JP7345151B1 - cancer treatment drug - Google Patents

Abstract

[Problem] To provide a cancer therapeutic drug that suppresses the proliferation of cancer cells. SOLUTION: A cancer therapeutic drug comprising a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof. [Selection diagram] None

Description

The present invention relates to a cancer therapeutic agent containing a compound having a specific structural formula or a pharmaceutically acceptable salt thereof. More specifically, it relates to a therapeutic agent for prostate cancer.

It has been a long time since cancer became the number one cause of death in Japan, and now more than 300,000 people die from cancer each year. Furthermore, it is estimated that one in two men and one in three women will develop cancer during their lifetime. The number of deaths from cancer is increasing worldwide, and according to the International Agency for Research on Cancer (IARC), an external research organization of the World Health Organization (WHO), the annual number of deaths from cancer will increase by 2030. It is predicted that the number will almost double from 2008 to 13.3 million.

Among cancers, prostate cancer is a cancer found in older men. Worldwide, prostate cancer is a cancer that occurs frequently, especially in Europe and the United States, and in the United States, it accounts for the highest number of morbidities and deaths among male cancers. Prostate cancer was originally not a very common cancer in Japan, but as the population ages, the incidence rate has increased rapidly, and recent statistics (National Cancer Center 2018 National Registered Incidence Data) ) is the most common cancer among Japanese men, and it is expected that the number will continue to increase.

Treatment methods for prostate cancer include "surgical therapy," "radiation therapy," and "hormone therapy." Among these, ``hormone therapy'' is a treatment method that can be used at any stage. This is because most prostate cancers grow under the influence of male hormones (androgens) secreted from the testes and adrenal glands, so by suppressing the secretion and function of androgens, it is possible to suppress the growth of prostate cancer cells. This is a treatment method that

This hormone therapy is effective while cancer cell growth is androgen-dependent, but after several years, prostate cancer becomes resistant to hormone therapy. Although it varies depending on when treatment is started and the stage of progression of prostate cancer, the average effect of initial hormone therapy is said to last three years. Cancer cells that have become hormone insensitive are more likely to invade and metastasize. Prostate cancer is particularly likely to metastasize to lymph nodes and bones (especially the spinal column and pelvic bones) (this is also called "metastatic prostate cancer"), and depending on the site of metastasis, it can be difficult to control, and it may take up to 5 years without recurrence. The survival rate is said to be 20% (5-year recurrence rate 80%) and 50% (mortality rate 50%) (see The Lancet Oncology (2013), 14, 149-158). Therefore, there has been a need for effective therapeutic methods for treating metastatic prostate cancer.
By the way, the expression of Activator of G-protein signaling 8 (hereinafter also referred to as "AGS8") has been observed in prostate cancer, and its expression level is higher in advanced cancer and metastatic prostate cancer, and the presence or absence of expression is also observed. It has been reported that it is correlated with life prognosis (see Non-Patent Document 1).

On the other hand, the inventors have previously shown that the interaction between AGS8 and G protein βγ subunit (hereinafter also referred to as "AGS8-Gβγ") plays an extremely important role in hypoxia-induced apoptosis of cardiomyocytes. We have reported that elucidating this and inhibiting this interaction could be a new approach to protecting myocardium from ischemic damage (see Non-Patent Document 2). Alternatively, it has been reported that peptides designed to inhibit the AGS8-Gβγ interaction inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis (see Non-Patent Document 3).

Dibash K.Das et al., “miR-1207-3p regulates the androgen receptor in prostate cancer via FNDC1/fibronectin”, Experimental Cell Research, 2016, 348(2):190-200 Motohiko Sato et al., “Protection of Cardiomyocytes from the Hypoxia-Mediated Injury by a Peptide Targeting the Activator of G-Protein Signaling 8”, PLos One, 2014, 9(3): e91980 Hisaki Hayashi et al., “Activator of G-protein signaling 8 is involved in VEGF-mediated signal processing during angiogenesis”, Journal of Cell Science, 2016, 129 (6): 1210-1222

Non-patent document 1 shows that AGS8 is expressed at a high rate in prostate cancer, especially metastatic prostate cancer, and non-patent document 3 states that the interaction between AGS8 and Gβγ is extremely important for the functional expression of AGS8. Although it has been suggested that it plays an important role, no specific treatment method for prostate cancer has been shown so far. Furthermore, a peptide that inhibits the AGS8-Gβγ interaction, as described in Non-Patent Document 3, has a problem that it is not suitable as a therapeutic agent because of its low stability in vivo.
Therefore, an object of the present invention is to provide a cancer therapeutic agent that inhibits the proliferation of cancer cells by inhibiting the AGS8-Gβγ interaction.

In order to solve the above problems, the present inventors considered that AGS8 would be a new target in cancer therapy, screened for compounds that inhibit the association of AGS8-Gβγ, and examined their effectiveness in detail. As a result, the inventors discovered that a specific compound has the effect of inhibiting the proliferation of cancer cells, leading to the completion of the present invention.
That is, the present invention has been made to solve the above-mentioned problems, and embodiments of the present invention may include the configurations listed below.

(1) A cancer therapeutic comprising a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof.
(2) The cancer therapeutic agent according to (1), further comprising a pharmaceutically acceptable excipient.
(3) The cancer therapeutic agent according to (1) or (2), wherein the cancer is prostate cancer.
(4) The cancer therapeutic agent according to any one of (1) to (3), wherein the cancer is metastatic prostate cancer.
(5) The cancer therapeutic agent according to any one of (1) to (4), wherein the cancer is a cancer accompanied by an increased expression level of AGS8 compared to human normal prostate epithelial cells.

According to the cancer therapeutic agent of the present invention, differentiation, proliferation and migration of cancer cells can be inhibited, and the proliferation of cancer cells can be specifically suppressed. Among cancers, it is effective in treating prostate cancer, especially androgen-independent prostate cancer, which is difficult to respond to hormone therapy. Since such androgen-independent cancers are metastatic, in other words, they are effective in treating metastatic prostate cancer. Furthermore, since the cancer therapeutic drug of the present invention has a mechanism of action of inhibiting the AGS8-Gβγ association, it is effective against cancer cells expressing AGS8, even in androgen-independent metastatic prostate cancer. Particularly effective. Furthermore, the cancer therapeutic agent of the present invention has low toxicity and causes less burden on patients. Furthermore, since it is transported into the blood without being broken down in the stomach or intestines, it has the advantage of being able to choose from a variety of administration methods, including oral administration, which is convenient for patients. Furthermore, since it is a low-molecular compound, manufacturing costs can be kept low, unlike biopharmaceuticals.

A diagram showing the mRNA expression level of AGS8 in PC-3 and DU145 cells relative to normal prostate epithelial cells. FIG. 3 is a diagram showing the results of confirmation of inhibition of AGS8-Gβγ subunit association by the cancer therapeutic drug of the present invention. FIG. 3 is a diagram showing the cytotoxicity of the cancer therapeutic agent of the present invention in PC-3 and DU-145 cells. FIG. 2 is a diagram showing the cell survival rate of DU-145 cells treated with the cancer therapeutic agent of the present invention. FIG. 3 is a diagram showing inhibition of cell migration by the cancer therapeutic agent of the present invention in DU-145 cells. FIG. 2 is a diagram showing the in vivo tumor formation suppressing effect of the cancer therapeutic agent of the present invention.

Examples of the present invention will be described in detail below, but the scope of the present invention is not limited to these examples.
<Activator of G-protein signaling 8 and cancer>

Trimeric G protein forms a heterotrimer consisting of three subunits α, β, and γ, functions as a molecular switch in living organisms, and plays an important role in physiological regulation. Among the subunits, the Gβ subunit and the Gγ subunit are stably linked, and the Gβγ complex functions as one subunit. Trimeric G proteins are activated by G protein-coupled receptors (GPCRs) on the cell surface and convert various external stimuli recognized by the GPCRs into intracellular signals.

Traditionally, G proteins have been considered to be molecular switches activated by receptors that recognize hormones, drugs, physical stimuli, etc., but in recent years, proteins other than receptors have activated G proteins, and this has been shown to be a pathophysiological regulatory mechanism. It has become clear that it is working. One of these proteins is Activator of G-protein signaling 8 (AGS8).

Our studies show that knockdown of AGS8 by small interfering RNA (siRNA) inhibits vascular endothelial growth factor (VEGF)-induced tube formation and VEGF-stimulated cell proliferation and migration. inhibited. Furthermore, a peptide that inhibits AGS8-Gβγ interaction inhibited vascular endothelial growth factor (VEGF)-induced angiogenesis (see Non-Patent Document 3). From this, the present inventors revealed that association with Gβγ is important for the function of AGS8. Furthermore, it has been reported that AGS8 is particularly expressed in prostate cancer cells among cancer cells, and is particularly expressed at high levels in metastatic prostate cancer cells, which are advanced prostate cancer (see Non-Patent Document 1). . It is known that advanced prostate cancer often becomes androgen independent. Therefore, the present inventors focused on the relationship between AGS8 and cancer, discovered a cancer therapeutic drug that targets AGS8 through screening, and completed the present invention. Specifically, they screened for compounds that inhibit the association of AGS8-Gβγ subunits, which is necessary for AGS8 to exert its functions, and confirmed their effectiveness.
<Cancer treatment drugs>

The cancer therapeutic agent of the present invention contains a compound represented by the following general formula or a pharmaceutically acceptable salt thereof. It is preferable to contain an effective amount of a compound represented by the following general formula or a salt thereof. (Hereinafter, the compound represented by the general formula below may also be referred to as an "AGS8 inhibitor.")
It may have a substituent as long as the above basic skeleton is maintained. The number of substituents may be one or more. When it has two or more substituents, the substituents may be the same or different.

Pharmaceutically acceptable salts refer to salts that do not cause undue toxicity, irritation, allergic reactions, etc. and are at least suitable for use in humans. Any basic salt such as alkali metal salt or alkaline earth metal salt, hydrochloride, sulfate, nitrate, acetate, citrate, tartrate, methanesulfonate may be used as long as it does not impair the effects of the present invention. - It may be an organic acid salt such as toluenesulfonate or a salt with an amino acid.

The above-mentioned compounds and salts thereof can be produced by known methods, or commercially available products can be obtained and used. It is preferable to use one that complies with the Japanese Pharmacopoeia. Because they can be synthesized through organic synthesis, manufacturing costs can be kept low, unlike biopharmaceuticals.

Since the cancer therapeutic agent of the present invention is a low molecular weight compound, it is stable in vivo. The peptide designed to inhibit the interaction between AGS8 and Gβγ, which is described in Non-Patent Document 3, is easily degraded by enzymes in the body when inoculated, and does not have the expected effect as a drug. Additionally, cell membranes generally act as a barrier between the inside and outside of cells, and biopolymers such as macromolecules and proteins and nucleic acids are highly hydrophilic and cannot pass through cell membranes. . Therefore, it is generally not easy to introduce any substance into cells for medical purposes. At the laboratory level, transfection methods are used to deliver proteins into cells. Various transfection reagents are commercially available to accommodate various conditions such as the type of cells to be transfected and the type of biomolecules to be transfected, but further improvements are required when considering transfection efficiency, cytotoxicity, and toxicity. The current situation is that this is desired.

For example, in Non-Patent Document 3, a transfection reagent called PLUSin (Polyplus) is used to deliver peptides to cells, but the transfection reagent used for peptides is based on lipophilic vesicles. This reagent is toxic to cell membranes and can be highly toxic to cells. In addition, when introducing a sample into cells using a transfection reagent, it is necessary to optimize various conditions such as the reagent and the number of cells to be introduced, and appropriate introduction can be achieved by meeting the optimization conditions. In in vivo administration, it is difficult to obtain uniform introduction conditions, resulting in non-uniformity in sample introduction.

On the other hand, since the cancer therapeutic agent of the present invention is a low-molecular compound, it easily passes through the cell membrane and is taken into cells. Since transfection reagents are not required, there is no need to worry about cell damage or toxicity caused by reagents.

Subjects to whom the cancer therapeutic agent of the present invention is administered are humans and non-human mammals in which cancer treatment is desired or required. Non-human mammals include, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, and include pet animals, livestock, and laboratory animals. Preferred subjects include humans.

The cancer therapeutic drug of the present invention contains the above-mentioned AGS8 inhibitor as an active ingredient, and optionally contains a non-toxic, inert, pharmaceutically acceptable excipient, such as a solid, semi-solid or liquid diluent. It is formulated by mixing with agents, dispersants, fillers and carriers. In addition, stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, flavoring preservatives, within a range that does not impair the effects of the present invention, Vehicles, saline, and other medicinal agents may be included as additives.
<Dosage form and dosage>

The dosage form of the cancer therapeutic agent of the present invention is not particularly limited, and includes oral administration preparations (tablets, coated tablets, powders, granules, capsules, liquid preparations, etc.), transrespiratory administration preparations, intraperitoneal administration preparations, and intraperitoneal administration preparations. Examples include preparations for intravenous administration, injections, suppositories, patches, ointments, etc., but preparations for oral administration, preparations for administration through the respiratory tract, and preparations for intravenous administration are preferred. Preparations for intravenous administration include intravenous injection preparations and drip intravenous injection preparations. For humans, formulations for oral administration or intravenous administration are preferred.

The dosage of the cancer therapeutic drug of the present invention can be adjusted appropriately taking into consideration the purpose of use, the subject to be administered, the sex, age, weight, and stage of cancer progression of the subject, but when administered to humans, It is preferable that the AGS8 inhibitor is contained in a dose of 1 to 10 mg/kg, preferably 3 to 10 mg/kg based on the patient's body weight. Within the above range, the effects of the present invention can be easily achieved, and toxicity is also low, resulting in fewer side effects. As for the administration regimen, the amount within the above range may be administered once a day, or intermittently every 1 to 2 days. Note that this dosage varies depending on various conditions, so a dosage or frequency of administration smaller than the above range may be sufficient, or a dosage or frequency of administration exceeding the above range may be necessary.

When administering the cancer therapeutic agent of the present invention, it may be administered in combination with other cancer therapeutic agents. For example, co-administration with the oral cancer treatments Xtandi (registered trademark) and Zytiga (registered trademark), the injectable drugs Taxotere (registered trademark) and Jevtana (registered trademark), and the bone metastasis treatment drug Xofigo (registered trademark). You may.
Concomitant administration means administration simultaneously with the administration of the cancer therapeutic agent of the present invention, or before and after administration of the cancer therapeutic agent of the present invention. Alternatively, the cancer therapeutic agent of the present invention and the other cancer therapeutic agents mentioned above can be mixed to form a single preparation.
<Applicable target>

The cancer therapeutic agent of the present invention can be used to treat, for example, prostate cancer, malignant melanoma, squamous cell carcinoma, basal cell carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung). cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer , colon cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney or renal cancer, vulvar cancer, thyroid cancer, eyelid tumor (including sebaceous gland cancer and basal cell carcinoma), conjunctiva tumors, orbital tumors (including lacrimal gland tumors), intraocular tumors (including retinoblastoma and choroidal melanoma), malignant lymphomas, ocular metastatic tumors, or various types of head and neck cancers (oral cancer, pharyngeal cancer). Applications include cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, nasal/sinus cancer, salivary gland cancer, thyroid cancer, etc.). Among these, application to prostate cancer is preferred, application to androgen-independent prostate cancer for which hormonal therapy is not effective is more preferred, and application to androgen-independent metastatic prostate cancer is even more preferred.

Androgen-independent prostate cancer refers to cancer that exhibits androgen dependence and has acquired resistance after a certain period of hormone therapy (androgen ablation therapy). In this specification, whether or not prostate cancer is androgen-independent is determined based on the criteria shown in the 2016 Prostate Cancer Treatment Guidelines (edited by the Japanese Urological Association, Medical Review Co., Ltd.). Cancer is determined to be resistant if the specific antigen value (e.g. PSA value) is 25% or more from the lowest value and the increase is at least a specific value (e.g. 2.0 ng/mL). Unless otherwise specified herein, if the PSA value is 25% or more from the lowest value and the increase is 2.0 ng/mL or more, it is determined to be androgen-independent prostate cancer.

When cancer becomes androgen independent, metastasis is often observed as the disease progresses. The degree of progression of cancer is called the stage, and is generally classified using the TMN classification (T (tumor): state of prostate cancer, N (nodes): presence or absence of lymph node metastasis, M (metastasis): distant metastasis). is used. These are performed using various image diagnoses. In this specification as well, whether prostate cancer has metastasized or not is diagnosed by various image diagnoses such as MRI, CT, and if bone metastasis is suspected, bone scintigraphy.

Since the cancer therapeutic drug of the present invention has a mechanism of action that inhibits the AGS8-Gβγ association, the expression level of AGS8 is increased in human normal prostate epithelial cells even in androgen-independent prostate cancer. It is effective against certain types of cancer. Incidentally, metastasis is often observed when the cell becomes androgen independent, but in this specification, as long as the expression level of AGS8 is increased in human normal prostate epithelial cells, it does not matter whether it is metastatic prostate cancer or not.

The expression level of AGS8 can be determined by any method as long as the expression level of AGS8 can be quantified in a specimen taken by biopsy or surgically resected, but methods such as quantitative real-time (qRT) PCR, microarray, etc. can be used. can be used. qRT-PCR can be performed using, for example, commercially available reagents and equipment. Specifically, mRNA is extracted from cells and cDNA is synthesized. Using the synthesized cDNA as a template, qRT-PCR can be performed using a real-time PCR device. The following primer sequences can be used: 5'-TTCCGTAACCCTCTCCCG-3' (sense) and 5'-AACCCACGATCAAGGTCCAC-3' (antisense).

The expression level of AGS8 is measured in cancer cells and human normal prostate epithelial cells, and if the expression ratio of mRNA in cancer cells to human normal prostate epithelial cells is 2 fold or more, the expression level of AGS8 in the present invention is increased. It can be said that it is cancer. Preferably it is 5 fold or more. More preferably, it is 100 fold or more.

The present invention will be specifically explained with reference to Examples shown below, but the present invention is not limited thereto.
(AGS8 inhibitor)

A pilot library was obtained from the RIKEN compound bank NPDepo, and was used to screen for compounds that have the activity of inhibiting the association of AGS8 and Gβγ subunit, thereby identifying the AGS8 inhibitor of the present invention. The identified AGS8 inhibitor was purchased from overseas suppliers (Asinex (Russia), Sundia MediTech Company (Shanghai, China)) through Namiki Shoji Co., Ltd., and used in the test.
(cell)

Androgen-independent metastatic cancer cells PC-3 (derived from bone metastatic prostate cancer cells) and DU-145 (derived from brain metastatic prostate cancer cells) were obtained from the Medical Cell Resource Center (Tohoku University Research Institute for Aging and Aging). RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 U/mL penicillin, and 100 μg/mL streptomycin (both GIBCO) in a humidified incubator at 5% CO ₂ , 95% O ₂ , 37°C. (Life Technologies, Inc.) for monolayer culture.

Human normal prostate epithelial cells (PrEC) were purchased from Lonza and incubated at 37°C in PrEGM Bullet Kit Medium (Lonza) supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin (both GIBCO). It was cultured in Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza. In this example, cells used in all experiments were grown to 60%-70% confluence.
(Confirmation test for AGS8 expression level)

AGS8 expression levels in PC-3, DU-145, and PrEC were measured using quantitative real-time (qRT) PCR. Specifically, total RNA was extracted from a suspension of cultured cells using the PureLink RNA Mini Kit (Ambion), and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qRT-PCR analysis was performed. qRT-PCR was performed using the Step One Plus real-time PCR system (Applied Biosystem) according to the protocol of TB Green Premix Ex Taq II (Takara Bio). Specific PCR primers were designed as follows.
The following primer sequences were used for human AGS8 real-time PCR.
5'-TTCCGTAACCCTCTCCCG-3' (sense) and
5'-AACCCACGATCAAGGTCCAC-3' (antisense)
The relative expression levels of AGS8 of PC-3 and DU-145 on human normal prostate epithelial cells (PrEC) were determined by the comparative Ct value method.

The results are shown in Figure 1. Compared to human normal prostate epithelial cells, androgen-independent metastatic cancer cells PC-3 express 5.7 folds of AGS8 and 240 folds of DU-145, and both cells express AGS8 more than human normal prostate epithelial cells. It can be seen that the expression level of .
(AGS8-Gβγ subunit association inhibition confirmation test)

It was confirmed by a pull-down assay whether the cancer therapeutic agent of the present invention inhibits the association of AGS8-Gβγ subunit. The gene encoding AGS8 (372 amino acids at the C-terminus of AGS8) was inserted into pGEX-4T vector (Amersham Biosciences) and expressed as a GST-tagged fusion protein in Escherichia coli (Esche-richia coli BL21, Amersham Biosciences). After disrupting the bacterial cells, the eluted GST-AGS8 was affinity purified using Glutathione-Sepharose 4B. The G protein β1γ2 subunit was synthesized in insect cells. Add 100 nM GST-AGS8, 10 nM Gβ1γ2, the cancer therapeutic agent of the present invention (final concentration 16 μg/ml), and a compound that does not inhibit the association of AGS8-Gβγ subunit (final concentration 16 μg/ml), and add 300 μl at 4°C. (20mM Tris-HCl, pH 7.4, 0.6mM EDTA, 1mM dithiothreitol, 70mM NaCl, 0.01% Lubrol, 10μm GDP, and 10mM MgCl2) for 18 hours. After the reaction, Glutathione-Sepharose 4B was added to the reaction tube to adsorb GST-AGS8C for 1 hour. Glutathione-Sepharose 4B was washed three times with the reaction solution to remove nonspecific adsorption, and the adsorbed proteins were eluted into Laemmli sample buffer. The entire sample was loaded into a well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to Immobilon-P PVDF membrane, labeled with anti-Gβ subunit antibody (1:1,000; AC-74; Sigma-Aldrich) as the primary antibody and HRP (horseradish peroxidase) as the secondary antibody, and then labeled with ImmunoStar. Detection was performed using an LD (Fujifilm Wako Pure Chemical Corp.), and images were analyzed using an Amersham Imager 600 system (GE Healthcare Life Sciences).

The results are shown in Figure 2. Input is a 1/50 volume mixed reaction solution containing GST-AGS8 and Gβ1γ2. GST-AGS8 coprecipitated Gβγ subunits, but in the presence of 16 μg/ml of the cancer therapeutic drug of the present invention (described as AGS8 inhibitor in Figure 2), the amount of Gβ subunits precipitated on the resin decreased. is decreasing. On the other hand, a compound that does not inhibit the association of AGS8-Gβγ subunits at the same concentration (denoted as a control group in Figure 2) did not affect the coprecipitation of Gβ subunits by GST-AGS8. Therefore, it was revealed that the cancer therapeutic agent of the present invention inhibits the association of AGS8-Gβγ subunit.
(Cytotoxicity test)

When cells are destroyed by the toxicity of the drug, the enzyme LDH is eluted into the culture medium. Therefore, the cytotoxicity of the cancer therapeutic agent of the present invention was evaluated by setting the concentration of LDH eluted under conditions where almost all cells would be damaged as 100%.

HUVEC or DU-145 cells were seeded in 96-well culture plates at a density of 6,000 cells/well. After culturing overnight, the cells were incubated for 48 hours in a medium containing the cancer therapeutic agent of the present invention (1, 3, 10, 13, 100 μg/mL). Cytotoxicity was evaluated by the amount of LDH released into the medium using an LDH assay kit (Dojindo Laboratories). The results obtained were quantified colorimetrically at absorbance at 450 nm using SpectraMax M3 (Molecular Devices).

The results are shown in Figure 3. Even when the cancer therapeutic agent of the present invention was administered, no concentration-dependent increase in LDH was observed in either normal cells or DU-145 cells, and no significant cytotoxicity was observed in the range of 1 to 100 μg/mL. I couldn't.
(DU-145 cell viability confirmation test)

The effect of the cancer therapeutic agent of the present invention on the survival rate of DU-145 cells was confirmed. DU-145 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 96-well plate and treated with different doses of the cancer therapeutic agent of the present invention. After 48 hours of treatment, cell viability was assessed using Cell Counting Kit-8 assay. Shown as mean ± SEM of 6 experiments.

The results are shown in Figure 4. When the cancer therapeutic drug of the present invention was administered, the cell viability of DU-145 cells significantly decreased (p<0.01) in a dose-dependent manner, indicating that cell proliferation was suppressed (one-way analysis of variance (ANOVA)). ).
(DU-145 cell migration inhibition test)

DU-145 suspended at 5×10 ⁴ cells/200 μl was seeded into fibronectin-coated transwell inserts with 8 μm pores (BD Biosciences) pretreated with 3% BSA. Cell migration was induced by adding 150 ng/ml PDGF-BB to the lower chamber of a 24-well plate. After 18 hours, remaining cells on the top of the filter were removed using a cotton swab. Transwell inserts were fixed with 4% PFA and stained with 1% crystal violet. As a control, phosphate buffered saline was used. A digital microscope image was randomly taken of the underside of each transwell insert, and stained cells were counted. Data were performed 7 times independently and expressed as mean ± SEM. (Analyzed using one-way analysis of variance (ANOVA))

The results are shown in Figure 5. When the AGS8 inhibitor, which is a cancer therapeutic drug of the present invention, was added, the number of migrating cells was significantly suppressed (p<0.01). Generally, cancer cells separate from the tissue in which they occur, break down the basement membrane, and invade surrounding tissues. The cancer cells then migrate within the blood vessels and are carried by the blood to other parts of the body, where they proliferate and metastasize. Since the cancer therapeutic agent of the present invention inhibits cancer cell migration, it is useful as a cancer cell metastasis inhibitor.
(In vivo tumor formation inhibition test)

Male BALB/c nude mice (5 weeks old) (Japan SLC Co., Ltd.) were obtained. DU-145 cells (4×10 ⁶ cells/200 μL) were suspended in serum-free RPMI-1640 medium, mixed with Matrigel (1:1 ratio), and implanted subcutaneously into the flank region of mice. The cancer therapeutic agent of the present invention was suspended in 0.9% physiological saline, and 10 μg and 30 μg of the suspension was subcutaneously injected into the tumor every day.
Every week, the DU-145 cell transplanted area was incised, the tumor was removed, and the tumor volume and weight were measured. Tumor volume (mm ³ ) was calculated by measuring the major axis and minor axis with calipers and calculating the major axis x (minor axis) ² /2 (n = 6 for each).

FIG. 6(A) shows typical tumors after administration of 0.1% physiological saline as a control and administration of 10 μg and 30 μg of the cancer therapeutic agent of the present invention for 1 to 4 weeks. FIG. 6 (B) shows the tumor volume after administering the control and the cancer therapeutic agent of the present invention for 1 to 4 weeks, and FIG. 6 (C) shows the tumor weight. Statistical differences between treatment groups at each time point were detected by two-way analysis of variance (ANOVA) and Tukey's correction test.
In controls, tumor growth was observed from week 1 to week 4. On the other hand, when the cancer therapeutic agent of the present invention was administered, the tumor volume and weight were significantly suppressed (**p <0.01). (Figure 6(A)-(C)). During the test, no abnormal appearance was observed in any of the mice, demonstrating the safety of the present invention.

The compound represented by general formula (1) or a pharmaceutically acceptable salt thereof can be applied to the treatment of cancer.

Claims (5)

A cancer therapeutic agent comprising a compound represented by general formula (1) or a pharmaceutically acceptable salt thereof.
The cancer therapeutic agent according to claim 1, further comprising a pharmaceutically acceptable excipient. The cancer therapeutic agent according to claim 1 or 2, wherein the cancer is prostate cancer. The cancer therapeutic agent according to claim 1 or 2 , wherein the cancer is metastatic prostate cancer. 3. The cancer therapeutic agent according to claim 1 , wherein the cancer is a cancer accompanied by an increased expression level of AGS8 compared to human normal prostate epithelial cells.