Patent Application
20120283120
The present invention relates to a novel variant androgen receptor, a method of screening for an agent for the prophylaxis and/or treatment of cancer using the variant, and the like.
At present, chemical castration (endocrine therapy) by a hormone medicament has been widely used for cancer treatment. Endocrine therapy for prostate cancer and the like utilizes the mechanism of suppressing the growth of cancer cells by cutting off the function of male sex hormone since it stimulates the growth of cancer cells.
Androgen is a generic term of steroid hormone having a male sex hormone action, and includes testosterone synthesized by the testis, dehydroepiandrosterone synthesized by the adrenal cortex and the like.
For testosterone that stimulates the growth of cancer cells to enter the cells and exhibit its action, testosterone needs to be converted to dihydrotestosterone and bind to an androgen receptor (hereinafter sometimes to be abbreviated as AR) in the nucleus of the cancer cells. An anti-androgenic agent binds to AR to prevent binding between dihydrotestosterone and AR, thereby exhibiting efficacy. It is known, however, when the treatment is continued, the effect disappears and the cancer starts to grow again (androgen independent prostate cancer; AIPC). An effective prophylactic or therapeutic method for relapsed prostate cancer that acquired androgen independence has not been developed yet. While the mechanism of relapsed prostate cancer relative to the endocrine therapy has not been elucidated yet, plural factors, such as emergence or increase of cells that acquired androgen hypersensitivity by amplification or overexpression of AR gene, capability of utilizing a substance other than androgen (e.g., glucocorticoid, anti-androgenic agent per se) as an agonist due to the mutation of AR and the like, are being clarified. For example, a mutant androgen receptor wherein flutamide and bicalutamide, which are anti-androgenic agents, act as agonists, was separated from patients with relapsed prostate cancer (e.g., see patent document 1).
Hu et al. (non-patent document 1) found 7 kinds of AR splice variants (AR-V1, AR-V2, AR-V3, AR-V4, AR-V5, AR-V6, AR-V7) lacking a ligand binding domain. However, whether these AR splice variants become treatment targets of AIPC has not been demonstrated. On the other hand, Guo et al. (non-patent document 2) found 3 kinds of AR splice variants lacking a ligand binding domain, and suggest that one kind thereof (AR3; having the same amino acid sequence as the above-mentioned AR-V7) can be a treatment target of AIPC. However, as a concrete image of a therapeutic drug with the AR splice variant as a target, an example using only an shRNA expressing lentivirus for the variant is merely shown. Thus, the possibility of a treatment with a single agent of siRNA or a low-molecular-weight compound that inhibits the function of AR splice variant, and a combined therapy with other kind of active ingredient or radiation therapy etc. has not been verified at all.
In addition, the presence of variant AR other than those mentioned above (ARv567es; non-patent document 4) and cDNA fragments capable of encoding variant AR (AR-V8, AR-V9, AR-V10, AR-V11; non-patent document 5) has also been reported.
Note that some compounds are known to inhibit the activity of the N-terminal domain of AR (patent document 2, non-patent document 3). However, whether or not they inhibit variant AR, represented by AR3 (AR-V7), which is inherently expressed by prostate cancer cells such as CWR22Rv1, VCaP, JDCaP/JDCaP-hr and the like, has not been verified.
Meanwhile, a 90 kDa heat shock protein (HSP90), which is a molecular chaperone, controls interaction, intracellular transport, degradation and function of various proteins that play an important role in the cell control, such as kinase, transcription factor, hormone receptor and the like, via enhancement of the folding of these proteins, thus playing multifaceted roles in the control of the growth and survival of the cells. Particularly, increased expression of HSP90 is seen in cancer cells, and the level thereof is correlated with the malignancy of the cancer. In addition, HSP90 inhibitor is expected to be a new type of anti-cancer agent, and its action to potentiate the effects of radiation and conventional anti-cancer agents is attracting attention.
While HSP90 inhibitors are known to promote degradation of mutant AR wherein a polyglutamine chain is abnormally elongated, which is a causative gene of spinobulbar muscular atrophy, their actions on mutant AR observed in relapsed androgen-independent prostate cancer and the above-mentioned variant AR remain to be elucidated.
A medicament effective for the prophylaxis or treatment of androgen-independent prostate cancer that relapsed after endocrine therapy, has been demanded. It is therefore an object of the present invention to identify a novel variant AR lacking ligand binding domain, clarify the involvement of the variant AR in relapsed androgen-independent prostate cancer, and provide a method of searching a novel prophylactic or therapeutic drug for relapsed androgen-independent prostate cancer, which uses the variant AR inhibitory activity as an index.
The present inventors have conducted unique studies and found that a C-terminally deficient variant AR emerges during the process of conversion of androgen dependent prostate cancer (ADPC) tumor line (JDCaP) into AIPC (JDCaP-hr). The 3′ terminal sequence of mRNA was cloned and the nucleotide sequence was analyzed. As a result, two kinds of variant AR clones (ARaiv1 and ARaiv2) wherein a part of the third intron (intron 3) is linked to immediately after the third exon (exon 3) of AR gene to generate a stop codon were identified, and translation products predicted from these were constitutively active molecules lacking the ligand binding domain. ORF cDNA of ARaiv1 was acquired from CWR22Rv1 cell and JDCaP-hr cell and the sequence was analyzed. As a result, ARaiv1 was found to contain a variant having a intact first exon, as well as three variants (ARaiv1Δ1-Δ3) lacking a part of the first exon (exon 1).
These 4 kinds of ARaiv1 were all confirmed to be constitutively active molecules by a reporter gene assay. ARaiv1 without deletion in the first exon has the same amino acid sequence as one of the AR splice variants reported by Hu et al. (non-patent document 1) and Guo et al. (non-patent document 2) (AR-V7 and AR3, respectively). It has been clarified that ARaiv1 is also highly expressed in other AIPC tumor lines, and its expression is increased in 7.9% of the clinical samples of prostate cancer.
Therefore, the present inventors have specifically knocked down ARaiv1 in ARaiv1 highly expressing tumor cell line to find that the growth of tumor cell is remarkably suppressed in the absence of androgen. It has also been shown that the growth of tumor cell is more remarkably suppressed when both the wild-type AR and ARaiv1 are knocked down than when either one alone is knocked down.
Moreover, it has been found by exposure of COS7 cells transfected with ARaiv1 and androgen responsive reporter plasmid to geldanamycin (HSP90 inhibitor) that the HSP90 inhibitor inhibits activation of reporter gene transcription by ARaiv1.
Furthermore, it has been found by exposure of JDCaP-hr cell to geldanamycin (HSP90 inhibitor) that the HSP90 inhibitor inhibits expression of AR and shows a higher expression inhibitory effect on variant AR lacking a ligand binding domain.
The present inventors have conducted further investigations based on these findings, and completed the present invention.
Accordingly, the present invention provides, as one embodiment,
[1-A] a method of screening for a substance for the prophylaxis or treatment of cancer, comprising
a step of contacting a cell comprising a nucleic acid encoding
a variant androgen receptor protein comprising the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12, and a reporter gene under control of an androgen responsive promoter with a test compound,
a step of measuring the expression level of the reporter gene, and
a step of selecting a compound that suppresses the expression of the reporter gene as compared to a control without contact with the test compound,
[2-A] a method of screening for a substance for the prophylaxis or treatment of cancer, comprising
a step of contacting a variant androgen receptor protein comprising the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12 with a test compound,
a step of measuring the activity of the protein, and
a step of selecting a compound that suppresses the activity of the protein as compared to a control without contact with the test compound,
[3-A] a variant androgen receptor protein comprising the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12,
[4-A] a nucleic acid encoding the protein of the above-mentioned [3-A],
[5-A] a cell transduced with the nucleic acid of the above-mentioned [4-A],
[6-A] a nucleic acid of any of the following (a)-(e),
(a) an antisense nucleic acid for a nucleic acid encoding the protein of the above-mentioned [3-A]
(b) siRNA for RNA encoding the protein of the above-mentioned [3-A]
(c) a nucleic acid capable of generating siRNA for RNA encoding the protein of the above-mentioned [3-A]
(d) miRNA for RNA encoding the protein of the above-mentioned [3-A]
(e) a nucleic acid capable of generating miRNA for RNA encoding the protein of the above-mentioned [3-A],
[7-A] a method of evaluating the efficacy of a substance for the prophylaxis or treatment of cancer, comprising a step of contacting a test compound with an animal or tissue thereof into which the nucleic acid of the above-mentioned [4-A] has been transduced, and measuring growth, differentiation or cell death, canceration or cancer growth of the tissue,
[8-A] a diagnostic reagent for cancer expressing a variant androgen receptor lacking a ligand binding domain, which comprises a substance that specifically recognizes the amino acid sequence shown by SEQ ID NO: 2 or the nucleotide sequence shown by SEQ ID NO: 14, and the like.
As another embodiment, the present invention provides [1-B] a function inhibitor of a variant androgen receptor lacking a ligand binding domain, comprising an HSP90 inhibitor, [2-B] the inhibitor of the above-mentioned [1-B], wherein the variant androgen receptor comprises a C-terminal amino acid sequence shown by SEQ ID NO: 1 or 2,
[3-B] the inhibitor of the above-mentioned [1-B], wherein the HSP90 inhibitor is geldanamycin or a derivative thereof,
[4-B] an agent for the prophylaxis or treatment of cancer, comprising a substance that inhibits a function of a variant androgen receptor lacking a ligand binding domain (particularly, a variant androgen receptor protein comprising the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12),
[5-B] the agent of the above-mentioned [4-B], wherein the variant androgen receptor comprises a C-terminal amino acid sequence shown by SEQ ID NO: 1 or 2,
[6-B] the agent of the above-mentioned [4-B], wherein the aforementioned substance is an expression inhibitor of the aforementioned receptor protein,
[7-B] the agent of the above-mentioned [6-B], wherein the aforementioned substance is any of the following (a)-(e),
(a) an antisense nucleic acid for a nucleic acid encoding the above-mentioned receptor protein
(b) siRNA for RNA encoding the above-mentioned receptor protein
(c) a nucleic acid capable of generating siRNA for RNA encoding the above-mentioned receptor protein
(d) miRNA for RNA encoding the above-mentioned receptor protein
(e) a nucleic acid capable of generating miRNA for RNA encoding the above-mentioned receptor protein
[8-B] the agent of the above-mentioned [7-B], wherein the target sequence of the aforementioned nucleic acid is the entire or partial fourth exon of mRNA encoding the aforementioned receptor protein,
[9-B] the agent of the above-mentioned [4-B], wherein the cancer is prostate cancer,
[10-B] the agent of the above-mentioned [4-B], wherein the cancer is androgen independent prostate cancer,
[11-B] the agent of the above-mentioned [4-B], wherein the cancer expresses the aforementioned receptor,
[12-B] the agent of the above-mentioned [11-B], which is combined with an androgen receptor signal inhibitor,
[13-B] a method for the prophylaxis or treatment of cancer in an animal, comprising administering an effective amount of a substance that inhibits a function of a variant androgen receptor lacking a ligand binding domain to the animal,
[14-B] use of a substance that inhibits a function of a variant androgen receptor lacking a ligand binding domain for the production of an agent for the prophylaxis or treatment of cancer,
[15-B] a diagnostic reagent for cancer expressing a variant androgen receptor lacking a ligand binding domain, which comprises a substance that specifically recognizes the amino acid sequence shown by SEQ ID NO: 1 or 2 or the nucleotide sequence shown by SEQ ID NO: 13 or 14,
[16-B] the agent of the above-mentioned [4-B], which is administered using the reagent of the above-mentioned [15-B] to a patient diagnosed with cancer expressing a variant androgen receptor lacking a ligand binding domain,
[17-B] a variant androgen receptor protein comprising the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12,
[18-B] a nucleic acid encoding the protein of the above-mentioned [17-B],
[19-B] a cell into which the nucleic acid of the above-mentioned [18-B] has been transfected,
[20-B] a method of screening for a substance for the prophylaxis or treatment of cancer, comprising contacting a test compound with a cell comprising the nucleic acid of the above-mentioned [18-B], and a reporter gene under control of an androgen responsive promoter, and measuring the expression level of the reporter gene,
[21-B] an animal transduced with the nucleic acid of the above-mentioned [18-B],
[22-B] a method of evaluating the efficacy of a substance for the prophylaxis or treatment of cancer, comprising contacting a test compound with an animal or tissue thereof into which the nucleic acid of the above-mentioned [18-B] has been transduced, and measuring growth, differentiation or cell death, canceration or cancer growth of the tissue, and the like.
Using the inhibition of transcription regulation by the novel variant AR of the present invention as an index, a substance effective for the prophylaxis or treatment of cancer expressing the variant AR can be searched for. In addition, detection of a C-terminal sequence specific to the variant AR or a sequence resulting from partial deletion of exon 1 enables diagnosis of cancer expressing the variant AR, thus making it possible to determine an appropriate administration subject for a substance obtained by the aforementioned search method.
Moreover, an HSP90 inhibitor can shut off AR signal that is mediated by the variant by inhibiting the function (expression and/or activity) of a ligand independent (ligand binding domain-deleted) variant AR which could be one factor of relapsed androgen-independent prostate cancer. Therefore, it is useful for the treatment and prevention of recurrence of cancer expressing the variant AR. Since other medicament that inhibits the function (expression and/or activity) of the variant AR can also inhibit transcription regulating action by the variant AR, it is similarly useful for the treatment and prevention of recurrence of cancer expressing the variant AR. Moreover, a combined use of a inhibitor of conventional AR signal via full-length AR with an expression/activity inhibitor of the above-mentioned variant AR affords an effect of increased efficacy and/or decreased side effects.
The variant AR (ARaiv) lacking a ligand binding domain in the present invention is a protein comprising a region at least necessary for retaining a transcriptional activation action in the amino acid sequence encoded by the first exon (5001-7731-position of NG—009014.1; 1-1616-position of SEQ ID NO: 15) of human AR gene (its nucleotide sequence is registered as NCBI RefSeq No. NG—009014.1 (GI:213385253)), and a region at least necessary for binding to a target gene in the amino acid sequence encoded by the first-third exons (the second and the third exon are the 104225-104376-position and the 146979-147095-position of NG—009014.1, respectively; the 1617-1768-position and the 1769-1885-position of SEQ ID NO: 15, respectively), and lacking a region necessary for binding to a ligand in the amino acid sequence encoded by the fourth-eighth exons (the 172371-172658-position, the 178447-178591-position, the 182802-182932-position, the 183796-183953-position and the 184655-185246-position of NG—009014.1, respectively; the 1886-2173-position, the 2174-2318-position, the 2319-2449-position, the 2450-2607-position and the 2608-2763-position of SEQ ID NO: 15, respectively). Preferably, ARaiv of the present invention includes a protein containing an amino acid sequence which is an amino acid sequence of full-length AR shown by SEQ ID NO: 16 wherein a C-terminal sequence shown by amino acid Nos. 625-920 is deleted and, instead, a C-terminal amino acid sequence shown by SEQ ID NO: 1 or 2 is added (i.e., amino acid sequence shown by SEQ ID NO: 4 (ARaiv1) or 6 (ARaiv2)), or a protein containing an amino acid sequence which is an amino acid sequence shown by SEQ ID NO: 4 or 6 wherein an amino acid sequence shown by amino acid Nos. 367-495 is entirely or partially deleted. In the present specification, the protein and peptide are described according to conventional peptide lettering, and the left end is N-terminal (amino terminal), and the right end is C-terminal (carboxyl terminal).
The amino acid sequence shown by the above-mentioned amino acid Nos. 367-495 corresponds to a region called transactivation unit 5 (TAU5) in the AR transcriptional activation domain (AF1), and is reported to inhibit androgen-dependent AR activity in androgen-dependent cancer cells, but potentiate androgen-independent AR activity in androgen-independent cancer cells (Cancer Res., 2007, 67: 10067-77). Even if TAU5 is completely deleted, a transcriptional activation action of AR is maintained at a level sufficient to promote growth of cancer cells. Specific examples of the TAU5-deleted ARaiv include a protein containing an amino acid sequence shown by SEQ ID NO: 8, 10 or 12 (ARaiv1Δ1, ARaiv1Δ2, ARaiv1Δ3, respectively) and the like.
The Gln repeat number of the polyglutamine region in the N-terminal sequence of AR shows individual difference (generally about 9-34 residues, repeat number in prostate cancer cells may decrease), and therefore, a protein containing an amino acid sequence which is the amino acid sequence shown by each of the above-mentioned SEQ ID NO, wherein the Gln repeat number of the polyglutamine region varies within a range generally observed, is also encompassed in the ARaiv of the present invention.
The ARaiv protein may be isolated and purified by a protein separation and purification technique known per se from human cell [e.g., hepatocyte, splenocyte, nerve cell, glial cell, pancreatic β cell, myelocyte, mesangial cell, Langerhans' cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell, fibroblast, fibrocyte, myocyte, adipocyte, immunocyte (e.g., macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, bone cell, osteoblast, osteoclast, mammary gland cell, interstitial cell, or a corresponding progenitor cell, stem cell or cancer cell thereof etc.] or any tissue in which these cells are present [e.g., brain or any portion of brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (e.g., smooth muscle, skeletal muscle), lung, gastrointestinal tract (e.g., large intestine and small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (e.g., white adipose tissue, brown adipose tissue) etc.] and the like, preferably cancer cells of prostate, testis, mammary gland, ovary, placenta, uterus and the like, more preferably relapsed androgen-independent prostate cancer cells. When, for example, an ARaiv protein is used for screening for a prophylactic or therapeutic drug for cancer, and the like, it may be a protein produced by chemical synthesis or biochemical synthesis in a cell-free translation system, or a recombinant protein produced by a transformant transduced with a nucleic acid having a nucleotide sequence encoding any of the above-mentioned amino acid sequences.
The ARaiv in the present invention also encompasses a protein containing any of the above-mentioned amino acid sequences, polymorphic or natural allele variant found in human (said mutation does not influence AR function), and ortholog in a mammal other than human (e.g., mouse, rat, monkey, dog, cat, bovine, horse, swine, sheep, goat, rabbit and the like).
Preferably, the ARaiv in the present invention is a protein containing an amino acid sequence shown by SEQ ID NO: 6, 8 or 10 (i.e., ARaiv1Δ1, ARaiv1Δ2 or ARaiv1Δ3), or a protein containing an amino acid sequence shown by SEQ ID NO: 12 (i.e., ARaiv2). These ARaivs are novel variant ARs identified in the present invention.
The present invention provides a method of screening for a substance for the prophylaxis and/or treatment of a disease involving variant AR, for example, variant AR highly expressing cancer, which uses, as an index, a function of the variant AR, particularly the above-mentioned novel ARaiv, namely, a transcription regulating action on an androgen responsive gene (e.g., transcriptional activation of prostate specific antigen (PSA) gene, TMPRSS2 gene and the like, or transcriptional repression of SERPIN B5 gene and the like), or a suppressive action on the expression of the variant AR genes.
As mentioned above, when expression and/or activity of ARaiv is inhibited, transcription regulation of an androgen responsive gene is suppressed, and AR signal is shut off to suppress cancer cell growth. Therefore, a compound that inhibits expression and/or activity of ARaiv can be used as a blocker of AR signal from ARaiv and as a prophylactic or therapeutic drug for cancer, particularly ARaiv highly expressing cancer (e.g., prostate cancer and the like), inter alia, relapsed androgen-independent prostate cancer.
Hence, an ARaiv protein or a cell that produces ARaiv can be used as a tool for screening for a substance for the prophylaxis or treatment of cancer, by using the expression level and/or activity (e.g., transcription regulation activity) of ARaiv as an index.
When a compound that inhibits the activity of ARaiv is screened for, the screening method includes (1) contacting an ARaiv protein or a cell capable of producing ARaiv with a test compound, and
(2) measuring the activity of the protein,
(3) selecting a compound that suppresses the activity of the protein as compared to the activity of a control without contact with the test compound.
A cell capable of producing ARaiv can be contacted with a test compound by, for example, cultivating the cell in the presence of the test compound. In this case, the cell cultivated in the absence of the test compound is used as a control, and the activities of ARaiv under the both conditions are compared to evaluate the ARaiv activity suppressive effect of the test compound.
Examples of the activity of ARaiv include androgen responsive gene transcription regulation activity, ARaiv protein nuclear translocation activity, and androgen response sequence (ARE)-binding activity of the gene.
The cell capable of producing ARaiv to be used for the above-mentioned screening method is not particularly limited as long as it is a human or other mammalian cell that inherently expresses ARaiv or a biological sample containing same (e.g., blood, tissue, organ etc.). Preferred are androgen independent cancer cell line or tumor line (e.g., cell line or tumor line of AIPC prostate cancer) and the like. When blood, tissue, organ and the like derived from a non-human animal are used, they may be isolated from the living organism and cultured, or a test compound may be administered to the living organism itself, and a biological sample may be isolated after lapse of a given time.
Examples of the cell capable of producing ARaiv include various transformants produced by a conventionally known genetic engineering technique. As a host, animal cells such as H4IIE-C3 cell, HepG2 cell, HEK293 cell, COS7 cell, CHO cell and the like are preferably used. As a host, prostate cancer cells that do not inherently express ARaiv protein, for example, LNCaP-FGC cell, PC-3 cell, DU-145 cell and the like, can also be used.
Specifically, the cell can be prepared by linking a DNA encoding ARaiv (i.e., DNA encoding a protein containing the amino acid sequence shown by SEQ ID NO: 4, 6, 8, 10 or 12, particularly SEQ ID NO: 6, 8, 10 or 12, specifically, DNA containing a nucleotide sequence shown by SEQ ID NO: 3, 5, 7, 9 or 11, particularly SEQ ID NO: 5, 7, 9 or 11 (CDS sequence), or a nucleotide sequence that hybridizes to a complementary chain of said nucleotide sequence under high stringent conditions, and encodes a polypeptide having an androgen responsive gene transcription regulating action equivalent to that of a protein comprising an amino acid sequence shown by SEQ ID NO: 4, 6, 8, 10 or 12, particularly SEQ ID NO: 6, 8, 10 or 12) to the downstream of a promoter in a suitable expression vector and transducing the vector into a host animal cell.
DNA encoding ARaiv can be cloned, for example, by synthesizing a suitable oligonucleotide as a probe or primer based on the nucleotide sequence shown by SEQ ID NO: 3 or 5, and by using a hybridization method or PCR method, from a cDNA or cDNA library derived from a cell or tissue that produces the aforementioned ARaiv. The hybridization can be performed, for example, by the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and the like. When a commercially available library is used, the hybridization can be performed according to the instructions of the manufacturer's protocol attached to the library.
The nucleotide sequence of the DNA can be converted according to a method known per se, such as the ODA-LA PCR method, the Gapped duplex method, or the Kunkel method, or a method based thereon, using a commonly known kit, for example, Mutan™-super Express Km (TAKARA SHUZO CO. LTD.), Mutan™-K (TAKARA SHUZO CO. LTD.) and the like. For example, a part or the entirety of the region that does not cause disappearance of the function of ARaiv even if deleted, such as TAU5 region and the like, may be deleted from a cloned DNA or the entirety or a part of the deleted region may be inserted into or added to a DNA encoding mutant ARaiv lacking a part of the region.
The cloned DNA can be used as is, or after digestion with a restriction endonuclease or addition of a linker as desired, depending on the purpose of its use. The DNA may have the translation initiation codon ATG at the 5′ end thereof, and the translation stop codon TAA, TGA or TAG at the 3′ end thereof. These translation initiation codons and translation stop codons can be added by using a suitable synthetic DNA adaptor.
As an expression vector, animal cell expression plasmid (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo); bacteriophage such as λphage and the like; animal virus vectors such as retrovirus, vaccinia virus, adenovirus, lentivirus and the like; and the like are used. The promoter may be any promoter, as long as it is appropriate for the host used to express the gene. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukaemia virus) LTR, HSV-TK (simple herpes virus thymidine kinase) promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferred.
Useful expression vectors include, in addition to the above, expression vectors that optionally comprises an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter also abbreviated as SV40 ori), and the like. As examples of the selection markers, the dihydrofolate reductase gene (hereinafter also abbreviated as dhfr) [methotrexate (MTX) resistance], the ampicillin resistance gene (hereinafter also abbreviated as ampr), the neomycin resistance gene (hereinafter also abbreviated as neor, G418 resistance), and the like can be mentioned. In particular, when a dhfr gene defective Chinese hamster cell is used and the dhfr gene is used as the selection marker, a target gene can also be selected using a thymidine-free medium.
When a nuclear translocation activity is measured as the ARaiv activity, the vector can also be designed such that ARaiv is expressed as a fusion protein with a reporter protein, so that nuclear translocation of the ARaiv protein can be easily detected. Examples of the reporter protein include fluorescence proteins such as GFP, YFP, BFP, RFP and the like, and the like.
An ARaiv expressing cell can be produced by transforming a host with an expression vector containing the above-mentioned DNA encoding ARaiv.
As the host, mammalian cells, for example, HepG2 cell, HEK293 cell, HeLa cell, LNCaP-FGC cell, PC-3 cell, DU-145 cell, human FL cell, monkey COS-7 cell, monkey Vero cell, Chinese hamster ovary cell (hereinafter to be abbreviated as CHO cell), dhfrgene defective CHO cell (hereinafter to be abbreviated as CHO(dhfr−) cell), mouse L cell, mouse AtT-20 cell, mouse myeloma cell, rat H4IIE-C3 cell, rat GH3 cell and the like can be used.
Transformation can be performed by a calcium phosphate coprecipitation method, a PEG method, an electroporation method, a microinjection method, a lipofection method and the like. For example, the method described in Saibo Kogaku, extra issue 8, Shin Saibo Kogaku Jikken Protocol, 263-267 (1995), published by Shujunsha, or Virology, 52, 456 (1973) can be used.
The transformed cell obtained as mentioned above, a mammalian cell inherently having an ability to produce ARaiv, and a tissue or organ containing the cell can be cultivated in a medium, for example, minimum essential medium (MEM) containing about 5-20% fetal bovine serum [Science, vol. 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, vol. 8, 396 (1959)], RPMI1640 medium [The Journal of the American Medical Association, vol. 1995, 19 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, vol. 73, 1 (1950)] and the like. The medium preferably has a pH of about 6-8. Culture is performed generally at about 30-40° C., and aeration and stirring may be added as necessary.
Examples of the test compound include protein, peptide, antibody, nonpeptidic compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract, plasma and the like, which may be novel or known.
A test compound can be contacted with the above-mentioned cell by, for example, adding the test compound to the above-mentioned medium and various buffers (e.g., HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetate buffer and the like), and incubating the cell for a given time. While the concentration of the test compound to be added varies depending on the kind of the compound (solubility, toxicity etc.), it is, for example, appropriately determined within the range of about 0.1 nM-about 10000 nM, preferably about 1 nM-about 1000 nM. Examples of the incubation time include about 10 min-about 24 hr.
When an ARaiv-producing cell is provided in the form of a non-human individual mammal, the condition of the individual animal is not particularly limited. For example, it may be a model animal such as a cancer bearing mouse and the like. Such cancer bearing mouse can be produced by a conventional method. Preferably, a transgenic non-human mammal transduced with a nucleic acid containing a protein coding sequence (CDS) of ARaiv, particularly, a nucleic acid containing a CDS sequence of ARaiv2 or any of ARaiv1Δ1-3 (i.e., CDS sequence encoding a protein containing the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12, specifically, a CDS sequence in a nucleotide sequence shown by SEQ ID NO: 5, 7, 9 or 11), in an expressible form can be used. Such transgenic animal can be produced by a conventional method.
While the rearing conditions of the animal to be used are not particularly limited, the animal is preferably reared in an environment of SPF grade or above. A test compound is contacted with the cell by the administration of the test compound to the animal individual. While the administration route is not particularly limited, for example, intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, tracheobronchial administration, rectal administration and the like can be mentioned. While the dose is not particularly limited, either, for example, a dose of about 0.5-20 mg/kg can be administered 1-5 times, preferably 1-3 times, per day for 1-14 days.
The ARaiv activity can be measured in the above-mentioned screening method by, for example, using the expression level of an androgen responsive gene (e.g., PSA gene, TMPRSS2 gene etc.) as an index. The expression level of the gene may be of an RNA level by using RT-PCR or Northernblot analysis, or of a protein level by using an antibody to a translation product thereof. The ARaiv activity is more preferably evaluated by introducing a DNA encoding a reporter protein under the control of an androgen responsive promoter (e.g., a promoter having a native androgen response sequence (ARE) such as PSA promoter and the like, a chimera promoter wherein ARE sequence is combined with an animal cell promoter such as SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like, and the like) and examining the expression of the reporter protein. The “PSA promoter” here means, in the 5′ upstream sequence of PSA gene, a region containing at least from the essential promoter region such as TATA box and the like to an ARE corresponding sequence in the upstream (in human PSA gene, AGAACAGCAAGTGCT; SEQ ID NO: 17), preferably, a region further containing up to 35 base pairs known as an androgen response region (ARR) in the further upstream [for human PSA gene, GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTG; SEQ ID NO: 18 (J. Biol. Chem., 271(11): 6379-6388, 1996)], more preferably a region containing about 6000 base pairs in the upstream of the transcription start point, and still more preferably a region containing about 600 base pairs in the upstream of the transcription start point. PSA promoter is useful since it reflects well the behavior of ARaiv in actual prostate cancer cell. When the activity of the promoter needs to be enhanced, plural (preferably 2-5, more preferably 2 or 3) promoters are preferably linked in tandem. As the reporter protein, luciferase, green fluorescence protein (GFP), β-galactosidase, chloramphenicol acetyltransferase, peroxidase, alkaline phosphatase and the like can be used. A DNA encoding a reporter protein can be inserted into the aforementioned vector for animal cell expression, and transduced according to the aforementioned gene transfer method.
When a cell that produces ARaiv is provided in the form of a non-human individual mammal, the activity of ARaiv can also be evaluated by using, as an index, the growth, differentiation, cell death or canceration, or cancer growth of the tissue (e.g., prostate tissue) collected from the animal. When such index is used, this screening system using ARaiv-producing non-human individual mammal is particularly useful not only for screening for a novel candidate substance for a prophylactic or therapeutic drug for cancer, but also an efficacy evaluation system for existing prophylactic or therapeutic drugs for cancer.
Examples of the test sample for activity measurement include:
a culture extract when the ARaiv-producing cell is provided in the form of a cell culture, tissue or organ culture; when the cell is provided as a non-human individual mammal, cell, tissue, organ extract separated from the individual, for example, homogenate of prostate, testis, mammary gland or tissue sections and the like; and the like.
When an isolated ARaiv protein is used in the above-mentioned screening method, a variant AR protein containing the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12 is preferably used as ARaiv. The protein may be isolated and purified from a cell or tissue that produces the protein, or chemically synthesized, or produced as a recombinant protein by genetic engineering.
The ARaiv can be contacted with a test compound in, for example, water or a suitable buffer. While the concentration of the test compound to be added varies depending on the kind of the compound (solubility, toxicity etc.), it is, for example, appropriately determined within the range of about 0.1 nM-about 10000 nM, preferably 1 nM-1000 nM. Examples of the incubation time include about 10 min-about 24 hr. The activity of ARaiv can be measured by, for example, a gel shift assay using an oligonucleotide containing a consensus ARE sequence (e.g., AGAACAnnnTGTTCT; SEQ ID NO: 32) of an androgen responsive gene (e.g., PSA gene, TMPRSS2 gene etc.) as a probe.
When, for example, in the above-mentioned screening method, the activity of ARaiv in the presence of a test compound is inhibited by not less than about 20%, preferably not less than 30%, more preferably not less than about 50%, as compared to the activity in the absence of the test compound, the test compound can be selected as a candidate for an ARaiv activity inhibitory substance, therefore, a substance for the prophylaxis or treatment of cancer, particularly ARaiv highly expressing cancer (e.g., prostate cancer etc.), inter alia, relapsed androgen-independent prostate cancer.
The present invention also provides a method of screening for a substance for the prophylaxis or treatment of cancer, comprising comparing expression of a variant AR in a cell having an ability to produce ARaiv, particularly novel ARaiv (variant AR protein containing the amino acid sequence shown by SEQ ID NO: 6, 8, 10 or 12), between in the presence and absence of a test compound. The cell to be used for this method, the kind of the test compound, the manner of contact between the test compound and the cell and the like are the same as in the above-mentioned method using the ARaiv activity as an index.
The expression level of ARaiv can be measured at the RNA level by detecting ARaiv mRNA by using a nucleic acid which can be hybridized with the aforementioned DNA encoding ARaiv under high stringent conditions, i.e., a nucleic acid which can be hybridized with a nucleotide sequence complementary to a nucleotide sequence shown by SEQ ID NO: 3, 5, 7, 9 or 11 under high stringent conditions. Alternatively, the expression level can also be measured at the protein level, by detecting ARaiv protein by using an antibody to AR or the below-mentioned antibody to ARaiv. When a cell inherently expressing ARaiv together with full-length AR, or a cell obtained by transducing a nucleic acid encoding ARaiv into a cell expressing full-length AR alone is used, mRNA or protein of full-length AR, and mRNA or protein of ARaiv need to be measured in a distinguished manner. A nucleic acid and an antibody capable of recognizing a region specific to ARaiv, which are used in the below-mentioned “diagnostic reagent of the present invention” can be used therefor. When measured at the protein level, ARaiv and AR can be measured in a distinguished manner based on the molecular weight of the band obtained by Western Blot using an antibody to AR.
When, for example, in the above-mentioned screening method, the expression level (mRNA amount or protein amount) of ARaiv in the presence of a test compound is inhibited by not less than about 20%, preferably not less than about 30%, more preferably not less than about 50%, as compared to the activity in the absence of the test compound, the test compound can be selected as a candidate for an ARaiv expression inhibitory substance, therefore, a substance for the prophylaxis or treatment of cancer, particularly ARaiv highly expressing cancer (e.g., prostate cancer etc.), inter alia, relapsed androgen-independent prostate cancer.
ARaiv is expressed in a subset of relapsed androgen-independent prostate cancer, and is a ligand (androgen, anti-androgenic agent) independent constitutively active molecule. Therefore, AR signal from this variant is considered to be at least one cause of the growth of androgen independent cancer cells in ARaiv expressing patients. Accordingly, a substance that inhibits the function of ARaiv is effective for the prophylaxis and/or treatment of ARaiv expressing cancer.
The “substance that inhibits function of variant AR(ARaiv) lacking a ligand binding domain” is largely divided into a substance that inhibits expression of ARaiv (expression inhibitor) and a substance that inhibits activity of ARaiv (activity inhibitor). The ARaiv expression inhibitor may act at any level from ARaiv transcription level, post-transcriptional regulation level, translation into protein level, post-translational modification level, ARaiv protein intracellular transport level, ARaiv protein degradation and the like. Therefore, examples of the ARaiv expression inhibitor include a substance inhibiting transcription of ARaiv, a substance inhibiting the processing from early transcription products to mRNA, a substance inhibiting transport of mRNA to cytoplasm, a substance promoting degradation of mRNA, a substance inhibiting translation from mRNA to protein, a substance inhibiting formation of the correct structure of ARaiv polypeptide, a substance inhibiting posttranslational modification of ARaiv polypeptide and the like. The substances acting at any of those levels are preferably used, but in the sense of directly inhibiting the production of ARaiv protein, a substance inhibiting translation from mRNA to protein is preferably employed. Moreover, a substance inhibiting selective splicing that produces ARaiv mRNA and a substance selectively promoting degradation of ARaiv mRNA are also preferable.
As a substance which can specifically inhibit translation from mRNA of ARaiv to protein, a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to the mRNA nucleotide sequence, or a part thereof, is preferably mentioned.
The “nucleotide sequence substantially complementary to the nucleotide sequence of ARaiv mRNA” refers to the nucleotide sequence having complementarity in an extent of inhibiting the translation by binding to a target sequence of the mRNA, under physiological conditions in the ARaiv expressing cell of a mammal. Specifically, it is, for example, the nucleotide sequence having not less than about 80% homology, preferably not less than about 90% homology, more preferably not less than about 95% homology, and particularly preferably not less than about 97% homology, to the nucleotide sequence completely complimentary to the mRNA nucleotide sequence (that is, nucleotide sequence of mRNA complementary strand) in the overlapping region.
The “homology of the nucleotide sequence” in the present invention can be calculated under the following conditions (an expectation value=10; gaps are allowed; filtering ═ON, match score=1; mismatch score=−3) using a homology scoring algorithm NCBI BLAST (National. Center for Biotechnology Information Basic Local Alignment Search Tool). As other algorithm for determining the homology of nucleotide sequence, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is incorporated in NBLAST and XBLAST program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [the algorithm is incorporated in the GAP program in GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is incorporated in ALIGN program (version 2.0) which is a part of the CGC sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is incorporated in the FASTA program in GCG software package] and the like can be mentioned, and they can be preferably used in a similar way.
More specifically, as a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of ARaiv mRNA, a nucleotide sequence complementary or substantially complementary to (a) a nucleotide sequence encoding a protein containing the amino acid sequence shown by SEQ ID NO: 4, 6, 8, 10 or 12, particularly SEQ ID NO: 6, 8, 10 or 12, more specifically, a nucleotide sequence shown by SEQ ID NO: 3, 5, 7, 9 or 11, particularly SEQ ID NO: 5, 7, 9 or 11, or (b) a nucleotide sequence that hybridizes to a complementary chain of said nucleotide sequence under high stringent conditions, and that encodes a polypeptide having an activity (e.g., androgen responsive gene transcription regulating action) equivalent to that of a protein comprising an amino acid sequence shown by SEQ ID NO: 4, 6, 8, 10 or 12, particularly SEQ ID NO: 6, 8, 10 or 12 can be mentioned.
“High-stringent conditions” refer to, for example, conditions involving a sodium salt concentration of about 19 to about 40 mM, preferably about 19 to about 20 mM, and a temperature of about 50 to about 70° C., preferably about 60 to about 65° C. In particular, a case wherein the sodium salt concentration is about 19 mM and the temperature is about 65° C. is preferred.
ARaiv mRNA is preferably human ARaiv2 mRNA containing a nucleotide sequence shown by SEQ ID NO: 5, or mRNA of human ARaiv1Δ1, ARaiv1Δ2 or ARaiv1Δ3 containing a nucleotide sequence shown by SEQ ID NO: 7, 9 or 11, respectively.
The “part of a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA” is not particularly limited as regards the length and position thereof as long as it can specifically bind to ARaiv mRNA and inhibit translation of protein from mRNA. From the aspect of sequence specificity, however, it contains not less than 10 bases, preferably not less than about 15 bases, more preferably not less than about 20 bases, of a region complementary or substantially complementary to a target sequence. In consideration of a simple synthesis, antigenic problem, and transitional problem in cell, oligonucleotide comprising about 10 to 40 bases, particularly about 15 to 30 bases is preferable. In addition, since ARaiv and normal splice variant (full-length AR) commonly have the 1st-3rd exons, ARaiv desirably contains the whole 4th exon of ARaiv (nucleotide sequence shown by SEQ ID NO: 13 (the 155642-157044-position of NCBI RefSeq No. NG—009014.1 (GI:213385253)) for ARaiv1, nucleotide sequence shown by SEQ ID NO: 14 (the 147929-149083-position of NG—009014.1) for ARaiv2) or a part thereof in at least a part of a target sequence, to specifically inhibit translation from ARaiv mRNA. However, since cancer cells express not only ARaiv mRNA but also mRNA encoding full-length AR, it is also preferable to set a region in the 1st-3rd exons as a target sequence, to shut off signaling from both ARs.
Since expression of full-length AR corresponding to TAU5-deleted ARaiv has not been confirmed, translation from the TAU5-deleted ARaiv mRNA may also be specifically inhibited by setting the linking site of 5′-side and 3′-side flaking sequences of a deleted region resulting from the deletion of the whole TAU5 region or a part thereof as a target sequence.
Specifically, as a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA, or a part thereof, for example, the nucleic acid of any of the following (a)-(e) is preferable.
(a) antisense nucleic acid for ARaiv mRNA
(b) siRNA for ARaiv mRNA
(c) nucleic acid capable of generating siRNA for ARaiv mRNA
(d) miRNA for ARaiv mRNA
(e) nucleic acid capable of generating miRNA for ARaiv mRNA
(a) Antisense Nucleic Acid for ARaiv mRNA
The “antisense nucleic acid for ARaiv mRNA” in the invention is a nucleic acid comprising a nucleotide sequence complimentary or substantially complimentary to the nucleotide sequence of mRNA or a part thereof, which has a function of inhibiting the protein synthesis by binding specifically with the target mRNA to form a stable duplex.
As the antisense nucleic acid, a polydeoxyribonucleotide containing 2-deoxy-D-ribose, a polyribonucleotide containing D-ribose, another type of polynucleotide that is an N-glycoside of the purine or pyrimidine base, or another polymer having a non-nucleotide backbone (e.g., commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or another polymer having a special bond (however, this polymer contains a nucleotide having a configuration that allows base pairing or base attachment as found in DNA and RNA) and the like can be mentioned. These may be double-stranded DNAs, single-stranded DNAs, double-stranded RNAs or single-stranded RNAs, or DNA:RNA hybrids, and may also be non-modified polynucleotides (or non-modified oligonucleotides), those having a known modification added thereto, for example, those with a label known in the relevant field, those with a cap, those methylated, those having 1 or more naturally occurring nucleotides substituted by analogues, those having an intramolecular nucleotide modification, for example, those having a non-charge bond (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate and the like), those having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate and the like), for example, those having a side chain group of a protein (nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine and the like), those having a side chain group of a sugar (e.g., monosaccharide and the like) and the like, those having an intercalating compound (e.g., acridine, soralen and the like), those containing a chelate compound (e.g., metals, radioactive metals, boron, oxidizing metals and the like), or those containing an alkylating agent, those having a modified bond (e.g., a anomer type nucleic acid and the like). Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing the purine and pyrimidine bases, but also those containing another modified heterocycle type base. These modified products may contain a methylated purine and pyrimidine, an acylated purine and pyrimidine, or another heterocycle. The modified nucleoside and the modified nucleotide may also have their sugar portion modified by, for example, substitution of 1 or more hydroxyl groups by a halogen, an aliphatic group and the like, or conversion to a functional group such as an ether or an amine.
As described above, the antisense nucleic acid may be DNA or RNA, or DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous RNase H, thereby directing the selective degradation of the target RNA.
Further, when not only ARaiv but also full-length AR are also the targets, the antisense nucleic acid may be a nucleic acid that inhibits the translation into a protein by hybridizing with mRNA of ARaiv, and it may as well as be the nucleic acid capable of forming a triplex by binding with the AR gene which is the double-stranded DNA and inhibiting the transcription into RNA (anti-gene).
The nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but may also include various chemical modifications in order to improve the stability (chemical and/or against enzyme) and specific activity (affinity with RNA). For example, to prevent digestion with a hydrolase such as nuclease, etc., the phosphate residue (phosphate) of each nucleotide that constitutes the antisense nucleic acid may be substituted with chemically modified phosphate residues, e.g., phosphorothioate (PS), methyl phosphonate, phosphorodithionate, etc. Also, a hydroxyl group at the 2′ position of the sugar (ribose) in each nucleotide may be replaced by —OR(R represents, e.g., CH3 (2′-O-Me), CH2CH2OCH3 (2′-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3. CH2CH2CN, etc.). Further, the base part (pyrimidine, purine) may be chemically modified, for example by the introduction of a methyl group or a cationic functional group to 5-position of the pyrimidine base or the replacement of a C2 carbonyl group with thiocarbonyl, etc.
The conformation of sugar in RNA is dominant in two of C2′-endo (S-form) and C3′-endo (N-form) and exists in equilibrium of those two in single-stranded RNA, but restricted in N-form in the case of a double strand. Accordingly, in order to provide a strong binding ability to the target RNA, BNA(LNA) (Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, J. S. et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003), which are RNA derivatives of which the sugar conformation is restricted into N-form by linking 2′-oxygen with 4′-carbon, are preferably employed.
The antisense oligonucleotide of the invention can be prepared by determining a target sequence of mRNA on the basis of a cDNA sequence information of ARaiv, and synthesizing its complementary sequence with the use of a commercially available DNA/RNA automatic synthesizer (Applied Biosystem company, Beckman company, etc.). In addition, the aforementioned antisense nucleic acids including various modifications can be chemically synthesized by a technique known per se.
(b) siRNA for ARaiv mRNA
In the present specification, a double-stranded RNA comprising oligoRNA complementary to ARaiv mRNA and a strand complementary to the oligoRNA, i.e., siRNA, is also defined to be included in a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA or a part thereof. The phenomenon of so-called RNA interference (RNAi) in which mRNA complementary to the RNA introduced is degraded by introducing short double-stranded RNA into a cell, is known to occur in nematode, an insect, plant, etc., but after it is confirmed that the phenomenon also occurs in animal cells [Nature, 411 (6836): 494-498 (2001)], it is widely used as an alternative technology of ribozyme. siRNA can be appropriately designed on the basis of nucleotide sequence information of target mRNA by using a commercially available software (e.g., RNAi Designer; Invitrogen corp.). In more specific, siRNAs used in EXAMPLES shown later are preferably exemplified as siRNA for ARaiv of the invention, but not limited by those.
The ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the antisense nucleic acid described above so as to improve the stability and specific activity. However, in the case of siRNA, introduction of minimal modified nucleoside allowing the RISC complex to function is necessary, since substitution of all ribonucleoside molecules in natural RNA by modified type may result in the loss of RNAi activity.
siRNA can be prepared according a process comprising synthesizing a sense strand and an antisense strand of target sequence on mRNA each with the DNA/RNA automatic synthesizer, denaturing in a suitable annealing-buffer solution at about 90 to 95° C. for about 1 minute, and annealing at about 30 to 70° C. for about 1 to 8 hours. In addition, siRNA can also be prepared by synthesizing short hairpin RNA (shRNA) which is the precursor of siRNA and cleaving the shRNA with the use of a dicer.
(c) Nucleic Acid Capable of Producing siRNA for ARaiv mRNA
In the present specification, a nucleic acid designed to be able to generate siRNA for the above-mentioned ARaiv mRNA in vivo is also defined to be included in a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA, or a part thereof. Such nucleic acid can be exemplified by aforementioned shRNA, an expression vector constructed in a manner to express the shRNA, or the like. The shRNA can be prepared by designing oligoRNA comprising a nucleotide sequence in which a sense strand and an antisense strand of the target sequence on mRNA are linked by a spacer sequence of a suitable length to form a loop-structure (for example, about 15 to 25 bases) intervened between the strands, and synthesizing the same by a DNA/RNA automatic synthesizer. The expression vector comprising a shRNA expression cassette can be prepared by first preparing a double-stranded DNA encoding the aforementioned shRNA by a conventional method and then inserting into a suitable expression vector. As the shRNA expression vector, those having Pol III promoter such as U6 or H1 can be used. In this case, shRNA transcribed in an animal cell in which the expression vector had been introduced forms a loop by itself, and then is processed by an endogeneous enzyme dicer or the like to form mature shRNA.
(d) miRNA for ARaiv mRNA
In the present specification, an endogenous single strand RNA having a short nucleotide sequence complementary to 3′-untranslated region of ARaiv mRNA, i.e., miRNA, is also defined to be included in a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA or a part thereof. More specifically, preferable examples of the miRNA for ARaiv of the present invention include, but are not limited to, hsa-mir-98, hsa-let-7a-1/2/3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1/2, hsa-let-7g, hsa-let-71, hsa-miR-125a-5p, hsa-miR-134, hsa-miR-149*, hsa-miR-491-5p, hsa-miR-574-5p and the like.
(e) Nucleic Acid Capable of Generating miRNA for ARaiv mRNA
In the present specification, a nucleic acid designed to be able to generate miRNA for the above-mentioned ARaiv mRNA in vivo (pre-miRNA, pri-miRNA etc.) is also defined to be included in a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA, or a part thereof.
Other preferred example of the nucleic acid comprising a nucleotide sequence complimentary or substantially complimentary to the nucleotide sequence of ARaiv mRNA or a part of the nucleotide sequence includes a ribozyme capable of specifically cleaving the internal coding region of the mRNA. The “ribozyme” is narrowly-defined as RNA having enzymatic activity for cleaving nucleic acid, but the present specification also includes DNA as long as there is a sequence specific enzymatic activity for cleaving nucleic acid. Ribozyme with mostly high-generality includes self-splicing RNA which can be found in infectious RNA such as viroid, a virusoid, etc., and hammer-head type or hairpin type are known. The hammer-head type exhibits enzymatic activity by about 40 bases, and it is possible to specifically cleave only the target mRNA by arranging several bases (about 10 bases in total) of both ends adjacent to the part having a hammer-head structure, so as to be complimentary to the desired cleavage site in mRNA. This type of ribozyme has a further advantage that genomic DNA is never targeted as its substrate is only RNA. When ARaiv mRNA forms itself a double-stranded structure, the target sequence can be formed into a single-strand by using hybrid ribozyme coupled with RNA motif derived from viral nucleic acid which can specifically bind to RNA helicase [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Also, in a case where ribozyme is used in the form of an expression vector having DNA which encodes the ribozyme, the ribozyme can be hybrid ribozyme further coupled with the sequence of modified tRNA so as to promote transport to cytoplasm of a transcription product [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
A nucleic acid comprising a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of ARaiv mRNA, or a part thereof can be supplied in a special form like liposome or microspheres, or can be given in an adduct form with a polycation like polylysine, which acts to neutralize the charge of the phosphate backbone, or a hydrophobic compound like a lipid that enhances the interaction with cellular membrane or increases nucleic acid uptake (e.g., phospholipid, cholesterol and the like). As lipids preferred for addition, cholesterol and derivatives thereof (e.g., cholesterylchloroformate, cholic acid and the like) can be mentioned. These can be attached to the 3′ end or the 5′ end of nucleic acid, and can be attached via a base, a sugar or an intramolecular nucleoside bond. As other groups, a capping group specifically arranged at the 3′ end or 5′ end of nucleic acid to prevent degradation by a nuclease such as exonuclease or RNase can be mentioned. As such a capping group, hydroxyl group protecting groups known in the relevant field, including glycols such as polyethylene glycol and tetraethylene glycol can be mentioned, which, however, are not to be construed as limitative.
[Medicament Containing Antisense Nucleic Acid, siRNA, Precursor Nucleic Acid Thereof, miRNA, Precursor Nucleic Acid Thereof]
An antisense nucleic acid that complementarily binds to an ARaiv transcription product and can suppress translation of protein from the transcription product, siRNA (or ribozyme) capable of cleaving an ARaiv transcription product by using a homologous (or complementary) nucleotide sequence in the ARaiv transcription product as a target, shRNA which is a precursor of the siRNA, miRNA that complementarily binds to an ARaiv transcription product and suppresses translation of protein from the transcription product or promotes degradation of the transcription product, and pre-miRNA which is a precursor of the miRNA and the like (hereinafter sometimes to be comprehensively referred to as “the nucleic acid of the present invention”) can be administered as they are in the form of liquid preparations, or as pharmaceutical compositions in suitable dosage forms to human or non-human mammals (e.g., mouse, rats, rabbits, sheep, swine, bovine, feline, canine, simian, etc.) orally or parenterally (e.g., intravascularly, subcutaneously, etc.).
When the nucleic acid of the present invention is used as an agent for the prophylaxis or treatment of relapsed prostate cancer, it can be prepared into a pharmaceutical preparation according to a method known per se and administered. That is, the nucleic acid of the present invention can be formulated singly, or formulated according to a conventional means after operably inserting into a suitable expression vector for mammalian cell such as retrovirus vector, adenovirus vector, adenovirus associated virus vector, lentivirus vector and the like. The nucleic acid can also be administered as it stands, or can be administered together with an auxiliary agent to assist its uptake, by a gene gun or through a catheter such as a catheter with a hydrogel. Alternatively, the antisense nucleic acid may be prepared into an aerosol, which is topically administered into the trachea as an inhaler.
Further for the purposes of improving pharmacokinetics, prolonging a half-life, and improving intracellular uptake efficiency, the nucleic acid described above may be prepared into a pharmaceutical preparation (injectable preparation) alone or together with a carrier such as liposome, etc. and the preparations may be administered intravenously, subcutaneously, or the like.
The pharmaceutical composition containing the nucleic acid of the present invention may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, a diluent or an excipient. Such a pharmaceutical composition is provided in the form of a pharmaceutical preparation suitable for oral or parenteral administration.
Examples of the composition for parenteral administration are injectable preparations, suppositories, etc. The injectable preparations may include dosage forms such as intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by methods publicly known. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying the nucleic acid of the present invention described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenated castor oil)], etc.
As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable thus prepared is usually filled in an appropriate ampoule. The suppository used for rectal administration may be prepared by blending the nucleic acid described above with a conventional base for suppository.
The composition for oral administration includes solid or liquid preparations, particularly, tablets (including dragees and film-coated tablets), pills, granules, powdery preparations, capsules (including soft capsules), syrup, emulsions, suspensions, etc. Such a composition is manufactured by publicly known methods and contains a carrier, a diluent or an excipient conventionally used in the field of pharmaceutical preparations. Examples of the carrier or excipient for tablets are lactose, starch, sucrose and magnesium stearate.
Advantageously, the pharmaceutical compositions for parenteral or oral use described above are prepared into pharmaceutical preparations with a unit dose formed to fit a dose of the active components. Such unit dose preparations include, for example, tablets, pills, capsules, injections (ampoules) and suppositories. The nucleic acid of the present invention is generally contained in 5 to 500 mg per dosage unit form, in 5 to 100 mg especially in the form of injection, and in 10 to 250 mg for the other forms.
The dose of the above-mentioned medicament comprising the nucleic acid of the present invention varies depending on the subject of administration, target disease, symptom, administration route and the like. When, for example, the medicament is used for the treatment or prophylaxis of prostate cancer in adult, the nucleic acid of the present invention is conveniently administered at, per one dose, generally about 0.01-20 mg/kg body weight, preferably about 0.1-10 mg/kg body weight, more preferably about 0.1-5 mg/kg body, weight, about 1-5 times, preferably 1-3 times, per day, by intravenous injection. When other parenteral or oral administration is employed, an amount similar thereto can be administered. When the symptom is particularly severe, the amount may be increased according to the symptom.
Each composition described above may further contain other active ingredients (e.g., HSP90 inhibitor, pyrvinium pamoate etc. to be mentioned later) as long as blending thereof does not cause any adverse effect on the nucleic acid of the present invention.
Furthermore, the nucleic acid of the present invention may be used in combination with other medicaments, for example, alkylating agents (e.g., cyclophosphamide, ifosfamide etc.), metabolic antagonists (e.g., methotrexate, 5-fluorouracil etc.), antitumor antibiotics (e.g., mitomycin, adriamycin etc.), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol etc.), cisplatin, carboplatin, etoposide, irinotecan, Taxotere, androgen lowering agents (e.g., LH-RH analogue (e.g., leuprorelin acetate etc.), LH-RH antagonists (e.g., degarelix)), androgen synthesis inhibitors (e.g., abiraterone etc.), anti-androgenic agents (e.g., flutamide, bicartamide etc.), synthetic adrenal cortex hormone drugs such as estrogen agents, prednisolone and the like, antibody drugs (e.g., trastuzumab, cetuximab, pertuzumab etc.), low-molecular-weight target drugs (e.g., lapatinib, imatinib, rapamycin, everolimus, temsirolimus etc.), cancer vaccine and the like. The nucleic acid and the above-mentioned medicament in the present invention can be administered to patients simultaneously or at different times.
In the present invention, ARaiv1 (here means a generic term of ARaiv containing C-terminal amino acid sequence shown by SEQ ID NO: 1) and ARaiv2 (here means a generic term of ARaiv containing C-terminal amino acid sequence shown by SEQ ID NO: 2) have a specific C-terminal amino acid sequence absent in full-length AR. Therefore, by examining the presence or absence of a protein having the amino acid sequence, or RNA containing a nucleotide sequence of the 4th exon of ARaiv1 and ARaiv2 encoding the amino acid sequence (SEQ ID NO: 13 and 14, respectively) in a cancer cell, whether the cancer cell expresses ARaiv1 or ARaiv2 can be determined.
Thus, the present invention also provides a diagnostic reagent for cancer that expresses ARaiv1 or ARaiv2, comprising a substance that specifically recognizes an amino acid sequence shown by SEQ ID NO: 1 or 2 or a nucleotide sequence shown by SEQ ID NO: 13 or 14. While the cancer patients to be the test subject are not particularly limited, they are preferably patients with prostate cancer, testis cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, colorectal cancer or pancreatic cancer, more preferably patients with relapsed androgen-independent prostate cancer. As the sample, biopsy and circulating tumor cell (CTC) can be used.
Examples of the substance that specifically recognizes an amino acid sequence shown by SEQ ID NO: 1 or 2 include an antibody for which the amino acid sequence is an epitope. Such antibody can be obtained by preparing a polyclonal antibody or monoclonal antibody according to a conventional method by chemically synthesizing a peptide comprising an amino acid sequence shown by SEQ ID NO: 1 or 2, immunizing a rabbit, mouse or the like with the peptide as an immunogen. ARaiv1 or ARaiv2 protein can be detected by an immunological method known per se (e.g., Western blotting, ELISA, immunohistochemistry, flow cytometry etc.). In addition, CTC detection system such as Cell Search System and the like may also be used in combination.
Examples of the substance that specifically recognizes a nucleotide sequence shown by SEQ ID NO: 13 or 14 include a nucleic acid containing a nucleotide sequence complementary to the whole or a part of the nucleotide sequence and/or a complementary chain thereof. Such nucleic acid can be obtained by chemical synthesis using a commercially available DNA/RNA automatic synthesizer. ARaiv1 or ARaiv2 mRNA can be detected by a nucleic acid detection method known per se such as Northern blotting, dot blotting, RT-PCR, in situ hybridization and the like. In addition, CTC detection system such as Cell Search System and the like can also be used in combination.
Patients judged to have cancer expressing ARaiv1 or ARaiv2 as a result of the above-mentioned diagnosis are preferable subjects of administration for the ARaiv expression/activity inhibitor of the present invention. Accordingly, the present invention also provides an agent for the prophylaxis or treatment of cancer containing an ARaiv expression/activity inhibitor, which is administered to patients diagnosed with cancer expressing ARaiv1 by using the above-mentioned diagnostic reagent.
The ARaiv expression inhibitor in the invention is not limited by the aforementioned nucleic acid comprising a nucleotide sequence complimentary or substantially complimentary to the nucleotide sequence of ARaiv mRNA or a part of the nucleotide sequence, and any other substances such as low-molecular-weight compounds may also be employed as long as it inhibits directly or indirectly the production of ARaiv protein. Examples of such low-molecular-weight compound include the following HSP90 inhibitor and the like.
The HSP90 inhibitor to be used in the present invention is not particularly limited as regards the pattern of HSP90 inhibition and the like as long as it can inhibit function of ARaiv, that is, a transcription regulating action of an androgen responsive gene (e.g., activation of transcription of prostate specific antigen (PSA) gene, TMPRSS2 gene and the like, suppression of transcription of SERPIN B5 gene and the like) and, for example, a medicament that binds to ATP binding pocket of HSP90 to inhibit its chaperone function (e.g., geldanamycin and a derivative thereof), a medicament that binds to HSP90 at a site other than the ATP binding pocket to inhibit its function (e.g., celastrol, gedunin and the like) and the like can be mentioned.
Examples of the geldanamycin derivative include compounds described in Tian Z Q et al., Bioorg. Med. Chem. 12 (2004) 5317-5329, U.S. Pat. No. 5,387,584, U.S. Pat. No. 4,261,989, U.S. Pat. No. 3,987,035, WO 00/03737, WO 02/079167, WO 99/21552, and WO 99/22761. Preferable examples of the geldanamycin derivative include a compound obtained by substituting the 17-position methoxy group of geldanamycin by an alkylamino group (17-alkylamino-17-demethoxy geldanamycin). Such compound is advantageous in that it can reduce the toxicity of geldanamycin. Specifically, for example, 17-allylamino-17-demethoxy geldanamycin (17-AAG), 17-(2-dimethylaminoethyl)amino-17-demethoxy geldanamycin (17-DMAG), 17-(4-(dimethylamino)butyl)amino-17-demethoxy geldanamycin, 17-(2-(carboxy)ethyl)amino-17-demethoxy geldanamycin, 17-(2-(N-methylethylamino)ethyl)amino-17-demethoxy geldanamycin, 17-(2-(pyrrolidin-1-yl)ethyl)amino-17-demethoxy geldanamycin, 17-(2-(piperazin-1-yl)ethyl)amino-demethoxy geldanamycin, 17-(4-(dimethylamino)butyl)amino-17-demethoxy geldanamycin and the like can be used.
Examples of other HSP90 inhibitor include, but are not limited to, radicicol and a derivative thereof, adenosine derivative PU3 (WO02/036075), a 1,3-dihydroxybenzene derivative wherein a pyrazole ring is bonded (WO03/055860, WO2004/050087, WO2004/056782), a 1,3-dihydroxybenzene derivative wherein an isoxazole ring is bonded (WO2004/072051), a 1,3-dihydroxybenzene derivative wherein a pyrazole ring having a nitrogen-containing substituent directly bonded at the nitrogen atom is bonded (JP-A-2006-306755), celastrol, gedunin (Cancer Cell, 2006 10(4): 321-30) and the like.
The HSP90 inhibitor may be in the form of a salt of any of the above-mentioned compounds. Examples of the salt include hydrohalic acid salt (specifically hydrofluoride, hydrochloride, hydrobromide, hydroiodide etc.), inorganic acid salt (specifically sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate etc.), organic carboxylic acid salt (specifically acetate, trifluoroacetate, oxalate, maleate, tartrate, fumarate, citrate etc.), organic sulfonate (specifically methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate etc.), amino acid salt (specifically aspartate, glutamate etc.), quaternary amine salt, alkali metal salt (specifically sodium salt, potassium salt etc.), alkaline earth metal salt (specifically magnesium salt, calcium salt etc.) and the like.
Since an HSP90 inhibitor can inhibit the function of ARaiv (transcription regulating action on androgen responsive gene), it can be used as a growth inhibitor for an ARaiv1 expressing cancer cell or an agent for the prophylaxis or treatment of ARaiv1 expressing cancer (e.g., colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, gastric cancer, liver cancer, biliarycancer, spleen cancer, kidney cancer, urinary bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor and the like, preferably, prostate cancer, testis cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, colorectal cancer, pancreatic cancer and the like, more preferably relapsed androgen-independent prostate cancer).
An ARaiv transcription regulating action inhibitor containing an HSP90 inhibitor can be administered directly as a liquid or a pharmaceutical composition in a suitable dosage form to a human or mammal (e.g., rat, mouse, rabbit, sheep, swine, bovine, cat, dog, monkey and the like) orally or parenterally (e.g., intravascular administration, subcutaneous administration and the like). The pharmaceutical composition to be used for the administration may contain an HSP90 inhibitor and a pharmacologically acceptable carrier, diluent or excipient.
Examples of the composition for parenteral administration are injectable preparations, suppositories, etc. The injectable preparations may include dosage forms such as intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by methods publicly known. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying HSP90 inhibitor in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mols) adduct of hydrogenated castor oil)], and the like. As an oily medium, for example, sesame oil, soybean oil and the like are used. It may be concurrently used with a solubilizing agent such as benzyl benzoate, benzyl alcohol and the like. A prepared injection is preferably filled in a suitable ampoule. A suppository to be used for rectal administration can also be prepared by mixing HSP90 inhibitor with a general base for suppository.
As a composition for oral administration, solid or liquid dosage form, specifically tablet (including sugar-coated tablet, film-coated tablet), pill, granule, powder, capsule (including soft capsule), syrup, emulsion, suspension and the like can be mentioned. Such composition is produced by a known method, and may contain a carrier, diluent or excipient generally used in the pharmaceutical field. As carrier or excipient for tablets, for example, lactose, starch, saccharose or magnesium stearate is used.
The above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in a dosage form of a dose unit suitable for the dose of an active ingredient. Examples of the dosage form for such dose unit include, for example, tablet, pill, capsule, injection (ampoule), and suppository. The HSP90 inhibitor is preferably contained in an amount of generally 5-500 mg per dose unit dosage form, particularly 5-100 mg for injection and 10-250 mg for other dosage form.
While the dose of the above-mentioned preparation containing the HSP90 inhibitor varies depending on the subject of administration, target disease, symptom, administration route and the like, for example, the HSP90 inhibitor is conveniently administered at, per one dose, generally about 0.01-20 mg/kg body weight, preferably about 0.1-10 mg/kg body weight, more preferably about 0.1-5 mg/kg body weight, for the treatment or prophylaxis of prostate cancer in adult, about 1 to 5 times, preferably about 1 to 3 times, per day by intravenous injection. In other parenteral administration and oral administration, the amounts similar thereto can be administered. When the symptom is particularly severe, the dose may be increased depending on the symptom.
The pharmaceutical composition containing an HSP90 inhibitor may contain other active ingredients as long as blending with an HSP90 inhibitor does not cause any unpreferable interaction.
Furthermore, the HSP90 inhibitor may be used in combination with other medicaments, for example, alkylating agents (e.g., cyclophosphamide, ifosfamide etc.), metabolic antagonists (e.g., methotrexate, 5-fluorouracil etc.), antitumor antibiotics (e.g., mitomycin, adriamycin etc.), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol etc.), cisplatin, carboplatin, etoposide, irinotecan, LH-RH analogues (e.g., leuprorelin acetate etc.), anti-androgenic agents (e.g., flutamide, bicartamide etc.), androgen synthesis inhibitors (e.g., abiraterone etc.), antibody drugs (e.g., trastuzumab, cetuximab etc.) and the like. The HSP90 inhibitor and the above-mentioned medicaments may be administered to patients simultaneously or at different times. When it is used in combination with an antibody drug, the HSP90 inhibitor and the antibody are conjugated by a method known per se to give a molecular target drug. The HSP90 inhibitor and the antibody are preferably bound via a linker. The linker contains, for example, substituted or unsubstituted aliphatic alkylene chain, and contains, at the both terminals, a group that can be bound to a functional group of the HSP90 inhibitor or antibody, for example, an N-hydroxysuccinimide group, an ester group, a thiol group, an imidecarbonate group, an aldehyde group and the like (Koutai Kougaku Nyumon, Chijin Shokan, 1994).
The ARaiv activity inhibitor in the present invention may be any as long as it inhibits ARaiv protein, once operably produced, from exhibiting an androgen responsive gene transcription regulating action and includes, for example, a substance that inhibits formation of an ARaiv-cofactor complex, a substance that inhibits binding of an ARaiv complex to a target gene, a substance that inhibits nuclear translocation of ARaiv, a substance that promotes degradation of ARaiv and the like.
In specific, the substance inhibiting the activity of ARaiv protein can be exemplified by antibodies against ARaiv protein. The antibody may be any of polyclonal antibody and monoclonal antibody. These antibodies can be manufactured in accordance with the method of manufacturing antibody or antisera, which is known per se. In this case, to obtain an antibody that specifically recognizes ARaiv from AR variants, it is desirable to use a partial peptide of a region encoded by the 4th exon of ARaiv, a junction region between 3rd exon and 4th exon as an immunogen, or manufacture a conformational antibody by a DNA immunization method. The isotype of the antibody is not particularly limited, but it is preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly subject to limitation, as long as it has at least a complementality determining region (CDR) for specifically recognizing and binding to the target antigen; in addition to the whole antibody molecule, the antibody may, for example, be a fragment such as Fab, Fab′, or F(ab′)2, a genetically engineered conjugate molecule such as scFv, scFv-Fc, minibody, or diabody, or a derivative thereof modified with a molecule having protein stabilizing action, such as polyethylene glycol (PEG), or the like, and the like.
In a preferred mode of embodiment, since the antibody to the ARaiv protein is used as a pharmaceutical product targeting humans as the subject of administration thereof, the antibody (preferably a monoclonal antibody) is an antibody whose risk of showing antigenicity when administered to a human has been reduced; to be specific, the antibody is a fully human antibody, a humanized antibody, a mouse-human chimeric antibody and the like, particularly preferably a fully human antibody. A humanized antibody and a chimeric antibody can be prepared by genetic engineering technology according to the usual method. Although a fully human antibody can also be produced from human-human (or -mouse) hybridoma, it is desirable to produce it using a human antibody-producing mouse or the phage display method in order to stably supply the antibody in large amounts at low costs.
Since ARaiv is a nuclear receptor variant and plays a key role in the transcription regulation of an androgen responsive gene, an ARaiv activity inhibitor is desirably a substance superior in intracellular transferability and nuclear translocation. Therefore, a more preferred substance for inhibiting the activity of ARaiv is a low-molecular-weight compound obeying Lipinski's Rule. Such compound can be obtained by, for example, the aforementioned screening method of the present invention. When the above-mentioned anti-ARaiv antibody is used as an ARaiv activity inhibitor, it can be encapsulated in a liposome to enhance intracellular transferability. Preferable liposome includes positively-charged liposome, positively-charged cholesterol, membrane permeable peptide-bound liposome and the like (Mamoru Nakanishi et al., protein nucleic acid enzyme 44:1590-1596 (1999), Shiro Niki, Kagaku to Seibutsu 43:649-653 (2005), Clinical Cancer Research 59:4325-4333 (1999) and the like). To is deliver an anti-ARaiv antibody selectively to the target cancer cell, moreover, the antibody can also be prepared as a bispecific antibody to a surface antigen molecule that is specifically highly expressed in the cancer cell.
Since the substance that inhibits a function of ARaiv in the present invention shuts off AR signal by inhibiting regulation of the transcription of an androgen responsive gene by ARaiv, it can be used as an agent for the prophylaxis and/or treatment of cancer, particularly ARaiv highly expressing cancer (e.g., colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, gastric cancer, liver cancer, biliarycancer, spleen cancer, kidney cancer, urinary bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, pancreatic cancer, brain tumor, blood tumor and the like, preferably, prostate cancer, testis cancer, breast cancer, uterine cancer, ovarian cancer, lung cancer, colorectal cancer, pancreatic cancer and the like, more preferably relapsed androgen-independent prostate cancer).
A medicament containing an antibody to ARaiv or a low-molecular-weight compound inhibiting ARaiv expression or activity (e.g., HSP90 inhibitor, pyrvinium pamoate etc.) can be administered directly as a liquid or a pharmaceutical composition in a suitable dosage form to a human or mammal (e.g., mouse, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like) orally or parenterally (e.g., intravascular administration, subcutaneous administration and the like).
The pharmaceutical composition comprising the above-mentioned antibody or low-molecular-weight compound may contain the above-mentioned antibody or low-molecular-weight compound or a salt thereof, and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration. Here, as the pharmacologically acceptable carrier, diluent and excipient, those similar to the carrier, diluent and excipient recited above for a preparation containing an HSP90 inhibitor can be preferably used.
The dose of the above-mentioned medicament containing the above-mentioned antibody or low-molecular-weight compound or a salt thereof varies depending on the subject of administration, target disease, symptom, administration route and the like. When used for the treatment or prophylaxis of prostate cancer in adult, the antibody or low-molecular-weight compound is preferably administered at, per one dose, generally about 0.01-20 mg/kg body weight, preferably about 0.1-10 mg/kg body weight, more preferably about 0.1-5 mg/kg body weight, about 1-5 times, preferably about 1-3 times, per day by intravenous injection. In other parenteral administration and oral administration, the amounts similar thereto can be administered. When the symptom is particularly severe, the dose may be increased depending on the symptom.
Each of the aforementioned compositions may contain other active ingredients as long as blending with the above-mentioned antibody or low-molecular-weight compound does not cause any unpreferable interaction.
Furthermore, the above-mentioned antibody or low-molecular-weight compound may be used in combination with other medicaments similar to those recited above for a medicament containing the nucleic acid of the present invention. The above-mentioned antibody or low-molecular-weight compound and other medicaments can be administered to patients simultaneously or at different times.
The substance that inhibits a function of ARaiv (i.e., the above-mentioned ARaiv expression or activity inhibitor) of the present invention can shut off at least the AR signal from ARaiv. In ARaiv expressing cancer cell, generally, full-length AR (androgen dependent AR) is also expressed simultaneously. As shown in the below-mentioned Examples, the ARaiv expressing cancer cell growth suppressive effect is higher by inhibition of both full-length AR and ARaiv than by specific inhibition of ARaiv alone. However, inhibition of not only ARaiv but also full-length AR by an ARaiv expression/activity inhibitor alone possibly requires increased doses. For example, since HSP90 is expressed in cancer cell as well as normal cell and responsible for various physiological functions, administration at a high dose is associated with feared expression of side effects.
Full-length AR has a ligand binding domain, and expresses AR activity in an androgen-dependent manner. Thus, transcription regulation of an androgen responsive gene can be inhibited by shutting off the supply of androgen itself, or co-presence of a substance that acts on AR competitively with androgen to shut off AR signal from the full-length AR. Therefore, by combining an AR signal inhibitor with the ARaiv expression/activity inhibitor of the present invention, the ARaiv expression/activity inhibitor can selectively act for inhibition of the ARaiv expression or activity. Consequently, the dose of the ARaiv expression/activity inhibitor can be reduced and the side effects can be decreased.
Accordingly, the present invention also provides an agent for the prophylaxis or treatment of ARaiv expressing cancer based on a combined use of an ARaiv expression/activity inhibitor of the present invention and an AR signal inhibitor. The subject of administration of the concomitant drug is not particularly limited as long as it is an animal having cancer expressing ARaiv. For example, it is an animal diagnosed using the diagnostic reagent of (4) below to have cancer expressing ARaiv.
While the ARaiv expression/activity inhibitor used here is not particularly limited as long as it is among those mentioned above, for example, a substance that specifically or preferentially inhibits expression or activity of ARaiv is preferable. In the case of an HSP90 inhibitor, for example, since it can exhibit an expression inhibitory effect at a lower concentration on ARaiv than on full-length AR, as shown in the below-mentioned Examples, HSP90 inhibitor is one preferable example of an ARaiv expression/activity inhibitor.
Examples of the AR signal inhibitor used here include, but are not limited to, androgen lowering agents (e.g., LH-RH analogue (e.g., leuprorelin acetate etc.), LH-RH antagonists (e.g., degarelix)), androgen synthesis inhibitors (e.g., abiraterone etc.), 5a reductase inhibitors (e.g., dutasteride etc.), AR antagonists (anti-androgen) (e.g., bicartamide, flutamide etc.), estrogen agent, etc., and the like.
In the above-mentioned combined use, the ARaiv expression/activity inhibitor can be administered to patients in an amount equal to or appropriately lower than the amount mentioned above for single administration. In addition, AR signal inhibitors can be each administered to patients in an amount equal to or appropriately lower than the dose known per se for single administration. The ARaiv expression/activity inhibitor and the AR signal inhibitor can be administered to patients simultaneously or at different times.
Abbreviations for bases, amino acids and the like used in the present specification are based on abbreviations specified by the IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations in common use in relevant fields. Some examples is are given below. When an enantiomer may be present in amino acid, it is of the L-configuration, unless otherwise stated.
DNA: Deoxyribonucleic acid
cDNA: Complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
RNA: Ribonucleic acid
mRNA: Messenger ribonucleic acid
dATP: Deoxyadenosine triphosphate
dTTP: Deoxythymidine triphosphate
dGTP: Deoxyguanosine triphosphate
dCTP: Deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: Sodium dodecyl sulfate
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: Methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: Histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: Pyroglutamic acid
Sec: Selenocysteine
The sequence identification numbers in the sequence listing of the present specification indicate the following sequences.
This shows a C-terminal amino acid sequence specific to ARaiv1.
This shows a C-terminal amino acid sequence specific to ARaiv2.
This shows the nucleotide sequence of ARaiv1 cDNA.
This shows the amino acid sequence of ARaiv1.
This shows the nucleotide sequence of ARaiv2 cDNA.
This shows the amino acid sequence of ARaiv2.
This shows the nucleotide sequence of ARaiv1Δ1 cDNA.
This shows the amino acid sequence of ARaiv1Δ1.
[SEQ ID NO: 9]
This shows the nucleotide sequence of ARaiv1Δ2 cDNA.
This shows the amino acid sequence of ARaiv1Δ2.
This shows the nucleotide sequence of ARaiv1Δ3 cDNA.
This shows the amino acid sequence of ARaiv1Δ3.
This shows the nucleotide sequence of the 4th exon of ARaiv1 mRNA.
This shows the nucleotide sequence of the 4th exon of ARaiv2 mRNA.
This shows the CDS sequence of wild-type AR.
This shows the amino acid sequence of wild-type AR.
This shows the ARE sequence in human PSA promoter.
[SEQ ID NO: 18]
This shows the ARE sequence in human PSA promoter.
This shows a consensus ARE sequence.
Refer to the Examples for SEQ ID NO: 19-31, 33-52.
Escherichia coli TOP10/pcDNA3.1-hARaiv1 harboring ARaiv1 clone obtained in Example 3 has been deposited at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since Jul. 15, 2009 (accession number: FERM BP-11144).
Escherichia coli TOP10/pcDNA3.1-hARaiv1Δ1 harboring ARaiv1Δ1 clone obtained in Example 3 has been deposited at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since Jul. 15, 2009 (accession number: FERM BP-11145).
Escherichia coli TOP10/pcDNA3.1-hARaiv1Δ2 harboring ARaiv1Δ2 clone obtained in Example 3 has been deposited at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since Jul. 15, 2009 (accession number: FERM BP-11146).
Escherichia coli TOP10/pcDNA3.1-hARaiv1Δ3 harboring ARaiv1Δ3 clone obtained in Example 3 has been deposited at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since Jul. 15, 2009 (accession number: FERM BP-11147).
Escherichia coli TOP10/pGEM-T easy-hARaiv2-3′ cDNA harboring ARaiv2 clone obtained in Example 2 has been deposited at the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since Jul. 15, 2009 (accession number: FERM BP-11148).
The present invention is explained in more detail in the following by referring to Examples and Reference Examples, which are not to be construed as limitative.
A biopsy sample obtained from the skin metastatic focus of prostate cancer patient was cut in thickness 2-3 mm, and the tumor fragments were subcutaneously transplanted to male nude mice (BALE-nu/nu, Charles River Japan). The tumor developed in the cancer-bearing mouse was named JDCaP tumor. The JDCaP tumor was maintained by repeated subcutaneous transplantation into the male nude mice. The JDCaP tumors were completely regressed by castrating the cancer-bearing mice, and the mice were reared for about 6 months to allow formation of relapsed tumor. The relapsed tumor was named JDCaP-hr (hormone refractory).
The JDCaP-hr cell line was established from JDCaP-hr tumor. The JDCaP-hr tumor was minced, and cultured in phenol red-free RPMI1640 (Invitrogen) containing 10% DCC-FBS. After several passages, the medium was changed to RPMI1640 containing 3% DCC-FBS and 100 ng/ml IGF (JRH Biosciences) for purification by suppressing the growth of mouse-derived stromal cells.
AR proteins in the JDCaP tumor and JDCaP-hr tumor were analyzed by Western blotting by using a specific antibody to the N-terminal (N-20) or C-terminal (C-19) portion of AR (Santa Cruz). As a loading control, β-actin was detected by using an AC-15 antibody (Sigma). The results are shown in
Total RNA was extracted from cultured cells and xenograft tumor of JDCaP-hr by using RNeasy Mini kit (Invitrogen) and RNase-Free DNase set (Qiagen), and 3′ rapid amplification of cDNA end (RACE) was performed using GeneRacer kit (Invitrogen). For primary PCR, a forward primer specific to AR exon 1 (5′-gcatggtgagcagagtgccctat-3′; SEQ ID NO: 19) and a reverse primer attached to the kit were used. Then, using a diluted solution of the primary PCR reaction mixture as a template, and a nested forward primer specific to exon 1 (5′-tactccggaccttacggggacat-3′; SEQ ID NO: 20) and a reverse primer attached to the kit, nested PCR was performed. The obtained 3′-RACE product was cloned, and the nucleotide sequence was analyzed by Big Dye Terminator v3.1 and ABI PRISM7700 (Applied Biosystems). As a result, two kinds of clones in which a part of AR intron 3 was linked to immediate downstream of AR exon 3 to produce an in-frame stop codon were found in addition to known AR 3′ sequence clone (
cDNA was prepared from JDCaP-hr RNA by using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Using PfuUltra II Fusion HS DNA Polymerase (Stratagene), a forward primer containing AR initiation codon (5′-atggaagtgcagttagggctgggaag-3′; SEQ ID NO: 21) and a reverse primer specific to exon 4b encoding the 3′ terminal of ARaiv1 transcript (5′-ggtctggtcattttgagatgcttgcaatt-3′; SEQ ID NO: 22), ARaiv1 ORF was amplified from the obtained cDNA. The obtained ORF fragment was cloned to a pGEM-T easy vector (Promega), and the sequence was analyzed. As a result, three kinds of clones having an in-frame defect in the TAU5 (transcriptional activation unit 5) region of exon 1 were obtained (
cDNA was prepared from RNA of CWR22Rv1 cell by using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Using PfuUltra II Fusion HS DNA Polymerase (Stratagene), a forward primer containing AR initiation codon (5′-atggaagtgcagttagggctgggaag-3′; SEQ ID NO: 21) and a reverse primer specific to exon 4b (5′-ggtctggtcattttgagatgcttgcaatt-3′; SEQ ID NO: 22), ARaiv1 ORF was amplified from the obtained cDNA. The obtained ORF fragment was cloned to a pGEM-T easy vector (Promega), and the sequence was analyzed. As a result, ORF clone of ARaiv1 having intact exon 1 and no defect on the N-terminal side was obtained (
The above-mentioned 4 kinds of ARaiv1 clones (ARaiv1 and ARaiv1Δ1, ARaiv1Δ2 and ARaiv1Δ3) were respectively subcloned to a pcDNA3.1 expression vector, and the transcription activity was examined by a reporter gene assay. The ARaiv1 clone expression vector and pGL3-2×PSA reporter were cotransfected into COS7 cell. After 24 hr, DHT or an AR antagonist bicalutamide, was added, and 24 hr later, a luciferase assay was performed. As a result, the 4 kinds of ARaiv1 clones all showed a DHT independent increase of the reporter activity (
The expression of ARaiv1 and AR in various prostate cancer cell lines was examined by a quantitative RT-PCR method. The quantitative RT-PCR was carried out using ABI PRISM7700 (Applied Biosystems), QuantiTect Probe RT-PCR kit (Qiagen) and gene specific primers and probes. The primers and probes used were AR (forward primer: 5′-aaatgttatgaagcagggatgactct-3′ (SEQ ID NO: 23); reverse primer: 5′-gcttctgggttgtctcctcagt-3′ (SEQ ID NO: 24); probe: 5′-FAM-actacaggaggaaggaga-MGB-3′ (SEQ ID NO: 25)), ARaiv1 (forward primer: 5′-catcttgtcgtcttcggaaatg-3′ (SEQ ID NO: 26); reverse primer: 5′-tgccaacccggaatttttct-3′ (SEQ ID NO: 27); probe: 5′-FAM-cagggatgactctggg-MGB-3′ (SEQ ID NO: 28)), and GAPDH (Hs99999905, TaqManR Gene Expression Assays, Applied Biosystems). As a result, high expression of ARaiv1 was found in JDCaP-hr cells, 22Rv1 cells and VCaP cells (
An siRNA targeting exon 4b specific to ARaiv1 (siARaiv1: 5′-gattgttcttgatcacata-3′ (SEQ ID NO: 29)) was designed and synthesized by Qiagen. In addition, an siRNA targeting exon 8 specific to AR (siAR: Hs_AR—6_HP Validated siRNA: 5′-ggaacucgaucguaucauu-3′ (SEQ ID NO: 30)) and an siRNA cocktail containing siRNA targeting exon 1 common to AR and ARaiv1 (siDual: OTP SMART pool, L-003400-00-0005) were purchased from Qiagen and Dharmacon, respectively. siAR, siARaiv1 or siDual was transfected into JDCaP-hr cells. RNA was extracted from the cells after 48 hr from the siRNA transfection, and the expression level of the target gene was analyzed by quantitative RT-PCR. As a result, it was confirmed that gene knockdown by each siRNA was carried out sufficiently efficiently (
siRNA targeting AR-specific exon 8 (siAR), siRNA targeting ARaiv1-specific exon 4b (siARaiv1) or siRNA targeting exon 1 common to the both (siDual) was transfected into JDCaP-hr cells, the cells were cultured for 5 days in an androgen-free medium added with DHT or DMSO, and the cell proliferation was measured (
Expression plasmids of shRNA targeting AR-specific exon 8 (shAR), shRNA targeting ARaiv1-specific exon 4b (shARaiv1) and shRNA targeting exon 1 common to the both (shDual) were constructed using siSTRIKE U6 hairpin cloning system (Promega). The target sequences of shAR and siARaiv1 were the same as those of the above-mentioned siAR and siARaiv1. As the target sequence of shRNA specific to exon 1 (shDual), the sequence of exon 1-specific siRNA (5′-gcagaaatggattgcactat-3′ (SEQ ID NO: 31)) contained in siDual was used.
The expression vector of shAR, siARaiv1 or shDual was transfected into 22Rv1 cells, and a colony formation assay was carried out under androgen-free conditions. The results are shown in
A homology analysis was carried out between the 3′-UTR nucleotide sequence of ARaiv1 and not less than 400 kinds of the nucleotide sequences of human miRNA. As a result, hsa-mir-98, hsa-let-7a-1/2/3, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f-1/2, hsa-let-7g, hsa-let-71, hsa-miR-125a-5p, hsa-miR-134, hsa-miR-149*, hsa-miR-491-5p, and hsa-miR-574-5p were considered to be able to bind to 3′-UTR of ARaiv1 (Tables 1-1 and 1-2).
5′] alignment
3′]
5′] alignment
3′]
Using Tissue Scan™ Real-Time, Prostate Cancer Array II (Origene) as cDNA of clinical prostate cancer, the expression levels of AR, ARaiv1 and β-actin were measured by quantitative RT-PCR. The quantitative RT-PCR was carried out using ABI PRISM7700 (Applied Biosystems), QuantiTect Probe RT-PCR kit (Qiagen) and gene specific primers and probes. The primers and probes used were AR (forward primer: 5′aaatgttatgaagcagggatgactct-3′ (SEQ ID NO: 23); reverse primer: 5′-gcttctgggttgtctcctcagt-3′ (SEQ ID NO: 24); probe: 5′-FAM-actacaggaggaaggaga-MGB-3′ (SEQ ID NO: 25)), ARaiv1 (forward primer: 5′-catcttgtcgtcttcggaaatg-3′ (SEQ ID NO: 26); reverse primer: 5′-tgccaacccggaatttttct-3′ (SEQ ID NO: 27); probe: 5′-FAM-cagggatgactctggg-MGB-3′ (SEQ ID NO: 28)), and β-actin (Human ACTB, Pre-Developed TaqManR Assay Reagents, PE Biosystems). The results are shown in
COS7 cells were seeded at 106 cells/6 cm diameter culture dish and, 24 hr later, 200 ng of ARaiv1 expression plasmid and 7 μg of 2×PSA reporter plasmid were transfected using lipofectoamine-2000. After 6 hr, the cells were detached therefrom, and seeded again in RPMI1640 medium containing 10% steroid-free fetal bovine serum in a 96 well culture plate. After 24 hr, geldanamycin or solvent DMSO was added and the culture was continued. After 24 hr, the luciferase activity was measured by Bright-Glo luciferase assay system (Promega). As a result, transcription activity of ARaiv1 was inhibited by geldanamycin (
Geldanamycin was added to JDCaP-hr cells, and an influence on the cell proliferation, ARaiv1 mRNA expression and ARaiv1 protein production were examined. It was clarified that geldanamycin remarkably reduced ARaiv1 mRNA and protein production (
Pyrvinium pamoate (PP) has been reported to inhibit the transcription activity of AR according to an action mechanism different from androgen antagonism. An influence of PP on the transcription activity of ARaiv1 was examined by a reporter gene assay. An empty vector or ARaiv1 expression vector was transfected into LNCaP-FGC with 2×PSA reporter, PP was added 24 hr later, and a luciferase assay was carried out 24 hr later. As a result, it was clarified that PP inhibits transcription by ARaiv1 in a concentration-dependent manner (
Pyrvinium pamoate (PP) was added to JDCaP-hr cells and an influence on the cell proliferation was examined. It was clarified that PP remarkably inhibits the growth of JDCaP-hr (
Using inhibition of transcriptional activation by ARaiv and the like as an index, a substance effective for the prophylaxis or treatment of ARaiv expressing cancer can be screened for. An antisense nucleic acid, siRNA, miRNA, or HSP90 inhibitor capable of specifically inhibiting expression of ARaiv and other substance that inhibits expression or activity of ARaiv are useful for the treatment and prevention of recurrence of ARaiv expressing cancer. In addition, detection of the C-terminal sequence of ARaiv enables diagnosis of ARaiv expressing cancer.
This application is based on a patent application No. 2009-225541 filed in Japan on Sep. 29, 2009, the contents of which are incorporated in full herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-225541 | Sep 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/066874 | 9/28/2010 | WO | 00 | 3/28/2012 |
