JP2021123560A - Claudin-2 binding low-molecular-weight compound having anticancer agent resistance improving action - Google Patents

Abstract

To create a cancer adjuvant therapy useful for cancer treatment and improve the outcomes of cancer treatment.SOLUTION: There is provided an agent that contains ledipasvir or a pharmaceutically acceptable salt thereof as an active ingredient and is useful for improving a hypoxic state of a cancer cell or enhancing the anticancer agent sensitivity of a cancer cell.SELECTED DRAWING: None

Description

The present invention relates to a claudin-2 (hereinafter sometimes abbreviated as "CLDN2") binding small molecule compound. More specifically, the present invention relates to a small molecule compound that binds to CLDN2 and is useful for the treatment of cancer and its use (therapeutic agent, etc.).

Acquisition of treatment resistance has become a major problem in cancer drug therapy. The causes have been reported to be the induction of drug excretion pumps, the induction of metabolic enzymes, the structural changes of target molecules, etc., but there are still many unclear points, and clinically usable therapeutic resistance overcoming drugs have not been developed. .. Recently, it has become clear that cancer cell aggregates are involved in treatment resistance. Cancer cells form aggregates in the living body, and it is difficult for many anticancer agents to act on the deep part of the aggregates. In addition, the deep part of the agglutinin is always in a stress state of hypoxia and malnutrition, which contributes to the acquisition of treatment resistance, malignant transformation, and recurrence. Therefore, it is expected that not only the development of anticancer agents that act on the protooncogene but also the development of new types of agents that improve the permeability of aggregates to anticancer agents and the stress state. By developing an adjunct therapeutic drug that enhances the permeability of anticancer drugs to the deep part of the agglutinin, it is expected that the therapeutic effect will be improved not only for existing anticancer drugs but also for anticancer drugs to be developed in the future.

Abnormal expression of the claudin (CLDN) subtype has been reported in many solid tumor tissues. CLDN is a cell membrane protein present at tight junctions of epithelial cells and endothelial cells. So far, our research group has reported that CLDN2, which is not expressed in normal human lung cells, is highly expressed in adenocarcinoma cells (Non-Patent Document 1). We also reported that the peptide DFYSP (SEQ ID NO: 6), which has the same structure as a part of the second extracellular loop of CLDN2, acts on CLDN2 expressed in adenocarcinoma cells and damages the cells (Non-Patent Document 2). ). On the other hand, the effect of CLDN2 on the formation of agglomerates and the sensitivity to anticancer drugs has not been reported in Japan and overseas, but the research group of the present inventors reduced the hypoxicity of agglomerates by knocking down CLDN2 expression. However, it was discovered that the sensitivity to anticancer drugs is enhanced (Non-Patent Document 3).

Ikari, A., Sato, T., Watanabe, R., Yamazaki, Y., and Sugatani, J. (2012) Increase in claudin-2 expression by an EGFR / MEK / ERK / c-Fos pathway in lung adenocarcinoma A549 cells. Biochim. Biophys. Acta 1823, 1110-1118 Ikari, A., Taga, S., Watanabe, R., Sato, T., Shimobaba, S., Sonoki, H., Endo, S., Matsunaga, T., Sakai, H., Yamaguchi, M., Yamazaki, Y., and Sugatani, J. (2015) Clathrin-dependent endocytosis of claudin-2 by DFYSP peptide causes lysosomal damage in lung adenocarcinoma A549 cells. Biochim. Biophys. Acta 1848, 2326-2336 Maruhashi R, Akizuki R, Sato T, Matsunaga T, Endo S, Yamaguchi M, Yamazaki Y, Sakai H, Ikari A. (2018) Elevation of sensitivity to anticancer agents of human lung adenocarcinoma A549 cells by knockdown of claudin-2 expression in monolayer and spheroid culture models. Biochim. Biophys. Acta 1865 (3), 470-479 Kinugasa, T., Huo, Q., Higashi, D., Shibaguchi, H., Kuroki, M., Tanaka, T., Futami, K., Yamashita, Y., Hachimine, K., Maekawa, S., Nabeshima, K., and Iwasaki, H. (2007) Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer. Anticancer Res. 27, 3729-3734 Weber, CR, Nalle, SC, Tretiakova, M., Rubin, DT, and Turner, JR (2008) Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation. Lab. Invest. 88, 1110-1120 Halasz, J., Holczbauer, A., Paska, C., Kovacs, M., Benyo, G., Verebely, T., Schaff, Z., and Kiss, A. (2006) Claudin-1 and claudin-2 differentiated fetal and embryonal components in human hepatoblastoma. Hum. Pathol. 37, 555-561 Luettig, J., Rosenthal, R., Barmeyer, C., and Schulzke, J. D. (2015) Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation. Tissue barriers 3, e977176

As described above, it was clarified that CLDN2 contributes to the resistance of cancer cells forming aggregates to anticancer drugs. Drugs that reduce the expression of CLDN2, which is highly expressed in cancer tissues, not only increase the sensitivity of anticancer drugs, but also suppress the malignant transformation of cancer cells and enable eradication of new types of adjuvant therapies. Can be expected to become. A main object of the present invention is to create such an adjuvant therapeutic agent and contribute to improving the treatment results of cancer.

While proceeding with the research in view of the above problems, the present inventors focused on the structure of CLDN2. Then, for the purpose of inhibiting the binding between CLDN2, we searched for compounds that are predicted to bind CLDN2 to the second extracellular loop by docking simulation. As a result of examining the expression-reducing effect and specificity of CLDN2 protein on the compounds selected by screening, we succeeded in identifying the extremely effective compound "ledipasvir". In addition, useful findings were obtained regarding its mechanism of action, and it was found that ledipasvir improves the hypoxic state (stress state) of cancer cells and enhances anticancer drug sensitivity. In other words, we have succeeded in finding a "substance that improves the hypoxic state of cancer cells and enhances the sensitivity to anticancer drugs" that is useful as an active ingredient of an adjunct therapeutic drug for cancer treatment.

By the way, CLDN2 is highly expressed in lung adenocarcinoma, colon cancer, liver cancer, etc. (Non-Patent Document 1, Non-Patent Documents 4 to 6), but Crohn's disease, ulcerative colitis, celiac disease, and HIV. Even in infection, the expression level of CLDN2 in the intestinal tract increases, causing symptoms such as diarrhea (Non-Patent Document 7). That is, high expression of CLDN2 is important for the cause (base) of various diseases and the formation of pathological conditions, and is a therapeutic target. From this, it is highly expected that ledipasvir, which is a compound that has been successfully identified, can be used and applied to the treatment of various diseases associated with high expression of CLDN2.

Based on the above results and considerations, the following inventions are provided.
[1] A drug containing ledipasvir or a pharmaceutically acceptable salt thereof as an active ingredient and effective for improving the hypoxic state of cancer cells and / or enhancing the sensitivity of cancer cells to anticancer drugs.
[2] A cancer adjuvant therapy drug containing the drug according to [1] and used in combination with an anticancer drug.
[3] The cancer adjuvant therapeutic agent according to [2], which is used for the treatment of cancer in which high expression of claudin-2 is observed.
[4] The cancer adjuvant therapy agent according to [3], wherein the cancer is lung adenocarcinoma, colon cancer, liver cancer or esophageal cancer.
[5] A step of administering a therapeutically effective amount of the cancer adjuvant therapeutic agent according to any one of [2] to [4] to a cancer patient treated with an anticancer drug is included. How to treat cancer.
[6] A therapeutic agent for a disease caused by or forming a pathological condition of claudin-2, which contains ledipasvir or a pharmaceutically acceptable salt thereof as an active ingredient.

The structure of CLDN2. A: The two-dimensional structure of CLDN2 is schematically shown. The amino terminus (1st) and carboxy terminus (230th) are intracellular. The extracellular second loop (145th to 159th) assumed as the binding site of the compound in this study is shown by a thick line. B: Amino acid sequence of CLDN2 (SEQ ID NO: 5). The amino acids in the second extracellular loop are shown in a frame. Effect of each compound on protein expression of CLDN2. A549 cells were treated with solvent, 1 or 10 μM compound for 24 hours and then the cells were harvested. The expression levels of CLDN2 and β-actin proteins were examined by Western blotting. The band strength was quantified and shown as a ratio to the vehicle. Effect of Compound 3 (# 3: Ledipasvir) on protein expression of CLDN2. A: A549 cells were treated with each concentration of Compound 3 for 24 hours, and the cells were collected. The expression levels of CLDN2 and β-actin proteins were examined by Western blotting. The band intensity was quantified and shown as a relative value to 0 μM. B: A549 cells were treated with 10 μM compound 3 for a specified time, and the cells were collected. The expression levels of CLDN2 and β-actin proteins were examined by Western blotting. The band intensity was quantified and shown as a relative value with respect to 0 hours. If there is a significant difference of less than 5% with respect to 0 μM or 0 hours, it is indicated by *, if it is less than 1%, it is indicated by **, and if there is no significant difference, it is indicated by NS. Effect of Compound 3 (# 3: Ledipasvir) on the mRNA expression and transcriptional activity of CLDN2. A: A549 cells were treated with compound 3 at each concentration for 6 hours, and RNA was extracted. After the reverse transcription reaction, the expression levels of CLDN2 and β-actin mRNA were examined by real-time PCR. The mRNA expression level is shown as a relative value to 0 μM. If there is no significant difference with respect to 0 μM, it is indicated by NS. B: Under transfection with the pGL4 luciferase vector in which the CLDN2 promoter was incorporated into 549 cells and the pRL-TK vector. After treatment with compound 3 at each concentration for 6 hours, transcriptional activity was measured. Transcription activity is shown as a relative value to 0 μM. If there is no significant difference with respect to 0 μM, it is indicated by NS. Effect of inhibitors on protein expression of CLDN2. A: A549 cells were treated with 10 μM compound 3 (# 3: ledipasvir) and 100 μM chloroquine (CQ) for 24 hours, and then the cells were recovered. The expression levels of CLDN2 and β-actin proteins were examined by Western blot method. The band intensity was quantified and shown as a relative value to 0 μM. B: A549 cells were treated with 10 μM compound 3, 10 μM lactacystin (LC) for 24 hours, and the cells were recovered. The expression levels of CLDN2 and β-actin proteins were examined by Western blot method. The band intensity was quantified and shown as a relative value to 0 μM. If it is less than 1% and there is a significant difference with respect to the solvent (vehicle), it is indicated by **, and if it is less than 1% and there is a significant difference with respect to the compound 3 treatment, it is indicated by ##. Effect of Compound 3 (# 3: Ledipasvir) on cell localization of CLDN2. A549 cells were treated with solvent (control), 10 μM compound 3, 100 μM chloroquin (CQ), 10 μM lactacystin (LC), and 5 μM monodansyl cadaverine (MDC) for 24 hours, and then fluorescent immunostaining was performed. It shows the cell localization of CLDN2, ZO-1, and nucleus (DAPI). Changes in intercellular permeability due to compound 3 (# 3: Ledipasvir). A: A549 cells cultured in transwells were treated with 0, 1, 5, and 10 μM compound 3 for 24 hours, and then the electrical resistance between top coats was measured using a Volt ohmmeter. B: After measuring the electrical resistance between the upper coatings, doxorubicin was added to the upper layer of the transwell, and after 30 minutes, the solution in the lower layer was recovered and the fluorescence intensity of doxorubicin was measured. If there is a significant difference of less than 1% with respect to 0 μM, it is indicated by **. Effect of short-chain peptides on spheroid size and cell viability. After culturing A549 cells on a round bottom plate for 72 hours, a solvent, 10 μM compound 3 (# 3: Ledipasvir) was added, and the cells were further cultured for 24 hours. The size was calculated based on the circumference of the spheroid and shown as a relative value to the solvent. After culturing A549 cells on a round bottom plate for 72 hours, 10 μM compound 3 and LOX-1 were added, and the cells were further cultured for 24 hours. The fluorescence intensity of LOX-1 was measured with a fluorescence microscope. After culturing A549 cells on a round bottom plate for 72 hours, 10 μM compound 3 was added, and the cells were further cultured for 24 hours. The cell viability was calculated based on the intracellular ATP concentration and shown as a relative value to the solvent. If there is a significant difference of less than 1% with respect to the solvent, it is indicated by **, and if there is no significant difference, it is indicated by NS. Effect of doxorubicin and compound 3 (# 3: ledipasvir) on spheroid cells. A: A549 cells were cultured on a round bottom plate for 72 hours, then a solvent, 10 μM compound 3 was added, and the cells were further cultured for 24 hours. Fluorescence images were taken 1 hour after the addition of each concentration of doxorubicin, and the fluorescence intensity of doxorubicin was calculated. The amount of doxorubicin accumulated is shown as a relative value to 0 μM. B: A549 cells were cultured on a round bottom plate for 72 hours, then a solvent, 10 μM compound 3 and each concentration of doxorubicin were added, and the cells were further cultured for 24 hours. The size was calculated based on the circumference of the spheroid and shown as a relative value to the solvent. C: A549 cells were cultured on a round bottom plate for 72 hours, 10 μM compound 3 and each concentration of doxorubicin were added, and the cells were further cultured for 24 hours. The cell viability was calculated based on the intracellular ATP concentration and shown as a relative value to the solvent. If it is less than 5% and there is a significant difference with respect to the solvent, it is indicated by *, and if it is less than 1%, it is indicated by **. Effect of cisplatin and SN-38 on cell viability of spheroids. A: A549 cells were cultured on a round bottom plate for 72 hours, then added with a solvent, 10 μM compound 3 (# 3: regipasvir), each concentration of cisplatin or SN-38 (active metabolite of irinotecan), and further cultured for 24 hours. .. The size was calculated based on the circumference of the spheroid and shown as a relative value to 0 μM. B: A549 cells were cultured on a round bottom plate for 72 hours, then the solvent, 10 μM compound 3, cisplatin or SN-38 at each concentration were added, and the cells were further cultured for 24 hours. The survival rate was calculated based on the ATP concentration of spheroids and shown as a relative value to 0 μM. If it is less than 5% and there is a significant difference with respect to Vehicle, it is indicated by *, and if it is less than 1% and there is a significant difference, it is indicated by **.

<Drugs targeting cancer cells and cancer adjuvant therapies>
The present invention relates to the use of the compound "ledipasvir" which has been found to bind to CLDN2 and reduce its expression (abundance). Although not bound by theory, cancer cells (typically cancer cells that make up cell aggregates) are treated with Regipasvir (Ledipasvir; Methyl {(1S) -1-[(1R, 3S, 4S)). -3- (5- {9,9-difluoro-7- [2-((6S) -5-{(2S) -2-[(methoxycarbonyl) amino] -3-methylbutanoyl} -5-azaspiro [2.4] hept-6-yl) -1H-imidazol-4-yl] -9H-fluoren-2-yl} -1H-benzimidazol-2-yl) -2-azabicyclo [2.2.1] heptane-2-carbonyl] -2 -Methylpropyl} carbamate) reduces the expression of CLDN2 at tight junctions between cells, improves the hypoxic state of cancer cells, suppresses malignant transformation of cancer cells, and suppresses the malignant transformation of cancer cells. Increased anticancer drug sensitivity. Applications that take advantage of this unique effect include ledipasvir or a pharmaceutically acceptable salt thereof (ie, as an active ingredient), improving the hypoxic state of cancer cells, and / or of cancer cells. Drugs that are effective in increasing anticancer drug sensitivity are provided. If the drug is used in combination with an anticancer drug, the efficacy of the anticancer drug can be enhanced, and the therapeutic effect or the therapeutic result can be expected to be improved. That is, the drug of the present invention is useful as a cancer adjuvant therapy drug, and is applied to cancer treatment together with an anticancer drug. In order to express a typical usage mode in which it is used in combination with an anticancer drug in cancer treatment (that is, it is used as a supplement in the administration of an anticancer drug), the medicament of the present invention is referred to as "cancer support". Although referred to as a "therapeutic agent", the medicament of the present invention can suppress the malignant transformation of cancer cells by improving the hypoxic state of the cancer cells, and in this respect, the cancer cells themselves. It can be said that it exerts a medicinal effect on. The structure of the Ledipasvir is shown below.

CLDN2 is one of the claudin family proteins involved in the formation of tight junctions between cells, is highly expressed in the proximal tubule, bile sac, and small intestine of the kidney, and is involved in the transport of sodium ions. It is an important molecule. Although there are several subtypes of CLDN (CLDN1, CLDN2, CLDN3, CLDN4, etc.), the active ingredient of the medicament of the present invention (ledipasvir or a pharmaceutically acceptable salt thereof) is highly selective for CLDN2. / Shows specificity.

The medicament of the present invention is highly useful in that it can be used to enhance the efficacy of various anticancer agents. On the other hand, according to the medicament of the present invention, it is clinically extremely important and meaningful to provide a promising therapeutic strategy for so-called treatment-resistant cancer in which an anticancer drug is difficult or ineffective. In addition, the combined use of the anticancer agent and the adjuvant therapeutic agent of the present invention can also enable the eradication of cancer, and its clinical value and significance are great.

In the present invention, the term "cancer" is broadly interpreted and used interchangeably with the term "malignant tumor". In addition, benign tumors, benign malignant borderline lesions, and malignant tumors may be comprehensively included before the pathological diagnosis is confirmed, that is, before either benign or malignant tumor is confirmed. could be. In general, cancer is called by the name of the organ that became the mother of its development or the name of the mother tissue that developed it. , Esophageal cancer, gastric cancer, small bowel cancer, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bile sac cancer, pancreatic cancer, lung cancer, breast cancer, thyroid cancer, adrenal cancer, pituitary tumor, pineapple tumor, uterine cancer, Ovarian cancer, vaginal cancer, bladder cancer, kidney cancer, prostate cancer, urinary tract cancer, retinoblastoma, conjunctival cancer, neuroblastoma, glioma, glioblastoma, skin cancer, Myelopathy, leukemia, malignant lymphoma, testicular tumor, osteosarcoma, horizontal print myoma, smooth myoma, hemangiosarcoma, liposarcoma, chondrosarcoma, Ewing's sarcoma, etc. Further, depending on the characteristics of the site of the developing organ, it is subdivided into upper / middle / hypopharyngeal cancer, upper / middle / lower esophageal cancer, gastric jet cancer, gastric pyloric cancer, cervical cancer, endometrial cancer, etc. , These are not limited and are included in the description as "cancer" of the present invention.

The medicament of the present invention is used for the treatment of cancer in which expression of CLDN2 is observed. Preferably, cancers with high expression of CLDN2 are treated. Examples of applicable cancers include lung adenocarcinoma, colon cancer, liver cancer, and esophageal cancer. High expression of CLDN2 has been confirmed in these cancers (see Non-Patent Documents 1, 4 and 6).

As the active ingredient of the medicament of the present invention, a pharmacologically acceptable salt of ledipasvir may be used. The "pharmacologically acceptable salt" is not particularly limited, and the use of various salts such as acid addition salts and amino acid addition salts is assumed. Examples of acid addition salts include hydrochlorides, sulfates, nitrates, phosphates, inorganic acid salts such as hydrobromide, acetates, maleates, fumarates, citrates, benzenesulfonates, Examples include organic acid salts such as benzoate, malate, oxalate, methanesulfonate, and tartrate. Examples of amino acid addition salts include glycine addition salt, phenylalanine addition salt, lysine addition salt, aspartic acid addition salt, and glutamic acid addition salt. Acetone adduct (Ledipasvir Acetonate) is one of the preferable active ingredients.

The pharmaceutical product of the present invention can be formulated according to a conventional method. When formulated, other components (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, surfactants) that are acceptable in the formulation Activator, lubricant, dilute agent, coating agent, sugar coating agent, flavoring agent, emulsifying / solubilizing agent, pH adjusting agent, isotonic agent, solubilizing agent, fragrance, coloring agent, solubilizing agent, physiology (Salt solution, etc.) can be contained. The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, liquids, suspensions, emulsions, jellies, injections, topical agents, inhalants, nasal drops, eye drops and loci. It is an agent. The medicament of the present invention contains an amount (that is, a therapeutically effective amount) of an active ingredient necessary for obtaining the expected therapeutic effect (or preventive effect). The amount of the active ingredient in the medicament of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set in the range of, for example, about 0.01% by weight to about 95% by weight so that a desired dose can be achieved. Normally, the medicament of the present invention is composed as a single preparation, but it may be a combination drug with an anticancer drug.

The medicament of the present invention is used in combination with an anticancer agent. When the drug of the present invention is used in combination with an anticancer drug, improvement in treatment results can be expected. Here, "improvement of treatment results" includes (1) increase of therapeutic effect, (2) improvement of response rate or efficacy rate, and (3) reduction or avoidance of side effects. The anticancer drug is not particularly limited. Examples of anticancer agents include platinum preparations such as cisplatin, nedaplatin, oxaliplatin, carboplatin, cyclophosphamide, iposphamide, nitrosourea, dacarbazine, temozolomid, nimustin, busulfan, melfaran, thiotepa, procarbazine, lanimustin, etc. Alkylating agents, enocitabine, carmofur, capecitabin, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, citarabin, citarabin ocphosphat, nerarabin, fluorouracil, fludarabin, pemetauximab , Hydroplasty antagonists such as hydroxycarbamide and mercaptopurine, antitumors such as mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idalubicin, pyrarubicin, pepromycin, mitoxantron, amurubicin, dinostatinstimalamar Cetuximab (registered trademark) , Trastuzumab (Herceptin®), Alemtuzumab (Campath®), Cetuximab (Erbitux®), Panitumumab (Vectibix®), Ofatumumab (Arzerra®), Denosumab (Ranmark) Ipilimumab (Yervoy®), Mogamurizumab (Poteligeo®), Pertuzumab (Perjeta®), Ofatumumab (Gazyva®), Ramsilumab (Cyramza®), Nivorumab (Opdivo®), Pembrolizumab (Keytruda®), Blincyto®, Unituxin®, Darzalex®, Portrazza® Antibody drugs such as (trademark)) and eroticuzumab (Empliciti (registered trademark)), gemtuzumab ofatumumab (Mylotarg (registered trader)) It is an antibody drug conjugate (ADC) such as brentuximab vedotin (Adcetris®), trastuzumab emtansine (Kadcyla®), and inotuzumab ozogamicin (BESPONSA®). It is also possible to use two or more anticancer agents in combination. The dose of the anticancer drug is the same as the amount used when it is used alone (that is, the usual amount). However, since it is expected that the efficacy of the anticancer drug will be increased by the combined use with the drug of the present invention, the dose may be set lower than the usual dose. A person skilled in the art can set the "normal usage amount" in consideration of the patient's medical condition, age, gender, body weight, and the like.

The active ingredient of the medicament of the present invention lowers the barrier function of tight junctions through the reduced expression of CLDN2. Therefore, the medicament of the present invention can be expected to enhance the intercellular permeability of an anticancer drug, and is not only effective for increasing the efficacy of a specific anticancer drug. That is, it is versatile or highly general, and can be used in combination with various anticancer agents. Therefore, in addition to the various existing anti-cancer agents exemplified above, it is naturally expected to be used in combination with an anti-cancer agent under development or an anti-cancer agent to be developed in the future.

The medicament of the present invention is administered to a subject (patient) at the same time as or at intervals of time with the anticancer drug. "Simultaneous" here does not require strict simultaneity. Therefore, it goes without saying that when the drug of the present invention and the anticancer drug are mixed and then administered to the subject, the administration of the two is carried out under conditions where there is no time lag, and of course, the other is immediately administered after the administration of one. The concept of "simultaneous" is also included in the case where both administrations are carried out under conditions where there is substantially no time difference.

The medicament of the present invention is targeted by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) depending on the dosage form. Applies. These administration routes are not exclusive to each other, and two or more arbitrarily selected administration routes can be used in combination (for example, intravenous injection or the like at the same time as oral administration or after a lapse of a predetermined time). Topical administration may be used instead of systemic administration. It may be administered so that the active ingredient is delivered specifically to the target tissue using a drug delivery system (DDS).

A further aspect of the present invention is a therapeutic method for cancer using the medicament of the present invention (preventive treatment is also included in the concept of therapeutic method). The treatment method of the present invention includes the step of administering the medicament of the present invention to a cancer patient who is treated with an anticancer agent. The dose of the medicament of the present invention may vary depending on the patient's symptoms, age, gender, body weight, etc., but those skilled in the art can appropriately set an appropriate dose. The administration schedule may correspond to the administration schedule of the anticancer drug.

<Application to other diseases>
As described above, the active ingredient of the medicament of the present invention has a characteristic action effect of directly binding to the second extracellular loop of CLDN2 and reducing the expression (abundance) of CLDN2 distributed in tight junctions between cells. Is shown. Therefore, it can exert its peculiar action and effect on diseases caused by high expression of CLDN2 or forming the pathological condition thereof, such as Crohn's disease, ulcerative colitis, celiac disease, and HIV infection. Therefore, the present invention also provides a therapeutic agent for various diseases associated with high expression of CLDN2, which contains the active ingredient of the medicament of the present invention.

CLDN2 is a molecule that is highly expressed in the proximal tubule, bile sac, and small intestine of the kidney in normal tissues and is involved in the transport of sodium ions, and plays an important physiological function. In addition, as mentioned above, it is involved in the onset and progression of Crohn's disease, ulcerative colitis, celiac disease, HIV infection, etc., as well as various cancers, and is the target of basic research and development of therapeutic agents or treatment methods. .. The active ingredient of the medicament of the present invention is also useful as a tool (research reagent) in such research and development.

The following studies were conducted with the aim of developing a new type of adjuvant therapy that suppresses the malignant transformation of cancer cells and enables eradication.

1. 1. Method (1) Culturing of lung adenocarcinoma cells A549 cells derived from human lung adenocarcinoma were cultured in a carbon dioxide incubator under _{37 ° C. and 5% CO 2 conditions.} Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum, 100 U / ml penicillin-G potassium, and 100 μg / ml streptomycin sulfate was used as the growth medium. Phosphate buffered saline (PBS, pH 7.4) containing 0.25% trypsin and 0.02% EDTA was used to detach the adherent cells.

(2) Western blot
^{A549 cells were seeded in 2 × 10 5} cells on a 60 mm dish and cultured for 72 hours. Small molecule compounds of each concentration were added to the serum-removed medium, and the cells were further cultured for 24 hours. After extracting the protein from the cells, the protein was separated by SDS-PAGE using 10 or 12.5% acrylamide gel. After transcription to the PVDF membrane, the protein expression level was examined using antibodies against CLDN2 and β-actin.

(3) Real-time PCR
Total RNA was extracted from A549 cells using the TRI reagent reagent. Single-stranded cDNA was prepared by incubating with ReverTraAce® at 37 ° C. for 15 minutes. Using the prepared cDNA as a template, CLDN2 primers (sense: 5'-ATTGTGACAGCAGTTGGCTT-3'(SEQ ID NO: 1), antisense: 5'-CTATAGATGTCAC-ACTGGGTGATG-3' (SEQ ID NO: 2)), β-actin primers ( Real-time PCR reaction was performed using sense: 5'-CCTGAGGCACTCTTCCAGCCTT-3'(SEQ ID NO: 3) and antisense: 5'-TGCGGATGTCCACGTCACACTTC-3' (SEQ ID NO: 4). The reaction conditions were initial denaturation: 95 ° C. for 60 seconds, denaturation: 95 ° C. for 15 seconds, annealing / elongation: 60 ° C. for 60 seconds, 40 cycles.

(4) Fluorescent immunostaining
^{A549 cells were seeded in 5 × 10 4} cells / 2 mL each on a 35 mm dish containing a cover glass and cultured for 72 hours. 100 μM short-chain peptide and various inhibitors were added to the serum-removed medium, and the cells were further cultured for 24 hours. After fixation with cold methanol, the cell membrane was permeated with PBS containing 0.2% Triton X-100. After blocking with 4% Block Ace, CLDN2 and ZO-1 antibody were reacted overnight at 4 ° C. After incubating for 1 hour with PBS containing Alexa 488 mouse secondary antibody, Alexa 548 rabbit secondary antibody, and DAPI, they were fixed on glass slides. Cell localization of CLDN2 and ZO-1 was observed using an LSM700 confocal laser scanning microscope (Zeiss).

(5) Evaluation of transcriptional activity
A549 cells were seeded in 24-well plates in ^{groups of 5 × 10 and 4} hours later, transfected with the pGL4 vector and the pRL-TK vector into which the CLDN2 promoter was introduced. After 48 hours, compound 3 was treated for 6 hours and cells were harvested. Transcriptional activity was measured using the Dual-Glo Luciferase Assay Kit (Promega).

(6) Evaluation of intercellular molecular permeability
A549 cells were seeded in transwells at a rate of 4 × 10 ⁴ cells / 100 μL and cultured for 72 hours. Compound 3 at each concentration was added to the serum-removed medium, and the cells were further cultured for 24 hours. The electrical resistance between the top coats was measured using a Volt ohmmeter (Millipore). In addition, a buffer containing 10 μM doxorubicin was added to the upper layer of the transwell, and after 30 minutes, the buffer in the lower layer was collected, and the fluorescence intensity of doxorubicin was measured with Infinite F200 Pro (Tecan). A calibration curve was prepared and the concentration of doxorubicin was calculated.

(7) Three-dimensional culture and measurement of cell viability
A549 cells were seeded on a 96-well round bottom plate (Sumitomo Bakelite) at a rate of 1 × 10 ⁵ cells / 100 μL and cultured for 72 hours. After treating Compound 3 for 24 hours, spheroids were photographed under an inverted microscope. The sizes were compared based on the circumference of the spheroid. After treatment of the hypoxic fluorescent probe LOX-1 (MBL) in the presence and absence of Compound 3 for 24 hours, the degree of hypoxia in the spheroid was evaluated. The cell viability was calculated based on the ATP concentration using the CellTiter-Glo 3D Cell Viability Assay Kit (Promega).

(8) Toxicity evaluation of anticancer drugs in spheroids
A549 cells were seeded on a 96-well round bottom plate at a ^{rate of 1 × 10 5} cells / 100 μL and cultured for 72 hours. After treating Compound 3 for 24 hours, each concentration of anticancer agent was added. In the case of doxorubicin, a fluorescence image was taken under a fluorescence microscope 1 hour after the addition, and the accumulation rate of doxorubicin in the spheroid was calculated. In addition, spheroids were collected 24 hours later, and the cell viability was calculated based on the ATP concentration using the CellTiter-Glo 3D Cell Viability Assay kit.

2. result
CLDN2 has a 4-transmembrane structure with carboxy-terminus and amino-terminus present in the cytoplasm (Fig. 1). Of the two extracellular loops, it has been reported that the second loop on the carboxy side (amino acids 145 to 159) is required for binding between CLDNs. For the purpose of inhibiting the binding between CLDN2, we searched for compounds that are predicted to bind CLDN2 to the second extracellular loop by docking simulation. Approximately 1700 compounds registered in the US Food and Drug Administration (FDA) drug master file were screened and 11 compounds with low binding energies were selected. When the expression-lowering effect of CLDN2 protein was examined by Western blotting, the effect of Compound 3 (trade name: Harbony, generic name: Ledipasvir) was the strongest (Fig. 2). Compound 3 reduced the expression level of CLDN2 protein in a concentration-dependent and time-dependent manner (Fig. 3). On the other hand, the expression level of CLDN1 did not change with the treatment of compound 3, suggesting that it acts selectively on CLDN2.

CLDN2 mRNA levels and transcriptional activity were measured to rule out the possibility of compound 3 affecting gene expression. As a result, the amount of CLDN2 mRNA and transcriptional activity did not decrease significantly (Fig. 4). The expression of CLDN2 was regulated by the MEK / ERK pathway and the PI3K / Akt pathway, but the phosphorylation amount of ERK and Akt was not changed by compound 3. These results suggest that inhibition at the transcriptional stage is not involved in the reduction of CLDN2 protein expression by Compound 3.

Previously, our research group reported that endocytosed CLDN2 was degraded by lysosomes by short-chain peptide treatment (Non-Patent Document 2 above). In order to elucidate whether compound 3 also induces a similar degradation mechanism, the effects of endocytosis inhibitors and lysosomal inhibitors were investigated. The reduction in CLDN2 protein expression by Compound 3 was inhibited by co-treatment with the lysosomal inhibitor chloroquine (CQ) or the clathrin-dependent endocytosis inhibitor monodansyl cadaverine (MDC) (Fig. 5). In addition, in the fluorescent immunostaining method, the red fluorescence of CLDN2 was confirmed at the adjacent site of the cell together with the green fluorescence of ZO-1, which is an adapter protein of tight junction (Fig. 6). Compound 3 reduced the red fluorescence of CLDN2 distributed at tight junctions. By co-treatment with CQ, CLDN2 was mainly accumulated intracellularly. In addition, CLDN2 was distributed at tight junctions even in the presence of compound 3 by co-treatment with MDC. These results suggest that compound 3 promotes endocytosis of CLDN2 via the clathrin-dependent pathway, and that CLDN2 taken up into cells is degraded by lysosomes.

CLDN2 forms a cation-permeable pore at tight junctions while forming a barrier to small molecule compounds. When the inter-coating electrical resistance value (TER), which is an index of ion permeability, was measured, TER was increased by compound 3 (Fig. 7). In addition, the amount of doxorubicin (anthracycline anticancer drug) transferred from the upper layer to the lower layer in the transwell was increased by Compound 3. These results suggest that Compound 3 enhances intercellular small molecule permeability through decreased CLDN2 expression.

In vivo, cancer cells move away from blood vessels to create a microenvironment, which reduces the concentration of anticancer drugs. Three-dimensional spheroid culture is widely used to perform tests that mimic the microenvironment. When the effect of Compound 3 on spheroid formation was examined, the size and cell viability of the spheroid did not change significantly, but the fluorescence intensity of the hypoxic fluorescent probe decreased (Fig. 8). Therefore, it was suggested that Compound 3 has an effect of reducing hypoxic stress inside the spheroid. Next, the cancer cells that formed spheroids were treated with doxorubicin, and the amount of doxorubicin accumulated in the spheroids was evaluated. Treatment with 0 to 20 μM doxorubicin increased the amount of doxorubicin accumulated in the spheroids in a concentration-dependent manner (Fig. 9). Furthermore, Compound 3 increased the amount of doxorubicin accumulated. In addition, the size of spheroids and cell viability decreased depending on the concentration of doxorubicin, and these effects were enhanced by Compound 3. Similarly, the reduction in cell viability due to cisplatin (a platinum-based anticancer drug) and SN-38 (an active metabolite of irinotecan, a topoisomerase inhibitor) was enhanced by Compound 3 (Fig. 10).

3. 3. Discussion 27 subtypes of CLDN have been reported in humans, and each subtype is tissue-specifically expressed (Cited Paper 1). CLDN2 is highly expressed as normal tissue in the proximal tubule, gallbladder, and small intestine of the kidney, and has been reported to be involved in the transport of sodium ions (cited papers 2 and 3). Although CLDN2 is not expressed in normal lung tissue, it is highly expressed in lung adenocarcinoma tissue (cited paper 4), so CLDN2 may be a new diagnostic marker for lung adenocarcinoma. In addition, since CLDN2 reduces the sensitivity of anticancer drugs due to the agglutination of cancer cells (corresponding to the microenvironment in the body), drugs that reduce the expression level of CLDN2 will become new cancer adjuvants. think. So far, no compound that directly binds to CLDN2 has been reported. It has been reported that CLDN has a 4-transmembrane structure, the first loop is required for ion selectivity (cited paper 5), and the second loop is required for binding between CLDNs (cited paper 6). .. In this study, we searched for a compound that binds to the second extracellular loop of CLDN2 and reduces the expression level of CLDN2 by docking simulation, and identified compound 3 (ledipasvir).

Treatment with compound 3 did not change the amount of CLDN2 mRNA and transcriptional activity, but decreased the amount of protein (Figs. 3 and 4). In addition, the decrease in CLDN2 protein level was inhibited by co-treatment with the clathrin-dependent endocytosis inhibitor monodansyl cadaberin (MDC) and the lysosomal inhibitor chloroquin (CQ) (Fig. 5). It was suggested that 3 promotes the degradation of CLDN2 distributed in tight junctions in endocytosis and lysosomes. We recently found that a short-chain peptide that binds to CLDN2 reduces its expression via endocytosis of CLDN2 (in press). In addition, it has been reported that short-chain peptides that are predicted to bind to CLDN3 and CLDN4, which are highly expressed in breast cancer cells, cause endocytosis of CLDN (Cited Paper 7). CLDN can be stably distributed by binding in a homo or hetero manner at tight junctions, but it is suggested that binding of another short-chain peptide or compound to the second loop makes it unstable and facilitates endocytosis.

In analysis with spheroids in three-dimensional culture, Compound 3 enhanced the reduction in cell viability due to doxorubicin, cisplatin, and SN-38 (active metabolite of irinotecan) (FIGS. 9 and 10). Therefore, Compound 3 is considered to be useful as an adjunct in the treatment of lung adenocarcinoma showing resistance to anticancer drugs. Although the specific mechanism for enhancing the anticancer effect is unknown, since compound 3 increased the transmembrane permeability of doxorubicin and the amount accumulated in the spheroid, the anticancer drug permeability into the spheroid was increased. It is suggested that the enhancement is involved. In addition, since Compound 3 reduced the hypoxic state inside the spheroid, it is suggested that the release from the stress state enhances the sensitivity of the anticancer drug to cancer cells. Since the regulatory mechanism of hypoxia inside spheroids is unknown, the mechanism of hypoxia induction by high expression of CLDN2 is for further study.

The following three points can be mentioned as the spillover effect of Compound 3 in the medical field.
(i) High expression of CLDN2 has been reported not only in lung adenocarcinoma (cited paper 4) but also in colorectal cancer, liver cancer, and esophageal cancer (cited papers 8 to 10). Therefore, the CLDN2-binding peptide developed in this study is considered to be effective not only for lung adenocarcinoma but also for cancer treatment of other organs.
(ii) It has been reported that in Crohn's disease, ulcerative colitis, celiac disease, and HIV infection, the expression level of CLDN2 in the intestinal tract increases, causing symptoms such as diarrhea (cited paper 11). Therefore, the CLDN2-binding peptide developed in this study is considered to be effective not only for cancer but also for the treatment of these diseases.
(iii) Since tight junctions serve as a barrier to drug absorption, CLDN2-binding peptides are considered to have the effect of improving the rate of drug absorption from the intestinal tract.

Since there are cancer tissues in which CLDN4, 6, 18 and the like are highly expressed, antibody drugs against these CLDNs are being developed. Antibody drugs have the advantages of high specificity, high in vivo stability, and high commonality in production and manufacturing methods, but have the following problems: that is, manufacturing takes time and cost, and membrane permeation. The low sex is mentioned. In particular, low membrane permeability is considered to be a major problem in cancer treatment. This is because cancer cells form a microenvironment (hypoxic, malnourished stress environment) that is not found in normal cells due to angiogenesis deficiency, and it is difficult to penetrate the antibody drug into the deep microenvironment. .. Furthermore, when epithelial cells form tight junctions, only molecules of about 500 Da or less can pass between cells, and antibodies with a molecular weight of about 150 kDa are difficult to pass. Compound 3 (ledipasvir) identified in this study reduces the barrier function of tight junctions by reducing the expression of CLDN2, so it can be expected to enhance the intercellular permeability of drugs including antibodies.

<Cited paper>
1. Turksen, K., and Troy, TC (2004) Barriers built on claudins. J. Cell Sci. 117, 2435-2447
2. Enck, AH, Berger, UV, and Yu, AS (2001) Claudin-2 is selectively expressed in proximal nephron in mouse kidney. Am. J. Physiol. Renal Physiol. 281, F966-974
3. Amasheh, S., Meiri, N., Gitter, AH, Schoneberg, T., Mankertz, J., Schulzke, JD, and Fromm, M. (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci. 115, 4969-4976
4. Ikari, A., Sato, T., Watanabe, R., Yamazaki, Y., and Sugatani, J. (2012) Increase in claudin-2 expression by an EGFR / MEK / ERK / c-Fos pathway in lung adenocarcinoma A549 cells. Biochim. Biophys. Acta 1823, 1110-1118
5. Colegio, OR, Van Itallie, CM, McCrea, HJ, Rahner, C., and Anderson, JM (2002) Claudins create charge-selective channels in the paracellular pathway between epithelial cells. American Journal of Physiology. Cell Physiology 283, C142-C147
6. Krause, G., Winkler, L., Mueller, SL, Haseloff, RF, Piontek, J., and Blasig, IE (2008) Structure and function of claudins. Biochim. Biophys. Acta 1778, 631-645
7. Baumgartner, HK, Beeman, N., Hodges, RS, and Neville, MC (2011) A D-peptide analog of the second extracellular loop of claudin-3 and -4 leads to mislocalized claudin and cellular apoptosis in mammary epithelial cells . Chem. Biol. Drug Des. 77, 124-136
8. Kinugasa, T., Huo, Q., Higashi, D., Shibaguchi, H., Kuroki, M., Tanaka, T., Futami, K., Yamashita, Y., Hachimine, K., Maekawa, S ., Nabeshima, K., and Iwasaki, H. (2007) Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer. Anticancer Res. 27, 3729-3734
9. Weber, CR, Nalle, SC, Tretiakova, M., Rubin, DT, and Turner, JR (2008) Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation. Lab. Invest. 88, 1110-1120
10. Halasz, J., Holczbauer, A., Paska, C., Kovacs, M., Benyo, G., Verebely, T., Schaff, Z., and Kiss, A. (2006) Claudin-1 and claudin -2 differentiated fetal and embryonal components in human hepatoblastoma. Hum. Pathol. 37, 555-561
11. Luettig, J., Rosenthal, R., Barmeyer, C., and Schulzke, JD (2015) Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation. Tissue barriers 3, e977176

The medicament of the present invention containing a low molecular weight compound exhibiting a characteristic physiological function of binding to CLDN2 and reducing its expression can improve the hypoxic state of cancer cells and make cancer cells susceptible to anticancer drugs. It is effective in promoting cancer and is useful as an adjunct therapeutic agent in cancer treatment. INDUSTRIAL APPLICABILITY According to the present invention, an effective treatment strategy can be provided particularly for a difficult-to-treat case showing resistance / resistance to an anticancer drug. The present invention can also contribute to reducing the amount of anticancer drug used and the type of anticancer drug (when two or more kinds of anticancer drugs are used in combination). On the other hand, CLDN2 is highly expressed in Crohn's disease, ulcerative colitis, celiac disease, HIV infection, etc., and the present invention is expected to be used for the treatment of these diseases.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

SEQ ID NO: 1, 3: Artificial sequence description: Sense primer SEQ ID NO: 2, 4: Artificial sequence description: Antisense primer SEQ ID NO: 6: Artificial sequence description: Peptide that mimics the second extracellular loop of Claudin-2

Claims (6)

A drug containing ledipasvir or a pharmaceutically acceptable salt thereof as an active ingredient and effective for improving the hypoxic state of cancer cells and / or enhancing the sensitivity of cancer cells to anticancer drugs. An adjuvant cancer therapeutic agent containing the agent according to claim 1 and used in combination with an anticancer agent. The cancer adjuvant therapeutic agent according to claim 2, which is used for the treatment of cancer having a high expression of claudin-2. The cancer adjuvant therapy according to claim 3, wherein the cancer is lung adenocarcinoma, colon cancer, liver cancer or esophageal cancer. A method for treating cancer, which comprises a step of administering a therapeutically effective amount of the cancer adjuvant therapeutic agent according to any one of claims 2 to 4 to a cancer patient who is treated with an anticancer agent. A therapeutic agent for a disease caused by or forming a pathological condition of claudin-2, which contains ledipasvir or a pharmaceutically acceptable salt thereof as an active ingredient.

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