Application of fluconazole in preparation of medicine for treating autism spectrum disorder

Abstract

The invention discloses an application of fluconazole in preparation of a medicine for treating autism spectrum disorder. Animal experiment research shows that fluconazole can improve the social disorder performance of an SHANK3 KO mouse, the number of dendritic small ridges of the prefrontal cortex of the SHANK3 KO mouse can be remarkably increased, the level of synaptic scaffold protein in the prefrontal cortex of the SHANK3 KO mouse can be remarkably improved, and the fluconazole has repairing and remodeling effects on synapses. Cell experiment research shows that fluconazole can reverse neurotoxicity caused by glutamic acid, so that cell activity is recovered to a relatively high level, and the fluconazole has concentration dependence; glutamic acid can enable nerve cells to lose normal cellular morphology, tree mutation is short, cell bodies become large, fluconazole can reverse the phenomenon, and the cellular morphology of neurons is recovered. Therefore, fluconazole shows a relieving effect on the core symptom of ASD, and has application potential and prospect as an ASD treatment drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of fluconazole in the preparation of a medicament for treating autism spectrum disorder, and belongs to the field of specific therapeutic activity of compounds. BACKGROUND

[0002] Autism spectrum disorder (ASD) is a relatively serious neurodevelopmental disorder, the core symptoms of which are social impairment and language communication impairment, in addition to some peripheral symptoms such as anxiety and irritability. The cause of ASD is currently unknown, and there is currently no effective treatment. Behavior and education intervention is an important part of ASD treatment. In terms of drugs, the FDA has currently approved drugs for ASD, including risperidone and aripiprazole, which are used to alleviate the peripheral symptoms of ASD.

[0003] Fluconazole, with the chemical name of α- (2, 4-difluorophenyl) -α- (1H-1, 2, 4-triazole-1-ylmethyl) -1H-1, 2, 4-triazole-1-yl ethanol, has a molecular formula of C 13 H 12 F2N6O, and belongs to imidazole antifungal drugs. It has a broad antifungal spectrum and a long plasma elimination half-life, and is clinically used as an antifungal drug for fungal infectious diseases such as systemic candidiasis and cryptococcosis. There is currently no relevant report on the use of fluconazole for treating ASD. SUMMARY

[0004] In view of the above prior art, the present application provides a new use of fluconazole, i.e. the use of fluconazole in treating autism spectrum disorder.

[0005] The present application is achieved by the following technical solutions: The use of fluconazole in the preparation of a medicament for treating autism spectrum disorder.

[0006] Further, the use of fluconazole alone as an effective ingredient in the preparation of a medicament for treating autism spectrum disorder.

[0007] The use of fluconazole in the preparation of a medicament for treating neurological diseases.

[0008] The use of fluconazole in the preparation of a medicament for treating mental diseases.

[0009] The animal experiment researches of the present application show that fluconazole can improve the social impairment performance of SHANK3 KO mice, can significantly increase the number of dendritic spines of the prefrontal cortex of SHANK3 KO mice, can significantly improve the level of synaptic scaffold proteins in the prefrontal cortex of SHANK3 KO mice, and has a repairing and remodeling effect on synapses. The cell experiment researches of the present application show that fluconazole can reverse the neurotoxicity caused by glutamate, so that the cell activity is restored to a higher level, and has a concentration-dependent effect; glutamate can make nerve cells lose normal cell morphology, dendrites become shorter, and cell bodies become larger, while fluconazole can reverse this phenomenon and restore the morphology of neuron cells. Therefore, fluconazole shows a relieving effect on the core symptoms of ASD, has application potential and prospect as an ASD treatment drug, and has application potential for treating neurological / psychiatric diseases (neurological diseases refer to a series of diseases caused by problems in the nervous system, nerve cells, brain tissue and other solid organs, and psychiatric diseases refer to problems in the thinking and spirit of patients). BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : Results of three-chamber social experiment (social activity), wherein, WT refers to a wild type mouse control group, Shank3 - / - : SHANK3 KO mouse control group, Shank3 - / - +Flu refers to a fluconazole group (i.e. SHANK3 KO mouse experimental group). The vertical coordinate is social activity, and the unit is second (s). “****” indicates that the difference is extremely significant (p<0.0001).

[0011] Figure 2 : Results of three-chamber social experiment (social novelty), wherein, WT refers to a wild type mouse control group, Shank3 - / - : SHANK3 KO mouse control group, Shank3 - / - +Flu refers to a fluconazole group (i.e. SHANK3 KO mouse experimental group). The vertical coordinate is social novelty, and the unit is second (s). “***” indicates that the difference is extremely significant (p<0.001), and “****” indicates that the difference is extremely significant (p<0.0001).

[0012] Figure 3 : Observation results of Golgi staining of the prefrontal cortex of SHANK3 KO mice, wherein, the upper graph is a model control group, and the lower graph is an experimental group.

[0013] Figure 4Statistical results of dendritic crest density in the prefrontal cortex of SHANK3 KO mice. The vertical axis represents the number of dendritic crests per 50 μm. "*" indicates a significant difference (p < 0.05).

[0014] Figure 5 Typical protein imprints of synaptic scaffold-related proteins.

[0015] Figure 6 The results of relative grayscale analysis of the proteoblot values ​​of Homer1 b / c, a protein associated with the synaptic scaffold, are shown below. WT refers to the wild-type mouse control group, SHANK3 KO refers to the SHANK3 KO mouse control group, and SHANK3KO+Flu refers to the fluconazole group (i.e., the SHANK3 KO mouse experimental group). "**" indicates a significant difference (p < 0.01), and "***" indicates an extremely significant difference (p < 0.001).

[0016] Figure 7 The results of relative grayscale analysis of the protein blot of PSD95, a protein associated with the synaptic scaffold, are shown below. WT refers to the wild-type mouse control group, SHANK3 KO refers to the SHANK3 KO mouse control group, and SHANK3KO+Flu refers to the fluconazole group (i.e., the SHANK3 KO mouse experimental group). "*" indicates a significant difference (p < 0.05).

[0017] Figure 8 The results of relative grayscale analysis of the protein blot of synaptic scaffold-related protein NR2B are shown below. WT refers to the wild-type mouse control group, SHANK3 KO refers to the SHANK3 KO mouse control group, and SHANK3KO+Flu refers to the fluconazole group (i.e., the SHANK3 KO mouse experimental group). "*" indicates a significant difference (p < 0.05), and "***" indicates an extremely significant difference (p < 0.001).

[0018] Figure 9 The results of CCK8 cell viability assay are shown, where "*" indicates a significant difference (p < 0.05) and "****" indicates an extremely significant difference (p < 0.0001).

[0019] Figure 10 The images show the morphology of nerve cells. The left image represents the control group, the middle image represents the experimental group, and the right image represents the treatment group. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0021] The instruments, reagents, materials involved in the following examples, if not specifically stated, are conventional instruments, reagents, materials already existing in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, if not specifically stated, are conventional experimental methods, detection methods already existing in the prior art.

[0022] Experiment 1 Therapeutic effect of fluconazole on ASD model mice SHANK3 KO mice (this strain of mice cannot express SHANK3 structural protein; SHANK3 structural protein is an important protein for the structure of neuronal synapses, and the absence of this protein will cause the mice to exhibit a series of neurological symptoms) are a classic and reliable ASD model mouse strain, which exhibits core symptoms of ASD including reduced social behavior and increased stereotyped behavior. Therefore, SHANK3 KO mice were administered fluconazole orally in this experiment, and behavioral experiments were used to assess whether ASD-like performance was improved.

[0023] (I) Experimental methods (1) Grouping and intervention methods Experimental group: SHANK3 KO mice (purchased from Jiangsu Jucu Yaoke Biotechnology Co., Ltd.) (6-week-old male mice, about 18-20 g) were used as the experimental group, a total of 8. Oral fluconazole (fluconazole capsules purchased from Pfizer Pharmaceuticals Co., Ltd., with the trade name "Dafukang", each 150 mg): dissolve the drug powder in the drinking water for mice, administer freely, the administration concentration is 0.5 mg / ml, and the administration lasts for 14 days. Normal feeding and daily management during the experiment.

[0024] Model control group: SHANK3 KO mice (6-week-old male mice, about 18-20 g) were used as the model control group, a total of 8. Oral drinking water was given, and the mice freely drank water for 14 days. Normal feeding and daily management during the experiment.

[0025] Negative control group: normal healthy wild type C57BL / 6 mice (purchased from Jiangsu Jucu Yaoke Biotechnology Co., Ltd.) (6-week-old male mice, about 18-20 g) were used as the negative control group, a total of 8. Oral drinking water was given, and the mice freely drank water for 14 days. Normal feeding and daily management during the experiment.

[0026] (2) Three-chamber social interaction experiment After 14 days of feeding, three groups of mice were subjected to three-chamber social experiment. Three days before the three-chamber social experiment, the experimenter stroked each mouse to be tested for 1 minute every day to ensure that the experimental mice did not show stress during the experiment. The experimental device was a 40 cm x 60 cm x 40 cm (length x width x height) rectangular transparent acrylic box, whose long side was separated into three small boxes by two transparent plates, with a passage connecting the three boxes, and each side placed a metal cage of the same size, which could accommodate one mouse. Before the experiment, the mice were placed in the behavior test room for 1 minute of adaptation. In the first stage of the experiment, one inanimate object (a block, referred to as Object-O) and one 6-week-old male stranger mouse (referred to as Stranger 1, S1) were placed in the two metal cages, respectively, and then the test mouse was allowed to freely move in the box for 5 minutes. After 5 minutes, the test mouse was immediately photographed and the number of times and duration of contact with the left and right metal cages in the next 10 minutes were recorded, i.e. the social activity of the mouse. In the second stage of the experiment, Object-O in the metal cage was replaced with another 6-week-old male stranger mouse (referred to as Stranger 2, S2), and then the test mouse was photographed and the number of times and duration of contact with the left and right metal cages in the next 10 minutes were recorded, i.e. social novelty.

[0027] (3) After the three-chamber social experiment, the mice were sacrificed, and the prefrontal cortex was isolated.

[0028] (4) The prefrontal cortex was homogenized in RIPA lysis buffer, then centrifuged and the supernatant was mixed with 5x SDS-PAGE Loading Buffer to prepare a protein sample. Then 10% SDS-PAGE gel was used for gel electrophoresis to separate proteins and transfer to PVDF membrane. After blocking with 5% skim milk powder for 1 hour, the corresponding antibody for detecting the target protein (Homer1 b / c, PSD95, NR2B) was incubated at 4°C for 16 hours. Then the corresponding species-specific secondary antibody labeled with HRP was incubated at room temperature for 1 hour, developed with ECL developing solution and photographed, and the band gray value was analyzed by ImageJ software. β-actin was used as the internal reference protein.

[0029] (5) Data processing Each data was measured three times and the average value was taken. Statistical data were subjected to p test.

[0030] (II) Experimental results (1) In the three-chamber social experiment, normal healthy mice showed stronger interest in new stranger mice, i.e. the social interest in Stranger-S was higher than that in Object-O; the interest in new stranger mice Stranger2-S2 was higher than that in known mice Stranger1-S1.

[0031] Results of the three-chamber social interaction experiment are shown in Figure 1 , Figure 2 . It can be seen that healthy mice (negative control group) have much higher social desire for Stranger-S than for Object-O, and have much higher social desire for new stranger mouse Stranger2-S2 than for known mouse Stranger1-S1. SHANK3 KO mice (model control group) have impaired social behavior, showing social desire for Stranger-S that is basically the same as for Object-O, and also showing lower social desire for new stranger mouse Stranger2-S2. Mice in the experimental group have much higher social desire for Stranger-S than for Object-O, and have much higher social desire for new stranger mouse Stranger2-S2 than for known mouse Stranger1-S1, showing social desire that is comparable to or even stronger than that of healthy mice. This shows that treatment with fluconazole can improve the social impairment performance of SHANK3 KO mice.

[0032] (2) The results of observation of Golgi staining of the prefrontal cortex of SHANK3 KO mice are shown in Figure 3 , and the statistical results of the density of dendritic spines in the prefrontal cortex of SHANK3 KO mice are shown in Figure 4 . SHANK3 KO mice (model control group) have lower dendritic spine density due to the absence of the classic synaptic scaffold protein SHANK3. The number of dendritic spines in the prefrontal cortex of SHANK3 KO mice (experimental group) that have received fluconazole treatment is significantly increased. This shows that fluconazole can promote synaptic repair and remodeling in SHANK3 KO mice, and thus improve their social behavior.

[0033] (3) Representative Western blot images of synaptic scaffold-related proteins are shown in Figure 5 , the relative gray value analysis results of synaptic scaffold-related protein Homer1 b / c are shown in Figure 6 , the relative gray value analysis results of synaptic scaffold-related protein PSD95 are shown in Figure 7 , and the relative gray value analysis results of synaptic scaffold-related protein NR2B are shown in Figure 8The SHANK3 KO mice (model control group) showed synaptic density disruption due to the loss of SHANK3 protein, and also showed a significant decrease in the levels of synaptic scaffold-related proteins such as Homer1, PSD95, etc. compared with wild-type mice, which is involved in the mechanism of the occurrence of their ASD-like performance. The levels of Homer1, PSD95, and NR2B in the experimental group of mice treated with fluconazole were restored. This indicates that fluconazole treatment can significantly improve the levels of synaptic scaffold proteins in the prefrontal cortex of SHANK3 KO mice, and again suggests the role of fluconazole in synaptic repair and remodeling from another perspective.

[0034] Experiment 2 Protective effect of fluconazole on glutamate-induced neurotoxicity (I) In this experiment, the neurotoxic effect of glutamate (L-Glu) on human neuroblastoma cell line SH-SY5Y was used to simulate the nerve damage of ASD patients, and CCK8 cell viability detection (used to evaluate cell activity) was used to evaluate whether fluconazole had a protective effect on glutamate-induced neurotoxicity.

[0035] The human neuroblastoma cell line SH-SY5Y (a human neuroblastoma cell line commonly used in neuro-oncology and neurobiology research, purchased from Shanghai Kuisai Biotechnology Co., Ltd.) was plated into a 96-well plate at 1 x 10 4 Each group had 8 wells, and the cells were incubated at 37°C for one day to rest. Then, fluconazole (fluconazole capsules were purchased from Pfizer Pharmaceuticals Co., Ltd., with the trade name "Dafukang", each containing 150 mg; when used, the drug powder was added) was added to the culture supernatant of experimental groups 2, 3, and 4 to make the concentrations 0.5, 1.0, and 5.0 μM, respectively, and pretreated for 24 hours. After the treatment was completed, all the culture supernatants were washed away, and DMEM complete medium was added to the blank group, and DMEM complete medium containing L-Glu at a concentration of 60 mM was added to experimental groups 1, 2, 3, and 4, respectively, for 24 hours. After the treatment was completed, all the culture supernatants were washed away, and CCK8 was added for cell viability detection. The absorbance value (OD value) was measured at 450 nm after 1 hour of reaction, and the cell activity was calculated.

[0036] The results of CCK8 cell viability detection are shown in Table 2. Figure 9Glutamate can induce excitotoxicity in nerve cells, causing the cell viability of nerve cells to decrease. The addition of glutamate to normal nerve cells can cause the cell viability of nerve cells to decrease significantly compared with the blank group. On this basis, the use of fluconazole for pretreatment can reverse the nerve toxicity caused by glutamate, restore the cell viability to a higher level, and this phenomenon is concentration-dependent, and with the increase of the concentration of fluconazole, the neuroprotective effect is stronger. Since the system is relatively simple, only containing cells, culture medium and drugs, it can prove that fluconazole directly acts on the cells themselves rather than indirectly.

[0037] (II) In this experiment, glutamate was used to induce nerve toxicity in mouse neuroblastoma cell line N2a, which simulates the nerve cell damage of ASD patients, to evaluate whether fluconazole has a protective effect on glutamate-induced nerve toxicity.

[0038] The mouse neuroblastoma cell line N2a (a mouse neuroblastoma cell line widely used in neurobiology, oncology and drug screening research, purchased from the American ATCC Biological Standard Resource Center) cells were plated into a confocal dish, incubated at 37°C for 1 day to rest, and then replaced with serum-free DMEM medium to replace the original DMEM complete medium. Incubate with serum-free DMEM medium for 24 hours to induce N2a cell differentiation (Note: N2a cell differentiation does not require other intervention, only needs to be cultured with serum-free DMEM medium), observe the N2a cell dendritic structure under white light microscope, which is the induction success. Then the induced N2a cells were set as the control group, and another group of experimental group and one group of treatment group were set. Add fluconazole (Dafukang powder) to the culture supernatant of the treatment group to make its concentration 5.0 μM, and pretreat for 24 hours. After the treatment, the culture supernatant of the experimental group (without fluconazole pretreatment) and the treatment group (with fluconazole pretreatment) was replaced with DMEM complete medium containing 60 mM glutamate, and treated for 24 hours to induce cell toxicity. Then the N2a cells in the three groups were subjected to immunofluorescence staining, and the cell morphology was observed, as shown in Figure 10 , red is the neuron maturation marker MAP2, and blue is the cell nucleus DAPI. It can be seen that the N2a cells induced by serum-free medium grow out obvious dendrites, and the cell body is small (see Figure 10 left figure), while the N2a cells lose their normal differentiated cell morphology after glutamate-induced excitotoxicity, the dendrites become shorter, and the cell body becomes larger (see Figure 10 middle figure). Fluconazole can also reverse this phenomenon and restore the morphology of N2a cells (see Figure 10 right figure).

[0039] The foregoing examples are provided to give the skilled person in the art complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications apparent to those skilled in the art are to be within the scope of the claims.

Claims

1. Use of fluconazole in the manufacture of a medicament for the treatment of autism spectrum disorders.

2. Use of fluconazole alone as an active ingredient in the manufacture of a medicament for the treatment of autism spectrum disorders.

3. Use of fluconazole in the manufacture of a medicament for the treatment of neurological diseases.

4. Use of fluconazole in the manufacture of a medicament for the treatment of psychiatric diseases.

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