Mutated androgen receptor, cancer cells expressing the same, method of constructing the same and use thereof

Technical Field

0001The present invention relates to a mutant androgen receptor derived from human prostate cancer cells, a human prostate cancer cell line that expresses it, a method of producing them, a method of screening an antiandrogen drug that exhibits proliferation-suppressing action even on prostate cancer in the androgen-independent stage using them and the like.

Background Art

0002For advanced prostate cancer with metastases, it is standard practice worldwide to conduct androgen suppression endocrine therapy as the first choice. Also, in localized prostate cancer without metastases, it was demonstrated that prostate cancer progression is delayed by conducting an endocrine therapy with an antiandrogen drug after radical prostatectomy or radiotherapy; endocrine therapy is spreading for localized prostate cancer as well.

0003Androgens generically refer to steroid hormones possessing male sex hormone actions; testosterone, which is synthesized in the testis, dehydroepiandrosterone and androstenedione, which are synthesized in the adrenal cortex, and the like can be mentioned. Because about 95% of androgens are secreted from the testis, surgical (orchiectomy) or chemical (estrogen, LHRH agonist and the like) castration for blocking testis-derived testosterone is frequently used as androgen suppression endocrine therapy. However, because the adrenal-cortex-derived testosterone precursor is converted to highly active dihydrotestosterone (DHT) in prostate cells, it is sometimes not negligible as a proliferative stimulation to androgen-hypersensitive cancer cells, which are considered to exist at a given ratio in prostate cancer lesions, even if their abundance is apparently only several percent. For this reason, MAB (Maximal Androgen Blockade) therapy, in which an antiandrogen drug (particularly a non-steroidal drug) that antagonistically inhibits the binding of androgens to their receptors in the prostate is used in combination with castration surgery, is also conducted.

0004On the other hand, no effective prophylactic/therapeutic method for androgen-independent relapsed cancers, which are experienced sooner or later in almost all patients in the advanced stage undergoing endocrine therapy, has been developed. Although the mechanism of cancer relapse to endocrine therapy has not been elucidated, a plurality of factors, such as development/increase of cells that have acquired androgen hypersensitivity as a result of androgen receptor (AR) gene amplification or overexpression, and non-androgen substances (for example, glucocorticoids and antiandrogen drugs themselves) becoming acting as agonists due to AR mutations, are suggested.

0005For example, some clinical findings have been reported concerning the relationship between cancer relapse after antiandrogen drug administration and AR mutations. That is, AR mutations were observed in 5 of 17 patients who experienced relapsed prostate cancer after an endocrine therapy with a combination of flutamide and castration, all of which mutations were missense mutations of the 877th amino acid (corresponding to amino acid number 882 in the amino acid sequence of SEQ ID NO: 2) (<nplcit id="ncit0001" npl-type="s"><text>Taplin et al., Cancer Res., 59: 2511-2515, 1999</text></nplcit>). On this type of mutant ARs, some antiandrogen drugs, including flutamide, conversely exhibited an action to stimulate cancer cell proliferation (<nplcit id="ncit0002" npl-type="s"><text>Veldscholte et al., Biochem. Biophys. Res. Commun., 173: 534-540, 1990</text></nplcit>). Also, although missense mutations of AR were identified in 3 of 11 biopsy samples from patients who experienced relapsed prostate cancer after an endocrine therapy with a combination of bicalutamide and surgical castration, all mutation sites were other than the 877th amino acid, which is a prevalent mutation site in flutamide-resistant relapsed cancers (<nplcit id="ncit0003" npl-type="s"><text>Haapala et al., Lab. Invest., 81: 1647-1651, 2001</text></nplcit>).

0006In developing a drug useful for the prophylaxis/treatment of androgen-independent cancers based on AR mutations, cancer cell lines that express such mutant ARs are very useful research tools. However, all mutant-AR-expressing cancer cells that have been reported to date were clinically obtained from relapsed cancers or pre-treatment prostate cancer; there is no case wherein a mutation was induced in AR-expressing cancer cells <i>in vitro</i> to establish a cancer cell line that expresses an AR having a mutation similar to those of clinically found mutant ARs derived from androgen-independent cancers. Also, mutant ARs clinically found in androgen-independent cancers mostly have a single mutation at the amino acid level; reported cases of ARs having amino acid mutations at 2 sites or more are not unavailable but are very rare.

0007Accordingly, the object of the present invention is to provide a method of producing in <i>vitro</i> a cancer cell line that expresses an AR having a mutation similar to those of clinically found mutated ARs derived from androgen-independent cancers, and the mutant AR and the cancer cell line that expresses it which are obtained by said method, and to provide a method of screening a drug effective in the prophylaxis/treatment of androgen-independent relapsed cancers to endocrine therapy using them.

Disclosure of the Invention

0008The present inventors conducted extensive investigations with the aim of accomplishing the above-described object, and found that when human prostate cells that are sensitive to a specified antiandrogen drug are cultured in the presence of said antiandrogen drug for a long period, a mutation to cause the antiandrogen drug to considerably lose potency or conversely exhibit agonist-like action is induced in an AR, and, as a result, the cells become an antiandrogen-drug-resistant, that is, androgen-independent, cancer and begin proliferation. Furthermore, the present inventors isolated the cDNA of the AR from cancer cells showing proliferation, analyzed the entire base sequence thereof, and found that the mutation site of the AR agrees well with that of a mutant AR clinically obtained from a relapsed cancer in a patient undergoing an endocrine therapy with an antiandrogen drug. The present inventors conducted further investigations based on these findings, which resulted in the completion of the present invention.

Brief Description of the Drawings

0009<ul id="ul0001" list-style="none"><li><figref idref="f0001">Figure 1</figref> is a drawing showing the effects of bicalutamide on the proliferation of LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells. LNCaP-caD11, LNCaP-cxD2 and LNCaP-FGC cells suspended in a medium (RPM11640 +10% DCC-FBS) were sown to 24-well plates at 40000 cells/plate, and, on the following day, bicalutamide was added. Three days after bicalutamide addition, cells were counted. Mean ± standard error, n=3.</li><li><figref idref="f0001">Figure 2</figref> is a drawing showing the effects of bicalutamide on the PSA production by LNCaP-caD11, LNCaP-cxD2 and LNCaP-FGC cells. LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells suspended in a medium (RPM11640 + 10% DCC-FBS) were sown to 24-well plates at 40000 cells/plate, and, on the following day, bicalutamide was added. Three days after bicalutamide addition, supernatant concentrations of PSA were measured. Mean ± standard error, n=3.</li><li><figref idref="f0002">Figure 3</figref> is a drawing showing the transcription-promoting activity of bicalutamide on wild type or mutant (W741C, W741L) ARs. A vector DNA incorporating each AR and a vector DNA having the luciferase gene linked downstream of an androgen-responsive promoter were co-transfected, and 0.01 or 1 µM bicalutamide was added, after which luciferase activity was measured. Mean ± standard error, n=4.</li><li><figref idref="f0002">Figure 4</figref> is a drawing showing the antagonistic action of Compound A on a W741C type mutant AR. A vector DNA incorporating the W741C type mutant AR and a vector DNA having the luciferase gene linked downstream of an androgen-responsive promoter were co-transfected, and 40nM DHT or both 40nM DHT and 10 µM Compound A were added, after which luciferase activity was measured. Mean ± standard error, n=16 for the only-DHT addition group and n=4 for the DHT+Compound A addition group.</li><li><figref idref="f0003">Figure 5</figref> is a drawing showing the transcription activation of a gene under the control of the PSA promoter by a wild type AR activated by DHT. A vector DNA incorporating the wild type AR and a vector DNA having the luciferase gene linked downstream of one PSA promoter or two tandemly ligated PSA promoters were co-transfected to COS-7 cells, and 1 µM DHT was added, after which luciferase activity was measured. Mean ± standard error, n=6.</li></ul>

0010The mutant AR of the present invention is a protein containing the amino acid sequence in which tryptophan at amino acid number 746 is substituted by leucine in the amino acid sequence represented by SEQ ID NO: 2 (<nplcit id="ncit0004" npl-type="s"><text>Proc. Natl. Acad. Sci. USA, 85: 7211-7215, 1988</text></nplcit>) (also abbreviated as "W746L"), or an amino acid sequence in which tryptophan at amino acid number 746 is substituted by leucine or cysteine and threonine at amino acid number 882 is substituted by alanine in the amino acid sequence represented by SEQ ID NO: 2 (also abbreviated as "W746L(C)+T882A").

0011The mutant AR of the present invention may be any protein derived from any cells (e.g., splenocytes, nerve cells, glial cells, beta cells of pancreas, bone marrow cells, mesangial cells, Langerhans' cells, epidermic cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells, immune cells (e.g., macrophage, T cells, B cells, natural killer cells, mast cells, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or interstitial cells, the corresponding precursor cells, stem cells, cancer cells, etc.), hemocyte type cells, or any tissues where such cells are present, e.g., brain or any region of the brain (e.g., olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital pole, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., large intestine and small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testicle, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. (particularly prostate) from mammals (e.g., human, guinea pig, rats, mouse, rabbit, swine, sheep, bovine, monkey, etc.). The mutant AR may also be a synthetic protein.

0012Throughout the present specification, proteins are represented in accordance with the conventional way of describing peptides, that is, the N-terminus (amino terminus) at the left hand side and the C-terminus (carboxyl terminus) at the right hand side. In the mutant AR of the present invention, the C-terminus may be usually in the form of a carboxyl group (-COOH), a carboxylate (-COO^-), an amide (-CONH₂) or an ester (-COOR).

0013Examples of the ester group shown by R include a C_1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C_3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc.; a C_6-12 aryl group such as phenyl, alpha -naphthyl, etc.; a C_7-14 aralkyl group such as a phenyl-C_1-2-alkyl group, e.g., benzyl, phenethyl, etc., or an α-naphthyl-C_1-2-alkyl group such as α-naphthylmethyl, etc.; and the like. In addition, pivaloyloxymethyl or the like, which is used widely as an ester for oral administration, may also be used.

0014When the mutant AR of the present invention has a carboxyl group (or a carboxylate) at a position other than the C-terminus, it may be amidated or esterified and such an amide or ester is also included within the mutant AR of the present invention. In this case, as examples of the ester, the above-described ester at the C-terminus and the like can be used.

0015Furthermore, examples of the mutant AR of the present invention include, referring to the above-described protein, those wherein the amino group of the methionine residue at the N-terminus is protected by a protecting group (for example, a C_1-6 acyl group such as a C_2-6 alkanoyl group such as a formyl group or acetyl, and the like), those wherein the glutamyl group resulting from cleavage on the N-terminus side <i>in vivo</i> is pyroglutamated, those wherein a substituent on the side chain of an amino acid in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group, guanidino group and the like) is protected by an appropriate protecting group (for example, a C_1-6 acyl group such as a C_2-6 alkanoyl group such as a formyl group or acetyl, and the like); or complex proteins such as what is called a glycoprotein, wherein a sugar chain is bound, and the like.

0016Specific examples of the mutant AR of the present invention include a human-derived (more preferably human prostate cancer-derived) AR containing W746L or W746L+T882A.

0017For salts of the mutant AR of the present invention, preferred are salts with physiologically acceptable acids or bases, especially physiologically acceptable acid addition salts. Examples of the salts include salts with, for example, inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid); salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

0018The mutant AR of the present invention or a salt thereof may be produced by isolating and purifying from animal cells having an ability of producing said mutant AR using a publicly known method for purifying a receptor protein. For example, it can be purified and isolated by homogenizing human or mammalian tissues followed by extraction with an acid or the like, and subjecting the extract to a combination of chromatography techniques such as reverse phase chromatography, ion exchange chromatography and the like.

0019Examples of animal cells having an ability of producing the mutant AR of the present invention include the aforementioned human or mammalian cells or tissues (particularly cells derived from a relapsed cancer of a prostate cancer patient receiving an antiandrogen drug (e.g., bicalutamide, or bicalutamide and flutamide)), an antiandrogen drug-resistant cancer cell line obtained by culturing a cancer cell line that expresses a normal AR or an AR having the T882A mutation (e.g., LNCaP cell line) in the presence of an antiandrogen drug (e.g., bicalutamide or an analogue thereof) followed by selection using cell proliferation as an index, and the like.

0020The mutant AR of the present invention or a salt thereof may also be produced by culturing a transformant transformed with a recombinant vector that contains a DNA encoding the mutant AR of the present invention described below. Alternatively, it may also be produced by the protein synthesis methods to be hereinafter described or modifications thereof, based on the amino acid sequence of W746L or W746L+T882A.

0021To synthesize the mutant AR of the present invention, or salts or amides thereof, commercially available resins that are used for protein synthesis may be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenyl acetamidomethyl resin, polyacrylamide resin, 4-(2',4'-dimethoxyphenylhydroxymethyl)phenoxy resin, 4-(2',4'-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Using these resins, amino acids in which α-amino groups and functional groups on the side chains are appropriately protected are condensed on the resin in the order of the sequence of the objective protein according to various condensation methods publicly known in the art. At the end of the reaction, the receptor protein is cut out from the resin and at the same time, the protecting groups are removed. Then, intramolecular disulfide bond-forming reaction is performed in a highly diluted solution to obtain the objective protein or its amides.

0022For condensation of the protected amino acids described above, a variety of activation reagents for protein synthesis may be used, and carbodiimides are particularly preferable. Examples of such carbodiimides include DCC, N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminoprolyl)carbodiimide, etc. For activation by these reagents, the protected amino acids in combination with a racemization inhibitor (e.g., HOBt, HOOBt) are added directly to the resin, or the protected amino acids are previously activated in the form of symmetric acid anhydrides, HOBt esters or HOOBt esters, followed by adding the thus activated protected amino acids to the resin.

0023Solvents suitable for use to activate the protected amino acids or condense with the resin may be chosen from solvents known to be usable for protein condensation reactions. Examples of such solvents are acid amides such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylene chloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.; sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine, dioxane, tetrahydrofuran, etc.; nitriles such as acetonitrile, propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.; and appropriate mixtures of these solvents. The reaction temperature is appropriately chosen from the range known to be applicable to protein binding reactions and is usually selected in the range of approximately -20°C to 50°C. The activated amino acid derivatives are used generally in an excess of 1.5 to 4 times. The condensation is examined by a test using the ninhydrin reaction; when the condensation is insufficient, the condensation can be completed by repeating the condensation reaction without removal of the protecting groups. When the condensation is yet insufficient even after repeating the reaction, unreacted amino acids are acetylated with acetic anhydride or acetylimidazole.

0024Examples of the protecting groups used to protect the amino groups of the starting compounds include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc.

0025A carboxyl group can be protected by, e.g., alkyl esterification (in the form of linear, branched or cyclic alkyl esters of the alkyl moiety such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification (e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.), phenacyl esterification, benzyloxycarbonyl hydrazidation, t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

0026The hydroxyl group of serine can be protected through, for example, its esterification or etherification. Examples of groups appropriately used for the esterification include a lower alkanoyl group, such as acetyl group, an aroyl group such as benzoyl group, and a group derived from carbonic acid such as benzyloxycarbonyl group, ethoxycarbonyl group, etc. Examples of a group appropriately used for the etherification include benzyl group, tetrahydropyranyl group, t-butyl group, etc.

0027Examples of groups for protecting the phenolic hydroxyl group of tyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl, etc.

0028Examples of groups used to protect the imidazole moiety of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

0029Examples of the activated carboxyl groups in the starting compounds include the corresponding acid anhydrides, azides, activated esters (esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)). As the activated amino acids, in which the amino groups are activated in the starting material, the corresponding phosphoric amides are employed.

0030To eliminate (split off) the protecting groups, there are used catalytic reduction under hydrogen gas flow in the presence of a catalyst such as Pd-black or Pd-carbon; an acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid or trifluoroacetic acid, or a mixture solution of these acids; a treatment with a base such as diisopropylethylamine, triethylamine, piperidine or piperazine; and reduction with sodium in liquid ammonia. The elimination of the protecting group by the acid treatment described above is carried out generally at a temperature of approximately -20°C to 40°C. In the acid treatment, it is efficient to add a cation scavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, 2,4-dinitrophenyl group known as the protecting group for the imidazole of histidine is removed by a treatment with thiophenol. Formyl group used as the protecting group of the indole of tryptophan is eliminated by the aforesaid acid treatment in the presence of 1,2-ethanedithiol or 1,4-butanedithiol, as well as by a treatment with an alkali such as a dilute sodium hydroxide solution and dilute ammonia.

0031Protection of functional groups that should not be involved in the reaction of the starting materials, protecting groups, elimination of the protecting groups and activation of functional groups involved in the reaction may be appropriately selected from publicly known groups and publicly known means.

0032In another method for obtaining the amides of the protein, for example, the α-carboxyl group of the carboxy terminal amino acid is first protected by amidation; the peptide (protein) chain is then extended from the amino group side to a desired length. Thereafter, a protein in which only the protecting group of the N-terminal α-amino group in the peptide chain has been eliminated from the protein and a protein in which only the protecting group of the C-terminal carboxyl group has been eliminated are prepared. Both proteins are condensed in a mixture of the solvents described above. The details of the condensation reaction are the same as described above. After the protected protein obtained by the condensation is purified, all the protecting groups are eliminated by the method described above to give the desired crude protein. This crude protein is purified by various known purification means. Lyophilization of the major fraction gives the amide of the desired protein.

0033To prepare the esterified protein, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to prepare the amino acid ester, which is followed by procedure similar to the preparation of the amidated protein above to give the ester form of the desired protein.

0034The mutant AR of the present invention can be produced by publicly known methods for peptide synthesis. For the methods for peptide synthesis, for example, either solid phase synthesis or liquid phase synthesis may be used. That is, the partial peptide or amino acids that can construct the mutant AR of the present invention or the partial peptide of the present invention are condensed with the remaining part. Where the product contains protecting groups, these protecting groups are removed to give the desired peptide. Publicly known methods for condensation and elimination of the protecting groups are described in (1)-(5) below: <ol id="ol0001" compact="compact"><li>(1) <nplcit id="ncit0005" npl-type="b"><text>M. Bodanszky &amp; M.A. Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966</text></nplcit>);</li><li>(2) <nplcit id="ncit0006" npl-type="b"><text>Schroeder &amp; Luebke: The Peptide, Academic Press, New York (1965</text></nplcit>);</li><li>(3) <nplcit id="ncit0007" npl-type="b"><text>Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics and experiments of peptide synthesis), published by Maruzen Co. (1975</text></nplcit>);</li><li>(4) <nplcit id="ncit0008" npl-type="s"><text>Haruaki Yajima &amp; Shunpei Sakakibara: Seikagaku Jikken Koza (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Proteins) IV, 205 (1977</text></nplcit>);</li><li>(5) <nplcit id="ncit0009" npl-type="b"><text>Haruaki Yajima, ed.: Zoku lyakuhin no Kaihatsu (A sequel to Development of Pharmaceuticals), Vol. 14, Peptide Synthesis, published by Hirokawa Shoten</text></nplcit>.</li></ol>

0035Also, after completion of the reaction, the mutant AR of the present invention may be purified and isolated using a combination of conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the mutant AR of the present invention obtained by the above methods is in a free form, it can be converted into an appropriate salt by a publicly known method; when it is obtained in a salt form, it can be converted into a free form by a publicly known method.

0036The polynucleotide encoding the mutant AR of the present invention may be any polynucleotide so long as it contains the base sequence (DNA or RNA, preferably DNA) encoding the mutant AR of the present invention described above. Such a polynucleotide may also be any one of DNA encoding the mutant AR of the present invention, RNA such as mRNA, etc., and may be double-stranded or single-stranded. Where the polynucleotide is double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA:RNA hybrid. Where the polynucleotide is single-stranded, it may be a sense strand (i.e., a coding strand) or an antisense strand (i.e., a non-coding strand).

0037Using the polynucleotide encoding the mutant AR of the present invention, mRNA of the mutant AR of the present invention can be quantified by, for example, the publicly known method published in <nplcit id="ncit0010" npl-type="s"><text>separate volume of Jikken Igaku 15 (7) "New PCR and its application" (1997</text></nplcit>) and the like, or by its modifications.

0038The DNA encoding the mutant AR of the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above and synthetic DNA. The vector to be used for the library may be any of bacteriophage, plasmid, cosmid and phagemid. The DNA may also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR) using the total RNA or mRNA fraction prepared from the cells and tissues described above.

0039Specifically, the DNA encoding the mutant AR of the present invention may be any DNA, as long as it is a DNA having a base sequence in which the base at base number 2237 is substituted by thymine in the base sequence represented by SEQ ID NO: 1 (G2237T) or a base sequence in which the base at base number 2237 is substituted by thymine and the base at base number 2644 is substituted by guanine in the base sequence represented by SEQ ID NO: 1 (G2237 T+A2644G).

0040For cloning of the DNA that completely encodes the mutant AR of the present invention, the DNA may be either amplified by PCR method using synthetic DNA primers containing a part of the base sequence encoding the mutant AR of the present invention, or selected by hybridizing the DNA inserted into an appropriate vector with a labeled DNA fragment or synthetic DNA that encodes a part or entire region of the mutant AR of the present invention. The hybridization can be carried out, for example, according to the method described in <nplcit id="ncit0011" npl-type="b"><text>Molecular Cloning, 2nd, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989</text></nplcit>. The hybridization may also be performed using commercially available library in accordance with the protocol described in the attached instructions.

0041Conversion of the base sequence of the DNA can be effected by publicly known methods such as the Gapped duplex method or the Kunkel method or its modification by using a publicly known kit available as Mutan^™-G or Mutan^™-K (both manufactured by Takara Shuzo Co., Ltd.).

0042The cloned DNA encoding the mutant AR can be used as it is, depending upon purpose or, if desired, after digestion with a restriction enzyme or after addition of a linker thereto. The DNA may contain ATG as a translation initiation codon at the 5' end thereof and may further contain TAA, TGA or TAG as a translation termination codon at the 3' end thereof. These translation initiation and termination codons may also be added by using an appropriate synthetic DNA adapter.

0043The expression vector for the mutant AR of the present invention can be manufactured, for example, by (a) excising the desired DNA fragment from the DNA encoding the mutant AR of the present invention, and then (b) ligating the DNA fragment with an appropriate expression vector downstream a promoter in the vector.

0044Examples of the vector include plasmids derived form E. coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19, pSH15), bacteriophages such as λ phage, etc., animal viruses such as retrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

0045The promoter used in the present invention may be any promoter if it matches well with a host to be used for gene expression. In the case of using animal cells as the host, examples of the promoter include SR α promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc.

0046Among them, CMV promoter or SRα promoter is preferably used. Where the host is bacteria of the genus Escherichia, preferred examples of the promoter include trp promoter, lac promoter, recA promoter, λP_L promoter, lpp promoter, etc. In the case of using bacteria of the genus Bacillus as the host, preferred example of the promoter are SPO1 promoter, SPO2 promoter and penP promoter. When yeast is used as the host, preferred examples of the promoter are PHOS promoter, PGK promoter, GAP promoter and ADH promoter. When insect cells are used as the host, preferred examples of the promoter include polyhedrin prompter and P10 promoter.

0047In addition to the foregoing examples, the expression vector may further optionally contain an enhancer, a splicing signal, a polyA addition signal, a selection marker, SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) etc. Examples of the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp^r), neomycin resistant gene (hereinafter sometimes abbreviated as Neo^r, G418 resistance), etc. In particular, when dhfr gene is used as the selection marker in CHO (dhfr^-) cells, selection can also be made on thymidine free media.

0048If necessary, a signal sequence that matches with a host is added to the N-terminus of the AR of the present invention. Examples of the signal sequence that can be used are PhoA signal sequence, OmpA signal sequence, etc. in the case of using bacteria of the genus Escherichia as the host; α-amylase signal sequence, subtilisin signal sequence, etc. in the case of using bacteria of the genus Bacillus as the host; MFα signal sequence, SUC2 signal sequence, etc. in the case of using yeast as the host; and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. in the case of using animal cells as the host, respectively.

0049Using the vector containing the DNA encoding the mutant AR of the present invention thus constructed, transformants can be manufactured.

0050Examples of the host, which may be employed, are bacteria belonging to the genus Escherichia, bacteria belonging to the genus Bacillus, yeast, insect cells, insects and animal cells, etc.

0051Specific examples of the bacteria belonging to the genus Escherichia include Escherichia coli K12 DH1 (<nplcit id="ncit0012" npl-type="s"><text>Proc. Natl. Acad. Sci. U.S.A., 60, 160 (1968</text></nplcit>)), JM103 (<nplcit id="ncit0013" npl-type="s"><text>Nucleic Acids Research, 9, 309 (1981</text></nplcit>)), JA221 (<nplcit id="ncit0014" npl-type="s"><text>Journal of Molecular Biology, 120, 517 (1978</text></nplcit>)), HB101 (<nplcit id="ncit0015" npl-type="s"><text>Journal of Molecular Biology, 41, 459 (1969</text></nplcit>)), C600 (<nplcit id="ncit0016" npl-type="s"><text>Genetics, 39, 440 (1954</text></nplcit>)), etc.

0052Examples of the bacteria belonging to the genus Bacillus include Bacillus subtilis MI114 (<nplcit id="ncit0017" npl-type="s"><text>Gene, 24, 255 (1983</text></nplcit>)), 207-21 (<nplcit id="ncit0018" npl-type="s"><text>Journal of Biochemistry, 95, 87 (1984</text></nplcit>)), etc.

0053Examples of yeast include Saccharomyces cereviseae AH22, AH22R^-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris, etc.

0054Examples of insect cells include, for the virus AcNPV, Spodoptera frugiperda cells (Sf cells), MG1 cells derived from mid-intestine of Trichoplusia ni, High Five^™ cells derived from egg of Trichoplusia ni, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, etc.; and for the virus BmNPV, Bombyx mori N cells (BmN cells), etc. are used. Examples of the Sf cell which can be used are Sf9 cells (ATCC CRL1711) and Sf21 cells (both cells are described in <nplcit id="ncit0019" npl-type="s"><text>Vaughn, J. L. et al., In Vivo, 13, 213-217 (1977</text></nplcit>).

0055As the insect, for example, a larva of silkworm can be used (<nplcit id="ncit0020" npl-type="s"><text>Maeda, et al., Nature, 315, 592 (1985</text></nplcit>)).

0056Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter referred to as CHO cells), dhfr gene deficient Chinese hamster cells CHO (hereinafter simply referred to as CHO(dhfr^-) cell), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc.

0057Bacteria belonging to the genus Escherichia can be transformed, for example, by the method described in <nplcit id="ncit0021" npl-type="s"><text>Proc. Natl. Acad. Sci. U.S.A., 69, 2110 (1972</text></nplcit>) or <nplcit id="ncit0022" npl-type="s"><text>Gene, 17, 107 (1982</text></nplcit>).

0058Bacteria belonging to the genus Bacillus can be transformed, for example, by the method described in <nplcit id="ncit0023" npl-type="s"><text>Molecular &amp; General Genetics, 168, 111 (1979</text></nplcit>).

0059Yeast can be transformed, for example, by the method described in <nplcit id="ncit0024" npl-type="s"><text>Methods in Enzymology, 194, 182-187 (1991</text></nplcit>), <nplcit id="ncit0025" npl-type="s"><text>Proc. Natl. Acad. Sci. U.S.A., 75, 1929 (1978</text></nplcit>), etc.

0060Insect cells or insects can be transformed, for example, according to the method described in <nplcit id="ncit0026" npl-type="s"><text>Bio/Technology, 6, 47-55(1988</text></nplcit>), etc.

0061Animal cells can be transformed, for example, according to the method described in <nplcit id="ncit0027" npl-type="b"><text>Saibo Kogaku (Cell Engineering), extra issue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering Experimental Protocol), 263-267 (1995), published by Shujunsha</text></nplcit>, or <nplcit id="ncit0028" npl-type="s"><text>Virology, 52, 456 (1973</text></nplcit>).

0062Thus, the transformant transformed with the expression vector containing the DNA encoding the AR can be obtained.

0063Where the host is bacteria belonging to the genus Escherichia or the genus Bacillus, the transformant can be appropriately cultured in a liquid medium which contains materials required for growth of the transformant such as carbon sources, nitrogen sources, inorganic materials, and so on. Examples of the carbon sources include glucose, dextrin, soluble starch, sucrose, etc. Examples of the nitrogen sources include inorganic or organic materials such as ammonium salts, nitrate salts, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract, etc. Examples of the inorganic materials are calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc. In addition, yeast extract, vitamins, growth promoting factors etc. may also be added to the medium. Preferably, pH of the medium is adjusted to about 5 to about 8.

0064A preferred example of the medium for cultivation of the bacteria belonging to the genus Escherichia is M9 medium supplemented with glucose and Casamino acids (<nplcit id="ncit0029" npl-type="b"><text>Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972</text></nplcit>). If necessary, a chemical such as 3β-indolylacrylic acid can be added to the medium thereby to activate the promoter efficiently.

0065Where the bacteria belonging to the genus Escherichia are used as the host, the transformant is usually cultivated at about 15°C to about 43°C for about 3 hours to about 24 hours. If necessary, the culture may be aerated or agitated.

0066Where the bacteria belonging to the genus Bacillus are used as the host, the transformant is cultivated generally at about 30°C to about 40°C for about 6 hours to about 24 hours. If necessary, the culture can be aerated or agitated.

0067Where yeast is used as the host, the transformant is cultivated, for example, in Burkholder's minimal medium (<nplcit id="ncit0030" npl-type="s"><text>Bostian, K. L. et al., Proc. Natl. Acad. Sci. U.S.A., 77, 4505 (1980</text></nplcit>)) or in SD medium supplemented with 0.5% Casamino acids (<nplcit id="ncit0031" npl-type="s"><text>Bitter, G. A. et al., Proc. Natl. Acad. Sci. U.S.A., 81, 5330 (1984</text></nplcit>)). Preferably, pH of the medium is adjusted to about 5 to about 8. In general, the transformant is cultivated at about 20°C to about 35°C for about 24 hours to about 72 hours. If necessary, the culture can be aerated or agitated.

0068Where insect cells or insects are used as the host, the transformant is cultivated in, for example, Grace's Insect Medium (<nplcit id="ncit0032" npl-type="s"><text>Grace, T. C. C., Nature, 195, 788 (1962</text></nplcit>)) to which an appropriate additive such as immobilized 10% bovine serum is added. Preferably, pH of the medium is adjusted to about 6.2 to about 6.4. Normally, the culture is performed at about 27°C for about 3 days to about 5 days and, if necessary, the culture can be aerated or agitated.

0069Where animal cells are employed as the host, the transformant is cultivated in, for example, MEM medium containing about 5% to about 20% fetal bovine serum (<nplcit id="ncit0033" npl-type="s"><text>Science, 122, 501 (1952</text></nplcit>)), DMEM medium (<nplcit id="ncit0034" npl-type="s"><text>Virology, 8, 396 (1959</text></nplcit>)), RPMI 1640 medium (<nplcit id="ncit0035" npl-type="s"><text>The Journal of the American Medical Association, 199, 519 (1967</text></nplcit>)), 199 medium <i>(</i><nplcit id="ncit0036" npl-type="s"><text>Proceeding of the Society for the Biological Medicine, 73, 1 (1950</text></nplcit>)), etc. Preferably, pH of the medium is adjusted to about 6 to about 8. The culture is usually done at about 30°C to about 40°C for about 15 hours to about 60 hours and, if necessary, the culture can be aerated or agitated.

0070As described above, the mutant AR of the present invention can be produced within the cell or out of the cell of the transformant.

0071The mutant AR of the present invention can be separated and purified from the culture described above by the following procedures.

0072When the mutant AR of the present invention is extracted from the cultured bacteria or cells, after cultivation these cells are collected by a publicly known method and suspended in a appropriate buffer. The bacteria or cells are then disrupted by publicly known methods such as ultrasonication, a treatment with lysozyme and/or freeze-thaw cycling, followed by centrifugation, filtration, etc. Thus, the crude extract of the mutant AR of the present invention can be obtained. The buffer used for the procedures may contain a protein modifier such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100^™, etc. When the mutant AR of the present invention is secreted in the culture, after completion of the cultivation the supernatant can be separated from the bacteria or cells to collect the supernatant by a publicly known method.

0073The AR of the present invention contained in the supernatant or the extract thus obtained can be purified by appropriately combining the publicly known methods for separation and purification. Such publicly known methods for separation and purification include a method utilizing difference in solubility such as salting out, solvent precipitation, etc.; a method utilizing mainly difference in molecular weight such as dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, etc.; a method utilizing difference in electric charge such as ion exchange chromatography, etc.; a method utilizing difference in specific affinity such as affinity chromatography, etc.; a method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography, etc.; a method utilizing difference in isoelectric point such as isoelectrofocusing electrophoresis; and the like.

0074When the mutant AR of the present invention thus obtained is in a free form, it can be converted into the salt form by publicly known methods or modifications thereof. On the other hand, when the mutant AR is obtained in the salt form, it can be converted into the free form or in the form of a different salt by publicly known methods or modifications thereof.

0075The mutant AR of the present invention produced by the recombinant can be treated, prior to or after the purification, with an appropriate protein modifying enzyme so that the mutant AR can be appropriately modified and partially removed a polypeptide. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase or the like.

0076The activity of the thus produced mutant AR of the present invention or salts thereof can be determined by a binding experiment to a labeled ligand, by an enzyme immunoassay using the antibody of the present invention (described in detail below) and the like.

0077A DNA that encodes the mutant AR of the present invention or a salt thereof, or an amide thereof or an ester thereof or a salt thereof can be used for (1) an agent for the prophylaxis and/or treatment of diseases that can be ameliorated by overexpression of the mutant AR of the present invention, (2) a genetic diagnostic agent, (3) a method of screening for a compound that alters the expression level of the mutant AR of the present invention or a salt thereof, (4) an agent for the prophylaxis and/or treatment of various diseases, which comprises a compound that alters the expression level of the mutant AR of the present invention, (5) a method of screening for a compound that alters the bindability of the mutant AR of the present invention and ligand (agonist, antagonist and the like), (6) an agent for the prophylaxis and/or treatment of various diseases, which comprises a compound that alters the bindability of the mutant AR of the present invention and ligand (agonist, antagonist), (7) quantitation of the mutant AR of the present invention, (8) a pharmaceutical agent comprising the antibody of the present invention, (9) a pharmaceutical agent comprising an antisense DNA, (10) production of a DNA-introduced animal, and the like.

0078Particularly, by a screening with a receptor binding assay system using an animal cell capable of expressing the mutant AR of the present invention or an expression system of a recombinant mutant AR, a compound that alters the bindability of a ligand for a human- or mammal-specific receptor (e.g., agonist, antagonist and the like) can be selected, and said agonist or antagonist can be used as an agent for the prophylaxis/treatment of various diseases, and the like.

0079The intended uses of the mutant AR of the present invention, the DNA that encodes the mutant AR of the present invention (hereinafter also abbreviated as the DNA of the present invention), are specifically described below.

(1) Prophylactic and/or therapeutic agent for diseases that can be ameliorated by overexpression of the mutant AR of the present invention

0080The mutant AR of the present invention is <b>characterized in that</b> bicalutamide or an analogue thereof, or further flutamide or an analogue thereof, acts thereon as an agonist. Because it is known that in prostate cancer, a stimulation of AR in excess conversely suppresses proliferation, by locally delivering (1) the mutant AR of the present invention or (2) the DNA of the present invention to cancer cells and allowing it in excess therein, it is possible to suppress the proliferation of antiandrogen drug-resistant cancer cells.

0081When the mutant AR of the present invention is used as the above-described prophylactic/therapeutic agent, it can be made into a preparation in a conventional manner.

0082On the other hand, where the DNA of the present invention is used as the above-described prophylactic/therapeutic agent, the DNA of the present invention itself is administered; alternatively, the DNA is inserted into an appropriate vector such as retrovirus vector, adenovirus vector, adenovirus-associated virus vector, etc. and then administered in a conventional manner. The DNA of the present invention may also be administered as naked DNA, or with adjuvants to assist its uptake by gene gun or through a catheter such as a catheter with a hydrogel.

0083For example, (1) the mutant AR of the present invention or b) the DNA of the present invention can be used orally, for example, in the form of tablets which may be sugar coated if necessary, capsules, elixirs, microcapsules etc., or parenterally in the form of injectable preparations such as a sterile solution and a suspension in water or with other pharmaceutically acceptable liquid. For example, these preparations can be manufactured by mixing (1) the mutant AR of the present invention or (2) the DNA of the present invention with a physiologically acceptable known carrier, a flavoring agent, an excipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc. in a unit dosage form required in a generally accepted manner that is applied to making pharmaceutical preparations. The effective component in the preparation is controlled in such a dose that an appropriate dose is obtained within the specified range given.

0084Additives miscible with tablets, capsules, etc. include a binder such as gelatin, corn starch, tragacanth and gum arabic, an excipient such as crystalline cellulose, a swelling agent such as corn starch, gelatin and alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose and saccharin, and a flavoring agent such as peppermint, akamono oil and cherry. When the unit dosage is in the form of capsules, liquid carriers such as oils and fats may further be used together with the additives described above. A sterile composition for injection may be formulated by conventional procedures used to make pharmaceutical compositions, e.g., by dissolving or suspending the active ingredients in a vehicle such as water for injection with a naturally occurring vegetable oil such as sesame oil and coconut oil, etc. to prepare the pharmaceutical composition. Examples of an aqueous medium for injection include physiological saline and an isotonic solution containing glucose and other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used in combination with an appropriate dissolution aid such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol and polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80^™ and HCO-50), etc. Examples of the oily medium include sesame oil and soybean oil, which may also be used in combination with a dissolution aid such as benzyl benzoate and benzyl alcohol.

0085The prophylactic/therapeutic agent described above may further be formulated with a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus-prepared liquid for injection is normally filled in an appropriate ampoule.

0086Since the thus obtained preparation is safe and low toxic, it can be administered to mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like).

0087The dose of the mutant AR of the present invention varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient, with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

0088The dose of the DNA of the present invention varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

(2) Genetic diagnostic reagent

0089By using the DNA of the present invention as a probe, the DNA or mRNA encoding the mutant AR of the present invention (i.e., an abnormality in a normal AR gene or mRNA) in mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like) can be detected. Therefore, it is useful as a genetic diagnostic agent for a cancer, particularly as a genetic diagnostic agent for the development, increase and the like of a cancer cell that has acquired resistance to an antiandrogen drug (e.g., bicalutamide, flutamide and the like).

0090The genetic diagnosis described above using the DNA of the present invention can be performed by, for example, the publicly known Northern hybridization assay or the PCR-SSCP assay (<nplcit id="ncit0037" npl-type="s"><text>Genomics, 5, 874-879 (1989</text></nplcit>); <nplcit id="ncit0038" npl-type="s"><text>Proceedings of the National Academy of Sciences of the United States of America, 86, 2766-2770 (1989</text></nplcit>)).

(3) Method of screening for a compound that alters the expression level of the mutant AR of the present invention

0091By using as a probe, the DNA of the present invention can be used for screening for a compound that alters the expression level of the mutant AR of the present invention.

0092That is, the present invention provides a method of screening for a compound that alters the expression level of the mutant AR of the present invention, which comprises measuring the amount of mRNA encoding the mutant AR of the present invention contained in, for example, (i) (1) blood, (2) specific organs, (3) tissues or cells isolated from the organs of non-human mammals (e.g., rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like), or in (ii) transformants, etc.

0093The amount of mRNA encoding mutant AR of the present invention can be specifically measured as follows. <ol id="ol0002"><li>(i) A non-human mammal (e.g., mouse, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like) having cells that express a normal AR or the mutant AR of the present invention receive administration of a drug or physical stress, and blood, specific organs, or tissues or cells isolated from the organs are obtained after a specified period of time. The mRNA encoding the mutant AR of the present invention contained in the thus obtained cells is extracted from the cells, for example, in a conventional manner and quantified using, e.g., TaqManPCR, or may also be analyzed by Northern blot technique by publicly known methods.</li><li>(ii) A Transformant that expresses the mutant AR of the present invention or an animal cell capable of expressing the mutant AR of the present invention is prepared according to the methods described above, and the mRNA encoding the AR of the present invention can be quantified and analyzed in the same manner.</li></ol>

0094Screening for a compound that alters the expression level of the mutant AR of the present invention can be coducted by the following procedures: <ol id="ol0003"><li>(i) To a non-human mammal having cells that express a normal AR or the mutant AR of the present invention, a test compound is administered at a specified period of time before (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, more preferably 1 hour to 6 hours before), at a specified time after (30 minutes to 3 days after, preferably 1 hour to 2 days after, more preferably 1 hour to 24 hours after), or simultaneously with a drug or physical stress. At a specified time (30 minute to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) after administration of the test compound, the amount of mRNA encoding the mutant AR of the present invention contained in the cells is quantified and analyzed; or</li><li>(ii) A test compound is mixed in a culture medium and a transformant or an animal cell capable of expressing the mutant AR of the present invention is cultured therein in a conventional manner. After a specified time (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 to 3 days), the amount of mRNA encoding the mutant AR of the present invention contained in the transformant or animal cell capable of expressing the mutant AR of the present invention can be quantified and analyzed.</li></ol>

0095The compound or its salt, which is obtainable by the screening method of the present invention, is a compound that alters the expression level of the mutant AR of the present invention, specifically, (a) a compound that excessively enhances a cell-stimulating activity (e.g., cell proliferation stimulation, PSA expression induction activity and the like) mediated by the mutant AR of the present invention by increasing the expression level of the mutant AR of the present invention, or (b) a compound that attenuates said cell-stimulating activity by decreasing the expression level of the mutant AR of the present invention.

0096The compound includes a peptide, protein, non-peptide compound, synthetic compound, and fermentation product. It may be a novel or known compound.

0097The compound that excessively enhances the cell-stimulating activity is capable of suppressing the proliferation of antiandrogen drug-resistant cancers by, for example, using it in combination with an antiandrogen drug, and is useful as a prophylactic/therapeutic agent for hormone-sensitive cancers (e.g., prostate cancer and the like) in the androgen-independent stage.

0098Since the compound that attenuates the cell-stimulating activity decreases the physiological activity of the mutant AR of the present invention, it is useful as a prophylactic/therapeutic agent for hormone-sensitive cancers (e.g., prostate cancer and the like) in the androgen-independent stage.

0099When the compound or its salt obtained by the screening method of the present invention is used as a pharmaceutical composition, the compound can be prepared into a pharmaceutical composition in a conventional manner. For example, in the same manner as the above-described pharmaceutical agent containing the mutant AR of the present invention, the compound can be made into tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like.

0100Since the thus obtained preparation is safe and low toxic, it can be administered to mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey, etc.).

0101The dose of the compound or its salt varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

(4) Prophylactic and/or therapeutic agent for various diseases, which comprises a compound that alters the expression level of the mutant AR of the present invention

0102A compound that alters the expression level of the mutant AR of the present invention can be used as a prophylactic/therapeutic agent for hormone-dependent and hormone-independent cancer (e.g., prostate cancer and the like). Said compound can be made into a preparation in a conventional manner.

0103For example, said compound can be used orally in the form of tablets which may be sugar coated if necessary, capsules, elixirs, microcapsules etc., or parenterally in the form of injectable preparations such as a sterile solution and a suspension in water or with other pharmaceutically acceptable liquid. For example, these preparations can be manufactured by mixing said compound with a physiologically acceptable known carrier, a flavoring agent, an excipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc. in a unit dosage form required in a generally accepted manner that is applied to making pharmaceutical preparations. The effective component in the preparation is controlled in such a dose that an appropriate dose is obtained within the specified range given.

0104Additives miscible with tablets, capsules, etc. include a binder such as gelatin, corn starch, tragacanth and gum arabic, an excipient such as crystalline cellulose, a swelling agent such as corn starch, gelatin and alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose and saccharin, and a flavoring agent such as peppermint, akamono oil and cherry. When the unit dosage is in the form of capsules, liquid carriers such as oils and fats may further be used together with the additives described above. A sterile composition for injection may be formulated by conventional procedures used to make pharmaceutical compositions, e.g., by dissolving or suspending the active ingredients in a vehicle such as water for injection with a naturally occurring vegetable oil such as sesame oil and coconut oil, etc. to prepare the pharmaceutical composition. Examples of an aqueous medium for injection include physiological saline and an isotonic solution containing glucose and other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used in combination with an appropriate dissolution aid such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol and polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80^™ and HCO-50), etc. Examples of the oily medium include sesame oil and soybean oil, which may also be used in combination with a dissolution aid such as benzyl benzoate and benzyl alcohol.

0105The prophylactic/therapeutic agent described above may further be formulated with a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus-prepared liquid for injection is normally filled in an appropriate ampoule.

0106Since the thus obtained preparation is safe and low toxic, it can be administered to mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like).

0107The dose of the compound or salt thereof varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

(5) Method of screening for a compound (agonist, antagonist and the like) that alters the bindability of the mutant AR of the present invention and a ligand

0108By using the mutant AR of the present invention, or by constructing an expression system for the recombinant mutant AR and using a receptor binding assay system with the above expression system, a compound that alters the bindability of a ligand and the mutant AR of the present invention (e.g., peptide, protein, non-peptide compound, synthetic compound, fermentation product, etc.), or a salt thereof, can be screened efficiently.

0109Such compounds include (a) a compound that has an activity to promote a cell-stimulating activity (e.g., cell proliferation activity, PSA expression induction activity and the like) via the mutant AR of the present invention (so-called agonist for the mutant AR of the present invention), (b) a compound that has no cell-stimulating activity (so-called antagonist for the mutant AR of the present invention), (c) a compound that enhances the binding force of a ligand and the mutant AR of the present invention, or (d) a compound that decreases the binding force of a ligand and the mutant AR of the present invention and the like.

0110That is, the present invention provides a method of screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention or a salt thereof, which comprises comparing (i) the case wherein the mutant AR of the present invention is brought into contact with a ligand, with (ii) the case wherein the mutant AR of the present invention is brought into contact with a ligand and a test compound.

0111The screening method of the present invention is characterized by comprising determining, for example, the amount bound of ligand (androgen) for the AR of the present invention, a cell-stimulating activity (e.g., cell proliferation activity, PSA expression induction activity) and the like, in cases (i) and (ii), and comparing both cases.

0112More specifically, the present invention provides: <ol id="ol0004" compact="compact"><li>(1) a method of screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention, or a salt thereof, which comprises measuring the amounts of a labeled ligand bound to the mutant AR of the present invention, when the labeled ligand is brought into contact with said mutant AR and when the labeled ligand and a test compound are brought into contact with said mutant AR, and comparing the both amounts,</li><li>(2) a method of screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention, or a salt thereof, which comprises measuring the amounts of labeled ligand bound to cells containing the mutant AR of the present invention (animal cells capable of expressing the mutant AR, and the like), when the labeled ligand is brought into contact with said cells and when the labeled ligand and a test compound are brought into contact with said cells, and comparing the both amounts,</li><li>(3) a method of screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention, or a salt thereof, which comprises measuring the amounts of labeled ligand bound to the mutant AR, when the labeled ligand is brought into contact with the mutant AR expressed by culturing a transformant containing the DNA of the present invention and when the labeled ligand and a test compound are brought into contact with the mutant AR of the present invention expressed by culturing a transformant containing the DNA of the present invention, and comparing the both amounts, and</li><li>(4) a method of screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention, or a salt thereof, which comprises measuring receptor-mediated cell-stimulating activities (e.g., cell proliferation activity, PSA expression induction activity and the like), when a compound that activates the mutant AR of the present invention (e.g., an androgen or an antiandrogen drug such as bicalutamide or flutamide, and the like) is brought into contact with cells containing the mutant AR of the present invention (animal cells capable of expressing the mutant AR) and when the compound that activates the mutant AR of the present invention and a test compound are brought into contact with said cells, and comparing the both activities.</li></ol>

0113Hereinafter, the screening method of the present invention is described specifically.

0114First, for the mutant AR of the present invention used for the screening method of the present invention, any one may be used so long as it contains the mutant AR of the present invention described above. The aforementioned animal cells capable of expressing the mutant AR of the present invention, and the like are preferred.

0115To manufacture the mutant AR of the present invention, the methods described above are used, and it is preferred to express the DNA of the present invention in mammalian and insect cells. For the DNA fragment encoding the objective protein region, the complementary DNA, but not necessarily limited thereto, is employed. For example, the gene fragments and synthetic DNA may also be used. To introduce a DNA fragment encoding the mutant AR of the present invention into host animal cells and efficiently express the DNA there, it is preferred to insert the DNA fragment downstream of a polyhedorin promoter of nuclear polyhedrosis virus (NPV) belonging to baculovirus hosted by insects, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, or SRa promoter. The amount and quality of the expressed receptor are examined by publicly known methods, for example, the method described in the literature [<nplcit id="ncit0039" npl-type="s"><text>Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992</text></nplcit>].

0116Therefore, in the screening method of the present invention, as a material that contains the mutant AR of the present invention, the mutant AR purified by publicly known methods or cells containing the mutant AR may be used.

0117In the screening method of the present invention, when cells containing the mutant AR of the present invention are used, the cells may be fixed with glutaraldehyde, formalin, etc. The cells can be fixed by publicly known methods.

0118The cells containing the mutant AR of the present invention are host cells that express said mutant AR. For the host cells, <i>Escherichia coli, Bacillus subtilis,</i> yeast, insect cells, animal cells and the like are preferred.

0119The amount of the mutant AR in the cells containing the mutant AR is preferably 10³ to 10⁸ molecules per cell, more preferably 10⁵ to 10⁷ molecules per cell. As the amount of expression increases, the ligand binding activity (specific activity) increases so that not only the highly sensitive screening system can be constructed but also large quantities of samples can be assayed with the same lot.

0120To perform the above-mentioned (1) to (3) to screen for a compound that alters the bindability of a ligand and the mutant AR of the present invention, for example, an appropriate mutant AR fraction and a labeled ligand are necessary.

0121The mutant AR fraction is preferably an AR fraction derived from cells expressing the mutant AR, which is obtained by a spontaneous or artificial mutation, or a recombinant mutant AR fraction having an activity equivalent thereto. Herein, equivalent activity is intended to mean an equivalent ligand binding activity, cell-stimulating activity or the like.

0122For the labeled ligand, a labeled ligand (androgen) and a labeled ligand analogue are used. For example, testosterone, DHT and the like labeled with [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. are used.

0123Specifically, to screen a compound that alters the bindabiliy of a ligand and the mutant AR of the present invention, first, the mutant AR standard is prepared by suspending cells containing the mutant AR of the present invention in a buffer appropriate for the screening. For the buffer, any buffer that does not interfere with the binding of the ligand to the mutant AR is usable and examples of such a buffer are phosphate buffer, Tris-hydrochloride buffer, etc., having pH of 4 to 10 (preferably pH of 6 to 8). To minimize a non-specific binding, a surfactant such as CHAPS, Tween-80^™ (Kao-Atlas Co.), digitonin, deoxycholate, etc. may be added to the buffer. To inhibit degradation of the receptor and ligands by proteases, protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratory, Co.), and pepstatin may be added. To 0.01 to 10 ml of the receptor solution, a given amount (5,000 to 500,000 cpm) of labeled ligand is added, and 10^-4 M - 10^-10 M of a test compound is simultaneously added to be co-present. To examine non-specific binding (NSB), a reaction tube containing an unlabeled test compound in large excess is also prepared. The reaction is carried out at approximately 0 to 50°C, preferably about 4 to 37°C for about 20 minutes to about 24 hours, preferably about 30 minutes to about 3 hours. After completion of the reaction, the reaction mixture is filtrated through glass fiber filter paper, etc. and washed with an appropriate volume of the same buffer. The residual radioactivity on the glass fiber filter paper is then measured by means of a liquid scintillation counter or γ-counter. Regarding the count obtained by subtracting the amount of non-specific binding (NSB) from the count obtained in the absence of any competitive substance (B₀) as 100%, when the amount of specific binding (B-NSB) is, for example, 50% or less, the test compound can be selected as a candidate substance having a potential of competitive inhibition.

0124Of the method of (4) above to screen for a compound that alters the bindability of a ligand and the mutant AR of the present invention, the most preferable mode is described in detail below.

0125Generally, the cells containing the mutant AR of the present invention are first cultured on a multi-well plate, etc. Prior to screening, the medium is replaced with fresh medium or with an appropriate non-cytotoxic buffer, followed by incubation for a given period of time in the presence of a test compound, etc. Subsequently, the extent of proliferation of the cells is evaluated, or the cells are extracted or the supernatant is recovered and the resulting product is quantified by appropriate procedures. Where it is difficult to detect the production of the index substance (e.g., PSA and the like) for the cell-stimulating activity due to a degrading enzyme contained in the cells, an inhibitor against such a degrading enzyme may be added prior to the assay.

0126Screening by assaying the cell-stimulating activity requires cells that have expressed an appropriate mutant AR. For the cells that have expressed the mutant AR of the present invention, cells expressing the mutant AR, which is obtained by a spontaneous or artificial mutation, the cell line expressing the recombinant mutant AR described above and the like are desirable.

0127The test compound includes peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract and the like. These compounds may be new compounds or known compounds.

0128The kit for screening for a compound that alters the bindability of a ligand and the mutant AR of the present invention is a kit, which comprises the mutant AR of the present invention or cells containing the mutant AR of the present invention (animal cells capable of expressing the mutant AR and the like), and the like.

0129Examples of the screening kit of the present invention are as follow.

1. Reagents for screening

(1) Buffer for measurement and washing

0130Hanks' balanced salt solution (Gibco) supplemented with 0.05% bovine serum albumin (Sigma).

0131The solution is sterilized by filtration through a 0.45 µm filter, and stored at 4°C or may be prepared at use.

(2) Standard mutant AR

0132CHO cells expressing the mutant AR of the present invention are passaged in a 12-well plate at a density of 5 x 10⁵ cells/well followed by culturing at 37°C under 5% CO₂ and 95% air for 2 days.

(3) Labeled ligands

0133A solution of DHT labeled with commercially available [³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. is stored at 4°C or -20°C, and diluted to 1 µM with the measurement buffer.

(4) Standard ligand solution

0134DHT is dissolved in and adjusted to 1 mM with PBS containing 0.1% bovine serum albumin (Sigma) and stored at -20°C.

2. Measurement method

0135<ol id="ol0005" compact="compact"><li>(1) CHO cells expressing the mutant AR of the present invention are cultured in a 12-well culture plate and washed twice with 1 ml of the measurement buffer, and 490 µl of the measurement buffer is added to each well.</li><li>(2) After adding 5 µl of 10^-3 - 10^-10 M test compound solution, 5 µl of a labeled ligand is added to the mixture, and the cells are incubated at room temperature for an hour. To determine the amount of the non-specific binding, 5 µl of the non-labeled ligand is added in place of the test compound.</li><li>(3) The reaction solution is removed, and the wells are washed 3 times with the washing buffer. The labeled ligand bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries, Ltd.)</li><li>(4) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman Co.), and the percent maximum binding (PMB) is calculated by the equation below [Equation 1]. <maths id="math0001" num="[Equation 1]"><math display="block"><mi>PMB</mi><mo mathvariant="normal">=</mo><mfenced open="[" close="]"><mfenced><mi mathvariant="normal">B</mi><mo mathvariant="normal">-</mo><mi>NSB</mi></mfenced><mo mathvariant="normal">/</mo><mfenced><msub><mi mathvariant="normal">B</mi><mn mathvariant="normal">0</mn></msub><mo mathvariant="normal">-</mo><mi>NSB</mi></mfenced></mfenced><mo mathvariant="normal">×</mo><mn mathvariant="normal">100</mn></math><img file="EP1550720B1_D0001.tif" /></maths> PMB: Percent maximum binding B: Value obtained in the presence of a test compound NSB: Non-specific binding Bo: Maximum binding</li></ol>

0136The compound or its salt, which is obtained using the screening method or the screening kit of the present invention, is a compound that alters the bindability of a ligand and the mutant AR of the present invention. Specifically, it is: (a) a compound that has the mutant AR-mediated cell-stimulating activity (e.g., cell proliferation activity, PSA production, etc.) (so-called agonist for the mutant AR of the present invention); (b) a compound having no cell stimulating-activity (so-called antagonist for the mutant AR of the present invention); (c) a compound that enhances the binding force between the ligand and the mutant AR of the present invention; or (d) a compound that attenuates the binding force between the ligand and the mutant AR of the present invention.

0137The compound includes a peptide, protein, non-peptide compound, synthetic compound, and fermentation product. It may be a novel or known compound.

0138Since antagonists to the mutant AR of the present invention can suppress the physiological activities of ligands (androgens) and agonists (some antiandrogen drugs such as bicalutamide and flutamide, and the like) for the mutant AR of the present invention, they are useful as a prophylactic/therapeutic agent for androgen-independent (antiandrogen drug-resistant) cancers, particularly prostate cancer.

0139A compound that attenuates the binding force between the ligand and the mutant AR of the present invention is useful as a safe and low toxic pharmaceutical agent (e.g., a prophylactic/therapeutic agent for androgen-independent (antiandrogen drug-resistant) cancers, particularly prostate cancer) for decreasing the physiological activities of a ligand (agonist) for the mutant AR of the present invention.

0140When the compound or salt thereof obtained by using the screening method or the screening kit of the present invention, is used as the aforementioned pharmaceutical composition, the pharmaceutical preparation can be obtained in a conventional manner. For example, in the same manner as the above-described pharmaceutical agent comprising the mutant AR of the present invention, the compound can be made into tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like.

0141Since the thus obtained preparation is safe and low toxic, it can be administered to mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like).

0142The dose of the compound or salt thereof varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

(6) Prophylactic and/or therapeutic agent for various diseases, which comprises a compound that alters the bindability of the mutant AR of the present invention and a ligand (agonist, antagonist)

0143Since antagonists for the mutant AR of the present invention can suppress the physiological activities for ligands (androgens) and agonists (some antiandrogen drugs such as bicalutamide and flutamide, and the like) for the mutant AR of the present invention, they can be used as a prophylactic/therapeutic agent for androgen-independent (antiandrogen drug-resistant) cancers, particularly prostate cancer. When said compound is used for the above-described diseases, it can be made into a preparation according to a conventional manner.

0144For example, said compound can be used orally in the form of tablets which may be sugar coated if necessary, capsules, elixirs, microcapsules etc., or parenterally in the form of injectable preparations such as a sterile solution and a suspension in water or with other pharmaceutically acceptable liquids. For example, these preparations can be manufactured by mixing said compound with a physiologically acceptable known carrier, a flavoring agent, an excipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc. in a unit dosage form required in a generally accepted manner that is applied to making pharmaceutical preparations. The effective component in the preparation is controlled in such a dose that an appropriate dose is obtained within the specified range given.

0145Additives miscible with tablets, capsules, etc. include a binder such as gelatin, corn starch, tragacanth and gum arabic, an excipient such as crystalline cellulose, a swelling agent such as corn starch, gelatin and alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose and saccharin, and a flavoring agent such as peppermint, akamono oil and cherry. When the unit dosage is in the form of capsules, liquid carriers such as, oils and fats may further be used together with the additives described above. A sterile composition for injection may be formulated by conventional procedures used to make pharmaceutical compositions, e.g., by dissolving or suspending the active ingredients in a vehicle such as water for injection with a naturally occurring vegetable oil such as sesame oil and coconut oil, etc. to prepare the pharmaceutical composition. Examples of an aqueous medium for injection include physiological saline and an isotonic solution containing glucose and other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used in combination with an appropriate dissolution aid such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol and polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80^™ and HCO-50), etc. Examples of the oily medium include sesame oil and soybean oil, which may also be used in combination with a dissolution aid such as benzyl benzoate and benzyl alcohol.

0146The prophylactic/therapeutic agent described above may further be formulated with a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus-prepared liquid for injection is normally filled in an appropriate ampoule.

0147Since the thus obtained preparation is safe and low toxic, it can be administered to mammals (e.g., human, rat, rabbit, sheep, swine, bovine, cat, dog, monkey and the like).

0148The dose of the compound or salt thereof varies depending on subject to be administered, organs to be administered, conditions, routes for administration, etc.; in oral administration, e.g., for the patient with cancer, the dose is normally about 0.1 mg to about 100 mg, preferably about 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mg per day (as 60 kg body weight). In parenteral administration, the single dose varies depending on subject to be administered, target organ, conditions, routes for administration, etc. but it is advantageous, e.g., for the patient with cancer, to administer the active ingredient intravenously in a daily dose of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg (as 60 kg body weight). For other animal species, the corresponding dose as converted per 60 kg body weight can be administered.

0149When bases, amino acids and the like are shown in abbreviations in the present specification and drawings, they are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or abbreviations conventionally used in the pertinent field; examples thereof are given below. When an amino acid can have an optical isomer, it is the L-configuration unless otherwise specified. <ul id="ul0002" list-style="none" compact="compact"><li>DNA: Deoxyribonucleic acid</li><li>cDNA: Complementary deoxyribonucleic acid</li><li>A: Adenine</li><li>T: Thymine</li><li>G: Guanine</li><li>C: Cytosine</li><li>RNA: Ribonucleic acid</li><li>mRNA: Messenger ribonucleic acid</li><li>dATP: Deoxyadenosine triphosphate</li><li>DTTP: Deoxythymidine triphosphate</li><li>dGTP: Deoxyguanosine triphosphate</li><li>dCTP: Deoxycytidine triphosphate</li><li>ATP: Adenosine triphosphate</li><li>EDTA: Ethylenediamine tetraacetate</li><li>SDS: Sodium dodecyl sulfate</li><li>Gly: Glycine</li><li>Ala: Alanine</li><li>Val: Valine</li><li>Leu: Leucine</li><li>Ile: Isoleucine</li><li>Ser: Serine</li><li>Thr: Threonine</li><li>Cys: Cysteine</li><li>Met: Methionine</li><li>Glu: Glutamic acid</li><li>Asp: Aspartic acid</li><li>Lys: Lysine</li><li>Arg: Arginine</li><li>His: Histidine</li><li>Phe: Phenylalanine</li><li>Tyr: Tyrosine</li><li>Trp: Tryptophan</li><li>Pro: Proline</li><li>Asn: Asparagine</li><li>GIn: Glutamine</li><li>pGlu: Pyroglutamic acid</li><li>Me: Methyl group</li><li>Et: Ethyl group</li><li>Bu: Butyl group</li><li>Ph: Phenyl group</li><li>TC: Thiazolidine-4 (R)-carboxamide group</li></ul>

0150Also, substituents, protecting groups and reagents often mentioned in the present specification are designated with the following symbols. <ul id="ul0003" list-style="none" compact="compact"><li>Tos: p-Toluenesulfonyl</li><li>CHO: Formyl</li><li>Bzl: Benzyl</li><li>Cl₂Bzl: 2,6-Dichlorobenzyl</li><li>Bom: Benzyloxymethyl</li><li>Z: Benzyloxycarbonyl</li><li>CI-Z: 2-Chlorobenzyloxycarbonyl</li><li>Br-Z: 2-Bromobenzyloxycarbonyl</li><li>Boc: t-Butoxycarbonyl</li><li>DNP: Dinitrophenol</li><li>Trt: Trityl</li><li>Bum: t-Butoxymethyl</li><li>Fmoc: N-9-fluorenylmetboxycarbonyl</li><li>HOBt: 1-Hydroxybenztriazole</li><li>HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine</li><li>HONB: 1-Hydroxy-5-norbomane-2,3-dicarboxyimide</li><li>DCC: N, N'-dicyclohexylcarbodiimide</li></ul>

0151Sequence identification numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] Shows the base sequence of cDNA of wild type human AR. [SEQ ID NO: 2] Shows the amino acid sequence of the wild type human AR. [SEQ ID NO: 3] Shows an ARE sequence in the human PSA promoter. [SEQ ID NO: 4] Shows an ARR sequence in the human PSA promoter. [SEQ ID NO: 5] Shows an oligonucleotide designed to act as a primer for amplifying the human PSA promoter. [SEQ ID NO: 6] Shows an oligonucleotide designed to act as a primer for amplifying the human PSA promoter.

Free Texts in Sequence Listing

0152SEQ ID NO: 3: ARE sequence in human PSA promoter.

0153SEQ ID NO: 4: ARR sequence in human PSA promoter.

0154SEQ ID NO: 5: Oligonucleotide designed to act as primer for amplifying human PSA promoter.

0155SEQ ID NO: 6: Oligonucleotide designed to act as primer for amplifying human PSA promoter.

Example

0156The present invention is described in more detail by means of the following Examples, which are not to be construed as limiting the scope of the present invention.

Example 1: Method of establishing the LNCaP-cxD cell line

0157When LNCaP-FGC (ATCC Number: CRL-1740) is cultured in a culture broth (RPMI1640 + 10% Dextran Charcoal (DCC)-Fetal Bovine Serum (FBS)) containing 0.1 and 1 µM bicalutamide (commercial name: Casodex), it does not proliferate initially. However, when cultivation was continued for 6 weeks to 13 weeks or more, two cell lines exhibiting proliferation were obtained. These cells were designated LNCaP-cxD11 any LNCaP-cxD2, respectively.

Example 2: Bicalutamide response of the LNCaP-cxD cell line

0158LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC were sown to 24-well plates at 40000 cells/mL/well, on the following day 0.1-30 µM bicalutamide was added, and 3 days after addition, cells were counted. Also, at this time, the concentration of androgen-dependently produced PSA (Prostatic Specific Antigen) in the culture supernatant was measured.

0159As a result, the proliferation of LNCaP-czD11 and LNCaP-cxD2 was significantly promoted by bicalutamide [<figref idref="f0001">Fig. 1</figref>]. On the other hand, in the parent line LNCaP-FGC, proliferation was significantly suppressed by bicalutamide [<figref idref="f0001">Fig. 1</figref>]. Also, PSA production was significantly promoted by bicalutamide in LNCaP-cxD11 and LNCaP-cxD2 and significantly suppressed by bicalutamide in the parent line [<figref idref="f0001">Fig. 2</figref>].

Example 3: Identification of mutant ARs

0160After total RNA was extracted from LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC, it was converted to cDNA, and the base sequences of the AR genes were analyzed by the PCR direct sequencing method. For LNCaP-cxD11, only a portion, the androgen-binding region, of the AR gene was subjected to sequence analysis.

0161As a result, in both LNCaP-cxD11 and LNCaP-cxD2 ARs, one mutation in the gene sequence accompanied by an amino acid mutation was present in the androgen-binding region. The TGG (tryptophan) of codon 746 (normally a designation system to write the total number of codons of AR as 919, in which system this codon corresponds to codon 741) was found to be mutated to TTG (leucine) and TUT (cysteine) in LNCaP-cxD11 and LNCaP-cxD2, respectively.

0162Example 4: Transcription promoting activity of bicalutamide on mutated type AR 5,000,000 cells of Cos-7 were sown to a 150 cm² flask and cultured in a culture broth (DMEM + 10% Dextran Charcoal (DCC)-Fetal Bovine Serum (FBS) + 2mM glutamine) for 24 hours, after which a vector DNA incorporating a wild type AR or a mutated type AR (W741C and W741L type mutations) and a vector DNA having the luciferase gene bound downstream of an androgen-responsive promoter were co-transfected by the liposome method. Two hours later, the medium was replaced with a fresh one and 3 hours of cultivation was conducted, after which 0.01 or 1 µM bicalutamide was added and further 24 hours of cultivation was conducted, after which luciferase activity was measured and the AR transcription activity of bicalutamide was examined.

0163As a result, bicalutamide exhibited almost no transcription activity on the wild type, whereas bicalutamide exhibited transcription-promoting activity on the W741C and W741L type mutant ARs [<figref idref="f0002">Fig. 3</figref>].

Example 5: Screening of compound exhibiting antagonist action on W741C type mutant AR

0164A compound that inhibits the W741C type mutant AR transcription activity of DHT (dihydrotestosterone), namely a compound that exhibits antagonist action on the W741C type mutant AR was searched for by the same co-transfection system as the assay system described in Example 4; as a result, Compound A [ethyl 2,5-dimethyl-4-(4-nitrophenyl)-1H-pyrrole-3-carboxylate (manufactured by Labotest)] was found [<figref idref="f0002">Fig. 4</figref>].

Reference Example 1: Construction of expression vector containing a luciferase gene under the control of a PSA promoter

0165After genomic DNA was extracted from LNCaP-FGC cells according to a conventional method, a PCR reaction was conducted with this as the template using 5'-GGAGCTCGAATTCCACATTGTTTGCTGCACGTTGG-3' (SEQ ID NO: 5) and 5'-CAAGCTTTGGGGCTGGGGAGCCTCCCCCAGGAGC-3' (SEQ ID NO: 6) as the primers. Using Pyrobest (TaKaRa) as the DNA polymerase, the PCR reaction was conducted in 25 cycles of heating at 94°C for 1 minute, followed by heating at 98°C for 5 seconds, at 59°C for 30 seconds, and at 72°C for 1 minute, with the Gene Amp PCR System 9700 (Applied Biosystems), to yield an about 650 bp DNA fragment containing the PSA (prostate-specific antigen) promoter. After this fragment was cleaved with restriction endonucleases Hind <b>III</b> (TaKaRa) and Sac I (TaKaRa), it was subjected to agarose gel electrophoresis, and a DNA fragment was recovered. The DNA fragment was mixed with the pGL3-Basic vector (Promega), previously digested with Hind <b>III</b> and Sac I, and ligated using the Ligation high (TOYOBO), and <i>Escherichia coli</i> DH5α competent cells were transformed, to yield the vector pGL3-PSA-Luc, which has the luciferase gene ligated downstream of the PSA promoter. Furthermore, after pGL3-PSA-Luc was cleaved with Hind III, blunting was conducted using a Blunting kit (TaKaRa), after which the blunt-ended fragment was cleaved with Kpn I (TaKaRa) and a fragment containing the PSA promoter was recovered. After the DNA fragment was cleaved with Sac I, blunting was conducted using a Blunting kit, and the blunt-ended fragment was ligated to Kpn I-cleaved pGL3-PSA-Luc, and <i>Escherichia coli</i> DH5α competent cells were transformed, to yield pGL3-2PSA-Luc, which has two PSA promoters ligated tandem.

Example 6: Reporter assay system using a PSA promoter

01665,000,000 cells of Cos-7 were sown to a 150 cm² flask and cultured in a culture broth (DMEM + 10% Dextran Charcoal (DCC)-Fetal Bovine Serum (FBS) + 2mM glutamine) for 24 hours, after which pGL3-PSA-Luc or pGL3-2PSA-Luc obtained in Reference Example 1 above and a vector DNA incorporating a wild type AR were co-transfected using SuperFect (Qiagen). Two hours later, the medium was replaced with a fresh one and 3 hours of cultivation was conducted, after which 1 µM DHT (5α-dihydrotestosterone) was added and further 24 hours of cultivation was conducted, after which luciferase activity was measured and transcription activity was examined. The results are shown in <figref idref="f0003">Fig. 5</figref>.

0167As is evident from <figref idref="f0003">Fig. 5</figref>, it was found possible to measure the effect of DHT on AR transcription activity using the PSA promoter. Furthermore, it was found possible to obtain more potent activity by tandemly linking the PSA promoter.

Industrial Applicability

0168By the method of the present invention, an effective prophylactic/therapeutic agent for androgen-independent relapsed prostate cancer can be screened. Also, development (relapse) of androgen-independent cancers can be detected early. Furthermore, by applying the new endocrine therapy introduced by the present invention, it is expected that the survival rate and period in prostate cancer is significantly improved.

SEQUENCE LISTING

0169<ul id="ul0004" list-style="none"><li><110> Takeda Chemical Industries, Ltd.</li><li><120> Mutated Androgen Receptor, Cancer Cells Expressing Same, Production Method Thereof and Use Thereof</li><li><130> 3056WOOP</li><li><150> <patcit id="pcit0001" dnum="JP2002162206A"><text>JP 2002-162206</text></patcit> <151> 2002-06-03</li><li><150> <patcit id="pcit0002" dnum="JP2002255612A"><text>JP 2002-255612</text></patcit> <151> 2002-08-30</li><li><160> 6</li><li><170> PatentIn version 3.1</li><li><210> 1 <211> 2775 <212> DNA <213> Homo sapiens</li><li><220> <221> CDS <222> (1).. (2775) <223></li><li><400> 1 <img file="EP1550720B1_D0002.tif" /><img file="EP1550720B1_D0003.tif" /><img file="EP1550720B1_D0004.tif" /><img file="EP1550720B1_D0005.tif" /><img file="EP1550720B1_D0006.tif" /><img file="EP1550720B1_D0007.tif" /></li><li><210> 2 <211> 924 <212> PRT <213> Homo sapiens</li><li><400> 2 <img file="EP1550720B1_D0008.tif" /><img file="EP1550720B1_D0009.tif" /><img file="EP1550720B1_D0010.tif" /><img file="EP1550720B1_D0011.tif" /></li><li><210> 3 <211> 15 <212> DNA <213> Homo sapiens</li><li><220> <221> misc_feature <223> ARE sequence in human PSA promoter.</li><li><400> 3 agaacagcaa gtgct    15</li><li><210> 4 <211> 35 <212> DNA <213> Homo sapiens</li><li><220> <221> misc_feature <223> ARR sequence in human PSA promoter.</li><li><400> 4 gtggtgcagg gatcagggag tctcacaatc tcctg    35</li><li><210> 5 <211> 35 <212> DNA <213> Artificial Sequence</li><li><220> <221> misc_feature <223> Oligonucleotide designed to act as primer for amplifying human PSA promoter.</li><li><400> 5 ggagctcgaa ttccacattg tttgctgcac gttgg    35</li><li><210> 6 <211> 34 <212> DNA <213> Artificial Sequence</li><li><220> <221> misc_feature <223> Oligonucleotide designed to act as primer for amplifying human PSA promoter.</li><li><400> 6 caagctttgg ggctggggag cctcccccag gage    34</li></ul>