Application of Trolox in preparation of medicine for treating toxicity caused by valproic acid

Abstract

The invention discloses application of Trolox in preparation of a medicine for treating toxicity caused by valproic acid, and belongs to the technical field of biological medicine. The invention discovers for the first time that Trolox has a new medicine application of preventing and / or treating developmental toxicity caused by valproic acid or redox level imbalance caused by valproic acid; furthermore, animal experiments prove that Trolox can relieve zebra fish development malformation caused by valproic acid and also can relieve zebra fish redox level imbalance caused by valproic acid; this provides a new solution for the prevention and / or treatment of developmental toxicity caused by valproic acid or redox level imbalance caused by valproic acid.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Trolox in the preparation of drugs for treating toxicity caused by valproic acid. Background Technology

[0002] Valproic acid (VPA) is a short-chain fatty acid and a broad-spectrum antiepileptic drug, primarily used for epilepsy, but also for mental illnesses such as bipolar disorder. Prenatal exposure to VPA increases the risk of birth defects, autism, ADHD, and neural tube defects in children, but its specific mechanism of action is currently unclear. Studies show that VPA induces redox imbalances in mammals with autism, and there are currently no drugs that can prevent VPA-induced developmental abnormalities and redox damage.

[0003] Trolox is a water-soluble vitamin E analog, chemically named 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. It is a common antioxidant used in biological or biochemical applications to reduce oxidative stress or damage.

[0004] However, there are currently no research reports on Trolox in the prevention and / or treatment of developmental toxicity and redox imbalance caused by VPA. Summary of the Invention

[0005] The purpose of this invention is to provide the use of Trolox in the preparation of medicaments for treating toxicity caused by valproic acid. This addresses the lack of effective methods in the prior art to prevent and / or treat developmental toxicity and redox imbalance caused by VPA.

[0006] In a first aspect, the present invention provides the use of Trolox in the preparation of medicaments for the prevention and / or treatment of toxicity caused by valproic acid, including developmental toxicity.

[0007] In a second aspect, the present invention provides the use of Trolox in the preparation of medicaments for the prevention and / or treatment of redox imbalances caused by valproic acid.

[0008] In this invention, the inventors have discovered for the first time that Trolox has a novel pharmaceutical use for preventing and / or treating developmental toxicity caused by valproic acid or redox imbalance caused by valproic acid; furthermore, Trolox can alleviate developmental malformations caused by valproic acid, and at the same time alleviate redox imbalance caused by valproic acid; this provides a new solution for the prevention and / or treatment of developmental toxicity caused by valproic acid or redox imbalance caused by valproic acid.

[0009] In some implementations, developmental toxicity includes developmental abnormalities; wherein developmental abnormalities include at least one of pericardial edema, heart rate instability, decreased embryo survival rate, decreased embryo hatching rate, and head malformation.

[0010] It is understood that developmental malformations can be conventional malformations in the prior art, and the present invention does not limit them. Furthermore, in the present invention, developmental malformations preferably include at least one of pericardial edema, heart rate imbalance, decreased embryo survival rate, decreased embryo hatching rate, and head malformation.

[0011] In some implementations, head deformity includes at least one of reduced head length, reduced jaw length, and reduced keratin cartilage length.

[0012] It is understood that the head deformity can be a conventional deformity in the prior art, and the present invention does not limit it. Furthermore, in the present invention, the head deformity preferably includes at least one of the following: reduced head length, reduced jaw length, and reduced horny cartilage length.

[0013] In some implementations, the redox imbalance caused by valproic acid includes an intraembryonic redox imbalance; wherein the intraembryonic redox imbalance includes at least one of a decrease in reactive oxygen species levels, an increase in antioxidant capacity, and an increase in nitrite levels.

[0014] It is understood that the redox imbalance in the embryo can be a conventional redox damage as described in the prior art, and the present invention does not limit this. Furthermore, in the present invention, the redox imbalance in the embryo preferably includes at least one of a decrease in reactive oxygen species levels, an increase in antioxidant capacity, and an increase in nitrite levels.

[0015] In some implementations, the effective concentration of Trolox is 2-3 μM, for example, it can be 2 μM, 2.2 μM, 2.5 μM, 2.7 μM, 3 μM or other values ​​within this range.

[0016] In some embodiments, the concentration of valproic acid in the toxicity caused by valproic acid and / or the redox imbalance caused by valproic acid is 1-40 μM, for example, it can be 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM or other values ​​within this range.

[0017] In a third aspect, the present invention provides a pharmaceutical composition for preventing and / or treating toxicity caused by valproic acid or redox imbalance caused by valproic acid, the pharmaceutical composition comprising Trolox.

[0018] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0019] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0020] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0021] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0022] The pharmaceutical compositions provided by this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0023] The beneficial effects of this invention are as follows: Unlike the prior art, this invention is the first to discover that Trolox has a novel pharmaceutical use for preventing and / or treating developmental toxicity or redox imbalance caused by valproic acid; furthermore, animal experiments have demonstrated that Trolox can alleviate developmental malformations in zebrafish caused by valproic acid, and also alleviate redox imbalance in zebrafish caused by valproic acid; this provides a new solution for the prevention and / or treatment of developmental toxicity or redox imbalance caused by valproic acid. Attached Figure Description

[0024] Figure 1 The following are statistical results of zebrafish embryo survival and hatching rates after VPA exposure in Example 1 of this invention; wherein, (A) are the survival rates of zebrafish embryos after exposure to different concentrations of VPA (0μM, 5μM, 10μM, 15μM, 20μM, 25μM, 30μM, 35μM and 40μM) for 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h; (B) are the hatching rates of zebrafish embryos after exposure to different concentrations of VPA (0μM, 5μM, 10μM, 15μM, 20μM, 25μM, 30μM, 35μM and 40μM) for 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h; and (C) is the median lethal concentration (LC50) of VPA at the end of exposure (120h) for zebrafish embryos. 50 The results are expressed as Mean ± SD; Figure 2 The images show the morphological changes in zebrafish at the VPA exposure endpoint (120 h) in Example 1 of this invention. (A) shows a microscopic side view of zebrafish larvae at the exposure endpoint (120 h) with different concentrations of VPA (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM). The red arrows in the magnified side view of the zebrafish larvae indicate the pericardial length. (B) shows the statistical results of the body length of zebrafish larvae after treatment with different concentrations of VPA. (C) shows the statistical results of the pericardial length of zebrafish after treatment with different concentrations of VPA. Data are expressed as Mean ± SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference; Figure 3The results show the head development changes of zebrafish at the VPA exposure endpoint (120h) in Example 1 of this invention; where (A) is a top view under a microscope after staining of zebrafish juveniles exposed to different concentrations of VPA (0μM, 5μM, 10μM, 20μM and 30μM), and the length of the mandible and the length of the horny cartilage in the skull of the zebrafish juveniles after staining are indicated by red arrows; (B) is the statistical results of LJL data after staining of zebrafish juveniles; (C) is the statistical results of CCL data after staining of zebrafish juveniles, n=8; data are expressed as Mean±SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference; Figure 4 This document presents the detection results of reactive oxygen species (ROS) levels, total antioxidant capacity (FRAP), nitrite, and nitrate levels in zebrafish juveniles exposed to different concentrations of VPA in Example 1 of this invention. (A)-(B) show representative ROS fluorescence staining images and quantitative analysis results of fluorescence intensity in zebrafish juveniles treated with different concentrations of VPA (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 35 μM) for 72 h, respectively. (C) shows the detection results of total antioxidant capacity in zebrafish after exposure to different concentrations of VPA during the mitotic phase for 120 h. Each group has n=8 data. Data are expressed as Mean ± SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference; Figure 5 Statistical results of pericardial length data of zebrafish exposed to different concentrations 120 h after Trolox pretreatment of zebrafish embryos in Example 2 of this invention; n=8 for each group; data are expressed as Mean±SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference; Figure 6This is the result of heart rate changes induced by VPA after Trolox pretreatment of zebrafish embryos in Example 2 of the present invention; wherein, (A) is the morphological changes of zebrafish in different concentration exposure groups (Control, 20 μM VPA, 20 μM VPA + 2 μM Trolox, 20 μM VPA + 3 μM Trolox) at 120 h; (B) is the statistical result of heart rate data of zebrafish in different concentration exposure groups at 120 h; n=8 for each group; data are expressed as Mean±SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference; Figure 7 This document presents the DCF-DA staining, total antioxidant capacity, nitrite, and nitrate levels of zebrafish exposed to different concentrations of Trolox after pretreatment of zebrafish embryos in Example 2 of this invention. (A)-(B) show representative ROS fluorescence staining images and quantitative analysis results of fluorescence intensity in zebrafish larvae at 72 h for different exposure groups (Control, 20 μM VPA, 20 μM VPA + 2 μM Trolox, 20 μM VPA + 3 μM Trolox), with n=8 for each group. (C), (D), and (E) show the changes in total antioxidant capacity, nitrite, and nitrate levels in zebrafish exposed to different concentrations at 120 h, with n=6 for each group. Data are expressed as Mean ± SD. p <0.05, p <0.01, p <0.0001 indicates a significant difference. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0027] Example 1: Developmental toxicity and redox imbalance in zebrafish caused by VPA exposure Referring to the paper "Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA): An aquatic model forautism" published by Chen et al., a developmental toxicity and redox imbalance model in zebrafish was constructed using VPA exposure. The exposure concentrations of VPA were determined to be 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, and 40 μM. The VPA stock solution (20 mM) was prepared by dissolving valproic acid standard powder in ddH2O and stored at -20°C protected from light. The VPA working solution was prepared by diluting the stock solution before experimental use. Drug exposure was selected during the cell division phase, specifically starting at 8 h and completing drug administration for all experimental groups before 10 h. Healthy embryos were selected under an inverted microscope at 6 h. During the experiment, zebrafish embryos were continuously exposed to ddH2O and different concentrations of VPA working solution, with the exposure endpoint at 120 h. Drug exposure was performed in six-well plates, with each well containing 5 mL of VPA working solution and 20 embryos. The working solution was changed every 24 hours and dead embryos were removed to maintain water quality and working solution concentration.

[0028] First, the effects of VPA exposure on the survival rate and hatching rate of zebrafish embryos and juveniles were investigated.

[0029] Specifically, to study the developmental abnormalities of zebrafish embryos and larvae caused by VPA exposure, the survival rate and hatching rate of zebrafish after VPA exposure were statistically analyzed every 12 hours using the method described above. Zebrafish embryos coagulated to a milky white color after death, appearing black under a microscope. Healthy embryos began to hatch gradually after 48 hours, indicated by the successful breakout of the embryonic membrane by the head or tail of the larvae. After death, the zebrafish larvae coagulated to a white color, and the cessation of heartbeat could be observed under a microscope. Using this standard, the number of surviving and hatching zebrafish embryos was counted at 12, 24, 36, 48, 60, 72, 84, and 96 hours after drug exposure. The survival rate (survival rate = current surviving number / total number of embryos × 100%) and hatching rate (hatching rate = number of hatched embryos / current surviving number × 100%) were calculated using the following formulas. The results are as follows: Figure 1 As shown.

[0030] from Figure 1As can be seen, compared with the control group, 5 μM VPA and 10 μM VPA had no significant effect on the survival rate of zebrafish throughout the entire process; however, starting from 72 h, the survival rate of the 15 μM VPA exposure group (64.17 ± 12.01%) changed significantly compared with the control group (86.43 ± 3.78%). p =0.0348); starting from 84h, 15μM ( p =0.0146), 20μM ( p =0.0057), 25μM ( p =0.0011), 30μM ( p <0.0001), 35μM ( p <0.0001) and 40μM ( p Compared with the control group, the survival rate of zebrafish embryos in the VPA exposure group (<0.0001) showed a significant change (<0.0001); and the survival rate of zebrafish embryos was concentration-dependent, decreasing with increasing VPA concentration. Figure 1 A); from the median lethal concentration (LC50) 50 As shown in the figure, the VPA concentration reaches the median lethal concentration (LC50) at 24.8 μM. 50 =24.8μM ( Figure 1 C); As can be seen from the hatching rate results, the zebrafish in the control group began to hatch at 36 hours; starting at 36 hours, 5 μM VPA had already affected the hatching rate of zebrafish embryos, and the difference was significant. p =0.0002), and the p values ​​of other VPA exposure groups and the control group were all less than 0.0001; by 72h, almost all zebrafish embryos had hatched. After 72h, unhatched zebrafish embryos were observed under an inverted microscope, and it was found that all unhatched zebrafish embryos had stopped beating after 72h. Figure 1 B); The above results indicate that exposure to VPA reduces the survival and hatching rate of zebrafish and causes heart rate imbalance.

[0031] Then, the effects of VPA exposure on zebrafish morphological development were investigated.

[0032] Specifically, to evaluate the effects of VPA exposure during the mitotic phase on zebrafish development, the body length and pericardial length of juvenile zebrafish were measured at the drug administration endpoint (120 h) using the method described above. Eight juveniles were randomly selected from each group and anesthetized with 0.004% tricaine before being photographed. Side-view images were taken under an inverted microscope at the same magnification, and ImageJ software was used to analyze deformities such as pericardial edema and changes in body length. The results are as follows: Figure 2 As shown.

[0033] from Figure 2 As can be seen, VPA can cause pericardial edema in juvenile zebrafish, and the pericardial edema becomes more and more severe as the VPA concentration increases. Figure 2 A); As can be seen from the statistical chart of zebrafish juveniles, VPA exposure had no significant effect on the body length development of zebrafish. Figure 2 B); Statistical analysis of zebrafish pericardial length shows that, compared with the control group (105.4±16.41 μm), except for the 10 μM exposure group (125.8±24.48 μm), all other exposure groups showed significant differences, reaching a maximum at 35 μM (194.8±24.37 μm). Figure 2 C). The above results indicate that VPA exposure causes pericardial edema in zebrafish from embryonic development to juvenile stage, exhibiting certain developmental toxicity, but has no effect on the body length of zebrafish.

[0034] Next, the effects of VPA exposure on zebrafish head development were investigated.

[0035] Specifically, to evaluate the effect of VPA exposure during mitotic phase on zebrafish head development, the above method was used to perform whole-body Alcian blue staining on zebrafish at the drug administration endpoint (120 h). Staining the skull blue allowed for clear visualization of the skull and head development. After staining, the zebrafish were placed on slides containing glycerol, and top-view images were taken using an inverted microscope. ImageJ software was used to analyze the lower jaw length (LJL) and ceratohyal cartilage length (CCL) of the zebrafish. The results are as follows: Figure 3 As shown.

[0036] from Figure 3 As can be seen, with the increase of VPA exposure concentration, the heads of juvenile zebrafish gradually become smaller, and the deformities become more and more serious. Figure 3 A); As can be seen from the statistical charts of LJL and CCL data, with the increase of VPA exposure concentration, the lower jaw length of zebrafish juveniles at the exposure endpoint ( Figure 3 B) and the length of the keratin cartilage ( Figure 3 C) The length of the lower jaw of zebrafish was decreasing; among them, a significant difference was observed starting from the 5 μM VPA exposure group compared to the control group (85.85 ± 9.368 μm). p =0.0278), and the length of the lower jaw tends to decrease with increasing VPA concentration; the length of the keratin cartilage in juvenile zebrafish is roughly the same. These results indicate that VPA exposure can cause head deformities in zebrafish.

[0037] Finally, the effects of VPA exposure on redox imbalance in zebrafish were investigated.

[0038] Specifically, the above method was used to detect the levels of reactive oxygen species (ROS) and total antioxidant capacity (FRAP) in zebrafish juveniles exposed to different concentrations of VPA (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 35 μM). The results are as follows: Figure 4 As shown.

[0039] The methods for determining reactive oxygen species levels and total antioxidant capacity are as follows: 1) Measurement of reactive oxygen species levels The levels of ROS in zebrafish juveniles exposed to different concentrations of VPA (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 35 μM) were detected using the 2-(3,6-diacetoxy-2,7-dichloro-9H-oxanthracene-9-yl)benzoic acid (DCF-DA) method. DCF-DA is a widely used fluorescent ROS probe with cell membrane permeability. DCF-DA itself is not fluorescent, but when it enters the cell, it can be decomposed by cellular lipases to produce 2',7'-dichlorodihydrofluorescein (DCFH). DCFH cannot freely penetrate the cell membrane, and thus DCFH is oxidized by ROS in the cell to the green fluorescent substance 2',7'-dichlorofluorescein (DCF), and its fluorescence intensity is proportional to the intracellular ROS level. Preparation of DCF-DA stock solution (10 mg / mL): Weigh 0.1 g of DCF-DA powder and dissolve it in 10 mL of DMSO. Mix thoroughly in the dark, dispense into individual containers, and store at -20°C for immediate use. Before use, dilute the stock solution 1000 times to obtain a 10 μg / mL DCF-DA working solution. Experiments were conducted on zebrafish exposed for 72 h. Eight juvenile zebrafish from each group were washed three times with ddH2O, then 5 mL of DCF-DA working solution (10 μg / mL) was added and incubated at 28°C in the dark for 30 min. After incubation, the fish were washed three times with ddH2O, anesthetized with 0.004% tricaine, and photographed under a stereofluorescence microscope. The fluorescence intensity of the images was analyzed using ImageJ software.

[0040] 2) Determination of total antioxidant capacity The antioxidant capacity levels in zebrafish exposed to different concentrations of VPA (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, and 35 μM) were determined using the Ferric Reducing Ability of Plasma (FRAP) method. This method utilizes the principle that antioxidants can react with Fe under acidic conditions. 3+ The -TPTZ reaction reduces it to blue Fe. 2+The -TPTZ complex exhibits maximum absorption at 593 nm, thus the total antioxidant capacity of the analyte can be indicated by the absorbance measured at 593 nm. The specific experimental steps are as follows: Solution preparation: Acetic acid buffer: Weigh 0.155 g of sodium acetate trihydrate and dissolve it in 40 mL of ddH2O. Add 0.8 mL of glacial acetic acid, adjust the pH to 4.5 with 0.1 M NaOH solution, and bring the volume to 50 mL with ddH2O. Tripyridyltriazine (TPTZ) solution: Weigh 0.0312 g of TPTZ and dissolve it in 10 mL of 40 mM HCl. 20 mM ferric chloride (FeCl3) solution: Weigh 0.054 g of ferric chloride hexahydrate and dissolve it in 10 mL of ddH2O. FRAP working solution: Mix 10 mL of acetate buffer + 1 mL of TPTZ solution + 1 mL of FeCl3 solution thoroughly and use within 3 hours.

[0041] Sample Preparation: At the dosing endpoint (120 h), six parallel samples were prepared for each concentration, with 10-20 zebrafish per sample. The zebrafish juveniles were washed three times with ddH2O. Homogenizing the juveniles on ice using a glass homogenizer at a ratio of 1 juvenile: 10 μL ddH2O for 1 min, followed by centrifugation at 4℃ and 14000 rpm for 5 min, followed by collection of the supernatant into a new 1.5 mL centrifuge tube. Part of the supernatant was used for protein concentration determination using the BCA method, and the remaining supernatant was used for FRAP level detection. Standard Curve: A standard curve was constructed using FeSO4·7H2O standards at different concentrations (30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, and 300 μM).

[0042] FRAP level detection: Mix 60 μL of supernatant with 300 μL of FRAP working solution thoroughly and incubate at room temperature in the dark for 5 min. After incubation, take a reading at 593 nm. Substitute the reading into the standard curve to calculate the concentration. The FRAP result is expressed as sample FRAP concentration / sample protein concentration.

[0043] from Figure 4 As can be seen, the fluorescence intensity is mainly concentrated in the head and heart of zebrafish juveniles, while the fluorescence intensity in the trunk and tail is weaker. Furthermore, the fluorescence intensity of ROS in zebrafish juveniles decreases with increasing VPA exposure concentration; when the VPA exposure concentration reaches 15 μM, the fluorescence intensity of ROS in zebrafish juveniles becomes almost invisible to the naked eye. Figure 4 A). Statistical analysis of ROS fluorescence intensity data from DCF-DA staining in juvenile fish of each exposure group shows that, compared with the control group (30.68±0.9137), the ROS fluorescence intensity of the 5μM VPA exposure group (37.26±6.978) was significantly increased. p=0.029), but starting from 10 μM VPA (18.24±6.028), the ROS fluorescence intensity in zebrafish juveniles showed a decreasing trend, and the differences were statistically significant. p The values ​​were all less than 0.001; the increase in ROS fluorescence intensity in the 5 μM VPA exposure group may be due to the low exposure concentration, which has not yet caused a serious redox imbalance, and the certain fluctuation in ROS fluorescence intensity is within an acceptable range; thus, the results indicate that, except for the 5 μM VPA exposure group, VPA exposure during the mitotic phase significantly reduced the ROS level in zebrafish larvae. Figure 4 B).

[0044] Furthermore, from Figure 4 As shown in Figure C, after VPA exposure, the total antioxidant capacity in zebrafish juveniles increased with increasing VPA concentration. Compared with the control group (28.90±2.657 μmol / mg), there was no significant change in the 5 μM VPA exposure group (29.19±1.369 μmol / mg). However, starting from the 10 μM VPA exposure group (33.18±2.173 μmol / mg), the antioxidant capacity in zebrafish juveniles showed an increasing trend, with significant differences, reaching a maximum at 25 μM VPA (38.16±1.373 μmol / mg). p <0.001). The subsequent 30 μM VPA exposure group (34.44 ± 2.797 μmol / mg, p The antioxidant capacity of the <0.001) and 35 μM VPA exposure groups (33.57±2.570 μmol / mg) was slightly decreased, but both were significantly different from the control group.

[0045] The above results indicate that VPA exposure increases FRAP levels in zebrafish, which corresponds to a decrease in ROS levels, suggesting that VPA exposure shifts the redox balance in juvenile zebrafish towards reducing stress (imbalance).

[0046] In summary, valproic acid exposure can induce symptoms in zebrafish such as pericardial edema, heart rate arrhythmia, decreased embryo survival rate, decreased embryo hatching rate, head malformation, and redox imbalance. Therefore, the developmental toxicity and redox imbalance model induced by valproic acid in zebrafish has been successfully established.

[0047] Example 2: Trolox for prevention and treatment of VPA-induced developmental toxicity and redox imbalance in zebrafish First, the effects of pretreatment of zebrafish embryos with Trolox on VPA-induced pericardial edema and heart rate imbalance were investigated.

[0048] Specifically, zebrafish embryos exposed to VPA during the cleavage stage develop pericardial edema at 120 hours. To investigate whether Trolox pretreatment of zebrafish embryos could alleviate VPA-induced developmental malformations in zebrafish larvae, this example employs a pre-administration of Trolox. Specifically, before the VPA immersion administration method described in Example 1 (8-10 hours), Trolox was administered for 3-4 hours. Then, changes in pericardial length and heart rate were statistically analyzed at the drug concentration exposure endpoint (120 hours). The results are as follows: Figure 5 and 6 As shown.

[0049] from Figure 5 and 6 As can be seen from the data, compared with the control group (39.95±5.738μm), the 20μM VPA exposure group (52.83±10.26μm) had significantly lower concentrations. p The pericardial length of the VPA-induced pericardial edema was significantly increased (=0.0078), consistent with the conclusion in Example 1. However, with increasing Trolox concentration, the pericardial edema induced by VPA was alleviated. Compared to the 20 μM VPA exposure group, the pericardial length of the 20 μM VPA + 2 μM Trolox exposure group (48.77 ± 6.737 μm) was slightly decreased, but not significantly different. However, the pericardial length of the 20 μM VPA + 3 μM Trolox exposure group (41.52 ± 7.847 μm) was significantly increased (=0.0078), consistent with the conclusion in Example 1. p The pericardial length (=0.034) was significantly reduced compared to the 20 μM VPA exposure group, therefore Trolox alleviated VPA-induced pericardial edema. Figure 5 and 6 A).

[0050] Furthermore, from Figure 6 As shown in Figure B, compared with the control group (158.1±11.73 times), the 20μM VPA exposure group (143.6±17.01 times) had significantly lower exposure rates. p Heart rate was significantly reduced (μM VPA ± 0.0408), and Trolox alleviated this process. Compared with the 20 μM VPA exposure group, the 20 μM VPA + 2 μM Trolox exposure group (147.9 ± 23.73) showed a significantly lower heart rate (μM VPA ± 0.0408), which was alleviated by Trolox. p The heart rate of the group exposed to 20 μM VPA + 3 μM Trolox (160.4 ± 28.14) increased slightly until the heart rate of the group exposed to 20 μM VPA + 3 μM Trolox (160.4 ± 28.14). p It was only when the value was 0.0110 that the increase was significant.

[0051] The above results indicate that Trolox can alleviate myocardial edema and heart rate imbalance in zebrafish induced by VPA.

[0052] Then, the effect of pretreatment of zebrafish embryos with Trolox on the redox imbalance induced by VPA was investigated.

[0053] Specifically, zebrafish embryos exposed to VPA during the cleavage stage showed a significant decrease in ROS and a significant increase in total antioxidant capacity 72 hours after hatching. To investigate whether Trolox pretreatment of zebrafish embryos could alleviate the redox imbalance induced by VPA in zebrafish, this example employed a pre-administration of Trolox. Specifically, before the VPA immersion administration method described in Example 1 (8-10 hours), Trolox was administered for 3-4 hours. Then, the reactive oxygen species levels, total antioxidant capacity, nitrite levels, and nitrate levels in the treated zebrafish larvae were measured using the method described in Example 1. The results are as follows: Figure 7 As shown.

[0054] The methods for determining nitrates and nitrites are as follows: The nitric oxide levels in zebrafish were assessed by measuring nitrate and nitrite levels using a nitric oxide analyzer. This method is based on the principle of chemiluminescence and utilizes I... 3- The solution or VCl3 solution in the NOA reaction vessel will remove NOx (NO2) from the sample. - Or NO3 - O3 is reduced to NO, and the produced NO is fed into a detector using N2 gas. Inside the detector, O3 reacts with NO to generate excited-state NO2. The excited-state NO2 transitions to the ground-state NO2 and releases photons. The light signal is captured and converted into an amplified electrical signal by a photomultiplier tube. The computer system receives the electrical signal and presents it as a peak. The specific experimental steps are as follows: Reagent preparation: 100mM NaNO2 standard solution: Weigh 0.069g NaNO2 into a 15mL centrifuge tube, add 10mL ddH2O and vortex to mix. Then dilute with ddH2O to 10µM, 8µM, 5µM, 3µM, 2µM and 1µM successively to prepare a standard curve; 100mM NaNO3 standard solution: Weigh 0.085g NaNO3 into a 15mL centrifuge tube, add 10mL ddH2O and vortex to mix. Then dilute with ddH2O and water successively to 10µM, 8µM, 5µM, 3µM, 2µM and 1µM to prepare a standard curve; 0.011g / mL I 3- Solution: Weigh 2.0g KI and 1.3g iodine into 250mL beakers, then add 140mL glacial acetic acid and 40mL ddH2O. Stir slowly with a glass rod until homogeneous, and I can be obtained in 30min. 3-The solutions were placed in brown reagent bottles; 1 mol / L HCl solution: Measure 9 mL of concentrated HCl solution and add ddH2O to make up to 100 mL to obtain 1 mol / mL HCl solution; 0.008 g / mL VCl3 solution: Weigh 0.4 g of VCl3 solid into a 100 mL beaker, add 25 mL of 1 mol / L HCl, mix well with a glass rod, filter through a 0.45 µm filter, seal and store away from light; 1 mol / L NaOH solution: Weigh 10 g of NaOH and dissolve it in 250 mL of ddH2O, vortex to mix well and store in a cool place.

[0055] Sample preparation: At the dosing endpoint (120 h), six parallel samples were prepared for each concentration, with 10-20 zebrafish per sample. The zebrafish juveniles were washed three times with ddH2O. Homogenizing the juveniles on ice using a glass homogenizer at a ratio of 1 juvenile: 10 μL ddH2O for 1 min was performed. After homogenization, the mixture was centrifuged at 4℃ and 14000 rpm for 5 min. The supernatant was then transferred to a new 1.5 mL centrifuge tube. Nitrite and nitrate level detection: 50 μL (nitrite) or 20 μL (nitrate) samples were analyzed using a nitric oxide analyzer. The results were analyzed using Origin software. The final concentration was obtained as the ratio of nitrate or nitrite concentration to protein concentration.

[0056] from Figure 7 As can be seen, compared with the control group (6.606±1.433), the 20μM VPA exposure group (3.258±0.4667) showed significantly lower levels of VPA. p The ROS content in zebrafish was significantly reduced (<0.0001), while Trolox could alleviate the decrease in ROS in zebrafish induced by VPA; compared with the 20 μM VPA exposure group, the 20 μM VPA + 2 μM Trolox exposure group (4.451±0.8936, p The ROS content increased slightly in the 20 μM VPA + 3 μM Trolox exposure group (5.537 ± 1.024), but decreased slightly in the 20 μM VPA + 3 μM Trolox exposure group (5.537 ± 1.024). p =0.0013) before a significant increase ( Figure 7 A and 7B).

[0057] Furthermore, from Figure 7 As shown in C, compared with the control group (81.68±23.33 μmol / mg), the 20 μM VPA exposure group (144.5±40.51 μmol / mg) showed significantly lower levels of volatile organic compounds (VPA). pThe total antioxidant capacity level of zebrafish was significantly increased (μM VPA = 0.0082), while Trolox was able to alleviate the increase in total antioxidant capacity level in zebrafish induced by VPA. Compared with the 20 μM VPA exposure group, the 20 μM VPA + 2 μM Trolox exposure group (130.1 ± 43.57 μmol / mg) showed a significant increase. p The total antioxidant capacity level (=0.5680) decreased slightly, but increased to 87.79±35.38 μmol / mg in the 20 μM VPA + 3 μM Trolox exposure group. p It was only when the value was 0.0372 that it decreased significantly.

[0058] Furthermore, from Figure 7 As can be seen in D and 7E, compared with the control group (0.2257±0.06728 μmol / mg), the 20 μM VPA exposure group (0.5932±0.3346 μmol / mg) showed significantly lower levels of VPA. p The nitrite level was significantly increased in the 20 μM VPA + 2 μM Trolox exposure group (0.2312 ± 0.1404 μmol / mg, 0.0411); compared with the 20 μM VPA exposure group, the nitrite level was significantly increased in the 20 μM VPA + 2 μM Trolox exposure group (0.2312 ± 0.1404 μmol / mg, 0.0411). p The nitrite level in the group with a concentration of 0.0449 μmol / mg protein was significantly decreased, but there was no significant difference compared with the control group; while the nitrite level in the group exposed to 20 μM VPA + 3 μM Trolox (0.2826 ± 0.1291 μmol / mg protein) was significantly decreased. p =0.0983) was critically significant compared to the 20 μM VPA exposure group ( p <0.1. Compared with the control group (2.634±0.9636 μmol / mg), the 20 μM VPA exposure group (0.5932±0.3346 μmol / mg) showed significantly lower levels. p The nitrate levels (=0.1785) showed an increasing trend, but the difference was not significant, and there were no significant differences between the concentration groups.

[0059] The above results indicate that Trolox can alleviate the redox imbalance in zebrafish caused by VPA.

[0060] In summary, this invention is the first to discover a novel pharmaceutical use for Trolox that can prevent and / or treat developmental toxicity or redox imbalance caused by valproic acid; furthermore, animal experiments have demonstrated that Trolox can alleviate developmental malformations in zebrafish caused by valproic acid, and also alleviate redox imbalance in zebrafish caused by valproic acid.

[0061] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0062] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of Trolox for the preparation of a medicament for the prevention and / or treatment of toxicity caused by valproic acid, characterized in that, The valproic acid-induced toxicity includes developmental toxicity.

2. Use of Trolox in the preparation of a medicament for preventing and / or treating redox imbalance induced by valproic acid.

3. Use according to claim 1, characterized in that, The developmental toxicity includes developmental malformation. The developmental malformation includes at least one of pericardial edema, heart rate imbalance, decreased embryo survival rate, decreased embryo hatching rate, and head malformation.

4. Use according to claim 3, characterized in that, The head malformation includes at least one of decreased head length, decreased mandible length, and decreased keratin cartilage length.

5. Use according to claim 2, characterized in that, The valproic acid-induced redox imbalance includes intraembryonic redox imbalance. The intraembryonic redox imbalance includes at least one of decreased reactive oxygen species level, increased antioxidant capacity, and increased nitrite level.

6. Use according to any one of claims 1 to 5, characterized in that, The effective concentration of the Trolox is 2-3 μM.

7. The use according to any one of claims 1 to 5, characterized in that, The concentration of valproic acid in the valproic acid-induced toxicity and / or the valproic acid-induced redox imbalance is 1-40 μM.

8. A pharmaceutical composition for preventing and / or treating toxicity caused by valproic acid or redox imbalance caused by valproic acid, characterized by, The pharmaceutical composition includes Trolox.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semi-solid preparation, and a liquid preparation.

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