A composition for targeted therapy of canine mammary carcinoma and use thereof

Abstract

The application discloses a composition for targeted treatment of canine breast cancer and application thereof, and the composition comprises W6134 and XY018 in a molar ratio of 1:(0.5-2). The new W6134 and XY018 composition provided by the application is used for targeted treatment of canine breast cancer, and to some extent, the occurrence of canine breast tumors is relieved. If the composition is put into actual production and use in a large amount, the loss caused by canine breast cancer can be directly saved, and especially when the molar ratio of the two is 1:1, the composition exhibits extremely strong cancer cell killing capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to a therapeutic drug for canine mammary carcinoma, in particular, to a composition for targeted treatment of canine mammary carcinoma and application thereof. BACKGROUND

[0002] Canine mammary tumors (CMTs) are a group of tumors with high heterogeneity, and cancer cells break through the basement membrane of the mammary gland by local infiltration, spread to the surrounding fat, muscle and connective tissue, destroy the integrity of the adjacent tissue structure, and seriously disrupt the normal physiological function of the body, posing a great threat to the health of dogs. In unspayed female dogs, it accounts for up to 50% to 70% of tumor proportion, and its incidence rate varies by region, especially in countries where spaying surgery is not commonly performed, the incidence rate of female mammary tumors is even as high as three times that of human females. Historical data shows that about half of CMTs are malignant.

[0003] In female dogs and women, breast cancer is the most common malignant tumor, and is also the main cause of global female cancer-related deaths. In the past two decades, the treatment of HBC has made significant progress, and the treatment strategy has gradually shifted to more precise molecular targeted individualized treatment, while striving to reduce the side effects of radiotherapy and chemotherapy.

[0004] Although surgery is the common first choice for treatment of HBC and CMC, the adjuvant treatment strategies are different. In HBC, adjuvant therapy has become routine, while in CMC, the effectiveness of adjuvant chemotherapy is still inconclusive. This leads to a relatively high mortality rate of CMC, and more than 40% of dogs die within a year after diagnosis. Therefore, it is urgent to explore new treatment options and targeted therapies. SUMMARY

[0005] In view of the above problems that canine mammary carcinoma lacks effective targeted therapy, the present application provides a composition for targeted treatment of canine mammary carcinoma and application thereof, which alleviates the occurrence of canine mammary tumors to some extent, and if a large amount is put into actual production and use, the loss caused by canine mammary carcinoma can be directly saved.

[0006] In order to achieve the above purpose, the present application provides a composition for targeted treatment of canine mammary carcinoma, which comprises W6134 and XY018 in a molar ratio of 1: (0.5-2).

[0007] The chemical structures of W6134 and XY018 used in the present application are as follows:

[0008]

[0009] W6134

[0010]

[0011] XY018

[0012] W6134 and XY018 as two specific nuclear receptor ROR gamma inhibitors, have been proved to be able to effectively induce autophagic death of canine mammary cancer cells, and the substance itself has no obvious toxic side effects, and is convenient to use.

[0013] Preferably, W6134 and XY018 are included in a molar ratio of 1:1. The composition in this ratio, compared with W6134 and XY018 used alone, has very strong canine mammary cancer cell toxicity and very strong killing ability.

[0014] The second aspect of the present application provides the use of the above-mentioned composition in the preparation of a medicament for treating canine mammary cancer.

[0015] The above composition needs to be diluted when used, which can be diluted by mixing with water, a salt solution of water or one or several of the oil phases using DMSO. The oil phase is one or both of DMSO and corn oil.

[0016] Specifically, when the medicament is used for treatment, the amount of W6134 used is 1-2 mg / kg (body weight).

[0017] Through the above technical solution, the present application achieves the following beneficial effects:

[0018] A new W6134 and XY018 composition is proposed in the present application, which is used for targeted treatment of canine mammary cancer, and to some extent relieves the occurrence of canine mammary tumors. If a large amount is put into actual production and use, the loss caused by canine mammary cancer can be directly saved. Especially when the molar ratio of the two is 1:1, it shows very strong cancer cell killing ability. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 To detect the 450nm absorbance of 7 different treatments by CCK8 kit prepared in Example 1;

[0020] Figure 2 To count the cells per milliliter of 7 different treatments;

[0021] Figure 3 To observe the cell growth of different treatment groups at 24h time point under microscope;

[0022] Figure 4a To screen differentially expressed genes of 4 treatments and transcriptome analysis of autophagy pathway KEGG enrichment analysis after W6134 and XY018 treatment;

[0023] Figure 4bTo screen for differentially expressed genes under four treatments and to perform transcriptomic analysis, GO enrichment analysis was performed on the autophagy pathway after treatment with W6134 and XY018.

[0024] Figure 4c To screen for differentially expressed genes under four treatments and to perform transcriptomic analysis, GSEA enrichment analysis was performed on the autophagy pathway after treatment with W6134 and XY018.

[0025] Figure 5 To observe the autophagy of cells in different treatment groups under a microscope at a 24-hour time point;

[0026] Figure 6 To record the changes in mouse body weight over 28 days at regular intervals;

[0027] Figure 7 The changes in average daily food intake of mice over 28 days;

[0028] Figure 8 This shows the changes in tumor weight in mice over 28 days. Detailed Implementation

[0029] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0030] Example 1

[0031] This embodiment verifies the roles of W6134 and XY018 in the process of killing cancer cells:

[0032] CCK8 cell proliferation and toxicity assays, cell counting, microscopic observation, transcriptome analysis, and autophagy staining were performed. All cells used were CMT-U27 (canine breast cancer cells). The cell seeding density during the experiments was 8 × 10⁶ cells / cm². 4 The absorbance was / mL, and the experiment was conducted using 96-well plates with an initial cell density of 5000 cells / well.

[0033] For the above experiment, the following 7 treatments were performed: control group, W6134 treatment group for 24 h (1 μM), XY018 treatment group for 24 h (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).

[0034] The obtained data is as follows Figures 1-5 As shown.

[0035] like Figure 1As shown, the combined use of W6134 and XY018 exhibited high cytotoxicity, with the combined treatment at a molar ratio of 1:1 showing the most significant effect.

[0036] like Figure 2 As shown, the addition of only 1 μM and 1 μM of W6134 and XY018 exhibited strong cytotoxicity, but the combined use was even more effective, especially when used in a 1:1 ratio, which killed up to 80% of cancer cells, demonstrating extremely strong killing ability.

[0037] like Figure 3 As shown, microscopic observation more directly reflects the cytotoxic effects of W6134 and XY018. At final concentrations of 1 μM and 1 μM for W6134 and XY018, cell death significantly increased.

[0038] Differentially expressed genes and transcriptome analyses were performed on four treatments: control group, W6134-treated group (1 μM), XY018-treated group (1 μM) for 24 h, and combined W6134 and XY018 treatment group (final concentrations of 1 μM and 1 μM). The autophagy pathway was significantly enriched after W6134 and XY018 treatment. Figures 4a-4c As can be seen from the three enrichment analyses of KEGG, GO, and GSEA, KEGG and GO analyses performed upregulation and downregulation enrichment analyses, while GSEA analysis showed that the autophagy pathway was significantly upregulated, and the common differentially expressed genes in the four groups were significantly enriched in the autophagy signaling pathway.

[0039] like Figure 5 As shown, microscopic observation more intuitively reflects the activation of autophagy, and the combined treatment group of W6134 and XY018 (final concentrations of 1 μM and 1 μM) showed significant effects.

[0040] Example 2

[0041] In this embodiment, a mixed solution of W6134 and XY018 was prepared and administered by gavage:

[0042] Establishment of a mouse breast cancer model: Immunodeficient mice aged 6-8 weeks were used. Breast cancer cells in the logarithmic growth phase with a viability greater than 90% were cultured and counted to adjust the cell density to 1×10⁻⁶. 6 -5×10 6 Cells / mL. Mix Matrigel reagent with cell suspension at a ratio of 4:1 (Matrigel: cell suspension) and inject 100 μL subcutaneously into the mammary gland of mice, with 6 replicates per group.

[0043] Dilution: Dilute W6134 and XY018 and then pulverize them for about 5-10 minutes using a non-contact ultrasonic cell disruptor to improve dissolution efficiency and make dissolution more complete.

[0044] Weigh sterile EP tubes on a balance and zero them. Add 6.4 μL of W6134 and XY018 solutions diluted with DMSO to each EP tube, with concentrations of 1 mg / kg (mouse body weight) and 1.07 mg / kg, respectively. Mix well, then add 200 μL of physiological saline and corn oil to a final volume of 500 μL. Mix again. Fix the mice, draw 100 μL of the mixture into a gavage needle, insert the needle into the back of the mouse's tongue, and administer the gavage. Continue this treatment for 28 days.

[0045] Figure 6 To record changes in mouse body weight over 28 days, four treatments were administered: a control group (tumor group), a W6134 supplementation level of 1 mg / kg, an XY018 supplementation level of 1.07 mg / kg, and a combined W6134 and XY018 treatment group (W6134 supplementation level of 1 mg / kg, XY018 supplementation level of 1.07 mg / kg), to elucidate the tumor-inhibiting effect. Figure 6 As shown, changes in mouse body weight reflect a more significant tumor-suppressive effect of the composition. Day 1, the first day of successful tumor model establishment and initial drug administration, is characterized by a noticeable tumor mass. Over time, mice in the W6134 and XY018 combined treatment group showed significant weight gain, indicating good health.

[0046] Figure 7 The study investigated the changes in average daily food intake in mice over 28 days. Four treatments were used: a control group (tumor group), a W6134 supplementation level of 1 mg / kg, an XY018 supplementation level of 1.07 mg / kg, and a combined W6134 and XY018 treatment group (W6134 supplementation level of 1 mg / kg, XY018 supplementation level of 1.07 mg / kg). Figure 7 As shown, the average daily food intake of mice reflects the significant tumor-suppressive effect of the composition. Day 1, the first day of successful establishment of the tumor model and the start of drug administration, showed obvious tumors. Over time, the mice in the W6134 and XY018 combined treatment group had good food intake, indicating that they were in good health.

[0047] Figure 8 To illustrate the changes in tumor weight in mice over 28 days, four treatments were used: a control group (tumor group), a W6134 supplementation level of 1 mg / kg, an XY018 supplementation level of 1.07 mg / kg, and a combined W6134 and XY018 treatment group (W6134 supplementation level of 1 mg / kg, XY018 supplementation level of 1.07 mg / kg). These treatments were designed to visually demonstrate the tumor-inhibiting effect. Figure 8 As shown, the combined use of W6134 and XY018 significantly reduced tumor weight.

[0048] In summary, the addition of W6134 and XY018 described in this invention can cause cytotoxic effects, and the combined effect of W6134 and XY018 (molar ratio of 1:1) is even better.

[0049] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0050] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0051] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A composition for targeted therapy of canine mammary cancer, characterized in that, The mixture includes W6134 and XY018 in a molar ratio of 1:(0.5~2), and the chemical structures of W6134 and XY018 are shown below: W6134， XY018.

2. The composition according to claim 1, characterized in that, This includes W6134 and XY018 with 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 mammary cancer.

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