JPWO2017159835A1 - Plasma cell tumor therapeutic agent - Google Patents

Abstract

An object of the present invention is to provide a pharmaceutical composition for treating plasma cell tumors, particularly multiple myeloma, a method for treating plasma cell tumors comprising administering the pharmaceutical composition, a DPP-8 inhibitor and bone marrow An object of the present invention is to provide a method for inducing apoptosis of myeloma cells, which comprises contacting with tumor cells. The above problems have been solved by a pharmaceutical composition comprising at least one agent that suppresses the activity of dipeptidyl peptidase (DPP) -8.

Description

  The present invention relates to a pharmaceutical composition for the treatment of a 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 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.

  As a therapeutic agent for multiple myeloma, an anticancer agent such as melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone has been conventionally used in combination. In recent years, 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. Has been developed. In addition, transplantation of hematopoietic stem cells can be mentioned as one of treatment options.

  Although the prognosis of multiple myeloma has improved considerably due to the appearance of the above-mentioned new drugs, there is still no report that it has been completely cured, and recently, the increase in secondary carcinogenesis due to treatment has become a problem. There is still a great need for new therapeutics.

  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 known to inactivate incretin, which is an intestinal hormone, due to its serine protease activity, and inactivation of incretin is known to cause suppression of insulin secretion. Therefore, DPP-4 inhibitors 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. For example, Non-Patent Document 1 discloses that suppression of CD26 / DPP-4 expression in the undifferentiated large cell lymphoma cell line Karpas 299 causes dephosphorylation of integrin β 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.

  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 cells, and structural similarity with DPP-4 is thought to play a role in T cell activation and immune function. However, its function has not been elucidated in practice.

Sato et al., Cancer Res 2005; 65 (15): 6950-6

  The object of the present invention is a pharmaceutical composition for treating plasma cell tumors, especially 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.

  In studying the relationship between hematologic diseases, particularly hematopoietic tumors, and proteins belonging to the DPP-4 family, the inventor has developed DPP-8 and myeloma cells from subjects with multiple myeloma. 9 was found to be significantly highly expressed. As a result of diligent research focusing on these findings, when vildagliptin, a DPP-4 inhibitor known to have DPP-8 / 9 inhibitory activity, is administered to myeloma cells, As a result of finding that the number of viable cells was significantly reduced and further researching, the present invention was completed.

That is, 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 of [1], wherein the agent that suppresses the activity of DPP-8 is a DPP-8 inhibitor.
[3] The pharmaceutical composition according to [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.

[5] The pharmaceutical composition of [1] to [4], further comprising a tumor cell targeting agent.
[6] The pharmaceutical composition of [5], wherein the targeting agent is an agent that specifically recognizes CD138.
[7] The pharmaceutical composition of [6], wherein the agent that specifically recognizes CD138 is an anti-CD138 antibody.
[8] The pharmaceutical composition of [1] to [7], wherein the plasma cell tumor is multiple myeloma.
[9] The pharmaceutical composition of [1] to [8], wherein the pharmaceutical composition is used for a subject for which conventional therapy is no longer effective.

[10] The pharmaceutical composition of [1] to [9], which is further used in combination with another agent for treating one or more plasma cell tumors.
[11] 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.

[12] A method for inducing apoptosis of myeloma cells, comprising suppressing DPP-8 activity in myeloma cells.
[13] The method for inducing apoptosis of myeloma cells according to [12], which comprises contacting a myeloma cell with an agent that suppresses the activity of DPP-8.
[14] 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.

[15] 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 cells are detected in the subject when the amount of DPP-8 in the biological sample is significantly greater than a reference value.
[16] 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) The said method including measuring the activity level of DPP-8 in the DPP-8 expression cell after contact with the said candidate compound.

  The present invention provides a pharmaceutical composition for treating plasma cell tumors that induces myeloma cells to apoptosis by a mechanism of action different from that conventionally known. Such 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. Furthermore, since the treatment of myeloma cells, including suppressing the activity of DPP-8, found by the present invention is a completely different mechanism of action from the pharmaceuticals or treatment methods currently known as effective treatment methods, 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 DPP-8 expression was confirmed in both bone marrow samples of subjects suffering from plasma cell tumor multiple myeloma and primary macroglobulinemia compared to healthy subject plasma cells. FIG. 2 is a graph showing the effect of inducing cell death on myeloma cells when various DPP-4 inhibitors are used. Among five types known as DPP-4 inhibitors, cell death-inducing effects were confirmed only in vildagliptin and saxagliptin, which are also known to inhibit DPP-8 / 9 together. 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 diagram confirming the cell death pattern when DPP-8 / 9 selective inhibitor 1G244 was added to myeloma cells. As both 7-ADD / Annexin V positive populations increased with increasing amounts of 1G244, it was confirmed that the induced cell death was apoptotic. 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. Since all the DPP-8 siRNAs showed a significant decrease in absorbance, it was found that the number of viable cells was significantly reduced. However, the DPP-9 siRNA had no change in absorbance and had an effect on the number of viable 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. The effect of bortezomib could be remarkably improved by adding a small amount of 1D244 to both the 1S cell line and the KMS-5 cell line. FIG. It is the graph which compared the increase in the tumor volume at the time of administering 1G244 to 1S cancer bearing mouse | mouth compared with an untreated group. A difference began to appear in the increase in tumor volume around day 12, and a significant difference was confirmed on day 24.

The present invention will be described in detail below.
In the present invention, 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. Examples of 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. In addition to agents that act directly, 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.

“Dipeptidyl peptidase-8” or “DPP-8” is a serine protease having a structure similar to dipeptidyl peptidase (DPP) -4, and belongs 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.
Gene (mRNA): NM_017743
Protein: NP_060213

  In the present invention, “plasma cell tumor” means a disease caused by abnormal proliferation 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.

  The present inventor has shown that 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.

<1> Pharmaceutical Composition of the Present Invention 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. 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 cells, and is not limited thereto. -8 inhibitors and agents that suppress the expression of DPP-8.

  Examples of 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. Is 2-amino-4- {4- [bis (4-fluorophenyl) methyl] piperazin-1-yl} -1- (2,3-dihydro-1H-isoindol-2-yl) butane-1,4 -DPP-8 / 9 selective inhibitors such as dione (also referred to as 1G244), Allo-Ile-isoindoline, DPP-4 inhibitors having DPP-8 / 9 inhibitory activity such as vildagliptin and saxagliptin It is done.

  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 gene and the like can be mentioned.

  In one aspect, 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. Any 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.

  In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier is present in the composition in a form that exhibits a function as a carrier. Examples of such forms include, but are not limited to, 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 Examples include 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.

  In the present invention, the members A and B are “operably connected” means that the members A and B are connected without impairing the essential functions of the respective members. Thus, for example, when a DPP-8 inhibitor and a polymer carrier are operably linked, the DPP-8 inhibitor does not impair its ability to inhibit the activity of DPP-8, and the polymer carrier does not They are connected to each other without impairing the protection and delivery functions. Here, 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. Moreover, as long as the member A and the member B do not impair the function, both members may be connected via another member C instead of the linker.

  In another embodiment, 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. Accordingly, in a preferred embodiment, the agent that suppresses the activity of DPP-8 and the targeting agent are operably linked. In such an embodiment, 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). The agent and the targeting agent can be operably linked. Linkers capable of operably linking drugs and targeting agents are known in the art (eg Hamann et, al., Bioconjug. Chem., 2002 Jan-Feb; 13 (1): 40-6 , DiJoseph et al, Blood. 2004 Mar 1; 103 (5): 1807-14), a person skilled in the art can appropriately select a linker according to the drug and the targeting agent employed.

  In another preferred embodiment, the pharmaceutical composition of the present invention comprises an agent (drug) that suppresses the activity of DPP-8, which is an active ingredient, a pharmaceutically acceptable carrier, and a targeting agent. In such embodiments, at least the targeting agent and the carrier are operably linked. When 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. When 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. Pharmaceutical compositions of such embodiments are known in the art (eg Ono et al., 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.

  In the present invention, “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). Refers to rapid, efficient and / or large volume delivery compared to the untargeted substance rather than to the target or tissue, i.e. enabling delivery of a target specific substance. By a substance is meant a substance that can thus be targeted when bound or reacted with the substance. In the case where 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. In general, 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. When used as a targeting agent in a pharmaceutical composition, an antibody (particularly a monoclonal antibody) is preferably used from the viewpoint of ease of preparation.

  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. Thus, 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 the molecule that specifically recognizes syndecan-1 include, but are not limited to, a syndecan-1 receptor molecule, an anti-CD138 antibody (particularly a monoclonal antibody), and the like. When used as a targeting agent in a pharmaceutical composition, an antibody (particularly a monoclonal antibody) is preferred as described above. Therefore, in the pharmaceutical composition of the present invention, more preferably, the targeting agent is an anti-CD138 antibody (particularly a monoclonal antibody).

  A “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. In addition, mass production of monoclonal immunoglobulin (M protein) 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. In recent years, the development of new drugs such as bortezomib and lenalidomide and the establishment of new therapies such as hematopoietic stem cell transplantation have greatly improved the prognosis, but there have been no cures yet. In addition, 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.

  As described above, 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.

  Here, “conventional therapy” means treatment by administration of any agent currently known to be effective for plasma cell tumors. Typically, such agents include, but are not limited to, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat and the like.

  As described above, 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. In fact, as shown in the Examples below, when the present inventors used a DPP-8 inhibitor, which is an active ingredient of the pharmaceutical composition of the present invention, and bortezomib, which is a therapeutic agent used in conventional therapy, myeloma It has been shown to exert a dramatic synergistic effect in the treatment of cells. Accordingly, in a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is used in combination with other agents for treating one or more plasma cell tumors.

  In the present invention, “another agent for treating a plasma cell tumor” means an agent administered in the above-mentioned “conventional therapy”. Thus, typically, but not limited to, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, dexamethasone, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat and the like.

  In the present invention, when an agent A and another agent B are “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.

<2> Method for Inducing Apoptosis of the Present Invention As described above, the present invention originated 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.

  Details of the mechanism of action by which apoptosis is induced by suppressing the activity of DPP-8 are unknown. However, it has been confirmed by the present inventors that 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.

  As a technique for suppressing the activity of DPP-8, any technique known to those skilled in the art as a technique for reducing the activity of a protein in a cell may be used, but typically the activity of DPP-8 is suppressed. This is accomplished by administering the agent and contacting it with myeloma cells. Therefore, in a preferred embodiment, the method for inducing apoptosis of the present invention comprises contacting an agent that suppresses the activity of DPP-8 with a myeloma cell. As an agent that suppresses the activity of DPP-8, any agent described herein can be used as such an agent.

<3> Myeloma cell detection method (examination method, diagnosis method)
As described above, the present inventors have newly found that DPP-8 is highly expressed in myeloma cells. Therefore, the present invention also provides a myeloma cell detection method (examination method, diagnosis method) based on the activity level of DPP-8.
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. It was first found by the present inventor 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. . As 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 activity level of DPP-8 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).

As the “activity level of DPP-8”, 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. Specifically, for example, 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. Specifically, 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.

  For the first time, the 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. Thus, for example, when the DPP-8 activity level of a healthy subject or a healthy tissue is used as a reference value, if the biological sample derived from the subject has a DPP-8 activity level that is significantly higher than the reference value, Is considered to have myeloma cells.

  “Significantly” increased or increased or “significantly” large, for example, the DPP-8 activity level in a sample from a test subject is preferably, for example, 2 times or more, 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”.

<4> Plasma cell tumor treatment method (therapy method)
The present invention also provides a method for treating a plasma cell tumor in a subject, comprising an effective amount of an agent that suppresses the activity of DPP-8 or a pharmaceutical composition of the present invention comprising the same in 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.

  Examples of the agent that suppresses the activity of DPP-8 or the pharmaceutical composition containing the agent used in the prevention / treatment method of the present invention include any of those described herein. 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 the like and a test in a model animal such as a mouse or 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.

  As a specific dose, for example, it is 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. As the administration method, any known appropriate administration method such as intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, intrathecal administration and the like can be used.

<5> Screening method for therapeutic agent for plasma cell tumor According to the present inventors, myeloma cells are induced to apoptosis by high expression of DPP-8 in myeloma cells and suppression of the activity of DPP-8 It was found that Therefore, by assessing the ability of a compound to suppress the activity of DPP-8, it can be determined whether it is useful as a plasma cell tumor therapeutic agent.

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. In addition, 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 engineered 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.
In the screening method of the present invention, 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.

In one embodiment, 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 ′) It 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”.

All patents, applications and other publications mentioned herein are hereby incorporated by reference in their entirety.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

Example 1. Expression of DPP-8 in myeloma cells In order to examine the relationship between myeloma development and DPP-8 expression, gene expression profiling data described in Gutierrez et al., Leukemia. 2007; 21: 541-9 was used. The data was reanalyzed.
Specifically, on the DataSet Browser page of Gene Expression Omnibus Datasets (http://www.ncbi.nlm.nih.gov/sites/GDSbrowser/) on the National Center for Biotechnology Information (NCBI) homepage The data set of GDS2643 which is the registration number of the data set of Gutierrez et al. Was called, and the data regarding DPP-8 gene were extracted from it. Expression of DPP-8 in normal bone marrow derived plasma cells (NPC) and bone marrow samples from multiple myeloma patients (MM) or primary macroglobulinemia patients (WM) by Student t test Compared.

  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 normal bone marrow. From this, it can be inferred that DPP-8 is specifically expressed in myeloma cells, and the association between plasma cell tumors and DPP-8 is suspected.

Example 2. DPP-8 inhibitor sensitivity of myeloma cells (1) Sensitivity to DPP-4 / 8/9 inhibitors In order to confirm the effects of DPP-4 family inhibitors on myeloma cells, they are DPP-4 inhibitors Verification was performed using vildagliptin, saxagliptin, sitagliptin, alogliptin and linagliptin.
1 × 10 ⁵ cells / 100 μL of myeloma cell line MM. 1S was seeded, and each of the above inhibitors at concentrations of 5 μM, 10 μM, 50 μM and 100 μM was added and cultured for 72 hours. Thereafter, the Premix WST-1 reagent was added and further cultured for 1 hour, and the number of viable cells was semi-quantified using a microplate reader.

  The results are shown in FIG. In the case of using vildagliptin and saxagliptin having inhibitory activity not only for DPP-4 but also for DPP-8 and 9, a significant decrease in the number of living cells was confirmed in a concentration-dependent manner. When sitagliptin, alogliptin and linagliptin, which have no inhibitory activity against -8 and 9, were used, no significant change was observed in the number of viable cells.

(2) Sensitivity to DPP-8 inhibitor Next, 1G244 which is an inhibitor that inhibits only DPP-8 / 9 was used, and the same test was performed by changing the cell type. Specifically, 1 × 10 ⁵ cells / 100 μL of various myeloma cells (Delta47, U266, KMS-5, RPMI8226 and MM.1S) are seeded per well of a 96-well microplate, and each 5 μM, 10 μM, DPP-8 / 9 inhibitor 1G244 at a concentration of 50 μM and 100 μM was added, respectively, and cultured for 72 hours. Thereafter, the Premix WST-1 reagent was added and further cultured for 1 hour, and the number of cells was semi-quantified using a microplate reader.

  The results are shown in FIG. MM. Although it showed the highest sensitivity in 1S, sensitivity was observed in all myeloma cell lines. This suggests that inhibition of DPP-8 / 9 specifically expressed in myeloma cells causes cell death regardless of the cell line.

Example 3 Analysis Example 2 of Cell Death Pattern In order to confirm that the cell death caused by the DPP-8 / 9 inhibitor was apoptosis, the following test was performed. 1 × 10 ⁶ myeloma cells (MM.1S and KMS-5) were added with various concentrations of DPP-8 / 9 inhibitor 1G244 and cultured for 48 hours, followed by anti-annexin V-PE antibody and 7-AAD. Staining was performed and the cell death pattern was determined by flow cytometry.

  The results are shown in FIG. MM. In both the 1S strain and the KMS-5 strain, administration of a DPP-8 inhibitor increased the number of both 7-AAD / Annexin V positive populations, and MM. 90% or more of 1S and 80% or more of KMS-5 were 7-AAD / Annexin V positive populations. This indicates that the cell death caused by the DPP-8 / 9 inhibitor is apoptosis.

Example 4 Examination using siRNA In order to examine whether DPP-8 inhibition or DPP-9 inhibition actually contributes to the induction of apoptosis, siRNA that specifically reduces DPP8 and DPP9 was tested.
As 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 ^™ siRNA negative control kit Medium Low GC was used. The siRNA of DPP-9 is model number HSS132085 (DPP9-1, SEQ ID NO: 4), HSS132060 (DPP9-2, SEQ ID NO: 5) and HSS132087 (DPP9-3, SEQ ID NO: 6), and the negative control is Stealth RNAi ^™ 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 absorbance was measured using a microplate reader.

  The results are shown in FIG. When DPP-8 expression was suppressed, cell death was significantly induced, whereas when DPP-9 expression was suppressed, there was almost no change from the negative control. Therefore, it was speculated that the gene involved in the growth of myeloma cells was DPP-8, not DPP-9.

Example 5. Combination test with bortezomib

  Bortezomib is a proteasome inhibitor that treats myeloma cells by a different mechanism of action than DPP-8 inhibition. Therefore, the combined use of bortezomib and DPP-8 inhibitor 1G244 was examined.

1 × 10 ⁵ cells / 100 μL of myeloma cells (MM.1S or KMS-5) are seeded per well of a 96-well microplate, and 0.5 μM or 5 μM DPP-8 / 9 inhibitor 1G244 and 20 nM, respectively. Alternatively, 40 nM bortezomib was added and cultured for 72 hours. Thereafter, the Premix WST-1 reagent was added and further cultured for 1 hour, and the number of cells was semi-quantified using a microplate reader.

  The results are shown in FIG. It was confirmed that the effects were clearly synergistically improved compared to the case where each drug was used alone. In particular, the effect of bortezomib could be remarkably improved by using 1G244 in combination with bortezomib in an amount that could hardly be confirmed with a single agent.

Example 6 In vivo tumor growth inhibition test The in vivo tumor growth inhibitory effect of DPP-8 inhibitor 1G244 was examined.
5 × 10 ⁶ myeloma cells MM. 1S was injected subcutaneously into NOG mice, and 50 μL of anti-asialo GM1 antibody was administered to further immunodeficiently to produce tumor-bearing model mice. When the tumor became palpable, the tumor volume was measured. The tumor volume was calculated by measuring the minor axis and major axis of the tumor, and the tumor volume = (minor axis) ² × (major axis) × 0.5. The tumor-bearing model mice were divided into an untreated group (n = 3) and a 1G244 administration group (n = 3), respectively, and phosphate buffered saline (PBS) was added to the 1G244 administration group as a negative control in the untreated group. 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.

  The 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. From this, 1G244 is MM. It was found to suppress the proliferation of 1S cells.

  The pharmaceutical composition of the present invention exhibits an effect by inducing apoptosis of myeloma cells by a new mechanism of action that has not been known before. In particular, since 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. In addition, it has been confirmed that 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.

Claims (16)

  A pharmaceutical composition for treating a plasma cell tumor, comprising at least one agent that suppresses the activity of dipeptidyl peptidase (DPP) -8.   The pharmaceutical composition according to claim 1, wherein the agent that suppresses the activity of DPP-8 is a DPP-8 inhibitor.   The pharmaceutical composition according to claim 1, wherein the agent that suppresses the activity of DPP-8 is an agent that suppresses the expression of the DPP-8 gene.   The pharmaceutical composition according to any one of claims 1 to 3, further comprising a pharmacologically acceptable carrier.   The pharmaceutical composition according to any one of claims 1 to 4, further comprising a tumor cell targeting agent.   The pharmaceutical composition according to claim 5, wherein the targeting agent is an agent that specifically recognizes CD138.   The pharmaceutical composition according to claim 6, wherein the agent that specifically recognizes CD138 is an anti-CD138 antibody.   The pharmaceutical composition according to any one of claims 1 to 7, wherein the plasma cell tumor is multiple myeloma.   9. The pharmaceutical composition according to any one of claims 1 to 8, characterized in that it is used for a subject for which conventional therapy is no longer effective.   Furthermore, the pharmaceutical composition as described in any one of Claims 1-9 used together with the other agent for treating 1 or 2 or more plasma cell tumors.   Another agent for treating a plasma cell tumor is selected from the group consisting of bortezomib, dexamethasone, melphalan, prednisolone, cyclophosphamide, vincristine, doxorubicin, thalidomide, lenalidomide, pomalidomide, carfilzomib and panobinostat Or the pharmaceutical composition of Claim 10 which is 2 or more types of agents.   A method for inducing apoptosis of a myeloma cell, comprising suppressing the activity of DPP-8 in the myeloma cell.   The method of inducing apoptosis of myeloma cells according to claim 12, comprising contacting the myeloma cells with an agent that suppresses the activity of DPP-8.   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:
(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 cells are detected in the subject when the amount of DPP-8 in the biological sample is significantly greater than a reference value. A method for screening an agent for use in the treatment of plasma cell tumors, comprising:
(A) contacting a candidate compound with a DPP-8-expressing cell;
(B) The said method including measuring the activity level of DPP-8 in the DPP-8 expression cell after contact with the said candidate compound.