Pharmaceutical composition for treating cancer

Abstract

The present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a nucleic acid that suppresses the expression of Chromosome 11 Open Reading Frame 97 (C11orf97). With the present invention, it becomes possible to treat cancer.

Description

Pharmaceutical compositions for the treatment of cancer

[0001] The present invention relates to a pharmaceutical composition for the treatment of cancer, which contains a substance that suppresses the expression of C11orf97.

[0002] Ferriclome is a cyclic protein of microorganism origin called a siderophore that possesses metal chelating ability. Ferriclome induces cell death in cancer cells such as colorectal cancer cells, gastric cancer cells, pancreatic cancer cells, and esophageal cancer cells, and exhibits antitumor effects in the mouse Xenograph model, while not affecting the proliferation of non-tumor cells. Therefore, it is expected to be used as an antitumor agent (for example, Non-Patent Documents 1-4, Patent Document 1).

[0003] While elucidating the mechanism of action of ferricome is expected to contribute to the development of new cancer treatments, the details of ferricome's target molecules and the activation of cell death signals mediated by these molecules remain unclear.

[0004] Konishi H, Fujiya M, Tanaka H, Ueno N, Moriichi K, Sasajima J, Ikuta K, Akutsu H, Tanabe H, Kohgo Y. Probiotic-derived ferrichrome inhibits colon cancer progression via JNK-mediated apoptosis. Nat Commun. 2016 Aug 10;7:12365. doi: 10.1038 / ncomms12365.Ijiri M, Fujiya M, Konishi H, Tanaka H, Ueno N, Kashima S, Moriichi K, Sasajima J, Ikuta K, Okumura T. Ferrichrome identified from Lactobacillus casei ATCC334 induces apoptosis through its iron-binding site in gastric cancer cells. Tumour Biol. 2017 Jun;39(6):1010428317711311. doi: 10.1177 / 1010428317711311.Kita A, Fujiya M, Konishi H, Tanaka H, Kashima S, Iwama T, Ijiri M, Murakami Y, Takauji S, Goto T, Sakatani A, Ando K, Ueno N, Ogawa N, Okumura T. Probiotic-derived ferrichrome inhibits the growth of refractory pancreatic cancer cells. Int J Oncol. 2020 Sep;57(3):721-732. doi: 10.3892 / ijo.2020.5096. Epub 2020 Jul 8.Kunogi T, Konishi H, Sakatani A, Moriichi K, Yamamura C, Yamamoto K, Kashima S, Ando K, Ueno N, Tanaka H, ​​Okumura T, Fujiya M. Probiotic-derived ferrichrome induces DDIT3-mediated antitumor effects in esophageal cancer cells. Heliyon. 2024 Mar 15;10(6):e28070. doi: 10.1016 / j.heliyon.2024.e28070.

[0005] WO2017 / 126626

[0006] This invention provides a novel treatment method for cancer.

[0007] The inventors have found that ferrichrome binds to C11orf97, and that nucleic acids that suppress the expression of C11orf97 have an antitumor effect.

[0008] This disclosure provides the following inventions: 1. A pharmaceutical composition for the treatment of cancer, comprising a nucleic acid that suppresses the expression of C11orf97 (Chromosome 11 Open Reading Frame 97). 2. The composition according to claim 1, wherein the nucleic acid that suppresses the expression of C11orf97 is an RNAi-inducing nucleic acid or an antisense nucleic acid for C11orf97. 3. The composition according to claim 2, wherein the RNAi-inducing nucleic acid comprises a nucleotide sequence complementary to a subsequence sequence of the nucleotide sequence shown in SEQ ID NO: 1, the length of which is 15 to 30 nucleotides. 4. The composition according to claim 2 or 3, wherein the RNAi-inducing nucleic acid is a nucleic acid comprising a double-stranded portion consisting of an oligonucleotide containing a nucleotide sequence shown in any of SEQ ID NOs: 2 to 5 and a complementary oligonucleotide. 5. The composition according to claim 4, wherein one or both of the oligonucleotides and the complementary oligonucleotide have an overhang of 1 to 4 nucleotides at their 3' ends. 6. A composition according to any one of claims 1 to 5, wherein at least one nucleotide in the nucleic acid is chemically modified. Claim 7. A pharmaceutical composition according to any one of claims 1 to 6, further comprising at least one other anticancer agent. Claim 8. A pharmaceutical composition according to any one of claims 1 to 7, for the treatment of a patient who is receiving treatment with at least one other anticancer agent, or who is scheduled to receive treatment with at least one other anticancer agent. Claim 9. A pharmaceutical composition according to claim 7 or 8, wherein at least one other anticancer agent is selected from the group consisting of fluorouracil, cisplatin, oxaliplatin, and irinotecan. Claim 10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the cancer is colorectal cancer, pancreatic cancer, or esophageal cancer. Claim 11. A kit for the treatment of cancer, comprising a nucleic acid that suppresses the expression of C11orf97 and at least one other anticancer agent. Claim 12. The kit according to claim 11, wherein at least one other anticancer agent is selected from the group consisting of fluorouracil, cisplatin, oxaliplatin, and irinotecan. Claim 13. The kit according to claim 11 or 12, wherein the cancer is colorectal cancer, pancreatic cancer, or esophageal cancer.

[0009] According to the present invention, cancer treatment becomes possible.

[0010] Figure 1 is a graph showing the cell proliferation of colorectal cancer cell lines treated with two types of siRNA against C11orf97 [siRNA#1 (hereinafter referred to as siC11orf97#1, siC11orf97-1, or #1), siRNA#2 (hereinafter referred to as siC11orf97#2, siC11orf97-2, or #2)] and random sequence RNA (hereinafter referred to as Scramble or Control). Figure 2 is a graph showing the C11orf97 mRNA expression levels in colorectal cancer cell lines treated with siRNA#1, siRNA#2, and random sequence RNA. Figure 3 is a photograph showing the appearance of the tumor transplant site in tumor-bearing mice administered with siRNA#1, siRNA#2, and random sequence RNA. Figure 4 is a graph showing the change in tumor volume in tumor-bearing mice treated with siRNA#1, siRNA#2, and random sequence RNA. Figure 5 is a graph showing the cell proliferation of various cancer cells treated with three types of siRNA against C11orf97 [siRNA#1, siRNA#2, siRNA#3 (indicated as #3 in the figure)] and random sequence RNA. Figure 6 is a graph showing the cell proliferation and C11orf97 mRNA expression levels of colorectal cancer cell lines treated with siRNA against C11orf97 [siRNA#4 (indicated as siC11orf97#4 in the figure)] and random sequence RNA. Figure 7 is a graph showing the change in tumor volume in tumor-bearing mice after co-administration of 5-FU and siRNA#2 (indicated as siC11orf97 in Figures 7-11) or random sequence RNA. Figure 8 is a graph showing the tumor mass weight of tumor-bearing mice co-administered with 5-FU and siRNA#2 or random sequence RNA. Figure 9 is a representative immunohistochemical staining image of tumor tissue from tumor-bearing mice co-administered with 5-FU and siRNA#2 or random sequence RNA. Figure 10 is a graph showing the number of cleared PARP-positive cells in tumor tissue from tumor-bearing mice treated with 5-FU and siRNA#2 or random sequence RNA. Figure 11 shows scored data regarding synergistic effects, obtained by analyzing the cell viability of colorectal cancer cell lines treated with siRNA#2 and 5-fluorouracil, cisplatin, oxaliplatin, or irinotecan using HSA Synergy Score software.Figure 12 shows a schematic diagram (A) illustrating the reaction of complex formation and biotinylation between compound 1, a ferrichrome derivative, and a protein that interacts with it (Target protein in the figure), as well as a graph (B) showing cell proliferation of colorectal cancer cell lines treated with ferrichrome, compounds 1 and 2. Figure 13 is an immunoblot showing complex formation between compound 1 and FLAG-added C11orf97.

[0011] [Definitions] The following descriptions may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges represented using "~" or "-" mean ranges that include the numbers at both ends as the upper and lower limits, respectively, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be combined in any way. All numbers include numbers with one less digit than the number that can be rounded to that number. For example, "1.0" includes numbers that can be rounded to that number, i.e., numbers between 0.95 and less than 1.05.

[0012] In this disclosure, the term “oligonucleotide” refers to an oligomer or polymer composed of nucleotides as monomers and can be used interchangeably with the terms “nucleic acid” and “polynucleotide.” Examples of oligonucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and chimeric nucleic acids that contain both deoxyribonucleotides and ribonucleotides as constituent units.

[0013] The terms "region" and "part" in relation to oligonucleotides are interchangeable and both refer to a single nucleotide or a sequence of nucleotides contained within an oligonucleotide.

[0014] In this disclosure, the term "complementarity" refers to the ability of two nucleotides to pair to form Watson-Crick base pairs (A-T base pairs, G-C base pairs, A-U base pairs). This term can be used for individual nucleotides or for nucleotide sequences. The percentage of complementarity between two nucleotide sequences is the proportion (%) of nucleotides that formed Watson-Crick base pairs to the total nucleotides in the nucleotide sequence, and 100% complementarity means that the two nucleotide sequences are complementary. In determining complementarity, chemically modified nucleotides are considered identical to unmodified nucleotides as long as they retain the ability to form Watson-Crick base pairs.

[0015] Nucleotide sequence identity refers to the percentage of identical nucleotides relative to all overlapping nucleotides in an optimal alignment calculated using an algorithm known in the art (preferably, the algorithm may consider introducing gaps into one or both sequences for optimal alignment). Identity can be calculated, for example, by aligning two nucleotide sequences using NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) with general settings (e.g., E-value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3, etc.).

[0016] [Nucleic acid that suppresses the expression of C11orf97] C11orf97 (Chromosome 11 Open Reading Frame 97; NCBI Gene ID: 643037) is a gene located in the region from 94512461 to 94532123 of human chromosome 11 (NCBI Reference Sequence: NC_000011.10, GRCh38.p14), and codes for a protein of unknown function. The nucleotide sequence of C11orf97 mRNA, the transcript of C11orf97, is registered as NCBI Reference Sequence: NM_001190462.2 (Sequence ID 1), and the amino acid sequence of the C11orf97 protein is registered as NCBI Reference Sequence: NP_001177391.1 (searched November 12, 2024).

[0017] In this disclosure, nucleic acids that suppress the expression of C11orf97 (hereinafter also referred to as expression-suppressing nucleic acids) mean nucleic acids that have the ability to suppress the expression of C11orf97, and include nucleic acids that can suppress the transcription of C11orf97, nucleic acids that can degrade C11orf97 mRNA or its precursor, and nucleic acids that can suppress protein translation from C11orf97 mRNA. Examples of expression-suppressing nucleic acids include RNAi-inducing nucleic acids and antisense nucleic acids.

[0018] In this disclosure, RNAi-inducing nucleic acid means a nucleic acid that, when introduced into a cell, has the ability to induce RNA interference with C11orf97 mRNA. In this disclosure, the target sequence of the RNAi-inducing nucleic acid is a subsequence of the nucleotide sequence of C11orf97 mRNA (e.g., the nucleotide sequence shown in SEQ ID NO: 1). The RNAi-inducing nucleic acid may include, or consist of, a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity with a nucleotide sequence complementary to the subsequence of the nucleotide sequence of C11orf97 mRNA (e.g., the nucleotide sequence shown in SEQ ID NO: 1), or a nucleotide sequence having 100% sequence identity (i.e., a nucleotide sequence complementary to the target sequence). The length of the target sequence of the subsequence may be 10-30 nucleotides, 13-30 nucleotides, 13-25 nucleotides, 15-30 nucleotides, 15-25 nucleotides, 15-23 nucleotides, 17-25 nucleotides, 17-23 nucleotides, 19-23 nucleotides, or 19-21 nucleotides. The length of the RNAi-inducing nucleic acid may be, for example, 15-30 nucleotides, 16-30 nucleotides, 17-30 nucleotides, 17-25 nucleotides, 19-25 nucleotides, 19-23 nucleotides, 21-25 nucleotides, or 21-23 nucleotides. Examples of RNAi-inducing nucleic acids include RNA such as siRNA and miRNA.

[0019] siRNA is a double-stranded RNA comprising a guide strand (antisense strand) complementary to the target sequence contained in the target mRNA, and a passenger strand (sense strand) complementary to the guide strand. In this disclosure, the target sequence of siRNA is a subsequence of the nucleotide sequence of C11orf97 mRNA (for example, the nucleotide sequence shown in Sequence ID No. 1). The guide strand of siRNA may contain, or consist of, a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% sequence identity with the nucleotide sequence complementary to the target sequence, and particularly preferably a nucleotide sequence having 100% sequence identity (i.e., a nucleotide sequence complementary to the target sequence). The length of the subsequence that becomes the target sequence is as described above with respect to the subsequence of RNAi-inducing nucleic acid, and is, for example, 15 to 30 nucleotides, preferably 17 to 25 nucleotides, more preferably 19 to 23 nucleotides, and even more preferably 19 to 21 nucleotides. The aforementioned subsequence can be selected, for example, from the nucleotide sequence shown in SEQ ID NO: 1, preferably from the range of nucleotides 260 to 672 of the nucleotide sequence shown in SEQ ID NO: 1. The length of the guide strand is as described above with respect to the length of the RNAi-inducing nucleic acid, and is, for example, 15 to 30 nucleotides, preferably 17 to 25 nucleotides, more preferably 19 to 25 nucleotides or 19 to 23 nucleotides, and even more preferably 21 to 25 nucleotides or 21 to 23 nucleotides.

[0020] In this disclosure, the nucleotide sequence of the siRNA passenger strand can be designed to include, or consist of, a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% sequence identity with a nucleotide sequence complementary to the guide strand, or particularly preferably include a nucleotide sequence having 100% sequence identity (i.e., a nucleotide sequence complementary to the guide strand), or consist of such a nucleotide sequence. The length of the passenger strand may be the same as or different from that of the guide strand. The length of the passenger strand is, for example, 15 to 30 nucleotides, preferably 17 to 25 nucleotides, more preferably 19 to 25 nucleotides or 19 to 23 nucleotides, and even more preferably 21 to 25 nucleotides or 21 to 23 nucleotides.

[0021] Examples of siRNAs that can be used in this disclosure include nucleic acids comprising a double-stranded portion consisting of an oligonucleotide (guide chain) having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and particularly preferably 100% sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 2 to 5, and an oligonucleotide (passenger chain) having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and particularly preferably 100% sequence identity with a nucleotide sequence complementary to the guide chain (i.e., a nucleotide sequence complementary to the guide chain), and double-stranded nucleic acids comprising the guide chain and the passenger chain.

[0022] The guide strand and the passenger strand may each independently have an additional sequence at their 3' end. The length of the additional sequence is, for example, 1 to 6 nucleotides, preferably 2 to 4 nucleotides. Examples of additional sequences include ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', uuuuu-3', etc.

[0023] The ends of the siRNA may be overhanging ends with an overhang (e.g., 1 to 4 nucleotides, preferably 2 to 3 nucleotides) attached to the 3' end of either or both strands, or they may be blunt ends. The siRNA may also form shRNA, which is a single-stranded oligonucleotide in which one end of the guide strand and one end of the passenger strand are linked via a linker (e.g., 5 to 25 nucleotides) capable of forming a loop structure, or it may form dumbbell-shaped siRNA, which is a circular oligonucleotide in which both ends of the guide strand and passenger strand are linked.

[0024] Methods for designing siRNA are known to those skilled in the art, and various design software or algorithms can be used to select a suitable siRNA nucleotide sequence from the C11orf97 mRNA nucleotide sequence.

[0025] Examples of siRNAs that can be used in this disclosure include nucleic acids comprising a double-stranded portion consisting of an oligonucleotide (guide chain) having at least 80% sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 2 to 5, and an oligonucleotide (passenger chain) complementary to the guide chain. Here, the sequence identity is preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and particularly preferably 100%.

[0026] miRNAs are single-stranded non-coding RNAs with a length of 16 to 30 nucleotides, mostly around 21 to 25 nucleotides, that have the ability to bind to the 3' UTR of a target mRNA and suppress translation. In this disclosure, miRNA also includes miRNA precursors (prim-miRNA, pre-miRNA). Methods for designing miRNAs are known to those skilled in the art, and various design software or algorithms can be used to select a suitable miRNA nucleotide sequence from the nucleotide sequence of C11orf97 mRNA. Examples of miRNAs that can be used in this disclosure include hsa-miR-5004-3p, hsa-miR-4284, hsa-miR-1248, hsa-miR-6868-3p, hsa-miR-4724-5p, hsa-miR-4678, hsa-miR-4291, hsa-miR-5571-5p, hsa-miR-922, hsa-miR-4438, and the like.

[0027] An antisense nucleic acid is a single-stranded oligonucleotide complementary to the transcript of the target gene (target mRNA or its precursor, the early transcript). In this disclosure, the antisense nucleic acid may be DNA, RNA, a DNA-RNA chimeric nucleic acid, or it may form a double helix with a complementary oligonucleotide. Furthermore, the antisense nucleic acid may bind to C11orf97 mRNA or its precursor and induce degradation by RNaseH (RNA degradation type), bind to the splice sequence of the C11orf97 mRNA precursor and regulate splicing (splice-regulating type), or bind to the translation initiation site or ribosome binding site of C11orf97 mRNA and inhibit translation (steric block type). In addition, the antisense nucleic acid may bind to C11orf97 on a chromosome in the form of double-stranded DNA to form a triple helix and inhibit transcription to RNA. The length of the antisense nucleic acid is, for example, 10 to 40 nucleotides, preferably 15 to 30 nucleotides.

[0028] Methods for designing miRNAs and antisense nucleic acids are known to those skilled in the art, and appropriate nucleotide sequences of miRNAs and antisense nucleic acids can be selected from the nucleotide sequence of C11orf97 mRNA or its precursor using various design software or algorithms.

[0029] In addition to natural deoxyribonucleotides and ribonucleotides, the expression-suppressing nucleic acid may further contain chemically modified nucleotides. Examples of chemically modified nucleotides include nucleotides in which the phosphate group is replaced with a chemically modified phosphate group such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, etc.; nucleotides in which the hydroxyl group at the 2'-position of the sugar (ribose) moiety is replaced with -OR (R is, for example, CH ３ (2'-O-Me), CH ２ CH ２ OCH ３ (2'-MOE), CH ２ CH ２ NHC(NH)NH ２ 、CH ２ CONHCH ３ 、CH ２ CH ２ CN, etc.); nucleotides in which the hydroxyl group at the 2'-position of the sugar (ribose) moiety is replaced with hydrogen (i.e., deoxyribonucleotides); artificial nucleic acids in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position of the sugar (ribose) moiety are crosslinked [e.g., LNA (Locked Nucleic Acid) in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position are crosslinked via a methylene group, ENA crosslinked via an ethylene group, BNA (Bridged Nucleic Acid) crosslinked via -CH ２ OCH ２ -, BNA crosslinked via -NR-CH ＣＯＣ - (R is methyl or a hydrogen atom), cMOE crosslinked via -CH ２ - (R is methyl or a hydrogen atom), BNA crosslinked via -CH ＮＣ 、-CH ２ (OCH ３ ), cMOE crosslinked via -CH ２ (CH ３cEt crosslinked via -), AmNA crosslinked via amide, and scpBNA crosslinked via methylene and having cyclopropane formed at the 6'-position, etc.]; nucleotides having a methyl group or a cationic functional group introduced at the 5-position of the pyrimidine base, nucleotides having the carbonyl group at the 2-position of the pyrimidine base substituted with a thiocarbonyl group, and the like.

[0030] The expression-suppressing nucleic acid can be artificially synthesized using gene recombination technology or chemical synthesis technology. Methods for gene recombination, chemical synthesis, synthesis of chemically modified nucleotides, and synthesis of nucleic acids containing them are well known to those skilled in the art.

[0031] Also, an expression vector of an expression-suppressing nucleic acid against C11orf97 is also included in substances having the ability to suppress the expression of C11orf97. The term "expression vector" refers to a vector equipped with a mechanism for expressing an incorporated polynucleotide in a cell. In an expression vector, the polynucleotide to be expressed is operably linked to a control sequence such as a promoter. The term "operably linked" means that a control sequence such as a promoter is placed in a position close to the polynucleotide to such an extent that it can affect the expression of the polynucleotide. For example, when a polynucleotide is operably linked to a promoter, it means that the polynucleotide is linked so that it can be expressed under the control of the promoter. Examples of expression vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, Sendai viruses, etc., and non-viral vectors such as plasmid vectors.

[0032] [Pharmaceutical composition] The present disclosure provides a pharmaceutical composition for the treatment of cancer, containing an expression-suppressing nucleic acid against C11orf97. The pharmaceutical composition of the present disclosure contains an effective amount of an expression-suppressing substance against C11orf97 for the treatment of cancer. The effective amount of the expression-suppressing substance against C11orf97 can be appropriately determined according to factors such as the usage method, age, sex, body weight, type of cancer, severity, and other factors of the subject.

[0033] As used herein, the term "treatment" encompasses all types of medically acceptable therapeutic interventions aimed at curing a disease or condition, achieving temporary remission, etc. Thus, the treatment of cancer encompasses various medically acceptable interventions for various purposes, including delaying or halting the progression of cancer, shrinking or disappearing of lesions, preventing metastasis or recurrence, etc.

[0034] Examples of cancers that can be treated with the pharmaceutical composition of the present disclosure include epithelial tumors (e.g., lung cancer, colorectal cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, cervical cancer, ovarian cancer, esophageal cancer, kidney cancer, bladder cancer), mesenchymal tumors (e.g., osteosarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma), hematopoietic tumors (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome), nervous system tumors (e.g., glioma, medulloblastoma, neuroblastoma, astrocytoma, meningioma, pituitary tumor, spinal cord tumor, schwannoma of the auditory nerve), pigment cell tumors (e.g., malignant melanoma, retinoblastoma), and mesotheliomas (e.g., pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma). The cancer is preferably colorectal cancer, pancreatic cancer, or esophageal cancer.

[0035] The expression-suppressing nucleic acid against C11orf97 has anti-cancer effects such as growth inhibition against cancer cells, induction of apoptosis against cancer cells, and reduction of tumor volume. This anti-cancer effect can be synergistically enhanced by combining it with other anti-cancer agents. The present disclosure provides a pharmaceutical for the treatment of cancer (hereinafter also referred to as a combination pharmaceutical) comprising a combination of an expression-suppressing nucleic acid against C11orf97 and at least one other anti-cancer agent.

[0036] The combination drug comprises a nucleic acid that inhibits the expression of C11orf97 as described above, and at least one other anticancer agent. The combination drug may also be in the form of a formulation containing both the nucleic acid that inhibits the expression of C11orf97 and at least one other anticancer agent. The combination drug may be a pharmaceutical composition for the treatment of cancer, containing the nucleic acid that inhibits the expression of C11orf97 and at least one other anticancer agent.

[0037] The combination drug may also be in the form of a combination of at least two formulations, each separately containing a nucleic acid that inhibits the expression of C11orf97 and at least one other anticancer agent. The combination drug may be a kit for the treatment of cancer comprising a pharmaceutical composition containing a nucleic acid that inhibits the expression of C11orf97 and at least one other anticancer agent. The nucleic acid that inhibits the expression of C11orf97 and at least one other anticancer agent contained in the kit may be administered together or separately, simultaneously or sequentially, to a subject requiring treatment for cancer, and there are no particular restrictions on the order and timing of administration of each formulation.

[0038] C11orf97 inhibitory nucleic acids intended for use in combination pharmaceuticals are also included in this disclosure. This disclosure provides a pharmaceutical composition for the treatment of cancer comprising a C11orf97 inhibitory nucleic acid, for the treatment of a subject receiving or scheduled to receive treatment with at least one other anticancer agent. In one embodiment, this disclosure also provides a pharmaceutical composition for the treatment of cancer comprising a C11orf97 inhibitory nucleic acid, wherein the treatment comprises the administration of at least one other anticancer agent.

[0039] Examples of other anticancer agents in this disclosure include antimetabolites, such as folic acid antimetabolites like methotrexate, pemetrexed, and pralatrexate; purine antimetabolites like mercaptopurine, thioguanine, fludarabine, cladribine, and clofarabine; cytarabine, fluorouracil (5-FU); preparations containing tegafur [UFT (tegafur / uracil), S-1 (tegafur / gimeracil / oteracil)]; capecitabine; gemcitabine. Pyrimidine antimetabolites such as trifluridine / tipiracil; alkylating agents, such as nitrogen mustards such as cyclophosphamide, ifosfamide, melphalan, chlorambucil, bendamustine, busulfan, procarbazine, thiotepa; nitrosoureas such as ranimustine, nimustine, carmustine, lomustine; triazenes such as dacarbazine, temozolomide; antitumor antibiotics, such as doxorubicin, epirubicin, Anthracycline antibiotics such as pirarubicin, acralubicin, amrubicin, and idarubicin; other antitumor antibiotics such as bleomycin, peplomycin, dactinomycin (actinomycin D), and mitomycin C; platinum-based drugs, such as cisplatin, carboplatin, oxaliplatin, and nedaplatin; topoisomerase inhibitors, such as topoisomerase I inhibitors like irinotecan and topotecan; etoposide, teniposide, and sobriety. Examples of anticancer agents include topoisomerase II inhibitors such as zoxane and mitoxantrone; microtubule inhibitors, such as vinca alkaloids like vincristine, vinblastine, vindesine, and vinorelbine; taxanes like paclitaxel, docetaxel, and cabazitaxel; and other microtubule inhibitors such as eribulin; molecularly targeted drugs such as cetuximab and bevacizumab; and immune checkpoint inhibitors such as nivolumab, atezolizumab, and ipilibumab. Other anticancer agents can preferably be selected from the group consisting of fluorouracil, cisplatin, oxaliplatin, and irinotecan.

[0040] The pharmaceutical compositions of this disclosure may include, in addition to a nucleic acid that inhibits the expression of C11orf97 and optionally at least one other anticancer agent, other pharmaceutically acceptable components, such as pharmaceutically acceptable excipients (buffers, stabilizers, preservatives, excipients, etc.), pharmaceutically acceptable media (water, saline, phosphate-buffered saline (PBS), etc.), and other agents for the treatment of cancer. The pharmaceutically acceptable excipients and media are well known to those skilled in the art and can be appropriately selected and used by those skilled in the art within the scope of their ordinary capacity.

[0041] Pharmaceutical compositions are also preferably administered using a drug delivery system (DDS). Materials used in DDSs include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses; lipid membrane structures such as liposomes or micelles (e.g., cationic lipid membrane structures), cationic lipids (e.g., lipofectamines), cationic polymers (e.g., polyethyleneimine), and nonviral carriers such as magnetic microparticles. This disclosure also provides pharmaceutical compositions in which an inhibitory nucleic acid is supported or encapsulated on such a DDS material, and inhibitory nucleic acids supported or encapsulated on such a DDS material.

[0042] The dosage form of the pharmaceutical composition is arbitrary and can be appropriately selected depending on the target site, type of cancer, etc. The dosage form of the pharmaceutical composition is preferably a parenteral preparation, such as an injection, intravenous drip, or topical preparation. The route of administration of the pharmaceutical composition is not particularly limited, but in the case of a parenteral preparation, examples include intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and local administration to the target site. The method of administration of the pharmaceutical composition may be continuous administration or rapid administration, and may include hydrodynamic administration.

[0043] The pharmaceutical compositions of this disclosure are administered to subjects suffering from cancer. These subjects include humans and non-human animals, which include mammals such as rodents (including mice, rats, hamsters, and guinea pigs), primates (including chimpanzees and rhesus monkeys), livestock (including pigs, cattle, goats, horses, and sheep), and companion animals (including dogs and cats). The pharmaceutical compositions are preferably administered to humans.

[0044] This disclosure provides a method for treating cancer, comprising administering to a subject with cancer the above-mentioned pharmaceutical composition containing a nucleic acid that suppresses the expression of C11orf97, or the above-mentioned pharmaceutical composition containing a nucleic acid that suppresses the expression of C11orf97 and at least one other anticancer agent. This disclosure provides a method for treating cancer, comprising administering to a subject with cancer the above-mentioned pharmaceutical composition containing a nucleic acid that suppresses the expression of C11orf97 and at least one other anticancer agent together or separately, simultaneously or sequentially. This disclosure provides a method for treating cancer, comprising administering to a subject who is receiving treatment with at least one other anticancer agent, or who is scheduled to receive treatment with at least one other anticancer agent, the above-mentioned pharmaceutical composition containing a nucleic acid that suppresses the expression of C11orf97.

[0045] This disclosure provides the use of C11orf97-repressor nucleic acids in the manufacture of pharmaceutical compositions for the treatment of cancer, the use of C11orf97-repressor nucleic acids and at least one other anticancer agent in the manufacture of pharmaceutical compositions for the treatment of cancer, the use of C11orf97-repressor nucleic acids for the treatment of cancer, and the use of C11orf97-repressor nucleic acids and at least one other anticancer agent for the treatment of cancer.

[0046] The present invention will be described in more detail by the following examples, but the present invention is not limited thereto.

[0047] [Materials and Methods] Cell lines and their cultured colorectal cancer cell lines HCT116 (ATCC) and SW620 (ECACC), the pancreatic cancer cell line Suit2 (JCRB), the esophageal cancer cell line OE33 (DS Pharma), and the human intestinal epithelial cell line HCEC-1CT (Eversyte) were used for evaluation.

[0048] HCT116 was cultured in McCoy's 5A (ThermoFisher Scientific), Suit2 in DMEM (Fujifilm Wako Pure Chemical Corporation), and SW620 and OE33 in RPMI1640 (Fujifilm Wako Pure Chemical Corporation), each cultured in a solution containing 10% FBS (Sigma-Aldrich), 50 U / mL penicillin (Fujifilm Wako Pure Chemical Corporation), and 50 μg / mL streptomycin (Fujifilm Wako Pure Chemical Corporation). HCEC-1CT is a liquid culture medium prepared by mixing DMEM (Sigma-Aldrich) Medium 199 Earle's (Sigma-Aldrich) in a 4:1 ratio with 2% Cosmic Calf Serum (cytiva) and 1× GlutaMAX (ThermoFisher) The cells were cultured using a solution containing Scientific, 20 ng / mL EGF (Sigma-Aldrich), 10 μg / mL insulin (Sigma-Aldrich), 2 μg / mL apotransferrin (Sigma-Aldrich), 5 nM sodium selenite (Sigma-Aldrich), 1 μg / mL hydrocortisone (Sigma-Aldrich), 50 U / mL penicillin (Fujifilm Wako Pure Chemical Industries, Ltd.), and 50 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0049] - Cancer-bearing model mouse: 2 x 10⁶ cells subcutaneously on the back of 6-8 week old male BALB / c nude mice (Jackson Laboratory, Inc.) ６ HCT116 cells were transplanted by injecting a suspension solution containing 50 μL of Matrigel (Becton Dickinson) in D-PBS(-).

[0050] Four types of siRNAs were used against siRNA C11orf97: [siRNA#1 (s55192, ThermoFisher Scientific), siRNA#2 (Hokkaido System Science), siRNA#3 (s227049, ThermoFisher Scientific)], siRNA#4 (Hokkaido System Science), and a random sequence RNA (Scramble RNA, Hokkaido System Science) as a control. All were purchased after annealing. The nucleotide sequences of each are shown in Table 1. In the table, the target sequence indicates the binding site of each siRNA antisense strand in the C11orf97 mRNA (NM_001190462.2) shown in Sequence ID No. 1. Both the sense strands and antisense strands of siRNA#1 to siRNA#4 have a 2-nucleotide overhang at the 3' end.

[0051] [Gene Transfer] Gene transfer into HCT116, Suit2, OE33, and HCEC-1CT was performed using the Reverse Transfer method with Lipofectamine RNAiMAX (ThermoFisher Scientific). Gene transfer into SW620 was performed using the Neon electroporation system (ThermoFisher Scientific). Gene transfer into tumor-bearing model mice was performed using the GENOMONE-Si transfer kit (Ishihara Sangyo Co., Ltd.) for siRNA#1 and random sequence RNA, and using AteloGene® Local Use "Quick Gelation" (Koken Co., Ltd.) for siRNA#2 and random sequence RNA.

[0052] [Measurement of antitumor activity by SRB assay] 0.75 × 10⁶ in a 96-well plate (Corning) ４Test cells were seeded to a density of cells per well (n=3 unless otherwise specified) and cultured for 24, 48, and 72 hours. After incubation, the plates were collected and fixed in 5% trichloroacetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.). After washing four times with pure water, the plates were dried at room temperature. 100 μL of 0.057 wt% SRB (Sigma-Aldrich) / 0.1% acetic acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well to stain the cells. The plates were then washed four times with 0.1% acetic acid and dried. The cell density at each incubation time was measured by measuring the OD at 510 nm when the stained cells were dissolved in 10 mM Tris buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) using a plate reader EnSpire (Perkin Elmer).

[0053] [Immunohistochemical Staining] Tumor tissue recovered from tumor-bearing model mice was fixed with 10% neutral formalin buffer (Muto Chemical Co., Ltd.) to prepare paraffin-embedded blocks. The blocks were sectioned to 4 μm and fixed onto immunohistochemical slides (Immunocort YS, Muto Chemical Co., Ltd.). Subsequently, deparaffinization and peroxidase blocking were performed, followed by antigen retrieval by autoclaving with Dako Target Retrievable Solution (DAKO). The slides were blocked in 2.5% Horse Serum for 20 minutes, and then reacted overnight at 4°C with anti-cleaved PARP antibody (Cell Signaling Technologies) diluted 1 / 100 in Horse Serum. Subsequently, the cells were washed three times for 3 minutes with 0.05% PBS-T, and reacted with Impress Horse Anti-Rabbit IgG Polymer Kit, Peroxidase (VECTOR) at room temperature for 30 minutes. After developing the color with Impact DAB Peroxidase Substrate Kit (VECTOR), the cells were stained with hematoxylin, mounted, and observed under a light microscope. The number of cleared PARP-positive cells was calculated using ImageJ.

[0054] [Statistical Analysis] Statistical analysis was performed using GraphPad Prism 6.02. One-way ANOVA was conducted, and the Post-hoc test was performed using Tukey's multiple comparison test. A p-value of < 0.05 was considered statistical significance. Data are shown as mean ± S.D.

[0055] [Example 1] Human intestinal epithelial cell line (HCEC-1CT) and colorectal cancer cell lines (HCT116, SW620) were used as test cells. siRNA (siRNA #1, siRNA #2) or random sequence RNA targeting C11orf97 was introduced, and antitumor activity was measured by SRB assay. Specifically, 200 μL of OptiMEM (ThermoFisher Scientific) warmed to 37°C was mixed with 2 μL of 10 μM siRNA #1, #2 or random sequence RNA and 2 μL of Lipofectamine RNAiMAX (ThermoFisher Scientific), and 20 μL of each mixture was added to 96-well plates (Corning). After incubation at room temperature for 10-20 minutes, 0.75 × 10⁶ was measured. ４ Test cells were added one by one, and plates were collected after 24, 48, and 72 hours to perform SRB assays. Gene expression suppression efficiency was quantified using the QuantStudio 3D digital PCR system (ThermoFisher). Specifically, 200 μL of OptiMEM (ThermoFisher Scientific) warmed to 37°C was mixed with 2 μL of 10 μM siRNA #1, #2, or random sequence RNA and 2 μL of Lipofectamine RNAiMAX (ThermoFisher Scientific), and 200 μL of this mixture was added to each 12-well plate (Corning). After incubation at room temperature for 10–20 minutes, 0.75 × 10⁶ was obtained. ５Test cells were added one by one, and after 24 hours, the plates were collected. Total RNA was recovered using the RNA Easy Mini kit (Quagen). The recovered 200 ng Total RNA was used to synthesize cDNA using the High-capacity cDNA Reverse Transcription kit (ThermoFisher Scientific), and the cDNA was quantified using Taqman Gene Expression Assays (Assay ID: Hs01375735_m1) with the QuantStudio 3D digital PCR system.

[0056] The results of the SRB assay are shown in Figure 1, and the gene expression levels of C11orf97 are shown in Figure 2. In all colorectal cancer cell lines, siRNA against C11orf97 suppressed C11orf97 expression and demonstrated strong antitumor activity. However, no significant inhibitory effect on cell proliferation was observed against HCEC-1CT.

[0057] [Example 2] 2 x 10 ６ In BALB / c nudes transplanted subcutaneously into the back of cells containing HCT116 cells, 100 pmol of siRNA #1 (n=5) or random sequence RNA (n=5) was administered daily starting the day after transplantation, or 600 pmol of siRNA #2 (n=5) or random sequence RNA (n=5) was administered directly to the transplantation site on the day after transplantation (D1) and on the 7th day after transplantation (D7). The growth of tumor masses at each transplantation site was observed for 10 days.

[0058] Figure 3 shows the appearance of the mouse 10 days after transplantation, and Figure 4 shows the change in tumor mass volume. As shown in Figures 3 and 4, siRNA against C11orf97 significantly inhibited tumor mass growth by transplanted cells.

[0059] [Example 3] Colorectal cancer cell line HCT116, pancreatic cancer cell line suit2, and esophageal cancer cell line OE33 were used as test cells. siRNA #1, #2, #3, and random sequence RNA were introduced, and the antitumor activity of each was tested by SRB assay. Specifically, 200 μL of 37°C OptiMEM (ThermoFisher Scientific) was mixed with 2 μL of 10 μM siRNA #1, #2, #3, or random sequence RNA and 2 μL of Lipofectamine RNAiMAX (ThermoFisher Scientific), and 20 μL of each was added to a 96-well plate. After incubation at room temperature for 10-20 minutes, 0.75 × 10⁶ ４ Test cells were added to each plate individually, and plates were collected after 24, 48, and 72 hours, followed by an SRB assay. siRNA against C11orf97 showed significant antitumor activity against all test cells (Figure 5).

[0060] Similarly, the colorectal cancer cell line HCT116 was used as the test cell line, and siRNA#4 and random sequence RNA were introduced, and their respective antitumor activities were tested by SRB assay. siRNA#4 also showed excellent antitumor activity (Figure 6, top). When the gene expression level of C11orf97 was measured using the same method as in Example 1, suppression of expression by siRNA#4 was confirmed (Figure 6, bottom).

[0061] [Example 4] 2 x 10 ６ BALB / c nude mice, in which HCT116 cells were transplanted subcutaneously into the back, were directly injected with 600 pmol of siRNA #2 (n=10) or random sequence RNA (n=10) at the transplantation site on the day after transplantation (D1) and on the 7th day after transplantation (D7). Mice administered with siRNA #2 or random sequence RNA were randomly divided into two groups (n=5), and 5-fluorofuracil (5-FU) (n=5) or PBS (n=5) were administered intraperitoneally daily for 8 days (D1 to D8), starting on the day after transplantation (D1). 5-FU (Sigma) was administered in a suspension prepared so that 100 μg was contained in 100 μL of PBS, resulting in a dose of 5 mg / kg body weight. PBS was administered in a volume equivalent to the dose of the 5-FU suspension.

[0062] During the study period, changes in tumor mass volume were measured. Additionally, tumor masses were collected on day D8 and their weight was measured. Figure 7 shows the changes in tumor mass volume, and Figure 8 shows the weight of the tumor mass. As shown in Figures 7 and 8, siRNA against C11orf97 significantly inhibited tumor growth more effectively than 5-FU monotherapy. Furthermore, the inhibitory effect on tumor growth was enhanced when siRNA was used in combination with 5-FU.

[0063] Using the recovered tumor mass, the number of cleared PARP-positive cells in the tumor tissue was quantified by immunohistochemical staining. Representative immunohistochemical staining images are shown in Figure 9, and the quantification results for the number of cleared PARP-positive cells are shown in Figure 10. Cleared PARP is a poly-ADP-ribose polymerase cleaved by caspase and is used as a marker of apoptosis. As shown in Figures 9 and 10, siRNA against C11orf97 increased the number of cleared PARP-positive cells to a higher level than 5-FU alone. Furthermore, a more significant increase in the number of positive cells was observed when used in combination with 5-FU.

[0064] [Example 5] The colon cancer cell line HCT116 was used as the test cell line. siRNA#2 was treated with other anticancer agents (5-FU, cisplatin, oxaliplatin, irinotecan), and the antitumor activity was tested by SRB assay. Specifically, siRNA#2 was added to 200 μL of 37°C OptiMEM at concentrations of 0 nM, 0.16 nM, 0.8 nM, 4 nM, and 20 nM, mixed, and 20 μL was added to each of the 96-well plates. After incubation at room temperature for 10-20 minutes, 0.75 × 10⁶ was measured. ４Each cell group was added to the test cells and cultured overnight. Then, 5-FU was added at concentrations of 0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL; cisplatin (Nichi-Iko) at concentrations of 0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL; oxaliplatin (Nipro) at concentrations of 0 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL; and irinotecan (Nippon Kayaku) at concentrations of 0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL. After this, the cells were cultured for a further two days, and cell viability was measured by SRB assay. The obtained viability data were analyzed for the combined effect using SynergyFinder Plus software. In the analysis, an HSA Synergy Score of -10 or less was evaluated as an antagonistic effect, -10 to 10 as an additive effect, and 10 or more as a synergistic effect.

[0065] The scored data is shown in Figure 11. siRNA targeting C11orf97 showed an HSA Synergy Score of 10 or higher in combination with any of the anticancer drugs used. This indicates that the combination of siRNA and anticancer drugs exhibits a synergistic effect.

[0066] [Test Example] To evaluate the binding of ferrichrome to C11orf97, compound 1, a ferrichrome derivative having a photoreactive diazirine group and an alkyne group, and a control compound 2, in which three hydroxyl groups of the ferrichrome portion of compound 1 are substituted with hydrogen, were synthesized. Proteins that interact with compound 1 or 2 react with the carbene generated from the diazirine group under light irradiation, crosslinking with the compound to form a complex. When this complex is reacted with biotin having an azide group, the alkyne group of the complex binds to the azide group of biotin, and the complex is biotin-labeled, enabling analysis using avidin-biotin interaction (Figure 12A). The structures of compounds 1 and 2 are shown below.

[0067] SRB assays using SW620 showed that compound 1 had antitumor activity equivalent to ferrichrome, while compound 2 did not have antitumor activity, confirming that derivatization did not affect the antitumor activity of ferrichrome (Figure 12B).

[0068] Furthermore, HCT116 cells transfected with an expression vector for FLAG-tagged C11orf97 were cultured, and cell lysates were prepared. After incubation of the cell lysates with compound 1 and compound 2, a crosslinking reaction was carried out under light irradiation. After the reaction, an azide-PEG3-biotin complex was added to biotin-label the compounds, and the biotin-labeled compounds were separated and eluted from the reaction solution by a pull-down assay using streptavidin beads. Analysis of the eluate by immunoblotting with an anti-FLAG antibody confirmed the presence of a complex between compound 1 and FLAG-tagged C11orf97 (Figure 13), indicating that ferrichrome binds to C11orf97.

Claims

1. A pharmaceutical composition for the treatment of cancer, containing a nucleic acid that suppresses the expression of C11orf97 (Chromosome 11 Open Reading Frame 97).

2. The composition according to claim 1, wherein the nucleic acid that suppresses the expression of C11orf97 is an RNAi-inducing nucleic acid or an antisense nucleic acid for C11orf97.

3. The composition according to claim 2, wherein the RNAi-inducing nucleic acid comprises a nucleotide sequence complementary to a subsequence of the nucleotide sequence shown in SEQ ID NO: 1, and the length of the subsequence is 15 to 30 nucleotides.

4. The composition according to claim 2, wherein the RNAi-inducing nucleic acid is a nucleic acid comprising a double-stranded portion consisting of an oligonucleotide having a nucleotide sequence shown in any of SEQ ID NOs: 2 to 5 and a complementary oligonucleotide.

5. The composition according to claim 4, wherein one or both of the oligonucleotides and complementary oligonucleotides have an overhang of 1 to 4 nucleotides added to their 3' ends.

6. The composition according to claim 1, wherein at least one nucleotide in the nucleic acid is chemically modified.

7. A pharmaceutical composition according to any one of claims 1 to 6, for the treatment of a patient who is receiving treatment with at least one other anticancer drug, or a patient who is scheduled to receive treatment with at least one other anticancer drug.

8. The pharmaceutical composition according to claim 7, wherein at least one other anticancer agent is selected from the group consisting of fluorouracil, cisplatin, oxaliplatin, and irinotecan.

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