WO2017159835A1 - Agent thérapeutique pour un néoplasme de plasmocytes - Google Patents

Agent thérapeutique pour un néoplasme de plasmocytes Download PDF

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WO2017159835A1
WO2017159835A1 PCT/JP2017/010828 JP2017010828W WO2017159835A1 WO 2017159835 A1 WO2017159835 A1 WO 2017159835A1 JP 2017010828 W JP2017010828 W JP 2017010828W WO 2017159835 A1 WO2017159835 A1 WO 2017159835A1
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dpp
agent
pharmaceutical composition
activity
cells
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PCT/JP2017/010828
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Japanese (ja)
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佐藤　勉
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北海道公立大学法人 札幌医科大学
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Priority to JP2018506035A priority Critical patent/JP7292702B2/ja
Publication of WO2017159835A1 publication Critical patent/WO2017159835A1/fr
Priority to JP2021156273A priority patent/JP2022023063A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the present invention relates to a pharmaceutical composition for the treatment of plasma cell tumor comprising an agent that suppresses the activity of dipeptidyl peptidase-8, a method for treating a plasma cell tumor using the pharmaceutical composition, and the like.
  • Multiple myeloma is one of the typical plasma cell tumors, and develops when plasma cells, which are a type of lymphocyte differentiated from bone marrow-derived hematopoietic stem cells, become tumors and proliferate abnormally. Symptoms range from bone lesions and hypercalcemia caused by osteoclast activation, concomitant infections due to decreased immune function, anemia associated with decreased hematopoietic ability, and kidney damage associated with abnormal immunoglobulin production. Across.
  • anticancer agents such as melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone have been used in combination.
  • thalidomide has been confirmed to be effective in multiple myeloma, and new drugs that show high efficacy in multiple myeloma, such as lenalidomide and pomalidomide, which are derivatives of thalidomide, and bortezomib, a proteasome inhibitor, have been added one after another.
  • lenalidomide and pomalidomide which are derivatives of thalidomide, and bortezomib, a proteasome inhibitor
  • transplantation of hematopoietic stem cells can be mentioned as one of treatment options.
  • Dipeptidyl peptidase-4 (DPP-4) is a cell surface glycoprotein having a molecular weight of 110 kDa. It has a function as a serine protease and is known to be expressed in cells of various tissues including T lymphocytes, endothelial cells, epithelial cells and the like. DPP-4 is responsible for inactivation of incretin, an intestinal hormone, due to its serine protease activity, and inactivation of incretin is known to result in suppression of insulin secretion. Therefore, inhibitors of DPP-4 are used as therapeutic agents for treating type 2 diabetes by promoting insulin secretion by maintaining incretin activity and thereby controlling blood glucose levels.
  • DPP-4 is known to be highly expressed in lymphoid tumor cells.
  • suppression of CD26 / DPP-4 expression in the undifferentiated large cell lymphoma cell line Karpas 299 causes dephosphorylation of integrin ⁇ 1 and p38 mitogen activated protein kinase (MAPK), It is described that it loses the ability to adhere to extracellular matrices such as fibronectin and type I collagen, and also loses its tumorigenicity.
  • MAPK mitogen activated protein kinase
  • DPP-4 In addition to DPP-4, the DPP-4 family includes DPP-8 and DPP-9 that are structurally similar to DPP-4.
  • DPP-8 is known to localize in the cell, and due to the structural similarity with DPP-4, it is thought that it may have some function in T cell activation and immune function. However, its function has not been elucidated in practice.
  • the object of the present invention is a pharmaceutical composition for the treatment of plasma cell tumors, in particular multiple myeloma, comprising a DPP-8 inhibitor and optionally other plasma cell tumors , Said pharmaceutical composition, method for treating plasma cell tumor comprising administering said pharmaceutical composition, method for inducing apoptosis of myeloma cells, comprising contacting DPP-8 inhibitor with myeloma cells And so on.
  • the present invention relates to the following: [1] A pharmaceutical composition for treating a plasma cell tumor, comprising at least one agent that suppresses the activity of dipeptidyl peptidase (DPP) -8. [2] The pharmaceutical composition according to [1], wherein the agent that suppresses the activity of DPP-8 is a DPP-8 inhibitor. [3] The pharmaceutical composition of [1], wherein the agent that suppresses the activity of DPP-8 is an agent that suppresses the expression of the DPP-8 gene. [4] The pharmaceutical composition of [1] to [3], further comprising a pharmacologically acceptable carrier.
  • DPP dipeptidyl peptidase
  • the pharmaceutical composition of [6], wherein the agent that specifically recognizes CD138 is an anti-CD138 antibody.
  • the pharmaceutical composition of [1] to [7], wherein the plasma cell tumor is multiple myeloma.
  • [10] The pharmaceutical composition of [1] to [9], which is used in combination with another agent for treating one or more plasma cell tumors.
  • Other agents for treating plasma cell tumors are selected from the group consisting of bortezomib, dexamethasone, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, thalidomide, lenalidomide, pomalidomide, carfilzomib and panobinostat
  • the pharmaceutical composition according to [10] which is one or more agents.
  • a method for inducing apoptosis of a myeloma cell comprising suppressing the activity of DPP-8 in the myeloma cell.
  • a method for diagnosing a plasma cell tumor in a subject comprising quantitatively measuring the activity level of DPP-8 in a biological sample obtained from the subject.
  • a method for detecting myeloma cells in a subject comprising the steps of: (A) quantitatively measuring the activity level of DPP-8 in the biological sample obtained from the subject, and (b) comparing the obtained activity level of DPP-8 with a reference value. Wherein the myeloma cell is detected in the subject when the amount of DPP-8 in the biological sample is significantly greater than a reference value.
  • a screening method for an agent for use in the treatment of a plasma cell tumor (A) contacting a candidate compound with a DPP-8 expressing cell; (B) measuring the activity level of DPP-8 in DPP-8 expressing cells after contact with the candidate compound.
  • the present invention provides a pharmaceutical composition for treating plasma cell tumors that induces myeloma cells to undergo apoptosis by a mechanism of action different from that conventionally known.
  • a pharmaceutical composition induces myeloma cells to apoptosis by suppressing the activity of DPP-8. Therefore, there is also a method for inducing myeloma cells to apoptosis using an agent that suppresses the activity of DPP-8. Also provided.
  • the treatment of myeloma cells, including inhibiting the activity of DPP-8, found by the present invention is a completely different mechanism of action from the pharmaceuticals or treatment methods known as effective treatment methods at present, A synergistic effect can be expected when used in combination with these treatments. Furthermore, an effect can be expected for a subject for whom these treatments are ineffective or no longer effective.
  • FIG. 1 is a graph comparing the expression level of DPP-8 in plasma cells of a healthy subject and the expression level of DPP-8 in a bone marrow sample of a subject suffering from multiple myeloma or primary macroglobulinemia. Significantly higher expression of DPP-8 was confirmed in both myeloma samples that were plasma cell tumors multiple myeloma and bone marrow samples from subjects suffering from primary macroglobulinemia compared to plasma cells in healthy subjects.
  • FIG. 2 is a graph showing the effect of inducing cell death on myeloma cells when various DPP-4 inhibitors are used.
  • FIG. 3 is a graph showing the cell death-inducing effect of 1G244, which is a DPP-8 / 9 selective inhibitor, against various myeloma cell lines. Although there was some difference in sensitivity, the cell death inducing effect was confirmed in all cell lines.
  • FIG. 4 is a view confirming the cell death pattern when DPP-8 / 9 selective inhibitor 1G244 was added to myeloma cells.
  • FIG. 5 is a graph showing the effect on myeloma cells when the expression of these genes in myeloma cells was suppressed using DPP-8 and DPP-9 siRNA. All of the DPP-8 siRNAs showed a significant decrease in absorbance, indicating that the number of viable cells was significantly decreased. However, the siRNA of DPP-9 had no change in absorbance and had an effect on the number of living cells. I knew it was n’t there.
  • FIG. 6 is a graph showing the synergistic effect of the combined use of bortezomib and 1G244. MM.
  • FIG. It is the graph which compared the increase in the tumor volume at the time of administering 1G244 to 1S cancer bearing mouse
  • the “agent that suppresses protein activity” means any agent that can reduce the level of protein activity in cells.
  • the reduction in the activity level may be temporary or permanent.
  • the agent that suppresses the activity of the protein does not necessarily have to act directly on the protein as long as the activity level of the protein can be reduced.
  • agents that suppress protein activity include, but are not limited to, for example, agents that bind to the active site of a protein, agents that bind to a protein to change its three-dimensional structure, and protein denaturants.
  • agents that inhibit transcription of mRNA encoding proteins, agents that inhibit translation of mRNA, and agents that reduce protein expression itself and agents that competitively inhibit protein activity are also included.
  • DPP-8 is a serine protease having a structure similar to dipeptidyl peptidase (DPP) -4, and is a protein belonging to the dipeptidyl peptidase-4 family. In the present invention, unless otherwise stated, typically means a protein or gene registered in Genebank under the following number.
  • plasma cell tumor means a disease caused by abnormal growth of plasma cells, particularly monoclonal plasma cells. Accordingly, the term “plasma cell tumor cell” means a plasma cell that is undergoing abnormal growth. Such “plasma cells undergoing abnormal growth” may be generally referred to as “myeloma cells”. Therefore, in the present invention, “plasma cell tumor cell” and “myeloma cell” are synonymous and used interchangeably.
  • DPP-8 and 9 of DPP-4 family are highly expressed in myeloma cells and that the activity of such DPP can be suppressed to induce myeloma cells to apoptosis. Newly found. The mechanism by which DPP-8 leads myeloma cells to apoptosis is not well understood, but some signaling is thought to be involved.
  • Induction of apoptosis by suppressing the activity of DPP-8 of the present invention causes myeloma cells to have a mechanism of action completely different from the effect on myeloma cells in the conventional treatment of plasma cell tumors such as multiple myeloma. It attacks and thus provides a completely new medicine and treatment for plasma cell tumors, especially multiple myeloma. Moreover, it can be expected to be very useful as a treatment that can be performed in combination with a conventional treatment or can be performed on a subject for which the conventional treatment is no longer effective.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition comprising at least one agent that suppresses the activity of dipeptidyl peptidase (DPP) -8.
  • DPP dipeptidyl peptidase
  • Such pharmaceutical compositions can typically be used for the treatment of plasma cell tumors.
  • the agent that suppresses the activity of DPP-8 may be any agent that can reduce the activity level of DPP-8 in the cell, and is not limited thereto. -8 inhibitors and agents that suppress the expression of DPP-8.
  • inhibitors of DPP-8 include, but are not limited to, Van der Veken et al., Bioorg Med Chem Lett. 2008 Jul 15; 18 (14): 4154-8, Van Goethem et al., Bioorg Med Chem Lett. 2008 Jul 15; 18 (14): 4159-62, Zhang et al., Mol Cancer Res. 2013 Dec; 11 (12): 1487-96, etc.
  • the agent that suppresses the expression of DPP-8 may be any agent that can suppress the transcription and / or translation of the DPP-8 gene, and is not limited thereto.
  • SiRNA for -8 genes may be any agent that can suppress the transcription and / or translation of the DPP-8 gene, and is not limited thereto.
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier in addition to an agent (drug) that suppresses the activity of DPP-8, which is an active ingredient.
  • a pharmaceutically acceptable carrier can be used as long as it can improve the bioavailability of the drug to be delivered and the delivery efficiency to a diseased site, but is not limited thereto. Examples thereof include a liposome carrier, a polymer carrier, and a micelle carrier.
  • a pharmaceutically acceptable carrier is present in the composition in a form that exhibits a function as a carrier.
  • Such forms include, but are not limited to, for example, forms in which an agent that suppresses the activity of DPP-8, which is an active ingredient, and a pharmaceutically acceptable carrier are operatively linked, or active ingredients And a form in which an agent that suppresses the activity of DPP-8 is encapsulated in capsule-like particles such as micro / nanocapsules, liposomes, and micelles composed of a pharmaceutically acceptable carrier.
  • “operably connected” between the member A and the member B means that the member A and the member B are connected without impairing the essential functions of the respective members.
  • the DPP-8 inhibitor does not impair its ability to inhibit the activity of DPP-8
  • the polymer carrier does not They are connected to each other without impairing the protection and delivery functions.
  • the term “linkage” means that they are linked through an arbitrary chemical bond or linker, and the chemical bond is not limited to this, for example, a covalent bond, an ionic bond, Includes hydrogen bonding and the like.
  • both members may be connected via another member C instead of the linker.
  • the pharmaceutical composition of the present invention further comprises a targeting agent that specifically targets tumor cells, particularly plasma cell tumor cells (myeloma cells).
  • the targeting agent is contained in the pharmaceutical composition in a form capable of specifically delivering an agent that suppresses the activity of DPP-8, which is an active ingredient, to tumor cells.
  • the agent that suppresses the activity of DPP-8 and the targeting agent are operably linked.
  • the activity of DPP-8 is suppressed by a linker such as, but not limited to, succinimidyl 6-hydrazinonicotinatoacetone hydrazone (SANH), succinimidyl 4-formylbenzoate (SFB), and the like.
  • a linker such as, but not limited to, succinimidyl 6-hydrazinonicotinatoacetone hydrazone (SANH), succinimidyl 4-formylbenzoate (SFB), and the like.
  • the agent and the targeting agent can be operably linked.
  • Linkers that can operably link drugs and targeting agents are known in the art (eg Hamann et, al., Bioconjug.gChem., 2002 Jan-Feb; 13 (1): 40-6 , DiJoseph et al, Blood. 2004 Mar 1; 103 (5): 1807-14, etc.), those skilled in the art can appropriately select a linker according to the drug and targeting agent employed.
  • the pharmaceutical composition of the present invention comprises an agent (drug) that suppresses the activity of DPP-8 as an active ingredient, a pharmaceutically acceptable carrier, and a targeting agent.
  • a pharmaceutically acceptable carrier for suppresses the activity of DPP-8 as an active ingredient
  • a pharmaceutically acceptable carrier for delivering a therapeutically active agent.
  • the targeting agent and the carrier are operably linked.
  • the carrier is a polymer or the like
  • the agent that suppresses the activity of DPP-8 may be further operably linked to the carrier operably linked to the targeting agent.
  • the carrier is a carrier that forms capsule particles such as liposomes or micelles, the surface may be encapsulated in the capsule particles modified with a targeting agent.
  • compositions of such embodiments are known in the art (eg Ono etOal., Blood Cancer J., 2014; 4: e180, etc.), and those skilled in the art will employ drugs, carriers and targeting The optimum mode can be appropriately selected according to the agent.
  • targeting refers to a cell that does not target a substance, such as a drug or drug delivery carrier, to a specific target, such as a specific cell or tissue (in the present invention, a tumor cell, preferably a myeloma cell).
  • a specific target such as a specific cell or tissue
  • a tumor cell in the present invention, a tumor cell, preferably a myeloma cell.
  • a substance is meant a substance that can thus be targeted when bound or reacted with the substance.
  • the targeting agent is in the form of a molecule, this is synonymous with “targeting molecule”.
  • the targeting agent that can be used in the present invention may be any agent that can specifically deliver a substance to tumor cells, preferably myeloma cells.
  • targeting is performed using a substance that recognizes a molecule specifically expressed in a target cell or tissue. Therefore, preferably, the targeting agent is a substance that specifically recognizes a molecule that is specifically expressed in tumor cells, particularly myeloma cells. Examples of substances that specifically recognize a specific molecule include monoclonal antibodies and ligand / receptors.
  • an antibody particularly a monoclonal antibody
  • Syndecan-1 (SDC1), also called CD138, is a transmembrane heparan sulfate proteoglycan present on the cell surface. Syndecan-1 has functions such as cell binding, signaling mediation, and cytoskeleton formation, and is also considered to play an important role in cell proliferation, migration, and interaction with extracellular matrix. It is known that this syndecan-1 is specifically expressed in mature plasma cells, and is also expressed on the cell surface of plasma cells derived from plasma cells (ie, plasmacytoma). Therefore, syndecan-1 (CD138) is considered to function as a suitable marker (target target molecule) for plasma cell tumor cells. Therefore, in a preferred embodiment of the pharmaceutical composition of the present invention, the targeting agent is an agent (or molecule) that specifically recognizes syndecan-1 (CD138).
  • Examples of molecules that specifically recognize syndecan-1 include, but are not limited to, syndecan-1 receptor molecules, anti-CD138 antibodies (particularly monoclonal antibodies), and the like.
  • an antibody particularly a monoclonal antibody
  • the targeting agent is an anti-CD138 antibody (particularly a monoclonal antibody).
  • Plasma cell tumor means a disease caused by abnormal growth of plasma cells, particularly monoclonal plasma cells, as described above.
  • Plasma cell tumors include multiple myeloma (MM), monoclonal globulinemia of unknown significance (MGUS), bone single plasmacytoma, extramedullary plasmacytoma, macroglobulinemia, plasma cells Sexual leukemia, heavy chain disease and the like are included.
  • MM multiple myeloma
  • MGUS monoclonal globulinemia of unknown significance
  • M protein monoclonal immunoglobulin due to abnormal proliferation of plasma cells causes amyloidosis and monoclonal immunoglobulin deposition as complications.
  • the plasma cell tumor treated with the pharmaceutical composition of the present invention is preferably a symptomatic plasma cell tumor.
  • a typical disease among symptomatic plasma cell tumors is multiple myeloma, which is very difficult to treat.
  • new drugs such as bortezomib and lenalidomide
  • new therapies such as hematopoietic stem cell transplantation
  • an increase in secondary carcinogenesis has become a problem due to treatment. Therefore, there is a demand for the establishment of a treatment method based on a new mechanism of action.
  • the present invention originates from the discovery of a completely new mechanism of action that, when the activity of DPP-8 is suppressed in myeloma cells, apoptosis of the myeloma cells is induced. Therefore, the pharmaceutical composition of the present invention can be suitably used particularly in the treatment of multiple myeloma.
  • the pharmaceutical composition of the present invention attacks myeloma cells by a new working mechanism that is completely different from conventional treatment methods. Therefore, it is expected that it can be used effectively even for subjects having myeloma cells that have acquired resistance to conventional therapy. Accordingly, in a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used for the treatment of plasma cell tumors in a subject where conventional therapy has become ineffective.
  • “conventional therapy” means treatment by administration of any agent currently known to be effective for plasma cell tumors.
  • agents include, but are not limited to, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat and the like.
  • the pharmaceutical composition of the present invention attacks myeloma cells by a new working mechanism that is completely different from conventional treatment methods, and therefore, a synergistic effect is expected when used in combination with conventional treatments. Is done.
  • a DPP-8 inhibitor which is an active ingredient of the pharmaceutical composition of the present invention
  • bortezomib which is a therapeutic agent used in conventional treatment
  • myeloma It has been shown to exert a dramatic synergistic effect in the treatment of cells.
  • the pharmaceutical composition of the present invention is used in combination with other agents for treating one or more plasma cell tumors.
  • another agent for treating a plasma cell tumor means an agent administered in the above-mentioned “conventional therapy”.
  • an agent administered in the above-mentioned “conventional therapy” typically, but not limited to, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat and the like.
  • agent A and another agent B when “used together” or “used together”, it means that the agent B is in an effective state while the agent A is effective. . Therefore, the agent B may be administered simultaneously with the administration of the agent A, or the agent B may be administered after a certain interval after the administration of the agent A. Further, the agent A and the agent B may be in the same dosage form or in different dosage forms. Furthermore, as long as the agent A or the agent B does not lose its effect, the agent A and the agent B may be mixed into one composition.
  • the present invention originates from the newly found that DPP-8 is highly expressed in myeloma cells, and DPP-8 in myeloma cells. It is based on the fact that the present inventor found for the first time that myeloma cells are induced by apoptosis when the enzyme activity of is inhibited. Accordingly, the present invention in one aspect relates to a method for inducing apoptosis of myeloma cells comprising inhibiting DPP-8 activity in myeloma cells. The method may be performed either in vivo or in vitro.
  • apoptosis of myeloma cells is induced in any of the cases where the activity of DPP-8 is suppressed by a plurality of methods in a plurality of myeloma cell lines.
  • the method for inducing apoptosis of the present invention comprises contacting an agent that suppresses the activity of DPP-8 with a myeloma cell.
  • an agent that suppresses the activity of DPP-8 any agent described herein can be used as such an agent.
  • the present invention also provides a myeloma cell detection method (test method, diagnostic method) based on the level of DPP-8 activity as a criterion.
  • the detection method (diagnostic method) of the present invention detects the presence or level of myeloma cells present in the biological sample by measuring the activity level of DPP-8 contained in the biological sample derived from the test subject. To be examined or diagnosed.
  • the inventor of the present application has first found that the presence or degree of myeloma cells can be detected, examined, or diagnosed based on the activity level of DPP-8.
  • the biological sample derived from the test subject is preferably a sample that can contain myeloma cells, and typically includes, but is not limited to, blood, bone marrow fluid, a part of a test tissue suspected of being a tumor, and the like. .
  • a technique for collecting a biological sample any technique known in the technical field can be used, and those skilled in the art can appropriately select according to the sample to be collected.
  • the detection (examination, diagnosis) method of the present invention detects (examines) whether or not the disease has been improved when a therapeutic agent is administered to improve the disease in a patient having a plasma cell tumor, for example. Diagnosis).
  • a particular embodiment of the detection (testing) method of the present invention comprises the following steps (a) and (b), and optionally (c): (A) quantitatively measuring the activity level of DPP-8 in a biological sample obtained from a subject, (B) comparing the DPP-8 activity level obtained in (a) with a reference value; (C) To determine the incidence of cancer based on the results of (b).
  • a particular embodiment of the diagnostic method of the present invention comprises the steps (a) and optionally (b) and (c).
  • any measurement value can be used as long as it can be measured quantitatively and can represent the amount of DPP-8 activity in the sample.
  • the expression level of the DPP-8 gene, the amount of DPP-8 protein, the activity level of the serine protease of DPP-8, and the like can be mentioned. Any method known in the art can be used to measure the activity level of DPP-8.
  • the enzyme activity in the sample is measured, for example, a method for measuring the expression level of DPP-8 such as proteome analysis, a method for measuring the amount of protein such as Western blotting, and an activity measurement method using a fluorescent substrate. The method of doing is mentioned.
  • the reference value to be compared with the activity level measured above may be the same amount as the amount measured as the activity level.
  • Reference values include, but are not limited to, for example, DPP-8 activity level in a sample obtained from a healthy tissue of a test subject (subject), DPP-8 activity level in the same sample obtained from a healthy subject The average value or statistical intermediate value of the DPP-8 activity level in a healthy subject population measured under uniform conditions in advance.
  • the present inventors have found that the expression of the DPP-8 gene is significantly increased in myeloma cells, and therefore the activity of the DPP-8 protein is significantly increased. Therefore, for example, when the DPP-8 activity level of a healthy subject or a healthy tissue is used as a reference value, if the subject-derived biological sample has a DPP-8 activity level significantly higher than the reference value, Is considered to have myeloma cells.
  • “Significantly” increased or increased or “significantly” large for example, that the DPP-8 activity level in a sample from a test subject is, for example, more than twice, preferably compared to those levels in a healthy subject Means three times more. Generally, when it is determined that the difference in values is significant as a result of statistical analysis known in the technical field, it is determined to be “significant”.
  • Plasma cell tumor treatment method (therapy method)
  • the present invention also provides a method for treating a plasma cell tumor in a subject, wherein an effective amount of an agent that suppresses the activity of DPP-8 or a pharmaceutical composition comprising the same is provided to a subject in need thereof. It also relates to said method comprising administering.
  • the “subject” in the present invention may be any individual organism as long as it can suffer from a plasma cell tumor, but preferably human and non-human mammals (eg, mouse, rat, guinea pig, hamster, etc.) Rodents, primates such as chimpanzees, cloven-hoofed animals such as cows, goats and sheep, terrestrial hogs such as horses, rabbits, dogs and cats), and more preferably human individuals.
  • human and non-human mammals eg, mouse, rat, guinea pig, hamster, etc.
  • Rodents primates such as chimpanzees, cloven-hoofed animals such as cows, goats and sheep, terrestrial hogs such as horses, rabbits, dogs and cats
  • primates such as chimpanzees
  • cloven-hoofed animals such as cows, goats and sheep
  • terrestrial hogs such as horses, rabbits, dogs and cats
  • Examples of the agent that suppresses the activity of DPP-8 used in the prevention / treatment method of the present invention or a pharmaceutical composition containing the agent include any of those described in the present specification.
  • the effective amount in the present invention is, for example, an amount that reduces the symptoms of plasma cell tumors and / or their complications, or delays or stops their progression, preferably plasma cell tumors and / or their concomitants The amount that suppresses or cures the disease. In addition, an amount that does not cause adverse effects exceeding the benefits of administration is preferred. Such an amount can be appropriately determined by an in vitro test using cultured cells or a model animal such as a mouse or a rat, and such a test method is well known to those skilled in the art.
  • the specific dose of the active ingredient depends on various conditions relating to the subject in need thereof, such as severity of symptoms, general health status of the subject, age, weight, subject sex, diet, timing and frequency of administration, It can be determined in consideration of the drug used in combination, the response to treatment, the dosage form, compliance with the treatment, and the like.
  • the specific dose is, for example, usually 0.01 to 100 mg, preferably 0.01 to 10 mg, more preferably 0.01 to 1 mg per kg body weight of the subject, and this is administered once a day. Is preferred.
  • any known appropriate administration method such as intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, intrathecal administration and the like can be used.
  • the screening method of the present invention includes the following (a) and (b): (A) contacting a candidate compound with a DPP-8 expressing cell; (B) measuring the activity level of DPP-8 in DPP-8 expressing cells after contact with the candidate compound.
  • the DPP-8 expressing cells used in the screening method of the present invention may be cells derived from living organisms or cultured cells. Further, it may be a cell that inherently expresses DPP-8 (for example, a mature plasma cell), or may be a cell that has been genetically manipulated to express DPP-8. Arbitrary cells can be used as the cell type, but eukaryotic cells are preferable, mammalian cells are more preferable, and human cells are most preferable.
  • the measurement of the activity level in (b) can be carried out by the same technique as the measurement of the activity level in the above detection (test, diagnosis) method.
  • the screening method of the present invention may further include the following (c):
  • (C) The activity level obtained in (b) is compared with a reference value, and if the activity level is significantly smaller than the reference value, the candidate compound is determined to be a plasma cell tumor therapeutic agent.
  • the “reference value” used in the screening method of the present invention means the DPP-8 activity level in the DPP-8 expressing cells. This may be a numerical value measured in advance, for example, for confirming the performance of the transformed cell, or as a previous step of (a) above;
  • (A ′) The method may further comprise measuring the activity level of DPP-8 in the DPP-8-expressing cell before contacting with the candidate compound.
  • “Significantly small” means, for example, that the activity level of DPP-8 after contact with the candidate compound is, for example, half or less, preferably 1/3 or less, compared to the reference value. Generally, when it is determined that the difference in values is significant as a result of statistical analysis known in the technical field, it is determined to be “significant”.
  • Example 1 Expression of DPP-8 in myeloma cells
  • NPC normal bone marrow derived plasma cells
  • MM multiple myeloma patients
  • WM primary macroglobulinemia patients
  • the results are shown in FIG.
  • the expression level of DPP-8 was significantly higher in both bone marrow samples from patients with multiple myeloma and cellular bone marrow samples from patients with primary macroglobulinemia compared to plasma cells from healthy bone marrow. From this, it can be inferred that DPP-8 is specifically expressed in myeloma cells, and the relationship between plasma cell tumors and DPP-8 is suspected.
  • DPP-8 inhibitor sensitivity of myeloma cells (1) Sensitivity to DPP-4 / 8/9 inhibitors
  • 1G244 which is an inhibitor that inhibits only DPP-8 / 9, was used to perform the same test while changing the cell type.
  • 1 ⁇ 10 5 cells / 100 ⁇ L of various myeloma cells (Delta47, U266, KMS-5, RPMI8226 and MM.1S) are seeded per well of a 96-well microplate, and each 5 ⁇ M, 10 ⁇ M, DPP-8 / 9 inhibitor 1G244 at a concentration of 50 ⁇ M and 100 ⁇ M was added, respectively, and cultured for 72 hours. Thereafter, Premix WST-1 reagent was added and the cells were further cultured for 1 hour, and the number of cells was semi-quantified using a microplate reader.
  • Example 3 Cell death pattern analysis
  • the following test was conducted to confirm that the cell death caused by the DPP-8 / 9 inhibitor was apoptosis. After adding 1 ⁇ 10 6 myeloma cells (MM.1S and KMS-5) with various concentrations of DPP-8 / 9 inhibitor 1G244 and culturing for 48 hours, anti-annexin V-PE antibody and 7-AAD were used. Staining was performed and the cell death pattern was determined by flow cytometry.
  • siRNA that specifically reduces DPP8 and DPP9 was tested.
  • siRNA Stealth siRNAs (Invitrogen) series was used.
  • the siRNA of DPP-8 is model number HSS123433 (DPP8-1, SEQ ID NO: 1), HSS123434 (DPP8-2, SEQ ID NO: 2) and HSS123435 (DPP8-3, SEQ ID NO: 3), and the negative control is Stealth RNAi TM siRNA negative control kit Medium Low GC was used.
  • the siRNA of DPP-9 is model number HSS132085 (DPP9-1, SEQ ID NO: 4), HSS132086 (DPP9-2, SEQ ID NO: 5) and HSS132087 (DPP9-3, SEQ ID NO: 6), and the negative control is Stealth RNAi TM siRNA negative control kit Medium Medium GC was used. 1 ⁇ 10 5 MM. After adding 20 nM siRNA to the 1S cell line and culturing for 72 hours, Premix WST-1 reagent was added and further cultured for 1 hour, and the absorbance was measured using a microplate reader.
  • Bortezomib is a proteasome inhibitor and treats myeloma cells by a mechanism of action different from DPP-8 inhibition. Therefore, the combined use of bortezomib and DPP-8 inhibitor 1G244 was examined.
  • 1 ⁇ 10 5 cells / 100 ⁇ L of myeloma cells are seeded per well of a 96-well microplate, and 0.5 ⁇ M or 5 ⁇ M DPP-8 / 9 inhibitor 1G244 and 20 nM, respectively.
  • 40 nM bortezomib was added and cultured for 72 hours. Thereafter, Premix WST-1 reagent was added and the cells were further cultured for 1 hour, and the number of cells was semi-quantified using a microplate reader.
  • PBS phosphate buffered saline
  • 30 mg / kg of 1G244 was injected subcutaneously every 3 days from the 3rd day.
  • the tumor volume of each group was measured every 3 days, and changes in the tumor volume from the first day of measurement were observed.
  • Results are shown in FIG.
  • the increase in tumor volume began to show a difference in value around day 12 of measurement, and on day 24 of measurement, the increase in tumor volume in the 1G244 administration group was significantly lower than that in the untreated group. For this reason, 1G244 is a MM. It was found to suppress the proliferation of 1S cells.
  • the pharmaceutical composition of the present invention exhibits an effect by inducing myeloma cells to apoptosis by a new mechanism of action that has not been previously known.
  • these pharmaceutical compositions are effective with different mechanisms of action from conventional plasma cell tumor therapeutic agents, they can be used effectively even for subjects who no longer receive the effects of conventional plasma cell tumor therapeutic agents. obtain.
  • a strong synergistic effect is exhibited when used in combination with conventional plasma cell tumor therapeutic agents, and in combination with conventional plasma cell tumor therapeutic agents where strong side effects are problematic, It can also be expected to reduce side effects.

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Abstract

L'objet de la présente invention est de fournir : une composition médicinale permettant de traiter un néoplasme de plasmocytes, en particulier un myélome multiple ; une méthode permettant de traiter un néoplasme de plasmocytes, ladite méthode consistant à administrer la composition médicinale précitée ; une méthode permettant d'induire l'apoptose d'une cellule de myélome, ladite méthode consistant à mettre en contact un inhibiteur de DPP-8 avec la cellule de myélome ; etc. L'objet précité a été atteint grâce à une composition médicinale qui comprend au moins un type d'inhibiteur de l'activité de la dipeptidyl peptidase (DPP)-8.
PCT/JP2017/010828 2016-03-18 2017-03-17 Agent thérapeutique pour un néoplasme de plasmocytes WO2017159835A1 (fr)

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