CN116602948A - Compound and composition for reducing drug resistance of bladder cancer cells and application of compound and composition - Google Patents
Compound and composition for reducing drug resistance of bladder cancer cells and application of compound and composition Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Urology & Nephrology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application provides a novel biguanide derivative capable of inhibiting bladder cancer, which has the structure as follows:wherein R is 1 Is C 5 Saturated alkyl of R 2 An electron withdrawing group substituted for F, cl, br, I or F, cl, br, I. The minimum concentration of metformin required for inhibiting tumor is 2mM, and conventional oral administration is adopted for the metformin, and the administration mode can lead to local effective drug concentration of bladder to be lower than therapeutic concentration, so that the effect of inhibiting bladder cancer growth cannot be achieved, and oral administration of dimethylBiguanides cause an increase in the lactic acid content in the blood, resulting in lactic acidosis. The novel biguanide derivative inhibits the drug resistance of bladder cancer cells, so that the sensitivity of the bladder cancer cells to therapeutic drugs such as octreotide and the like is improved, and the effective treatment of the bladder cancer cells is promoted.
Description
Technical Field
The application relates to a compound and a composition for treating bladder cancer cells and application thereof, in particular to a compound and a composition for reducing drug resistance of bladder cancer cells and application thereof.
Background
Bladder cancer refers to a malignant tumor occurring on the mucosa of the bladder, and is the most common malignant tumor of the urinary system. According to global cancer data statistics reported by WHO in 2020, the incidence of bladder cancer is the tenth, and new cases and mortality in men are the first ten. Clinical data continues to grow with the incidence of bladder cancer. Bladder cancer is classified into a non-myogenic invasive type and a myogenic invasive type. In recent years, various treatment methods are endless, and the main treatment methods at present are surgical treatment such as laser, electrotomy, total bladder excision and the like, but all the treatment methods have higher recurrence rate and serious adverse reaction. And the patient also needs to keep drug infusion after the operation treatment to prevent recurrence. And since 30 years ago bcg immune conservation therapy was developed, few new therapies have been discovered. And global is currently facing the problem of BCG shortage. It is therefore important to find new drugs for the treatment of bladder cancer.
Biguanides based on metformin are the first-line drug for treating type 2 diabetes clinically at present, and in recent years, the biguanides have remarkable anti-tumor effect in-vitro experiments of tumor cells and in mouse experiments. Although there are a number of experimental results currently demonstrating that metformin can significantly treat bladder cancer, there are studies showing the prognostic impact of metformin in combination with the EGFR channel blocker gefitinib on NSCLC in combination with type 2 diabetics. The experimental result shows that the metformin prolongs the survival period and delays the development of gefitinib acquired drug resistance. The metformin combined with erlotinib also has good inhibition effect on human lung cancer cells. However, the clinical treatment of bladder cancer with metformin has not been approved so far. The most important reason is that the minimum concentration required by the metformin to inhibit the tumor is 2mM, and the conventional oral administration is adopted for the metformin, so that the effective drug concentration in the local part of the bladder is lower than the therapeutic concentration, the effect of inhibiting the growth of bladder cancer cannot be achieved, and the lactic acid content in blood is increased by oral administration of the metformin, so that lactic acid poisoning is caused.
Disclosure of Invention
The application 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.
The application provides a novel biguanide derivative capable of inhibiting bladder cancer, which has the structure as follows:
wherein R is 1 Is C 5 Saturated alkyl of R 2 An electron withdrawing group substituted for F, cl, br, I or F, cl, br, I.
Further, the R 1 Is C 5 Straight chain saturated alkyl groups of (a).
Further, the R 2 The substitution position of (C) is para to the benzene ring.
Further, the R 2 Is F substituted alkoxy.
Further, the R 2 Is trifluoromethoxy.
In another aspect, the application also discloses a composition for treating bladder cancer, comprising the novel biguanide derivative and at least one drug for treating bladder cancer.
Further, the composition further comprises octreotide.
Still further, the molar ratio of the octreotide to the novel biguanide derivative is (0.1-10): 1
The third aspect of the application also discloses the use of the above composition for the treatment of bladder cancer.
Further, the bladder cancer is EGFR-highly expressed bladder cancer.
Compared with the prior art, the application adopts the compound with the metformin structure to inhibit the drug resistance of bladder cancer cells, thereby improving the sensitivity of the bladder cancer cells to therapeutic drugs such as the octreotide and the like, and promoting the effective treatment of the bladder cancer cells.
Drawings
Fig. 1 shows the inhibition of J82, T24 bladder cancer cell lines by 5C at various concentrations (< P <0.05, < P <0.01, < P <0.001, n=3);
fig. 2 shows the inhibition of J82, T24 bladder cancer cell lines by octenib at various concentrations (< P <0.05, < P <0.01, < P <0.001, n=3);
FIG. 3 shows the combined effect of MTT assay 5C with octreotide in T24, J82 bladder cancer cells;
FIG. 4 is a graph showing the single use and combination of 5C and Orientinib with EGFR expression.
FIG. 4 shows the protein expression of p-EGFR, p-Akt, p-Erk1/2 and β -actin in each group of bladder cancer cells. P <0.05, P <0.01, P <0.001. N=3).
Detailed Description
In order that those skilled in the art will better understand the present application, a technical solution of the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments.
The embodiment of the application discloses a novel biguanide derivative capable of inhibiting bladder cancer, which has the structure as follows:
wherein R is 1 Is C 5 Saturated alkyl of R 2 An electron withdrawing group substituted for F, cl, br, I or F, cl, br, I.
Optionally, the R 1 Is C 5 Straight chain saturated alkyl groups of (a).
Optionally, the R 2 The substitution position of (C) is para to the benzene ring.
Optionally, the R 2 Is F substituted alkoxy.
In particular, said R 2 Is trifluoromethoxy.
The specific chemical formula of the novel biguanide derivative capable of inhibiting bladder cancer is
The embodiment of the application also discloses a composition for treating bladder cancer, which comprises the compound and at least one drug for treating bladder cancer.
Optionally, the composition comprises octreotide.
Wherein, the person skilled in the art can use other similar inhibitors besides the Ornitinib based on actual needs, and can also use various inhibitor mixed medicines including the Ornitinib.
The embodiment of the application also discloses application of the composition, and the composition is used for treating bladder cancer.
Alternatively, the bladder cancer is EGFR-highly expressed bladder cancer.
Among these, bladder cancer is specifically EGFR-highly expressed bladder cancer.
To further illustrate the specific effects of embodiments of the present application, the following will apply(hereinafter referred to as 5C) as an example, comparative experiments were conducted as follows.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
The test on bladder cancer cells by taking the compound 5C of the embodiment of the application shows that the 5C has obvious inhibition effect on J82 and T24 cancer cells as shown in figure 1. The results of testing the bladder cancer cells by using the octreotide are shown in figure 2, and the octreotide has obvious inhibition effect on J82 and T24 cancer cells.
Example 1: in vitro antitumor combined action of 5C and octenib on bladder cancer cells
The MTT method is respectively arranged on the T24 cell strain and the J82 cell strain of the bladder cancer, and the 5C cell strain and the Ornitinib cell strain are combined, so that the in vitro anti-tumor effect of the combined combination is compared. 5C selected 5 concentrations, octenib selected 5 concentrations, and CI values were calculated.
The specific operation is as follows:
s101, adding 10% fetal bovine serum solution into a 5A culture medium, and inoculating T24 and J82 cells into a 96-well plate at a density of 6000 cells/hole/180 mu L;
s102, at 37 ℃,5% CO 2 Culturing for 24 hours under the condition;
s103, diluting the medicines with different concentrations with fresh culture medium, and adding the test liquid medicine into the culture plate with the cells according to the addition amount of 20 mu L per hole; wherein, a single group containing only the octenib and a combined group containing the octenib and 5C are adopted, the combined group has two large groups, the specific concentration of 5C in the two large groups is 1 mu M and 2 mu M respectively, the concentration of the octenib in the solution is adjusted, and at least three compound holes are arranged in each concentration to reduce experimental errors so as to test the inhibition of the single group and the combined group on cells under different concentrations; a control group (no cell added with medicine) and a blank group (no cell added with medicine and no medicine added with only culture medium) are additionally arranged;
s104, at 37 ℃,5% CO 2 After 72h of incubation under the conditions of (2) MTT reagent (2 mg/mL) was added at 50. Mu.L/well volume, followed byIncubation is continued for 5h;
s105, removing the culture medium, adding 150 mu L of dimethyl sulfoxide, shaking and uniformly mixing for 15min, detecting the OD value of each hole by an enzyme-labeling instrument, and detecting the wavelength of 490nm; from the OD value readings, cell viability was calculated and IC was calculated using Grapad Prism 6 50 The method comprises the steps of carrying out a first treatment on the surface of the The CI value is calculated using CompuSyn software.
As shown in FIG. 3, 5C and Ornitinib have strong synergistic inhibition in bladder cancer cells, and CI < 0.5, indicating strong synergy.
Example 2: the anti-tumor effects of 5C and Ornitinib alone and in combination were compared in bladder cancer cells J82 and T24 using a clonogenic assay
S201, respectively inoculating 1500T 24 cells and J82 cells into a 24-hole plate, uniformly mixing 0.5mL of culture solution, and arranging 3 compound holes in each group;
s202, culturing for 24 hours, diluting the medicine with fresh culture medium after the cells are attached, and adding 500 mu L of the medicine into the culture plate with the added cells; the concentration of the single group containing only 5C was 1. Mu.M, the concentration of the single group containing only octenib was 1. Mu.M, and the combined group was formed by mixing 250. Mu.L of 5C+1. Mu.L of octenib having a concentration of 10. Mu.M, and the administration time was set to 7 days;
s203, at 37 ℃,5% CO 2 Culturing continuously for 8 days under the condition, and stopping culturing until the clone grows to 1-2 mm in diameter;
s204, discarding the supernatant, washing with PBS for 2 times, adding 2mL of 10% paraformaldehyde into each hole, and fixing for 15min;
s205, removing the fixing solution, and dyeing for 15min by 0.5mL of crystal violet dye solution;
s206, washing off the dyeing liquid with clear water, airing at room temperature, photographing, detecting the light absorption value by adopting an enzyme-labeled instrument, and the wavelength is 550nm.
The results are shown in figure 3, where the two-drug combination further inhibited tumor cell proliferation compared to 5C or octreotide alone.
Example 3: western blot detection of expression level of p-EGFR and downstream signal channels thereof
The EGFR family is an important class of tyrosine kinase receptors. They control proliferation, differentiation and migration of cells by mediating extracellular signaling, and EGFR is highly expressed in various tumor tissues such as bladder cancer, head and neck cancer, lung cancer, intestinal cancer, etc. To further explore the antiproliferative mechanisms of 5C, octenib and combination drugs on bladder cancer, the present examples investigated their relationship to EGFR signaling pathways. The cell samples treated by different drugs are subjected to detection of proliferation-related proteins through WB experiments, and the results show that 5C and Ornitinib inhibit EGFR phosphorylation, and the combined effect of the two is more obvious. Meanwhile, research shows that abnormal activation of the downstream PI3K/Akt/mTOR pathway and RAS/RAF/Erk pathway of EGFR can promote the growth and proliferation of cells. The influence of the difference on Akt and Erk1/2 is further studied.
The test comprises the following specific steps:
s301, setting four groups (a 5C single group, an octenib single group and a 5 C+octenib combined group in sequence, wherein a non-drug treatment group is set as a control group), the concentration of the 5C single group at the cell level is 1 mu M, the concentration of the octenib single group is 1 mu M, and the 5 C+octenib combined group is formed by mixing 1 mu M of 5C solution and 1 mu M of octenib solution in the same volume;
s302, after the medicine is processed for a proper time (two medicines are added simultaneously), collecting cell extract proteins, and detecting the expression level of related proteins by western blot.
The results are shown in FIG. 4, which shows that both the compound 5C treated group and the Ornitinib can down-regulate the phosphorylation levels of Akt and Erk1/2, and the combined group can significantly down-regulate the phosphorylation levels of Akt and Erk1/2 (FIG. 4). These above results indicate that: 5C and octenib inhibit bladder cancer cell growth by down-regulating EGFR and its downstream signaling pathways.
In summary, the embodiment of the application selects the combination of the Ornitinib and 5C for use, and MTT detection on the influence of the MTT detection on the cell viability shows that the Ornitinib and 5C can jointly inhibit the proliferation of bladder cancer cells. The cloning result shows that the combination of the two medicines has more remarkable inhibition effect compared with the single medicine. WB assays have also been found to enhance inhibition of EGFR and its downstream pathway associated protein Akt, erk activation levels. The EGRF pathway was shown to play an important role in the inhibition of bladder cancer with octenib and 5C. In summary, 5C down-regulates EGFR and its downstream signaling pathways to inhibit bladder cancer cell growth, and the combination enhances the effects of both, suggesting that 5C in combination with octenib is a potential treatment regimen for bladder cancer.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the specific embodiments of the present application after reading the present specification, and these modifications and variations do not depart from the scope of the application as claimed in the pending claims.
Claims (10)
1. A novel biguanide derivative capable of inhibiting bladder cancer, wherein the compound has the structure:
wherein R is 1 Is C 5 Saturated alkyl of R 2 An electron withdrawing group substituted for F, cl, br, I or F, cl, br, I.
2. A novel biguanide derivative for inhibiting bladder cancer according to claim 1, wherein R is 1 Is C 5 Straight chain saturated alkyl groups of (a).
3. A novel biguanide derivative for inhibiting bladder cancer according to claim 1, wherein R is 2 The substitution position of (C) is para to the benzene ring.
4. A novel biguanide derivative for inhibiting bladder cancer according to any one of claims 1 to 3, wherein R 2 Is F substituted alkoxy.
5. A kind of cocoa according to claim 4Novel biguanide derivatives for inhibiting bladder cancer characterized in that R 2 Is trifluoromethoxy.
6. A composition for the treatment of bladder cancer, comprising the novel biguanide derivative of any one of claims 1 to 4 and at least one drug useful for the treatment of bladder cancer.
7. The composition for treating bladder cancer according to claim 6, wherein the composition further comprises octenib.
8. The composition for treating bladder cancer according to claim 7, wherein the molar ratio of the octenib to the novel biguanide derivative is (0.1-10): 1.
9. use of a composition according to any one of claims 6 to 8 for the treatment of bladder cancer.
10. The use of claim 9, wherein the bladder cancer is EGFR-highly expressed bladder cancer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110357795A (en) * | 2019-07-26 | 2019-10-22 | 湖南师范大学 | A kind of Biguanide derivative, pharmaceutical composition, preparation method and its application in preparation of anti-tumor drugs |
| CN115947673A (en) * | 2022-12-19 | 2023-04-11 | 湖南岳靶生物医药有限公司 | Chemotherapeutics sensitizer, chemotherapeutics composition and application of chemotherapeutics sensitizer and chemotherapeutics composition |
| CN115957203A (en) * | 2022-12-19 | 2023-04-14 | 湖南岳靶生物医药有限公司 | Compound and composition for reducing drug resistance of bladder cancer cells and application of compound and composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110357795A (en) * | 2019-07-26 | 2019-10-22 | 湖南师范大学 | A kind of Biguanide derivative, pharmaceutical composition, preparation method and its application in preparation of anti-tumor drugs |
| CN115947673A (en) * | 2022-12-19 | 2023-04-11 | 湖南岳靶生物医药有限公司 | Chemotherapeutics sensitizer, chemotherapeutics composition and application of chemotherapeutics sensitizer and chemotherapeutics composition |
| CN115957203A (en) * | 2022-12-19 | 2023-04-14 | 湖南岳靶生物医药有限公司 | Compound and composition for reducing drug resistance of bladder cancer cells and application of compound and composition |
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