JP2011184325A - Cancer therapeutic agent using inhibition of hcp-1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effect-reinforcing agent of a cancer therapeutic agent and an anticancer agent. <P>SOLUTION: The effect-reinforcing agent of a cancer therapeutic agent or an anticancer agent includes a substance to inhibit the expression of the gene coding HCP-1 or a substance to inhibit the function of HCP-1 protein. As the substance to inhibit the expression of the gene coding HCP-1, at least one kind selected from the group consisting of an antisense nucleic acid against the gene, a decoy nucleic acid, a microRNA, an shRNA, an siRNA, and an aptamer is used, and as the substance to inhibit the function of HCP-1 protein, an antibody to the HCP-1 protein is used. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for enhancing the therapeutic effect of a cancer chemotherapeutic agent by inhibiting the function of HCP-1 protein, and an enhancer.

The present inventors have previously found for the first time in the world that autofluorescent material is formed in association with the formation of gastric mucosal hemorrhagic lesions in rats, and that the fluorescent material is porphyrin (Matsui H et al. Dig Dis Sci. 1994 Jan; 39 (1): 116-23: Non-patent document 1).
Photodynamic therapy (PDT) has been implemented in Japan as a treatment for digestive organ cancer, and is still being implemented today. PDT is a treatment method that induces cancer cell-specific apoptosis by generating active oxygen by the reaction of porphyrin and laser, utilizing the phenomenon of cancer-specific porphyrin accumulation. The drugs administered in this treatment method are roughly classified into two types. One is aminolevulinic acid (ALA) which is a precursor, and the other is a pigment (porphyrins and chlorins) having a porphine ring structure.

Regarding the mechanism of the accumulation phenomenon of the above-mentioned cancer-specific porphyrin ring structure, several reports have been made as follows.
(i) Fluorescence is up-regulated by crosslinking with albumin and iron-binding globulin (Hamblin MR et al., J Photochem Photobiol B. 26, 45-56, 1994: Non-Patent Document 2).
(ii) Hyaluronic acid has a sensitizing effect on porphyrin uptake (Kakehashi A et al., Ophthalmic Res. 26, 51-9, 1994: Non-Patent Document 3)
(iii) Plasma factor hemopexin that binds to porphyrin forms a complex with hematoporphyrin and accumulates in cells (Sery TW et al., Cancer Res. 39, 96-100, 1979: Non-patent document 4) )
(iv) Fluorescence accumulates predominantly in cancer cells by the action of the LDL receptor (Misawa J et al., J Dermatol Sci. 40, 59-61, 2005: Non-Patent Document 5).
(v) Mutation of MDR inhibits porphyrin excretion (Gibson SL et al., Cancer Res. 54 (10), 2556-9, 1994: Non-Patent Document 6).

However, scientific reports are difficult to explain in any of the reports, and they have not been regarded as theories.
On the other hand, the inventor has been interested in the accumulation phenomenon of this cancer-specific porphyrin for many years and has tried to elucidate it. As a result, we found that the mechanism inherent to cancer by the above-mentioned ALA administration is brought about by inactivation of ferrochelatase, the final enzyme of heme synthesis by high concentrations of NO in cancer cells (Yamamoto F et. Al., BBRC 353, 541-546, 2007: Non-patent document 7).
In addition, the present inventor has focused on Heme carrier protein 1 (HCP-1), a protein that selectively transports heme, which is one of porphin ring structures. HCP-1 is a protein isolated from the mouse duodenum in 1996 (Diatchenko L et. Al., PNAS, 93, 6025-6030, 1996: Non-patent document 8). In 2005, this protein was reported to be a transport carrier for porphine ring structures containing heme in the human duodenum (Shayeghi.M et. Al., Cell, 122, 789-801, 2005: Non-patent document 9). ). In addition, in 2006, HCP-1 was also reported to be a carrier of folic acid in the human duodenum (Qiu.A et. Al., Cell, 127, 917-928, 2006: Non-Patent Document 10).

Matsui H et al. Dig Dis Sci. 1994 Jan; 39 (1): 116-23 Hamblin MR et al., J Photochem Photobiol B. 26, 45-56, 1994 Kakehashi A et al., Ophthalmic Res. 26, 51-9, 1994 Sery TW et al., Cancer Res. 39, 96-100, 1979 Misawa J et al., J Dermatol Sci. 40, 59-61, 2005 Gibson SL et al., Cancer Res. 54 (10), 2556-9, 1994 Yamamoto F et.al., BBRC 353, 541-546, 2007 Diatchenko L et.al., PNAS, 93, 6025-6030, 1996 Shayeghi.M et.al., Cell, 122, 789-801, 2005 Qiu.A et. Al., Cell, 127, 917-928, 2006

  An object of the present invention is to provide a cancer therapeutic agent and an anti-cancer agent effect enhancer containing a substance that inhibits the function of HCP-1 protein or the expression of a gene encoding HCP-1.

The present inventor believes that if HCP-1 is expressed specifically in cancer, not only heme but also a porphin ring structure such as porphyrin may be transported into the cancer cell. The line RGM-1 was established by itself, and the expression of HCP-1 was examined in four types of cells, the cancer-like mutant RGK-1, whose cell line is the parent strain, and human gastric cancer-derived cultured cell types MKN45 and AGS. As a result, it was found that (i) HCP-1 expression was enhanced in cancer cells, and (ii) hematoporphyrin uptake into cancer cells was reduced by HCP-1 knockdown (4 ^th Soel National University Bungdang Hospital PDT Colloquium, 2009, July 8 ^th ).

 As a result of intensive studies to solve the above problems, the present inventor has found that inhibition of HCP-1 can efficiently kill cancer cells, and has led to the completion of the present invention.

That is, the present invention is as follows.
(1) A cancer therapeutic agent comprising a substance that inhibits expression of a gene encoding HCP-1 or a substance that inhibits the function of HCP-1 protein.
(2) An anticancer agent effect enhancer comprising a substance that inhibits the expression of a gene encoding HCP-1 or a substance that inhibits the function of HCP-1 protein.
In the cancer therapeutic agent or effect enhancer of the present invention, the substance that inhibits the expression of the gene encoding HCP-1 is, for example, a group consisting of siRNA, shRNA, microRNA, antisense nucleic acid, decoy nucleic acid and aptamer for the gene And at least one selected from. Examples of the substance that inhibits the function of the HCP-1 protein include an antibody against the HCP-1 protein or a porphine ring structure.

  INDUSTRIAL APPLICABILITY According to the present invention, there is provided an effect enhancer for cancer therapeutic agents and anticancer agents (for example, anticancer agents such as chemotherapeutic agents) containing a substance that inhibits the function of HCP-1 protein or the expression of a gene encoding the protein . The therapeutic agent and potentiator of the present invention can enhance toxicity against various cancers and efficiently kill cancer cells. For example, in cancer cells in which HCP-1 is expressed, the effect of the anticancer agent is enhanced. Therefore, the enhancer of the present invention is extremely useful for the treatment of cancers that express HCP-1.

  Hereinafter, the present invention will be described in detail.

It is a figure which shows the arrangement | sequence of HCP-1 siRNA. A cocktail in which equal amounts of all three sequences were mixed was used in the experiment (B-Bridge siTrio). It is a figure which shows the effect of siRNA with respect to HCP-1. HCP-1 siRNA (left) and scrambled RNA (right) were stained with anti-HCP-1 antibody (Abcam, Rabbit polyclonal antibody) (using VECTASTAIN ABC-AP Rabbit IgG kit). Secondary staining used Vector Red. The expression of HCP-1 is clearly suppressed by siRNA. It is a figure which shows the effect of siRNA with respect to HCP-1. The absorbance at 450 nm of each treated cell was measured (reference: 630 nm). There was a significant difference in the anti-cancer drug non-administration group, indicating that HCP-1 is important for maintaining cancer function and homeostasis. The cell viability on the vertical axis represents the absorbance in the MTT assay. It is a figure which shows the effect of siRNA with respect to HCP-1. Based on the results obtained by the TC-1 method (modified MTT assay), the absorbance of the anticancer agent non-administered group was taken as 1, and the absorbance of each treated cell was divided. In the CDDP-administered and 5-FU-administered groups, the cytotoxicity of the anticancer agent is significantly enhanced by the inhibition of HCP-1. The ADR administration group also has an enhanced cytotoxic effect.

1. Outline The present invention is a therapeutic agent for cancer and an effect enhancer for anticancer agents, including a substance that inhibits HCP-1 (protein or gene). Based on previous findings, it is possible to conclude that HCP-1 is specifically expressed in cancer and transports heme and folic acid into cells. Heme and folic acid are important substances involved in cell redox, detoxification, cell proliferation, and the like. Therefore, the present inventor thought that by inhibiting the action of this cancer-specific protein, cancer-selective cytotoxicity could be exhibited, and cancer cells of anticancer agents depending on the presence or absence of HCP-1 inhibition. Differences in toxicity were examined.
As a result, it has been found that by inhibiting the function or expression of the HCP-1 gene or protein, cancer can be treated and the effect of anticancer agents (for example, chemotherapeutic agents) is enhanced. That is, the present invention is characterized in that the cytotoxicity of an anticancer agent is enhanced by inhibiting the action of HCP-1 by a method that does not specifically limit the method, such as an interfering RNA, an antibody, or a competitive inhibitor.

An ideal anticancer drug is one that shows anticancer adoption only in cancer cells, but almost all drugs are also toxic to normal cells. Anticancer agents are ideal as the difference between anticancer activity and toxicity to normal cells increases. The present invention utilizes the fact that HCP-1 is expressed specifically in cancer and enhances the toxicity of anticancer agents.
In the present invention, by using HCP-1 RNAi, the expression of the HCP-1 gene can be inhibited, and the cancer therapeutic effect and the therapeutic effect of the anticancer agent can be enhanced. In addition, the cytotoxicity against cancer cells can be selectively enhanced by administering an antibody or nucleic acid inhibitor against HCP-1 alone, simultaneously with an anticancer agent, or before or after administration of an anticancer agent. Examples of HCP-1 inhibitors include porphin ring structures (hematoporphyrin derivatives or talaporphyrin sodium) that are taken into cancer cells via HCP-1.

2. HCP-1
As described above, Heme carrier protein 1 (HCP-1) is a protein that selectively transports heme, which is one of the porphine ring structures, and is a transporter that takes in folic acid in the human duodenum by cotransporting with protons. It is known.

In the present invention, cancer treatment is performed by targeting the gene encoding HCP-1 protein or HCP-1. Hereinafter, the term “HCP-1” in the present specification may be used to mean both the HCP-1 protein and the gene encoding HCP-1 for convenience.
The amino acid sequence of the HCP-1 protein and the base sequence of the gene encoding HCP-1 are known (NCBI), and the accession numbers are shown below.
Gene sequence: NM_080669.3 (SEQ ID NO: 1)
Amino acid sequence: NP_542400.2 (SEQ ID NO: 2)

3. HCP-1 function inhibitor The present invention refers to inhibiting the expression of a gene encoding HCP-1 and inhibiting the function of HCP-1 protein as "HCP-1 function inhibition", and these inhibitors Is called “HCP-1 function inhibitor”.

“Inhibition of HCP-1 function” refers to inhibiting HCP-1 activation by acting on HCP-1, inhibiting expression of HCP-1 on cells, and inhibiting production of HCP-1. This means that the signal transduction of HCP-1 is inhibited, or the transport function of HCP-1 is inhibited.
Therefore, as long as the function of HCP-1 is inhibited, the “HCP-1 function inhibitor” is not limited. For example, as an inhibitor of HCP-1 function, an antibody against HCP-1, an inhibitory nucleic acid against a gene encoding HCP-1 (also referred to as “HCP-1 gene”) (eg, siRNA, shRNA or aptamer, antisense nucleic acid, decoy) Nucleic acid, microRNA), porphine ring structure (hematoporphyrin derivative, or talaporphyrin sodium) and the like.

3-1. Inhibitory nucleic acid against HCP-1 gene
An inhibitory nucleic acid for the HCP-1 gene means a nucleic acid that suppresses its gene function or gene expression, for example, a nucleic acid used for RNA interference (microRNA, shRNA, siRNA), an antisense nucleic acid, a decoy nucleic acid, or An aptamer etc. are mentioned. These inhibitory nucleic acids can suppress the expression of the gene. The base sequence of the HCP-1 gene to be inhibited is known as described above, and sequence information can be obtained for each. In the present invention, the base sequence of the HCP-1 gene is shown in SEQ ID NO: 1, but not only the coding region of HCP-1 but also a non-coding region can be used.

(i) RNA interference In the present invention, gene expression can be inhibited using RNA interference (RNAi) against the HCP-1 gene. Examples of such nucleic acids used as RNAi include siRNA, microRNA (miRNA), and shRNA.
siRNA (small interfering RNA) molecules are various RNAs corresponding to the target HCP-1 gene. Examples of such RNA include mRNA, post-transcriptionally modified RNA of the HCP-1 gene, and the like.
In general, the target sequence on the mRNA can be selected from a cDNA sequence corresponding to the mRNA. However, the present invention is not limited to this region.
siRNA molecules can be designed based on criteria well known in the art. For example, as the target segment of the target mRNA, a continuous 15-30 base, preferably 19-25 base segment, preferably starting with AA, TA, GA or CA can be selected. The GC ratio of siRNA molecules is 30-70%, preferably 35-55%.
siRNA can be generated as a single-stranded hairpin RNA molecule that folds on its own nucleic acid to generate a double-stranded portion. siRNA molecules can be obtained by conventional chemical synthesis, but can also be produced biologically using expression vectors.
In order to introduce siRNA into cells, a method of linking synthesized siRNA to plasmid DNA and introducing it into cells, a method of annealing double-stranded RNA, and the like can be employed.

  The present invention can also use shRNA to produce RNAi effects. shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a partial region of a single strand forms a complementary strand with another region.

shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is set as sequence A and the complementary strand to sequence A is set as sequence B, these sequences are linked in the order of sequence A, spacer, and sequence B so that they are present in one RNA strand. Design to be 45-60 bases in length. The length of the spacer is not particularly limited.
Sequence A is the sequence of a partial region of the target HCP-1 gene, and the target region is not particularly limited, and any region can be a candidate. The length of the sequence A is 19 to 25 bases, preferably 19 to 21 bases.

Furthermore, the present invention can inhibit the expression of the HCP-1 gene using microRNA. MicroRNA (miRNA) is a single-stranded RNA with a length of about 20-25 bases that exists in cells, and is considered to have a function to regulate the expression of other genes (non-coding RNA) It is a kind of. miRNAs are produced as a nucleic acid that forms a hairpin structure that is produced by being processed when transcribed into RNA and suppresses the expression of a target sequence.
Since miRNA is also an inhibitory nucleic acid based on RNAi, it can be designed and synthesized according to shRNA or siRNA.

(ii) Antisense nucleic acid In another embodiment of the invention, an antisense nucleic acid can be used to inhibit the expression of the HCP-1 gene. An antisense nucleic acid is a single-stranded nucleic acid sequence (either RNA or DNA) that can bind to the mRNA or DNA sequence of the HCP-1 gene. The length of the antisense nucleic acid sequence is at least 14 nucleotides, preferably 14-100 nucleotides. The antisense nucleic acid binds to the gene sequence to form a duplex, and suppresses transcription or translation of the HCP-1 gene.
Antisense nucleic acids can be produced using chemical synthesis methods or biochemical synthesis methods known in the art. For example, a nucleic acid synthesis method using a commonly used DNA synthesizer can be used. Antisense nucleic acids are introduced into cancer cells that express HCP-1 by various gene transfection methods such as DNA transfection or electroporation.

(iii) Decoy nucleic acid In another embodiment of the present invention, the expression of the HCP-1 gene can be inhibited by using a decoy nucleic acid called a decoy nucleic acid.
A decoy nucleic acid is a nucleic acid that suppresses HCP-1 gene expression by binding to a transcription factor of the HCP-1 gene or HCP-1 receptor gene and suppressing the promoter activity, and includes a binding site for the transcription factor It means a short decoy nucleic acid. When this nucleic acid is introduced into cancer cells, transcription factors bind to the nucleic acid. Thereby, the binding of the transcription factor to the genome binding site is competitively inhibited, and as a result, the expression of the transcription factor is suppressed.

The preferred decoy nucleic acid of the present invention includes, for example, a nucleic acid that binds to the promoter of the HCP-1 gene, or a nucleic acid that binds to the promoter of HCP-1 mRNA or a factor present upstream of HCP-1. The decoy nucleic acid can be designed as a single strand or a double strand based on the promoter sequence of the HCP-1 gene. The length of the decoy nucleic acid is not particularly limited, and is 15 to 60 bases, preferably 20 to 30 bases.
The decoy nucleic acid may be DNA or RNA, and may include a modified nucleic acid.
The decoy nucleic acid used in the present invention can be produced using a chemical synthesis method or a biochemical synthesis method known in the art. For example, a nucleic acid synthesis method using a DNA synthesizer generally used in gene recombination techniques can be used. In addition, after isolating or synthesizing a base sequence serving as a template, a PCR method or a gene amplification method using a cloning vector can also be used. Furthermore, in order to obtain a more stable decoy nucleic acid in a cell, chemical modification such as alkylation or acylation can be added to a base or the like.

  For analysis of promoter transcriptional activity when decoy nucleic acid is used, a commonly performed luciferase assay, gel shift assay, RT-PCR, or the like can be employed. Kits for performing these assays are also commercially available (eg, promega dual luciferase assay kit).

(iv) Aptamer Furthermore, the present invention can inhibit the expression of the HCP-1 gene using an aptamer.
Aptamers are synthetic DNA or RNA molecules and peptide molecules capable of specifically binding to a target substance, and can be chemically synthesized in a test tube in a short time. Therefore, it can be obtained based on the base sequence shown in SEQ ID NO: 1 or by an evolutionary engineering method known as in vitro selection method or SELEX method.
Since nucleic acid aptamers are rapidly decomposed and removed by nucleases in the bloodstream, it is preferable to extend the half-life by performing molecular modification with a polyethylene glycol (PEG) chain or the like as necessary.

3-2. Antibody to HCP-1 An antibody against HCP-1 contained in the therapeutic agent or enhancer of the present invention can be prepared as follows.

(i) Preparation of polyclonal antibody In preparing the above antibody, first, an HCP-1 protein to be used as an immunogen (antigen) is prepared. For example, HCP-1 can be produced by genetic engineering based on the amino acid sequence shown in SEQ ID NO: 1. Alternatively, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having HCP-1 activity can also be used. “HCP-1 activity” means the transporter activity of a porphine ring structure. For example, the transporter activity is measured by an experiment of uptake of heme radiolabeled with central iron or an experiment of uptake of [ ³ H] -labeled folic acid. be able to.
In addition, the amino acid sequence information of HCP-1 protein can be obtained from the NCBI database. As the purified HCP-1 antibody, a commercially available product can also be used (for example, manufactured by Abcam).

  Next, the animal is immunized with the obtained purified HCP-1. Immunization is performed by administering a solution containing purified HCP-1 to a mammal (eg, mouse, rat, rabbit, guinea pig, goat, etc.). Administration is mainly performed by injecting intravenously, subcutaneously or intraperitoneally. An adjuvant can be appropriately added as necessary. Subsequently, serum (antiserum) obtained by immunization is collected. The collection of serum is not limited, and it can be collected from blood of an immunized animal 1 to 28 days after the last administration date according to a conventional method. The target antiserum is screened from the collected antisera using a known immunoassay. The antibody contained in the target antiserum by the above screening is a polyclonal antibody. When the antibody needs to be purified, for example, known purification methods such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel chromatography and the like can be used alone or in combination of two or more.

(ii) Preparation of monoclonal antibody The preparation of the antigen and its solution and the immunization method can be performed in the same manner as in the preparation of the polyclonal antibody.

The collection of antibody-producing cells (antibody-producing cells) obtained by the above immunization is not limited, but is preferably performed, for example, 1 to 14 days after the last administration, more preferably 2 to 4 days . As antibody-producing cells, for example, spleen cells, lymph node cells, peripheral blood cells and the like are preferable, and spleen cells are more preferable. A fused cell (hybridoma) is obtained by performing cell fusion between the collected antibody-producing cells and myeloma cells.
As myeloma cells, cell lines that are generally available in mammals such as mice, for example, have drug selectivity, and cannot grow in a HAT selection medium (medium containing hypoxanthine, aminopterin and thymine) in an unfused state. However, cell lines that can grow in a state fused with antibody-producing cells can be mentioned. As mouse myeloma cells, for example, PAI, P3X63-Ag.8.U1 (P3U1), NS-I and the like can be used. The cell fusion is performed, for example, by mixing antibody-producing cells and myeloma cells in a culture medium for animal cell culture such as RPMI-1640 medium and performing a fusion reaction. The mixing ratio of antibody-producing cells and myeloma cells is arbitrary, for example 5: 1.

  In general, the fusion reaction is preferably performed in the presence of a cell fusion promoter, and as the promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons or the like can be used. In addition, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electroporation.

  The cells after the fusion reaction are cultured using, for example, a HAT selection medium. After the above culture, cells in which growth on the HAT selection medium is observed become fused cells (hybridomas).

  The target hybridoma is screened from the hybridoma obtained by the above culture. The screening method is not particularly limited, and can be performed by ELISA or the like. Establishment (cloning) of the target hybridoma by the above screening can be performed by selecting colonies derived from one cell by culturing using a limiting dilution method or the like.

  As a method for collecting a monoclonal antibody from the hybridoma obtained by the above cloning, a cell culture method or ascites formation method can be generally employed. These methods are well known in the art. In addition, when purification of the antibody is required at the time of collection or after collection of the monoclonal antibody, known purification methods such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel chromatography, etc. are used alone or They can be combined as appropriate.

In the present invention, humanized antibodies, human antibodies, and fragments of the antibodies can also be used.
A humanized antibody is a monoclonal antibody produced by genetic engineering. Specifically, a part or all of the complementarity determining region (CDR) of the hypervariable region is derived from a mouse monoclonal antibody. It is an antibody whose complementarity-determining region is a region, whose framework region (FR) is a variable region derived from human immunoglobulin, and whose constant region is a constant region derived from human immunoglobulin. Human-type antibodies can be prepared by methods well known in the art.
A human antibody refers to an antibody in which all regions constituting an immunoglobulin are derived from a gene encoding human immunoglobulin. A human antibody can be produced by a method well known in the art, for example, by preparing a transgenic animal that produces a human antibody and immunizing the transgenic animal with an antigen.
The antibody fragment means a partial fragment containing the antigen-binding region of the antibody, specifically, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), scFv (single chain Fv), and dsFv (disulphide stabilized Fv).

3-3. Porphine Ring Structure In the present invention, a porphine ring structure can be used to inhibit the function of the HCP-1 protein. Porphine ring structures that have already been approved and clinically used in more than two countries as pharmaceuticals include hematoporphyrin derivatives (Photofurin (registered trademark): Wyeth), or talaporphyrin sodium (resaphyrin (registered trademark): Meiji Confectionery).
These porphine ring structures are freely available in Japan as drugs already approved by the Ministry of Health, Labor and Welfare.

4). In the present invention, an inhibitor of HCP-1 can be used as a cancer therapeutic agent (pharmaceutical composition for cancer treatment) or as an effect enhancer of an anticancer agent.
The patient for whom the therapeutic agent and the enhancer of the present invention are used is a human or non-human mammal. When a test subject is a human, it is preferably various cancer patients. Non-human mammals include, for example, pets, zoo animals, livestock, and the like, and specifically include cats, dogs, horses, cows, pigs, goats, sheep, primates, and the like.
The cancer to be treated is not particularly limited as long as it expresses HCP-1 on the cell surface. For example, digestive system, respiratory system, cranial nervous system, dermatological system, urological system, maternal and female Various cancers included in departments, blood systems and the like can be mentioned.
Specifically, brain cancer, head and neck cancer, cervical cancer, oral cancer, pharyngeal cancer, esophageal cancer, pancreatic cancer, gastric cancer, biliary tract cancer, cancer of the small intestine or duodenum, colon cancer, lung cancer, bladder cancer, kidney cancer, liver cancer, Prostate cancer, breast cancer, uterine cancer (including cervical cancer, endometrial cancer), ovarian cancer, thyroid cancer, various sarcomas (eg, osteosarcoma, chondrosarcoma, Kaposi sarcoma, myoma, angiosarcoma, fibrosarcoma, etc.), Malignant lymphoma (including Hodgkin lymphoma, non-Hodgkin lymphoma), leukemia (eg, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL ), Lymphoma, multiple myeloma (MM), myelodysplastic syndrome, etc.), skin cancer and melanoma.

The therapeutic agent or enhancer of the present invention (hereinafter referred to as “therapeutic agent”) can be applied to the affected area as it is, or any known method, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection, or nasal cavity Alternatively, it can be introduced into a living body by inhalation from the lung, oral administration, intravascular administration using a catheter or the like.
Furthermore, the therapeutic agent of the present invention can be used in combination with other anticancer agents (chemotherapeutic agents, etc.). In this case, the therapeutic agent of the present invention and other antitumor agents can be administered simultaneously, or a method of administering one after the other and the other after a predetermined time can be employed.

The therapeutic agent and the like of the present invention can be administered orally, such as tablets, capsules, powders, granules, pills, solutions, syrups, injections, external preparations, suppositories, depending on the mode of oral or parenteral administration. It can be in the form of a parenteral agent such as an agent or eye drops.
When used as a parenteral preparation, its form is not generally limited. For example, any of intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. There may be. In the case of various injections, for example, it can be provided in the state of a unit dose ampoule or a multi-dose container, or in the form of a lyophilized powder that is re-dissolved in a solution at the time of use. In addition to the active ingredients described above, the parenteral preparations can contain various known excipients and additives according to various forms within a range that does not impair the effects of the active ingredients. For example, in the case of various injections, water, glycerol, propylene glycol, polyethylene glycol and the like can be mentioned.
When used as an oral preparation, its form is generally not limited, and for example, any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. Alternatively, it may be a dry product which is redissolved when used. In addition to the above-mentioned active ingredient, the oral preparation can contain various known excipients and additives according to various forms as long as the effects of the active ingredient are not impaired. For example, binder (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, etc.), filler (lactose, sugar, corn starch, potato starch, calcium phosphate, sorbitol, glycine, etc.), lubricant (magnesium stearate, talc, Polyethylene glycol, silica, etc.), disintegrating agents (various starches, etc.), wetting agents (sodium lauryl sulfate, etc.).

  Furthermore, the therapeutic agent and the like of the present invention can be in the form of a solvate. Examples of solvates include hydrates and non-hydrates, and hydrates are preferred. Examples of the solvent include water, alcohol (eg, methanol, ethanol, n-propanol, etc.), dimethylformamide and the like.

  Furthermore, the therapeutic agent or the like of the present invention may be one that forms a pharmacologically acceptable salt with an acid or base. Examples of the salt with an acid include inorganic acid salts such as hydrochloride, hydrobromide, sulfate and phosphate, or formic acid, acetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, and tartaric acid. And organic acid salts such as stearic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid. Examples of the salt with a base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N, N′- Examples thereof include organic base salts such as dibenzylethylenediamine, arginine and lysine, and ammonium salts. Furthermore, the therapeutic agent of the present invention may be crystalline or non-crystalline, and when a crystalline polymorph exists, it may be a single substance or a mixture of any of those crystalline forms. Good.

  The dosage of the therapeutic agent of the present invention is not limited to the patient's age, weight, height, sex, clinical state, past treatment history, and accumulation of the therapeutic agent of the present invention at the site and extent of the disease to be treated. It can be appropriately changed depending on the degree and speed.

  The dose of the therapeutic agent of the present invention is generally determined in consideration of the compounding ratio of the active ingredient (HCP-1 gene inhibitory nucleic acid, anti-HCP-1 antibody or porphyrin derivative) in the preparation ( It can be appropriately set in consideration of the age, weight, type of disease or progress of the patient, administration route, number of administrations, administration period, etc.

  The parenteral dosage (per day) is not limited. For example, in the case of various injections, the above-mentioned active ingredient (in the case of siRNA) is generally 10 μg / kg of the subject (patient) body weight. ˜100 mg / day, preferably 50 μg to 50 mg / day.

The dose (per day) of the oral preparation is generally 100 μg to 2000 mg / day, preferably 1 mg to 1 g / day, per 1 kg body weight of the application subject (patient) of the above-mentioned active ingredient.
For example, when siRNA, shRNA or miRNA is administered, the effective amount is not particularly limited as long as it is an amount sufficient to cause RNAi-mediated degradation of the target mRNA. One skilled in the art can readily determine the effective amount to be administered to a subject taking into account factors such as the subject's height and weight, age, sex, route of administration, or local or systemic administration. In general, the siRNA, shRNA or miRNA is at an intracellular concentration of about 50 pM to about 100 nM, preferably 1 nM to 10 nM, for parenteral administration (eg, intravenous injection).

  In another aspect of the present invention, the therapeutic effect of the anticancer agent can be enhanced by using the therapeutic agent of the present invention in combination with an anticancer agent (for example, a cancer chemotherapeutic agent). “Enhancement” means that the reduction rate of cancer or the reduction rate of cancer cells, which is the therapeutic effect of an anticancer agent, is at least 10% or more than when used alone.

The anticancer agent used in the present invention is not particularly limited, and the following can be mentioned depending on the type of activity.
Alkylation activity: Carbocon, busulfan, nimustine, cyclophosphamide, etc. Antimetabolite activity: methotrexate, mercaptopurine, cytarabine, fluorouracil, tegafur, etc. Antibiotics: actinomycin D, bleomycin, adriamycin, mitomycin, bleomycin, etc. Microtubule inhibitory activity: Docetaxel, paclitaxel, vinorelbine, vincristine, vinblastine, etc. Topoisomerase inhibitory activity: irinotecan, podophyllotoxin derivatives, etoposide, etc. Angiogenesis inhibitory activity: bevacizumab (Avastin), sorafenib (Nexabal), sunitinib (Sutentin), etc. When using a porphine ring structure as a therapeutic agent of the present invention, etc., the porphine ring structure is administered to a patient, Comprising the step of irradiating light when the derivative is accumulated in cancerous tissue or cancer cell in a mammal. In this case, you may use together with the said anticancer agent, and according to the administration schedule of an anticancer agent, administration of the said porphine ring structure and light irradiation can be performed. Therefore, the timing of administration of the anticancer agent and the timing of administration of the porphine ring structure and light irradiation are not particularly limited, and both can be treated simultaneously, or one can be treated first and the other after.

5. Kit The present invention provides a kit for treating cancer comprising an HCP-1 expression inhibitor and an anticancer agent. The kit of the present invention contains an anticancer agent in addition to the above-mentioned HCP-1 expression inhibitor. Therefore, the kit of the present invention can be used for the combined use of the anticancer agent and the HCP-1 expression inhibitor of the present invention for the purpose of enhancing the anticancer effect of the anticancer agent.
The kit of the present invention may further contain a buffer solution, a lysis solution, a syringe, an instruction manual, a package insert, and the like. In the instruction manual or package insert, a combination for using the HCP-1 expression-inhibiting substance and the anticancer agent in combination can be described, and as a form or mixture in which separate substances are used at the time of administration For the form, usage, dosage and the like can be described.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<Experiment method>
Cells Rat gastric mucosal cell carcinoma-like mutant RGK-36 was used. This cell line is a mutant strain having cancer-like properties established from the rat normal gastric mucosa cultured cell line RGM-1 established by the inventor using the carcinogen MNNG (Shimokawa O, Matsui H, et al. In Vitro Cell Dev Biol Anim. 44: 26-30. 2008).
Medium DMEM / F12 (1: 1) medium (w / t 10% FBS) without antibiotics was used.
Culture conditions
2 × 10 ⁴ cells were seeded in a 96-well plate and cultured in a medium of 100 μL / well for 24 hours.
siRNA
A 4 pg / well HCP-1 siRNA cocktail (in which the sequence is as follows) was used, or scrambled RNA (sequence: random) was used as a control.
Sense strand
(i) ccaaaguccacgaggcuuuTT (SEQ ID NO: 3)
(ii) ggguacuggagaagguuaaTT (SEQ ID NO: 4)
(iii) gaguaaaggagauggcauuTT (SEQ ID NO: 5)
Antisense strand
(i) aaagccucguggacuuuggTT (SEQ ID NO: 6)
(ii) uuaaccuucuccaguacccTT (SEQ ID NO: 7)
(iii) aaugccaucuccuuuacucTT (SEQ ID NO: 8)
Transfection LIPO TRUST 0.5 μL / well manufactured by Hokkaido System Science Co. was used. After adding 4 pg of siRNA to 10 μL of serum-free medium and mixing, LIPO TRUST 0.5 μL was added and mixed. These were incubated for 20 minutes at room temperature. Once cultured, the medium was removed, mixed with 100 μL / well of serum-containing medium, and administered to each well. The cells were cultured for 48 hours.
Cytotoxicity test The cultured medium was discarded and washed once with PBS. 1, 5, 10 μg / mL adriamycin (ADR) (manufactured by Wako, doxorubicin hydrochloride), cisplatin (CDDP) (manufactured by Wako), 1,10 μg / mL fluorouracil (5-FU) (manufactured by Wako) Administered. ADR and CDDP were dissolved in DMSO and 5-FU was dissolved in DMF, and these were used after being diluted 1,000 times or more. After administration, the cells were cultured for 24 hours.
The cultured medium was removed and washed twice with PBS. Administer 50 μL of medium and 10 μL of Tetra Color-1 (TetraColor ONE-Cell Proliferation Assay System, manufactured by Seikagaku Biobusiness Co., Ltd.) After incubation for a time, the absorbance at 450 nm was measured. (Reference: 630nm)
<Result>
The administration of siRNA clearly suppressed the expression of HCP-1 as shown in FIG. FIG. 2 shows the effect of siRNA on the expression level of HCP-1 in RGK36.

Furthermore, the result of the obtained cytotoxicity test was as shown in FIG. 3 (n = 6). FIG. 3 shows the cancer toxicity enhancing effect of various anticancer agents by siRNA of HCP-1.
In addition, the cell viability (absorbance) of each anticancer agent non-administered group was set to 1, and the ratio of cell viability when the anticancer agent was added was taken (n = 6). The results are shown in FIG.
These results indicate that HCP-1 siRNA significantly enhances the toxicity of ADR, CDDP, and 5-FU to cancer cells compared to the non-specific RNA group.

  The HCP-1 inhibitor of the present invention is useful as a therapeutic agent for cancer and an effect enhancer for anticancer agents.

Sequence number 3: Synthetic DNA / RNA
Sequence number 4: Synthetic DNA / RNA
Sequence number 5: Synthetic DNA / RNA
Sequence number 6: Synthetic DNA / RNA
Sequence number 7: Synthetic DNA / RNA
Sequence number 8: Synthetic DNA / RNA

Claims (6)

A cancer therapeutic agent comprising a substance that inhibits the expression of a gene encoding HCP-1 or a substance that inhibits the function of HCP-1 protein. 2. The substance that inhibits the expression of a gene encoding HCP-1 is at least one selected from the group consisting of siRNA, shRNA, microRNA, antisense nucleic acid, decoy nucleic acid, and aptamer against the gene. Therapeutic agent. The therapeutic agent according to claim 1, wherein the substance that inhibits the function of HCP-1 protein is an antibody against HCP-1 protein or a porphine ring structure. An agent for enhancing the effect of an anticancer agent, comprising a substance that inhibits expression of a gene encoding HCP-1 or a substance that inhibits the function of HCP-1 protein. 5. The substance that inhibits the expression of a gene encoding HCP-1 is at least one selected from the group consisting of siRNA, shRNA, microRNA, antisense nucleic acid, decoy nucleic acid, and aptamer against the gene. Enhancer. The enhancer according to claim 4, wherein the substance that inhibits the function of HCP-1 protein is an antibody against HCP-1 protein or a porphine ring structure.