CN116370474A

CN116370474A - Application of mefloquine in preparation of drugs for treating sporomycetosis

Info

Publication number: CN116370474A
Application number: CN202310315506.0A
Authority: CN
Inventors: 张振颖; 韩昌旭; 陈岩
Original assignee: Shenzhen Hospital University of Hong Kong
Current assignee: Shenzhen Hospital University of Hong Kong
Priority date: 2023-03-29
Filing date: 2023-03-29
Publication date: 2023-07-04

Abstract

The invention discloses application of mefloquine in preparing a medicine for treating sporotrichosis. The invention finds a new application for the old medicine mefloquine. According to the invention, the abaA gene is used as a target spot for searching new drugs, batch molecular docking is performed in a small molecular database which is obtained by FDA and marketed, and mefloquine is screened out from 1615 small molecules. Mefloquine can inhibit the sporotrichosis globosa and the shenkesporotrichosis, can inhibit the phase inversion of the sporotrichosis globosa and has a certain treatment effect on sporotrichosis, and is very likely to become a novel medicament for treating sporotrichosis with prospect. The invention provides a new idea for other invasive mycoses.

Description

Application of mefloquine in preparation of drugs for treating sporomycetosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel application of a small molecular medicine mefloquine in treating sporotrichosis.

Background

Sporomyces is a chronic fungal infection of the epidermis and subcutaneous tissue caused by the sporomyces complex. The main pathogenic subtypes in the sporosilk complex are sporosilk globosum, sporosilk narrowly defined and sporosilk brazil. Spore rhizobacteria are often present in soil and plant debris, so patients are often the job of agriculture, flowers and wood-working personnel for gardeners and the like. Some people with low immunity are prone to disseminated sporotrichosis, and serious people can also endanger life. The current drug for the treatment of sporomycetosis is mainly Itraconazole (Itraconazole). However, the problem of drug resistance of invasive mycosis is increasingly severe at present, and the drug resistance of sporotrichosis also appears, so that the search for new small-molecule drugs for treating sporotrichosis is not slow. One feature of sporotrichosis is that it is a biphasic fungus that exists in the form of mycelia in the environment (25 ℃), in which state it is non-pathogenic; after entering the human body (37 ℃) the sporotrichosis turns into a yeast phase, the state of which is pathogenic. Therefore, the related genes in the phase inversion process of the sporotrichosis can influence the toxicity of the sporotrichosis, so that the research of the therapeutic drugs by taking the related genes in the phase inversion process of the sporotrichosis as targets has practical significance.

As is well known, the market of new drugs on average takes a lot of time and development costs and requires the passing of strict safety tests by departments such as the national food and drug administration and other regulatory authorities. Many new drug developments are eventually abandoned due to their safety issues. While some "old drugs" have been found to have unusual therapeutic effects on other diseases in addition to the diseases with corresponding symptoms, and have low side effects. Therefore, the new use of old medicines for finding new use for the existing medicines is increasingly valued and adopted by a plurality of international pharmaceutical enterprises. And compared with the new compound, the new compound saves a great deal of time and money cost for analyzing pharmacokinetics and pharmacology toxicology.

Mefloquine (english name: mefloquine) has the following structural formula:

mefloquine is a drug that kills plasmodium erythrocyte inner trophozoites. Has long-acting plasmodium inhibiting effect, and can be used as antimalarial and for treating drug-resistant plasmodium.

Disclosure of Invention

In order to solve the technical problems, the abaA gene is used as a target spot for searching a new drug by a new application method of an old drug, batch molecular docking is carried out in a small molecular database which is obtained by FDA and marketed, and finally mefloquine is screened out by analyzing the binding energy, efficacy, price and side effect of small molecules screened out of 1615 small molecules.

The technical scheme adopted by the invention is as follows: use of mefloquine alone or in combination with other drugs for the treatment of sporozoites for the preparation of a drug for the treatment of sporozoites.

Further, the sporotrichosis is sporotrichosis caused by sporotrichum, sporotrichum and/or sporotrichum.

Further, the mefloquine is applied to the preparation of medicines for inhibiting the growth of candida globosa yeast.

Still further, the MIC value of the mefloquine inhibiting sporozoites was 12.5. Mu.g/mL and the MBC value was 25. Mu.g/mL.

Further, the dosage of mefloquine for inhibiting sporotrichosis is 3.8 mg/kg-20 mg/kg.

Further, the mefloquine is applied to the preparation of medicines for inhibiting the growth of candida shenkesii yeast phase.

Still further, the MIC value of the Mefloquine inhibiting sporozoites was 6.25 μg/mL and the MBC value was 25 μg/mL.

Further, the dosage of the mefloquine for inhibiting the sporozoites is 3.8 mg/kg-20 mg/kg.

The beneficial effects of the invention are as follows:

1. the invention finds a new application for the old medicine mefloquine. According to the invention, the abaA gene is used as a target spot for searching new drugs, batch molecular docking is performed in a small molecular database which is obtained by FDA and marketed, and mefloquine is screened out from 1615 small molecules. Mefloquine can inhibit the sporotrichosis globosa and the shenkesporotrichosis, can inhibit the phase inversion of the sporotrichosis globosa and has a certain treatment effect on sporotrichosis, and is very likely to become a novel medicament for treating sporotrichosis with prospect. The invention provides a new idea for other invasive mycoses.

2. In-vitro test on inhibiting spherical spore silk bacteria and inhibiting Shenkezhi spore silk bacteria is carried out on the screened mefloquine, and the result shows that the mefloquine has an inhibiting effect on the spherical spore silk bacteria and influences the phase inversion of the spherical spore silk bacteria, so that in order to further verify whether the mefloquine has antibacterial broad spectrum, the sensitivity of the mefloquine on the Shenkezhi spore silk bacteria is tested, and the result shows that the mefloquine small-molecule drug has an obvious inhibiting effect on the growth and the phase inversion of the Shenkezhi spore silk bacteria.

3. In order to further verify that mefloquine has an inhibition effect on the sporotrichosis globosa and the trichosporotrichosis lanuginosus, the invention determines a growth curve and a minimum inhibition concentration MIC of the mefloquine for inhibiting the sporotrichosis globosa and the trichosporotrichosis lanuginosus and a minimum sterilization concentration MBC, the result of the growth curve shows that the mefloquine inhibits the growth of the sporotrichosis globosa and the trichosporotrichosis lanuginosus, and simultaneously determines that the minimum inhibition concentration MIC of the mefloquine for inhibiting the sporotrichosis globosa is 12.5 mug/mL and the minimum sterilization concentration MBC is 25 mug/mL. The minimum inhibitory concentration MIC of Mefloquine for inhibiting the sporozoite is 6.25 mug/mL, and the minimum bactericidal concentration MBC is 25 mug/mL. The mefloquine drug has higher safety than antibiotics, the single dosage is lower than that of itraconazole which is currently used, and because the fungal resistance is increasingly severe, new small molecular drugs are urgently needed for treating mycosis, the mefloquine small molecular drugs have wide prospect for treating sporotrichosis.

4. According to the invention, the treatment effect of the small molecular medicine mefloquine on sporotrichosis is verified at animal level, and the results of HE staining and PAS staining of skin lesions show that the small molecular medicine can effectively inhibit or even kill sporotrichosis, reduce inflammatory cell infiltration in skin, and show that mefloquine has a certain treatment effect on sporotrichosis.

5. The small molecular medicine mefloquine provided by the invention has a certain treatment effect on sporotrichosis, and because the medicine is marketed in batches for treating other symptoms, compared with a novel compound, a great amount of time and money cost for pharmacokinetic and pharmacology toxicology analysis are saved, and the mefloquine has great potential for being used as a novel medicine for treating sporotrichosis.

Drawings

FIG. 1 shows the antifungal activity of dutasteride against sporozoite in vitro.

FIG. 2 shows the antifungal activity of eltrombopag against sporozoite in vitro.

FIG. 3 shows the antifungal activity of mefloquine against sporotrichosis globosa.

FIG. 4 is a graph showing the inhibition of growth of Mefloquine by sporozoites.

FIG. 5 shows the antifungal activity of mefloquine against sporozoite in vitro.

FIG. 6 is a graph of mefloquine inhibition of sporozoites growth.

FIG. 7 shows the results of mouse modeling.

FIG. 8 shows the result of purification culture of pus at the affected part of mice.

FIG. 9 shows the results of HE staining for skin lesions in mice.

FIG. 10 shows the result of PAS staining for skin lesions in mice.

Detailed Description

In previous studies, the abaA gene was up-regulated during the transformation from hyphal phase to yeast phase in S.patens, and the deletion of this gene resulted in a decrease in the resistance of S.patens to various stresses, which indicated that this gene was critical for the transformation of S.patens, so that it was reasonably assumed that this gene was also critical for the transformation of S.sphaeroides. Therefore, the invention performs batch molecular docking on the FDA batched small molecule database aiming at the DNA binding domain of the AbaA protein, and screens small molecule drugs capable of targeting the AbaA.

According to the invention, the three-dimensional structure of the AbaA protein is firstly predicted through bioinformatics, the DNA binding site of the AbaA gene is found, the predicted three-dimensional structure is subjected to quality scoring, the DNA binding site of the AbaA gene is selected from the structure with highest model quality, batch molecular docking is carried out in a small molecular database which is obtained by FDA and marketed, and 3 candidate small molecular drugs, namely dutasteride (Avodart), eltrombopag (Eltrombap) and Mefloquine, are finally selected from the small molecules screened out from 1615 small molecules through analysis of binding energy, efficacy, price and side effect, and then the next research is carried out.

In vitro antifungal Activity test of candidate Small molecule drugs against Sporotrichum globosum

The method comprises the following steps:

1. in an ultra clean bench, three sections of sporotrichosis globosa were streaked onto SDA medium with an inoculating loop and incubated for 4d at 25 ℃.

2. The spores of the cultured sporotrichosis globosa were rinsed with 1mL of sterile distilled water by pipetting, and the spore solution was transferred to 100mL of SDA medium and cultured at 28 ℃ for 4d at 150 r.

3. After microscopic examination, the cultured bacterial liquid is transferred to a 50mL centrifuge tube by a 5mL pipetting gun, and centrifuged at 4 ℃ and 10000r for 5min. The supernatant was discarded, 5mL of BHI medium was added, and the mixture was gently whipped and mixed with a pipette.

4. 5 bottles of 20mL BHI broth were used, labeled (1) control, (2) DMSO 100. Mu.L, (3) DMSO 200. Mu.L, (4) mefloquine 50. Mu.g/mL, and (5) mefloquine 100. Mu.g/mL, respectively.

5. 1mL of the bacterial liquid obtained in the step 3 is added into each bottle of BHI liquid culture medium.

6. 10mg of mefloquine was dissolved in 1mL of DMSO.

7. Add 100 μl DMSO to vial (2); 200. Mu.L of DMSO was added to the flask (3); adding 100 mu LDMSO and 100 mu L of the medicament prepared in the step 6 into a bottle (4) to obtain a final concentration of 50 mu g/mL; to the flask (5) were added 200. Mu.L of DMSO and 200. Mu.L of the agent prepared in step 6, at a final concentration of 100. Mu.g/mL.

8. Step 7 was incubated in a shaker at 37℃and 180r, observed and photographed with an inverted fluorescence microscope every 12h, and 10 positions were randomly photographed for each group.

9. All the above experiments were repeated three times and the results are shown in fig. 3.

The drugs dutasteride and eltrombopag were operated as above and the results are shown in figures 1 and 2.

The result of the antifungal activity of dutasteride on the outside of the spherical spore silk fungus body is shown in figure 1, and compared with a control group and a DMSO solvent control group, the dutasteride is not obviously changed from 12h to 96h of hyphae and spores after being added, thus indicating that the spherical spore silk fungus is insensitive to dutasteride.

As shown in figure 2, compared with the control group and DMSO solvent control group, the anti-fungus activity result of the eltrombopag on the outside of the spherical spore silk fungus body is shown in the figure, since the eltrombopag drug is orange in color, the mycelium of the spherical spore silk fungus is dyed to orange by the drug, and no other obvious change exists, so that the spherical spore silk fungus is insensitive to the eltrombopag.

The results of the extracellular antifungal activity of mefloquine against sporozoites are shown in fig. 3, which shows that mefloquine inhibited the growth and phase inversion of sporozoites from 12h compared to the control and DMSO solvent control.

Growth curve determination of mefloquine inhibiting sporotrichosis

1. 12 bottles of 20mL of BHI liquid medium after high temperature and high pressure sterilization were prepared, and each 3 bottles were a group, which was labeled Control, DMSO, itraconazole and mefloquine, respectively.

2. 100mL of the 5-day culture of 5-day sporotrichosis globosa at 25℃for 4-day culture was transferred to a 50mL sterile centrifuge tube at 8000r for 5min, the supernatant was discarded, the pellet was homogenized with BHI liquid medium sterilized at high temperature under high pressure, and 200. Mu.L of the bacterial suspension was added to each flask in step 1.

3. The preparation of a drug working solution was carried out by taking 10mg of Itraconazole and mefloquine in 1mL of DMSO, respectively, and using them as the drug working solution.

4. 100. Mu.L of the above-mentioned pharmaceutical working solution was added to the Itriconazole and mefloquine groups, respectively, to a final concentration of 50. Mu.g/mL.

5. Culturing at 37deg.C 150r, and measuring OD at 12h intervals using 96-well plate from 0h ₆₂₅ Absorbance below. Three replicates were taken per vial.

6. The data were processed and the results are shown in fig. 4.

The growth curve of Mefloquine inhibition for F.sphaeroides is shown in FIG. 4, and the results show that the addition of Mefloquine significantly inhibited the growth of F.sphaeroides compared to the control and solvent control.

MIC and MBC assays of mefloquine inhibition sporotrichosis

In order to study the minimum concentration of mefloquine to inhibit sporotrichosis globosus, minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of mefloquine were measured using a micro checkerboard dilution method, and the experimental design was referenced to CLSI and modified.

1. Preparing the medicine: 5mg of mefloquine is taken and dissolved in 1mL of DMSO, and the mixture is mixed by shaking for standby. 20 mu L of mother liquor is taken and prepared into 100 mu g/mL of mefloquine working solution in 980mL of high-temperature and high-pressure sterilized BHI liquid culture medium for standby.

2. Preparing a bacterial suspension: filtering and centrifuging the liquid of the spherical spore silk fungus cultured for 4d at 25 ℃, re-suspending spores by using a BHI liquid culture medium sterilized at high temperature and high pressure, and adjusting the concentration of spores to be 1 multiplied by 10 ⁵ 。

3. A sterile 96-well plate was placed in an ultra clean bench, 200. Mu.L of mefloquine working solution was added to well 1, 100. Mu.L of BHI liquid medium was added to each of wells 2-11, and 200. Mu.L of BHI liquid medium was added to well 12 as a negative control.

4. And (3) sucking 100 mu L of mefloquine working solution in the hole 1 to the hole 2, slowly whipping and mixing the mefloquine working solution in the hole 2 to the hole 3, slowly whipping and mixing the mefloquine working solution in the hole 2 until the hole 10 is reached, discarding 100 mu L sucked after uniformly mixing the hole 10, and finally adding 100 mu L of the bacterial liquid prepared in the step 2 into each hole. The above operation was repeated 3 rows as three sets of parallelism.

4. The 96-well plate was incubated in an incubator at 37℃for 72 hours.

5. At OD with enzyme labeling instrument ₆₃₀ Absorbance was measured and analyzed for Minimum Inhibitory Concentration (MIC).

6. 36 plates were prepared after autoclaving at high temperature, 40. Mu.L of the bacterial liquid in step 4 was spread, incubated at 37℃for 10 days, and the Minimum Bactericidal Concentration (MBC) was observed.

7. The data were processed and the results are shown in Table 1.

TABLE 1 MIC and MBC of mefloquine inhibition of F.sphaeroides

The MIC and MBC results of the Mefloquine inhibition on the spherical spore silk fungus are shown in Table 1, wherein the minimum inhibitory concentration MIC of the Mefloquine inhibition on the spherical spore silk fungus is 12.5 mug/mL, and the minimum bactericidal concentration MBC is 25 mug/mL.

Test of in vitro antifungal Activity of mefloquine against Sporothrix

To investigate whether mefloquine also has an inhibitory effect on other genotypes of sporotrichosis, a test was performed on the susceptibility of sporotrichosis to mefloquine. The method is the same as the step (one), except that the sporotrichosis globosa is replaced by the sporotrichosis lanuginosus.

The results of the anti-fungal activity of mefloquine on the outside of the Shenkea spore fungus are shown in FIG. 5, and compared with the control group and the DMSO solvent control group, mefloquine inhibits the growth of the Shenkea spore fungus from 12 hours, and the control group and the solvent control group have phase inversion phenomenon by 72 hours, while the group added with mefloquine has no phase inversion.

Determination of growth curve of Mefloquine inhibiting sporotrichosis suicola

The method is the same as the second step except that the sporotrichosis sphaericus is replaced by the sporotrichosis schneider. The results are shown in FIG. 6.

The growth curve of mefloquine for inhibiting the sporozoites is shown in fig. 6, and the result shows that the addition of mefloquine significantly inhibits the growth of the sporozoites compared with the control group.

MIC and MBC determination of Mefloquine inhibition on sporozoites

The method is the same as the step (III), except that the sporotrichosis globosa is replaced by the sporotrichosis lanuginosus. The results are shown in Table 2.

TABLE 2 MIC and MBC of Mefloquine inhibition of sporozoites

The MIC and MBC results of the Mefloquine inhibition on the sporozoites are shown in Table 2, the minimum inhibitory concentration MIC of the Mefloquine inhibition on the sporozoites is 6.25 mug/mL, and the minimum bactericidal concentration MBC is 25 mug/mL.

Seventh, animal level detection of therapeutic effect of small molecule drug mefloquine on sporotrichosis

1. Experimental animals: the experimental animals selected are SPF-grade KM mice, the day-old animals are three weeks, the male animals are about 20g in weight. Laboratory mice were purchased from Liaoning Changsheng Biotechnology Co., ltd, and the license number was SCXK 2020-0001. The experiment is strictly operated in compliance with the guidelines for the raising and administration of laboratory animals.

2. Experimental grouping:

the 32 KM mice were randomly divided into 4 groups of 8, and the grouping is shown in Table 3. The feeding conditions are as follows: the raising temperature is 24+/-2 ℃, the humidity is 44% -49%, and the ventilation times are 9-12 times/hour.

TABLE 3 Table 3

3. Immunosuppression of animals before and after vaccination

All groups of mice were given 1 injection of hydrocortisone at 7d intervals before inoculation after one week of acclimatization, 20 mg/kg/time, 1 injection every two days after 1 week, until molding was successful.

4. Preparation of bacterial suspension

Washing sporomyces lanuginosus spores with high-temperature high-pressure sterilized SDB culture medium, transferring to SDB at 25deg.C 150rp for culturing for 10 days, transferring bacterial film and mycelium pellet into 50mL centrifuge tube, placing on vortex oscillator, oscillating to disperse bacterial film and mycelium pellet, mixing, separating spores from mycelium into liquid culture medium, filtering to remove mycelium and impurities with cell filter screen, repeating the steps for 2 times, transferring filtrate into aseptic centrifuge tube, centrifuging at 8000r/min for 5min, discarding supernatant, adding 20mL sterile physiological saline, suspending with pipetting gun, centrifuging again, repeating the steps for 3 times, and adjusting bacterial suspension concentration to 1×10 with blood cell counting plate ⁸ The spores/mL can contain a small amount of hypha, but the ratio of the spores to the hypha is adjusted to be 100 as much as possible: 1.

5. experimental infection

All groups of mice were dehaired from the abdominal skin with a razor prior to injection of the spore wire suspension, and 0.1mL of spore wire suspension was injected intradermally with a 1mL syringe at the dehaired site on one side, approximately 1X 10 ⁷ And (3) spores.

6. Administration of drugs

Dosing solution preparation:

itraconazole group: taking 168mg of positive drug itraconazole in 7mL of 0.5% sodium carboxymethyl cellulose, and carrying out ultrasonic treatment for 20min; each mouse was perfused with 0.1mL/40g.

Mefloquine 3.8mg/kg group: taking 10.64mg of the medicine into 7mL of 0.5% sodium carboxymethyl cellulose, and carrying out ultrasonic treatment for 20min; each mouse was perfused with 0.1mL/40g.

Mefloquine 20mg/kg group: taking 56mg of the medicine in 7mL of 0.5% sodium carboxymethylcellulose, and carrying out ultrasonic treatment for 20min; each mouse was perfused with 0.1mL/40g.

Control group was 0.1mL of sodium carboxymethyl cellulose per lavage.

7. Skin damage HE, PAS staining

At the end of experimental administration, cervical vertebrae of all groups of mice are killed, soaked in 5% carbolic acid solution for 5 minutes, washed 3 times with sterile distilled water sterilized at high temperature and high pressure, placed on a sterile dissecting plate, fixed in 4% paraformaldehyde solution under sterile conditions after shearing skin damage, and the sample is sent to Liaoning Ji Jia organism for HE and PAS staining.

The results of the molding of the mice are shown in FIG. 7, the part of the intradermal injection bacterial liquid of the abdomen of the mice has skin lesions with different degrees, some of the mice grow out of the nodules, some of the nodules are grabbed by the stomach and flow out of pus, the pus is dipped by a cotton swab sterilized at high temperature, 40 mu L of the solution is diluted by sterile distilled water and evenly coated on SDA culture medium, and the culture is carried out for 4 days at 25 ℃, as shown in FIG. 8, the sporomyces lanuginosus grows out, and the sporomyces lanuginosus is verified by microscopic examination.

As can be seen from FIG. 7, the mice were judged to be sporotrichosis by analyzing the morphology of skin lesions and observing them under a microscope after the mice were injected with bacterial liquid intradermally, and the skin began to develop inflammation and nodules one week after the mice were injected with bacterial liquid intradermally. The sporomyces disease is most serious in the third week, at this time, after the administration of the medicine by gastric lavage, the surface nodules of the skin of the control group still exist, but the nodules of the positive medicine itraconazole group and mefloquine group become small and scab are improved, at this time, skin lesions are taken for HE and PAS staining, and the inflammatory factor condition and the fungal infection condition of the skin lesions are observed and analyzed.

As shown in fig. 9, the Mock group is a HE staining result of healthy skin, and it can be seen from the staining result that the control group has a large number of light blue punctate large nuclei, which are various inflammatory cells, in a circular shape or an oval shape, compared with the Mock group. Compared with the control group, the positive medicines itraconazole and mefloquine have lighter inflammatory infiltration than the control group.

Mouse PAS staining As shown in FIG. 10, mock group was PAS staining results of healthy skin, and from the staining results, it can be seen that the control group had a large number of mauve wire positive reactions, which were Shenkesporotrichosis, compared with Mock group. Compared with a control group, the purple red positive reaction of the positive drug itraconazole and the small molecular drug mefloquine is occasionally generated, but is far less than that of the control group, which shows that the antibacterial and bactericidal effects of the small molecular drug mefloquine screened by the invention are obvious.

Claims

1. Use of mefloquine alone or in combination with other drugs for the treatment of sporozoites for the preparation of a drug for the treatment of sporozoites.

2. The use according to claim 1, wherein the sporotrichosis is sporotrichosis caused by sporotrichosis sphaericus, sporotrichosis suicola and/or sporotrichosis brasiliensis.

3. The use according to claim 2, characterized in that said mefloquine is used for the preparation of a medicament for inhibiting the growth of candida globosa phases.

4. The use according to claim 3, wherein the mefloquine inhibits sporozoites with a MIC value of 12.5 μg/mL and an MBC value of 25 μg/mL.

5. The use according to claim 3, characterized in that the mefloquine inhibiting sporotrichosis dose is 3.8mg/kg to 20mg/kg.

6. The use according to claim 2, characterized in that said mefloquine is used for the preparation of a medicament for inhibiting the growth of candida shenkeatica phases.

7. The use according to claim 6, wherein the MIC value of mefloquine for inhibiting sporozoites is 6.25 μg/mL and MBC value is 25 μg/mL.

8. The use according to claim 6, wherein the dose of mefloquine to inhibit the sporozoites is 3.8mg/kg to 20mg/kg.