of PP6 on keratinocyte proliferation

[0133] In this embodiment, a human Pp6 overexpression plasmid was constructed using PC-DNA3.1 as the vector plasmid and transfected into HaCaT cells. The expression of Pp6 and cell proliferation were analyzed. The steps are as follows:

[0134] (1) Using PC-DNA3.1 as the vector plasmid, construct a human Pp6 overexpression plasmid.

[0135] (2) HaCaT cells were seeded into 24-well plates at a density of 100,000 cells / well and 1 mL of culture medium was added.

[0136] (3) After 24 hours, under the condition of cell adhesion, Pp6 overexpression plasmid and control vector plasmid were transfected into HaCaT cells using Lipo2000 as transfection reagent.

[0137] (4) After 48 hours, the treated cells were divided into two portions.

[0138] (5) Extract proteins from cells and detect the expression of Pp6 in cells using Western blotting.

[0139] (6) Another batch was seeded into 96-well plates at a density of 10,000 cells / well, and the cell proliferation was detected by CCK-8 after 24 hours.

[0140] The results showed that, compared with HaCaT cells transfected with the control plasmid, the protein level of Pp6 in HaCaT cells transfected with the Pp6 overexpression plasmid was significantly increased. Figure 7 ), while the cell proliferation capacity decreased significantly ( Figure 8 The results suggest that upregulation of Pp6 can inhibit the proliferation of keratinocytes, thus playing a positive role in the treatment of psoriasis.

[0141] Example 6: Expression of PP6 in the skin of psoriasis patients and normal individuals.

[0142] In this embodiment, immunohistochemical staining was performed on the skin of psoriasis patients and normal individuals to analyze the expression of PP6. The steps are as follows:

[0143] (1) Paraffin sections from normal individuals and psoriasis patients were dewaxed and hydrated sequentially using xylene and ethanol of varying concentrations.

[0144] (2) Prepare an antigen retrieval solution containing citric acid and sodium citrate, and perform antigen retrieval on the slides by heating.

[0145] (3) Inactivate endogenous enzymes and block endogenous biotin.

[0146] (4) Dilute BSA (bovine serum albumin) with PBS buffer to prepare a blocking solution containing 5% BSA and block the sections.

[0147] (5) Dilute the PP6 antibody with antibody diluent at a ratio of 1:100 and drop it onto the slide.

[0148] (6) The antibody and the slide were incubated overnight at 4°C, and then washed three times with PBS.

[0149] (7) Incubate at room temperature using a biotin-labeled second antibody.

[0150] (8) After 20 minutes, wash three times with PBS.

[0151] (9) Prepare the color development solution and incubate the slices for 1 minute.

[0152] (10) Remove the developing solution, perform hematoxylin staining, and mount the slide.

[0153] The results showed that, compared with normal individuals, the expression of PP6 in the skin of psoriasis patients was significantly reduced. Figure 9 The results suggest that restoring PP6 in keratinocytes may be an effective treatment for psoriasis.

[0154] Example 7: Analysis of the inhibitory effect of compound L1 on psoriasis in animals

[0155] 1. Model preparation and medication

[0156] In this embodiment, an imiquimod (IMQ)-induced psoriasis animal model was prepared, and histological and immunoblotting analyses of the skin of IMQ-induced mice were performed using solvent control (anhydrous ethanol), or L-menthol, or L1 treatment.

[0157] (1) IMQ cream was applied to the skin of the mouse ear at a dose of 30 mg per mouse per day for 7 consecutive days to obtain an IMQ-induced psoriasis animal model.

[0158] (2) Dissolve L-menthol or L1 in anhydrous ethanol to obtain a solution of the same molar concentration (16 mol / L).

[0159] (3) On the third day of IMQ modeling (the third day after IMQ application), apply solvent control (anhydrous ethanol), or L-menthol, or L1 solution to the skin of the mouse ear at a dose of 20 μL per mouse per day, and continue for 5 days.

[0160] Further animal-level analysis was then conducted.

[0161] 2. Analysis of symptoms of animal ear diseases

[0162] On day 8 after step (3) above, observe the condition of the skin behind the ears of the animals (mice).

[0163] The results showed that L1 effectively reduced scaling and erythema in an IMQ-induced psoriasis model, and its effect was significantly better than that of L-menthol. Figure 10 ).

[0164] 3. Analysis of animal epidermal thickness and dermal cell infiltration count

[0165] After the aforementioned step (3), the mice were euthanized, the skin was cut off, a portion of it was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

[0166] HE staining showed that L1 effectively inhibited epidermal proliferation, reduced epidermal thickness, and decreased dermal inflammatory cell infiltration in an IMQ-induced psoriasis model. Figure 10 (See image below).

[0167] The HE-stained sections were photographed using a microscope, and the epidermal thickness and dermal cell infiltration count in the images were analyzed using Photoshop.

[0168] Analysis of epidermal thickness and dermal cell infiltration count showed that L1 significantly reduced epidermal hyperplasia in an IMQ-induced psoriasis model, and its effect was significantly superior to L-menthol. Figure 11 NS indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0169] Furthermore, L1 significantly reduced inflammatory cell infiltration in an IMQ-induced psoriasis model. Figure 12 NS indicates no significant difference; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001).

[0170] 4. Pp6 expression analysis

[0171] Following step (3) above, the mice were euthanized, their skin was removed, and another portion of the skin was minced with scissors, frozen in liquid nitrogen, and then ground using a tissue homogenizer. The skin was lysed with tissue lysis buffer, and proteins were extracted. The content of Pp6 was detected using Western blotting.

[0172] Immunoblotting analysis of Pp6 levels showed that L1 significantly upregulated Pp6 protein levels in the skin of IMQ-induced mice, and its upregulation ability was significantly superior to that of L-menthol. Figure 13 ).

[0173] The above results indicate that L1 has a very good therapeutic effect on IMQ-induced psoriasis models, and has a more ideal effect than L-menthol.

[0174] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. Use of the compound of formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof, for the preparation of pharmaceutical compositions for the relief or treatment of psoriasis; in, R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, or halogen.

2. Use of the compound of formula (I) as claimed in claim 1, or its isomers, solvates, or precursors, or pharmaceutically acceptable salts thereof, characterized in that, The pharmaceutical composition is also used for: Upregulates Pp6 expression in an inflammatory environment; Downregulates ARG1 expression in an inflammatory environment; Preferably, Pp6 and ARG1 are included in the signal path, with ARG1 located downstream of Pp6.

3. Use of the compound of formula (I) as claimed in claim 2, or its isomers, solvates, or precursors, or pharmaceutically acceptable salts thereof, characterized in that, The pharmaceutical composition is also used for: Reduce dermal inflammatory cell infiltration in psoriatic lesions; Reduce the thickness of the epidermis in the psoriatic lesion area, or inhibit the epidermal proliferation in the psoriatic lesion area; Reduce skin scaling in psoriasis lesions; Reduce skin erythema in the psoriasis lesion area.

4. The compound of formula (I) or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof; in, R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, or halogen.

5. The compound of formula (I) as claimed in claim 4, or an isomer, solvate, or precursor thereof, or a pharmaceutically acceptable salt thereof, characterized in that, R1 or R2 is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl; preferably, R1 or R2 is independently hydrogen.

6. A method for preparing a compound of formula (I), wherein R1 or R2 is each independently hydrogen; The method includes performing the following reaction:

7. A method for preparing a pharmaceutical composition for relieving or treating psoriasis, comprising mixing a compound of formula (I) as claimed in claim 4 or 5, or an isomer thereof, a solvate or a precursor thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier.

8. A pharmaceutical composition comprising: a compound of formula (I) as claimed in claim 4 or 5, or an isomer, solvate, or precursor thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is a topical dosage form; or The dosage forms of the pharmaceutical composition include: solutions, suspensions, ointments, gels, foams, sprays, powders, or creams.

10. A medicine box comprising: The compound of formula (I) as claimed in claim 4 or 5, or its isomers, solvates or precursors, or pharmaceutically acceptable salts thereof; or The pharmaceutical composition according to claim 7 or 8.

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