Application of benzoyl guanidine derivative in preparation of medicine for treating and / or preventing chronic inflammatory diseases

Abstract

The invention discloses an application of a benzoylguanidine derivative in preparation of a medicine for treating and / or preventing chronic inflammatory diseases. According to the invention, the benzoylguanidine derivative is prepared into a hydrogel dosage form, and the hydrogel dosage form acts on a psoriasis affected part in a smearing manner, so that psoriasis-like skin lesion symptoms such as skin scales, erythema and epidermis thickening can be relieved, skin inflammatory cell infiltration and epidermis thickness can be relieved, spleen inflammatory cell infiltration can be relieved, and abnormal activity of a hair growth center can be improved; the traditional Chinese medicine composition reduces inflammatory reactions such as spleen index, reduces the level of inflammatory factors in plasma and spleen, and shows a good psoriasis treatment effect. Therefore, the benzoylguanidine derivative and the hydrogel preparation thereof have the remarkable advantages of safety, few toxic and side effects and no pollution of drug residues, and have a remarkable treatment effect and potential for realizing medical transformation.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of a benzoylguanidine derivative in the preparation of a drug for treating and / or preventing chronic inflammatory diseases. Background Art

[0002] Psoriasis, commonly known as psoriasis vulgaris, is a chronic and recurrent inflammatory skin disease, and its typical clinical manifestations are well-defined erythema, rashes, plaques, scales, etc. The pathological characteristics of psoriasis are hyperplasia of keratinocytes and infiltration of T cells, macrophages, dendritic cells and neutrophils. The currently recognized pathogenesis is a chronic inflammatory response centered on IL-23 / IL-17. IL-23 participates in the process of initial T cell differentiation into Th17 cells. Subsequently, Th17 cells secrete IL-17A, IL-17F, IL-6, IL-22, IL-26 and TNF-α. Th17 is the key driving factor for the onset of psoriasis. Modern research shows that the immune response of psoriasis includes enhanced activation of T cells and myeloid cells, and simultaneously upregulates TNF-α and IL-23, IL-17 and IL-6. Therefore, psoriasis can be treated by regulating Th17 and then inhibiting the secretion of related inflammatory factors. IL-17 is secreted by Th17 cells, promotes the transmission of other cytokines, and directly promotes activated keratinocytes to produce more chemokines, participating in the inflammatory response of psoriasis. The association between IL-17 and the onset of psoriasis is the most direct. Due to its long disease cycle, easy recurrence and inability to be cured, psoriasis seriously affects the quality of life of patients.

[0003] Currently, the main directions of drug research and development for psoriasis at home and abroad are biological agents such as IL-12 / 23p40 antibody, IL-17A antibody, TNFα antibody, small molecule chemical drugs such as PDE4 inhibitors, JAK inhibitors and Nrf2 stimulants, and traditional Chinese medicine compound preparations. Due to defects such as large side effects, poor efficacy, easy recurrence and high price of these drugs, so far, there is still a lack of specific treatment methods and effective drugs for the treatment of psoriasis. Therefore, it is of great significance to find a new drug with good efficacy and small side effects for the treatment of psoriasis. Summary of the Invention

[0004] Aiming at the above problems existing in the prior art and overcoming the deficiencies of the prior art, the purpose of the present invention is to provide the application of a benzoylguanidine derivative in the preparation of a drug for treating and / or preventing chronic inflammatory diseases.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions to be realized:

[0006] The present invention provides the application of a benzoylguanidine derivative and its new crystal form in the preparation of a drug for treating and / or preventing chronic inflammatory diseases.

[0007] Furthermore: the chronic inflammatory diseases include psoriasis.

[0008] Furthermore, the psoriasis includes psoriasis caused by IMQ.

[0009] Furthermore, the psoriasis caused by the IMQ is mainly characterized by increased scales on the skin, erythema coverage, and epidermal thickening.

[0010] Furthermore: the benzoylguanidine derivatives include compounds ML-1, ML-5, and their novel crystal forms, with the following structural formulas:

[0011] Furthermore, the preparation and characterization of benzoylguanidine derivatives, including compounds ML-1, ML-5 and their novel crystal forms, are provided.

[0012] Furthermore, the drug contains 0.01% to 5% by weight of benzoylguanidine derivatives and their new crystal forms, which are for external use, administered transdermally, with a dosage of 3.75 mg / kg to 7.5 mg / kg per dose.

[0013] The benzoylguanidine derivative reduces inflammatory cell infiltration and promotes the recovery of splenic trabecular structure.

[0014] The benzoyl guanidine derivative reduces CD4 in splenic lymphocytes. + IL-17A + The expression of Th17 cells inhibits the secretion of cytokines by Th17 cells.

[0015] Furthermore, the topical dosage form of the drug is a hydrogel preparation.

[0016] Furthermore, by weight percentage, the components of the hydrogel formulation include: 0.01% to 5% benzoylguanidine derivatives and their new crystal forms, 0.5% to 40% matrix material, 2% to 40% humectant, 0.1% to 1% preservative, 0.1% to 2% pH adjuster, 0.5% to 10% penetration enhancer, and the balance being water.

[0017] Furthermore, the matrix material is at least one of Carbomer 940, Carbomer 941, Carbomer 910, Carbomer 934, Carbomer 1342 and Poloxamer.

[0018] Furthermore: the humectant is at least one of glycerin and betaine; the pH adjuster is triethanolamine or sodium hydroxide.

[0019] Furthermore, the preservative is at least one of sodium benzoate, benzalkonium bromide, and ethylparaben.

[0020] Furthermore, the penetration enhancer is at least one of laurocapram, peppermint oil, and ethanol.

[0021] Further: The preparation method of the hydrogel formulation is as follows: (1) Weigh the matrix material into water and let it stand at room temperature to allow it to swell fully; (2) Add a humectant and stir thoroughly; (3) Add benzoyl guanidine derivative in small amounts several times and stir thoroughly; (4) Add a preservative and continue stirring thoroughly, add a pH adjuster to adjust the pH to 7, add the remaining purified water to the required amount, mix thoroughly, and obtain the hydrogel formulation of benzoyl guanidine derivative.

[0022] Furthermore: the hydrogel formulation is applied topically or systemically to inflamed skin.

[0023] Furthermore, the hydrogel formulation is administered once daily, twice daily, three times daily, once every two days, once every three days, or once weekly.

[0024] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] This invention demonstrates through a series of experiments that benzoylguanidine derivatives and their novel crystalline hydrogel formulations can effectively protect against imiquimod (IMQ)-induced psoriasis-like skin lesions in mice, improve skin scaling, erythema, thickening, and abnormal epidermal differentiation. Simultaneously, it reduces inflammatory cell infiltration and epidermal thickness in the skin, reduces inflammatory cell infiltration in the spleen, improves abnormal germinal center activity, reduces the spleen index and other inflammatory responses, lowers the level of inflammatory factors, and exerts a certain anti-inflammatory effect. The experimental research of this invention clarifies that benzoylguanidine derivatives and their novel crystalline hydrogel formulations have significant efficacy in psoriasis models, are safe, highly effective, and have no side effects. Furthermore, the production process is simple, showing potential for medical translation and contributing to expanding the potential application value of benzoylguanidine derivative topical formulations in other various inflammatory skin diseases. Attached Figure Description

[0026] Figure 1 The PXRD pattern of the benzoylguanidine derivative ML-5-CS;

[0027] Figure 2 The DSC spectrum of the benzoylguanidine derivative ML-5-CS;

[0028] Figure 3 The TG spectrum of the benzoylguanidine derivative ML-5-CS;

[0029] Figure 4 The image shows the IR spectrum of the benzoylguanidine derivative ML-5-CS.

[0030] Figure 5 The results of staining of histopathological sections of the skin tissue on the back of psoriatic mice after topical application of a benzoylguanidine derivative hydrogel preparation.

[0031] Figure 6 The effect of topical application of benzoylguanidine derivative hydrogel formulation on spleen function in psoriatic mice;

[0032] Figure 7 To investigate the effect of topical application of a benzoylguanidine derivative hydrogel formulation on the levels of inflammatory factors in the plasma and spleen of psoriatic mice;

[0033] Figure 8 To investigate the effect of topical application of benzoylguanidine derivative hydrogel formulation on CD4 in the spleen of psoriatic mice + IL-17A + The effect of Th17 cell expression. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0035] Examples 4 to 10 are provided below to illustrate the effect of benzoylguanidine derivatives on skin lesions in an imiquimod-induced mouse psoriasis model, evaluate the therapeutic effect of benzoylguanidine derivatives on this model, and explore the potential mechanism of benzoylguanidine derivatives in treating psoriasis.

[0036] Example 1: Synthesis of ML-5, a benzoylguanidine derivative methyl ester compound, and preparation and spectral identification of its crystal form.

[0037] 1. Synthesis of the representative compound 4-guanidinobutane-3-cyclopropylmethoxy-4-difluoromethoxybenzyl ester

[0038] Using commercially available 3-cyclopropylmethoxy-4-difluoromethoxybenzoic acid as the starting material, 35 g was dissolved in 100 mL of anhydrous dichloromethane. The solution was stirred until fully dissolved, and then 60 g of dimethylaminopyridine p-toluenesulfonate was added in portions. After the solution became clear, 32.5 g of Boc-protected 4-guanidino-1-butanol was added and stirred until dissolved. Then, 38.5 g of diisopropylcarboimide was added, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC to determine completion. Insoluble matter was first filtered off, followed by vacuum distillation to remove dichloromethane. Pre-frozen ice-cold methanol was added until white crystals precipitated. Approximately 25.3 g of a white powdery crystalline precursor was obtained by filtration. 4.0 mM of the precursor compound was added to 10 mL of a 1:1 mixture of dichloromethane and trifluoroacetic acid. The mixture was reacted at room temperature for 5 h, and then TLC was used to determine if the reaction was complete. Dichloromethane was removed by vacuum distillation. After washing three times with petroleum ether, an appropriate amount of saturated NaHCO3 solution was added to adjust the pH to 8-11, causing a solid to precipitate. The solid was then filtered to obtain 4-guanidinobutane-3-cyclopropylmethoxy-4-difluoromethoxybenzyl ester, which is the representative product ML-5. The 1H and 1C NMR spectra are as follows:

[0039]

[0040] 1H NMR spectrum of compound ML-5: 1 H NMR (400 MHz, DMSO-d6 ) δ 8.29 (s, 1H), 7.58 (s,2H), 7.41 (s, 2H), 7.06 (s, 2H), 6.60 (s, 4H), 6.05 (s, 2H), 4.40 (s, 2H), 4.30 (s, 2H), 3.94 (s, 4H), 3.57 (s, 2H), 1.83 (s, 2H), 1.50 (s, 2H), 1.10 (s, 1H), 0.47 (s, 2H), 0.23 (s, 2H).

[0041] ML-5 carbon spectrum: 13 C NMR (100 MHz, DMSO-d6) δ 166.37 (s), 156.86 (s), 153.61 (s), 149.68 (s), 125.82 (s), 124.84 (s), 124.56 (s), 119.05 (s), 118.56 (s), 74.00 (s), 66.74 (s), 41.46 (s), 27.93 (s), 27.36 (s), 10.70 (s), 7.85 (s).

[0042] 2. Preparation of ML-5 oxalic acid crystal form

[0043] Compound ML-5 was mixed with oxalic acid at a molar ratio of 1:1 in a flask. Isopropanol was added to completely dissolve the powder mixture. The mixture was heated and stirred for 2–4 h, then filtered while hot. After the filtrate was allowed to stand and cool to room temperature, a solid phase precipitated to obtain ML5-CS, with the following structural formula:

[0044] .

[0045] PXRD pattern of ML-5 oxalic acid crystal form ( Figure 1 The characteristic peaks mainly appear at 2-Theta of 9.63°, 10.08°, 12.34°, 13.81°, 15.15°, 16.67°, 19.27°, 19.85°, 20.38°, 25.25°, 26.56°, 26.94°, and 27.88°. The DSC of the ML-5 oxalic acid crystal form... Figure 2 The results show a sharp endothermic peak at around 162 °C, indicating that it melts at 162 °C and the sample has high purity. It also decomposes and releases heat at around 263 °C.

[0046] ML-5 oxalic acid crystal form TG ( Figure 3 The results indicate that the ML-5 oxalic acid crystal form rapidly decomposes and loses weight at around 160 °C, with no significant mass loss before 100 °C, which corresponds to the previous DSC results. The IR of ML-5 oxalate... Figure 4 The main spectral characteristic peak appears at 3427.59 cm⁻¹. -1 1714.44 cm -1 1644.05 cm -1 1508.87 cm -1 1471.71 cm -1 1431.12cm -1 1407.12 cm -1 1386.87 cm -1 1291.19 cm -1 1204.73 cm -1 1137.54 cm -1 1047.79 cm -1 1025.62 cm -1 1007.66 cm -1 878.54 cm -1 764.14 cm -1 571.53 cm -1 etc.

[0047] Example 2: Synthesis of ML-1, a benzylguanidine derivative benzamide compound, and its new crystalline form fumarate

[0048] 1. Synthesis of the representative compound 3-cyclopentanoxy-N-4-guanidinobutane-4-methoxybenzamine

[0049] Add 2.83 g NaH to 50 mL of dry tetrahydrofuran, then slowly add Boc-protected 4-guanidino-1-butylamine (78.2 mM), and react at room temperature for 1.5 hours. Then dissolve 3-cyclopentanoxy-4-methoxybenzoic acid (33.1 mM) in 200 mL of dry tetrahydrofuran, and slowly add 8.6 mL of SOCl2 dropwise under ice bath conditions. After the addition is complete, reflux for 6 hours, evaporate the solvent, dissolve the solvent in 65 mL of anhydrous tetrahydrofuran, and slowly add it dropwise to the above reaction system at room temperature. React at 50 °C for 6 hours. After cooling to room temperature, 300 mL of water was added, and the mixture was extracted three times with 150 mL of acetate. The combined organic phases were dried over anhydrous sodium sulfate. Then, 12 mL of a 1:1 mixture of dichloromethane and trifluoroacetic acid was added, and the mixture was reacted at room temperature for 6 h. TLC was used to determine whether the deprotection was complete. The solvent was then removed by vacuum evaporation to obtain the crude product. The crude product was recrystallized from ethyl acetate to obtain the representative compound 3-cyclopentanoxy-N-4-guanidinobutane-4-methoxybenzamide, i.e., compound ML-1. Its 1H and 1C NMR spectra are as follows:

[0050]

[0051] 1H NMR spectrum of compound ML-1: 1 H NMR (400 MHz, DMSO-d6 ) δ 7.61 (s, 1H), 7.49 (s,1H), 7.02 (s, 1H), 6.63 (s, 2H), 6.15 (s, 1H), 5.94 (s, 1H), 4.46 (s, 1H), 4.36 (s, 1H), 3.83 (s, 3H), 3.58 (s, 2H), 3.30 (s, 2H), 2.06 (s, 1H), 1.78(s, 1H), 1.71 (s, 1H), 1.61 (s, 1H), 1.55 (s, 2H), 1.49 (s, 2H).

[0052] ML-1 carbon spectrum of compound: 13C NMR (100 MHz, DMSO-d6) δ 166.82 (s), 156.86 (s), 153.51 (s), 148.80 (s), 128.31 (s), 122.37 (s), 116.54 (s), 113.88 (s), 83.94(s), 56.83 (s), 41.49 (d, J = 7.6 Hz), 33.43 (s), 27.39 (s), 24.08 (s).

[0053] 2. Preparation of ML-1 fumaric acid crystal form

[0054] Mix ML-1 and fumaric acid at a molar ratio of 1:1 in a round-bottom flask. Add 8 mL of methanol to the mixture, heat and stir in a 55°C water bath for 8 hours, then filter while hot. After the filtrate is allowed to cool to room temperature, a solid phase precipitates. Collect the solid phase to obtain ML1-fumaric acid eutectic, the structural formula of which is shown below:

[0055] .

[0056] Example 3: Preparation of benzoylguanidine derivative hydrogel formulation

[0057] The benzoylguanidine derivative described in this embodiment is compound ML-5-CS prepared in Example 2, and it will be used as an example for illustration.

[0058] This embodiment provides the following hydrogel formulations:

[0059]

[0060] The preparation process of the new oxalate crystal form hydrogel formulation of benzoylguanidine derivative in this embodiment is as follows:

[0061] (1) Weigh an appropriate amount of carbomer into an appropriate amount of water and let it stand at room temperature to allow the carbomer to fully swell.

[0062] (2) Add glycerol to the solution from step (1) and stir thoroughly.

[0063] (3) Weigh out the benzoylguanidine derivative and add it to the solution in step (2) in small amounts several times, stirring thoroughly until homogeneous;

[0064] (4) Add appropriate amount of sodium benzoate and menthol to the solution in step (3), continue to stir evenly, add appropriate amount of triethanolamine to adjust pH=7, add the remaining purified water to the required amount, mix evenly, and obtain benzoylguanidine derivative gel preparation.

[0065] Example 4: Performance testing of benzoylguanidine derivative gel formulation

[0066] 1. Particle size detection:

[0067] Experimental method: Take appropriate amounts of the hydrogel formulations 1-5 prepared in Example 3, coat them on glass slides, observe them under a microscope, and measure the particle size distribution.

[0068] Experimental results: No particulate matter was found in any of the formulation samples.

[0069] 2. Skin adhesion test:

[0070] Experimental method: Take an appropriate amount of the hydrogel preparation 1-5 prepared in Example 3, apply it to the back of the hand, shake it vigorously 10 times, and observe the peeling situation.

[0071] Experimental results: None of the formulation samples detached, and all samples showed good skin adhesion.

[0072] The above results show that the benzoylguanidine derivative hydrogel formulation prepared by the present invention has a uniform and delicate matrix, no particulate matter, moderate viscosity, is easy to apply to the skin, and has good adhesion.

[0073] Example 5: Construction of an imiquimod-induced mouse model of psoriasis-like skin lesions and activity evaluation of a new crystalline form of benzoylguanidine derivative.

[0074] Experimental Methods: Male BALB / c mice were randomly divided into three groups according to body weight: a control group (no treatment), a model group (IMQ), and a drug group (IMQ + 7.5 mg / kg of drug-treated hydrogel preparation), with 5 mice in each group. Two days before modeling, skin preparation was performed. Except for the control group, 62.5 mg of 5% IMQ was applied to the prepared area daily for 7 consecutive days to induce psoriasis-like skin lesions. In the drug treatment group, drug treatment was administered to the prepared area starting on the third day of modeling, i.e., benzoylguanidine derivative hydrogel preparation was applied to the lesion site at different doses for 5 consecutive days. The degree of skin lesions on the backs of mice was observed in three aspects: scaling, skin thickening, and erythema. The therapeutic effect of the drugs was recorded by photograph. The degree of skin lesions on the backs of mice was scored according to the "Psoriasis Area and Severity Index (PASI)" criteria: 0 points, no symptoms; 1 point, mild symptoms; 2 points, moderate symptoms; 3 points, moderate to severe symptoms; 4 points, severe symptoms. The total PASI score was calculated to characterize the incidence of psoriasis-like skin lesions and the therapeutic effect of the drugs. Mice were euthanized on the morning of the 8th day of the experiment, and blood, skin, and spleen samples were collected for subsequent testing.

[0075] Experimental Results: As shown in Tables 1, 2, and 3, after applying IMQ to the back skin of mice for 7 days, obvious scaling, thickening, and erythematous lesions were observed on the back skin of the mice. Topical application of the benzoylguanidine derivative and its novel crystalline form hydrogel formulation of this invention significantly improved these lesion symptoms. These results indicate that topical application of the benzoylguanidine derivative and its novel crystalline form can repair imiquimod-induced psoriatic skin lesions in mice, with the novel crystalline form showing superior efficacy.

[0076] Table 1: PASI Total Score (Rating Range 0-12)

[0077]

[0078] Table 2: Erythema (scoring range 0-2.5)

[0079]

[0080] Table 3: Scales (scoring range 0-4)

[0081]

[0082] Example 6: Effects of benzoylguanidine derivatives and their novel crystal forms on the histopathology of dorsal skin tissue in imiquimod-induced psoriatic mice

[0083] Experimental Methods: At the end of the experiment in Example 5, animal skin was collected. A small portion of the skin from the same location on each mouse was cut for HE staining, and the histopathological changes were observed under a microscope. The cut portion was placed in a 1.5 mL EP tube of 4% paraformaldehyde, fixed, stained with hematoxylin and eosin, and the sections were photographed and analyzed under a microscope.

[0084] Experimental results: such as Figure 5 As shown, the control group had a clear and intact epidermis with a thinner stratum corneum and more orderly and compact cell arrangement, showing no obvious abnormalities. In the model group, a dark purple layer was clearly visible on the outermost layer, with abnormal proliferation of stratum corneum cells and incomplete keratinization. The stratum spinosum thickened, and significant inflammatory cell infiltration was observed, forming pathological features similar to human psoriasis. After treatment with the benzoylguanidine derivative novel crystalline hydrogel formulation, the epidermal cell thickness decreased significantly, the epidermal thickness was significantly reduced, and the inflammatory infiltration gradually disappeared, approaching the normal control group.

[0085] Example 7: Effects of a novel benzoylguanidine derivative on spleen function in imiquimod-induced psoriatic mice

[0086] Experimental methods: At the end of the experiment, the spleens of the animals were harvested, photographed, and a small portion of the spleen from the same location in each mouse was taken for HE staining. The histopathological changes were observed under a microscope. The cut portions were placed in 1.5 mL EP tubes of 4% paraformaldehyde, fixed, stained with hematoxylin and eosin, and the sections were photographed and analyzed under a microscope.

[0087] Experimental results: such as Figure 6 As shown, imiquimod caused proliferation of fibrous tissue and inflammatory cell infiltration in the spleen capsule of mice, disordered splenic trabeculae structure, disappearance of nodular splenic corpuscles, active and unevenly distributed germinal centers, disappearance of the central artery, and patchy proliferation of lymphocytes in the red pulp area, with most blood sinuses dilated and congested; spleen imaging showed splenomegaly in the model group, indicating increased inflammatory response. Topical application of a novel crystalline form of benzoylguanidine derivative hydrogel reduced inflammatory cell infiltration, restored normal splenic trabeculae structure, revealed a structurally normal central artery, and decreased germinal center activity; the spleen was significantly smaller, approaching the size of the normal control group. In conclusion, this indicates that the novel crystalline form of benzoylguanidine derivative can regulate the inflammatory response caused by psoriasis and exert a certain anti-inflammatory effect.

[0088] Example 8: Effect of a novel benzoylguanidine derivative on inflammatory cytokine levels in imiquimod-induced psoriatic mice

[0089] Experimental methods: At the end of the experiment, the spleens of the animals were harvested. A small portion of the spleen from the same location in each mouse was cut off and ground. The levels of inflammatory factors (IL-17A, IL-23, TNF-α, IL-6) in the supernatant were detected by ELISA. Whole blood was collected, plasma was separated, and the levels of inflammatory factors (IL-17A, IL-23) in the plasma were detected by ELISA.

[0090] Experimental results: such as Figure 7 As shown, the levels of inflammatory factors in the spleen and plasma were detected by ELISA. The results showed that imiquimod significantly increased the levels of IL-17A, IL-23, TNF-α, and IL-6 in the spleen of mice, while topical application of the novel crystalline form of benzoylguanidine derivative significantly reduced the expression levels of these factors. Similarly, imiquimod significantly increased the levels of IL-17A and IL-23 in the plasma of mice, while topical application of the novel crystalline form of benzoylguanidine derivative significantly reduced the expression levels of these factors. In conclusion, the novel crystalline form of benzoylguanidine derivative can regulate the inflammatory response induced by psoriasis and exert a certain anti-inflammatory effect.

[0091] Example 9: The effect of a novel crystalline form of benzoylguanidine derivative on CD4+ in the spleen of imiquimod-induced psoriatic mice + IL-17A + Effects of Th17 cell expression

[0092] Experimental Methods: At the end of the experiment, spleens were harvested from animals. A small portion of fresh spleen was taken from the same location in each mouse's spleen and ground using a 100µm cell filter. The mixture was then washed with PBS and filtered. Red blood cell lysis buffer was added to lyse the splenic lymphocytes, which were then isolated and stained. Antibodies against FVS510 (Fixable Viability Stain 510), CD4-FITC, CD8a-PerCp, and IL-17A-AF647 were added to the lymphocytes for staining. When staining with IL-17A-AF647, the cells were fixed and permeated using Cytofix / Cytoperm™ Plus. Flow cytometry was used to analyze the cells.

[0093] Experimental Results: Th17 cells enhance the inflammatory process and produce various cytokines, including IL-17A, IL-22, and IL-23. These cytokines participate in abnormal follicular proliferation, lymphocyte infiltration, and other inflammatory immune-mediated processes. Furthermore, increased pro-inflammatory factors can migrate to skin tissue via the bloodstream, recruiting or activating inflammatory cells, such as neutrophils and macrophages, thereby manifesting or exacerbating psoriatic pathology. Therefore, cytokines are considered key regulators mediating the pathogenesis of psoriasis and are effective targets for psoriasis treatment. Th17 cells play a crucial role in the pathogenesis of psoriasis, and IL-17A is primarily produced by Th17 cells. Figure 8 As shown, flow cytometry results revealed that IMQ-induced CD4+ levels in the spleen of psoriatic mice... + IL-17A + The proportion of [specific cells] was higher than that of the control group. Compared with the model group, topical application of the novel crystalline hydrogel of benzoylguanidine derivative significantly increased CD4 count in mouse spleens. + IL-17A + The proportion was significantly reduced. This suggests that the new crystalline form of benzoylguanidine derivatives can reduce CD4+ in mouse spleen lymphocytes. + IL-17A + The expression of Th17 cells inhibits the secretion of cytokines by Th17 cells in psoriasis patients, thereby exerting an anti-inflammatory effect.

[0094] In conclusion, benzoylguanidine derivatives and their novel crystalline forms possess significant medicinal value in the treatment of psoriasis. Hydrogel formulations made with benzoylguanidine derivatives as the active ingredient can effectively improve symptoms of psoriatic skin characterized by increased scaling, erythema, and epidermal thickening caused by IMQ (inflammation-induced erythema), reduce inflammatory cell infiltration and epidermal thickness, while also reducing inflammatory cell infiltration in the spleen, improving abnormally active germinal centers, lowering the spleen index, and exhibiting anti-inflammatory effects by reducing inflammatory factor levels. Therefore, benzoylguanidine derivatives and their novel crystalline forms in hydrogel formulations possess good medical and market economic value.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The use of a benzoylguanidine derivative in the preparation of a medicament for the treatment and / or prevention of chronic inflammatory diseases.

2. The application according to claim 1, characterized in that: The chronic inflammatory diseases mentioned include psoriasis.

3. The application according to claim 1, characterized in that: The benzoylguanidine derivatives include compounds ML-1, ML-5, and their novel crystal forms, with the following structural formulas: 。 4. The application according to claim 1, characterized in that: The drug contains 0.01% to 5% by weight of a benzoylguanidine derivative or its oxalate or its fumarate neocrystalline form.

5. The application according to claim 1, characterized in that: The benzoylguanidine derivative reduces inflammatory cell infiltration and promotes the recovery of splenic trabecular structure. The benzoylguanidine derivative also reduces CD4 count in splenic lymphocytes. + IL-17A + The expression of Th17 cells inhibits the secretion of cytokines by Th17 cells.

6. The application according to claim 1, characterized in that: The drug is a topical dosage form, administered transdermally, with a dosage of 3.75 mg / kg to 7.5 mg / kg per application.

7. The application according to claim 6, characterized in that: The topical dosage form of the drug is a hydrogel preparation.

8. The application according to claim 7, characterized in that: The hydrogel formulation comprises, by weight percentage: 0.01% to 5% benzoyl guanidine derivative, 0.5% to 40% matrix material, 2% to 40% humectant, 0.1% to 1% preservative, 0.1% to 2% pH adjuster, 0.5% to 10% penetration enhancer, and the balance being water.

9. The application according to claim 8, characterized in that: The matrix material is at least one of carbomer 940, carbomer 941, carbomer 910, carbomer 934, carbomer 1342 and poloxamer; the humectant is at least one of glycerin and betaine; and the pH adjuster is triethanolamine or sodium hydroxide.

10. The application according to claim 8, characterized in that: The preservative is at least one of sodium benzoate, benzalkonium bromide, and ethylparaben, and the penetration enhancer is at least one of laurocapram, peppermint oil, and ethanol.

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