Composition for targeted therapy of canine breast cancer and application thereof

Abstract

The invention discloses a composition for targeted therapy of canine breast cancer and application of the composition. The composition comprises W6134 and XY018 in a molar ratio of 1: (0.5-2). The novel composition of W6134 and XY018 provided by the invention is used for targeted therapy of canine breast cancer, the occurrence of canine breast cancer is relieved to a certain extent, if a large amount of composition is put into actual production and use, the loss caused by canine breast cancer can be directly rescued, and especially when the molar ratio of W6134 and XY018 is 1: 1, the composition shows extremely strong cancer cell killing ability.

Description

Technical Field

[0001] The invention relates to a therapeutic drug for canine breast cancer, and in particular to a composition for targeted treatment of canine breast cancer and an application thereof. Background Art

[0002] Canine mammary tumors (CMTs) are a group of highly heterogeneous tumors. Cancer cells break through the mammary basement membrane through local infiltration and spread to the surrounding fat, muscle and connective tissue, destroying the structural integrity of adjacent tissues, seriously disrupting the body's normal physiological functions, and posing a great threat to dog health. In intact female dogs, CMTs account for up to 50% to 70% of tumors, and their incidence varies from region to region. In countries where sterilization surgery is not common, the incidence of mammary tumors in female dogs is even three times that of human women. Historical data show that about half of CMTs are malignant.

[0003] Breast cancer is the most common malignancy in both female dogs and women, and is the leading cause of cancer-related death in women worldwide. Over the past two decades, significant progress has been made in the treatment of HBC, with treatment strategies gradually shifting to more precise molecular targeted personalized therapies while striving to reduce the side effects of radiotherapy and chemotherapy.

[0004] Although surgery is the first-line treatment for both HBC and CMC, the adjuvant treatment strategies for the two are different. In HBC, adjuvant therapy has become routine, while in CMC, the effectiveness of adjuvant chemotherapy is still uncertain. This results in a relatively high mortality rate in CMC, with more than 40% of affected dogs dying within one year of diagnosis. Therefore, there is an urgent need to explore new treatment options and targeted therapies. Summary of the invention

[0005] In response to the problem of lack of effective targeted therapy for canine breast cancer, the present invention provides a composition for targeted treatment of canine breast cancer and its application. The composition alleviates the occurrence of canine mammary tumors to a certain extent. If it is put into actual production and use in large quantities, it can directly save the losses caused by canine breast cancer.

[0006] In order to achieve the above object, the present invention provides a composition for targeted treatment of canine breast cancer, comprising W6134 and XY018 in a molar ratio of 1: (0.5-2).

[0007] The chemical structures of W6134 and XY018 used in the present invention are shown below:

[0008] W6134

[0009] XY018 W6134 and XY018 are two specific nuclear receptor RORγ inhibitors. It has been proven that they can effectively induce autophagic death of canine breast cancer cells. The substances themselves have no obvious toxic side effects and are easy to use.

[0010] Preferably, the molar ratio of W6134 and XY018 is 1: 1. Compared with the use of W6134 and XY018 alone, the composition at this ratio exhibits extremely strong cytotoxicity to canine breast cancer cells and has extremely strong killing ability.

[0011] The second aspect of the present invention provides the use of the above-mentioned composition in the preparation of a drug for treating canine breast cancer.

[0012] The above composition needs to be diluted when used, and can be diluted by mixing it with water, a salt solution of water or an oil phase or more using DMSO. The oil phase is one or two of DMSO and corn oil.

[0013] Specifically, when using the drug for treatment, the dosage of W6134 is 1~2 mg / kg (body weight).

[0014] Through the above technical solution, the present invention achieves the following beneficial effects: The novel W6134 and XY018 composition proposed by the present invention is used for targeted treatment of canine breast cancer, which alleviates the occurrence of canine mammary tumors to a certain extent. If it is put into actual production and use in large quantities, it can directly save the losses caused by canine breast cancer. Especially when the molar ratio of the two is 1:1, it shows a very strong cancer cell killing ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The absorbance at 450 nm under 7 different treatments was detected by the CCK8 kit prepared in Example 1; Figure 2 Cell counts per milliliter were performed for the seven different treatments; Figure 3 To observe the cell growth of different treatment groups at 24h under microscope; Figure 4a To screen the differentially expressed genes among the four treatments and transcriptome analysis, KEGG enrichment analysis of the autophagy pathway after W6134 and XY018 treatment was performed; Figure 4b To screen the differentially expressed genes among the four treatments and to analyze the transcriptome, GO enrichment analysis of the autophagy pathway after W6134 and XY018 treatment was performed; Figure 4c To screen the differentially expressed genes among the four treatments and to analyze the transcriptome, GSEA enrichment analysis of the autophagy pathway after W6134 and XY018 treatment was performed; Figure 5 The microscopic observation of the autophagy of cells in different treatment groups at 24 h. Figure 6 To regularly record the weight changes of mice within 28 days; Figure 7 This is the change in the average daily food intake of mice within 28 days; Figure 8 The figure shows the changes in mouse tumor weight within 28 days. DETAILED DESCRIPTION

[0016] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0017] Example 1 This example verifies the role of W6134 and XY018 in killing cancer cells: CCK8 cell proliferation toxicity detection, cell counting, microscopy observation, transcriptome analysis, and cell autophagy staining were performed respectively, and the cells used were all CMT-U27 (canine breast cancer cells). The cell count plating density during the experiment was 8×10 4 / mL absorbance, the experiment used 96-well plates, and the initial cell density was 5000 cells / well.

[0018] For the above experiment, the following seven treatments were performed: control group, W6134 treatment group (1 μM), XY018 treatment group (1 μM) and W6134 and XY018 combined treatment group for 24 h (final molar concentrations were 1 μM:0.5 μM, 1 μM:1 μM, 1 μM:1.5 μM, 1 μM:2 μM).

[0019] The data obtained is as follows Figure 1~Figure 5 shown.

[0020] like Figure 1 As shown, the combination of W6134 and XY018 showed higher cytotoxicity, and the combined treatment effect was most significant when the molar ratio was 1:1.

[0021] like Figure 2 As shown, adding only 1μM and 1μM of W6134 and XY018 showed strong cytotoxicity, but the combined group had better effect, especially when used in a 1:1 combination, killing cancer cells by 80%, with extremely strong killing ability.

[0022] like Figure 3As shown, microscopic observation more intuitively reflects the cytotoxic effects of W6134 and XY018. When the final concentrations of W6134 and XY018 are 1 μM and 1 μM, cell death increases significantly.

[0023] The differentially expressed genes and transcriptome analysis of the four treatments were performed in the control group, W6134 treatment 24h group (1μM), XY018 treatment 24h group (1μM) and W6134 and XY018 combined treatment group (final concentrations of 1μM and 1μM). The cell autophagy pathway was significantly enriched after W6134 and XY018 treatment. Figure 4a~Figure 4c It can be seen that among the three enrichment analyses of KEGG, GO, and GSEA, KEGG and GO analyses performed up-regulation and down-regulation enrichment analyses, and GSEA analysis showed that the autophagy pathway was significantly up-regulated, and the common differentially expressed genes of the four groups were significantly enriched in the autophagy signaling pathway.

[0024] like Figure 5 As shown, microscopic observation more intuitively reflects that cellular autophagy is activated, and the combined treatment group of W6134 and XY018 (final concentrations of 1μM and 1μM) has a significant effect.

[0025] Example 2 In this example, a mixed solution of W6134 and XY018 was prepared and intragastrically administered: Establishment of mouse breast cancer model: Prepare 6-8 week-old immunodeficient mice, culture breast cancer cells in the logarithmic growth phase with viability greater than 90%, and count and adjust the cell density to 1×10 6 -5×10 6 Matrigel reagent was mixed with cell suspension at a ratio of 4:1 (Matrigel: cell suspension), and 100 μL was injected subcutaneously into the mammary gland of mice. Six replicates were set up in one group.

[0026] Dilution: Dilute W6134 and XY018, and crush them for about 5 to 10 minutes using a non-contact ultrasonic cell crusher to improve the dissolution efficiency and make the dissolution more complete.

[0027] Place the sterile EP tube on the balance and weigh it to zero. Add 6.4 μL of W6134 and XY018 solutions diluted with DMSO to the EP tubes, with concentrations of 1 mg / kg (mouse body weight) and 1.07 mg / kg, respectively. Mix well and add 200 μL of saline and corn oil to 500 μL, and mix again. Fix the mouse, extract 100 μL of the mixed solution with a gavage needle, insert the needle into the back of the mouse tongue, perform gavage and continue treatment for 28 days.

[0028] Figure 6In order to regularly record the weight changes of mice within 28 days, there are 4 treatments, namely the control group (tumor group), W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg, and W6134 and XY018 combined treatment group (W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg), to illustrate the tumor inhibition effect. Figure 6 As shown in the figure, the weight change of mice reflects that the tumor inhibition effect of the combination is more significant. Day 1 is the first day of drug administration after the tumor model is successfully established, which is manifested by obvious masses. Over time, the weight of mice in the combined use group of W6134 and XY018 increased significantly, indicating that they are in good health.

[0029] Figure 7 The figure shows the changes in the average daily food intake of mice within 28 days. There are 4 treatments, namely the control group (tumor group), W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg, and W6134 and XY018 combined treatment group (W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg). Figure 7 As shown, the average daily food intake of mice reflects that the tumor inhibition effect of the combination is significant. Day 1 is the first day of drug administration after the tumor model is successfully established, which is manifested by obvious lumps. As time goes by, the mice in the W6134 and XY018 combination group have a good appetite, indicating that they are in good health.

[0030] Figure 8 The changes in tumor weight of mice within 28 days were shown in 4 treatments, namely the control group (tumor group), W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg, and W6134 and XY018 combined treatment group (W6134 added at 1 mg / kg, XY018 added at 1.07 mg / kg), which were used to intuitively illustrate the tumor inhibition effect. Figure 8 As shown, the combination of W6134 and XY018 significantly reduced tumor weight.

[0031] In summary, the addition of W6134 and XY018 described in the present invention can cause toxic effects on cells, and the combination of W6134 and XY018 (molar ratio of 1:1) has a better effect.

[0032] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0033] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0034] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A composition for targeted treatment of canine breast cancer, characterized in that: It includes W6134 and XY018 in a molar ratio of 1:(0.5~2).

2. The composition according to claim 1, characterized in that It includes W6134 and XY018 in a molar ratio of 1:

1.

3. Use of the composition according to claim 1 or 2 in the preparation of a medicament for treating canine breast cancer.

4. The use according to claim 3, characterized in that: When the drug is used for treatment, the dosage of W6134 is 1-2 mg / kg.

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