Use of mefloquine for the preparation of a medicament for the treatment of sporotrichosis

Abstract

The application discloses application of mefloquine in preparation of a medicine for treating sporotrichosis. The application finds a new use of the old medicine mefloquine. The application takes abaA gene as a target for finding new drugs, carries out batch molecular docking in a small molecule database approved by FDA, and screens mefloquine from 1615 small molecules. The mefloquine can inhibit Sporothrix globosa and Sporothrix schenckii, can also inhibit phase conversion of the Sporothrix globosa and the Sporothrix schenckii, and has certain therapeutic effect on sporotrichosis, and is extremely likely to become a promising new medicine for treating sporotrichosis. The application provides a new idea for other invasive fungal diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically a new use of the small molecule drug mefloquine in the treatment of sporotrichosis. Background Technology

[0002] Sporothrix 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*, and *Sporothrix brasiliensis*. Sporothrix is ​​commonly found in soil and plant debris, so patients are often gardeners and other workers in agriculture, floriculture, and timber harvesting. Some immunocompromised individuals are also susceptible to disseminated sporothrix, which can be life-threatening in severe cases. Currently, itraconazole is the primary drug used to treat sporothrix. However, the problem of drug resistance in invasive fungal diseases is becoming increasingly serious, and drug resistance has also emerged in sporothrix. Therefore, the search for new small-molecule drugs to treat sporothrix is ​​urgent. A characteristic of Sporothrix is ​​that it is a dimorphic fungus; in the environment (25°C), it exists as mycelium, in which state it is non-pathogenic; after entering the human body (37°C), Sporothrix transforms into a yeast phase, at which point it becomes pathogenic. Therefore, genes related to the phase transition process of Sporothrix can affect the virulence of Sporothrix. Thus, it is of practical significance to study therapeutic drugs by targeting genes related to the phase transition process of Sporothrix.

[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 by national food and drug administrations and other regulatory bodies. Many new drug development projects are ultimately abandoned due to safety concerns. Meanwhile, some "old drugs" have been discovered incidentally to not only treat the symptoms of their respective diseases but also demonstrate 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 numerous international pharmaceutical companies. Furthermore, compared to developing new compounds, drug repurposing saves considerable time and money on pharmacokinetic and toxicological analyses.

[0004] Mefloquine has the following structural formula:

[0005]

[0006] Mefloquine is a drug that kills the intraerythrocytic trophozoites of Plasmodium. It has a long-lasting inhibitory effect on Plasmodium and can be used as an antimalarial drug, as well as for the treatment of drug-resistant Plasmodium. 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, mefloquine was ultimately identified.

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

[0009] Furthermore, the sporotrichosis is a sporotrichosis caused by Sporothrix globosa, Sporothrix schenckii, and / or Sporothrix brasiliensis.

[0010] Furthermore, the use of mefloquine in the preparation of drugs that inhibit the growth of the yeast phase of Sporothrix globosum.

[0011] Furthermore, the MIC value of the mefloquine for inhibiting Sporothrix globosum is 12.5 μg / mL, and the MBC value is 25 μg / mL.

[0012] Furthermore, the dosage of mefloquine that inhibits Sporothrix globosum is 3.8 mg / kg to 20 mg / kg.

[0013] Furthermore, the use of mefloquine in the preparation of a drug that inhibits the growth of the yeast phase of Sporothrix schenckii.

[0014] Furthermore, the MIC value of the mefloquine for inhibiting Sporothrix schenckii is 6.25 μg / mL, and the MBC value is 25 μg / mL.

[0015] Furthermore, the dosage of mefloquine that inhibits Sporothrix schenckii is 3.8 mg / kg to 20 mg / kg.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention provides a new application for the existing drug mefloquine. Using the abaA gene as a target for new drug discovery, this invention performs batch molecular docking in a database of FDA-approved small molecules, screening mefloquine from 1615 small molecules. Mefloquine 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. This invention also provides new insights into other invasive fungal diseases.

[0018] 2. In this invention, the screened mefloquine was subjected to in vitro inhibition tests on Sporothrix schenckii and Sporothrix chenckii. The results showed that the drug mefloquine had an inhibitory effect on Sporothrix schenckii and affected the phase transition of Sporothrix schenckii. In order to further verify whether mefloquine has a broad spectrum of antibacterial activity, the sensitivity of mefloquine to Sporothrix chenckii was tested. The results showed that the small molecule drug mefloquine also had a significant inhibitory effect on the growth and phase transition of Sporothrix chenckii.

[0019] 3. To further verify the inhibitory effect of mefloquine on *Sporothrix globosa* and *Sporothrix schenckii*, this invention determined the growth curves, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of mefloquine against these fungi. The growth curve results showed that mefloquine inhibited the growth of both *Sporothrix globosa* and *Sporothrix schenckii*. The MIC for mefloquine against *Sporothrix globosa* was determined to be 12.5 μg / mL, and the MBC was 25 μg / mL. The MIC for mefloquine against *Sporothrix schenckii* was 6.25 μg / mL, and the MBC was 25 μg / mL. Mefloquine 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, mefloquine, as a small-molecule drug, shows great promise for treating sporothrix infections.

[0020] 4. This invention has verified the therapeutic effect of the small molecule drug mefloquine on sporotrichosis at the animal level. The results of HE staining and PAS staining of skin lesions show that the small molecule drug can effectively inhibit or even kill sporotrichosis and reduce the infiltration of inflammatory cells in the skin, indicating that mefloquine has a certain therapeutic effect on sporotrichosis.

[0021] 5. The small molecule drug mefloquine 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. Mefloquine has great potential as a new drug for the treatment of sporotrichosis. Attached Figure Description

[0022] Figure 1 This refers to the in vitro antifungal activity of dutasteride against Sporothrix globosa.

[0023] Figure 2 This refers to the in vitro antifungal activity of eltrombopag against Sporothrix globosa.

[0024] Figure 3 This refers to the in vitro antifungal activity of mefloquine against Sporothrix globosum.

[0025] Figure 4The curve shows the inhibition of Sporothrix globosum growth by mefloquine.

[0026] Figure 5 It is the in vitro antifungal activity of mefloquine against Sporothrix schenckii.

[0027] Figure 6 The curve shows the inhibition of Sporothrix schenckii growth by mefloquine.

[0028] Figure 7 This is the result of mouse modeling.

[0029] Figure 8 These are the results of purified culture of pus from the lesion site in mice.

[0030] Figure 9 These are the results of HE staining of mouse skin lesions.

[0031] Figure 10 These are the results of PAS staining of mouse skin lesions. Detailed Implementation

[0032] 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.

[0033] 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 marketed small molecules. From 1615 small molecules screened, three candidate small molecule drugs—dutasteride (Avodart), eltrombopag, and mefloquine—were ultimately selected through analysis of their binding energy, efficacy, price, and side effects for further research.

[0034] (I) In vitro antifungal activity test of candidate small molecule drugs against Sporothrix globosum

[0035] The method is as follows:

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 4. Take 5 bottles of 20mL BHI liquid culture medium and label them as ① control, ② DMSO 100μL, ③ DMSO 200μL, ④ mefloquine 50μg / mL, and ⑤ mefloquine 100μg / mL.

[0040] 5. Add 1 mL of the bacterial culture obtained in step 3 to each bottle of BHI liquid culture medium.

[0041] 6. Dissolve 10 mg of mefloquine in 1 mL of DMSO.

[0042] 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.

[0043] 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.

[0044] 9. All the above experiments were repeated three times, and the results were as follows: Figure 3 .

[0045] The procedures for using dutasteride and eltrombopag were the same as above, and the results were as follows. Figure 1 and Figure 2 .

[0046] 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.

[0047] 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.

[0048] Results of the in vitro antifungal activity of mefloquine against Sporothrix globosum are as follows: Figure 3 As shown, compared with the control group and the DMSO solvent control group, mefloquine inhibited the growth and phase transition of Sporothrix globosa starting from 12 h.

[0049] (II) Determination of the growth curve of mefloquine inhibiting Sporothrix globosum

[0050] 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 mefloquine, respectively.

[0051] 2. Transfer 100 mL of Sporothrix globosum 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.

[0052] 3. Prepare the working solutions: Take 10 mg of Itraconazole and mefloquine respectively and dissolve them in 1 mL of DMSO.

[0053] 4. Add 100 μL of the above-mentioned drug working solution to each of the Itraconazole and mefloquine groups, with a final concentration of 50 μg / mL.

[0054] 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.

[0055] 6. Process the data; the results are as follows: Figure 4 .

[0056] The growth curve of mefloquine inhibiting Sporothrix globosum is as follows: Figure 4 As shown in the results, compared with the control group and the solvent control group, the addition of mefloquine significantly inhibited the growth of Sporothrix globosa.

[0057] (III) MIC and MBC determination of mefloquine in inhibiting Sporothrix globosum

[0058] To investigate the minimum concentration of mefloquine that inhibits Sporothrix globosum, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of mefloquine were measured using the micro-checkerboard dilution method. The experimental design was based on CLSI but modified.

[0059] 1. Drug preparation: Dissolve 5 mg of mefloquine in 1 mL of DMSO, shake to mix, and set aside. Take 20 μL of the stock solution and prepare a 100 μg / mL mefloquine working solution in 980 mL of autoclaved BHI liquid medium, and set aside.

[0060] 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 .

[0061] 3. Take a sterile 96-well plate in a clean bench, add 200 μL of mefloquine 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.

[0062] 4. Transfer 100 μL of the mefloquine working solution from well 1 to well 2 and mix slowly by suction. Transfer 100 μL of the mefloquine 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.

[0063] 4. Place the 96-well plate in an incubator at 37°C and incubate for 72 hours.

[0064] 5. Use an ELISA reader to analyze the OD... 630 The absorbance was measured to analyze the minimum inhibitory concentration (MIC).

[0065] 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).

[0066] 7. Process the data, and the results are shown in Table 1.

[0067] Table 1. MIC and MBC of mefloquine against Sporothrix globosum

[0068]

[0069]

[0070] The MIC and MBC results of mefloquine against Sporothrix glomeratus are shown in Table 1. The minimum inhibitory concentration (MIC) of mefloquine against Sporothrix glomeratus was 12.5 μg / mL, and the minimum bactericidal concentration (MBC) was 25 μg / mL.

[0071] (iv) In vitro antifungal activity test of mefloquine against Sporothrix schenckii

[0072] To investigate whether mefloquine also has an inhibitory effect on other genotypes of Sporothrix, the sensitivity of Sporothrix schenckii to mefloquine was tested. The method and procedure were the same as in (I), except that Sporothrix schenckii was replaced with Sporothrix schenckii.

[0073] Results of the in vitro antifungal activity of mefloquine against Sporothrix schenckii are as follows: Figure 5 As shown, compared with the control group and the DMSO solvent control group, mefloquine inhibited the growth of Sporothrix schenckii starting from 12h. By 72h, the control group and the solvent control group showed phase inversion, while the group with added mefloquine did not show phase inversion.

[0074] (V) Determination of the growth curve of mefloquine inhibiting Sporothrix schenckii.

[0075] 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 6 .

[0076] The growth curve of mefloquine inhibiting Sporothrix schenckii is as follows: Figure 6 As shown in the results, compared with the control group, the addition of mefloquine significantly inhibited the growth of Sporothrix schenckii.

[0077] (vi) MIC and MBC determination of mefloquine in inhibiting Sporothrix schenckii

[0078] 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.

[0079] Table 2. MIC and MBC of mefloquine against Sporothrix schenckii

[0080]

[0081] The MIC and MBC results of mefloquine against Sporothrix schenckii are shown in Table 2. The minimum inhibitory concentration (MIC) of mefloquine against Sporothrix schenckii was 6.25 μg / mL, and the minimum bactericidal concentration (MBC) was 25 μg / mL.

[0082] (vii) Animal-level testing of the therapeutic effect of the small molecule drug mefloquine on sporotrichosis

[0083] 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".

[0084] 2. Experimental Groups:

[0085] 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.

[0086] Table 3

[0087]

[0088] 3. Immunosuppression in animals before and after vaccination

[0089] 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.

[0090] 4. Preparation of bacterial suspension

[0091] 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.

[0092] 5. Experimental infection

[0093] 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.

[0094] 6. Administration of medication

[0095] Preparation of drug delivery solution:

[0096] 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.

[0097] Mefloquine 3.8 mg / kg group: 10.64 mg of the drug 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.

[0098] Mefloquine 20mg / kg group: 56mg 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.

[0099] Each animal in the control group was given 0.1 mL of sodium carboxymethyl cellulose by gavage.

[0100] 7. HE and PAS staining of skin lesions

[0101] 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.

[0102] Mouse modeling results as follows Figure 7 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 8 As shown, Schenck's s ...

[0103] from Figure 7 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 positive control group (itaconazole and mefloquine) 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.

[0104] Mouse skin lesions stained with HE as follows Figure 9 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 mefloquine) was less severe.

[0105] Mouse PAS staining Figure 10As 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 mefloquine treatment groups, but far fewer than in the control group, indicating that the mefloquine, a small-molecule drug screened in this invention, has a significant antibacterial and bactericidal effect.

Claims

1. The use of mefloquine, alone or in combination with other drugs for the treatment of sporotrichosis, for the preparation of a drug for the treatment of sporotrichosis. The sporotrichosis is sporotrichosis caused by Sporothrix globosa and / or Sporothrix schenckii.

2. Use according to claim 1, characterized in that, The mefloquine inhibits the growth of the yeast phase of Sporothrix globosa.

3. Use according to claim 1, characterized in that, The mefloquine inhibits Sporothrix globosa with MIC value of 12.5 μg / mL and MBC value of 25 μg / mL.

4. Use according to claim 3, characterized in that, The mefloquine inhibits Sporothrix globosa in a dose of 3.8 mg / kg to 20 mg / kg.

5. The use according to claim 1, characterized in that, The mefloquine inhibits the growth of the yeast phase of Sporothrix schenckii.

6. Use according to claim 1, characterized in that, The mefloquine inhibits Sporothrix schenckii with MIC value of 6.25 μg / mL and MBC value of 25 μg / mL.

7. Use according to claim 6, characterized in that, The mefloquine inhibits Sporothrix schenckii in a dose of 3.8 mg / kg to 20 mg / kg.

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