Use of sodium pentaborate pentahydrate as chemotherapeutic agent

Abstract

The present invention relates to the use of a sodium pentaborate pentahydrate rich in boron isotope (10B, 11B) as a chemotherapeutic agent due to its anti-cancer effect on kidney cancer. Results of experimental studies carried out within the range of the invention show that although the number of kidney cancer cells is reduced by 50%, the effect occurs by retarding the cell cycle and slowing down cell proliferation, so that the boron isotope-rich sodium pentaborate pentahydrate can be used as a chemotherapeutic agent in cancer treatment.

Description

Technical Field

[0001] The present invention relates to a boron isotope-rich 10 B. 11 B) Use of sodium pentaborate pentahydrate as a chemotherapeutic agent due to its anticancer effect on renal cancer cell lines. Background Art

[0002] The kidneys are a pair of organs located in the upper abdomen below and behind the chest on either side that produce urine. The urine produced is transferred to the bladder through two thin tubes (called ureters). The main function of the kidneys is to remove harmful substances produced by metabolic activities and excess water through urine. The kidneys also play a role in blood production and blood pressure regulation.

[0003] Renal cell carcinoma usually develops between the ages of 50 and 70 and is three times more common in men than in women. Renal cell carcinoma begins in the tissues of the kidneys that filter blood and produce urine. Although growing kidney cancer most often spreads to organs such as the colon, liver, and pancreas, it can also metastasize to distant organs. Many factors, such as smoking, obesity, high blood pressure, and dialysis can increase the risk of kidney cancer.

[0004] Although surgery is the standard treatment for kidney cancer, treatment requires evaluation of the tumor's location, size, metastasis, stage of the disease, and the patient's physical condition (age, weight, etc.), using one or more methods such as surgery, radiation therapy, immunotherapy, and chemotherapy.

[0005] Using chemotherapy to kill cancer cells has very limited effects against kidney cancer, although it is effective against many other cancers. This makes it critical to develop effective chemotherapeutic drugs for the treatment of kidney cancer. In addition to being used before surgery to reduce the size and metastasis of a tumor, chemotherapy can also be used after surgery to kill any remaining cancer cells. Chemotherapy can also be used alone as an effective anticancer agent.

[0006] All studies in the prior art used boron in the form of boric acid and phenylboronic acid, and their effects on renal cancer have not been previously investigated. Within the scope of the present application, boron isotopes enriched in boron ( 10 B. 11 B) of sodium pentaborate pentahydrate derivatives, improve its efficacy, and study its efficacy against renal cancer. Within the scope of this patent, it is expected that the boron isotope-rich ( 10 B. 11 B) provides protection for the selective anticancer effect of sodium pentaborate pentahydrate derivatives on cancer cells and their use in single or combined therapy.

[0007] WO 2020020086014 (Yeditepe University, known technical application) discloses the use of lead nanoparticles in the treatment of cancer. The boron derivatives of this patent are in the form of salts, and the boron derivatives in the patent are heavy metal compounds. There are no similarities in production technology and mechanism of action. Summary of the invention

[0008] The purpose of the present invention is to enrich the boron isotope ( 10 B. 11 B) Anticancer activity of sodium pentaborate pentahydrate (NaB) as a chemotherapeutic agent against renal cancer cells, reducing the number of renal cancer cells by 50%.

[0009] Another object of the present invention is to use sodium pentaborate pentahydrate (NaB) to treat renal cancer by delaying the cell cycle and slowing down the cell proliferation of cancer cells. DETAILED DESCRIPTION

[0010] The use of sodium pentaborate pentahydrate as a chemotherapeutic agent for the purpose of the present invention is illustrated in the accompanying drawings, wherein:

[0011] Figure 1 is a graphical illustration of the effect of sodium pentaborate pentahydrate on the viability of ACHN renal cancer cells (colorimetric tetrazolium (MTS) viability assay).

[0012] Figure 2 Is a graphical illustration of the effect of sodium pentaborate pentahydrate on the viability of A498 renal cancer cells (colorimetric tetrazolium (MTS) viability assay).

[0013] Figure 3 Is a graphical illustration of the effect of sodium pentaborate pentahydrate on the viability of RPTEC healthy kidney cells (colorimetric tetrazolium (MTS) viability assay).

[0014] Figure 4 is a graphical illustration of the effect of sodium pentaborate pentahydrate on ACHN renal cancer cell death via apoptosis (Annexin V assay).

[0015] Figure 5 is a graphical illustration of the effect of sodium pentaborate pentahydrate on A498 renal cancer cell death via apoptosis (Annexin V assay).

[0016] Figure 6 is a graphical illustration of the effect of sodium pentaborate pentahydrate on RPTEC healthy kidney cell death via apoptosis (Annexin V assay).

[0017] Figure 7is a graphical illustration of the effect of sodium pentaborate pentahydrate on cell division of ACHN renal cancer cells (propidium iodide staining) (*p<0.05).

[0018] Figure 8 is a graphical illustration of the effect of sodium pentaborate pentahydrate on cell division of A498 renal cancer cells (propidium iodide staining) (*p<0.05, ***p<0.0001).

[0019] Fig. 9 is a graphic illustration of the effect of sodium pentaborate pentahydrate on cell division of RPTEC healthy kidney cells (propidium iodide staining).

[0020] Fig.10 is a microscopic illustration of the effect of sodium pentaborate pentahydrate on the proliferation of ACHN and A498 renal cancer cells (EdU staining, confocal microscopy images).

[0021] The present invention relates to a boron isotope-rich 10 B. 11 B) as a chemotherapeutic agent for the treatment of cancer (renal cancer) through its anticancer activity against cancer cells (in particular renal cancer cells discussed within the scope of the present invention). Within the scope of experimental studies carried out during the development of the present invention, it has been observed that boron isotopes ( 10 B. 11 B) sodium pentaborate pentahydrate (NaB) delays the cell cycle and slows cell proliferation in cancer cells.

[0022] Experimental studies

[0023] In experimental studies carried out within the scope of the present invention, in order to determine the boron isotope-enriched ( 10 B. 11 B) of sodium pentaborate pentahydrate (NaB), respectively, the following:

[0024] - Weigh sodium pentaborate pentahydrate powder at the desired concentration / mL, dissolve it in the medium in which the cells are grown, and sterilize it by filtering through a 0.22 μm filter immediately before starting the experiment so that the solution is fresh,

[0025] - adding the solution at a ratio of 500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 2500 μg / ml, 3000 μg / ml, 3500 μg / ml for renal cancer cells (ACHN, A498), and at a ratio of 500 μg / ml, 1000 μg / ml, 2500 μg / ml, 5000 μg / ml, 7000 μg / ml for healthy renal cells (RPTEC),

[0026] - incubation at 37°C with 5% (v / v) CO2 and 95% (v / v) air from hour 0 to hour 72,

[0027] - Colorimetric tetrazolium (MTS) viability assay at the end of 24, 48 and 72 hours (day 1, 2 and 3),

[0028] - Determination of the half maximal inhibitory concentration (IC50) at the end of 72 hours,

[0029] - The IC50 values ​​determined for cancer cells were applied to healthy cells (RPTEC) and cancer cells (ACHN and A498) under the above incubation conditions for 72 hours and apoptosis and cell cycle were evaluated at the end of 72 hours.

[0030] The chemotherapeutic agent of the present invention comprises sodium pentaborate pentahydrate and exhibits apoptotic effect on renal cancer cells. Sodium pentaborate pentahydrate has a molecular weight of 295.107 g / mol and a chemical formula of B5H 10 NaO 13 Compound. The effective dose of NaB for renal cancer was determined by colorimetric tetrazolium (MTS) viability assay for renal cancer cells (ACHN and A498) and healthy kidney cells (RPTEC). MTS (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxyl-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) is a colorimetric assay based on tetrazolium salts. The assay measures the enzyme activity that reduces MTS to purple formazan. The cell cultures incubated for two hours were read by measuring the absorbance at a wavelength of 490nm by ELISA. The NaB compound was dissolved in a culture medium suitable for the cells used (a 30-second vortex was applied after adding the agent) at different concentrations (at a ratio of 500 μg / ml, 1000 μg / ml, 1500 μg / ml, 2000 μg / ml, 2500 μg / ml, 3000 μg / ml, 3500 μg / ml for renal cancer cells, and at a ratio of 500 μg / ml, 1000 μg / ml, 2500 μg / ml, 5000 μg / ml, 7000 μg / ml for healthy renal cells), filtered through a 0.22 micron filter, and sterilized. The MTS assay was performed at 24, 48, and 72 hours, and the concentration of dead cells at the IC50 dose was determined accordingly, and the next step was continued at these doses. The doses (median TD) that killed 50% of renal cancer cells at 24, 48, and 72 hours are shown in Tables 1 and 2. Figure 1 , Figure 2 , Figure 3As a result of the assay, the doses that killed 50% of renal cancer cells were determined to be 1400 μg / ml (4.7 mM) and 1550 μg / ml (5.2 mM) for A498 and ACHN cell lines, respectively. Figure 1 and Figure 2 For the RPTEC cell line, a dose of 1550 μg / ml was found to be suitable for use, which was observed to be harmless to healthy cells and was the highest dose determined for cancer cells ( Figure 3 ).

[0031] Table 1. IC50 determined by MTS results performed at 24, 48 and 72 hours.

[0032] 24 hours 48 hours 72 hours ACHN >7000μg / ml 2000μg / ml 1550μg / ml A498 >7000μg / ml 1900μg / ml 1400μg / ml

[0033] Annexin V was used to determine whether the mortality rate of renal cancer cells treated with the agent of the dose determined by the MTS assay depends on apoptosis. In normal cells, phosphatidylserine (PS) (a type of lipid) is present on the surface of the cell membrane. During apoptosis, the PS present in the cell membrane is transferred to the outer surface of the cell membrane. This transfer does not destroy the integrity of the cell membrane. Annexin V binds to the PS transferred to the outer surface of the cell, making apoptotic cells visible and measured by flow cytometry. Since Annexin V binding can also be observed on the surface of necrotic cells, propidium iodide (red fluorescence) that only stains dead cells is added as a second stain. Cells stained with Annexin V-FITC (green fluorescence) and propidium iodide (red fluorescence) simultaneously allow living cells (FITC-PI-), early apoptotic cells (FITC+PI-) and late apoptotic or necrotic cells (FITC+PI+) to be distinguished from each other. Cells labeled with markers conjugated with Annexin V-FITCH were analyzed by flow cytometry.

[0034] After propidium iodide staining, the cell division phase of renal cancer cells treated with the agent determined by the MTS assay was measured by flow cytometry. Propidium iodide selectively binds between bases in nucleic acids, thereby becoming a mediator molecule between bases and fluorescent molecules. Measured based on fluorescence intensity. For example, cells that have not divided or are about to divide (i.e., in the G1 phase) have 1 copy of DNA, so they fluoresce at 1X intensity, while cells that are dividing in the G2 / M phase have 2 copies of DNA, so they fluoresce at 2X intensity.

[0035] Cell proliferation analysis of renal cancer cells treated with the dose of the agent determined by the MTS assay was performed by EdU (ethynyl-2'-deoxyuridine) assay. The EdU stain binds to the newly formed replicating DNA molecules during the DNA synthesis phase, enabling quantitative comparison of the cell populations at the DNA synthesis phase after NaB treatment. For this assay, 10uM EdU was applied to cells cultured on a slide placed in a 6-well plate. After incubation, the cells were fixed with 3.7% formaldehyde diluted in PBS for 15 minutes and then washed with 3% BSA also diluted in PBS. Cell membrane permeability was increased by treatment with 0.5% nonionic surfactant diluted in PBS for 20 minutes at room temperature. After finally washing with 3% BSA solution, the reaction mixture was added to the cells. The cells were incubated at room temperature for 30 minutes, and then EdU stained cells that were actively synthesizing DNA were observed under a confocal microscope.

[0036] In this study, boron isotope-enriched ( 10 B. 11 Effects of sodium pentaborate pentahydrate (NaB) as a chemotherapeutic agent on renal cancer cells. 10 B. 11 B) and applied it to culture medium containing healthy cells and cancer cells, and observed its effects on cell viability, cell cycle and proliferation.

[0037] According to the results obtained, it was observed that the compound selectively causes programmed cell death in cancer cells ( Figure 4-Figure 6 ), in addition to this effect, the compound also blocks the cell cycle ( Figure 7-Figure 9 ) and slow cell proliferation ( Fig.10 ). Therefore, it has been shown that boron isotope enrichment ( 10 B. 11 Sodium pentaborate pentahydrate of B) can be used as a chemotherapeutic agent for the treatment of cancer, sarcoma, leukemia, multiple myeloma, lymphoma, melanoma, brain and spinal cord tumors, germ cell tumors and neuroendocrine tumors.

Claims

1. A boron isotope-rich 10 B. 11 B) sodium pentaborate pentahydrate (NaB), is used as a chemotherapeutic agent for the treatment of cancer due to its anticancer activity.

2. The boron isotope-rich ( 10 B. 11 B) Sodium pentaborate pentahydrate (NaB) as a chemotherapeutic agent for the treatment of renal cancer.

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