Pharmaceutical applications of myricetrin diselenide in tumors

Abstract

The application discloses a drug application of a di-myrictione diselenide in tumor treatment. Experiments prove that the di-myrictione diselenide has a tumor treatment prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the medical technology field, in particular to a pharmaceutical application of dimyricetin-yl-diselenide. BACKGROUND

[0002] Selenium is an essential trace element for human body. The Chinese Nutrition Society also lists selenium as one of the 15 nutrients essential for human body. A large number of clinical experiments at home and abroad show that selenium deficiency in human body can cause functional disorders of some important organs and lead to many serious diseases. Research shows that appropriate selenium supplementation in low-selenium or selenium-deficient populations can not only prevent the occurrence of tumors, liver diseases and the like, but also improve the immune capacity of the body, maintain the normal functions of important organs such as heart, liver, lung and stomach, and prevent the occurrence of senile cardiovascular and cerebrovascular diseases.

[0003] Myricetin is a flavonoid compound widely existing in myrica bark and leaves, and has many pharmacological activities such as anti-inflammatory, anti-tumor, anti-mutation, prevention of dental caries, antioxidant, elimination of free radicals in the body and the like.

[0004] Diselenide has important antioxidant effect, has the activity of simulating glutathione peroxidase (GSH-PX), and some diselenides also have the activities of anti-tumor, antibacterial, sterilization and disinfection.

[0005] Dimyricetin-yl-diselenide (DMS) has the molecular formula C 30 H 18 O 16 Se2, a molecular weight of 792.37, and a property of light yellow powder, is a new compound discovered by the applicant. SUMMARY

[0006] The application provides a new use of dimyricetin-yl-diselenide.

[0007] The application adopts the following technical scheme:

[0008] The application of dimyricetin-yl-diselenide in the preparation of a drug for treating tumors.

[0009] A method for inhibiting the growth of tumor cells in vitro in a non-therapeutic manner, comprising the step of adding dimyricetin-yl-diselenide to a tumor cell culture system to inhibit the growth of tumor cells.

[0010] The application has the following beneficial effects:

[0011] Dimyricetin-yl-diselenide has been proved by experiments to have the prospect of treating tumors. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0013] Appendix Figure 1 - Appendix Figure 7 These are the results of in vitro cell experiments. They include the Cpds IC50 values ​​of T24, MBT-2, HCC1954, EMT-6, HCT116, CT-26.WT, MKN45, MFC, 786-O, Renca, Hep G2, H22, HCC827, LL / 2(LLC1), A375, B16-F10, PANC-1, Panc 02, DU145, RM-1, and SK-OV-3 cells.

[0014] Figure 8 These are results from animal experiments.

[0015] Figure 9 These are results from animal experiments. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0017] Example 1: In vitro cell experiments

[0018] 1) Cell lines

[0019]

[0020]

[0021] 2) Experimental materials and reagents

[0022] DMEM, Product No.: 10-013-CV, Batch No.: 01222005;

[0023] FBS, Product No.: 10099-141C, Batch No.: 2544621CP;

[0024] 96-hole experimental flat plate Product number: 062096, Batch number: 2022010601;

[0025] Cell Titer-Glo (CTG) Promega, Cat. No. DD1101-03, Lot No. 7E522L1;

[0026] 3) Instruments

[0027] Centrifuge Thermo Fisher legend MACH 1.6R;

[0028] Inverted microscope Nikon, Model: TS100;

[0029] Biosafety cabinet Thermo MSC-Advantage;

[0030] CO2 incubator Thermo HERA cell 150;

[0031] 4) Test drugs

[0032]

[0033] 5) Cell seeding

[0034] Cells in exponential growth phase were collected and counted by cell counter. The cell suspension was adjusted to the appropriate concentration with the corresponding medium. 90 μL of cell suspension was added to each well of a 96-well cell culture plate, 3 replicates, containing 5000 cells / well, and placed in a 37°C, 5% CO2 incubator overnight.

[0035] The next day, 10 μL of di-Myricetin-based diselenide and cisplatin were added to the corresponding wells, with the highest final concentration of di-Myricetin-based diselenide being 300 μg / ml (i.e. 379 μM) and the highest final concentration of cisplatin being 300 μM, with 3 dilution factors, for a total of 9 concentrations.

[0036] After 72 h, the cell viability was detected by CTG.

[0037] 6) Data analysis

[0038] GraphPad Prism 8.0 software was used to draw the S-shaped dose-survival curve and calculate the IC50 value using log(inhibitor) vs. response - Variable slope (four parameters).

[0039] 7) Test results

[0040]

[0041]

[0042] The results show that the myricetrin-based diselenide has obvious inhibitory effect on tumor cells in vitro, and the inhibitory effect on some cells even exceeds that of the positive drug.

[0043] Example 2: In vivo subcutaneous transplanted tumor efficacy study

[0044] 1) Test substance

[0045] Positive control: Cisplatin for injection Supplier: Pengli Biology Producer: Qilu Pharmaceutical (Hainan) Co., Ltd. Property: Freeze-dried agent Batch number: FA2A1024B Specification: 20 mg / bottle Storage condition: Keep away from light and seal Expiration date: 2024.11.03

[0046] Target compound: Diyangmeisu-based diselenide Test substance batch number: 210701R Supplier: Shanghai Aiqi Pharmaceutical Technology Co., Ltd. Effect: Test substance Property: Yellow powder Storage condition: Seal and refrigerate

[0047] 2) Reagents

[0048]

[0049]

[0050] 3) Instruments

[0051]

[0052] 4) Experimental process

[0053] a) Animals and receiving, evaluation

[0054] Animal species and strain: BALB / c mice

[0055] Dosing record: No history of administration

[0056] Gender and age: Female, 6-7 weeks old

[0057] Breeding party / supplier: Shanghai Jihui Experimental Animal Breeding Co., Ltd. or other qualified suppliers

[0058] Experimental institution: Penglai Biological Animal Room

[0059] Acclimation period: 3-7 days

[0060] Number: 42

[0061] Room: SPF area room

[0062] Indoor temperature: 20-26℃

[0063] Indoor relative humidity: 40-70%

[0064] Light: Eye light illumination, 12 hours of illumination (08:00-20:00) and 12 hours of no illumination

[0065] Animal feeding: 2-6 per cage (same administration group)

[0066] Food: ad libitum access to feed (irradiated sterilization, Jiangsu Cooperation Pharmaceutical Biological Engineering Co., Ltd., China)

[0067] Water: ad libitum access to drinking water (tap water treated by reverse osmosis or autoclaving and acidified)

[0068] BALB / c mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. for H22 efficacy experiment.

[0069] All animals were free of specific pathogens and were approximately 5-6 weeks old when they arrived at the Penglai Biological animal room. The animal operation procedures of the experimental protocol will be approved by the Penglai Biological IACUC (Institutional Animal Care and Use Committee) before the experiment begins.

[0070] Upon arrival at Penglai Biological, the animal room staff will transfer the animals from the transport packaging to the mouse cage and inspect each animal. The inspection range includes appearance, limbs and cavities, etc., as well as whether there are any abnormal behaviors when the animal is still or moving. The adaptation period is 3-7 days.

[0071] b) Cell culture and inoculation

[0072] The H22 cell line used in this experiment was cultured in RPMI-1640 medium with 10% FBS in a CO2 incubator at 37°C. Before the cells were continuously cultured for ten generations, about 3x10 6 H22 cells were suspended in PBS and inoculated subcutaneously in each mouse at a volume of 100 uL. Before inoculation, the mice were previously anesthetized with 3-4% isoflurane.

[0073] c) Animal grouping and dosing

[0074] When the tumor grew to an average of about 50-80 mm 3 in size, the tumor-bearing mice were randomly divided into 4 groups according to body weight and tumor size, with 8 mice in each group. The day of grouping and dosing was defined as day 0. The grouping and dosing scheme are shown in the table below.

[0075]

[0076] N: number of animals per group

[0077] Dosing volume: adjusted according to animal body weight 10 uL / g

[0078] Test article vehicle: 20% DMSO + 60% PEG400 + 20% 10 mM Acetic buffer (pH 4.5)

[0079] d) Drug preparation

[0080]

[0081] e) Assay

[0082] Animal weight: The body weight of mice was measured and recorded twice a week after grouping.

[0083] Tumor volume: The tumor volume was measured twice a week after grouping for 4 consecutive weeks. The calculation method of tumor volume (V) is as follows: V = (length x width 2 ) / 2. The calculation method of relative tumor volume (RTV) of each nude mouse is: RTV = Vt / V0, wherein Vt is the measured volume per day, and V0 is the volume at the beginning of treatment.

[0084] Relative tumor proliferation rate (T / C%): used to evaluate the effect of drug anti-tumor activity, relative tumor proliferation rate T / C (%) = average RTV of treatment group (T) / average RTV of negative control group (C) x 100%.

[0085] Tumor inhibition rate (TGI%): TGI% = (1-T / C) x 100%; wherein T is the average relative tumor volume of the treatment group, and C is the average relative tumor volume of the negative control group.

[0086] Tumor weight: at the end of the experiment, all tumors were collected, weighed and photographed. Tumor weight inhibition rate (%) = (average tumor weight of negative control group - average tumor weight of treatment group) / average tumor weight of negative control group x 100%

[0087] 5) Statistical analysis

[0088] The results will be presented in the form of mean ± S.E.M. Comparison between two groups will be tested by Dunnett's multi-comparison test. If p < 0.05, it is considered to have statistically significant difference.

[0089] 6) Results

[0090] After administration, the tumor volume measurement results of different groups of mice are as follows:

[0091]

[0092] TGI 0 days 3 days 5 days 7 days 10 days 12 days 14 days G2 / -3.49％ 6.13％ 22.78％ 30.23％ 36.46％ 39.56％ G3 / 9.88％ 5.58％ 34.02％ 41.86％ 38.64％ 60.23％ G4 / 29.01％ 19.28％ 32.35％ 39.14％ 29.67％ 43.53％

[0093] The results show that the myricetin-based diselenide has obvious inhibitory effect on hepatocarcinoma tumor model in vivo, and the inhibitory effect of the 40 mg / kg dose group on tumor in vivo is better than that of the positive (cisplatin). By comparing the inhibitory effects of G3 and G4 (40 mg / kg, 20 mg / kg), the inhibitory effect on tumor is more significant as the administration dose increases.

[0094] The above merely provides an example of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the scope of claims of the present application.

Claims

1. The use of myricetin-based diselenyl ether in the preparation of a drug for treating tumors, wherein the tumor is bladder transitional cell carcinoma, breast cancer, renal cancer, melanoma, pancreatic cancer, or ovarian cancer.

2. The application as described in claim 1, characterized in that, The drug is used to treat or is administered to mammals.

3. The application as described in claim 2, characterized in that, The mammal in question is either a rodent or a human.

4. A method for inhibiting tumor cell growth in vitro without therapeutic effect, characterized in that, The procedure includes adding myricetin-based diselenyl ether to a tumor cell culture system to inhibit tumor cell growth, wherein the tumor is bladder transitional cell carcinoma, breast cancer, kidney cancer, melanoma, pancreatic cancer, or ovarian cancer.

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