Use of Sodium Pentaborate Pentahydrate as a Chemotherapeutic Agent

Abstract

The present invention relates to the use of sodium pentaborate pentahydrate enriched with boron isotopes ( 10 B, 11 B) as a chemotherapeutic agent due to its anti-cancer effect against renal cancer. As a result of experimental studies conducted within the scope of the present invention, the number of renal cancer cells decreased by 50%, and this effect was caused by arresting the cell cycle and delaying cell proliferation. Therefore, it was shown that sodium pentaborate pentahydrate enriched with boron isotopes can be used as a chemotherapeutic agent in cancer treatment.

Description

Technical Field

[0001] The present invention relates to the use of sodium pentaborate pentahydrate enriched with boron isotopes ( 10 B, 11 B) as a chemotherapeutic agent with an anti-cancer effect against renal cancer cell lines. 10 B, 11 B) for use as a chemotherapeutic agent having an anti-cancer effect against renal cancer cell lines.

Background Art

[0002] The kidneys are a pair of organs located under the back of the upper abdomen and on both sides of the rib cage, and produce urine. The produced urine is transported to the bladder through two thin tubes called ureters. The main function of the kidneys is to remove harmful substances and excess water produced by metabolic activities through urine. It also plays a role in blood production and blood pressure regulation.

[0003] Renal cell carcinoma usually develops between the ages of 50 and 70, is three times more common in men than in women, and originates from the tissues of the kidneys that filter blood and produce urine. Progressive renal cancer most commonly spreads to organs such as the large intestine, liver, and pancreas, but can also metastasize to distant organs. Many factors such as smoking, obesity, hypertension, and dialysis can increase the risk of developing renal cancer.

[0004] Surgery is the standard method in the treatment of renal cancer, but the location, size, metastasis, and stage of the disease, as well as the patient's physical condition (age, weight, etc.) are evaluated for treatment, and one or more methods such as surgical treatment, radiotherapy, immunotherapy, and chemotherapy are used.

[0005] The use of chemotherapy to kill cancer cells, even if effective in many other cancers, has extremely limited effects on renal cancer. This has made the development of effective chemotherapeutic agents for the treatment of renal cancer important. Chemotherapy is used not only to reduce the size and metastasis of tumors before surgery, but may also be used after surgery to kill remaining cancer cells. It can also be used alone as an effective anti-cancer drug.

[0006] In all prior art studies, boron in the form of boric acid and phenylboronic acid has been used, and its effect on renal cancer has not been studied so far. Within the scope of the present invention, a sodium pentaborate pentahydrate derivative enriched with boron isotopes ( 10 B, 11 B) was used to increase its effectiveness and conduct research on its effectiveness against renal cancer. Within the scope of this patent, it is desired to protect the selective anti-cancer effect of the sodium pentaborate pentahydrate derivative enriched with boron isotopes ( 10 B, 11 B) against cancer cells and its use in single or combined treatments.

[0007] As a known technical application, WO2020020086014 (Yeditepe University) discloses the use of lead nanoparticles in the treatment of cancer. The boron derivative of this patent is in the form of a salt, and the boron derivative in that patent is a heavy metal compound. There are no similarities in terms of manufacturing techniques and mechanisms of action.

Summary of the Invention

[0008] An object of the present invention is to reduce the number of renal cancer cells by 50% through the anti-cancer activity of sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ( 10 B, 11 B) as a chemotherapeutic agent against renal cancer cells.

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

Modes for Carrying Out the Invention

[0010] The use of "sodium pentaborate pentahydrate as a chemotherapeutic agent" implemented to achieve the object of the present invention is described in the accompanying drawings.

Brief Description of the Drawings

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[0012] The present invention relates to the use of sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ([ 10 B, 11 B) as a chemotherapeutic agent for the treatment of cancer (renal cancer) (in particular, renal cancer cells are described within the scope of the present invention) due to its anti-cancer activity against cancer cells. Within the scope of the experimental studies conducted during the development of the present invention, sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ( 10 B, 11 B) was observed to delay the cell cycle and cell proliferation in cancer cells. 10 B, 11 B) 10 B, 11 B)

Example

[0013] (Experimental study) In the experimental studies conducted within the scope of the present invention to determine the activity of sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ( 10 B, 11 B) used as a chemotherapeutic agent within the scope of the present invention, the following were each carried out: 10 B, 11 B) · Weigh powdered sodium pentaborate pentahydrate at the desired concentration per 1 mL, dissolve it in the medium for growing cells, sterilize it by filtering through a 0.22 μm filter immediately before the start of the experiment, and make the solution fresh. · Add the solution separately 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 500 μg / ml, 1000 μg / ml, 2500 μg / ml, 5000 μg / ml, 7000 μg / ml for healthy renal cells (RPTEC). · Incubate at 37 °C, with an air ratio of 5% (v / v) CO 2 and 95% (v / v) from 0 hours to 72 hours at the end. · Perform a colorimetric tetrazolium (MTS) viability assay at the end of 24, 48, and 72 hours (days 1, 2, and 3). · Determine the half-maximal inhibitory concentration (IC50) at the end of 72 hours. Apply the determined IC50 value to healthy cells (RPTEC) and cancer cells (ACHN and A498) under the above incubation conditions for 72 hours, and evaluate apoptosis and the cell cycle at the end of 72 hours.

[0014] The chemotherapeutic agent of the present invention contains sodium pentaborate pentahydrate and exhibits an apoptotic effect on renal cancer cells. Sodium pentaborate pentahydrate has a molecular weight of 295.107 g / mol and a chemical formula of B 5 H 10 NaO 13It is a compound. The effective dose of NaB against renal cancer was determined by a colorimetric tetrazolium (MTS) viability assay on renal cancer cells (ACHN and A498) and healthy renal cells (RPTEC). MTS (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) is a colorimetric assay based on a tetrazolium salt. In this assay, the enzyme activity that reduces MTS to purple formazan is measured. The cell culture incubated for 2 hours is read by measuring the absorbance at a wavelength of 490 nm using ELISA. The NaB compound is dissolved in a medium suitable for the cells used at various 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 500 μg / ml, 1000 μg / ml, 2500 μg / ml, 5000 μg / ml, 7000 μg / ml for healthy renal cells), vortexed for 30 seconds after the addition of the substance, filtered through a 0.22 μm filter, and sterilized. The MTS assay is performed at 24, 48, and 72 hours, and as a result, the concentration of dead cells at the IC50 is determined, and the next step is continued at these doses. The doses (TD median) that killed 50% of renal cancer cells at 24, 48, and 72 hours are shown in Table 1 and Figures 1, 2, and 3. As 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 the A498 and ACHN cell lines, respectively (Figures 1 and 2). In the case of the RPTEC cell line, it was observed to be harmless to healthy cells, and a dose of 1550 μg / ml, which is the highest dose determined for cancer cells, was found to be appropriate for use (Figure 3).

Table 1

[0015] Determine whether the mortality of renal cancer cells treated with the drug at the dose determined by the MTS assay is dependent on apoptosis by the annexin V assay. 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 moves to the outer surface of the cell membrane. This translocation does not disrupt the integrity of the cell membrane. Annexin V binds to the PS translocated to the outer surface of the cell, thereby visualizing apoptotic cells and measuring them by flow cytometry. Since annexin V binding can also be observed on the surface of necrotic cells, propidium iodide (red fluorescence), which stains only dead cells, is added as a second stain. Cells simultaneously stained with annexin V-FITC (green fluorescence) and propidium iodide (red fluorescence) can distinguish viable cells (FITC-PI-), early apoptotic cells (FITC+PI-), and late apoptotic or necrotic cells (FITC+PI+) from each other. Analyze the cells labeled with the annexin V-FITCH conjugate marker by flow cytometry.

[0016] Measure the stage of cell division of renal cancer cells treated with the drug at the dose determined by the MTS assay by flow cytometry after propidium iodide staining. Propidium iodide selectively binds between the bases in nucleic acids and serves as a mediator molecule between the base and the fluorescent molecule. The assay is performed based on the fluorescence intensity. For example, cells that are not dividing or are preparing for division (i.e., G1 phase) have 1 copy of DNA and thus fluoresce at 1-fold intensity, while cells in the dividing G2 / M phase have 2 copies of DNA and thus fluoresce at 2-fold intensity.

[0017] Cell proliferation analysis of renal cancer cells treated with drugs at doses determined by MTS assay is performed by EdU (ethynyl-2'-deoxyuridine) assay. EdU staining binds to newly formed replicated DNA molecules during the DNA synthesis phase, enabling a quantitative comparison of cell populations in the DNA synthesis phase as a result of NaB treatment. In this assay, 10 μM of EdU is applied to cells cultured on slides placed in 6-well plates. After incubation, the cells are fixed with 3.7% formaldehyde diluted in PBS for 15 minutes and then washed with 3% BSA diluted in PBS. Cell membrane permeability is increased by treating with 0.5% non-ionic surfactant diluted in PBS at room temperature for 20 minutes. After a final wash with 3% BSA solution, the reaction cocktail is added to the cells. The cells are incubated at room temperature for 30 minutes, and EdU-stained cells actively synthesizing DNA are observed with a confocal microscope.

[0018] In this study, the effect of sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ( 10 B, 11 B) as a chemotherapeutic agent against renal cancer cells was investigated in a laboratory environment. Compounds of sodium pentaborate pentahydrate enriched with boron isotopes ( 10 B, 11 B) were prepared at various concentrations and applied to media containing healthy and cancer cells, and the effects on viability, cell cycle, and proliferation were observed.

[0019] Consistent with the results obtained, it was observed that cancer cells were selectively induced to programmed cell death (Figs. 4 - 6), and in addition to this effect, the cell cycle was arrested (Figs. 7 - 9) and cell proliferation was slowed (Fig. 10). Therefore, it was shown that sodium pentaborate pentahydrate enriched with boron isotopes ( 10 B, 11 B) can be used as a chemotherapeutic agent in the treatment of cancer, sarcoma, leukemia, multiple myeloma, lymphoma, melanoma, brain and spinal cord tumors, germ cell tumors, and neuroendocrine tumors.

Claims

1. Sodium pentaborate pentahydrate (NaB) enriched with boron isotopes ( 10 B, 11 B) for use as a chemotherapeutic agent for the treatment of cancer by anti-cancer activity.

2. Sodium pentaborate pentahydrate (NaB) enriched with the boron isotope ( 10 B, 11 B) according to claim 1 for the treatment of renal cancer.

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