JP7045047B2 - Antitumor effect enhancer for oncolytic virus - Google Patents

Description

The present invention relates to an oncolytic virus antitumor effect enhancer administered in combination with an oncolytic virus.

In recent years, in medical treatment, research on therapy using an oncolytic virus that infects tumor cancer cells and causes the death of the tumor cancer cells is progressing. For example, a method for treating cancer using an oncolytic virus, which encodes a bipartite molecule containing a single-chain variable fragment (scFv) specific for a cell surface molecule and scFv specific for a tumor antigen (see Patent Document 1). ) Is disclosed.

Research is also underway to enhance the therapeutic effect of oncolytic viruses. For example, (a) introducing an effective amount of an oncolytic virus capable of selectively killing solid tumor cells into or near the solid tumor, and (b) at or near the solid tumor site. Disclosed is a method of treating or ameliorating a solid tumor of interest (see Patent Document 2), which comprises a step of suppressing a local immune response in the subject. Furthermore, for multiple myeloma in humans, a method in which leovirus and a histone deacetylase (HDAC) inhibitor are used in combination (see Patent Document 3 and Non-Patent Document 1), and for human gastric cancer. A method of using leovirus in combination with an antibody drug, trastumab, which binds to HER2 protein (see Non-Patent Document 2) is disclosed.

The above-mentioned treatment method using an oncolytic virus shows a certain antitumor effect on some tumors in both medical and veterinary medicine, but it is not completely cured, and the search for a combination drug showing a synergistic effect is proceeding. Has been done.

Special Table 2016-512199 Japanese Unexamined Patent Publication No. 2009-525989 Japanese Unexamined Patent Publication No. 2017-515847

Stiff, A. et al. Histone Deacetylase Inhibitors Enhance theTherapeutic Potential of Reovirus in Multiple Myeloma. Mol. Cancer Ther. 15, 830-841 (2016). Hamano, S. et al. Oncolytic reovirus combined with trastuzumabenhances antitumor efficacy through TRAIL signaling in human HER2-positive gastric cancer cells. Cancer Lett. 356, 846-854 (2015).

An object of the present invention is to search for a substance that enhances the antitumor effect of an oncolytic virus in oncolytic virus therapy.

The present inventors screened drugs that enhance the antitumor effect of oncolytic leovirus on canine malignant melanoma cell lines using a standard inhibitor kit transferred from the chemotherapy infrastructure support activity. As a result, it was found that the combined use of an ATM (Ataxia telangiectasia mutated) kinase inhibitor and an oncolytic leovirus enhances the antitumor effect on the oncolytic leovirus, and the present invention has been developed. completed.

That is, the present invention is as shown in the following (1) to (6).
(1) An agent for enhancing the antitumor effect of an oncolytic virus, which comprises an ATM kinase inhibitor as an active ingredient and is administered in combination with an oncolytic virus.
(2) The ATM kinase inhibitor is (2R, 6S-rel) -2,6-dimethyl-N- [5- [6- (4-morpholinyl) -4-oxo-4H-pyran-2-yl]- 9H-thioxanthene-2-yl] -4-morpholinacetamide or 2-morpholin-4-yl-6-thianthrene-1-yl-pyran-4-one, or pharmaceutically acceptable salts thereof. The agent for enhancing the antitumor effect of the oncolytic virus according to the above (1), which is characterized by the above.
(3) The agent for enhancing the antitumor effect of the oncolytic virus according to (1) or (2) above, wherein the oncolytic virus is an oncolytic leovirus.
(4) The agent for enhancing the antitumor effect of the oncolytic virus according to any one of (1) to (3) above, wherein the tumor is a canine tumor.
(5) The agent for enhancing the antitumor effect of the oncolytic virus according to any one of (1) to (4) above, wherein the tumor is malignant melanoma.
(6) A kit for preventing or treating a malignant tumor (cancer) containing the oncolytic virus antitumor effect enhancer according to any one of (1) to (5) above and the oncolytic virus.

By using the antitumor effect enhancer of the oncolytic virus of the present invention, it is possible to enhance the antitumor effect by using it in combination with the oncolytic virus.

We investigated the enhancement of cytotoxic effects by culturing four canine malignant melanoma cell lines (CMeC1, KMeC, LMeC, CMGD2) by co-administering the oncolytic leovirus with the ATM kinase inhibitor KU60019. The result. This is the result of investigating the enhancement of the cytotoxic effect by culturing a human tumor cell line (A549) in combination with an oncolytic leovirus and an ATM kinase inhibitor. It is a figure which shows the result of having measured the cell number at each stage of the cell cycle when the malignant melanoma cell line CMeC1 of a dog was cultured by co-administering a leovirus and an ATM kinase inhibitor. It is a figure which shows the result of having investigated the involvement of caspase in a cytotoxicity by culturing a canine malignant melanoma cell line CMeC1 or KMeC by administering a leovirus, an ATM kinase inhibitor and / or a Pan-caspase inhibitor. It is a figure which shows the detection result of the virus structure protein by the Western Blotting method in the case of culturing the malignant melanoma cell line CMeC1 of canine by co-administering leovirus and ATM kinase inhibitor. It is a figure which shows the result of having investigated the amount of virus production in the culture supernatant when the canine malignant melanoma cell line CMeC1 was cultured by co-administering a leovirus and an ATM kinase inhibitor.

The antitumor effect enhancer of the present invention is not particularly limited as long as it contains an ATM kinase inhibitor as an active ingredient and is administered in combination with an oncolytic virus. Here, ATM is chromosome 11q22 in the case of humans. In .3, it is located at the 5th chromosome in the case of dogs and is the responsible gene for Ataxia telangiectasia.

The ATM kinase inhibitor may be any substance that inhibits the activation of ATM, for example, serine at positions 367, 1893, and 1981 in the amino acid sequence of human ATM shown in SEQ ID NO: 1 or the dog shown in SEQ ID NO: 2. Examples thereof include substances that inhibit the phosphorylation of serine at positions 367, 1894, and 1985 in the amino acid sequence of ATM, and substances that inhibit kinase activity by competing with ATP specifically for ATM. The substance may be a low molecular weight compound, an antibody, an antibody fragment, a nucleic acid, or an aptamer, but is preferably a low molecular weight compound, and is, for example, (2R, 6S-rel) -2, represented by the following formula (I). 6-Dimethyl-N- [5- [6- (4-morpholinyl) -4-oxo-4H-pyran-2-yl] -9H-thioxanthene-2-yl] -4-morpholinacetmid ((2R) , 6S-rel) -2,6-Dimethyl-N- [5- [6- (4-morpholinyl) -4-oxo-4H-pyran-2-yl] -9H-thioxanthen-2-yl] -4- morpholineacetamide: CAS No: 925701-46-8, 2-Morpholine-4-yl-6-thianthrene-1-yl-pyran-4-one (2-Morpholin-4-yl-6-) shown in formula (II) thianthren-1-yl-pyran-4-one: CAS No: 587871-26-9), or pharmaceutically acceptable salts thereof can be preferably mentioned. The low molecular weight compounds are commercially available. The formula (I) is sold under the trade name of KU60019, and the formula (II) is sold under the trade name of KU55933.

The above (2R, 6S-rel) -2,6-dimethyl-N- [5- [6- (4-morpholinyl) -4-oxo-4H-pyran-2-yl] -9H-thioxanthene-2-yl] ] As pharmacologically acceptable salts of -4-morpholinacetamide and 2-morpholin-4-yl-6-thiantolen-1-yl-pyran-4-one, for example, sodium salt, potassium salt and the like. Alkaline metal salts, alkaline earth metal salts such as calcium salt and magnesium salt, transition metal salts such as zinc salt and the like can be mentioned.

Inhibition of kinase activity in human and canine ATMs by ATM kinase inhibitors can be measured by known methods, such as the method of Kim et al. (Kim, S., Lim, D., Christine, E., Kastan, MB). & Canman, CE Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members. J. Biol. Chem. 274, 37538-37543 (1999)) ..

The antitumor effect in the present invention means an action of killing malignant tumor cells.

The oncolytic virus in the present invention hardly proliferates even when infected with normal cells, but proliferates when infected with malignant tumor cells and has an ability to kill malignant tumor cells (malignant tumor cell damage). It is well known in the art as meaning a virus and is reviewed, for example, in Molecular Therapy, Vol. 18, No. 2, February 2010, pp. 233-234. The oncolytic virus is not particularly limited as long as it has the ability to infect malignant tumor cells and kill malignant tumor cells, but it is not particularly limited, but oncolytic leovirus, oncolytic adenovirus, oncolytic measles virus, and oncolytic virus. Simple herpes virus, oncolytic Newcastle disease virus, oncolytic bovine hemorrhoid virus, oncolytic mumps virus, oncolytic coxsackie virus, oncolytic vaccinia virus and the like can be mentioned, and oncolytic leovirus is preferable. , It is more preferably an oncolytic type 3 leovirus.

Examples of the combined use include a method of administering at the same time in time and a method of administering at almost the same time. The above-mentioned "almost simultaneous administration" can be mentioned, for example, administration within 24 hours, more preferably within 8 hours, and even more preferably within 1 to 4 hours with each other.

The antitumor effect enhancer of the present invention can be provided as a single preparation together with an attached document or the like to enhance the antitumor effect of the oncolytic virus when administered in combination with the oncolytic virus. It is also possible to prepare a mixed preparation of the antitumor effect enhancer of the present invention and an oncolytic virus.

The kit for treating or preventing a malignant tumor of the present invention is particularly limited as long as it is a kit containing as a combination (combination preparation) of individual preparations containing an ATM kinase inhibitor and an oncolytic virus as active ingredients. However, an attached document or the like describing a method for treating or preventing a malignant tumor by the above kit may be included, and when the kit for treating or preventing such a malignant tumor is used, an oncolytic virus antitumor is used. It is possible to enhance the effect.

Mammals or mammalian cells can be preferably mentioned as the administration target of the antitumor effect enhancer of the present invention and the kit for treating or preventing the malignant tumor of the present invention, and among such mammals, dogs. , Humans, mice, rats, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, monkeys, chimpanzees, and dogs, or humans can be particularly preferred.

Examples of the dosage form of each combination preparation of the antitumor effect enhancer of the present invention and the kit for treating or preventing the malignant tumor of the present invention include powders, granules, tablets, solutions, emulsions and suspensions. , Syrups, capsules, suppositories, injections and the like. These formulations may be an appropriate pharmaceutically acceptable carrier, such as a solvent, an excipient, a binder, a solubilizing agent, a suspending agent, an emulsifier, an isotonic agent, as required by the formulation. Manufactured by blending optional components such as buffers, stabilizers, soothing agents, preservatives, antioxidants, colorants, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, and diluents. be able to.

The method for administering the antitumor effect enhancer of the present invention and each component of the kit for treating or preventing the malignant tumor of the present invention is the method for enhancing the antitumor effect of the present invention or treating or preventing the malignant tumor of the present invention. It is not particularly limited as long as it has enhancement, but oral administration or parenteral administration such as rectal administration, oral administration, subcutaneous administration, intramuscular administration, intravenous administration, etc. can be mentioned, and continuous infusion therapy with a heavy burden is performed. Oral administration is preferred because it is not necessary.

The daily dose of the antitumor effect enhancer of the present invention and the antitumor effect enhancer in the kit for treating or preventing a malignant tumor of the present invention includes the antitumor effect enhancing effect, prevention of malignant tumor, or It is not particularly limited as long as it has a therapeutic effect, but when converted into an ATM kinase inhibitor which is an active ingredient, for example, in the case of a human adult, the body weight is 0.01 to 50 mg / kg, preferably 0.1 to 30 mg / kg. The body weight, more preferably 1 to 20 mg / kg body weight, can be mentioned, and in the case of an adult dog, 0.01 to 50 mg / kg body weight, preferably 0.1 to 20 mg / kg body weight, more preferably 1 to 1 to 1 to 20 mg / kg body weight. The body weight of 10 mg / kg can be raised. Further, it may be administered once a day, or may be administered in a plurality of times such as twice, three times, five times and the like. Further, the antitumor effect enhancer of the present invention to be administered may be used alone or in combination of two or more.

On the other hand, the daily dose of the oncolytic virus is not particularly limited, but in the case of a human adult, for example, 1 × 10 ² to 1 × 10 ¹⁵ pfu / kg body weight, preferably 1 × 10 ³ to 1. It is × 10 ¹³ pfu / kg body weight, and in the case of dogs, it is preferably 1 × 10 ² to 1 × 10 ¹⁵ pfu / kg body weight, preferably 1 × 10 ² to 1 × 10 ¹⁵ pfu / kg body weight. .. Further, it may be administered once a day, or may be administered in a plurality of times such as twice, three times, five times and the like. Further, the oncolytic virus to be administered may be one kind or a combination of two or more kinds.

The tumors in the antitumor effect enhancer of the present invention and the kit for treating or preventing malignant tumors of the present invention include malignant melanoma, lymphoma, obesity cell tumor, breast tumor, osteosarcoma, lung cancer, bladder cancer, head and neck. Partial cancer and the like can be mentioned, and malignant melanoma can be preferably mentioned.

As another aspect of the antitumor effect enhancer of the present invention, 1) a method for enhancing the antitumor effect of an oncolytic virus, which comprises administering an ATM kinase inhibitor to a subject in combination with an oncolytic virus. And 2) an ATM kinase inhibitor to be administered in combination with an oncolytic virus and used as an oncolytic effect enhancer of the oncolytic virus, and 3) an oncolytic virus of an ATM kinase inhibitor. In combination, it can be used in the preparation of an oncolytic virus antitumor effect enhancer to be administered to an ATM kinase inhibitor.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

<Screening>
The present inventors screened a drug that enhances oncolytic leovirus cytotoxicity against canine malignant melanoma cell lines using a standard inhibitor kit transferred from the Chemotherapy Infrastructure Support Activity (Ministry of Education, Culture, Sports, Science and Technology). It was carried out by the following method.

The canine malignant melanoma cell line CMeC1 was inoculated in RPMI-1640 growth medium in 5 × 10 ³ cells and cultured at 37 ° C. for 2 days. Next, the oncolytic leovirus type 3 Dearing strain GMP grade Reolysin (registered trademark) (obtained from ONCOLYTICS BIOTECH) and the 285 inhibitors included in the standard inhibitor kit were each concentrated in MOI (multiplicity of infection). : Number of viruses per cell) 100 and 10 μM were added and cultured at 37 ° C. for 2 days. The number of cells after culturing was measured using a Cell counting kit-8 (manufactured by Dojin Chemical Research Institute). Of the 285 inhibitors, those with low cytotoxic activity with the inhibitor alone and enhanced cytotoxic activity compared with oncolytic leovirus alone when used in combination with oncolytic leovirus. As a criterion, ATM (Ataxia telangiectasia mutated) kinase inhibitor KU55933 was selected. The ATM kinase inhibitor included in the standard inhibitor kit was KU55933, but since KU60019, which has higher specificity, was sold as a generic drug, KU60019 was used in the experiments after screening.

<Cytotoxicity enhancing effect>
We investigated the enhancement of the cytotoxic effect of the ATM kinase inhibitor on the four canine malignant melanoma cell lines (CMeC1, KMeC, LMeC, CMGD2) and human tumor cell line (A549). Canine malignant melanoma cell lines were seeded in RPMI-1640 growth medium in 5 × 10 ³ cells and cultured at 37 ° C. for 2 days. Next, the above oncolytic leovirus (obtained from ONCOLYTICS BIOTECH) was added with MOI100 (MOI10 for A549 and CMGD2) and the ATM kinase inhibitor KU60019 at concentrations of 0, 1.25, and 2.5 μM 2 The cells were cultured at 37 ° C. for one day. The number of cells after culturing was measured using a Cell counting kit-8 (manufactured by Dojin Chemical Research Institute). The results are shown in FIGS. 1 and 2. In FIGS. 1 and 2, the horizontal axis is the concentration of the ATM kinase inhibitor KU60019, the vertical axis is the cell survival rate (ratio when 100% is the case without the inhibitor in Mock), and Mock is the oncolytic leovirus. The case without addition is shown.

As shown in FIGS. 1 and 2, the ATM kinase inhibitor KU60019 alone has no cytotoxic effect in any of the cell lines, and the combination of the ATM kinase inhibitor KU60019 and the oncolytic leovirus causes tumor lysis. It was revealed that the cytotoxic effect of sex leovirus was enhanced.

<Analysis of cytotoxic enhancement mechanism-cell cycle>
In order to elucidate the mechanism of enhancement of cell damage by the combined use of ATM kinase inhibitor and oncolytic leovirus, the relationship with the cell cycle was investigated. The canine malignant melanoma cell line CMeC1 was cultured on MOI100 of the above oncolytic leovirus (obtained from ONCOLYTICS BIOTECH) and 2.5 μM ATM kinase inhibitor KU60019 for 36 hours or 48 hours at 37 ° C. Trypsin EDTA was added to the plate to which the CMeC1 strain was adhered, and the cells were cultured at 37 ° C. for 5 minutes, collected in a tube, washed with PBS, stained with PI, and the number of cells at each stage of the cell cycle was measured by flow cytometry. .. The results are shown in FIG.

As shown in FIG. 3, the number of cells in the G0 phase and the G2 / M phase was increased by adding ATM kinase inhibition. From this point, it can be seen that the enhancement of cell damage observed by the combined use of an ATM kinase inhibitor with oncolytic leovirus is due to cell death and cell cycle arrest (increase in the number of cells in the G2 / M phase). It became clear.

<Analysis of cytotoxic enhancement mechanism-depending on caspase>
In order to elucidate the mechanism of enhancement of cell damage by the combined use of ATM kinase inhibitor and oncolytic leovirus, the relationship with caspase involved in apoptosis was investigated. The canine malignant melanoma cell line CMeC1 or KMeC was pretreated with DMSO (vehicle) or Z-VAD-FMK (Calbiochem), a 50 μM Pan-caspase inhibitor, for 1 hour at 37 ° C. Next, (1) DMSO, (2) 2.5 μM ATM kinase inhibitor KU60019, (3) MOI100 oncolytic leovirus (obtained from ONCOLYTICS BIOTECH), (4) 2.5 μM ATM kinase inhibitor. The oncolytic leovirus of the agent KU60019 + MOI100 was added and cultured at 37 ° C. for 2 days. The number of cells after culturing was measured using trypan blue staining (manufactured by Nacalai Tesque), and a relative value was calculated when 100 was used for Vehicle + DMSO. The results are shown in FIG.

As shown in FIG. 4, the combined use of the Pan-caspase inhibitor Z-VAD-FMK with an ATM kinase inhibitor and an oncolytic leovirus completely suppressed cell death. Therefore, it was revealed that the cell death caused by the combined use of the ATM kinase inhibitor and the oncolytic leovirus is a caspase-dependent cell death.

<Analysis of cytotoxic enhancement mechanism-virus proliferation>
In order to elucidate the mechanism of enhancement of cell damage by the combined use of ATM kinase inhibitor and oncolytic leovirus, the relationship with viral proliferation was investigated. The canine malignant melanoma cell line CMeC1 was cultured on RPMI-1640 growth medium for 2 days at 37 ° C. Next, the oncolytic leovirus was added to MOI100 and the ATM kinase inhibitor KU60019 to 2.5 sheets μM, and the cells were cultured at 37 ° C. for 24 hours or 48 hours. After that, protein extraction treatment was performed to detect viral structural proteins by the Western Blotting method, and the amount of virus produced in the culture supernatant was determined by the TCID50 (Tissue Culture Infectious Dose) method using L929 cells for the culture supernatant after 48 hours. Examined. FIG. 5 shows the results of detection of viral structural proteins by the Western Blotting method, and FIG. 6 shows the results of examining the amount of virus produced in the culture supernatant. In FIG. 6, the vertical axis shows the amount of virus increase when the amount of virus before addition is 1. As shown in FIGS. 5 and 6, it was clarified that the combined use of the ATM kinase inhibitor KU60019 and the oncolytic leovirus increased the proliferation and production of the oncolytic leovirus.

The present invention can be used in the treatment of tumors as a combination therapy method for enhancing the antitumor effect of oncolytic leovirus therapy, and should be used as standard when performing oncolytic leovirus therapy. Can be done.

Claims (4)

An oncolytic leovirus antitumor effect enhancer characterized by having an ATM kinase inhibitor as an active ingredient and administered in combination with an oncolytic leovirus , the ATM kinase inhibitor is (2R). , 6S-rel) -2,6-dimethyl-N- [5- [6- (4-morpholinyl) -4-oxo-4H-pyran-2-yl] -9H-thioxanthene-2-yl] -4 -Morphorin acetomid or 2-morpholin-4-yl-6-thiantolen-1-yl-pyran-4-one, or pharmaceutically acceptable salts thereof, or ATM kinase activity bound to ATM An oncolytic leovirus antitumor effect enhancer, which is an aptamer that inhibits . The agent for enhancing the antitumor effect of an oncolytic leovirus according to claim 1 , wherein the tumor is a canine tumor. The agent for enhancing the antitumor effect of an oncolytic leovirus according to claim 1 or 2 , wherein the tumor is malignant melanoma. A kit for preventing or treating a malignant tumor containing the oncolytic effect enhancer of the oncolytic leovirus according to any one of claims 1 to 3 and the oncolytic leovirus.

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