WO2005053690A1

WO2005053690A1 - Use of benzimidazole derivatives for the manufacture of a medicament for treating a cancer

Info

Publication number: WO2005053690A1
Application number: PCT/KR2004/003209
Authority: WO
Inventors: Ki-Baik Hahm; Marie Yeo; Jong Eun Lee
Original assignee: Ki-Baik Hahm; Marie Yeo; Jong Eun Lee
Priority date: 2003-12-08
Filing date: 2004-12-08
Publication date: 2005-06-16
Also published as: KR20050055415A

Abstract

There is provided a use of benzimidazole derivatives for the manufacture of a medicament for treating a cancer.

Description

USE OF BENZIMIDAZOLE DERIVATIVES FOR THE MANUFACTURE OF A MEDICAMENT FOR TREATING A CANCER

Field of the Invention

The present invention relates to a use of benzimidazole derivatives for the manufacture of a medicament for treating a cancer.

Background of the Invention

Most anticancer agents do not exert their anticancer activity specific to cancer cells, but also damage normal cells, especially tissue cells which undergo active cell divisions, to induce many side effects, such as hypofiinction of bone marrow, gastroenteric trouble, and depilation. Accordingly, local therapy including operation, radiotherapy, and a combination of chemotherapy and immunotherapy has been used to treat cancers. For such therapy, a selective anticancer drug, which can selectively destroy cancer cells without harming normal cells, is needed, and the development thereof has been the focal point of current cancer research. Proton pump inhibitors (PPIs) which function to inhibit the secretion of gastric acid have been developed to cure human gastric acid-related diseases, such as gastric ulcer and duodenal ulcer. Various compounds having proton pump-inhibitory activity have been reported, e.g., benzimidazole derivatives such as pantoprazole (European Patent No. 166,287), omeprazole (European Patent No. 5,129), lansoprazole (European Patent No. 174,726), and leminoprazole (UK Patent No. 2,163,747). These patents merely disclose that the benzimidazole derivatives function to strongly inhibit the secretion of gastric acid, but are totally silent about their anticancer activity in suppressing the growth and in inducing apoptosis of cancer cells.

Summary of the Invention

Accordingly, it is an object of the present invention to provide a use of benzimidazole derivatives having proton pump-inhibitory activities for the manufacture of a medicament for treating a cancer with high specificity. In accordance with one aspect of the present invention, there is provided a use of a compound of formula ( I ) for the manufacture of a medicament for treating a cancer:

wherein, R.¹ and R² are each independently hydrogen, methoxy, difhioromethoxy, ethoxy or ethoxycarbonyl; and R³ to R⁵ are each independently hydrogen, methyl, methoxy, ethoxy or trifluoroethoxy.

Brief Description of the Drawings The above object and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Fig. 1: The cell viability changes of cancer cells (Kato HI, AGS, MKN-45, MKN-28, SNU-601 and SNU-1) and normal cells (RGM-1, IEC-6 and COS-1) with the change in pH of the culture solution; Fig. 2: Immunofluorescent staining photographs showing the expression of α-subunit of the proton pump in cancer cells (AGS and MKN-45) and normal cells (RGM-1 and IEC-6); Fig. 3: The cell viabilities of cancer cells (MKN-45, MKN-28, AGS, Kato-1, SNU-601 and SNU-1) (A) and normal cells (RGM-1, IEC-6 and COS-1) (B) after pantoprazole injection; Fig. 4a: A gel photograph showing the mode of genomic DNA fragmentation in cancer cell (MKN-45) and normal cell (RGM-1) after pantoprazole injection; Fig. 4b: Western-blotting photographs showing the mode of cleavage of poly (ADP-ribose) polymerase by caspase-3 in cancer cell (MKN-45) and normal cell (RGM-1) after pantoprazole injection; Fig. 4c: Pictures showing the changes in the phosphatidylserine distribution and membrane permeability in cancer cell (MKN-45) and normal cell (RGM-1) after pantoprazole injection; Fig. 5a: The tumor cell growth rate of a xenografted human stomach cancer in athymic nude mice after intra-peritoneally (IP) or intra-tumorally (IT) injecting pantoprazole into the cancer; Fig. 5b: The change in the shape and size of a xenografted human stomach cancer in athymic nude mice after intra-peritoneally (IP) or intra-tumorally (IT) injecting pantoprazole into the cancer; Fig. 5c: Microscopic photographs of the cross section of a xenografted human stomach cancer in athymic nude mice after intra-peritoneally (IP) or intra-tumorally (IT) injecting pantoprazole into the cancer; and Fig. 5d: Fluorescent microscopic photographs of the cross section of a xenografted of human stomach cancer in athymic nude mice after intra-peritoneally (IP) or intra-tumorally (IT) injecting pantoprazole into the cancer.

Detailed Description of the Invention The present inventors have found that cancer cells are more resistant to acidic condition than normal cells (see Fig. 1). This result suggests that the proton pumps in cancer cells are more efficient than normal cells in dispersing proton. Immunocytochemical staining with an anti-proton pump antibody indeed shows that proton pumps are more abundant in cancer cells than normal cells (see Fig. 2). Accordingly, there has existed a possibility that cancer cells could be selectively destroyed by inhibiting the proton pumps, and the administration of a benzimidazole derivative, e.g., pantoprazole, a well known proton pump inhibitor, to cancer cells and normal cells has revealed that growth inhibition and cell destruction take place in cancer cells, but not in normal cells. Accordingly, the present invention provides a use of a compound of formula ( I ) for the manufacture of a medicament for treating a cancer:

wherein, R¹ and R² are each independently hydrogen, methoxy, difluoromethoxy, ethoxy or ethoxycarbonyl; and R to R are each independently hydrogen, methyl, methoxy, ethoxy or trifluoroethoxy. Further, the present invention provides an anticancer agent comprising a compound of formula ( I ) as an active ingredient. Moreover, the present invention provides a method for treating a cancer comprising administering a compound of formula ( I ) with an effective amount to a cancer patient. Preferred exemplary compounds of the compound of formula ( I ) in accordance with the present invention are:

5 -difluoromethoxy-2 [ [(3 ,4-dimethoxy-2-pyridinyl)methyl] sulfmyl] - 1 H- benzimidazole (pantoprazole); 5-methoxy-2[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-l H-benzimidazole (omeprazole); 2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridinyl]methyl]sulfϊnyl]-lH -benzimidazole (lansoprazole); 2-[[(2-pyridinyl)methyl]sulfinyl]-lH-benzimidazole (timoprazole); 5 -ethoxycarbonyl-6-methyl-2 [ [(3 -methyl-2-pyridinyl)methyl] sulfmyl] - 1 H-benzimidazole (picoprazole); and rameprazole. Among them, pantoprazole is most preferred.

Pantoprazole, omeprazole, and lansoprazole can be represented by structural formula ( LI ) to (IV), respectively.

The compound of formula ( I ) in accordance with the present invention shows an anticancer effect, i.e., selective growth inhibition and apoptosis of cancer cells. For example, in the normal cells, there is little difference in cell viability between the group of cells treated with pantoprazole and the group of cells not treated (see Fig. 3(B)). In the cancer cells, however, the cell viability of the group of cancer cells treated with pantoprazole declines sharply with decreasing pH, while no significant change in the cell viability occurs in cancer cells received no injection of pantoprazole (see Fig. 3(A)). Apoptosis is one of the genetically programmed cell death, and cells undergoing apoptosis have typical events such as DNA fragmentation and changes in the phospholipids distribution and membrane permeability. In addition, a caspase cascade is typically activated in the event of apoptosis, and, especially, caspase-3 is activated to cleave its substrate, poly (ADP-ribose) polymerase (PARP). Therefore, activation of caspase-3 may be a major determinant for the process of apoptosis and an important indicator for the identification of apoptosis. These events of apoptosis indeed occur in cancer cells treated with the compound of formula ( I ) of the present invention. First, the compound of formula ( I ) of the present invention induces DNA fragmentation in cancer cells (see Fig. 4a); the cleavage of PARP by activated caspase-3 (see Fig. 4b); and the change in the phospholipids distribution and membrane permeability in cancer cells (see Fig. 4c). Further, the compound of formula ( I ) exhibits marked anticancer activity against a xenografted human stomach cancer in athymic nude mice. For example, the tumor size of a xenografted human stomach cancer in mice is reduced by 77.4 % 22 days after intra-peritoneally injecting pantoprazole, and by 44.68 %, when pantoprazole is injected intra-tumorally (IT) (see Figs. 5a and 5b). Thus, the compounds of the present invention having proton pump-inhibitory activity exhibit anticancer activity specific to cancer cells by inducing apoptosis, and the compounds of the present invention can be advantageously employed in the manufacture of a medicament for treating cancers, especially a stomach cancer.

The compound of the present invention can be administered via oral and parenteral, and can be used as a form of a general pharmaceutical agent. That is, the above compounds can be formulated into pharmaceutical preparation using common fillers, extenders, binders, wetting agents, disintegrants as an excipient. A solid preparation for oral administration includes powder, granule, tablet and coated preparation, and these can be prepared by mixing crude drag extracts and at least one of excipients, e.g., starch, sodium carbonate, sucrose, lactose, gelatin and the like. Further, lubricants can be added such as magnesium styrate and talc. A liquid preparation for oral administration includes suspension, solution, emulsion, syrup and the like, and additionally includes diluents such as water, liquid paraffin and excipients, e.g., wetting agents, flavoring agents, aromatic agents, preservatives and the like. A preparation for parenteral administration includes sterilized aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized agent and suppository. Non-aqueous solution and suspension may be vegetable oils such as propyleneglycol, polyethyleneglycol and olive oil, and injectable ester like ethylolate. Suppository is based on witepsol, macrogol, Tween 61, cacao fat, laurin fat, glycerol, gelatin and the like. The effective amount of the compound of the present invention can be determined according to an age and body weight of each individual, and, preferably, a daily dose may be 0.6 mg/kg. The following examples are intended to further illustrate the present invention without limiting its scope.

Example 1: Identification of characteristics of cancer cells

(1-1) Cell culture Human stomach cancer cells (AGS, KatoHI, MKN-45 and MKN-28), human fibroblast (COS-1), normal mouse gastric epithelial cell (RGM-1) and normal mouse enteroepithelial cell (IEC-6) were obtained from ATCC (The American Type Culture Collection); and Korean stomach cancer cells (SNU-1 and SNU-601) were obtained from KCLB (Korean Cell Line Bank). AGS, KatoIH, MKN-28, MKN-45, COS-1, SNU-601 and SNU-1 were each cultured in RPMI 1640 medium (Gibco BRL); RGM-1, in DMEM-F12 medium containing 10 % FBS (fetal bovine serum) and 100 units/ penicillin; and IEC-6, in DMEM-high glucose medium (Gibco BRL) containing 10 % bovine insulin.

(1-2) Identification of resistance to acid of cancer cells and immunocytochemical staining

The cells obtained in Example (1-1) were cultured in a medium having various pH, i.e., 7.4, 6.9, 6.5, 5.9, 5.4 and cell viability thereof was investigated by employing MTT assay. As shown in Fig. 1, the cell viability of normal cells (RGM-1, IEC-6 and COS-1) declined with decreasing pH, declining precipitously at below pH 5.9. In contrast, the cell viability of cancer cells declined gently. Human stomach cancer cells (AGS and MKN-45) and normal mouse cells (RGM- 1 and IEC-6) were each cultured for 24 hours in cell culture dishes. The cells were fixed with methanol, and then treated with 1/100 diluted anti-proton pump α-subunit antibody (Santa Cruz Biotechnology) for 2 hours. Thereafter, the cells were allowed to react with Cy3 -conjugated secondary antibody (Zymed) for 1 hour, and observed under a fluorescent microscope. Immunocytochemical staining with the anti-proton pump antibody indicated that more proton pumps were presented in cancer cells than in normal cells (see Fig. 2), and that the proton pumps were extensively expressed in the membrane and cytoplasm of cancer cells.

Example 2: The growth inhibition of cancer cells

The growth inhibition of cancer cells by pantoprazole, a benzimidazole derivative well known as a proton pump inhibitor, was examined as follows. Cancer cells and normal cells were respectively treated with 50 mM of pantoprazole (Aitana, German; Pacific pharmaceuticals Co, Ltd., South Korea), and cultured in a medium at various pH for 24 hours; and the cell viability thereof was examined by MTT assay. In case of the normal cells, there was little difference in the cell viability between the group of cells treated with pantoprazole and the group of cells not treated (see Fig. 3(B)). In case of cancer cells, however, the cell viability in the group of cells treated with pantoprazole declined sharply with decreasing pH, while there was no significant change in the cell viability in the group of cells not treated with pantoprazole (see Fig. 3(A)). This result shows that pantoprazole is converted to an active form under an acidic condition, and inhibits the growth of cancer cells in its active form at low pH. Example 3: Identification of apoptosis of cancer cells

Apoptotic events, such as DNA fragmentation, caspase-3 activation and cleavage of PARP (poly (ADP-ribose) polymerase), and the change of phospholipids distribution and membrane permeability, were examined for the cancer cells treated with the compound of the present invention.

(3-1) Observation of genomic DNA fragmentation RGM-1 and MKN-45 cells were divided into two groups, and one group was treated with pantoprazole leaving the other group untreated. Each of four groups was cultured in a medium for 24 hours at pH 7.4 and pH 5.4. Subsequently, the cells were lysed by treatment with cell lysis buffer (10 mM Tris, 5 mM EDTA and 1 % Triton X-100) for 15 minutes. After centrifugation, the supernatant was taken and reacted with 0.1 mg/mi of proteinase K at 37 ^°C for 1 hour. The nucleic acids were then isolated by adding equal amount of phenol/chloroform to the supernatant and inducing precipitation thereof by adding ethanol and 0.3 M of acetic acid. The precipitated nucleic acids were dissolved in TE buffer, treated with RNase, and subjected to electrophoresis in 1.8 % agarose gel. The results were examined for genomic DNA fragmentation. As shown in Fig. 4a, no genomic DNA fragmentation was observed for RGM-1 (normal cell) regardless of the pH change or pantoprazole treatment. In contrast, extensive genomic DNA fragmentation was observed for MKN-45 (human stomach cancer cell) treated with pantoprazole. This discloses the fact that pantoprazole specifically induces apoptosis of cancer cells. (3-2) Observation of caspase-3 activation and cleavage of PARP

The activation of caspase-3, a proteinase playing an important role in apoptosis and the cleavage of the substrate thereof, PARP, was examined by western blotting analysis. After RGM-1 and MKN-45 cells were treated with 0.3 mM and 0.6 mM of pantoprazole for 16 hours, respectively, the cells were lysed to extract proteins, followed by electrophoresing the extracted proteins in 12 % and 8 % of SDS-PAGE, respectively. The electrophoresed proteins were transferred to a PVDF membrane by employing a semi-dry transfer (hoeffer). The membrane was blocked with 5 % skim milk for 1 hour and allowed to react with 1/1000 diluted anti-caspase-3 antibody and anti-PARP antibody (Santa Craze Biotechnology) for 16 hours at 4 ^°C . The membrane was washed with TBST, treated with 1/2000 diluted secondary antibody for 1 hour, and observed through ECL (enhanced chemiluminescence). As shown in Fig. 4b, the amount of procaspase-3, a proenzyme form of caspase-3, was dependant on the administered amount of pantoprazole; and,

PARP in MKN-45 (cancer cell) was cleaved. This result shows that pantoprazole selectively activates the apoptosis enzyme caspase-3 in cancer cells.

(3-3) Observation of the change of phospholipids distribution and membrane permeability Apoptosis is accompanied by various unique cellular events; for example, the change in the membrane permeability and the migration of phosphatidylserine of phospholipids constituting cellular membranes to the outer membrane of cytoplasm (J Immunol Meth (1995) 184: 39-51; J Exp Med 182: 1545-1556). Examined, accordingly, was whether or not such unique cellular events can be observed in the cells undergoing apoptosis induced by pantoprazole. RGM-1 and MKN-45 cells were divided into two groups, and one group was treated with 0.5 mM pantoprazole, leaving the other group untreated. The cells were cultured in a medium for 16 hours. Subsequently, the cells were treated with a staining solution (BD Biosciences) containing Annexin V -FITC and PI (propidium iodide) for 15 minutes, and observed under a phase-contrast microscope. As shown in Fig. 4c, much more stained cells were observed in MKN-45 cells than in RGM-1 cells. Accordingly, it was confirmed that pantoprazole induces the change of phopholipids distribution in the early stage of apoptosis, specifically in cancer cells.

Example 4: Anticancer activity in mouse

The effect of pantoprazole on the growth of a xenograft of human stomach cancer on athymic nude mice was examined. First, 5X 10⁷ cells of MKN-45 cells in PBS was injected hypodermically into the back of each mouse of a group of mice at an amount of 5X 10⁶ cells/site and the mice were separated into 3 groups. The mice in Group 1 were each injected with PBS intra-tumorally once a day after 14 days of MKN-45 cell injection; the mice in Group 2 were each injected with 0.4 mg/kg pantoprazole intra-peritoneally once a day after 1 day of MKN-45 cell injection; and the mice in Group 3 were each injected with 0.4 mg/kg pantoprazole intra-tumorally once a day after 14 days of MKN-45 cell injection. The volume of the tumor in each mouse was calculated by measuring the long and short arms of the tumor with a clipper every two days. The tumor tissues were isolated from each mouse after 22 days of MKN-45 cell injection, and subjected to hematoxylin-eosin (H&E) staining and TUNEL staining. As a result, it was found that on the 22nd day after grafting, the tumor size of the mouse treated with intra-peritoneal (IP) injection of pantoprazole was reduced by 77.4 % compared with that of the control (Group 1), and the tumor size of the mouse treated with intra-tumoral (IT) injection of pantoprazole was smaller by 44.68 % than that of the control (Group 1) (see Fig. 5 a and 5b). H&E staining analysis thus shows that remarkable decrease in the size of tumor tissue took place in Group 2 and Group 3 as the result of pantoprazole treatment and that cell necrosis was proceeding in the center of the tissue (see Fig. 5c). In addition, the TUNEL staining analysis also demonstrated that apoptosis was induced in Group 2 and 3 (see Fig. 5d).

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

What is claimed is:

1. A use of a compound of formula ( I ) for the manuracture of a medicament for treating a cancer:

wherein, R and R are each independently hydrogen, methoxy, difluoromethoxy, ethoxy or ethoxycarbonyl; and R to R are each independently hydrogen, methyl, methoxy, ethoxy or trifluoroethoxy.

2. The use of claim 1, wherein the compound is selected from the group consisting of pantoprazole, omeprazole, lansoprazole, timoprazole, picoprazole and rameprazole.

3. The use of claim 2, wherein the compound is pantoprazole.

4. The use of claim 1, wherein the cancer is selected from the group consisting of stomach cancer, liver cancer, brain tumor, breast cancer and skin cancer.

5. The use of claim 4, wherein the cancer is stomach cancer.