P-GP inhibitor synthetic compounds that increase chemotherapy sensitivity

Abstract

The invention relates to three synthetic compounds that inhibit drug efflux from cells due to P-glycoprotein (P-gp) encoded by the ABCB1 gene, whose expression is increased in cancer cells, and increase the sensitivity of cells to doxorubicin.

Description

[0001] P-GP INHIBITOR SYNTHETIC COMPOUNDS THAT INCREASE CHEMOTHERAPY SENSITIVITY

[0002] Technical Field

[0003] The invention relates to three new organic molecules that increase the amount of doxorubicin passing to target cells by inhibiting drug efflux from cells (through inhibition of the relevant protein) due to P-glycoprotein (P-gp) encoded by the ABCB1 gene, the expression of which is increased in cancer cells.

[0004] State of the Art

[0005] Drug resistance developed against chemotherapeutic agents is an important clinical problem in anti-cancer drug treatment. Natural (intrinsic) or acquired resistance, defined as "multidrug resistance (MDR)", causes failure in cancer chemotherapy. Drug resistance causes an increase in mortality and morbidity, a decrease in quality of life, and an increase in treatment costs in cancer patients. Chemotherapy requires the administration of one or more cytotoxic drugs and varies depending on the type of cancer. However, it is common for patients not to respond to treatment during chemotherapy. Tumor cells with multidrug resistance phenotype can be resistant to many drugs that differ structurally and functionally. Drug efflux out of the cell with ATP binding cassette (ABC) transporter protein pumps causes a decrease in intracellular drug accumulation and thus the toxic effects of the drug, and constitutes an important component of the MDR phenotype. ABC transporter protein pumps obtain the energy necessary for transportation by binding ATP. Among the species, their preserved structures consist of at least four parts: two transmembrane domains (TMD) and two nucleotide binding domains (NPD). Efflux pump P-glycoprotein (P-gp), which is encoded by the ABCB1 / MDR1 (multidrug resistance 1) gene in the ABC transporter protein family and is responsible for the elimination of harmful toxins under normal physiological conditions, is a transmembrane protein consisting of 1280 amino acids of 170 kDa. P-gp is found in normal intestinal, liver, kidney, placental tissues, and blood-brain barrier. A high amount of P-gp expression was detected in many hematological malignancies and tumors and was associated with the clinical course of the patients. P-gp protein interacts with more than one molecule depending on its physical structure, causing it to be effluxed out of the cell. This property of P-gp protein is defined as "polyspecificity" and has more than 300 substrates. Reversal of drug resistance due to P-gp protein is one of the important issues in the field of cancer research. Today, there are P-gp inhibitors classified as 3 generations. The first-generation modulators identified are calcium channel inhibitors (verapamil), calmodulin antagonists, steroidal agents, protein kinase C inhibitors, immunosuppressive agents (cyclosporine A), antibiotics (erythromycin), antimalarial agents (quinine), psychotropic phenothiazines and indole alkaloids (fluphenazine and reserpine), steroid hormones and anti-steroids (progesterone and tamoxifen), detergents (cremophore EL) and surfactants. Second-generation P-gp inhibitors were obtained by structural modification of valspodar (PSC 833), biricodar citrate (VX-710) and dexverapamil first-generation P-gp inhibitors. Quantitative structure-activity relationships (QSAR) analysis and combined chemistry approaches were used for the design of third generation P-gp inhibitors, depending on the inability of the first two generations of P-gp inhibitors to be included in clinical use and their disadvantages. Tariquidars (XR9576), zosuquidars (LY335979), laniquidars (R101933), and elacridar (F12091) are the most important compounds in their class. Tariquidars (XR9576) and elacridar (GF120918) are 3rd generation P-gp inhibitors that inhibit pump function specifically and effectively, at nanomolar concentrations, by showing high affinity for P-gp.

[0006] First-generation P-gp inhibitors have low affinity and low transporter selectivity. Their potential for clinical use is low due to their high toxicity and cardiovascular adverse effects at the concentrations required for effective P-gp inhibition. Second-generation and third-generation modulators are basically synthesized agents to reduce the high cytotoxic effect of first-generation modulators. Second-generation P-gp inhibitors have relatively high potential, P-gp selectivity and low toxicity. Thanks to QSAR approaches, the strength and specificity of third-generation P-gp inhibitors were further improved, and these inhibitors showed lower affinity for the specific cytochrome P450 family from liver enzymes than the concentrations requested by regulators such as the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA). Many different studies have shown that the resistance developed against drugs such as doxorubicin, vinblastine, and paclitaxel is reversed with 3rd generation inhibitors. However, in phase III clinical trials on patients with non-small cell lung cancer (NSCLC), tariquidars, one of the 3rd generation inhibitors, were found to be highly toxic. However, due to the high potential and effective properties of tariquidars, different molecules such as tariquidars were synthesized to optimize the reversal activity of multidrug resistance and reduce the toxic effect. For P-gp ABC transporter protein inhibition, synthetic or semi -synthetic modulators are gradually tried for problems such as high inhibition effect, metabolism by P450 and change in the pharmacokinetics of anti cancer agents.

[0007] Therefore, new synthetic compounds that inhibit drug efflux from cells due to P -glycoprotein (P-gp) encoded by the ABCB1 gene, whose expression is increased in cancer cells, and increase the sensitivity of cells to doxorubicin, are needed to overcome the abovementioned deficiencies.

[0008] The invention in the Turkish patent application No. 2023 / 019403, which is in the known state of the art, is a radioactive molecule used in the treatment of breast cancer, characterized in that said radioactive molecule is radioactive magnesium salts with changed Mg-24, Mg-25 and Mg-26 isotope ratios by exposure to radioactive radiation.

[0009] Brief Description of the Invention

[0010] The object of the present invention is to obtain three synthetic compounds that inhibit drug efflux from cells due to P-glycoprotein encoded by the ABCB1 gene, whose expression is increased in cancer cells, and increase the sensitivity of cells to doxorubicin.

[0011] Another object of the present invention is to realize synthetic compounds that reverse drug resistance and increase drug sensitivity by P-gp inhibition in doxorubicin-resistant breast cancer cells.

[0012] Detailed Description of the Invention

[0013] The present invention has bicyclic structures with P-gp inhibitor imidazole ring that inhibits drug efflux from cells due to P-glycoprotein encoded by the ABCB1 gene, whose expression is increased in cancer cells, and increases the sensitivity of cells to doxorubicin, that is, chemotherapy: Ml

[0014] They are synthetic compounds with molecular formula Ml, M2 and M3.

[0015] The synthetic compound with the molecular formula Ml, which is the subject of the invention, reduces cell viability by 50% in the concentration range of 530-760 pM in doxorubicin-resistant and sensitive breast carcinoma cells with active P-gp protein in 48 and 72 hours (inhibitory concentration 50; IC50). Ml molecular formula synthetic compounds have fluorescent properties (Ex: 488nm, Em: FL1; 530 / 30nm, FL2; 585 / 40nm, FL3; 670LP). The synthetic compound with the molecular formula Ml accumulates less in resistant cells with active P-gp protein than in sensitive cells. When the synthetic compound with the molecular formula Ml, P-gp protein inhibitor is used, its accumulation in resistant cells with P-gp protein increases. When the synthetic compound with the molecular formula Ml is administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment. The synthetic compound with the molecular formula Ml, when co-administered with doxorubicin for 14 days, reduces the ability of resistant cells to form colonies. The synthetic compound with the molecular formula Ml causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp at the end of 72 hours.

[0016] The synthetic compound with the molecular formula M2, which is the subject of the invention, reduces cell viability by 50% in the concentration range of 70-130 pM in doxorubicin-resistant and sensitive breast carcinoma cells in 48 and 72 hours (inhibitory concentration 50; IC50). The synthetic compound with the molecular formula M2 has a synergistic effect when co-administered with doxorubicin. The synthetic compound with the molecular formula M2 increases the intracellular accumulation of the P-gp substrate when administered to resistant cells with P-gp protein for 30 minutes (IC50). When the synthetic compound with the molecular formula M2 is administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment, and these results show that P-gp pump activity is prevented. The synthetic compound with the molecular formula M2, when co-administered with doxorubicin for 14 days, reduces the ability of resistant cells to form colonies. The synthetic compound with the molecular formula M2 causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp at the end of 72 hours.

[0017] The synthetic compound with the molecular formula M3, which is the subject of the invention, reduces cell viability by 50% in the concentration range of 240-420 pM in doxorubicin-resistant and sensitive breast carcinoma cells in 48 and 72 hours (inhibitory concentration 50; IC50). The synthetic compound with the molecular formula M3 has a higher cytotoxic effect in resistant cells than in sensitive cells. The synthetic compound with the molecular formula M3 has an 'additive' effect when co-administered with doxorubicin. The synthetic compound with the molecular formula M3 increases the intracellular accumulation of the P-gp substrate when administered to resistant cells with P-gp protein for 30 minutes (IC50). When the synthetic compound with the molecular formula M3 is administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment, and these results show that P-gp pump activity is prevented. The synthetic compound with the molecular formula M3, when coadministered with doxorubicin for 14 days, reduces the ability of resistant cells to form colonies. The synthetic compound with the molecular formula M3 causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp at the end of 72 hours.

[0018] It is possible to develop a wide range of applications for the "P-gp Inhibitor Synthetic Compounds Increasing the Sensitivity of Chemotherapy" of the invention around these basic concepts, and the invention cannot be limited to the examples described herein, mainly as specified in the claims.

Claims

CLAIMS1. Bicyclic structures comprising an imidazole ring that act as P-gp inhibitors, thereby preventing the efflux of drugs from cells via P-glycoprotein encoded by the ABCB1 gene, which is overexpressed in cancer cells, and increasing the sensitivity of cells to doxorubicin, i.e., chemotherapy;synthetic compounds characterized by having molecular formula Ml, M2 and M3.

2. The synthetic compound with the molecular formula Ml according to claim 1, characterized in that it reduces cell viability by 50% (inhibitory concentration 50; IC50) at concentrations ranging from 530 to 760 pM in 48- and 72-hour treatments in doxorubicin- resistant and sensitive breast carcinoma cells expressing active P-gp protein.

3. The synthetic compound with the molecular formula Ml according to claim 1 or 2, characterized in that it is fluorescent and accumulates less in resistant cells with active P- gp protein compared to sensitive cells.

4. The synthetic compound with the molecular formula Ml according to any one of the preceding claims, characterized in that, when the P-gp protein inhibitor is used, its accumulation in resistant cells with P-gp protein increases.

5. The synthetic compound with the molecular formula Ml according to any one of the preceding claims, characterized in that, when administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment.

6. The synthetic compound with the molecular formula Ml according to any one of the preceding claims, characterized in that, when co-administered with doxorubicin for 14 days, it reduces the ability of resistant cells to form colonies.

7. The synthetic compound with the molecular formula Ml according to any one of the preceding claims, characterized in that it causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp after 72 hours of administration.

8. The synthetic compound with the molecular formula M2 according to claim 1, characterized in that it reduces cell viability by 50% (inhibitory concentration 50; IC50) at concentrations ranging from 70 to 130 pM in 48- and 72-hour treatments in doxorubicin- resistant and sensitive breast carcinoma cells.

9. The synthetic compound with the molecular formula M2 according to claim 1 or 8, characterized in that, when co-administered with doxorubicin, it has a synergistic effect.

10. The synthetic compound with the molecular formula M2 according to any one of claims 1, 8 or 9, characterized in that, when administered to resistant cells with P-gp protein for 30 minutes (IC50), it increases the intracellular accumulation of the P-gp substrate.

11. The synthetic compound with the molecular formula M2 according to any one of claims 1 or 8 to 10, characterized in that, when administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment and P-gp pump activity is inhibited based on these results.

12. The synthetic compound with the molecular formula M2 according to any one of claims 1 or 8 to 11, characterized in that, when co-administered with doxorubicin for 14 days, it reduces the ability of resistant cells to form colonies.

13. The synthetic compound with the molecular formula M2 according to any one of claims 1 or 8 to 12, characterized in that it causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp after 72 hours of administration.

14. The synthetic compound with the molecular formula M3 according to claim 1, characterized in that it reduces cell viability by 50% (inhibitory concentration 50; IC50) at concentrations ranging from 240 to 420 pM in 48- and 72-hour treatments in doxorubicin- resistant sensitive breast carcinoma cells.

15. The synthetic compound with the molecular formula M3 according to claim 1 or 14, characterized in that it has a higher cytotoxic effect in resistant cells compared to sensitive cells.

16. The synthetic compound with the molecular formula M3 according to any one of claims 1, 14 or 15, characterized in that, when administered to resistant cells with P-gp protein for 30 minutes (IC50), it increases the intracellular accumulation of the P-gp substrate.

17. The synthetic compound with the molecular formula M3 according to any one of claims 1 or 14 to 16, characterized in that, when administered to the cells for 30 minutes, the accumulation of doxorubicin in the cells increases compared to the cells without treatment and P-gp pump activity is inhibited based on these results.

18. The synthetic compound with the molecular formula M3 according to any one of claims 1 or 14 to 17, characterized in that, when co-administered with doxorubicin for 14 days, it reduces the ability of resistant cells to form colonies.

19. The synthetic compound with the molecular formula M3 according to any one of claims 1 or 14 to 18, characterized in that it causes a decrease in the expression of the MDR1 / ABCB1 gene encoding P-gp after 72 hours of administration.

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