JP2022071454A - Pharmaceutical composition and kit for treating rb1 positive cancer - Google Patents

Abstract

To provide a technique for effectively treating RB Transcriptional Corepressor 1 (RB1) positive cancer.SOLUTION: A pharmaceutical composition for treating RB1 positive cancer contains a combination of a cyclin-dependent kinase (CDK) 4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor as active ingredients. A method and a kit for testing the effectiveness of administering the pharmaceutical composition to a cancer patient include: a process of inspecting whether or not a cancer cell derived from a cancer patient is RB1-positive, in which the cancer cell indicating RB1-positive shows that administration of the pharmaceutical composition is effective for treating the cancer patient.SELECTED DRAWING: None

Description

The present invention relates to pharmaceutical compositions and kits for the treatment of RB1-positive cancers. More specifically, the present invention relates to a pharmaceutical composition for treating RB1-positive cancer, a kit for treating RB1-positive cancer, a method for testing the efficacy of administration of a drug to a cancer patient, and an administration of a drug to a cancer patient. Regarding kits for testing efficacy.

Currently, the efficacy of single-agent administration of cyclin-dependent kinase (CDK) 4 and 6 inhibitors for various types of cancer including hepatocellular carcinoma is being investigated in Phase II clinical trials (Non-Patent Document 1). ..

Schettini F., et al., CDK 4/6 Inhibitors as Single Agent in Advanced Solid Tumors, Front Oncol., 8, 608, 2018.

However, in vitro and in vivo studies, the inventors have shown that single agent administration of CDK4 and 6 inhibitors to cancer cell lines is effective but not optimal. An object of the present invention is to provide a technique for effectively treating RB1-positive cancer.

The present invention includes the following aspects.
[1] A combination of a cyclin-dependent kinase (CDK) 4 and 6 inhibitor and an NF-κB signal transduction pathway inhibitor or an AKT inhibitor as an active ingredient in RB Transcrictional Corepressor 1 (RB1) -positive cancer. Therapeutic pharmaceutical composition.
[2] The CDK4 and 6 inhibitors are palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1111441-98-3), abemaciclib (CAS number: 1231929-97-7), trilaciclib (CAS). The pharmaceutical composition according to [1], which is number: 1374743-00-6) or relocyclib (CAS number: 1628256-23-4).
[3] A combination of a CDK4 and 6 inhibitor and an NF-κB signal transduction pathway inhibitor is contained as an active ingredient, and the NF-κB signal transduction pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor. , [1] or [2].
[4] The NF-κB signaling pathway inhibitor is an IKKβ inhibitor, and the IKKβ inhibitor is Bay11-7082 (CAS number: 1954-67-7), IMD-0354 (CAS number: 978-62-). 1), IMD-1041 (CAS number: 1073666-73-5), IMD-2560, TPCA1 (CAS number: 507475-17-4), BOT-64 (CAS number: 113760-29-5), BMS345541 (CAS) Number: 445430-58-0), SC-514 (CAS number: 354812-17-2), IKK-16 (CAS number: 1186195-62-9), Elchiprotafib (CAS number: 251303-04-5) ) And Bay65-1942 (HCl salt) (CAS No .: 600734-06-3), the pharmaceutical composition according to [3].
[5] The NF-κB signaling pathway inhibitor is an NF-κB inhibitor, and the NF-κB inhibitor is QNZ (CAS number: 545380-34-5), JSH-23 (CAS number: 749886-). 87-1), GYY4137 (CAS number: 106740-09-4), p-XSC (CAS number: 85539-83-9), CV3988 (CAS number: 85703-73-7), Prostaglandin E2 (CAS number) : 363-24-6), LY294002 (CAS number: 154447-36-6), Wortmannin (CAS number: 1954-26-7), Mesalamine (CAS number: 89-57-6), Loriplum (CAS number: 85416) -75-7), gallic acid (CAS number: 149-91-7), anacardic acid (CAS number: 16611-84-0), MG-115 (CAS number: 133407-86-0), MG-132 ( CAS Number: 133407-82-6), Lactacistin (CAS Number: 133343-34-7), Epoxomecin (CAS Number: 134381-21-8), Parthenolide (CAS Number: 20554-84-1), Calfilzomib (CAS Number: 20554-84-1) Number: 868540-17-4), Voltezomib (CAS number: 179324-69-7), MLN-4924 (CAS number: 905579-51-3), Delanzomib (CAS number: 847499-27-8), Ixazomib (CAS number: CAS) The pharmaceutical composition according to [3], which is selected from the group consisting of numbers: 123908-20-3), marizomib (CAS number: 437742-34-2) and oprosomib (CAS number: 935888-69-0).
[6] A combination of CDK4 and 6 inhibitors and an AKT inhibitor is contained as an active ingredient, and the AKT inhibitors are Akt Inhibitor X (CAS number: 925681-41-0) and Ipatasertib (CAS number: 1001264-). 89-6), Capiva Celtibu (CAS number: 1143532-39-1), Miraceltibu (CAS number: 1313881-70-7), Afresertib (CAS number: 1047644-62-1), Ipatasertib (CAS number: 1047634-65-) 0), the pharmaceutical composition according to [1] or [2], which is selected from the group consisting of tricilibine phosphate (CAS number: 61966-08-3) and MK2206 (CAS number: 1032350-13-2).
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the RB1-positive cancer is hepatocellular carcinoma, breast cancer, lung cancer or colon cancer.
[8] A kit for treating RB1-positive cancer, which comprises cyclin-dependent kinase (CDK) 4 and 6 inhibitors, and an NF-κB signaling pathway inhibitor or AKT inhibitor.
[9] A step of examining whether or not a cancer cell derived from a cancer patient is RB1 positive is included, and the fact that the cancer cell is RB1 positive is used for treating the cancer patient with CDK4 and 6 inhibitors, and , A method for testing the effectiveness of administration of the combination to a cancer patient, showing that administration of the combination of NF-κB signaling pathway inhibitor or AKT inhibitor is effective.
[10] A primer set for amplifying genomic DNA of RB1 gene, a probe for genomic DNA of RB1 gene, a primer set for cDNA amplification of RB1 gene, a probe for mRNA of RB1 gene, or a specific binding substance for RB1 protein. Cancer cells derived from cancer patients are used for testing whether or not they are RB1 positive, and the fact that the cancer cells are RB1 positive can be used to treat the cancer patients with CDK4 and 6 inhibitors, and NF-. A kit for testing the effectiveness of administration of the combination to the cancer patient, which shows that administration of the combination of a κB signaling pathway inhibitor or an AKT inhibitor is effective.

According to the present invention, it is possible to provide a technique for effectively treating RB1-positive cancer.

It is a schematic diagram explaining the action of CDK4 and 6 inhibitors on RB1. (A) is a schematic diagram illustrating a procedure for producing a QKO mouse. (B) is a representative photograph of a tumor formed in the liver of a QKO mouse. (A) is a graph showing the result of flow cytometry in Experimental Example 2. (B) is a graph created based on the result of (a). It is a graph which shows the result of the screening in Experimental Example 3. 6 is a graph showing the survival rate of cells in the study using Bay11-7082 in Experimental Example 4. It is a graph created based on the result of flow cytometry in the study using Bay11-7082 in Experimental Example 4. It is a graph created based on the result of flow cytometry in the study using IMD-0354 in Experimental Example 4. It is a graph prepared based on the result of the flow cytometry in the study using shRNA for IKKβ in Experimental Example 4. It is a graph prepared based on the result of the flow cytometry in the examination using the primary hepatocellular carcinoma cell (QKO PHCC) in Experimental Example 4. It is a graph prepared based on the result of the flow cytometry in the study using Huh7 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using Li-7 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using H1975 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using HCC1143 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using T47D cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using HCT-116 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the study using DLD-1 cells in Experimental Example 5. It is a graph prepared based on the result of the flow cytometry in the examination which used the ribociclib and Bay11-7082 of Experimental Example 5 together. It is a graph prepared based on the result of the flow cytometry in the study which used abemaciclib and Bay11-7082 of Experimental Example 5 in combination. 6 is a graph created based on the result of flow cytometry in Experimental Example 6. It is a graph created based on the result of flow cytometry in the study using HepG2 cells in Experimental Example 7. It is a graph prepared based on the result of the flow cytometry in the study using SW480 cells in Experimental Example 7. It is a graph prepared based on the result of the flow cytometry in the study using HCT-116 cells in Experimental Example 7. It is a graph prepared based on the result of the flow cytometry in the study using DLD-1 cells in Experimental Example 7. It is a graph created based on the result of flow cytometry in the study using A549 cells in Experimental Example 7. 6 is a graph created based on the result of flow cytometry in Experimental Example 8. It is a graph which shows the time-dependent change of the tumor volume in the examination by the cancer-bearing mouse model transplanted with HepG2 cells of Experimental Example 9. It is a graph which shows the time-dependent change of the tumor volume in the examination by the cancer-bearing mouse model transplanted with Li-7 cells of Experimental Example 9. It is a graph which shows the time-dependent change of the tumor volume in the examination by the cancer-bearing mouse model transplanted with the QKO PHCC cell which expresses a mouse Rb1 in a tetracycline-inducible manner of Experimental Example 9.

[Notation of gene name and protein name]
As used herein, human genes and proteins are represented by uppercase alphabets. In the mouse gene, the first letter is represented by an uppercase alphabet and the subsequent letters are represented by a lowercase alphabet. In addition, mouse protein shall be represented by an uppercase alphabet. However, in some cases, human genes and mouse genes may be represented without strict distinction. In addition, human protein and mouse protein may be represented without strict distinction.

[Pharmaceutical composition or kit]
In one embodiment, the present invention provides a pharmaceutical composition for treating RB1-positive cancer, which comprises a combination of a CDK4 and 6 inhibitor and an NF-κB signal transduction pathway inhibitor or an AKT inhibitor as an active ingredient. do.

The pharmaceutical composition may be a pharmaceutical composition in which a CDK4 and 6 inhibitor and an NF-κB signal transduction pathway inhibitor are mixed. Alternatively, the pharmaceutical composition may be a pharmaceutical composition in which a CDK4 and 6 inhibitor and an AKT inhibitor are mixed.

Further, in one embodiment, the CDK4 and 6 inhibitors and the NF-κB signal transduction pathway inhibitor or AKT inhibitor are not mixed and are contained in separate containers to form a therapeutic kit. You may be doing it. For therapeutic kits, these drugs are not mixed, but are administered in combination.

The therapeutic kit may include a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor. Alternatively, the therapeutic kit may include a combination of CDK4 and 6 inhibitors and an AKT inhibitor.

As described below in the Examples, we used CDK4 and 6 inhibitors in combination with NF-κB signaling pathway inhibitors or AKT inhibitors to generate RB1-positive cancer cells in vitro and in vivo. It was revealed that it can be effectively killed.

FIG. 1 is a schematic diagram illustrating the action of CDK4 and 6 inhibitors on RB1. RB1 is dephosphorylated immediately after mitosis. Activated CDK4 and 6 monophosphorylate RB1. When RB1 is monophosphorylated, the CDK2 complex achieves complete phosphorylation of RB1. Inhibition of CDK4 and 6 activity inhibits monophosphorylation of RB1. As a result, RB1 will be maintained for a long period of time in a non-phosphorylated state. As a result, not only cell cycle arrest but also aging, apoptosis, enhancement of immunogenicity and the like are induced.

The RB1-positive cancer means a cancer in which the cancer cells constituting the tumor express RB1. The RB1 expressed by the RB1-positive cancer may have a mutation, but it is preferable that the function of the wild-type RB1 described above is maintained, and the wild-type RB1 is preferable. Specific RB1-positive cancers include hepatocellular carcinoma, breast cancer, lung cancer, colon cancer and the like.

The accession number of the cDNA of human RB1 is NM_000321.3. The accession number of the human RB1 protein is NP_000312.2. The accession number of the cDNA of mouse Rb1 is NM_009029.2. The accession number of the mouse RB1 protein is NP_033055.2.

In the pharmaceutical composition of the present embodiment, as the CDK4 and 6 inhibitor, a drug that inhibits the activity of the complex consisting of CDK4 and 6 and cyclin D can be used. The CDK4 and 6 inhibitors may be small molecule compounds or may be nucleic acids that are inhibitory to CDK4, CDK6 or cyclin D. Examples of the inhibitory nucleic acid include shRNA, siRNA and the like.

More specific CDK4 and 6 inhibitors include palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1121441-98-3), abemaciclib (CAS number: 1231929-97-7), trilaciclib (CAS number: 1231929-97-7). CAS number: 1374734-00-6), relocyclib (CAS number: 1628256-23-4) and the like can be mentioned. One of these may be used alone, or two or more thereof may be mixed and used.

Further, as the NF-κB signal transduction pathway inhibitor, a drug that blocks the NF-κB signal transduction pathway can be used.

The NF-κB signaling pathway is the phosphorylation and ubiquitination of IκB of NF-κB in which IκB kinase (IKK) consisting of IKKα, IKKβ and IKKγ (NEMO) binds to and inactivates IκB. It is a pathway that leads to degradation and, as a result, NF-κB translocates from the cytoplasm to the nucleus and activates gene transcription.

The NF-κB signaling pathway inhibitor may be, for example, an IKKβ inhibitor or an NF-κB inhibitor. One of these may be used alone, or two or more thereof may be mixed and used.

The NF-κB signaling pathway inhibitor may be a small molecule compound or an inhibitory nucleic acid against IKKα, IKKβ, IKKγ (NEMO), IκB, NF-κB and the like. Examples of the inhibitory nucleic acid include shRNA, siRNA and the like.

Specific IKKβ inhibitors include Bay11-7082 (CAS number: 1954-67-7), IMD-0354 (CAS number: 978-62-1), and IMD-1041 (CAS number: 1073666-73-5). , IMD-2560, TPCA1 (CAS number: 507475-17-4), BOT-64 (CAS number: 113760-29-5), BMS345541 (CAS number: 445430-58-0), SC-514 (CAS number:: 354812-17-2), IKK-16 (CAS number: 1186195-62-9), Elchiprotafib (CAS number: 251303-04-5), Bay65-1942 (HCl salt) (CAS number: 600734-06). -3) and the like.

Specific examples of the NF-κB inhibitor include QNZ (CAS number: 545380-34-5), JSH-23 (CAS number: 794886-87-1), and GYY4137 (CAS number: 106740-09-4). , P-XSC (CAS number: 85539-83-9), CV3988 (CAS number: 85703-73-7), Prostaglandin E2 (CAS number: 363-24-6), LY294002 (CAS number: 154447-36) -6), Wortmannin (CAS number: 1954-26-7), mesalamine (CAS number: 89-57-6), loliplum (CAS number: 85416-75-7), gallic acid (CAS number: 149-91-). 7), Anacardic acid (CAS number: 16611-84-0), MG-115 (CAS number: 133407-86-0), MG-132 (CAS number: 133407-82-6), lactacystin (CAS number:: 133343-34-7), epoxomicin (CAS number: 134381-21-8), parthenolide (CAS number: 20554-84-1), calfilzomib (CAS number: 868540-17-4), voltezomib (CAS number: 179324-4). 69-7), MLN-4924 (CAS number: 905579-51-3), delanzomib (CAS number: 847499-27-8), ixazomib (CAS number: 1239908-20-3), marizomib (CAS number: 437742-). 34-2) and oprozomib (CAS number: 935888-69-0) and the like.

The pharmaceutical composition of the present embodiment may contain a combination of a CDK4 and 6 inhibitor and an AKT inhibitor as an active ingredient. The CDK4 and 6 inhibitors are the same as those described above. The AKT inhibitor may be a small molecule compound or a nucleic acid that inhibits AKT. Examples of the inhibitory nucleic acid include shRNA, siRNA and the like.

Examples of AKT inhibitors include Akt Inhibitor X (CAS number: 925681-41-0), Ipatasertib (CAS number: 1001264-89-6), Capivaceltib (CAS number: 1143532-39-1), and Miraceltib (CAS number: 1313881). -70-7), Afresertib (CAS number: 1047644-62-1), Ipatasertib (CAS number: 1047634-65-0), Trisiribin phosphate (CAS number: 61966-08-3), MK2206 (CAS number: 1032350) -13-2) and the like.

The CDK4 and 6 inhibitors and the NF-κB signaling pathway inhibitor or AKT inhibitor may be mixed with a pharmaceutically acceptable carrier and formulated as a pharmaceutical composition.

Alternatively, the CDK4 and 6 inhibitors, the NF-κB signaling pathway inhibitor, and the AKT inhibitor are mixed with a pharmaceutically acceptable carrier and individually formulated as a pharmaceutical composition to form a therapeutic kit. You may be doing it.

The pharmaceutical composition is preferably formulated into a dosage form that is used orally or a dosage form that is used parenterally. The pharmaceutical composition may be in a dosage form used orally or in a dosage form used parenterally. Dosage forms used orally include, for example, tablets, capsules, elixirs, microcapsules and the like. Dosage forms used parenterally include, for example, injections, inhalants, suppositories, patches and the like.

Pharmaceutically acceptable carriers include, for example, solvents such as sterile water and physiological saline; binders such as gelatin, cornstarch, tragant gum and arabic rubber, excipients such as crystalline cellulose; swelling agents such as alginic acid and the like. Can be mentioned.

The pharmaceutical composition may further contain additives. Additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavoring agents such as peppermint and red mono oil; stabilizers of benzyl alcohol and phenol; buffers such as phosphate and sodium acetate. Agents; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.

The pharmaceutical composition can be formulated by appropriately combining the above carriers and additives and mixing them in a generally accepted unit dose form required for pharmaceutical practice.

Administration to patients is performed by methods known to those skilled in the art, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc., intranasally, transbronchially, intramuscularly, transdermally, or orally. sell. The dose varies depending on the weight, age, symptoms, administration method, etc. of the patient, but those skilled in the art can appropriately select an appropriate dose.

The doses of the CDK4 and 6 inhibitors vary depending on the patient's symptoms, etc., but in the case of oral administration, generally in adults (assuming a body weight of 60 kg), about 75 to 400 mg per day, for example, about 75 to 125 mg, For example, it is considered appropriate to administer about 100 to 400 mg once a day or in several divided doses. When administered parenterally, the dose varies depending on the patient's symptoms, target organs, administration method, etc., but when systemic administration is performed, it is generally per day for adults (assuming a body weight of 60 kg). It is considered appropriate to administer about 30 to 200 mg, for example, about 30 to 60 mg, for example, about 50 to 200 mg once every 1 to 10 days. In the case of local administration, in general, for adults (assuming a body weight of 60 kg), about 7.5 to 40 mg per day, for example, about 7.5 to 12.5 mg, for example, about 10 to 40 mg, 1 to 10 It is considered appropriate to administer about once a day.

The dose of the NF-κB signal transduction pathway inhibitor varies depending on the patient's symptoms, etc., but in the case of oral administration, generally in adults (assuming a body weight of 60 kg), about 50 to 900 mg per day, for example, about 300. It is considered appropriate to administer to 900 mg, for example, about 2 to 5 mg once a day or in several divided doses. When administered parenterally, the dose varies depending on the patient's symptoms, target organs, administration method, etc., but when systemic administration is performed, it is generally per day for adults (assuming a body weight of 60 kg). It is considered appropriate to administer about 2 to 200 mg, for example, about 2 to 5 mg, for example, about 50 to 200 mg once every 1 to 10 days. In the case of local administration, in general, for adults (assuming a body weight of 60 kg), about 2 to 100 mg per day, for example, about 2 to 50 mg, for example, about 10 to 100 mg, is administered about once every 1 to 10 days. It is considered appropriate to do so.

The dose of the AKT inhibitor varies depending on the patient's symptoms and the like, but in the case of oral administration, generally in adults (assuming a body weight of 60 kg), about 200 to 600 mg per day, for example, about 200 to 400 mg, for example, about. It is considered appropriate to administer about 300 to 600 mg once a day or in several divided doses. When administered parenterally, the dose varies depending on the patient's symptoms, target organs, administration method, etc., but when systemic administration is performed, it is generally per day for adults (assuming a body weight of 60 kg). It is considered appropriate to administer about 100 to 800 mg, for example, about 100 to 500 mg, for example, about 400 to 800 mg once every 1 to 10 days. In the case of local administration, in general, for adults (assuming a body weight of 60 kg), about 10 to 100 mg per day, for example, about 10 to 25 mg, for example, about 30 to 100 mg, is administered about once every 1 to 10 days. It is considered appropriate to do so.

[Method of testing the effectiveness of administration to cancer patients]
In one embodiment, the present invention comprises a step of inspecting whether or not a cancer cell derived from a cancer patient is RB1 positive, and that the cancer cell is RB1 positive is used in the treatment of the cancer patient, CDK4. And 6 inhibitors, and a method for testing the effectiveness of administration of the combination to a cancer patient, showing that administration of the combination of NF-κB signaling pathway inhibitor or AKT inhibitor is effective.

As will be described later in the Examples, when the cancer cells are RB1 positive, the cancer cells are effectively killed by administration of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor. Clarified that it can be done.

When a cancer cell is RB1 positive, it means that the cancer cell expresses RB1. The RB1 expressed by the cancer cells may have a mutation, but it is preferable that the function of the wild-type RB1 described above is maintained, and it is preferable that the RB1 is a wild-type RB1.

Whether or not a cancer cell is RB1 positive can be determined, for example, by PCR amplification of the RB1 locus on the genomic DNA of the cancer cell, PCR amplification of the cDNA of the RB1 gene, detection by a microarray of the mRNA of the RB1 gene, ELISA of the RB1 protein, and the like. It can be performed by western blotting, immunochemical staining and the like. The above PCR amplification products may be sequenced to analyze for the presence of mutations in the RB1 gene.

[Kit for testing the effectiveness of administration of the above combination to cancer patients]
In one embodiment, the invention is specific for a primer set for genomic DNA amplification of RB1 gene, a probe for genomic DNA of RB1 gene, a primer set for cDNA amplification of RB1 gene, a probe for mRNA of RB1 gene, or a specific for RB1 protein. It is used to test whether cancer cells derived from a cancer patient, including a binding substance, are RB1 positive, and the fact that the cancer cells are RB1 positive inhibits CDK4 and 6 in the treatment of the cancer patient. Provided are a kit for testing the effectiveness of administration of the combination to the cancer patient, showing that the combination of the agent and the NF-κB signaling pathway inhibitor or AKT inhibitor is effective.

The kit of this embodiment is suitable for carrying out the above-mentioned method for testing the effectiveness of administration of a combination of CDK4 and 6 inhibitors and an NF-κB signal transduction pathway inhibitor or AKT inhibitor to a cancer patient. Can be used for.

(Primer set for genomic DNA amplification)
The kit of this embodiment may include a primer set for amplifying genomic DNA of the RB1 gene. The base sequence of the primer set is not particularly limited as long as it can amplify the RB1 gene on the genome.

When the genomic DNA of a cancer cell derived from a cancer patient is amplified with a primer set for amplifying the genomic DNA of the RB1 gene, amplification of the RB1 gene is observed when the cancer cell carries the RB1 gene on the genomic DNA.

When cancer cells derived from cancer patients carry the RB1 gene, it is effective to administer a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or AKT inhibitor to the cancer patients. It can be determined that there is.

(Probe for genomic DNA)
The kit of this embodiment may include a probe for genomic DNA of the RB1 gene. The base sequence of the probe is not particularly limited as long as the RB1 gene on the genome can be detected. The probe may constitute a nucleic acid array.

When a probe for the genomic DNA of the RB1 gene is hybridized to the genomic DNA of a cancer cell derived from a cancer patient, if the cancer cell carries the RB1 gene on the genomic DNA, the probe hybridizes and the RB1 gene is used. The presence can be detected.

When cancer cells derived from cancer patients carry the RB1 gene, it is effective to administer a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or AKT inhibitor to the cancer patients. It can be determined that there is.

(Primer set for cDNA amplification)
The kit of this embodiment may include a primer set for cDNA amplification of the RB1 gene. The base sequence of the primer set is not particularly limited as long as it can amplify the cDNA of the RB1 gene.

When RNA was extracted from cancer cells derived from a cancer patient and amplified by RT-PCR using a primer set for cDNA amplification of the RB1 gene, if the cancer cells expressed the mRNA of the RB1 gene, the cDNA of the RB1 gene was expressed. Amplification is observed.

When cancer cells derived from a cancer patient express RB1 gene mRNA, administration of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient is performed. It can be judged to be valid.

(Probe for mRNA)
The kit of this embodiment may include a probe for mRNA of the RB1 gene. The base sequence of the probe is not particularly limited as long as it can detect the mRNA of the RB1 gene. The probe may constitute a nucleic acid array.

When RNA is extracted from cancer cells derived from a cancer patient and a probe for RB1 gene mRNA is hybridized, if the cancer cell expresses RB1 gene mRNA, the probe hybridizes and RB1 gene mRNA. Can be detected.

When cancer cells derived from a cancer patient express RB1 gene mRNA, administration of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient is performed. It can be judged to be valid.

(Specific binding substance)
The kit of this embodiment may contain a specific binding agent for the RB1 protein. Specific binding substances include antibodies, antibody fragments, aptamers and the like. Examples of the antibody fragment include F (ab') ₂ , Fab', Fab, Fv, scFv and the like. The specific binding substance is not particularly limited as long as the RB1 protein can be detected. The specific binding substance may be bound to a solid phase to form, for example, a flow strip, an array, or the like.

The presence of the RB1 protein can be detected by extracting the protein from cancer cells derived from a cancer patient and performing ELISA or Western blotting using a specific binding substance for the RB1 protein. Alternatively, the presence of RB1 protein can be detected by subjecting a section of cancer tissue derived from a cancer patient to immunochemical staining with a specific binding agent for RB1 protein.

When cancer cells derived from cancer patients express RB1 protein, it is effective to administer a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or AKT inhibitor to cancer patients. It can be determined that there is.

[Other embodiments]
In one embodiment, the invention presents a step of testing whether a patient-derived cancer cell is RB1 positive, and if the cancer cell is RB1 positive, a CDK4 and 6 inhibitor, and a CDK4 and 6 inhibitor to the patient. , A method of treating cancer, comprising the step of administering a combination of an NF-κB signaling pathway inhibitor or an AKT inhibitor.

In one embodiment, the invention provides the use of a combination of CDK4 and 6 inhibitors and NF-κB signaling pathway inhibitors or AKT inhibitors for the treatment of RB1-positive cancers.

In one embodiment, the invention is a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or AKT inhibitor for the manufacture of a therapeutic pharmaceutical composition or therapeutic kit for RB1-positive cancer. Provide use of.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. In the following examples, experiments using mice were performed according to a protocol (AP-153426) approved by the Animal Care and Use Committee of Kanazawa University.

[Experimental Example 1]
(Inactivation of the RB pathway induced the development of hepatocellular carcinoma)
In the RB family, there are RB1, RB Transcrictional Corepressor Like 1 (RBL1), and RB Transcrictional Corepressor Like 2 (RBL2).

In this experimental example, in a mouse in which the RBL1 gene is deleted and the Rb1 gene and the Rbl2 gene can be conditionally knocked out (hereinafter, may be referred to as “TKO mouse”), the Tumor Protein P53 (Trp53) gene is further deleted. A lost mouse (hereinafter, may be referred to as "QKO mouse") was used.

As shown in FIG. 2 (a), Rb1 ^{lox / lox} ; Rbl1 ^{− / −} ; Rbl2 ^{lox / lox} ; Trp53 ^{− / −} (QKO) mice were subjected to Cre recombinase, green fluorescent protein (GFP), and puromycin-resistant protein ( PiggyBac transposon vector construct (pCMV-Puro-Cre-IRES2-GFP) having a gene encoding Puro) in tandem and an expression vector (pCMV-hyPBase) encoding a transpozese are injected into the tail vein by hydrodynamic injection. bottom.

As a result, fluorescence of GFP was observed in the liver 3 days after the injection. Then, as shown in FIG. 2 (b), tumor formation was typically observed in the liver 2.5 months after the injection. In FIG. 2 (b), the arrows indicate tumors.

Primary hepatocellular carcinoma cells (hereinafter sometimes referred to as "QKO PHCC") collected from tumors showed high expression of Ki67 and α-fetoprotein (AFP). This is a characteristic of hepatocellular carcinoma. From the above results, it was clarified that the inactivation of the RB pathway induces the onset of hepatocellular carcinoma.

[Experimental Example 2]
(Administration of CDK4 / 6 inhibitors is not optimal)
RB7LP is a variant of RB1 and is not phosphorylated. RB7LP lacks the N-terminal domain of RB1, but retains all of pockets A, B, and C so that it can bind to proteins with E2F and LxCxE motifs. However, seven serine residues known to be phosphorylated have been replaced with non-phosphorylated amino acids. The amino acid sequence of RB7LP is shown in SEQ ID NO: 1.

Therefore, RB7LP can mimic the RB1-dependent effects of CDK4 / 6 inhibitors. Therefore, RB7LP was expressed in hepatocellular carcinoma cells to examine the induction of apoptosis.

Specifically, a tetracycline-induced RB7LP expression vector (pTRE3G-puro-RB7LP-GFP) was introduced into HepG2, which is a human liver cancer cell line. These cells (hereinafter, may be referred to as "HepG2-pTRE3G-puro-RB7LP-GFP cells") express RB7LP by adding doxycycline to the medium.

Subsequently, 1 μg / mL doxycycline was added to the medium of HepG2-pTRE3G-puro-RB7LP-GFP cells and incubated for 3 days. Also, for comparison, cells similarly incubated without the addition of doxycycline were used.

Subsequently, the Annexin V-positive cells of each cell were stained with the eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI).

Subsequently, each cell was analyzed using FACS Canto II (BD Bioscience). A cell having a high annexin V fluorescence value and a low PI fluorescence value is a cell in the early stage of apoptosis, and a cell having both annexin V fluorescence value and a PI fluorescence value is a late apoptosis cell.

FIG. 3A is a graph showing the results of flow cytometry. In FIG. 3 (a), "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. The horizontal axis shows the fluorescence intensity of Annexin V, and the vertical axis shows the fluorescence intensity of PI.

FIG. 3B is a graph created based on the result of FIG. 3A. In FIG. 3 (b), the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis. In FIG. 3 (b), "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "***" indicates that there is a significant difference in P <0.001 as a result of the t-test of unpaired two-sided students.

As a result, it was clarified that the induction of apoptosis by the induction of RB7LP expression was insufficient. This result indicates that the induction of apoptosis by administration of the CDK4 / 6 inhibitor to cancer cells is insufficient.

[Experimental Example 3]
(Screening of compounds that enhance the effect of RB7LP)
Compounds that specifically kill HepG2-pTRE3G-puro-RB7LP-GFP cells in the presence of doxycycline were screened from the compound library.

As a compound library, a commercially available library containing 80 kinds of kinase inhibitors (product name "SCREEN-WELL Kinase Inhibitor Library", model number "BML-2832", Enzo Life Science Co., Ltd.) was used.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded in 96-well plates at 20,000 cells / well, exposed to each compound at a final concentration of 1 μM in the presence or absence of 1 μg / mL doxycycline, and incubated for 24 hours. Subsequently, the survival rate of each cell was measured using a commercially available kit (product name "Cell reagent SF", Nacalai Tesque, Inc.).

FIG. 4 is a graph showing the results of screening. The vertical axis of the graph shows the ratio represented by the following formula (F1). In the following formula (F1), "DOX (+) cells" indicate cells exposed to the compound in the presence of doxycycline, and "DOX (-) cells" indicate cells exposed to the compound in the absence of doxycycline.
Percentage = DOX (-) cell viability / DOX (+) cell viability ... (F1)

The horizontal axis indicates the compound number. The higher the value on the vertical axis, the more DOX (+) cell-specific cytotoxicity is indicated. As a result, it was revealed that the IKKβ inhibitor Bay11-7082 (CAS number: 1954-67-7) has DOX (+) cell-specific cytotoxicity. That is, it was revealed that Bay11-7082 enhances the cytotoxicity of HepG2 cells in which RB7LP was induced.

[Experimental Example 4]
(Expression of RB7LP and examination of effect of IKKβ inhibitor)
The cytotoxicity of HepG2 cells and primary hepatocellular carcinoma cells (QKO PHCC) was investigated by inducing the expression of RB7LP and adding an IKKβ inhibitor. As the IKKβ inhibitor, shRNA for Bay11-7082, IMD-0354 (CAS number: 978-62-1) and IKKβ was used.

<< Examination using Bay11-7082 >>
HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded in a 6-well plate and exposed to Bay11-7082 at a final concentration of 0,5,10,15,20,25 μM in the presence or absence of 1 μg / mL doxycycline for 24 hours. Incubated. Subsequently, the survival rate of each cell was measured using a commercially available kit (product name "Cell reagent SF", Nacalai Tesque, Inc.).

FIG. 5 is a graph showing the survival rate of each cell. As a result, it was revealed that Bay11-7082 of 10 to 20 μM showed the most synergistic effect on the induction of RB7LP expression.

Subsequently, for HepG2-pTRE3G-puro-RB7LP-GFP cells exposed to 15 μM Bay11-7082 for 24 hours, eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) was used in the same manner as in Experimental Example 2. Used to stain Annexin V-positive cells. In addition, dead cells were stained with propidium iodide (PI).

Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis. FIG. 6 is a graph created based on the results of flow cytometry. In FIG. 6, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 6, "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "BAY-" indicates the result in the absence of Bay11-7082, and "BAY +" indicates the result in the presence of Bay11-7082 having a final concentration of 15 μM. Further, "Z-VAD-" indicates that it is the result in the absence of Z-VAD-FMK (catalog number "3188-v", Peptide Institute, Ltd.), which is a caspase inhibitor, and "Z-". "VAD +" indicates the result in the presence of Z-VAD-FMK with a final concentration of 20 μM. Further, "*" and "*****" indicate that there is a significant difference at P <0.05 and P <0.0001, respectively, as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

The results revealed that more than 80% of HepG2-pTRE3G-puro-RB7LP-GFP cells exposed to 15 μM Bay11-7082 in the presence of doxycycline developed apoptosis. In addition, this apoptosis was significantly suppressed by the addition of Z-VAD-FMK.

This result further supports that Bay11-7082 significantly enhances the induction of apoptosis in HepG2 cells that have induced RB7LP expression.

<< Examination using IMD-0354 >>
Subsequently, a similar study was conducted using IMD-0354 (CAS No .: 978-62-1), which is an IKKβ inhibitor, instead of Bay11-7082.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded on a 6-well plate, exposed to IMD-0354 at a final concentration of 15 μM in the presence or absence of 1 μg / mL doxycycline, and incubated for 24 hours.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 7 is a graph created based on the results of flow cytometry. In FIG. 7, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 7, "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "IMD-" indicates the result in the absence of IMD-0354, and "IMD +" indicates the result in the presence of IMD-0354 having a final concentration of 15 μM. In addition, "**" and "*****" indicate that there is a significant difference at P <0.01 and P <0.0001, respectively, as a result of the bidirectional ANOVA and the subsequent Tukey's post-test. ..

As a result, it was revealed that IMD-0354 significantly enhanced the induction of apoptosis of HepG2-pTRE3G-puro-RB7LP-GFP cells that induced the expression of RB7LP in the presence of doxycycline. That is, it was clarified that IMD-0354 also showed the same effect as Bay11-7082.

<< Examination using shRNA for IKKβ >>
Subsequently, IKKβ knockdown was performed instead of the addition of the IKKβ inhibitor, and it was examined whether or not the induction of apoptosis of HepG2-pTRE3G-puro-RB7LP-GFP cells was enhanced.

As a shRNA for IKKβ, a commercially available shRNA expression vector (catalog number “# SHCLNG-NM_001556”, “TRCN000018917”, “TRCN00000018915”, “TRCN00000018916”, “TRCN00000018918”) was replaced with a puromycin resistance gene. used.

As a result of introducing four types of shRNA into HepG2-pTRE3G-puro-RB7LP-GFP cells and confirming the expression of IKKβ by Western blotting, it was confirmed that IKKβ can be knocked down using any of the shRNAs. Therefore, one of these shRNAs was used in the next experiment.

HepG2-pTRE3G-puro-RB7LP-GFP cells with or without an shRNA expression vector for IKKβ were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded on a 6-well plate and incubated for an additional 24 hours in the presence or absence of 1 μg / mL doxycycline.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 8 is a graph created based on the results of flow cytometry. In FIG. 8, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 8, "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "SHRNA-" indicates the result of cells into which shRNA has not been introduced into IKKβ, and "shRNA +" indicates the result of cells into which shRNA has been introduced into IKKβ. Further, "*****" indicates that there is a significant difference at P <0.0001 as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

As a result, it was revealed that knockdown of IKKβ significantly enhanced the induction of apoptosis of HepG2-pTRE3G-puro-RB7LP-GFP cells that induced the expression of RB7LP in the presence of doxycycline. This result further supports that inhibition of IKKβ function significantly enhances the induction of apoptosis in HepG2 cells that have induced RB7LP expression.

<< Examination using primary hepatocellular carcinoma cells (QKO PHCC) >>
Instead of HepG2-pTRE3G-puro-RB7LP-GFP cells, QKO PHCC collected in Experimental Example 1 was used for the study.

Cells in which an RB7LP expression vector (plenti6.3-RB7LP) has been introduced into QKO PHCC (hereinafter, may be referred to as “RB7LP (+) QKO PHCC cells”) and a LacZ expression vector (plenti6.3 / V5-LacZ). ) Was introduced (hereinafter, may be referred to as "RB7LP (-) QKO PHCC cell").

Subsequently, RB7LP (+) QKO PHCC cells and RB7LP (−) QKO PHCC cells were exposed to Bay11-7082 at a final concentration of 15 μM and incubated for 24 hours.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 9 is a graph created based on the results of flow cytometry. In FIG. 9, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 9, "RB7LP-" indicates the result of RB7LP (-) QKO PHCC cells, and "RB7LP +" indicates the result of RB7LP (+) QKO PHCC cells. Further, "BAY-" indicates the result in the absence of Bay11-7082, and "BAY +" indicates the result in the presence of Bay11-7082 having a final concentration of 15 μM. Further, "*" and "*****" indicate that there is a significant difference at P <0.05 and P <0.0001, respectively, as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

As a result, it was revealed that Bay11-7082 significantly enhanced the induction of apoptosis of QKO PHCC cells expressing RB7LP. That is, the same results as in HepG2 cells were shown in QKO PHCC cells.

[Experimental Example 5]
(Examination of combined effects of CDK4 and 6 inhibitors and IKKβ inhibitors)
After incubating various cancer cell lines in the presence of CDK4 and 6 inhibitors and IKKβ inhibitors, eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) in the same manner as in Experimental Example 2. Was used to stain annexin V-positive cells. In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

The cancer cell lines include Huh7, Li-7 and HepG2, which are human liver cancer cell lines, H1975, which is a human lung cancer cell line, HCC1143 and T47D, which are human breast cancer cell lines, and HCT-, which is a human colon cancer cell line. 116 and DLD-1 were used. All of these cells are known to be RB1 positive.

As the CDK4 and 6 inhibitors, palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3) or abemaciclib (CAS number: 1231929-97-7) was used. In addition, Bay11-7082 was used as the IKKβ inhibitor.

<< Combined use of palbociclib and Bay11-7082 >>
Huh7 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. Li-7 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 15 μM. H1975 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. HCC1143 cells were incubated for 4 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. T47D cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. HCT-116 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 8 μM and Bay11-7082 at a final concentration of 15 μM. DLD-1 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 8 μM and Bay11-7082 at a final concentration of 15 μM.

FIGS. 10 to 16 are graphs created based on the results of flow cytometry. In FIGS. 10 to 16, the sum of the ratio of cells in the early stage of apoptosis (annexin V positive, PI negative) and the ratio of cells in the late stage of apoptosis (annexin V positive, PI positive) was defined as the ratio of cells having undergone apoptosis.

FIG. 10 shows the results of Huh7 cells, FIG. 11 shows the results of Li-7 cells, FIG. 12 shows the results of H1975 cells, FIG. 13 shows the results of HCC1143 cells, and FIG. 14 shows the results of T47D cells. Yes, FIG. 15 is the result of HCT-116 cells and FIG. 16 is the result of DLD-1 cells.

In FIGS. 10 to 16, "Palbo.-" Indicates the result in the absence of palbociclib, and "Palbo. +" Indicates the result in the presence of palbociclib. Further, "BAY-" indicates that the result is in the absence of Bay11-7082, and "BAY +" indicates that the result is in the presence of Bay11-7082. In addition, "ns.", "*", "**" and "*****" have no significant difference as a result of the two-way ANOVA and the subsequent Tukey's post-test, P <0. It is shown that there is a significant difference at 0.05, P <0.01 and P <0.0001.

As a result, it was clarified that the induction of apoptosis was significantly enhanced by the combined use of the CDK4 and 6 inhibitors and the IKKβ inhibitor in any of the cancer cell lines. This result shows that the combined use of the CDK4 and 6 inhibitors and the IKKβ inhibitor is effective not only in hepatocellular carcinoma but also in lung cancer, breast cancer and colon cancer.

<< Combined use of ribociclib and Bay11-7082 >>
Ribociclib was used as a CDK4 and 6 inhibitor. HepG2 cells were incubated for 24 hours in the presence or absence of a final concentration of 8 μM ribociclib and a final concentration of 15 μM Bay11-7082.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 17 is a graph created based on the results of flow cytometry. In FIG. 17, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 17, “Ribo. −” indicates the result in the absence of ribociclib, and “Ribo. +” Indicates the result in the presence of ribociclib. Further, "BAY-" indicates that the result is in the absence of Bay11-7082, and "BAY +" indicates that the result is in the presence of Bay11-7082. In addition, "ns." And "*****" have no significant difference as a result of the two-way ANOVA and the subsequent Tukey's post-test, and there is a significant difference at P <0.0001. Is shown.

As a result, it was clarified that even when ribociclib was used as a CDK4 and 6 inhibitor, the induction of apoptosis was significantly enhanced by the combined use with the IKKβ inhibitor.

<< Combined use of abemaciclib and Bay11-7082 >>
Abemaciclib was used as a CDK4 and 6 inhibitor. HepG2 cells were incubated for 24 hours in the presence or absence of abemaciclib at a final concentration of 6 μM and Bay11-7082 at a final concentration of 10 μM.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 18 is a graph created based on the results of flow cytometry. In FIG. 18, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells having undergone apoptosis.

In FIG. 18, “Abema. −” indicates the result in the absence of abemaciclib, and “Abema. +” Indicates the result in the presence of abemaciclib. Further, "BAY-" indicates that the result is in the absence of Bay11-7082, and "BAY +" indicates that the result is in the presence of Bay11-7082. Further, "*****" indicates that there is a significant difference at P <0.0001 as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

As a result, it was clarified that even when abemaciclib was used as a CDK4 and 6 inhibitor, the induction of apoptosis was significantly enhanced by the combined use with the IKKβ inhibitor.

[Experimental Example 6]
(Expression of RB7LP and examination of effect of NF-κB inhibitor)
Using an NF-κB inhibitor instead of an IKKβ inhibitor, the induction of RB7LP expression and the induction of apoptosis by the addition of an NF-κB inhibitor were investigated. QNZ (CAS No .: 545380-34-5) was used as an NF-κB inhibitor.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded on a 6-well plate, exposed to QNZ at a final concentration of 50 nM in the presence or absence of 1 μg / mL doxycycline, and incubated for 24 hours.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 19 is a graph created based on the results of flow cytometry. In FIG. 19, the total of the proportion of cells in the early stage of apoptosis (annexin V positive, PI negative) and the proportion of cells in the late apoptosis stage (annexin V positive, PI positive) was defined as the proportion of cells that had undergone apoptosis.

In FIG. 19, "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "QNZ-" indicates that the result is in the absence of QNZ, and "QNZ +" indicates that the result is in the presence of QNZ. In addition, "**" and "*****" indicate that there is a significant difference at P <0.01 and P <0.0001, respectively, as a result of the bidirectional ANOVA and the subsequent Tukey's post-test. ..

As a result, it was revealed that QNZ significantly enhanced the induction of apoptosis of HepG2-pTRE3G-puro-RB7LP-GFP cells that induced the expression of RB7LP in the presence of doxycycline. That is, it was clarified that the NF-κB inhibitor also shows the same effect as the IKKβ inhibitor.

[Experimental Example 7]
(Examination of combined effects of CDK4 and 6 inhibitors and AKT inhibitors)
A study was conducted using an AKT inhibitor instead of an IKKβ inhibitor. After incubating various cancer cell lines in the presence of CDK4 and 6 inhibitors and AKT inhibitors, eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermofisher Scientific) was performed in the same manner as in Experimental Example 2. Was used to stain Annexin V-positive cells. In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

As the cancer cell line, HepG2, which is a human liver cancer cell line, SW480, HCT-116 and DLD-1, which are human colon cancer cell lines, and A549, which is a human lung cancer cell line, were used.

Palbociclib (CAS No .: 571190-30-2) was used as the CDK4 and 6 inhibitor. Further, as an AKT inhibitor, Akt Inhibitor X (CAS number: 925681-41-0) was used.

HepG2 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 25 μM. SW480 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. HCT-116 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. DLD-1 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. A549 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM.

FIGS. 20 to 24 are graphs created based on the results of flow cytometry. In FIGS. 20 to 24, the sum of the ratio of cells in the early stage of apoptosis (annexin V positive, PI negative) and the ratio of cells in the late stage of apoptosis (annexin V positive, PI positive) was defined as the ratio of cells having undergone apoptosis.

FIG. 20 is the result of HepG2 cells, FIG. 21 is the result of SW480 cells, FIG. 22 is the result of HCT-116 cells, FIG. 23 is the result of DLD-1 cells, and FIG. 24 is the result of A549 cells. The result.

In FIGS. 20 to 24, "Palbo.-" Indicates the result in the absence of palbociclib, and "Palbo. +" Indicates the result in the presence of palbociclib. Further, "CAS-" indicates that the result is the result in the absence of Akt Inhibitor X, and "CAS +" indicates that the result is the result in the presence of Akt Inhibitor X. In addition, "*", "***" and "*****" are the results of the bidirectional ANOVA and the subsequent Tukey's post-test, P <0.05, P <0.001 and P <0, respectively. At .0001, there is a significant difference.

As a result, it was clarified that the induction of apoptosis was significantly enhanced by the combined use of the CDK4 and 6 inhibitors and the AKT inhibitor in any of the cancer cell lines. This result shows that the combined use of the CDK4 and 6 inhibitors and the AKT inhibitor is effective not only in hepatocellular carcinoma but also in colon cancer and lung cancer.

[Experimental Example 8]
(Investigation of the effect of RB1 expression on the combined effect of CDK4 and 6 inhibitors and IKKβ inhibitors)
The effect of RB1 expression on the combined effects of CDK4 and 6 inhibitors and IKKβ inhibitors was investigated.

First, a tetracycline-inducible Rb1 expression vector was introduced into QKO PHCC cells. These cells (hereinafter, may be referred to as "QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells") express Rb1 by adding doxycycline to the medium.

Subsequently, QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells were cultured for 3 days in the presence or absence of 1 μg / mL doxycycline. Subsequently, each cell was seeded in a 6-well plate in the presence or absence of 1 μg / mL doxicycline, in the presence or absence of parvocyclib at a final concentration of 10 μM, in the presence or absence of Bay11-7082 at a final concentration of 15 μM or Incubated for 24 hours in the absence.

Subsequently, in the same manner as in Experimental Example 2, annexin V-positive cells were stained with eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometric analysis was performed to measure the proportion of cells that developed apoptosis.

FIG. 25 is a graph created based on the results of flow cytometry. In FIG. 25, the sum of the ratio of cells in the early stage of apoptosis (annexin V positive, PI negative) and the ratio of cells in the late stage of apoptosis (annexin V positive, PI positive) was defined as the ratio of cells having undergone apoptosis.

In FIG. 25, "DOX-" indicates the result in the absence of doxycycline, and "DOX +" indicates the result of adding doxycycline. Further, "Palbo.-" Indicates that the result is in the absence of palbociclib, and "Palbo. +" Indicates that the result is in the presence of palbociclib. Further, "BAY-" indicates that the result is in the absence of Bay11-7082, and "BAY +" indicates that the result is in the presence of Bay11-7082. In addition, "**" and "*****" indicate that there is a significant difference at P <0.01 and P <0.0001, respectively, as a result of the bidirectional ANOVA and the subsequent Tukey's post-test. ..

As a result, in the absence of Rb1, the combined use of palbociclib and Bay11-7082 had a negligible effect of inducing apoptosis. On the other hand, in the presence of Rb1, it was revealed that the induction of apoptosis by the combined use of palbociclib and Bay11-7082 was significantly enhanced. This result indicates that the presence or absence of RB1 in cancer cells is a marker indicating the effectiveness of the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors.

[Experimental Example 9]
(Examination of combined effect of CDK4 / 6 inhibitor and IKKβ inhibitor in vivo)
A CDK4 / 6 inhibitor and an IKKβ inhibitor were administered to a cancer-bearing mouse model, and changes in tumor volume over time were measured.

The cancer-bearing mouse model includes a cancer-bearing mouse model transplanted with HepG2 cells, a cancer-bearing mouse model transplanted with Li-7 cells, and QKO PHCC cells (QKO-PHCC-pTRE3G-blast-) that express mouse Rb1 in a tetracycline-inducible manner. A cancer-bearing mouse model transplanted with mouse Rb1-mcherry cells) was used.

Palbociclib (CAS No .: 571190-30-2) was used as the CDK4 and 6 inhibitor. In addition, Bay11-7082 was used as the IKKβ inhibitor.

<< Examination using a cancer-bearing mouse model transplanted with HepG2 cells >>
^Six 1 × 10 HepG2 cells were transplanted subcutaneously into the back of a KSN / slc mouse (6 weeks old, male) together with Matrigel (catalog number “354234”, Corning Inc.) to prepare a cancer-bearing mouse model. After about 4 weeks, after the tumor size reached about 200 mm3 ^, administration of palbociclib and Bay11-7082 to the mice in the test group was started. Palbociclib was administered daily by gavage at a dose of 80 mg / Kg. Bay11-7082 was dissolved in corn oil and administered by subcutaneous injection at a dose of 20 mg / Kg every 2 days. In addition, vehicle (corn oil or L-sodium lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n = 5).

FIG. 26 is a graph showing changes over time in tumor volume of mice in each group. In FIG. 26, the vertical axis shows the rate of increase in tumor volume (%), and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 26, "Palbo.-" Indicates that the result was a mouse that did not receive palbociclib, and "Palbo. +" Indicates that the result was a mouse that received palbociclib. Further, "BAY-" indicates the result of the mouse to which Bay11-7082 was not administered, and "BAY +" indicates the result of the mouse to which Bay11-7082 was administered. In addition, "*", "*****" and "*****" are the results of the bidirectional ANOVA and the subsequent Tukey's post-test, P <0.05, P <0.01 and P <0, respectively. At .0001, there is a significant difference.

As a result, the increase in tumor volume was slightly suppressed by the administration of palbociclib alone as compared with the non-administered group. It was also revealed that the combined administration of palbociclib and Bay11-7082 almost completely suppressed the increase in tumor volume. In addition, there was no significant difference in the body weight of the mice in each group. This result shows that the combination of CDK4 and 6 inhibitors and IKKβ inhibitor is also effective in vivo.

<< Examination using a cancer-bearing mouse model transplanted with Li-7 cells >>
^Six 1 × 10 Li-7 cells were transplanted subcutaneously into the back of a KSN / slc mouse (6 weeks old, male) together with Matrigel (catalog number “354234”, Corning Inc.) to prepare a cancer-bearing mouse model. After about 5 weeks, after the tumor size reached about 200 mm3 ^, administration of palbociclib and Bay11-7082 to the mice in the test group was started. Palbociclib was administered daily by gavage at a dose of 80 mg / Kg. Bay11-7082 was dissolved in corn oil and administered by subcutaneous injection at a dose of 20 mg / Kg every 2 days. In addition, vehicle (corn oil or L-sodium lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n = 5).

FIG. 27 is a graph showing changes over time in tumor volume of mice in each group. In FIG. 27, the vertical axis shows the rate of increase in tumor volume (%), and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 27, "Palbo.-" Indicates that the result was a mouse that did not receive palbociclib, and "Palbo. +" Indicates that the result was a mouse that received palbociclib. Further, "BAY-" indicates the result of the mouse to which Bay11-7082 was not administered, and "BAY +" indicates the result of the mouse to which Bay11-7082 was administered. Further, "*****" indicates that there is a significant difference at P <0.0001 as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

As a result, the increase in tumor volume was significantly suppressed by the administration of palbociclib alone as compared with the non-administered group. It was also revealed that the combined administration of palbociclib and Bay11-7082 significantly reduced the tumor volume. In addition, there was no significant difference in the body weight of the mice in each group. This result indicates that the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is effective in vivo even in tumors derived from Li-7 cells.

<< Examination by a cancer-bearing mouse model transplanted with QKO PHCC cells expressing mouse Rb1 in a tetracycline-inducible manner >>
QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells 5 × 10 ⁵ cells were transplanted subcutaneously into the back of KSN / slc mice (6 weeks old, male) together with Matrigel (catalog number “354234”, Corning Inc.) and carried. A cancer mouse model was created. After the tumor size reached about 200 mm ³ , mice in the test group were treated with doxycycline, palbociclib and Bay11-7082.

Doxycycline (50 mg / Kg) and palbociclib (80 mg / Kg) were administered daily by oral gavage. Bay11-7082 was dissolved in corn oil and administered by subcutaneous injection at a dose of 20 mg / Kg every 2 days. In addition, vehicle (0.9% NaCl, corn oil or L-sodium lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n = 5).

FIG. 28 is a graph showing changes over time in tumor volume of mice in each group. In FIG. 28, the vertical axis shows the rate of increase in tumor volume (%), and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 28, "DOX-" indicates the result of mice not treated with doxycycline, and "DOX +" indicates the result of mice treated with doxycycline. Further, "Palbo.-" Indicates that the result was a mouse that did not receive palbociclib, and "Palbo. +" Indicates that the result was a mouse that received palbociclib. Further, "BAY-" indicates the result of the mouse to which Bay11-7082 was not administered, and "BAY +" indicates the result of the mouse to which Bay11-7082 was administered. Further, "*****" indicates that there is a significant difference at P <0.0001 as a result of the bidirectional ANOVA and the subsequent Tukey's post-test.

As a result, the increase in tumor volume was significantly suppressed by the administration of palbociclib alone as compared with the non-administered group. It was also revealed that the combined administration of palbociclib and Bay11-7082 significantly reduced the tumor volume. In addition, there was no significant difference in the body weight of the mice in each group. This result indicates that the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is effective in vivo even in tumors derived from Li-7 cells.

As a result, in the absence of Rb1, the combined administration of palbociclib and Bay11-7082 had a negligible effect of suppressing the increase in tumor volume. On the other hand, in the presence of Rb1, a significant decrease in tumor volume was observed by the combined administration of palbociclib and Bay11-7082. This result further supports that the presence or absence of RB1 in cancer cells is a marker indicating the effectiveness of the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors.

According to the present invention, it is possible to provide a technique for effectively treating RB1-positive cancer.

Claims (10)

A therapeutic agent for RB Transcrictional Corepressor 1 (RB1) -positive cancer containing a combination of a cyclin-dependent kinase (CDK) 4 and 6 inhibitor and an NF-κB signal transduction pathway inhibitor or an AKT inhibitor as an active ingredient. Composition. The CDK4 and 6 inhibitors are palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3), abemaciclib (CAS number: 1231929-97-7), trilaciclib (CAS number: 1374743). -00-6) or relocyclib (CAS No. 1628256-23-4), according to claim 1. Claimed that a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor is contained as an active ingredient, and the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor. The pharmaceutical composition according to 1 or 2. The NF-κB signaling pathway inhibitor is an IKKβ inhibitor,
The IKKβ inhibitors are Bay11-7082 (CAS number: 1954-67-7), IMD-0354 (CAS number: 978-62-1), IMD-1041 (CAS number: 1073666-73-5), IMD-. 2560, TPCA1 (CAS number: 507475-17-4), BOT-64 (CAS number: 113760-29-5), BMS345541 (CAS number: 445430-58-0), SC-514 (CAS number: 354812-17) -2), IKK-16 (CAS number: 1186195-62-9), Elchiprotafib (CAS number: 251303-04-5) and Bay65-1942 (HCl salt) (CAS number: 600734-06-3). The pharmaceutical composition according to claim 3, which is selected from the group consisting of. The NF-κB signaling pathway inhibitor is an NF-κB inhibitor,
The NF-κB inhibitors are QNZ (CAS number: 545380-34-5), JSH-23 (CAS number: 794886-87-1), GYY4137 (CAS number: 106740-09-4), p-XSC ( CAS Number: 85539-83-9), CV3988 (CAS Number: 85703-73-7), Prostaglandin E2 (CAS Number: 363-24-6), LY294002 (CAS Number: 154447-36-6), Wortmannin (CAS Number: 1954-26-7), Mesalamine (CAS Number: 89-57-6), Loriplum (CAS Number: 85416-75-7), Asbestosic Acid (CAS Number: 149-91-7), Anacardic Acid (CAS Number: 1661-84-0), MG-115 (CAS Number: 133407-86-0), MG-132 (CAS Number: 133407-82-6), Lactacystin (CAS Number: 133343-34-7) ), Epoxomicin (CAS number: 134381-21-8), parthenolide (CAS number: 20554-84-1), calfilzomib (CAS number: 868540-17-4), voltezomib (CAS number: 179324-69-7), MLN-4924 (CAS number: 905579-53-1-3), delanzomib (CAS number: 847499-27-8), ixazomib (CAS number: 1239908-20-3), marizomib (CAS number: 437742-34-2) and The pharmaceutical composition according to claim 3, which is selected from the group consisting of oprosomib (CAS No .: 935888-69-0). It contains a combination of CDK4 and 6 inhibitors and AKT inhibitor as an active ingredient.
The AKT inhibitors are Akt Inhibitor X (CAS number: 925681-41-0), Ipatasertib (CAS number: 1001264-89-6), Capivaceltib (CAS number: 1143532-39-1), Miraceltib (CAS number: 1313881). -70-7), Afresertib (CAS number: 1047644-62-1), Ipatasertib (CAS number: 1047634-65-0), Trisiribin phosphate (CAS number: 61966-08-3) and MK2206 (CAS number: 1032350) The pharmaceutical composition according to claim 1 or 2, which is selected from the group consisting of -13-2). The pharmaceutical composition according to any one of claims 1 to 6, wherein the RB1-positive cancer is hepatocellular carcinoma, breast cancer, lung cancer or colon cancer. A therapeutic kit for RB1-positive cancers comprising cyclin-dependent kinase (CDK) 4 and 6 inhibitors and NF-κB signaling pathway inhibitors or AKT inhibitors. Includes a step of testing whether cancer cells derived from a cancer patient are RB1 positive.
The fact that the cancer cells are RB1 positive indicates that administration of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor is effective for the treatment of the cancer patient. A method for testing the effectiveness of administration of the combination to a cancer patient. Primer set for genomic DNA amplification of RB1 gene,
Probe for genomic DNA of RB1 gene,
Primer set for cDNA amplification of RB1 gene,
Includes a probe for mRNA of the RB1 gene, or a specific binding agent for the RB1 protein.
Used to test whether cancer cells derived from cancer patients are RB1 positive,
The fact that the cancer cells are RB1 positive indicates that administration of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor is effective for the treatment of the cancer patient. , A kit for testing the efficacy of administration of the combination to the cancer patient.

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