Antidepressant compound 4-methyl-6-methoxycoumarin

Abstract

The invention relates to an anti-depression compound 4-methyl-6-methoxy coumarin, and belongs to the technical field of biological medicines. Depression is the most common mental disorder in the world and is also a main reason for disability. The development of a novel anti-depression drug which is based on a nerve plasticity mechanism, takes effect quickly, has a lasting curative effect and is high in safety, and particularly has important clinical requirements and technical necessity for an effective treatment means for refractory depression. Traditional Chinese medicine monomers and a per2- / -zebra fish depression model are adopted for anti-depression drug screening, and finally it is found that 10 mM of 4-methyl-6-methoxycoumarin has the anti-depression characteristic and has the positive treatment effect on the exploration behavior, social interaction and sleep-awakening period of per2- / -zebra fish. The method can effectively rescue and reverse depression-like phenotypes observed in a per2- / -zebra fish circadian rhythmic depression model, and can restore the balance of important gene expression.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an antidepressant compound, 4-methyl-6-methoxycoumarin. Background Technology

[0002] Depression is a common mental health disorder affecting millions worldwide. Traditional antidepressants, such as serotonin selective reuptake inhibitors (SSRIs) (e.g., fluoxetine, citalopram, escitalopram, paroxetine, and sertraline) and serotonin-norepinephrine reuptake inhibitors (SNRIs) (e.g., venlafaxine, norvenlafaxine, and duloxetine), are commonly used treatments. Antidepressants are reported to act on the pathophysiological systems of the central nervous system (CNS), including monoamine neurotransmission, the hypothalamic-pituitary-adrenal (HPA) axis, neurotrophic factors, and the number and function of synapses. Antidepressants modulate neurotransmitters such as serotonin and norepinephrine; however, their efficacy varies, with only about 50-60% of patients showing a adequate response. Furthermore, nearly one-third of patients exhibit treatment resistance, highlighting the importance of alternative therapies. The differences in antidepressant efficacy are attributed to pharmacological differences, including chemical structure and metabolic efficiency. Previous studies have shown that behavioral responses to antidepressants depend on both the drug administration regimen and the function of the cyclic adenosine monophosphate response element-binding protein (CREB) in the hippocampus. Given the current inadequacy of antidepressants approved by the U.S. Food and Drug Administration (FDA), there is an urgent need to identify and develop novel, effective antidepressants with fewer toxic side effects. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the lack of effective antidepressant drugs in the prior art and to provide a new treatment strategy, namely, using the traditional Chinese medicine monomer 4-methyl-6-methoxycoumarin for antidepressant treatment, which has high safety.

[0004] The first object of the present invention is to provide the use of 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof in the preparation of mood-improving products.

[0005] Furthermore, the pharmaceutically acceptable derivatives of 4-methyl-6-methoxycoumarin are in the form of pharmaceutically acceptable salts.

[0006] Furthermore, the mood-improving products include, but are not limited to, antidepressants.

[0007] Furthermore, the antidepressant also includes pharmaceutically acceptable excipients.

[0008] Furthermore, the antidepressant is composed of 0.1-100% 4-methyl-6-methoxycoumarin or its pharmaceutically acceptable derivatives and 99.9-0% pharmaceutical excipients.

[0009] Furthermore, the pharmaceutical excipients include pharmaceutically acceptable salts, excipients, or carriers.

[0010] Furthermore, the pharmaceutical excipients include, but are not limited to, fillers, binders, disintegrants, lubricants, flow aids, wetting agents, effervescent agents, colorants, sweeteners, flavorings, preservatives, dispersants, film-forming agents, plasticizers, pore-forming agents, light-blocking agents, retardants, solvents, etc.

[0011] Furthermore, the dosage forms of the antidepressant include, but are not limited to, capsules, tablets, oral preparations, microcapsule preparations, injections, powders, sprays, ointments, etc.

[0012] Furthermore, when the antidepressant is in solution form, the concentration range of 4-methyl-6-methoxycoumarin or its pharmaceutically acceptable derivative is 5-55 mM, with 10 mM being the most preferred.

[0013] Furthermore, the antidepressant is a drug that improves social behavior and / or sleep-wake cycles.

[0014] Furthermore, the antidepressant is a drug that upregulates the expression of per2 and the sleep-inducing gene sik3.

[0015] A second object of the present invention is to provide a pharmaceutical composition for antidepressant purposes, comprising 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof.

[0016] A third object of the present invention is to provide an antidepressant article comprising a container containing an individually packaged formulation of: a formulation containing 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof; or a formulation containing a conventional antidepressant drug.

[0017] Furthermore, traditional antidepressants include, but are not limited to, serotonin selective reuptake inhibitors and / or serotonin-norepinephrine reuptake inhibitors.

[0018] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0019] In this invention, the monomer 4-methyl-6-methoxycoumarin, screened and identified from a traditional Chinese medicine monomer library, is effective against per2. - / - Zebrafish exploratory behavior, social interaction, and sleep-wake cycles have positive therapeutic effects and can restore the balance of important gene expression. Therefore, this monomer can effectively rescue and reverse the expression of genes in per2. - / -A depression-like phenotype was observed in a zebrafish circadian rhythmic depression model, and neurotransmitter balance was restored by downregulating cortisol and inflammation-related genes. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Figure 1 Behavioral screening of monomers in traditional Chinese medicine.

[0022] Figure 2 4-Methyl-6-methoxycoumarin, a novel traditional Chinese medicine monomer with antidepressant effects, can rescue and reverse per2... - / - Depressive-like behavior in zebrafish.

[0023] Figure 3 The gene expression status after treatment with 4-methyl-6-methoxycoumarin. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0025] The solution involved in this invention is as follows:

[0026] Initially, 1119 traditional Chinese medicine (TCM) monomers were screened to identify novel antidepressant monomers. 174 TCM monomers identified as having rescue capabilities comparable to wild-type zebrafish were further classified according to their bioactive components or parts. The results showed that 19% of the compounds belonged to terpenes. Among the 174 identified TCM monomers, flavonoids, phenols, alkaloids, and phenylpropanoids were other significant categories. Furthermore, these 174 TCM monomers were further classified according to their predicted bioactivity and functional properties. Due to the multi-target nature of TCM, most of these compounds were predicted to possess one or more bioactivities. The classification results showed that most of the 174 TCM monomers possessed anti-inflammatory, antioxidant, and anticancer properties.

[0027] Finally, after multiple behavioral screening experiments, 4-methyl-6-methoxycoumarin was identified as an effective antidepressant. It is extracted from the rhizome of Angelica dahurica.

[0028] Example

[0029] 1. Drug screening methods

[0030] (1) Zebrafish rearing

[0031] Wild-type zebrafish and per2 - / -Adult and juvenile mutant fish were used for behavioral and drug screening experiments. Adult zebrafish were fed brine shrimp in the morning and evening, and dry pellets at noon each day. The water temperature was maintained at 28.5 ± 1°C, the pH was 7.2 - 7.6, and the light cycle of the fish tank was 14 hours of light / 10 hours of darkness, i.e., 14L:10D.

[0032] (2) Zebrafish mating and embryo collection

[0033] The night before, a pair of adult male and female zebrafish were placed in a mating tank, with a transparent partition separating the male and female. The next morning, after turning on the lights in the aquarium, the partition was removed. At least one hour later, the embryos were collected and placed in E3 culture medium. Unfertilized embryos were removed, and the remaining embryos were placed in a light-and-dark incubator (14L: 10D, 300 lux) at 28.5°C. Zebrafish fry complete development 6-7 days after fertilization.

[0034] (3) Zebrafish behavioral experiment

[0035] In zebrafish behavior detection, wild-type and per2 types were compared. - / - The social, exploratory, and sleep-wake cycle behaviors of mutant zebrafish were evaluated. The experimental instruments used for these measurements were the Zebrafish Automated Tracking Software EthoVision XT 12 (Noldus, Netherlands) and ZebraLab (Viewpoint, France). Both the Noldus and Viewpoint instruments have an internal high-speed infrared camera that captures the juvenile movement parameters set for each well. Six days post-fertilization, zebrafish juveniles were placed in 6-well plates for social behavior measurements, 24-well plates for exploratory behavior measurements, or 48-well plates for sleep behavior measurements, with one juvenile per well. For the drug-treated groups, pretreatment was performed before placement in the wells. The zebrafish behavioral tracking system was then set, with illumination conditions set to 300 lux, and monitoring times determined according to the specific experiments.

[0036] (4) Drug screening

[0037] A library of 1119 monomers from traditional Chinese medicine (product number L6810, TargetMol) was screened to identify novel monomers capable of rescuing the depressive-like phenotype in per2 mutant zebrafish. The drugs used were dissolved at a concentration of 10 mM in 100 μL of dimethyl sulfoxide (DMSO).

[0038] ① Zebrafish embryos and juveniles were cultured in a constant-temperature incubator with a light-dark cycle of 14 hours:10 hours and a temperature of 28.5 ℃. Five days (5 dpf) or six days (6 dpf) after fertilization, the zebrafish juveniles were placed into their respective well plates, with only one juvenile per well. Before conducting behavioral assessments, the zebrafish were pretreated with a traditional Chinese medicine compound at least one hour in advance.

[0039] ② During drug treatment, according to the experimental groups, 2 μL of a 10 mM drug was added to 2 mL of water in a perforated plate containing zebrafish. These were wild-type (WT) and per2... - / - A control group of mutant zebrafish was set up using DMSO treatment.

[0040] ③ One hour after treatment, zebrafish were placed in the holes of a perforated plate and subjected to various behavioral assessments under the required conditions using a Noldus or Viewpoint instrument, and monitored for specific time periods: 10 minutes for social behavior monitoring, 30 minutes for exploratory behavior monitoring, and 72 hours for sleep-wake cycle behavior monitoring.

[0041] (5) Results and Data Analysis

[0042] Quantitative data analysis was performed using GraphPad Prism 8. Normality was first assessed for all datasets to determine appropriate statistical tests. Student's t-tests were used for comparisons between two normally distributed groups. One-way or two-way ANOVA was used for comparisons between multiple groups, with Tukey's post-hoc analysis test employed. Unless otherwise stated, error bars in all graphs represent standard deviations (SD). ns indicates no significant difference; P < 0.05 indicates a significant difference (*); P < 0.01 indicates a more significant difference (**); P < 0.001 indicates a very significant difference (***); P < 0.0001 indicates an extremely significant difference (****).

[0043] 2. Drug screening results and validation

[0044] (1) Behavioral screening of Chinese herbal monomers

[0045] After preliminary screening of 1119 monomers, 372 traditional Chinese medicine monomers were observed to have similar antidepressant effects. Figure 1 A), because they can save and improve per2 - / - Zebrafish locomotor activity. Of these 372 monomers, 81 could enhance the locomotor activity of treated per2. - / -Zebrafish showed significantly higher activity than wild-type (WT), while 117 species, although capable of increasing per2% activity in treated individuals, were significantly more active. - / - The zebrafish were active, but below the average level of wild-type zebrafish. Figure 1 B). Interestingly, 174 of the screened herbal monomers were able to enable per2 - / - The zebrafish's locomotor activity has increased to a level roughly equivalent to that of the wild type. Figure 1 B).

[0046] Further screening and analysis were conducted on 174 monomers of traditional Chinese medicine to study their effects on per2 - / - Antidepressant-like improvements in exploratory behavior, social behavior, and sleep-wake cycle in zebrafish. Results showed that 117 compounds could improve per2... - / - Zebrafish locomotion and enhanced exploratory behavior. Further screening of these 117 monomers revealed that 32 were able to restore per2 - / - The sleep-wake cycle of zebrafish. Further screening of these 32 monomers for social behavior revealed that 4-methyl-6-methoxycoumarin could improve and enhance per2... - / - Social behavior in zebrafish. Importantly, 4-methyl-6-methoxycoumarin improved all behavioral parameters tested. 4-methyl-6-methoxycoumarin ( Figure 1 E) is extracted from the rhizome of Angelica dahurica and has been used for other medicinal purposes. However, there is currently no record of 4-methyl-6-methoxycoumarin being used to treat depression.

[0047] (2) The novel antidepressant-like traditional Chinese medicine monomer 4-methyl-6-methoxycoumarin can rescue and reverse the depressive-like behavior of per2 gene knockout zebrafish.

[0048] Based on experiments and data analysis, we selected 4-methyl-6-methoxycoumarin, a traditional Chinese medicine monomer compound with the most significant rescue effect. This monomer can increase motility and significantly enhance the per2 treatment. - / - Exploratory behavior of zebrafish Figure 2 A). With unprocessed per2 - / - Compared to zebrafish, treated zebrafish spent more time in the exploration area of ​​the test device. Figure 2 BC), and the wall preference index increased significantly ( Figure 2 D). Furthermore, we enhanced the per2 of this herbal monomer. - / - The social preferences and interaction abilities of zebrafish were examined. The results showed that 4-methyl-6-methoxycoumarin could enhance per2...- / - Social behavior and interaction of zebrafish ( Figure 2 EG). When individuals of the same species are added to the test apparatus, compared with untreated per2... - / - Compared to zebrafish, per2 treated with 4-methyl-6-methoxycoumarin - / - Zebrafish spent more time in the social cue zone. These results suggest that 4-methyl-6-methoxycoumarin possesses antidepressant-like abilities and can rescue per2... - / - Phenotypes of depressive-like decreased exploratory ability and social avoidance behavior in zebrafish.

[0049] Five days after fertilization (5 dpf), per2 was given - / - Zebrafish were treated with 4-methyl-6-methoxycoumarin, and their sleep-wake cycles were observed and analyzed. Results showed that, compared to untreated per2... - / - Compared to zebrafish ( Figure 2 H), 4-methyl-6-methoxycoumarin can rescue and restore treated per2 - / - The fragmentation and reduction of nighttime sleep in zebrafish.

[0050] per2 treated with 4-methyl-6-methoxycoumarin - / - An assessment of the sleep-wake structure in zebrafish showed differences compared to untreated per2 - / - Compared to zebrafish, this herbal monomer significantly increased per2 - / - Zebrafish sleep at night and reduce their daytime sleep. Figure 2 IM). Furthermore, treatment with 4-methyl-6-methoxycoumarin significantly improved per2. - / - Zebrafish exhibited reduced frequency and duration of sleep episodes, increased sleep transitions, and prolonged sleep latency. Therefore, 4-methyl-6-methoxycoumarin can improve per2... - / - The zebrafish exhibited reduced and fragmented sleep patterns, and this led to the treatment of per2... - / - The zebrafish's sleep-wake cycle returned to normal.

[0051] Importantly, the novel antidepressant-like herbal monomer 4-methyl-6-methoxycoumarin can upregulate the expression of per2 and the sleep-inducing gene sik3, and restore the balance of the hypothalamus-pituitary-adrenal (HPA) axis through upregulation of glucocorticoid receptor (GR). Figure 3 A, B, H). Subsequently, in zebrafish treated with traditional Chinese medicine monomers, significant downregulation of cortisol-inducing genes crha, crhb, pomca, and pomcb, as well as the inflammatory gene tnfa, was found. Figure 3(CG). In addition, this traditional Chinese medicine monomer can also rescue the depressive-like phenotype in reserpine-induced depressed zebrafish.

[0052] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof in the manufacture of a mood improving product.

2. Use according to claim 1, characterized in that, 4-methyl-6-methoxycoumarin pharmaceutically acceptable derivative is in the form of a pharmaceutically acceptable salt.

3. Use according to claim 1, characterized in that, The mood improving product is an antidepressant.

4. Use according to claim 3, characterized in that, The antidepressant further comprises a pharmaceutically acceptable excipient; the antidepressant consists of 0.1-100% of 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof and 99.9-0% of a pharmaceutical excipient; wherein: The pharmaceutical excipient comprises at least one of a filler, a binder, a disintegrant, a lubricant, a glidant, a wetting agent, an effervescent agent, a colorant, a sweetener, an aroma, a preservative, a dispersant, a film forming agent, a plasticizer, a pore forming agent, an opacifier, a retarder, a solvent.

5. Use according to claim 3, characterized in that, The antidepressant is in the form of a capsule, a tablet, an oral preparation, a microcapsule preparation, an injection, a powder, a spray or an ointment.

6. Use according to claim 3, characterized in that, When the antidepressant is in the form of a solution, the concentration of 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof is in the range of 5-55 mM.

7. Use according to claim 3, characterized in that, The antidepressant is a drug for improving social behavior and / or sleep-wake cycle.

8. Use according to claim 3, characterized in that, The antidepressant is a drug for up-regulating the expression of per2 and sleep-inducing gene sik3.

9. An antidepressant article, characterized by comprising: The antidepressant is a drug for up-regulating the expression of per2 and sleep-inducing gene sik3.

10. The antidepressant article according to claim 9, characterized in that The container comprises a preparation comprising 4-methyl-6-methoxycoumarin or a pharmaceutically acceptable derivative thereof; a preparation comprising a conventional antidepressant. The conventional antidepressant is a serotonin selective reuptake inhibitor and / or a serotonin-norepinephrine reuptake inhibitor.

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