CN114028401B

CN114028401B - Application of pratenstat and fluorouracil in preparation of anti-tumor pharmaceutical composition

Info

Publication number: CN114028401B
Application number: CN202111502344.9A
Authority: CN
Inventors: 汪洋; 何庆瑜; 梁晓玲
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-09-29
Filing date: 2021-12-09
Publication date: 2023-02-14
Anticipated expiration: 2041-12-09
Also published as: CN114028401A

Abstract

The invention discloses application of pratenstat and fluorouracil in preparation of an anti-tumor pharmaceutical composition. The research of the invention finds that the combination of the two drugs of the pratenstat and the fluorouracil can inhibit the growth of colorectal cancer, provides a new application of the combination of the two drugs of the pratenstat and the fluorouracil, and provides a new drug source for the adjuvant therapy of cancer.

Description

Application of pratenstat and fluorouracil in preparation of anti-tumor pharmaceutical composition

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of pratenstat and fluorouracil in preparation of an anti-tumor medicine composition.

Background

Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs) are essential enzymes that regulate the dynamic balance of histone acetylation and deacetylation. Dysregulation of histone acetylation and deacetylation is associated with the progression of cancer. Aberrant expression of HDACs is associated with a variety of cancer types, including intestinal cancer, gastric cancer, and acute myeloid leukemia. The pratenstat is a novel oral hydroxyhydroxamate HDACI, has good pharmacokinetic, physicochemical and pharmaceutical properties, and has the following structural formula:

fluorouracil is a pyrimidine analog type of antimetabolite with broad activity against solid tumors, either alone or in combination with chemotherapeutic regimens. Fluorouracil is a novel multi-targeted oral drug for the treatment of tumors, and is considered to be one of the most effective chemotherapeutic drugs for the treatment of CRC. However, it is immediately after oral administration capable of being metabolized by dihydropyrimidine dehydrogenase in the intestinal wall, greatly hindering its efficacy. In addition, clinical application of fluorouracil has not been fully utilized due to drug resistance of tumor cells. In addition, due to the high metabolic rate of fluorouracil, large doses need to be administered continuously and regularly. For these reasons, intravenous fluorouracil is more suitable for clinical patients than oral treatment, while reducing adverse drug reactions, such as severe cardiac, neurological and gastrointestinal side effects. The structural formula is shown as the figure:

there are reports showing that preclinical and clinical studies demonstrate anti-tumor activity of pratenstat in hematological malignancies. It has also been reported in the literature that pratenstat is able to inhibit breast cancer cell growth.

At present, no article exists for showing the function of the combination of the two drugs of the pralatte and the fluorouracil in the treatment of colorectal cancer, and no patent application exists.

Disclosure of Invention

The invention aims to provide a new application of pratenstat and fluorouracil in preparing an anti-tumor drug composition, and provides a new drug source idea for the adjuvant therapy of cancer. The invention combines in vitro and in vivo experiments, and the result shows that the combination of the two drugs of the pratenstat and the fluorouracil can inhibit the growth of colorectal cancer.

In order to achieve the purpose, the invention adopts the following technical scheme: application of pratenstat and fluorouracil in preparing antitumor pharmaceutical composition is provided.

Preferably, the anti-tumor pharmaceutical composition is an anti-colorectal cancer pharmaceutical composition.

Preferably, the mass part ratio of the pratenstat to the fluorouracil in the anti-tumor pharmaceutical composition is 2.5: (2-4).

Preferably, the pratenstat and fluorouracil exert an antitumor effect by inhibiting clonogenic capacity and cellular activity of colorectal cancer cells, and inducing apoptosis in colorectal cancer cells.

Preferably, the anti-tumor pharmaceutical composition is an oral preparation or an injection preparation.

Preferably, the oral formulations include tablets, capsules, powders, granules and film-coated tablets.

Preferably, the injection preparation includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavity injection formulations.

Compared with the prior art, the invention has the following beneficial effects:

the invention detects the colorectal cancer cell activity, proliferation, apoptosis and the like through a WST-1 cytotoxicity experiment, a clone formation and a cell apoptosis flow detection experiment, and proves that the combined use of the pratenstat and the fluorouracil can effectively inhibit the colorectal cancer cell activity, promote the apoptosis of the colorectal cancer cell and finally achieve the effect of effectively treating the colorectal cancer. Meanwhile, in-vivo animal experiments prove that the combination of the pratenstat and the fluorouracil can obviously enhance the capability of inhibiting colorectal cancer cell neoplasia, and does not generate drug toxicity to organisms.

Drawings

FIG. 1 is a drawing; schematic diagram of the influence of the combination of two drugs of pralatta and fluorouracil on the cloning of colorectal cancer cells.

FIG. 2 is a diagram: schematic representation of the effect of combination of both pratenstat and fluorouracil on colorectal cancer cell proliferation.

FIG. 3 is a diagram of: schematic representation of the effect of combination of both pratenstat and fluorouracil on apoptosis of colorectal cancer cells.

FIG. 4 shows: schematic representation of the effect of combination of both pratenstat and fluorouracil on the tumorigenicity of colorectal cancer cells in nude mice.

FIG. 5 is a diagram: schematic representation of HE staining of heart, liver, spleen, lung and kidney tissues of nude mice after the combination of two drugs of pratenstat and fluorouracil.

FIG. 6 is a diagram of: the result of biochemical detection of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in serum of nude mice is shown schematically after the combination of two drugs of Prelatta and fluorouracil.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

The apoptosis test uses Annexin V-FITC apoptosis kit for detection, and all reagents are provided by Kaikyi bio corporation kit.

Example 1 in vitro experiments

1. Experimental method

Colorectal cancer cells (HCT 116 and HT29 cells) are treated by different drug combinations, and then the cell activity is detected by clone formation and apoptosis and a WST-1 cytotoxicity test detection experiment, so that the influence of the drugs of different combinations on the proliferation of the liver cancer cells is researched.

1.1 clone formation experiment: cultured HCT116, HT29 cells were seeded into 6-well plates, with three replicate wells of approximately 500 cells per well, and after adherent growth of cells, 0.5 μ M of pratenstat, 0.4 μ M of fluorouracil, and 0.5 μ M of pratenstat in combination with fluorouracil 0.4 μ M were added to each replicate well. After the cells grow to form macroscopic cloning points, the cells are taken out and gently rinsed twice by PBS, paraformaldehyde is added for fixation, and 0.01% crystal violet dye is used for dyeing for 15min. After the crystal violet dye on the cell surface was washed away with water gently, a scan photograph was taken with a scanner.

1.2WST-1 cytotoxicity assay: cultured HCT116, HT29 cells were seeded into 96-well plates in triplicate wells of about 3000 cells per well, in a control blank well group without added cells in medium, a trestat group (0.25 μ M), a fluorouracil group (0.2 μ M and 0.4 μ M), and a pharmaceutical composition group (trestat 0.25 μ M + fluorouracil 0.2 μ M and trestat 0.25 μ M + fluorouracil 0.4 μ M). The activity of the cells was measured by CCK-8 assay, the cells after the drug treatment were taken out at 24h and 48h time points, WST-1 was added to the cultured cells at 10% working concentration, incubated at 37 ℃ for 1.5h, and absorbance was measured at 450nm with a microplate reader.

1.3 apoptosis assay: HCT-116, HT29 cells were seeded into 6-well plates (5X 105 cells/well), treated with different concentrations of Prelata, fluorouracil, and cells, including culture supernatant cells, were collected after 48 hours of incubation. Washing twice by PBS, centrifuging at 4 ℃,800g and 3min to remove supernatant, adding 500 mu l Binding buffer to resuspend cells, adding 5 mu l Annexin V-FITC dye, incubating for 15min in a dark place at room temperature, adding 5 mu l PI dye before loading, gently mixing, analyzing signals of Annexin V-FITC and PI of the stained cells by a flow cytometer, and analyzing the obtained result by Flowjo software to obtain the result of the apoptosis rate.

2. Results of the experiment

2.1 clonogenic and toxicity experiments on cells

The experimental results are shown in figures 1-2, the combination of pratenstat and fluorouracil has better effects of inhibiting the clone formation of colorectal cancer cells and inhibiting the activity of colorectal cancer cells, and compared with single-component pratenstat and single-component fluorouracil, the combined drug of pratenstat and fluorouracil provided by the invention has more excellent capabilities of inhibiting the clone formation of colorectal cancer cells and inhibiting the activity of cells.

2.2 apoptosis assay

The results of the experiment are shown in fig. 3, table 1 and table 2.

TABLE 1 colorectal cancer HCT116 apoptosis test results

TABLE 2 colorectal cancer HT29 apoptosis assay results

From fig. 3, table 1 and table 2, it can be seen that the combination of two drugs of pratenstat and fluorouracil can effectively increase the apoptosis rate of colorectal cancer cells, and simultaneously, compared with the single-component administration of pratenstat and fluorouracil, the apoptosis rate of colorectal cancer cells is greatly increased.

Example 2 animal experiments

1. Experimental methods

1. 24 female nude mice (balb/c-nu/nu) with age of 6 weeks, 6 control groups and 18 experimental groups are selected to construct a subcutaneous tumor model: (1) Colorectal cancer cells HT29 were resuspended in PBS buffer and mixed with matrigel at 1 ⁶ (ii) HT29 cells; (2) Before the experiment, a nude mouse is anesthetized, the anesthesia degree is evaluated through painless and painful stimulation, and the nude mouse is determined to be in an anesthetic state; (3) The resuspended cells were then removed by a 25G needle microinjector and the nude mice were injected intraperitoneally. (4) drug treatment: ten days after subcutaneous injection, administration was started by intraperitoneal injection. In the experiment, 3 drug combinations are set, namely, 25mg/kg of pratenstat, 20mg/kg of fluorouracil and 25mg/kg of pratenstat plus 20mg/kg of fluorouracil, and 6 drugs are respectively administered twice per week. Prlestat is dissolved in 5% Tween 80+10% DMSO +30% polyethylene glycol. Fluorouracil was dissolved in PBS. Each mouse was dosed with 200. Mu.L each time. Tumor size and nude mouse body weight were measured for each administration. After 3 weeks, the nude mice were euthanized and the tumors were removed.

HE staining

2.1 Paraffin section preparation: the nude mouse transplanted tumor was first isolated and fixed with 4% paraformaldehyde for 2 days, and then water in the tumor tissue was gradually removed by an automatic dehydrator according to ethanol of low to high concentration, and then the tumor tissue was cleaned by soaking in xylene as a clearing agent. The treated tumor tissue blocks were immersed in paraffin at 58 ℃ and then cooled and embedded. The thickness of the paraffin section was kept between 5 and 6 μm to complete the patch, which was then dried in an oven at 45 ℃ to remove moisture.

2.2 dyeing: firstly, staining the section with hematoxylin for 5min, washing with running water for 10min, performing color separation with 1% hydrochloric acid alcohol until the cell nucleus is blue, and soaking in 1% ammonia alcohol. Then, the mixture was dehydrated and stained with 0.5% eosin alcohol for 5min. And then, removing water of the dyed slices step by using an automatic dehydrator according to ethanol with low concentration to high concentration, dehydrating by using dimethylbenzene, finally sealing the slices by using neutral resin, and performing microscopic examination, photographing and archiving.

2. Results of the experiment

The results of the experiment are shown in fig. 4 and 5. Fig. 4 shows the experimental results of subcutaneous tumor formation in nude mice, and it can be known from the experimental results of the pharmaceutical composition group of pratenstat and fluorouracil and the blank control group in fig. 4 that the pharmaceutical composition of pratenstat and fluorouracil of the present invention can effectively inhibit the growth of tumor cells, and simultaneously has stronger ability of inhibiting the tumor formation of colorectal cancer cells in nude mice compared with single component of pratenstat and fluorouracil. FIG. 5 is a schematic view of HE staining of heart, liver, spleen, lung and kidney tissues of nude mice, and FIG. 6 is a biochemical assay of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in sera of nude mice. From FIG. 5 and FIG. 6, it can be seen that there is no abnormal change in the tissues of the heart, liver, spleen, lung and kidney of nude mice after the combination of the two drugs of Prelatta and fluorouracil, and further, it is proved that the combined use of the Prelatta and fluorouracil of the present invention does not generate drug toxicity.

The experiments can show that the invention carries out the detection of the activity, proliferation, apoptosis and the like of the colorectal cancer cells through a WST-1 cytotoxicity experiment, a clone formation and apoptosis flow detection experiment, and proves that the invention can effectively inhibit the activity of the colorectal cancer cells and promote the apoptosis of the colorectal cancer cells by jointly using the pratenstat and the fluorouracil, finally achieves the effect of effectively treating the colorectal cancer, simultaneously carries out corresponding animal experiments, and further proves that the invention can obviously enhance the capability of inhibiting the colorectal cancer cell from forming tumor by jointly using the pratenstat and the fluorouracil, and does not generate drug toxicity to organisms.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. The application of the pralatte and the fluorouracil in preparing an anti-tumor pharmaceutical composition, wherein the anti-tumor pharmaceutical composition is an anti-colorectal cancer pharmaceutical composition;

the mass part ratio of the pralatta to the fluorouracil in the anti-tumor drug composition is 2.5: (2-4).

2. The use of claim 1, wherein the anti-tumor pharmaceutical composition is an oral formulation or an injectable formulation.

3. Use according to claim 2, wherein the oral formulation is selected from the group consisting of tablets, capsules, powders and granules.

4. The use according to claim 2, wherein the injectable formulation is selected from the group consisting of intravenous, intramuscular, subcutaneous, intradermal and intracavitary formulations.