Use of small molecule drug azelastine in preparation of medicine for treating sporotrichosis

Abstract

The application discloses application of a small-molecule drug azelastine in preparation of a medicine for treating sporotrichosis. The application takes abaA gene as a target for finding a new drug, performs batch molecular docking on a small-molecule database which has been approved by FDA and marketed, and screens out azelastine. The azelastine can inhibit Sporothrix globosa and Sporothrix schenckii, can also inhibit phase conversion of the Sporothrix globosa and the Sporothrix schenckii, and has a certain therapeutic effect on sporotrichosis, and is extremely likely to become a promising new drug for treating sporotrichosis. In the research process, it is also found that the small-molecule drug azelastine can have a broad-spectrum bacteriostatic effect. Since the drug is approved for marketing for treating other symptoms, compared with a new compound, a large amount of time and money cost for analyzing pharmacokinetics and pharmacology and toxicology is saved, and the azelastine has great potential as a new drug for treating sporotrichosis.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically a novel use of the small molecule drug azelastine in the treatment of sporotrichosis. Background Technology

[0002] Sporothrixeibrio is a chronic fungal infection of the epidermis and subcutaneous tissues caused by the Sporothrix complex. The main pathogenic subtypes within the Sporothrix complex are *Sporothrix globosa*, *Sporothrix schenckii* (narrowly defined), and *Sporothrix brasiliensis*. Currently, research on *Sporothrix globosa* is limited, with few related literature and clinical studies. Sporothrix is ​​commonly found in soil and plant debris, so patients are often gardeners and other workers in agriculture, floriculture, and timber harvesting. Individuals with weakened immune systems are also susceptible to disseminated sporothrixeibrio, which can be life-threatening in severe cases. Currently, itraconazole is the primary drug used to treat sporothrixeibrio. However, the problem of drug resistance in invasive fungal diseases is becoming increasingly serious, and drug resistance has also emerged in sporothrixeibrio, making the search for new small-molecule drugs for treating sporothrixeibrio urgent.

[0003] As is well known, the market launch of new drugs typically requires significant time and research and development costs, and necessitates rigorous safety testing. However, some "old drugs" have been discovered incidentally to not only treat the symptoms of their respective diseases but also to produce remarkable therapeutic effects on other ailments with minimal side effects. Therefore, drug repurposing, finding new applications for existing drugs, is increasingly valued and adopted by many international pharmaceutical companies. Furthermore, compared to developing new compounds, drug repurposing saves considerable time and money on pharmacokinetic and toxicological analyses.

[0004] Azelastine has the following structural formula:

[0005]

[0006] Azelastine is primarily used to prevent and treat bronchial asthma and allergic rhinitis. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention employs a drug repurposing approach, using the abaA gene as a target for new drug discovery. It involves batch molecular docking within an FDA-approved database of marketed small molecules. By analyzing the binding energy, efficacy, price, and side effects of 1615 small molecules selected, azelastine was ultimately identified.

[0008] The technical solution adopted in this invention is as follows: the application of the small molecule drug azelastine, alone or in combination with other drugs for treating sporotrichosis, in the preparation of drugs for treating sporotrichosis.

[0009] Furthermore, the sporotrichosis is sporotrichosis caused by *Sporotrichia coli*.

[0010] Furthermore, the application of the small molecule drug azelastine in the preparation of a drug that inhibits the growth of the yeast phase of Sporothrix globosa.

[0011] Furthermore, the minimum inhibitory concentration (MIC) of the small molecule drug azelastine against Sporothrix spheroides is 25 μg / mL, and the minimum bactericidal concentration (MBC) is 50 μg / mL.

[0012] Furthermore, the dosage of the small molecule drug azelastine to inhibit Sporothrix globosum is 3 mg / kg to 6 mg / kg.

[0013] Furthermore, the sporotrichosis is sporotrichosis caused by *Sporotrichia schenckii*.

[0014] Furthermore, the application of the small molecule drug azelastine in the preparation of a drug that inhibits the growth of the yeast phase of Sporothrix schenckii.

[0015] Furthermore, the minimum inhibitory concentration (MIC) of the small molecule drug azelastine against Sporothrix schenckii is 6.25 μg / mL, and the minimum bactericidal concentration (MBC) is 50 μg / mL.

[0016] Furthermore, the dosage of the small molecule drug azelastine to inhibit Sporothrix schenckii is 3 mg / kg to 6 mg / kg.

[0017] Furthermore, the sporotrichosis is sporotrichosis caused by *Sporotrichia brasiliensis*.

[0018] The beneficial effects of this invention are:

[0019] 1. This invention conducted in vitro inhibition tests on the screened small molecule drug azelastine against *Sporothrix globosa* and *Sporothrix schenckii*. The results showed that azelastine inhibited *Sporothrix globosa* and affected its phase transition. To further verify whether azelastine has a broad-spectrum antibacterial activity, its sensitivity to *Sporothrix schenckii* was tested. The results showed that azelastine also significantly inhibited the growth and phase transition of *Sporothrix schenckii*. To verify whether the drug only inhibits the growth of the yeast phase of *Sporothrix globosa*, experiments were conducted to observe whether azelastine inhibited the growth of the mycelial phase of *Sporothrix globosa*. The results showed that azelastine could not inhibit the growth of the mycelial phase of *Sporothrix globosa*, indicating that the abaA gene in *Sporothrix globosa* is likely also differentially expressed in the yeast phase.

[0020] 2. This invention determined the growth curves, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of azelastine against *Sporothrix globosa* and *Sporothrix schenckii*. The growth curve results showed that azelastine inhibited the growth of both *Sporothrix globosa* and *Sporothrix schenckii*. The MIC for *Sporothrix globosa* was determined to be 25 μg / mL, and the MBC to be 50 μg / mL. The MIC for *Sporothrix schenckii* was 6.25 μg / mL, and the MBC to be 50 μg / mL. Although the measured MIC and MBC values ​​do not show the advantage of low-dose treatment compared to other antifungal drugs, azelastine has a higher safety profile than antibiotics, and its dosage when used alone is lower than that of itraconazole, currently used. Furthermore, given the increasing severity of fungal resistance, there is an urgent need for new small-molecule drugs to treat fungal diseases. Therefore, azelastine, a small-molecule drug, shows great promise as a treatment for sporothrix infections.

[0021] 3. This invention verified the therapeutic effect of the small molecule drug azelastine on sporothrix infection at the animal level. The results of HE staining and PAS staining of skin lesions showed that the small molecule drug can effectively inhibit or even kill Sporothrix and reduce the infiltration of inflammatory cells in the skin, indicating that azelastine has a certain therapeutic effect on sporothrix infection.

[0022] 4. This invention provides a new application for the existing drug azelastine. Using the abaA gene as a target for new drug discovery, this invention conducted batch molecular docking in a database of FDA-approved small molecules, screening out azelastine from 1615 small molecules. Azelastine can inhibit *Sporothrix globosa* and *Sporothrix schenckii*, while also inhibiting their phase transition and exhibiting some therapeutic effect on sporotrichosis, making it a promising new drug for treating sporotrichosis. During the research, it was also discovered that azelastine, a small molecule drug, may have broad-spectrum antibacterial effects; therefore, this invention can also provide new insights for other invasive fungal diseases.

[0023] 5. The small molecule drug azelastine provided by this invention has a certain therapeutic effect on sporotrichosis. Since this drug was approved for marketing to treat other symptoms, it saves a lot of time and money on pharmacokinetic and pharmacological toxicology analysis compared with new compounds. Azelastine has great potential as a new drug for the treatment of sporotrichosis. Attached Figure Description

[0024] Figure 1 Dutasteride exhibits in vitro antifungal activity against Sporothrix globosa;

[0025] Note: The dutasteride test concentrations were 50 μg / mL and 100 μg / mL. The concentration selected in the figure is 100 μg / mL. The amount of DMSO added corresponds to the amount of dutasteride added.

[0026] Figure 2 It is the in vitro antifungal activity of eltrombopag against Sporothrix spheroides;

[0027] Note: The eltrombopag test concentrations were 50 μg / mL and 100 μg / mL; the concentration selected in the figure is 100 μg / mL. The amount of DMSO added corresponds to the amount of eltrombopag added.

[0028] Figure 3 This demonstrates the in vitro antifungal activity of azelastine against Sporothrix globosa;

[0029] Note: The concentrations for azelastine testing were 50 μg / mL and 100 μg / mL; the concentration selected in the figure is 100 μg / mL. The amount of DMSO added corresponds to the amount of azelastine added.

[0030] Figure 4 It is the in vitro antifungal activity of azelastine against the hyphal phase of Sporothrix globosa.

[0031] Figure 5 The curve of azelastine inhibiting the growth of Sporothrix globosum;

[0032] Note: The DMSO group was the solvent control group, and the Itraconazole group was the positive control group. *, P < 0.05; **, P < 0.01.

[0033] Figure 6 It is the in vitro antifungal activity of azelastine against Sporothrix schenckii;

[0034] Note: The concentrations for the azelastine test were 50 μg / mL and 100 μg / mL. The concentration selected in the figure is 100 μg / mL.

[0035] Figure 7 The curve of azelastine inhibiting the growth of Sporothrix schenckii;

[0036] Note: The DMSO group was the solvent control group, and the Itraconazole group was the positive control group. **, P < 0.01; ***, P < 0.001.

[0037] Figure 8 This is the result of mouse modeling.

[0038] Figure 9 These are the results of purified culture of pus from the lesion site in mice.

[0039] Figure 10 These are the results of HE staining of mouse skin lesions.

[0040] Figure 11 These are the results of PAS staining of mouse skin lesions. Detailed Implementation

[0041] One characteristic of Sporothrix is ​​that it is a dimorphic fungus. In the environment (25°C), it exists as mycelium, in which it is non-pathogenic; after entering the human body (37°C), Sporothrix transforms into a yeast phase, in which it becomes pathogenic. Therefore, the genes related to this phase transition process of Sporothrix affect its virulence, which becomes the key direction for the selection of targets in this invention.

[0042] Previous studies have shown that the abaA gene is upregulated during the transition from the hyphal phase to the yeast phase in *Sporothrix schenckii*, and the deletion of this gene leads to a decrease in the resistance of *Sporothrix schenckii* to various stresses. These results indicate that this gene is crucial for phase transition in *Sporothrix schenckii*, so it is reasonable to speculate that this gene is also crucial for phase transition in *Sporothrix globosa*. Therefore, this invention performs batch molecular docking of the DNA-binding domain of the AbaA protein against an FDA-approved small molecule database to screen for small molecule drugs that can target AbaA.

[0043] This invention first uses bioinformatics to predict the three-dimensional structure of the AbaA protein, locates the DNA binding site of the abaA gene, scores the quality of the predicted three-dimensional structure, selects the structure with the highest model quality to extract its DNA binding site, and performs batch molecular docking in a database of FDA-approved small molecules. From 1615 small molecules screened, three candidate small molecule drugs—dutasteride (Avodart), eltrombopag, and azelastine—were ultimately selected through analysis of their binding energy, efficacy, price, and side effects for further research.

[0044] (I) In vitro antifungal activity test of candidate small molecule drugs against Sporothrix globosum

[0045] The method is as follows:

[0046] 1. In a clean bench, use an inoculation loop to streak three zones of Sporothrix globosum onto SDA medium and incubate at 25°C for 4 days.

[0047] 2. Use a pipette to draw 1 mL of sterile distilled water to rinse the cultured Sporothrix spores, transfer the spore solution to 100 mL of SDA medium, and incubate at 28°C and 150 rpm for 4 days.

[0048] 3. After microscopic examination, transfer the cultured bacterial solution to a 50 mL centrifuge tube using a 5 mL pipette, and centrifuge at 10,000 rpm for 5 min at 4 °C. Discard the supernatant, add 5 mL of BHI medium, and gently mix with a pipette.

[0049] 4. Take 5 bottles of 20mL BHI liquid culture medium and label them as ① control, ② DMSO 100μL, ③ DMSO 200μL, ④ azelastine 50μg / mL, and ⑤ azelastine 100μg / mL, respectively.

[0050] 5. Add 1 mL of the bacterial culture obtained in step 3 to each bottle of BHI liquid culture medium.

[0051] 6. Dissolve 10 mg of azelastine in 1 mL of DMSO.

[0052] 7. Add 100 μL of DMSO to bottle ②; add 200 μL of DMSO to bottle ③; add 100 μL of DMSO and 100 μL of the reagent prepared in step 6 to bottle ④, with a final concentration of 50 μg / mL; add 200 μL of DMSO and 200 μL of the reagent prepared in step 6 to bottle ⑤, with a final concentration of 100 μg / mL.

[0053] 8. Place the product from step 7 in a shaker at 37°C and 180 rpm and incubate it. Observe and photograph it every 12 hours using an inverted fluorescence microscope. Take 10 random photos of each location in each group.

[0054] 9. All the above experiments were repeated three times, and the results were as follows: Figure 3 .

[0055] The procedures for using dutasteride and eltrombopag were the same as above, and the results were as follows. Figure 1 and Figure 2 .

[0056] Results of the in vitro antifungal activity of dutasteride against Sporothrix globosum are as follows: Figure 1 As shown, compared with the control group and the DMSO solvent control group, there were no significant changes in hyphae and spores from 12h to 96h after the addition of dutasteride, indicating that Sporothrix globosa is not sensitive to dutasteride.

[0057] Results of eltrombopag's in vitro antifungal activity against Sporothrix globosum: Figure 2 As shown, compared with the control group and the DMSO solvent control group, since eltrombopag itself is orange in color, apart from the drug staining the hyphae of *Sporothrix globosa* orange, there were no other obvious changes, indicating that *Sporothrix globosa* is not sensitive to eltrombopag.

[0058] Results of azelastine's in vitro antifungal activity against Sporothrix globosa: Figure 3As shown, compared with the control group and the DMSO solvent control group, it can be seen that from the 12th hour onwards, after the addition of azelastine, the spores on the hyphae of Sporothrix globosa detached and stopped growing. Azelastine significantly inhibited the growth of Sporothrix globosa. By the 96th hour, the control group and the DMSO solvent control group began to undergo phase inversion, while the group with added azelastine did not undergo phase inversion. This indicates that azelastine inhibited the growth and phase inversion of Sporothrix globosa.

[0059] To determine whether the drug azelastine only inhibited the proliferation of the yeast phase of Sporothrix globosa, a set of experiments was designed to observe the mycelial growth conditions.

[0060] 1. Take 5 bottles of 20mL SDB culture medium and label them as ① Control 1 (incubated at 25℃), ② Control 2 (incubated at 37℃), ③ DMSO 100μL, ④ Azelastine 50μg / mL (incubated at 25℃), and ⑤ Azelastine 50μg / mL (incubated at 37℃).

[0061] 2. Add 1 mL of Sporothrix globosum liquid culture for 4 days to ① and incubate at 25℃; add 1 mL of Sporothrix globosum liquid culture for 4 days to ② and incubate at 37℃; add 1 mL of Sporothrix globosum liquid culture for 4 days and 100 μL of DMSO to ③; add 1 mL of Sporothrix globosum liquid culture for 4 days and 100 μL of azelastine to ④ and incubate at 25℃; add 1 mL of Sporothrix globosum liquid culture for 4 days and 100 μL of azelastine to ⑤ and incubate at 37℃.

[0062] The results are as follows Figure 4 Compared with the control group and the solvent control group, the addition of azelastine at 25℃ resulted in almost no change in mycelial morphology and spore production. However, the addition of azelastine at 37℃ significantly inhibited the growth of *Sporothrix globosa*. This indicates that azelastine inhibits the growth of the yeast phase of *Sporothrix globosa* but has no significant effect on the mycelial phase.

[0063] (II) Determination of the growth curve of azelastine inhibiting Sporothrix globosum

[0064] 1. Prepare 12 bottles of 20mL BHI liquid culture medium after high temperature and high pressure sterilization. Make three bottles into a group and label them as Control, DMSO, Itraconazole, and Azelastine, respectively.

[0065] 2. Transfer 100 mL of Sporothrix spheroides cultured at 25℃ and 150 rpm for 4 days to a 50 mL sterile centrifuge tube and centrifuge at 8000 rpm for 5 min. Discard the supernatant, mix the precipitate with BHI liquid medium sterilized by high temperature and high pressure, and add 200 μL of bacterial suspension to each bottle in step 1.

[0066] 3. Prepare working solutions by dissolving 10 mg of Itraconazole and azelastine in 1 mL of DMSO.

[0067] 4. Add 100 μL of the above-mentioned drug working solution to each of the DMSO, Itraconazole, and azelastine groups to achieve a final concentration of 50 μg / mL.

[0068] 5. Incubate at 37℃ and 150 rpm, starting from hour 0, and measure the OD value every 12 hours using a 96-well plate. 625 The absorbance was measured. Each vial of bacterial culture was tested three times for replication.

[0069] 6. Process the data; the results are as follows: Figure 5 .

[0070] The growth curve of azelastine inhibiting Sporothrix globosum is as follows: Figure 5 As shown in the results, the addition of azelastine significantly inhibited the growth of Sporothrix globosa compared with the control group and the solvent control group.

[0071] (III) Determination of MIC and MBC of azelastine in inhibiting Sporothrix globosum

[0072] To investigate the minimum inhibitory concentration (MIC) of azelastine against Sporothrix globosa, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of azelastine were measured using the micro-checkerboard dilution method. The experimental design was based on CLSI but modified.

[0073] 1. Drug preparation: Dissolve 5 mg of azelastine in 1 mL of DMSO, shake well, and set aside. Take 20 μL of the stock solution and prepare a 100 μg / mL azelastine working solution in 980 mL of autoclaved BHI medium, and set aside.

[0074] 2. Preparation of bacterial suspension: The mycelial suspension of *Sporothrix globosa* cultured at 25℃ for 4 days was filtered and centrifuged. The spores were resuspended in autoclaved BHI liquid medium, and the spore concentration was adjusted to 1×10⁻⁶. 5 .

[0075] 3. Take a sterile 96-well plate in a clean bench, add 200 μL of azelastine working solution to well 1, add 100 μL of BHI liquid culture medium to wells 2-11, and add 200 μL of BHI liquid culture medium to well 12 as a negative control.

[0076] 4. Transfer 100 μL of azelastine working solution from well 1 to well 2 and mix slowly by suction. Transfer 100 μL of azelastine working solution from well 2 to well 3 and mix slowly by suction. Repeat this process until well 10. Discard the 100 μL transferred from well 10 after mixing. Finally, add 100 μL of the bacterial culture prepared in step 2 to each well. Repeat the above steps three times to form three parallel sets.

[0077] 4. Place the 96-well plate in an incubator at 37°C and incubate for 72 hours.

[0078] 5. Use an ELISA reader to analyze the OD... 630 The absorbance was measured to analyze the minimum inhibitory concentration (MIC).

[0079] 6. Prepare 36 autoclaved petri dishes, take 40 μL of the bacterial solution from step 4, spread it on the petri dish, incubate at 37℃ for 10 days, and observe its minimum bactericidal concentration (MBC).

[0080] 7. Process the data, and the results are shown in Table 1.

[0081] Table 1. MIC and MBC of azelastine in inhibiting Sporothrix globosum

[0082]

[0083] The MIC and MBC results of azelastine for inhibiting Sporothrix globosum are shown in Table 1. The minimum inhibitory concentration (MIC) of azelastine for inhibiting Sporothrix globosum was 25 μg / mL, and the minimum bactericidal concentration (MBC) was 50 μg / mL.

[0084] (iv) In vitro antifungal activity test of azelastine against Sporothrix schenckii

[0085] To investigate whether azelastine also has an inhibitory effect on other genotypes of Sporothrix, the sensitivity of Sporothrix schenckii to azelastine was tested. The method and procedure were the same as in (I), except that Sporothrix schenckii was replaced with Sporothrix schenckii.

[0086] Results of azelastine's in vitro antifungal activity against Sporothrix schenckii: Figure 6 As shown, compared with the control group and the DMSO solvent control group, it can be seen that Sporothrix schenckii was significantly inhibited 24 hours after the addition of azelastine. By 48 hours, the control group and the DMSO solvent control group began to undergo phase inversion and became rod-shaped, while the group with azelastine did not show phase inversion. This indicates that azelastine can also inhibit the growth and phase inversion of Sporothrix schenckii.

[0087] (V) Determination of the growth curve of azelastine inhibiting Sporothrix schenckii

[0088] The method and steps are the same as in (II), except that *Sporothrix globosa* is replaced with *Sporothrix schenckii*. The results are as follows: Figure 7 .

[0089] The growth curve of azelastine inhibiting Sporothrix schenckii is as follows: Figure 7 As shown in the results, the addition of azelastine significantly inhibited the growth of Sporothrix schenckii compared with the control group.

[0090] (vi) Determination of MIC and MBC of azelastine in inhibiting Sporothrix schenckii

[0091] The method and steps are the same as in (III), except that *Sporothrix globosa* is replaced with *Sporothrix schenckii*. The results are shown in Table 2.

[0092] Table 2. MIC and MBC of azelastine in inhibiting Sporothrix schenckii.

[0093]

[0094] The MIC and MBC results of azelastine for inhibiting Sporothrix schenckii are shown in Table 2. The minimum inhibitory concentration (MIC) of azelastine for inhibiting Sporothrix schenckii was 6.25 μg / mL, and the minimum bactericidal concentration (MBC) was 50 μg / mL.

[0095] (vii) Animal-level testing of the therapeutic effect of the small molecule drug azelastine on sporothrix.

[0096] 1. Laboratory Animals: The laboratory animals used were SPF-grade KM mice, three weeks old, male, and weighing approximately 20g. The mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number SCXK(Liaoning)2020-0001. This experiment was conducted in strict accordance with the "Guidelines for the Breeding, Management and Use of Laboratory Animals".

[0097] 2. Experimental Groups:

[0098] Thirty-two KM mice were randomly divided into four groups of eight each, as shown in Table 3. The rearing conditions were: temperature 24±2℃, humidity 44%–49%, and ventilation 9–12 times per hour.

[0099] Table 3

[0100]

[0101]

[0102] 3. Immunosuppression in animals before and after vaccination

[0103] All groups of mice were given intraperitoneal injections of hydrocortisone solution every other day for 7 days before vaccination, starting one week after acclimatization. After one week, the injections were changed to once every two days until the model was successfully established and the drugs were started.

[0104] 4. Preparation of bacterial suspension

[0105] Wash the Schenck's spores with autoclaved SDB medium and transfer them to SDB for 10 days at 25°C and 150 rpm. Once the mycelial membrane and hyphal aggregates have fully formed, transfer the culture to 50 mL centrifuge tubes and vortex to disperse and mix the mycelial membrane and aggregates, allowing the spores to detach from the hyphae into the liquid culture medium. Filter the solution through a cell filter to remove hyphae and impurities. Repeat this step twice. Transfer the filtrate to sterile centrifuge tubes and centrifuge at 8000 rpm for 5 minutes. Discard the supernatant, add 20 mL of sterile saline, resuspend the spores with a pipette, and centrifuge again. Repeat this step three times. Adjust the concentration of the bacterial suspension to 1 × 10⁻⁶ using a hemocytometer. 8 One spore / mL, which may contain a small amount of hyphae, but the ratio of spores to hyphae should be adjusted to 100:1 as much as possible.

[0106] 5. Experimental infection

[0107] Before injection of Sporothrix sp. suspension, the abdominal skin of all mice in all groups was shaved with a razor. 0.1 mL of Sporothrix sp. suspension was then injected intradermally into one shaved area using a 1 mL syringe, containing approximately 1 × 10⁻⁶ mg / mL of the suspension. 7 One spore.

[0108] 6. Administration of medication

[0109] Preparation of drug delivery solution:

[0110] Itraconazole group: 168 mg of the positive control drug itraconazole was added to 7 mL of 0.5% sodium carboxymethyl cellulose and sonicated for 20 min; each mouse was gavaged with 0.1 mL / 40 g.

[0111] Azelastine 3mg / kg group: 8.4mg of the drug was placed in 7mL of 0.5% sodium carboxymethyl cellulose and sonicated for 20min; each mouse was gavaged with 0.1mL / 40g.

[0112] Azelastine 6mg / kg group: 16.8mg of the drug was placed in 7mL of 0.5% sodium carboxymethyl cellulose and sonicated for 20min; each mouse was gavaged with 0.1mL / 40g.

[0113] Each animal in the control group was given 0.1 mL of sodium carboxymethyl cellulose by gavage.

[0114] 7. HE and PAS staining of skin lesions

[0115] At the end of the experiment, all mice in all groups were euthanized due to cervical dislocation. They were then immersed in 5% carbolic acid solution for 5 minutes, rinsed three times with sterile distilled water sterilized by high temperature and high pressure, placed on a sterile dissecting plate, and under sterile conditions, the skin lesions were cut off and fixed in 4% paraformaldehyde solution. The samples were sent to Liaoning Jijia Biotechnology for HE and PAS staining.

[0116] Mouse modeling results as follows Figure 8 As shown, mice injected intradermally with the bacterial solution showed varying degrees of skin lesions, some developing nodules, and others expelling pus after gavage. The pus was collected with a sterilized cotton swab, diluted with sterile distilled water, and 40 μL was evenly spread onto SDA medium. The medium was incubated at 25°C for 4 days. The results are as follows. Figure 9 As shown, Schenck's s ...

[0117] from Figure 8 As observed, inflammation and nodules appeared on the skin of mice one week after intradermal injection of the bacterial solution. Based on the phenotype, analysis of the lesion morphology, and microscopic observation after bacterial separation, it was determined to be sporotrichosis. Sporotrichosis was most severe in the third week. At this time, oral administration was administered. After 10 days, nodules were still observed on the skin surface of the control group, while the nodules in the itraconazole and azelastine positive control groups showed reduction in size and improvement in crusting. At this point, skin lesions were collected for HE and PAS staining to observe and analyze the inflammatory factors and fungal infection status.

[0118] Mouse skin lesions stained with HE as follows Figure 10 As shown, the Mock group contains HE staining results of healthy skin. The staining results reveal that, compared to the Mock group, the control group has numerous large, round or oval, pale blue punctate cell nuclei, which are various inflammatory cells. Compared to the control group, the inflammatory infiltration in the positive control groups (itchraconazole and azelastine) was less pronounced.

[0119] Mouse PAS staining Figure 11 As shown, the Mock group represents the PAS staining results of healthy skin. The staining results reveal that, compared to the Mock group, the control group exhibited a large number of purplish-red filamentous positive reactions, which are *Sporothrix schenckii*. Compared to the control group, the purplish-red positive reactions occasionally appeared in the itraconazole and azelastine treatment groups, but far fewer than in the control group, indicating that the azelastine, a small molecule drug screened in this invention, has a significant antibacterial and bactericidal effect.

Claims

1. Use of the small molecule drug azelastine for the manufacture of a medicament for the treatment of sporotrichosis, which is sporotrichosis caused by Sporothrix globosa or sporotrichosis caused by Sporothrix schenckii.

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