An anticancer preparation containing sodium pentaborate pentahydrate, curcumin and piperine for use in the treatment of hepatocellular carcinoma

Abstract

The present invention relates to a preparation containing sodium pentaborate, curcumin and piperine as active substances and its use in the treatment of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to a preparation containing sodium pentaborate, curcumin and piperine as active substances and its use in the treatment of hepatocellular carcinoma.

Background Art

[0002] Hepatocellular carcinoma is the most common primary malignant liver tumor, which originates from hepatocytes and usually occurs in the context of chronic liver diseases caused by viral hepatitis. It is the fifth most common cancer worldwide and the third most common cause of cancer death. It has been observed that the incidence and prevalence of hepatocellular carcinoma are rapidly increasing in our country along with the increase in chronic hepatitis [1-4]. The majority of cases of hepatocellular carcinoma occur in the context of chronic liver diseases, and cirrhosis is the first risk factor for hepatocellular carcinoma regardless of the etiology of liver diseases. It is estimated that one-third of cirrhotic patients will develop liver cancer during their lifetime, and an annual incidence rate of 1-8% has been reported in long-term follow-up studies. The incidence of hepatocellular carcinoma is seen to be lower in cirrhosis associated with alcoholic and non-alcoholic steatohepatitis than in active viral hepatitis, but its incidence is seen to be higher than 1.5% in the etiology of cirrhosis [5, 6].

[0003] There is currently no effective treatment method for hepatocellular carcinoma. In patients with early tumors, liver transplantation or surgical resection is performed, and for patients with advanced tumors, chemotherapy and / or radiotherapy are the current treatment modalities. Although liver transplantation and resection are the only treatment measures, these two therapies can only be performed in a small percentage of patients. One of the reasons is that hepatocellular carcinoma is often diagnosed at an inoperable stage. Recurrence after resection is observed in more than 80% of patients [7].

[0004] Boron derivatives are encountered today in many fields. The research on the use of boron derivatives as anticancer agents in cancer treatment has become a promising new application area for cancer treatment [8, 9]. Boron derivatives have been shown to be effective in hepatocellular carcinoma. For example, tests have been conducted in which cancer cells in hepatocellular carcinoma can be selectively captured and destroyed by boron neutron capture therapy [10, 11]. Furthermore, microarray experiments were performed on the HepG2 cell line, which is a cell line of hepatocellular carcinoma, treated with boric acid, a boron derivative, to examine the affected pathways. It was revealed that boric acid treatment induced an anticancer mechanism

[12] . Bortezomib, an FDA-approved proteasome inhibitor used in cancer treatment, is also a derivative of pyrazine and boric acid. It has also shown clinical success in the treatment of multiple myeloma and mantle cell lymphoma, as well as colon cancer, lung cancer, breast cancer, prostate cancer, and hepatocellular carcinoma. Despite all these clinical successes, there are limitations to this drug as some patients treated with bortezomib relapse with cancer or do not respond to the treatment at all. Resistance to bortezomib has been observed in some solid tumors [13 - 17].

[0005] Sodium pentaborate pentahydrate (Na2B 10 O 16 ·5H2O, 18%B; NaB: B5H 10 NaO 13 ) is known to promote wound healing, but a certain test has shown that it has anticancer activity in lung cancer [18 - 19]. Furthermore, in tests conducted using the hepatocellular carcinoma Hep3B cell line, it was shown that sodium pentaborate pentahydrate reduced the proliferation of Hep3B cells and brought about changes in metabolism through the activation of the SIRT3 gene

[20] .

[0006] Curcumin is a hydrophobic polyphenol naturally extracted from the rhizome of the turmeric plant. Curcumin has a wide range of metabolic effects, including antioxidant, anti-inflammatory, antiviral, anti-angiogenic, antimicrobial, and anticancer activities. Several clinical trials have classified curcumin as a potential chemopreventive and chemotherapeutic agent. Curcumin is a non-toxic substance but exhibits low bioavailability. This problem has been observed to be eliminated when curcumin treatment is carried out with some secondary agents [21 - 23].

[0007] Piperine is another polyphenol isolated from black long pepper and is characterized by its unique properties. This not only improves the existing anticancer activity of curcumin but also increases the bioavailability of curcumin. Co-administration of curcumin and piperine has been shown to increase the anticancer activity of curcumin in hepatocellular carcinoma, colorectal, leukemia, and breast cancer [24 - 27].

[0008] The emergence of drug resistance mechanisms necessitates new drug derivatives.

[0009] Currently, FDA-approved drugs for the treatment of hepatocellular carcinoma include sorafenib, bevacizumab, cabozantinib, and regorafenib (ClinicalTrials.gov; cancer.gov). These drugs, which are part of the typical treatment, have problems in terms of resistance during the course of the disease and do not have a significant positive effect on survival.

[0010] The patent application number EP1790349A1, known in the current state of the art, discloses the treatment of viral or bacterial infections with pentahydrated sodium pentaborate {NaB5O2·5H2O}. However, it does not mention the action of the components in the treatment of diseases and / or their combined use.

[0011] By combining agents that activate different mechanisms, it is possible to prevent cancer cells from becoming resistant. Furthermore, since the doses delivered in the drug composite are lower than the optimal doses delivered individually, the cytotoxic effects of each drug are eliminated.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Non-Patent Document 1

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Summary of the Invention

[0014] The present invention relates to a ternary complex containing a boron derivative and at least two polyphenol active substances for use in the treatment of hepatocellular carcinoma. Here, the boron derivative is preferably sodium pentaborate, and the polyphenol active substances are curcumin and piperine.

[0015] The present invention relates to a pharmaceutical preparation containing sodium pentaborate pentahydrate, curcumin and piperine, or a pharmaceutically acceptable salt or derivative thereof, which provides additional advantages to the field.

[0016] The main object of the present invention relates to a pharmaceutical preparation containing sodium pentaborate pentahydrate, curcumin and piperine, or a pharmaceutically acceptable salt or derivative thereof, which is used in the treatment of hepatocellular carcinoma.

[0017] The present invention relates to producing a synergistic effect by combining agents that activate different mechanisms (cell pathways). That is, it prevents cancer cells from becoming resistant. Furthermore, since the dosage delivered in the agent composite is lower than the optimal dosage delivered alone, the cytotoxic effects of each agent are eliminated.

Brief Description of the Drawings

[0018] The anti-cancer preparation containing sodium pentaborate pentahydrate, curcumin, and piperine for use in the treatment of hepatocellular carcinoma, developed to achieve the object of the present invention, will be described in the following attached drawings.

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Mode for Carrying Out the Invention

[0028] The object of the present invention relates to the use of a complex of sodium borate pentahydrate, curcumin, and piperine as an active substance for the treatment of liver cancer.

[0029] In the present invention, the term "use as a complex" means to use together in the same formulation, or in separate formulations, simultaneously or sequentially.

[0030] In a preferred embodiment of the present invention, the liver cancer is non-viral liver cancer or viral liver cancer. Preferably, this is hepatocellular carcinoma, which is a type of cancer.

[0031] Another object of the present invention relates to a preparation containing sodium borate pentahydrate, curcumin and piperine for use as a medicament for the treatment of hepatocellular carcinoma. This ternary complex has a higher medicinal efficacy in the treatment of hepatocellular carcinoma compared to the administration of sodium borate pentahydrate, curcumin and piperine in the same amount and alone, or the use of any two of these binary complexes.

[0032] The present invention shows a synergistic effect on hepatocellular carcinoma cell lines (Hep3B and HepG2) when administered as a complex of sodium borate pentahydrate, curcumin and piperine (Figures 1 and 2). The fact that the index of the complex is less than 1 (CI < 1) supports this synergistic effect.

[0033] The highest percentage of cell death leading to apoptosis in HepG2 and Hep3B cells administered with sodium borate pentahydrate, curcumin and piperine was observed in the administration of the ternary complex (Figure 4).

[0034] The present invention relates to a preparation containing sodium borate pentahydrate, curcumin and piperine or at least one pharmaceutically acceptable salt or derivative thereof (such as solvate, hydrate, polymorph or amorphous form, etc.).

[0035] The present invention relates to a preparation containing a therapeutically effective amount of sodium borate pentahydrate, curcumin and piperine or a pharmaceutically acceptable salt or derivative thereof, and at least one pharmaceutically acceptable excipient.

[0036] As used herein, the expression "therapeutically effective amount" means the dose of a drug that achieves a specific pharmacological response and for which purpose the drug is administered to a patient in need of such treatment.

[0037] According to a preferred embodiment, the amount of sodium pentaborate pentahydrate in the total preparation is between 1700 and 8500 μM (micromoles) in concentration, more preferably between 1700 and 2500 μM. Preferably, this concentration is 1700 μM in the total preparation.

[0038] According to a preferred embodiment, the amount of curcumin in the total preparation is between 5 and 500 μM (micromoles) in concentration. Preferably, this concentration is 30 μM in the total preparation.

[0039] According to a preferred embodiment, the amount of piperine in the total preparation is between 1 and 150 μM (micromoles) in concentration. Preferably, this concentration is 6 μM in the total preparation.

[0040] In a preferred embodiment of the present invention, 1700 μM of sodium pentaborate pentahydrate, 30 μM of curcumin and 6 μM of piperine were used.

[0041] The administration route of the preparation of the present invention is systemic or local. The systemic administration routes are parenteral, inhalation, enteral, and transdermal. Oral administration is preferred, and the preparation is an oral preparation.

[0042] The dosage form of the preparation of the present invention is in the form of a solid (tablet, capsule, dry powder for suspension, sachet, etc.), semi-solid or liquid (suspension, solution, emulsion, etc.).

[0043] Since the medicinal effects (concentration per dose) brought about by the ternary complex of the present invention are lower than the optimal medicinal effects of each drug alone, the cytotoxic effects caused by each drug are eliminated.

[0044] In non-cancer (HUVEC) cells, the cell inhibition rate of the sodium pentaborate pentahydrate, curcumin, piperine complex was lower than that of cancer cells at 48 hours (Figure 3).

[0045] When interpreting dysregulated genes functionally, they are found to be genes that are important for molecular functions, biological processes, and functions of cellular contents in cells. Furthermore, pathway analysis of these genes showed that the gene expression levels of genes effective in cancer-related pathways such as the p53, apoptosis, ferroptosis, MAPK, and FoxO signaling pathways changed (Figs. 6, 7, 8, 9).

[0046] The pharmaceutical preparations of the present invention can be prepared using standard techniques or manufacturing methods known in the art.

[0047] The following examples are presented to better illustrate the requirements of the present invention, and the requirements of the present invention are not limited by these examples.

Example

[0048] [Example 1] Cell Culture Cell lines of human hepatocellular carcinoma, HepG2 (HB-8065, ATCC), Hep3B (HB-8064, ATCC), and HUVEC (CRL-1730) cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) growth medium (2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin) containing 10% heat-inactivated FBS with 5% CO2 at 37 °C in a humid atmosphere. The cells are passaged at 3-day intervals.

[0049] [Example 2] Cell Viability Assay (Administration of Sodium Borate Pentahydrate, Curcumin, and Piperine to Cell Lines of Hepatocellular Carcinoma) HepG2 and Hep3B cells are seeded at 5000 cells / well in a 96-well culture dish (Corning Glasswork, Corning, NY) in DMEM medium containing 10% FBS and 1% PSA in the medium, and then 5 sodium borate pentahydrate (NaB:B5H 10 NaO 13) Curcumin (Sigma, C1386), and piperine (Sigma, P49007) were administered. After 24, 48, and 72 hours, cell viability was measured. The cell viability assay was performed according to the manufacturer's protocol by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method (CellTiter96 Aqueous One Solution; Promega, Southampton, UK). After a 2-hour incubation time, the cell viability test was performed by measuring at 490 nm using an ELISA microplate reader (Bio-tek ELx800, USA). The IC 50 value at 48 hours was calculated. Thus, the IC 50 value for sodium borate pentahydrate was 7664.5 μM and for curcumin was 44.8 μM for HepG2. Thus, the IC 50 value for sodium borate pentahydrate was 6561 μM and for curcumin was 41.4 μM for Hep3B cells.

[0050] The dose of piperine was increased up to 150 μM, but the IC 50 value was not reached. The cells have no toxic effect on themselves. In the formulation, piperine is used as a secondary agent to enhance the bioabsorption of curcumin.

[0051] [Example 3] Apoptotic cell death HepG2 and Hep3B cells were cultured at 200,000 cells / well in 6-well plates in DMEM medium containing 10% FBS and 1% PSA. Sodium pentaborate pentahydrate at 2500 μM, curcumin at 30 μM, and piperine at 6 μM were administered to HepG2. Sodium pentaborate pentahydrate at 1700 μM, curcumin at 30 μM, and piperine at 6 μM were administered to Hep3B cells. After 48 hours, floating and adherent cells were collected. Cells were then incubated with Annexin V and PI staining agents for 15 minutes according to the manufacturer's protocol of the AnnexinV / FLOUS (Roche) apoptosis cell death kit. All samples were analyzed at a cell count of 10,000 using FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA).

[0052] [Example 4] Composite Index The degree of drug interaction is measured using the composite index (CI). The concentration of inhibition in the composite is the sum of the ratios of the doses of each drug to the doses of each drug used alone

[28] . If the drug composite shows a synergistic effect, the result of the CI analysis should be CI < 1. The formalization is as follows.

[0053] [Equation 1] CI = [(D)1 / (Dx)1] + [(D)2 / (Dx)2] (Dx)1, (Dx)2: Individual IC 50 values (D)1, (D)2: Concentrations of substances in the composite

[0054] Accordingly, for the composite of sodium pentaborate pentahydrate and curcumin used as an anticancer agent in the HepG2 and Hep3B cell lines, CI < 1. The problem regarding the low bioabsorbability of curcumin could be overcome by using piperine as a secondary drug.

[0055] [Example 5] RNA Sequencing According to the results of the experiment, total RNA isolation was performed in triplicate from HepG2 cells with or without administration of a complex of 2500 μM sodium pentaborate pentahydrate, 30 μM curcumin, and 6 μM piperine, and from Hep3B cells with or without administration of a complex of 1700 μM sodium pentaborate pentahydrate, 30 μM curcumin, and 6 μM piperine, at the optimal dose obtained in 48 hours, using the Qiagen RNeasy Mini kit. After measuring the quality of the RNA, the RNA was sent to Eurofins, Germany for RNA sequencing. cDNA libraries for mRNA sequencing were prepared from each sample, and sequencing was performed in 2x150 bp (base) and paired-end on a Novaseq 6000 obtained from Illumina. As a result of the sequencing, at least 60 million reads were obtained from each library

[0056] [Example 6] Bioinformatics analysis Quality control of the reads obtained as a result of array determination was performed using the FastQC program. The reads were mapped to the human reference genome (GRCh38) using the STAR program (version 2.7.0). The mapped reads were merged with the Cufflinks program (version 2.2.1) by using the default parameters. Genes that were differentially expressed between cells administered with sodium pentaborate pentahydrate, curcumin, and piperine and untreated cells as a negative control were determined by Cuffdiff (version 2.2.1). Based on a statistical value of P value < 0.05 and fold change (FC) > 2 and < -2 in HepG2 cells administered with sodium pentaborate pentahydrate, curcumin, and piperine when compared to the negative control, a total of 1187 genes were found to be dysregulated, of which 723 had increased expression levels and 644 had decreased expression levels. When evaluating the results of the analysis performed using Hep3B cells, a total of 2097 genes were found to be dysregulated, of which 1453 had increased expression levels and 770 had decreased expression levels. Further bioinformatics analysis was continued using these genes expressed in HepG2 and Hep3B.

[0057] [Example 7] Gene Ontology and Pathway Analysis Genes showing different expression levels (FC > 2 and FC < -2, p < 0.05) in HepG2 and Hep3B cell lines were identified and grouped by mapping them to subgroups of molecular function, cellular component, and biological function in the Gene Ontology database. Furthermore, pathway analysis was performed in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database and for which pathways these differentially expressed genes functioned.

Claims

1. A formulation comprising the use of sodium pentaborate pentahydrate, curcumin and piperine as an active substance for use in the treatment of liver cancer.

2. The formulation according to claim 1, characterized in that it is used in the treatment of non-viral liver cancer or viral liver cancer.

3. The formulation according to claim 2, characterized in that the liver cancer is hepatocellular carcinoma.

4. The formulation according to any one of the preceding claims, characterized in that it contains a therapeutically effective amount of an active substance of sodium pentaborate pentahydrate, curcumin and piperine or at least one pharmaceutically acceptable salt or derivative thereof.

5. The formulation according to any one of the preceding claims, characterized in that it contains at least one pharmaceutically acceptable excipient.

6. The formulation according to any one of the preceding claims, characterized in that the amount of sodium pentaborate pentahydrate in the total formulation is between 1700 and 8500 μM in concentration, preferably 1700 μM.

7. The formulation according to any one of the preceding claims, characterized in that the amount of curcumin in the total formulation is between 5 and 50 μM in concentration, preferably 30 μM.

8. The formulation according to any one of the preceding claims, characterized in that the amount of piperine in the total formulation is between 1 and 150 μM in concentration, preferably 6 μM.

9. The formulation according to any one of the preceding claims, characterized in that it contains 1700 μM of sodium pentaborate pentahydrate, 30 μM of curcumin and 6 μM of piperine.

10. The formulation according to any one of the preceding claims, characterized in that the administration route of the formulation is systemic or local.

11. The formulation according to any one of the preceding claims, characterized in that the administration route of the formulation is oral administration.

12. The formulation according to any one of the preceding claims, characterized in that the dosage form is in the form of a solid, semi-solid or liquid.

13. The formulation according to any one of the preceding claims, characterized in that the formulation according to any one of claims 1 to 12 is used in the manufacture of a medicament for use in the treatment of liver diseases.

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