JP2006327980A - Antileukemic activity improver - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antileukemic activity improver or apoptosis activity improver capable of improving antileukemic activity, especially apoptosis activity, of antileukemic compositions, and to provide an antileukemic agent, a leukemic cell remover, a combination therapy of leukemia and a method for removing leukemic cells, using the improver. <P>SOLUTION: The peptide having amino acid sequence represented by sequence No.1 has improving effect on antileukemic activity of an antileukemic composition. Accordingly, medicines containing the peptide as an active ingredient is useful as an antileukemic activity improver, and the combination of the peptide and the antileukemic composition is useful for an antileukemic agent, a leukemic cell remover, a combination therapy of leukemia and a method for removing leukemic cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an apoptosis activity enhancer, an anti-leukemia activity enhancer, an anti-leukemia drug, a leukemia cell removal agent, a leukemia cell combination therapy, and a leukemia cell removal method.

Conventionally, daunorubicin, cytarabine (cytosine arabinoside: Ara-C), 6-MP (mercaptopurine), prednisone, hydroxycarbamide, vincristine, melphalan, adriamycin, dexamethasone, retinoic acid, camptothecin, etc. are effective as anti-leukemia compositions What was made into an ingredient is known. However, since some anti-leukemic compositions cause serious side effects, in recent years, the development of chemical substances that reduce the side effects and enhance the anti-leukemic activity of the anti-leukemic compositions, and combination therapy using these chemical substances, Attempts have been made to treat leukemia. Examples of such chemical substances include difaranizole A (see Patent Document 1) capable of enhancing the physiological activity of retinoic acid, and anti-VLA-4 antibody capable of enhancing the apoptosis-inducing activity of the anti-leukemic composition. (See Non-Patent Document 1) and the like have been developed.
JP 11-302194 A Nat Med. 2003, 9 (9), 1158-1165

  The present invention relates to a novel anti-leukemia activity enhancer or apoptotic activity enhancer capable of enhancing the anti-leukemia activity, particularly apoptotic activity of the anti-leukemia composition, as well as an anti-leukemia drug, leukemia cell removing agent using the same, The object is to provide a combination therapy for leukemia and a method for removing leukemia cells.

  Conventionally, as a substance that enhances the anticancer activity of a chemotherapeutic agent (for example, doxorubicin, vincristine, actinomycin D, etc.), FNIII14, which is a peptide derived from fibronectin, is known. For cancer cells derived from solid tumors such as A375 cells derived from malignant melanoma, ACHN cells derived from liver cancer, GI-1 cells derived from gliosarcoma, GT3TKB cells derived from gastric cancer, PC-3 cells derived from prostate cancer It has been reported that apoptosis can be efficiently induced (see WO 01/8698 pamphlet).

  However, it was not clear whether the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “FNIII14”) has an effect of enhancing the apoptosis-inducing activity of leukemia cells of the anti-leukemic composition. Therefore, the present inventors examined whether FNIII14 has an effect of enhancing the apoptosis-inducing activity of leukemia cells of the anti-leukemic composition, as in the method described in WO 01/8698. See Example 1). As a result, it was revealed that FNIII14 does not enhance the apoptosis-inducing activity of the anti-leukemic composition against leukemia cells.

  However, as a result of intensive efforts, the present inventors have revealed that FNIII14 can enhance the apoptosis-inducing activity of leukemia cells of anti-leukemic composition AraC in a container coated with stromal cells or fibronectin.

  Next, the present inventors examined whether FNIII14 and an anti-leukemia composition were useful for vertebrates suffering from leukemia in order to examine whether or not FNIII14 and FNIII14 were combined with an acute myeloid leukemia (AML) model mouse. The anti-leukemia composition was administered intraperitoneally and the combined effect of FNIII14 and the anti-leukemia composition on the model mice was investigated. However, the survival time was significantly increased compared to the single use of the anti-leukemia composition. Not (see Example 3). When FNIII14 was administered intraperitoneally, FNIII14 was decomposed by some peptidase in vivo and the effect of FNIII14 could not be obtained. In the following experiments, FNIII14 was administered intravenously.

  By the way, the progress of anti-leukemia composition and supportive therapy has improved the complete tolerance (CR) rate of AML to about 80%, but it has many recurrences and the long-term survival rate is about 30-40%. In order to improve, the development of recurrence prevention methods is required. This recurrence is said to be caused by a small amount of AML cells (minimal residual disease (MRD)) remaining in the CR bone marrow. In order to investigate whether a combination therapy of FNIII14 and an anti-leukemia composition is effective in killing residual AML cells in CR bone marrow, human leukemia cells, U937 cells, were used for MRD. A model mouse was prepared (Example 4: see FIG. 4B), the anti-leukemia composition AraC and FNIII14 were intravenously administered into the abdominal cavity, and the combined effect of AraC and FNIII14 on the MRD model mouse was examined. As a result, it has been clarified that FNIII14 enhances the effect of AraC and significantly prolongs the survival period of MRD model mice by co-administration with AraC (see Example 5). This indicated that FNIII14 was used as a highly effective leukemia therapeutic agent for use in combination with an anti-leukemic composition.

  In addition, the present inventors used a mouse in which the survival time was significantly prolonged by the combined use of the anti-leukemic composition and FNIII14, and determined whether or not U937 cell-derived human Alu sequences exist in each organ by PCR. When examined, human Alu sequences derived from U937 cells were not detected in these organs (Example 4: see FIG. 4C). Therefore, FNIII14 not only promotes the effects of AraC and significantly prolongs the survival of AML mice, but also can treat leukemia cells including leukemia cells by killing leukemia cells. Was suggested. Thus, the present inventors have completed the present invention.

  That is, the apoptotic activity enhancer according to the present invention is an apoptotic activity enhancer that enhances the apoptotic activity of the anti-leukemic composition, and contains a peptide having the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient. The apoptotic activity enhancer may be, for example, a preparation for intravenous administration.

  The anti-leukemia activity enhancer according to the present invention is an anti-leukemia activity enhancer that enhances the anti-leukemia activity of the anti-leukemia composition, and contains a peptide having the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient. . The anti-leukemia activity enhancer is preferably a formulation for intravenous administration.

  The anti-leukemic drug according to the present invention is a combination of a peptide having the amino acid sequence shown in SEQ ID NO: 1 and an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide. The anti-leukemic agent according to the present invention is particularly useful for inducing apoptosis of leukemia cells in the bone marrow. The peptide is preferably administered by an intravenous route. The anti-leukemic drug according to the present invention may be a combination of a peptide having the amino acid sequence represented by SEQ ID NO: 1 and an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide, A kit comprising a drug containing a peptide and the anti-leukemic composition may be used.

  Furthermore, the leukemia cell removing agent according to the present invention is a leukemia cell removing agent that induces apoptosis in leukemia cells cultured in a container coated with stromal cells or an extracellular matrix thereof and removes leukemia cells, SEQ ID NO: 1. A peptide having the amino acid sequence shown in 1 is combined with an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide. The leukemia cell removing agent according to the present invention may be a combination agent of a peptide having the amino acid sequence represented by SEQ ID NO: 1 and an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide, A kit comprising a drug containing the peptide and the anti-leukemic composition may be used.

  The leukemia combination therapy according to the present invention includes a step of administering to a vertebrate other than a human a peptide having the amino acid sequence shown in SEQ ID NO: 1 and an anti-leukemia composition whose anti-leukemic activity is enhanced by the peptide. . This leukemia combination therapy is particularly useful for inducing apoptosis of leukemia cells in the bone marrow. The peptide is preferably administered intravenously.

  In addition, the method for removing leukemia cells according to the present invention includes a peptide having the amino acid sequence shown in SEQ ID NO: 1 in a leukemia cell cultured in a container coated with stromal cells or extracellular matrix thereof, Acting with an anti-leukemic composition with enhanced activity.

  In addition, as said peptide used in this invention, the peptide which consists of an amino acid sequence shown by sequence number 2 can be mentioned, for example. In addition, the anti-leukemic activity of the above-described anti-leukemic composition may be caused by, for example, induction of apoptosis on leukemia cells. Furthermore, as the anti-leukemic composition, for example, cytarabine (cytosine arabinoside: Ara-C), daunorubicin, or the like can be used.

  According to the present invention, a novel anti-leukemia activity enhancer or apoptotic activity enhancer capable of enhancing the anti-leukemia activity, particularly apoptotic activity of the anti-leukemia composition, and an anti-leukemia drug and leukemia cell removal using the same An agent, a combination therapy for leukemia, and a method for removing leukemia cells can be provided.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Pharmacological action of peptide having amino acid sequence shown in SEQ ID NO: 1 ==
In order to enhance the action of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (TEATITGLEPGTEYTIYVIAL), FNIII14 (SEQ ID NO: 2: TEATITGLEPGTEYTIYVIALC) added with Cys at the C-terminus of the peptide so that the two peptides can be linked by a disulfide bond. ) Has the effect of enhancing the apoptosis-inducing activity of the anti-leukemic composition against leukemia cells cultured in a container coated with an extracellular matrix such as stromal cells or fibronectin, as described above. Therefore, it can be said that the peptide having the amino acid sequence shown in SEQ ID NOs: 1 and 2 is useful as an anti-leukemic activity enhancer that enhances the anti-leukemic activity resulting from the induction of apoptosis of the anti-leukemic composition.

  Furthermore, when leukemia cells cultured in a container coated with an extracellular matrix such as stromal cells or fibronectin are allowed to react with FNIII14 and an anti-leukemia composition, apoptosis can be efficiently induced in the leukemia cells. The peptide having the amino acid sequence shown in No. 1 and the anti-leukemic composition are considered useful for removing leukemia cells in the blood. As a result, leukemia cells in blood collected for blood transfusion can be removed, so that blood infection of leukemia can be prevented and blood for transfusion ensuring safety can be provided. . Moreover, since leukemia cells in the blood extracted from the living body can be removed, the treated blood can be returned to the living body.

  Further, as described above, it was shown that FNIII14 was intravenously administered to leukemia model animals to enhance the anti-leukemic activity of the administered anti-leukemia composition. On the other hand, it became clear that even when intraperitoneally administered FNIII14 and an anti-leukemic composition to a leukemia model animal, the anti-leukemic activity of the anti-leukemic composition was not enhanced. Therefore, FNIII14 can be directly administered intravenously, or a conjugate of FNIII14 and a polymer carrier (eg, a known carrier such as KLH, BSA, MAP core, polylysine, polyethyleneimine, polytamine, chitosan, synthetic peptide, dendrimer). It is considered that the effect of FNIII14 can be obtained by administering a liposome encapsulating FNIII14 or administering a liposome encapsulating FNIII14. Therefore, a drug containing a peptide having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient is useful as an anti-leukemia activity enhancer that enhances the anti-leukemia activity of an anti-leukemia composition in vivo. The combination with a leukemia composition is useful as an anti-leukemia drug, particularly an anti-MRD drug.

  In the present embodiment, the leukemia is not particularly limited as long as it is a leukemia caused by leukemia cells. For example, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute Leukemia caused by VLA-4 positive leukemia cells such as lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), B cell chronic lymphocytic leukemia (B-CLL) is preferred.

  Examples of the anti-leukemic composition include chlorambucil, daunorubicin (DNR), hydroxycarbamide, cytarabine (AraC), camptothecin, etoposide (VP-16), 6-MP (mercaptopurine), vincristine (VCR), enocitabine (Behenoyl AraC, BHAC), cytarabine oxophosphate (YNK-01), methotrexate (MTX), idarubicin (IDR), doxorubicin (DXR), aclarubicin (ACR), cyclophosphamide (CY), busulfan, nymustine ( ACNU), ranimustine (MCNU), vindesine (VDS), corticosteroids (eg, prednisone, prednisolone, dexamethasone, etc.), mitozantrone (MIT), sobuzoxane (MST-16), fludarabine, pentostatin, L-asparaginase, retinoic acid (ATRA), Tomivaro Effective substances such as (Am80), arsenous acid, imatinib (STI571), interferon alpha (IFN), and other substances that are conventionally used as therapeutic agents for leukemia, useful as leukemia therapeutic agents, or combinations of two or more thereof The thing contained as a component can be mentioned.

  The peptide having the amino acid sequence shown in SEQ ID NO: 1 only needs to contain the amino acid sequence, and any other sequence may be used. Examples of such peptides include FNIII14 and fibronectin to which all or a part of FNIII14 and fibronectin (see GenBank Accession number AB191261) and a tag (for example, His tag, GST tag) are bound.

  In the above description, the case where the peptide having the amino acid sequence represented by SEQ ID NO: 1 is used has been described. However, in the present invention, an expression vector containing a polynucleotide encoding the peptide, the expression vector is contained, Cells that secrete the peptide and bacteria or viruses that infect the cell and secrete the peptide outside the cell can also be used in place of the peptide in anti-leukemic activity enhancers and anti-leukemic agents.

  In addition, a complex of a plasmid capable of expressing the peptide having the amino acid sequence shown in SEQ ID NO: 1 and a polymer carrier can also carry the plasmid to the target tissue and release the drug there. The complex can be used in place of the peptide. Examples of the polymer carrier used in this case include, but are not limited to, polylysine, polyethyleneimine, porotamine, chitosan, synthetic peptide, and dendrimer. In particular, a conjugate of polylysine and an antibody against a surface antigen expressed specifically in a target tissue can efficiently carry the plasmid to the target tissue and send the plasmid into the target cell (see JP-A-7-216000). ).

  In addition, since a liposome encapsulating a plasmid capable of expressing a peptide having the amino acid sequence shown in SEQ ID NO: 1 can also carry the plasmid to the target tissue and release the drug there, The liposome can be used. In this case, the polynucleotide can be introduced into the target organ or tissue by using Trans IT In Vivo Gene Delivery System (trade name of TaKaRa) or the like. In addition, since the polymer micelle has excellent characteristics as a drug carrier, the polymer micelle encapsulating the peptide or polynucleotide can also use the polymer micelle instead of the peptide.

== Method for producing peptide having amino acid sequence shown in SEQ ID NO: 1 ==
The peptide having the amino acid sequence shown in SEQ ID NO: 1 can be synthesized organically based on the sequence information. In addition, a polynucleotide encoding a peptide having the amino acid sequence shown in SEQ ID NO: 1 is inserted into an expression vector having an appropriate enhancer / promoter, and this recombinant vector is used for bacteria such as Escherichia coli and Salmonella, yeast, animal cells It may be introduced and expressed. Alternatively, mRNA obtained by in vitro transcription of this recombinant vector may be translated by an in vitro translation system using rabbit reticulocyte extract, Escherichia coli S30 extract, malt extract, wheat embryo extract or the like. Good.

  Here, when preparing an expression vector, a fusion polynucleotide obtained by fusing a polynucleotide encoding a tag such as a His tag or GST tag and a polynucleotide encoding a peptide having the amino acid sequence shown in SEQ ID NO: 1 is used as the vector More preferably, it is inserted into and expressed as a fusion protein. This is because the target peptide can be easily purified using the tag. The tag can be removed at the final stage, and only the peptide having the amino acid sequence shown in SEQ ID NO: 1 can be purified by HPLC (high-performance (-speed) liquid chromatography) or the like.

  In addition, in order to secrete the peptide having the amino acid sequence shown in SEQ ID NO: 1 to the outside of the cell, a recombinant vector capable of expressing the peptide added with a signal peptide has an appropriate enhancer / promoter as described above. It can be prepared by inserting a polynucleotide encoding a peptide having the amino acid sequence shown in SEQ ID NO: 1 into an expression vector. In addition, cells (recombinant cells) containing the recombinant vector, bacteria or viruses for infecting cells in the living body and secreting the peptide outside the cell can be produced as follows. . For example, an animal cell, a bacterium, or a virus is infected with the recombinant vector using an electroporation method, a microinjection method, a lipofection method, a virus vector such as an adenovirus or a retrovirus, or calcium. It can be prepared by introduction by a transfection method or the like. When bacteria or viruses are used, it is preferable that the bacteria and viruses are detoxified in advance so as not to cause toxicity and side effects. Examples thereof include retroviruses and adenoviruses that have been infected so that they do not replicate.

== Administration of a peptide having the amino acid sequence shown in SEQ ID NO: 1 ==
A drug containing the peptide having the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient, a peptide having the amino acid sequence shown in SEQ ID NO: 1 and an anti-leukemia composition whose anti-leukemia activity is enhanced by the peptide When the combination drug is administered to a human or non-human vertebrate who has developed leukemia, it may be administered directly to the vicinity of the affected area to be introduced, but it may be administered orally, intravenously, intraperitoneally, etc. It may be administered orally. In addition, when administering the peptide which has an amino acid sequence shown by sequence number 1 with respect to a human or a non-human vertebrate, it is preferable to administer intravenously. Moreover, the anti-leukemia composition used as an anti-leukemia drug may be administered to the same site or a different site as the peptide having the amino acid sequence shown in SEQ ID NO: 1.

  When the above drugs and compositions are administered orally, they may be formulated into tablets, capsules, granules, powders, syrups, etc. When administered intraperitoneally or intravenously, injections, infusions A preparation such as an agent may be used. In addition, the said formulation is manufactured by a conventional method using the conventionally used formulation additive (For example, an excipient | filler, binder, a lubricant, a disintegrating agent, a corrigent, a solvent, a stabilizer, etc.). be able to.

  Hereinafter, it demonstrates in detail using an Example.

[Example 1]
According to the method described in the literature (J Biol Chem. 266, 8807-13, 1991), each well of a 96-well plate was coated at a concentration of 5 μg / ml of human plasma fibronectin, and bovine serum albumin (manufactured by Sigma) was coated. Blocked. Thereafter, a culture solution containing no serum (a culture solution in which Dulbecco's modified Eagle medium (DMEM) and Ham F12 medium were mixed at a volume ratio of 1: 1 (manufactured by Nissui)); hereinafter referred to as “DMEM / Ham medium” Mouse melanoma cell line B16-BL6 (2 × 10 ⁴ cell) or human leukemia cell line K562 (1.5 × 10 ⁴ cell) suspended in the well, and incubator at 37 ° C. and 5% CO ₂ Incubated for 2 hours. After the culture, the supernatant was removed, and vinblastine (manufactured by Wako Pure Chemical Industries, Ltd.) was adjusted to various concentrations as shown in FIG. 1, and FNIII14 (see SEQ ID NO: 1) was 50 μg / mg. The solution was added to make ml, and further cultured for 19 hours. As a control, a culture prepared by adding only vinblastine was prepared. After that, the culture medium was replaced with DMEM / Ham medium containing 10% fetal bovine serum (URH BIOSCIENCES) and further cultured overnight. After fixing the cells with 5% formaldehyde (Wako Pure Chemical Industries), apoptosis was performed. The cells that had undergone the washing were washed away with PBS. Viable cells attached to the plate are stained with 0.5% crystal violet solution, washed with water, and the cells are lysed with 1% SDS to extract the dye, and the absorbance (Spectra Rainbow Thermo, manufactured by Wako Pure Chemical Industries, Ltd.) is used for absorbance ( The ratio of viable cells was calculated by measuring (OD595nm). The result is shown in FIG.

  As shown in FIG. 1, the use of FNIII14 significantly enhanced the apoptosis-inducing activity of vincristine on B16-BL6 cells, but no significant effect was observed on K562 cells.

[Example 2]
The effects of anti-leukemic composition and FNIII14 on leukemia cells cultured in vessels coated with stromal cells or extracellular matrix fibronectin were investigated.

6-well plate coated with fibronectin containing human acute myeloid leukemia (AML) cell line U937 (5 × 10 ⁶ cells; American Type Culture Collection) in RPMI 1640 medium containing 10% FCS (Becton Dickinson Labware) And cultured in an incubator at 37 ° C. and 5% CO ₂ for 24 hours to adhere the cells to fibronectin. Thereafter, 50 or 100 μg / mL FNIII14, 10 μg / mL anti-VLA-4 antibody (VLA-4 Ab) and the like were added and cultured for 1 hour, and 10 ⁻⁶ M AraC (cytarabine) was added and cultured for 24 hours ( As a control, a culture prepared without adding FNIII14, VLA-4 Ab, AraC or the like was prepared. After the culture, the cell viability was measured by the trypan blue extracellular elimination test method. The result is shown in FIG.

  As shown in FIG. 2, FNIII14 was found to enhance the apoptosis-inducing activity of AraC against U936 cells, similar to VLA-4 Ab. Thus, it has been shown that FNIII14 can enhance the apoptosis-inducing activity of anti-leukemic compositions against leukemia cells cultured in containers coated with extracellular matrix such as stromal cells or fibronectin.

[Example 3]
Next, in order to investigate whether a combination therapy of FNIII14 and an anti-leukemia composition is useful for vertebrates suffering from leukemia, an acute myeloid leukemia (AML) model mouse was prepared, and FNIII14 against the model mouse was prepared. And the combined use effect of the anti-leukemic composition were examined.

U937 cells (5 × 10 ⁶ cells) were injected from tail vein into 6-10 week-old SCID (CB-17 / lcrCrj-scid / scid) mice (Charles River Japan Laboratories) treated with whole body irradiation (4Gy) The AML model mice that show symptoms of acute leukemia such as paralysis of the lower limbs were prepared in about 3 weeks. To this AML model mouse, 1.0 mL of PBS in which AraC (20 mg), FNIII14 (1 mg), VLA-4 Ab (1 mg), etc. were dissolved was intraperitoneally administered, and the average survival time of each administration group was measured (note that control) As a group, those not administered with AraC, FNIII14, VLA-4 Ab, etc. were prepared.) The result is shown in FIG.

  As shown in FIG. 3, the survival period of the AraC administration group was significantly prolonged as compared with the control group. Further, when VLA-4 Ab was further administered in addition to AraC, the survival period was further prolonged (AraC + VLA-4 Ab administration group). However, in the group in which FNIII14 was further administered in addition to AraC (AraC + FNIII14 administration group and AraC + FNIII14 + VLA-4 Ab administration group), there was no significant difference in survival time compared to the AraC administration group. .

[Example 4]
In this example, MRD model mice were prepared by transplanting human AML cells, and whether or not human AML cells were extinct by administration of an anti-leukemic composition and FNIII14 was examined using the presence of human Alu sequences as an index. .

<MRD model mouse production>
Similar to the method described in Example 3, U937 cells were injected into the tail vein of SCID mice and transplanted. In order to examine whether or not U937 cells infiltrated each organ of the mouse on the 7th and 14th day of transplantation, the human ALU sequence was amplified by the polymerase chain reaction (PCR) method. The PCR of human ALU sequence was performed at 94 ° C. for 1 minute using 5′-CACCTGTAATCCCAGCAGTTT-3 ′ (FP1: SEQ ID NO: 3) and 5′-CGCGATCTCGGCTCACTGCA-3 ′ (RP1: SEQ ID NO: 4) as primers (denaturation). ), 60 ° C. × 45 seconds (annealing), and 72 ° C. × 1 minute (extension).

  After the PCR reaction, the amplified DNA was electrophoresed on a 3% agarose gel and stained with ethidium bromide to examine the presence of the Alu sequence. Note that mouse erythropoietin receptor (EPO-R) was used as a house keeping gene, and PCR was performed using 5′-TTGGCCTCAAAGCCCAGGCCA-3 ′ (FP2: SEQ ID NO: 5) and 5′-CACATAGCCGGGATGCAGAGG-3 ′ (RP2: SEQ ID NO: 6). ) As a primer, 27 cycles were performed at 94 ° C. × 1 minute (denaturation), 65 ° C. × 2 minutes (annealing), and 72 ° C. × 4 minutes (extension). FIG. 4A shows the detection results of human ALU sequences in each organ of mice on the 14th day of transplantation, and FIG. 4B shows the detection results of human ALU sequences in each organ of mice on the 7th day of transplantation.

  As shown in FIG. 4A, since human ALU sequences were detected in each organ of mice on the 14th day of transplantation, it was found that U937 cells infiltrated into these organs. On the other hand, as shown in FIG. 4B, since the human ALU sequence was detected only in the bone marrow in the mice on the seventh day of transplantation, it became clear that U937 cells were present only in the bone marrow. This revealed that the mice on the seventh day of transplantation are useful as a model for bone marrow MRD.

<Effect of combined use of anti-leukemia composition and FNIII14 on MRD>
The mice on the seventh day of transplantation (MRD model mice) were tested in the same manner as in Example 3 except that 200 μL of PBS in which FNIII14 (1 mg) was dissolved were intravenously administered. The effect of combined use with FNIII14 was examined. When FNIII14 was not administered, only PBS was intravenously administered. The result is shown in FIG.

  As shown in FIG. 5, PBS (intravenous administration) + AraC (intraperitoneal administration) administration group includes PBS (intravenous administration) + physiological saline (intraperitoneal administration) administration group, FNIII14 (intravenous administration) + Survival was significantly extended compared to the saline (intraperitoneal administration) group, and the FNIII14 (intravenous administration) + AraC (intraperitoneal administration) group was PBS (intravenous administration) + AraC (peritoneal administration). Internal administration) The survival period was further extended as compared with the administration group, and all mice were alive at the 62nd day after administration. Therefore, the PCR method was performed in the same manner as described above to confirm the presence or absence of the human ALU sequence in each organ of the mouse surviving on the 62nd day after administration. The result is shown in FIG. 4C.

  As shown in FIG. 4C, by administering FNIII14 intravenously and AraC intraperitoneally, DNA of human ALU sequence was not detected in all organs examined including bone marrow. Based on the above, FNIII14 not only enhances the effect of AraC administered in combination by intravenous administration, significantly prolongs the survival period of AML mice, but also treats leukemia and MRD caused by VLA4-positive leukemia cells It was suggested to be useful as

In one Example of this invention, it is a figure which shows the result of having investigated the influence which FNIII14 has on the apoptosis induction activity with respect to B16-BL6 cell or K562 cell of vincristine. In one Example of this invention, it is a figure which shows the result of having investigated the influence which FNIII14 has on the apoptosis induction activity of AraC with respect to the leukemia cell cultured in the 6 well plate coated with fibronectin. In one Example of this invention, it is a figure which shows the result of having investigated the effect which AraC, FNIII14, VLA-4 Ab, etc. have on the survival of a leukemia model mouse. In one example of the present invention, on day 7 (A) and day 14 (B) after transplantation of human leukemia cells into mice, or in combination with AraC and FNIII14 in MRD model mice, 62 It is a figure which shows the result of having investigated the presence of the human ALU sequence in each organ by PCR method on the day (C). In one Example of this invention, it is a figure which shows the result of having investigated the effect which AraC, FNIII14, etc. have on the survival of a MRD model mouse.

Claims (33)

An apoptotic activity enhancer that enhances the apoptotic activity of an anti-leukemic composition comprising:
An apoptotic activity enhancer comprising a peptide having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.   The apoptotic activity enhancer according to claim 1, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   The agent for enhancing apoptosis activity according to claim 1 or 2, which is a preparation for intravenous administration.   The said anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C), The apoptosis activity enhancer in any one of Claims 1-3 characterized by the above-mentioned. An anti-leukemia activity enhancer that enhances the anti-leukemia activity of the anti-leukemia composition,
An anti-leukemic activity enhancer comprising a peptide having the amino acid sequence shown in SEQ ID NO: 1 as an active ingredient.   6. The anti-leukemia activity enhancer according to claim 5, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   The anti-leukemic activity enhancer according to claim 5 or 6, wherein the preparation is for intravenous administration.   The anti-leukemic activity enhancer according to any one of claims 5 to 7, wherein the anti-leukemic activity of the anti-leukemic composition results from apoptosis induction on leukemia cells.   The anti-leukemic activity enhancer according to any one of claims 5 to 8, wherein the anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C).   An anti-leukemia drug comprising a combination of a peptide having the amino acid sequence shown in SEQ ID NO: 1 and an anti-leukemia composition whose anti-leukemia activity is enhanced by the peptide.   The anti-leukemic drug according to claim 10, wherein the peptide is a peptide having an amino acid sequence represented by SEQ ID NO: 2.   The anti-leukemic agent according to claim 10 or 11, wherein the anti-leukemic activity is caused by induction of apoptosis of leukemia cells.   The anti-leukemia drug according to claim 12, which induces apoptosis in leukemia cells in bone marrow.   The anti-leukemic agent according to claim 13, wherein the peptide is administered by an intravenous route.   The anti-leukemic drug according to claim 10, wherein the anti-leukemic drug is a combination drug.   The anti-leukemia drug according to any one of claims 10 to 14, wherein the anti-leukemia drug is a kit comprising a drug containing the peptide and the anti-leukemia composition.   The anti-leukemic composition according to any one of claims 10 to 16, wherein the anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C). A leukemia cell removing agent that induces apoptosis in leukemia cells cultured in a container coated with stromal cells or an extracellular matrix thereof to remove leukemia cells,
A leukemia cell removing agent comprising a combination of a peptide having the amino acid sequence represented by SEQ ID NO: 1 and an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide.   The leukemia cell removing agent according to claim 18, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   The leukemia cell removing agent according to claim 18 or 19, wherein the anti-leukemic activity is caused by induction of apoptosis of leukemia cells.   The leukemia cell removing agent according to any one of claims 18 to 20, wherein the blood treatment agent is a combination drug.   The leukemia cell removing agent according to any one of claims 18 to 20, wherein the blood treatment agent is a kit comprising a drug containing the peptide and the anti-leukemic composition.   The leukemia cell removing agent according to any one of claims 18 to 22, wherein the anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C).   A combination therapy for leukemia, comprising administering a peptide having the amino acid sequence represented by SEQ ID NO: 1 and an anti-leukemia composition whose anti-leukemia activity is enhanced by the peptide to vertebrates other than humans.   The combination therapy for leukemia according to claim 24, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   26. The combination therapy for leukemia according to claim 24 or 25, wherein the anti-leukemic activity results from apoptosis induction on leukemia cells.   27. The combination therapy for leukemia according to claim 26, wherein apoptosis is induced in leukemia cells in the bone marrow.   The leukemia combination therapy according to any one of claims 24 to 27, wherein the peptide is administered intravenously.   29. The combination therapy for leukemia according to any one of claims 24 to 28, wherein the anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C).   A peptide having the amino acid sequence shown in SEQ ID NO: 1 and an anti-leukemic composition whose anti-leukemic activity is enhanced by the peptide are allowed to act on leukemia cells cultured in a container coated with stromal cells or an extracellular matrix thereof. And a method for removing leukemia cells.   The method for removing leukemia cells according to claim 30, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.   32. The method for removing leukemia cells according to claim 30 or 31, wherein the anti-leukemic activity is caused by induction of apoptosis of leukemia cells. The method for removing leukemia cells according to any one of claims 30 to 32, wherein the anti-leukemic composition is cytarabine (cytosine arabinoside: Ara-C).