WO2020018037A2

WO2020018037A2 - A chemotherapeutic agent comprising a combination of alexidine dihydrochloride and sodium pentaborate pentahydrate

Info

Publication number: WO2020018037A2
Application number: PCT/TR2019/050152
Authority: WO
Inventors: Fikrettin Sahin; Ezgi KASIKCI; Esra AYDEMİR ÇOBAN
Original assignee: Yeditepe Universitesi
Current assignee: Yeditepe Universitesi
Priority date: 2018-03-12
Filing date: 2019-03-11
Publication date: 2020-01-23
Anticipated expiration: 2020-09-12
Also published as: WO2020018037A3; US11510943B2; JP7277476B2; US20210015854A1; CN112040956A; EP3765037A2; CN112040956B; EP3765037A4; JP2021525223A; CA3093548A1

Abstract

The present invention relates to a chemotherapeutic agent comprising a combination of alexidine dihydrochloride and sodium pentaborate pentahydrate. Alexidine dihydrochloride solution and sodium pentaborate pentahydrate compound were prepared at different concentrations and combined. Upon injecting them to the media containing healthy cells and cancer cells, their effects on cell viability were observed. In accordance with the obtained results; it was observed that when alexidine dihydrochloride solution at concentrations not harming healthy cells is combined with sodium pentaborate pentahydrate, pancreatic cancer cell viability decreased 10% more compared to use of alexidine dihydrochloride alone, and it was realized that this combination can be used as a chemotherapeutic agent for treatment of pancreatic cancer.

Description

A CHEMOTHERAPEUTIC AGENT COMPRISING A COMBINATION OF ALEXIDINE DIHYDROCHLORIDE AND SODIUM PENTABORATE

PENTAHYDRATE

Field of the Invention

The present invention relates to the effect of the combination of alexidine dihydrochloride and sodium pentaborate pentahydrate on pancreatic cancer.

Background of the Invention

The pancreas is an organ which is approximately 15 cm long and located in the rearmost part of the abdomen, and whose front surface is completely covered with the stomach, duodenum and large intestine. It is known that pancreatic cancer has the least response to treatment among cancer types. The pessimistic predictions about the results of the surgical treatment of pancreatic cancer left their place to relatively better results after the eighties.

Although the etiology in pancreatic cancer is not known exactly, some risk factors have been identified. These are factors such as: age (increased risk after age 50), gender (increased risk of pancreatic cancer in men), smoking (30% of pancreatic cancer cases are thought to be associated with smoking), diet (it is difficult to formulate an idea about diet and exercise, however it is considered that consumption of fruits, vegetables, and fiber food reduce the risk, while consumption of meat and fatty products increase it).

The exact location of the tumor in the pancreas, the stage of the disease, the physical state of the patient (age, weight, etc.) are evaluated for the treatment of pancreatic cancer; and then one or more selected from the methods of surgical treatment, radiotherapy and chemotherapy is/are used.

Chemotherapy is the use of anti-cancer drugs to kill cancer cells. Pancreatic cancers can be treated with drug treatment called chemotherapy taking into consideration the general conditions of the patients before and after surgery. Chemotherapy can be applied to shrink tumors prior to surgery or as a primary treatment in combination with radiotherapy in place of surgery. Surgery and radiotherapy are not useful in advanced stage spread disease. It is known that the patients' quality of life can be significantly improved by applying chemotherapy to this group of patients.

The United States patent document no. US9399032B2, known in the art, discloses that a group of chemicals, including alexidine dihydrochloride, selectively inhibits cell growth upon being dissolved in galactose.

It is disclosed in the abstract of the article titled“Potential use of alexidine dihydrochloride as an apoptosis-promoting anticancer agent” in the book named Molecular Cancer Therapeutics published by Kenneth W. Yip, Emma Ito, Xinliang Mao, P.Y. Billie Au, David W. Hedley, Joseph D. Mocanu, Carlo Bastianutto, Aaron Schimmer and Fei-Fei Fiu published on September 2006, that alexidine dihydrochloride is used for cancer treatment.

It is disclosed in the article titled“Increased efficiency for performing colony formation assays in 96- well plates: novel applications to combination therapies and high-throughput screening” in the book named Bio Techniques published on February 2008 by David Katz, Emma Ito, Ken S. Fau, Joseph D. Mocanu, Carlo Bastianutto, Aaron D. Schimmer, and Fei-Fei Fiu, that alexidine dihydrochloride can be used in anti-cancer applications. Summary of the Invention

The objective of the present invention is to increase the therapeutic effect of the product produced by adding sodium pentaborate pentahydrate to the alexidine dihydrochloride compound by 10 to 20%.

Another objective of the present invention is to use the combination of alexidine dihydrochloride and sodium pentaborate pentahydrate as a chemotherapeutic agent for pancreatic cancer treatment.

Detailed Description of the Invention

The present invention is a chemotherapeutic agent which is used in pancreatic cancer treatment and is obtained by the following steps:

- weighing 2.9 mg of alexidine dihydrochloride compound,

- preparing alexidine dihydrochloride solution with concentrations of 200 mM, 100 mM, 50 pM, 25 pM, 10 pM, 5 pM, 2.5 pM, 1.25 pM, 0.6 pM, 0.3 pM, 0.15 pM, 0.078 pM and 0.039 pM respectively by adding dimethylsulfoxide solution into alexidine dihydrochloride,

- adding the solutions separately into the medium containing pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l), pancreatic healthy cells (hTert-HPNE) and healthy intervertebral disc cells (HNPC) such that dimethylsulfoxide ratio is less than 1/1000,

- incubating at 24, 48 and 72 hours respectively in incubators with 85% moisture at a temperature of 37°C which are the normal cell culture conditions,

- performing colorimetric tetrazolium salt (MTS) viability assay, - preparing 2,5mM alexidine dihydrochloride incubated at 48 hours which is determined in the light of the results of the analysis to give the optimum result,

- preparing sodium pentaborate pentahydrate solutions at concentrations of IOOOmM, 500mM, 250mM, IOOmM, respectively,

- applying the prepared sodium pentaborate pentahydrate solutions to the media of pancreatic healthy cells (hTert-HPNE), healthy intervertebral disc cells (HNPC), human foreskin mesenchymal stem cells (hFSSCs), human umbilical vein endothelial cells (HUVEC) and pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l) in incubators with 85% moisture at a temperature of 37°C which are the normal cell culture conditions,

- performing colorimetric tetrazolium salt (MTS) viability assay in the medium containing sodium pentaborate pentahydrate solutions and the cells,

- preparing sodium pentaborate pentahydrate solutions at concentrations of IOOOmM, 500mM, 250mM, IOOmM, respectively, and applying them together with alexidine dihydrochloride to the media containing pancreatic healthy cells (hTert-HPNE), healthy intervertebral disc cells (HNPC), human foreskin mesenchymal stem cells (hFSSCs), human umbilical vein endothelial cells (HUVEC) and pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l) respectively in incubators with 85% moisture at a temperature of 37°C which are the normal cell culture conditions,

- performing colorimetric tetrazolium salt (MTS) viability assay in the medium containing solutions of alexidine dihydrochloride and sodium pentaborate pentahydrate solutions and the cells.

The chemotherapeutic agent of the present invention comprises a combination of alexidine dihydrochloride and sodium pentaborate pentahydrate and induces apoptosis on pancreatic cancer cells. Alexidine dihydrochloride is a compound having a molecular weight of 581.71 gram/mol and the chemical formula of C26H56N10 · 2HC1 and it is dissolved in dimethylsulfoxide of 10 mg/ml or above. The effective dose of alexidine dihydrochloride on pancreatic cancer is determined by conducting colorimetric tetrazolium salt (MTS) viability assay on pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l), pancreatic healthy cells (hTert-HPNE) and healthy intervertebral disc cells (HNPC).

Alexidine dihydrochloride was dissolved in dimethylsulfoxide to prepare an alexidine dihydrochloride solution at concentrations of 200 mM, 100 pM, 50 pM, 25 pM, 10 pM, 5pM, 2.5 pM, 1.25 pM, 0.6 pM, 0.3 pM, 0.15 pM, 0.078pM and 0.039pM; and was added to the medium containing pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l), pancreatic healthy cells (hTert-HPNE) and healthy intervertebral disc cells (HNPC) such that dimethylsulfoxide ratio is less than 1/1000. As a result of the colorimetric tetrazolium salt (MTS) viability assay performed after incubation of 72 hours, it was determined that the healthy pancreas cell and the healthy intervertebral disc cells were not affected, however viability of the pancreatic cancer cells was reduced to 50% by 2.5 pM which is the median toxic dose (TD50) and 48 hours of incubation.

In order to increase the chemotherapeutic effect of the alexidine dihydrochloride solution, it is planned to add sodium pentaborate pentahydrate compound to the solution. Sodium pentaborate pentahydrate is a compound having a molecular weight of 295.107 grams / mol and the chemical formula of BsHmNaOr,.

The sodium pentaborate pentahydrate compound was dissolved in the appropriate medium for the cell that was used and filtered to enable sterilization. Sodium pentaborate pentahydrate compound at concentrations used is dissolved instantly in the medium without requiring any additional solvent addition. The normal pH value of the medium was 7.4 and did not form sodium pentaborate pentahydrate precipitate for 72 hours. Then, sodium pentaborate pentahydrate was prepared at concentrations of 1000 pM, 500 pM, 250 pM, 100 pM, respectively, and was injected to the medium containing the pancreatic healthy cell (hTert-HPNE), healthy intervertebral disc cell (HNPC), human foreskin mesenchymal stem cell (hFSSCs) and human umbilical vein endothelial cell (HUVEC), and then was incubated for 48 hours. Then toxicity analysis (colorimetric tetrazolium salt (MTS) viability assay) was performed. The solution of sodium pentaborate pentahydrate at a concentration of 1000 mM incubated for 48 hours gave the optimum result with an average vitality of 70%.

Then, 2.5 pM alexidine dihydrochloride was added into the sodium pentaborate pentahydrate solution prepared at concentrations of 1000 pM, 500 pM, 250 pM, 100 pM, respectively, and was injected to the medium containing the pancreatic healthy cell (hTert-HPNE), healthy intervertebral disc cell (HNPC), human foreskin mesenchymal stem cell (hFSSCs) and human umbilical vein endothelial cell (HUVEC), and then was incubated for 48 hours. Then toxicity analysis (colorimetric tetrazolium salt (MTS) viability assay) was performed. As a result of the analysis, it was determined that the optimum combination result that does not harm the healthy cells are the combinations of 250 pM and 100 pM sodium pentaborate pentahydrate and 2.5 pM alexidine dihydrochloride.

Upon preparing solutions of the same concentration, adding them to the medium containing pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l) and performing colorimetric tetrazolium (MTS) vitality assay; it was observed that the combination of 250 pM sodium pentaborate pentahydrate and 2.5 pM alexidine dihydrochloride, which is the non-lethal dose for the healthy cells, decreased the viability of pancreatic cancer cells by more than 50% upon 48 hours of incubation. This value is 10-20% more than the chemotherapeutic effect of alexidine dihydrochloride alone.

It was observed that the solution of alexidine dihydrochloride and sodium pentaborate pentahydrate prepared in the medium started to lose its effect at 4°C, at periods of time longer than 72 hours. The solution should be freshly prepared and used at once as a chemotherapeutic agent.

Experimental Analysis Data

In the graphs showing experimental analysis data, “NAB” is used as the abbreviation for sodium pentaborate pentahydrate and “AD” is used as the abbreviation for alexidine dihydrochloride. Negative control is abbreviated as “NC” and denotes that the cell’s medium is used alone.

Figure 1 shows the graph indicating the effect of alexidine dihydrochloride on the viability of healthy cells and pancreatic cancer cells (colorimetric tetrazolium (MTS) viability assay).

In the related graph; viabilities of the healthy pancreatic cells (hTert-HPNE), healthy intervertebral disc cells (HNPC) and pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l) incubated for 48 hours with AD were analyzed. In the light of the results obtained; it was observed that 48-hour incubation with AD at a concentration of 2.5 mM did not harm the healthy cells, however decreased the viability of the pancreatic cancer cells to 53% for MIA PaCa-2, 58% for AsPc-l and 45% for Psn-l.

Figure 2 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the cellular viability of healthy human foreskin stem cells (hFSSCs). In the related graph, the viability profiles of healthy human foreskin stem cells (hFSSCs) were observed upon 48-hour of incubation with NAB alone and combination thereof with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on hFSSC cells was low (85% viability).

Figure 3 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of healthy human umbilical vein endothelial cell (HUVEC).

In the related graph, the viability profiles of healthy human umbilical vein endothelial cells were observed upon 48-hour of incubation with NAB alone and in combination with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on HUVEC cells was low (94% viability).

Figure 4 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of healthy intervertebral disc cell (HPNC).

In the related graph, the viability profiles of healthy intervertebral disc cell were observed as a result of 48-hour incubation of NAB alone and in combination with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on HPNC cells was low (86% viability).

Figure 5 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of healthy human pancreatic cell (hTert-HPNE).

In the related graph, the viability profiles of healthy human pancreatic cell were observed as a result of 48-hour incubation of NAB alone and in combination with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on HPNC cells was low (89% viability).

As a result of the analysis performed on healthy cells with NAB alone and in combination with AD, it was determined that the dose to be used on healthy cells and cancer cells was the combination 250 mM NAB and 2.5 mM AD. This dose and the other doses used on healthy cells were injected on pancreatic cancer cells and their effects within a period of 48 hours were observed.

Figure 6 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of pancreatic cancer cell (MIA PaCa-2).

In the related graph, the viability profiles of pancreatic cancer cell (MIA PaCa-2) were observed as a result of 48-hour incubation of NAB alone and in combination with AD. In accordance with the obtained results, it was determined that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on MIA PaCa-2 cells was higher than the effect of the same dose of AD alone (41%). While application of AD alone decreased viability of MIA PaCa-2 cells to 53%, when NAB and AD were applied in combination, an extra 12% decrease was observed in viability.

Figure 7 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of pancreatic cancer cell (Psn-l).

In the related graph, the viability profiles of pancreatic cancer cell (Psn-l) were observed as a result of 48-hour incubation of NAB alone and in combination with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 mM AD on Psn-l cells was higher than the effect of the same dose of AD alone (41.60%). While application of AD alone decreased viability of Psn-l cells to 51.6%, when NAB and AD were applied in combination, an extra 10% decrease was observed in viability.

Figure 8 shows the graph of the effect of NAB alone and in combination with AD at the end of 48-hour incubation on the viability of pancreatic cancer cell (AsPc- 1).

In the related graph, the viability profiles of pancreatic cancer cell (AsPc-l) were observed as a result of 48-hour incubation of NAB alone and in combination with AD. In accordance with the obtained results, it was observed that the viability destroying effect of combination of 250 mM NAB and 2.5 pM AD on AsPc-l cells was higher than the effect of the same dose of AD alone (39.2%). While application of AD alone decreased viability of AsPC-l cells to 58.3%, when NAB and AD were applied in combination, an extra 19% decrease was observed in viability.

As a result of the analyses conducted, it was proved that alexidine dihydrochloride has chemotherapeutic activity on pancreatic cancer cells. It was found that by 2.5 pM of AD which is the median toxic dose (TD50) for pancreatic cancer cells do not kill the healthy cells. In the light of the information found, it can be used as a chemotherapeutic agent in pancreatic cancer treatment.

With the invention, a combination of alexidine dihydrochloride and sodium pentaborate pentahydrate can be obtained and used as a chemotherapeutic agent in the treatment of pancreatic cancer. With this combination, the viability of pancreatic cancer cells was reduced at least 10% more.

Claims

1. A chemotherapeutic agent produced with the combination of alexidine dihydrochloride and sodium pentaborate pentahydrate compound used for treatment of pancreatic cancer.

2. Chemotherapeutic agent according to Claim 1, which is used in pancreatic cancer treatment and is obtained by the steps of

- weighing the alexidine dihydrochloride compound,

- adding dimethylsulfoxide compound therein and preparing a stock solution at a concentration of 10 mM,

- storing the stock solution at a temperature of -20°C at an unilluminated environment until time of use,

- supplying sodium pentaborate pentahydrate solution and by dissolving it in the medium, forming a sodium pentaborate pentahydrate solution at a concentration of 10 pM,

- diluting the solution of sodium pentaborate pentahydrate dissolved in the medium to a concentration of 250 pM,

- filtering and sterilizing the solution diluted to 250 pM and

- adding alexidine dihydrochloride into sodium pentaborate pentahydrate solution having a concentration of 250 pM such that the final concentration will be 2.5 pM.

3. Chemotherapeutic agent according to Claim 1 comprising 2.9 mg alexidine dihydrochloride compound.

4. Chemotherapeutic agent according to Claim 1 obtained by adding 0.5 ml dimethylsulfoxide compound into alexidine dihydrochloride compound.

5. Chemotherapeutic agent according to Claim 1 comprising 2.5 mg sodium pentaborate pentahydrate compound.

6. Chemotherapeutic agent according to Claim 1 which can be stored for 72 hours at a temperature of 4°C without degradation.

7. Chemotherapeutic agent according to Claim 1 which decreases viability of pancreatic cancer cells (MIA PaCa-2, AsPc-l and Psn-l) by 60-80%.

8. Chemotherapeutic agent according to Claim 1 which decreases viability of healthy pancreatic cells (hTert-HPNE) by 10-15%.

9. Chemotherapeutic agent according to Claim 1 which decreases viability of healthy intervertebral disc cells (HPNC) by 10-15%.

10. Chemotherapeutic agent according to Claim 1 which decreases viability of healthy pancreatic cells (hTert-HPNE) by 10-15%.

11. Chemotherapeutic agent according to Claim 1 which decreases viability of healthy umbilical vein endothelial cells (HUVEC) by 5-10%.