A compound, composition and application thereof for reducing drug resistance of bladder cancer cells

Abstract

The present invention provides a novel biguanide derivative that can inhibit bladder cancer, and the compound structure is: #imgabs0# wherein R1 is a saturated alkyl group of C5, and R2 is an electron-withdrawing group substituted by F, Cl, Br, I or F, Cl, Br, I. The minimum concentration required for metformin to inhibit tumors is 2mM, and metformin is mostly administered orally in a traditional manner. This mode of administration will cause the effective drug concentration in the local bladder to be lower than the therapeutic concentration, and the effect of inhibiting the growth of bladder cancer cannot be achieved. In addition, oral administration of metformin will increase the lactic acid content in the blood, causing lactic acidosis. The novel biguanide derivative of the present invention inhibits the drug resistance of bladder cancer cells, thereby increasing the sensitivity of bladder cancer cells to therapeutic drugs such as osimertinib, thereby promoting the effective treatment of bladder cancer cells.

Description

Technical Field

[0001] The present invention relates to a compound, a composition and an application thereof for treating bladder cancer cells, and in particular to a compound, a composition and an application thereof for reducing the drug resistance of bladder cancer cells. Background Art

[0002] Bladder cancer refers to a malignant tumor that occurs on the bladder mucosa and is the most common malignant tumor of the urinary system. According to the 2020 global cancer data statistics reported by the WHO, the incidence of bladder cancer ranks tenth, and the new cases and mortality rates in men are both in the top ten. Clinical data As the incidence of bladder cancer continues to grow. Bladder cancer is divided into non-muscle invasive and muscle invasive types. In recent years, various treatment methods have emerged in an endless stream. At present, the main treatment measures are surgical treatment, such as laser, electroresection, total cystectomy, etc., but various treatment methods have a high recurrence rate and serious adverse reactions. And after surgical treatment, patients also need to insist on drug infusion to prevent recurrence. And since the development of BCG immunoconservative treatment more than 30 years ago, few new treatments have been discovered. And the world is currently facing a shortage of BCG. Therefore, it is very important to find new drugs to treat bladder cancer.

[0003] Biguanides, mainly metformin, are currently the first-line drugs for the clinical treatment of type 2 diabetes. In recent years, biguanides have been found to have significant anti-tumor effects in in vitro tumor cell experiments and mouse experiments. Although there are currently a large number of experimental results confirming that metformin can significantly treat bladder cancer, there are also studies showing the effect of metformin combined with EGFR channel blocker gefitinib on the prognosis of patients with NSCLC and type 2 diabetes. The experimental results show that metformin prolongs survival and delays the process of acquired resistance to gefitinib. Metformin combined with erlotinib also shows a good inhibitory effect on human lung cancer cells. However, so far, the clinical use of metformin for the treatment of bladder cancer has not been approved. The main reason is that the minimum concentration required for metformin to inhibit tumors is 2mM, and metformin is mostly administered orally. This mode of administration will make the local effective drug concentration in the bladder lower than the therapeutic concentration, and it cannot achieve the effect of inhibiting the growth of bladder cancer. In addition, oral metformin will increase the lactic acid content in the blood, causing lactic acidosis. Summary of the invention

[0004] The present invention provides a novel biguanide derivative capable of inhibiting bladder cancer, a composition and application thereof, so as to improve the sensitivity of bladder cancer to anticancer drugs.

[0005] The present invention provides a novel biguanide derivative capable of inhibiting bladder cancer, wherein the compound structure is:

[0006] Where R1 C 5 The saturated alkyl group, R 2 It is an electron withdrawing group substituted with F, Cl, Br, I or F, Cl, Br, I.

[0007] Furthermore, the R 1 C 5 A straight chain saturated alkyl group.

[0008] Furthermore, the R 2 The substitution position is the para position of the benzene ring.

[0009] Furthermore, the R 2 It is an alkoxy group substituted by F.

[0010] Furthermore, the R 2 The substituent is trifluoromethoxy.

[0011] Another aspect of the present invention discloses a composition for treating bladder cancer, comprising the novel biguanide derivatives mentioned above, and the composition further comprises at least one drug that can be used for treating bladder cancer.

[0012] Furthermore, the composition also includes osimertinib.

[0013] Furthermore, the molar ratio of osimertinib to the novel biguanide derivative is (0.1-10): 1

[0014] The third aspect of the present invention also discloses an application of the above composition, wherein the composition is used for treating bladder cancer.

[0015] Furthermore, the bladder cancer is EGFR highly expressed bladder cancer.

[0016] Compared with the prior art, the present invention uses a compound with a metformin structure to inhibit the drug resistance of bladder cancer cells, thereby increasing the sensitivity of bladder cancer cells to therapeutic drugs such as osimertinib, thereby promoting the effective treatment of bladder cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The inhibitory effects of 5C at different concentrations on J82 and T24 bladder cancer cell lines (*P<0.05, **P<0.01. ***P<0.001, n=3);

[0018] Figure 2 The inhibitory effects of osimertinib on J82 and T24 bladder cancer cell lines at different concentrations (*P<0.05, **P<0.01. ***P<0.001, n=3);

[0019] Figure 3MTT was used to detect the combined effect of 5C and osimertinib in T24 and J82 bladder cancer cells;

[0020] Figure 4 This is a graph showing the relationship between 5C and osimertinib used alone or in combination and EGFR expression.

[0021] in, Figure 4 The protein expressions of p-EGFR, p-Akt, p-Erk1 / 2 and β-actin in bladder cancer cells of each group (*P<0.05, **P<0.01. ***P<0.001. n=3). DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0023] The present invention discloses a novel biguanide derivative capable of inhibiting bladder cancer, wherein the compound structure is:

[0024] Where R 1 C 5 The saturated alkyl group, R 2 It is an electron withdrawing group substituted with F, Cl, Br, I or F, Cl, Br, I.

[0025] Optionally, the R 1 C 5 A straight chain saturated alkyl group.

[0026] Optionally, the R 2 The substitution position is the para position of the benzene ring.

[0027] Optionally, the R 2 It is an alkoxy group substituted by F.

[0028] In particular, the R 2 The substituent is trifluoromethoxy.

[0029] The specific chemical formula of the novel biguanide derivatives that can inhibit bladder cancer in the embodiment of the present invention is

[0030]

[0031] The embodiment of the present invention also discloses a composition for treating bladder cancer. The composition comprises the above compound and at least one drug that can be used for treating bladder cancer.

[0032] Optionally, the composition comprises osimertinib.

[0033] Among them, those skilled in the art may use other similar inhibitors besides osimertinib based on actual needs, or may use a mixed drug of multiple inhibitors including osimertinib.

[0034] The embodiment of the present invention also discloses an application of the above composition, wherein the composition is used for treating bladder cancer.

[0035] Optionally, the bladder cancer is EGFR highly expressed bladder cancer.

[0036] Among them, bladder cancer is specifically EGFR highly expressed bladder cancer.

[0037] To further illustrate the specific effects of the embodiments of the present invention, (hereinafter referred to as 5C) as an example, a comparative experiment is carried out as follows.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0039] The compound 5C of the present invention was tested on bladder cancer cells. The results are as follows: Figure 1 As shown in Figure 5C has a significant inhibitory effect on J82 and T24 cancer cells. Osimertinib was used to test bladder cancer cells. The results are as follows Figure 2 As shown, osimertinib has a significant inhibitory effect on J82 and T24 cancer cells.

[0040] Example 1: Antitumor effect of 5C and osimertinib on bladder cancer cells in vitro

[0041] The MTT method was used on bladder cancer T24 and J82 cell lines to compare the in vitro antitumor effects of 5C and osimertinib alone and in combination. Five concentrations of 5C and five concentrations of osimertinib were selected, and the CI values ​​were calculated.

[0042] The specific operations are as follows:

[0043] S101. Add 10% fetal bovine serum solution to 5A medium, and seed T24 and J82 cells in a 96-well plate at a density of 6000 cells / well / 180 μL;

[0044] S102. At 37°C, 5% CO 2 Cultured for 24 h under the same conditions;

[0045] S103. After diluting the drugs of different concentrations with fresh culture medium, the test drug solution was added to the culture plate with cells at an addition volume of 20 μL per well; wherein, a single-use group containing only osimertinib and a combination group containing osimertinib and 5C were used, and the combination group had two major groups, and the specific concentrations of 5C in the two major groups were 1 μM and 2 μM, respectively. The concentration of osimertinib in the solution was adjusted, and at least three replicate wells were set for each concentration to reduce experimental errors, so as to test the inhibitory effects of osimertinib on cells at different concentrations in the single-use group and the combination group; a control group (cells were added but no drug was added) and a blank group (no cells or drug was added, only culture medium was included) were also set up;

[0046] S104. At 37°C, 5% CO 2 After culturing for 72 h under the conditions of , MTT reagent (2 mg / mL) was added at a volume of 50 μL / well and incubated for another 5 h;

[0047] S105. Remove the culture medium, add 150 μL dimethyl sulfoxide, shake and mix for 15 min, and detect the OD value of each well with an ELISA reader at a wavelength of 490 nm. Calculate the cell survival rate based on the OD value readings, and use Grapad Prism 6 to calculate the IC 50 ; CI values ​​were calculated using CompuSyn software.

[0048] The results are as follows Figure 3 As shown, 5C and osimertinib have a strong synergistic inhibitory effect in bladder cancer cells, and the CI value is less than 0.5, indicating a strong synergistic effect.

[0049] Example 2: Comparison of the anti-tumor effects of 5C and osimertinib alone and in combination in bladder cancer cells J82 and T24 using a clone formation assay

[0050] S201. 1500 T24 and J82 cells were inoculated into 24-well plates respectively, mixed with 0.5 mL of culture medium, and 3 replicate wells were set up for each group;

[0051] S202. After culturing for 24 hours, after the cells have attached to the wall, dilute the drug with fresh culture medium and add 500 μL per well to the culture plate with cells added; the concentration of the single-use group containing only 5C is 1 μM, the concentration of the single-use group containing only osimertinib is 1 μM, and the combination group is composed of 250 μL of 5C with a concentration of 1 μM + 1 μL of osimertinib with a concentration of 10 μM, and the drug administration time is set to 7 days;

[0052] S203. At 37°C, 5% CO 2 The culture was continued for 8 days under the same conditions, and the culture was terminated when the clones grew to a diameter of 1-2 mm.

[0053] S204. Discard the supernatant, wash twice with PBS, add 2 mL of 10% paraformaldehyde to each well, and fix for 15 min;

[0054] S205. Remove the fixative and stain with 0.5 mL of crystal violet stain for 15 min;

[0055] S206. Wash off the staining solution with clean water, dry at room temperature, take a photo and use an enzyme-labeled instrument to detect the absorbance at a wavelength of 550nm.

[0056] The results are as follows Figure 3 As shown, compared with 5C or osimertinib alone, the combination of the two drugs further inhibited tumor cell proliferation.

[0057] Example 3: Western blot detection of the expression level of p-EGFR and its downstream signaling pathway

[0058] The EGFR family is an important class of tyrosine kinase receptors. They control cell proliferation, differentiation and migration by mediating the response of extracellular signals, and EGFR is highly expressed in various tumor tissues such as bladder cancer, head and neck cancer, lung cancer, and intestinal cancer. In order to further explore the anti-proliferation mechanism of 5C, osimertinib and combined medication for bladder cancer, the embodiment of the present invention studies its relationship with the EGFR signaling pathway. The cell samples treated with different drugs were tested for proliferation-related proteins by WB experiments. The results showed that both 5C and osimertinib inhibited the phosphorylation of EGFR, and the effect of the combination of the two was more significant. At the same time, studies have shown that abnormal activation of the downstream PI3K / Akt / mTOR pathway and RAS / RAF / Erk pathway of EGFR can promote cell growth and proliferation. The effects of different medications on Akt and Erk1 / 2 were further studied.

[0059] The specific steps of the test are as follows:

[0060] S301. Four groups were set up (the 5C alone group, the osimertinib alone group, the 5C + osimertinib combination group, and a drug-free group was set up as a control group), the concentration of the 5C alone group at the cellular level was 1 μM, the concentration of the osimertinib alone group was 1 μM, and the 5C + osimertinib combination group was prepared by mixing the same volume of 1 μM 5C solution and 1 μM osimertinib solution;

[0061] S302. After the drug treatment for an appropriate period of time (adding two drugs at the same time), the cells were collected to extract proteins, and the expression levels of related proteins were detected by western blot.

[0062] The results are as follows Figure 4 As shown, the results showed that both the compound 5C treatment group and osimertinib could downregulate the phosphorylation levels of Akt and Erk1 / 2, while the combination group could significantly downregulate the phosphorylation levels of Akt and Erk1 / 2 ( Figure 4 ). The above results indicate that 5C and osimertinib inhibit the growth of bladder cancer cells by downregulating EGFR and its downstream signaling pathways.

[0063] In summary, the embodiment of the present invention selects osimertinib and 5C for combined use, and the MTT test for the effect on cell viability shows that osimertinib can inhibit the proliferation of bladder cancer cells together with 5C. The cloning results show that the combination of the two drugs has a more significant inhibitory effect than single use. WB detection also found that the combination of the two can enhance the inhibition of the activation level of EGFR and its downstream pathway-related proteins Akt and Erk. It shows that the EGRF pathway plays an important role in the inhibition of bladder cancer by osimertinib and 5C. In short, 5C and osimertinib downregulate EGFR and its downstream signaling pathways to inhibit the growth of bladder cancer cells, and the combination can enhance the effects of the two, indicating that 5C combined with osimertinib is a potential treatment for bladder cancer.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that after reading the specification of this application, the technicians can still modify or replace the specific implementation mode of the present invention with equivalents, but these modifications or changes do not deviate from the scope of protection of the pending claims of the present application.

Claims

1. A composition for treating bladder cancer, characterized in that: The composition includes a biguanide derivative and osimertinib; The biguanide derivative structure is: The molar ratio of osimertinib to the biguanide derivative is (2-8): (1-2).

2. An application of the composition according to claim 1, characterized in that: The composition is used for preparing medicine for treating bladder cancer.

3. The application according to claim 2, characterized in that: The bladder cancer is EGFR highly expressed bladder cancer.

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