Application and application of pemetrexed in preparation of medicine for preventing and treating biological clock disorder

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application and application of pemetrexed in prevention and treatment of biological clock disorder. Pemetrexed can be directly combined with biological clock protein Bmal1, the amplitude of cell biological rhythm is remarkably inhibited, time difference of mice can be adjusted more quickly, biological clock disorder is effectively relieved, and pemetrexed has a certain treatment effect on biological clock disorder.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically to an application and use of pemetrexed in the preparation of a drug for preventing and treating circadian rhythm disorders. Background Art

[0002] Pemetrexed was first approved by the U.S. Food and Drug Administration (FDA) in February 2004 for the treatment of inoperable malignant pleural mesothelioma (MPM) in combination with cisplatin. It is the first FDA-approved drug for the treatment of malignant pleural mesothelioma. In August 2004, the U.S. FDA approved it for second-line treatment of non-small cell lung cancer (NSCLC). In September 2008, the U.S. FDA and the European Union approved it for first-line treatment of non-squamous non-small cell carcinoma. In 2009, the U.S. FDA and the European Union approved it for maintenance treatment of non-squamous non-small cell carcinoma. Pemetrexed is activated intracellularly by poly-γ-glutamylation, which is essential for its antiproliferative activity. Polyglutamic acid derivatives mainly inhibit three key enzymes of intracellular folate metabolism, namely thymidylate synthase (TYMS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT), among which TYMS is the most relevant target. However, there are no reports or patents on its use in treating circadian rhythm disorders. Therefore, on this basis, we continue to study new uses of Pemetrexed and expand its application areas.

[0003] There are many factors that lead to circadian rhythm disorders, which can be divided into external factors and internal factors: external factors are mainly due to regular light intervention; regular eating behavior and exercise are also effective non-light timing factors; temperature and sound also have a certain impact on peripheral oscillators; the therapeutic effects of some drugs may also be related to regulating circadian rhythms. In daily life, the factors that lead to circadian rhythm disorders are mainly environmental factors, such as jet lag, working late at night, shift work, work and study pressure, and the popularity of smartphones. Circadian rhythm disorders can also lead to cardiovascular diseases, tumor formation, and metabolic and immune diseases. It can be seen that alleviating circadian rhythm disorders is a scientific problem that people urgently need to overcome, but currently in the research on alleviating biological clock disorders, no drug has been widely recognized in the clinic. Therefore, the development of therapeutic drugs to alleviate biological clock disorders is of great significance for preventing and treating and / or alleviating biological clock disorders. Summary of the invention

[0004] The present invention provides an application of pemetrexed in the preparation of a drug for preventing and treating circadian rhythm disorders, so as to provide a new approach for preventing and / or alleviating circadian rhythm disorders and a new drug use of pemetrexed.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a use of pemetrexed in the preparation of a drug for preventing and treating circadian rhythm disorders, wherein the drug comprises pemetrexed.

[0006] In a preferred embodiment, the content of pemetrexed in the drug is 100 mg / kg.

[0007] In a preferred embodiment, the biological clock disorder is caused by light or non-light timing factors.

[0008] In a preferred embodiment, the dosage form of the drug is selected from a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0009] In a preferred embodiment, the drug is an injection.

[0010] In a preferred embodiment, the injection is administered by intracavitary injection.

[0011] In a preferred embodiment, the injection further comprises a pharmaceutically acceptable carrier.

[0012] The second aspect of the present invention provides a use of the above-mentioned pemetrexed in the preparation of a drug for treating circadian rhythm disorders, for preventing and / or alleviating circadian rhythm disorders.

[0013] The purpose of the present invention is to provide a new medicinal use of pemetrexed.

[0014] The new drug use of pemetrexed provided by the present invention is a drug for preventing and / or alleviating biological clock disorders.

[0015] The present invention also provides a medicine for preventing and / or alleviating biological clock disorders, wherein the medicine contains pemetrexed original medicine and its salts, esters and hydrates.

[0016] The above drugs can be introduced into the body by injection or chemically mediated methods such as intramuscular, intradermal, subcutaneous, or intravenous; or can be mixed or encapsulated with other substances and then introduced into the body.

[0017] One or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0018] The above medicines can be prepared in the form of injections. The above medicines can be prepared according to conventional methods in the pharmaceutical field.

[0019] Definition of terms

[0020] The term “non-light timing factors” refers to temperature, social interactions, exercise, eating patterns, etc.

[0021] The term "intestinal dosage form" refers to a dosage form in which a drug preparation enters the gastrointestinal tract after oral administration and exerts a local effect or a systemic effect through absorption, such as commonly used powders, tablets, granules, capsules, solutions, emulsions, suspensions, etc.

[0022] The term "parenteral dosage form" refers to all other dosage forms except oral administration routes, which can act locally at the site of administration or exert systemic effects after absorption. For example, the commonly used parenteral dosage form is an injectable dosage form, such as an injection, including multiple injection routes such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavitary injection.

[0023] The term "prevention" includes suppressing and delaying the onset of a disease, and includes not only prevention before the development of a disease but also prevention of recurrence of a disease after treatment.

[0024] The terms "treat," ...

[0025] Beneficial effects of the present invention

[0026] The experimental results of the present invention show that pemetrexed can directly bind to the circadian clock protein Bmal1 and significantly inhibit Bmal1 luc U2OS and Per2 luc The amplitude of the biological rhythm of U2OS cells can effectively regulate biological rhythms. In addition, mice injected with Pemetrexed were able to adapt to the new time difference on the 5th day, while mice in the control group needed 9 to 10 days. Injection of 100 mg / kg of Pemetrexed allowed mice to adjust to the time difference faster, effectively alleviating the biological clock disorder of mice. The above results show that Pemetrexed has an excellent therapeutic effect on alleviating biological clock disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the Bmal1:Clock dimer protein-protein binding pocket.

[0028] Figure 2 It is Pemetrexed's regulation of cellular biological rhythms.

[0029] Figure 3 It is the regulation of mouse behavior by Pemetrexed.

[0030] Figure 4 This is the site where Pemetrexed binds to the Bmal1 protein.

[0031] Figure 5This is the result of the surface plasmon resonance binding analysis experiment between Pemetrexed and Bmal1 protein. DETAILED DESCRIPTION

[0032] The present invention is described below by specific examples, but the present invention is not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0033] The present invention provides a new pharmaceutical use of pemetrexed. The new pharmaceutical use of pemetrexed provided by the present invention is to alleviate the effect of pemetrexed on circadian rhythm disorders. The experimental results of the present invention show that pemetrexed can directly bind to the Bmal1 protein and significantly inhibit the expression of Bmal1. luc U2OS and Per2 luc The amplitude of U2OS cells; at the same time, it can make mice adjust to jet lag faster. The above results show that pemetrexed has an excellent therapeutic effect on alleviating circadian rhythm disorders.

[0034] Example 1 Screening of small molecule compounds based on the crystal structure of receptor Bmal1:Clock protein

[0035] The different biological rhythm phenomena of the body are all controlled by biological rhythm genes, among which the core biological clock genes are Bmal1 and Clock. Bmal1 and Clock both have bHLH-PAS structures, and they can bind to form heterodimers. Figure 1 As shown, Figure 1 The green color in the middle is Bmal1 protein, and the blue color is Clock protein. When Bmal1 and Clock form a heterodimer, there is a druggable binding pocket in the crystal structure of the binding site of the two. The crystal structure of the binding site is used as a receptor, and the AutoDock Vina and FIPSDock tools are used to perform virtual screening in the FDA-listed and pharmacopoeia compound library. In the docking results, the screened small molecule compounds are ranked from high to low according to the binding energy, and the top 20 small molecule compounds are selected for in vitro cell bioluminescence detection to obtain the experimental results. The screened small molecule compounds and their docking scores are shown in Table 1.

[0036] Table 1 Information on the top 20 small molecule compounds in the FIPSDock docking results

[0037]

[0038] Example 2 Cellular bioluminescence detection of small molecules that can affect cell biological rhythms

[0039] Since Bmal1 plays a core regulatory role in the biological clock system, we first tested the effect of the small molecule compounds obtained by virtual screening on the rhythmic expression of the Bmal1 gene at the cellular level. The U2OS cell line (human osteosarcoma cell) was treated with the screened small molecule compounds. This cell line is commonly used to study biological rhythms. There is a fluorescent protein driven by the Bmal1 gene in the cell, called Bmal1 luc U2OS cells (Bmal1-luciferase labeled human osteosarcoma cells). Under the action of the substrate luciferin, the cells can produce fluorescence of a specific wavelength. The bioluminescent signal of the cells can be detected in real time in the Lumicycle instrument (biorhythmic photometric analysis system), thereby achieving the purpose of real-time monitoring of Bmal1 gene expression.

[0040] U2OS cells were inoculated into 35mm cell culture dishes, and the experiment could be started when the cells grew to a density of about 80%. 10% FBS, 1% penicillin / streptomycin, 0.1mM luciferin and 100nM dexamethasone were added to the DMEM medium without phenol red, and the small molecule compounds obtained by virtual screening (as shown in Table 1) were added respectively, and stored in the dark. 2ml of freshly prepared bioluminescence detection medium was added to each culture dish, and the cells were transferred to the Lumicycle instrument. The parameters of the instrument were adjusted to detect the cell bioluminescence every 5 minutes for a total of 96 hours. During the experiment, the culture environment of the cells was maintained at 37°C and the CO2 concentration was 5%.

[0041] In the above cell experiments, it was found that the drug pemetrexed (Catalog No.: HY-10820) had a significant effect on the biological rhythm of U2OS cells. luc U2OS cell bioluminescence results Figure 2 As shown in A, Pemetrexed significantly reduced the amplitude of Bmal1 expression, and the amplitude decreased more significantly with the increase of Pemetrexed drug concentration. Figure 2 B is Bmal1 luc Statistical graph of the amplitude change of Bmal1 expression in U2OS cells after Pemetrexed treatment. Amplitude is a key parameter that determines the circadian phenotype of an organism. A change in amplitude can be considered as a change in the circadian phenotype of an organism. Per2 (Period Circadian Protein Homolog 2) is a clock gene downstream of Bmal1, and its expression is directly regulated by Bmal1. Therefore, the effect of Pemetrexed on Per2 was detected. luc The bioluminescence of U2OS cells was detected. Figure 2As shown in C and 2D, Pemetrexed also significantly reduced the amplitude of Per2 expression. luc Effect of Bmal1 on U2OS cell bioluminescence luc The results of U2OS cells are consistent. The above cell experiments show that Pemetrexed can significantly change the biological rhythm of cells.

[0042] Example 3 Pemetrexed makes mice adjust to jet lag faster

[0043] In order to explore compounds that can adjust the biological rhythm of mice, we first selected compounds that can affect the biological rhythm of cells for in vivo experiments in mice. This study used the +8 time difference model to induce circadian rhythm disorders in mice, moving the lights off time forward by 8 hours, which also simulates the phenomenon of crossing 8 time zones eastward in human society. Before the formal experiment began, the experimental mice were placed in a light-controlled isolation cabinet to adapt for a week, and mice with normal movement were selected for the experiment. Figure 3 As shown, for 3 consecutive days, mice were injected with Pemetrexed (100 mg / kg) by intraperitoneal injection 1 hour before turning off the lights, and the control group was injected with the same volume of normal saline. After the third administration, the mouse circadian rhythm disorder model was established to detect the speed at which the mice adapted to the time difference. During the entire experiment, the running wheel system continuously recorded the behavioral activities of the mice. Mice injected with Pemetrexed were able to adapt to the new time difference on the 5th day, while the mice in the control group needed 9 to 10 days. After the injection of Pemetrexed, the mice adjusted to the time difference faster.

[0044] like Figure 3 Shown is the regulation of mouse behavior by Pemetrexed. Figure 3 A and 3B are two-point representative graphs of mouse running wheel activity. The values ​​on the horizontal axis are 2 light time cycles, 24+24 hours, and each row on the vertical axis represents 1 day. Figure 3 The black tiny bar in the middle indicates that the mouse is moving, the yellow area represents the light-on time period, and the gray area represents the light-off time period. The red dot indicates the time point (1 hour before turning off the light) when the mouse was intraperitoneally injected with Pemetrexed (100 mg / kg) or saline; Figure 3 C is the statistical analysis of the time when mice started to move every day. One-way ANOVA test was used.

[0045] Example 4 Pymol software analysis of the binding site between Pemetrexed and Bmal1:Clock dimer

[0046] Through computer virtual screening (Example 1) and in vitro and in vivo experimental screening (Example 2, Example 3), it was found that the drug Pemetrexed can regulate biological rhythms. Next, it is necessary to further explore the site where Pemetrexed binds to the Bmal1:Clock dimer. Figure 4 As shown in the figure, the binding sites of Pemetrexed and Bmal1:Clock dimer were analyzed by Pymol software. Pemetrexed mainly hydrogen-bonded with Leu320, Ser322, Val440, Met338 and Ala329 amino acids of Bmal1 protein, and had no effect on Clock. The above results show that Pemetrexed reduces the amplitude of the cell biological clock by directly binding to Bmal1 protein.

[0047] Example 5 Biacore analysis of the affinity strength of Pemetrexed to Bmal1

[0048] Through Pymol software analysis, we preliminarily verified that Pemetrexed interacted directly with Bmal1 protein. In order to further analyze the strength of the interaction between Pemetrexed and Bmal1, this study used the principle of surface plasmon resonance to analyze the affinity strength of Pemetrexed and the target protein Bmal1.

[0049] like Figure 5 The figure shows the steady-state analysis fitting diagram between Bmal1 protein and Pemetrexed drug. This experiment is based on a high-throughput molecular interaction analysis system based on surface plasmon resonance (SPR) technology. First, we fixed the purified Bmal1 protein to the CM5 chip, configured the drug concentration of Pemetrexed to 0, 0.048828μM, 0.097656μM, 0.195313μM, 0.390625μM, 0.78125μM, 1.56μM, a total of 7 gradients, set Pemetrexed as the mobile phase, and used a single-cycle detection method to analyze the affinity strength of Pemetrexed and Bmal1 to be KD=1.18×10 -6 M, where the KD value is the concentration corresponding to the RU value (1 picogram square millimeter contains protein mass) reaching 1 / 2RMAX, which is the affinity between the ligand and the receptor. Therefore, through the results of the surface plasmon resonance binding analysis of Pemetrexed and Bmal1 protein, it can be concluded that Pemetrexed has a strong affinity for Bmal1, further confirming that Pemetrexed targets and binds to Bmal1 protein.

[0050] In summary, the results of cell experiments show that Pemetrexed can make Bmal1 luc / Per2 luc The amplitude of the U2OS cell rhythm decreased, and as the concentration of the compound increased, the amplitude decreased more significantly; further from the animal experimental results, it can be seen that Pemetrexed can make mice adapt to jet lag faster; Pymol software and Biacore analysis further verified that Pemetrexed directly binds to the Bmal1 protein. In summary, the experiments show that Pemetrexed has an excellent therapeutic effect on alleviating circadian rhythm disorders by directly binding to the Bmal1 protein.

[0051] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. The use of pemetrexed in the preparation of a drug for preventing and treating circadian rhythm disorders, characterized in that: The drugs include pemetrexed.

2. The use according to claim 1, characterized in that: The content of pemetrexed in the medicine is 100 mg / kg.

3. The use according to claim 1, characterized in that: The biological clock disorder is caused by light or non-light timing factors.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is selected from a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

5. The use according to any one of claims 1 to 3, characterized in that: The medicine is an injection.

6. The use according to claim 5, characterized in that: The injection method of the injection is intracavitary injection.

7. The use according to claim 5, characterized in that: The injection further comprises a pharmaceutically acceptable carrier.

8. Use of the pemetrexed according to any one of claims 1 to 7 in the preparation of a drug for preventing and treating circadian rhythm disorders, characterized in that: Used to prevent and / or alleviate biological clock disorders.

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