Home / Hair Loss Treatments / Treatment Patents / Manipulation of androgen receptors in treatment of hair loss Manipulation of androgen receptors in treatment of hair loss United States Patent 5,556,956 Roy, et al. Sept. 17, 1996 Methods and compositions relating to the androgen receptor gene and uses thereof. Inventors: Roy; Arun K. (San Antonio, TX); Chatterjee; Bandana (San Antonio, TX). Assignee: Board of Regents, The University of Texas System (Austin, TX). Appl. No: 149,096 Filed: Nov. 4, 1993 Related U.S. Application Data: Intl. Cl.: C07H 21/00, C07H 21/02, C07H 21/04 Current U.S. Cl.: 536/24.1; 536/23.1; 536/24.3; 536/24.31 Field of Search: 536/24.1, 24.31, 24.3, 23.1; 514/44 Abstract: Disclosed are methods and compositions, including antisense and antigene constructs and pharmaceutical formulations thereof, for use in regulating androgen receptor gene expression. The promoter activity of the androgen receptor gene is herein shown to include a critical upstream acting domain with a unique sequence, the unique nucleic acid sequence corresponding to positions 1697 and 2084, particularly between positions 1697 and 1717, as defined in SEQ ID NO:1. The AR gene promoter sequence, herein characterized as having a a particular nucleotide sequence defined between nucleotides 1697 and 2084 of SEQ ID NO:1, is characterized as important in the regulation of androgen receptor gene expression. Oligonucleotides and triple helix forming oligonucleotides (TFO) that bind to this region and that have nucleic acid sequences corresponding particularly to the region between positions 1697 and 2084 of the androgen receptor gene promoter region provide specific and potent inhibition of AR promoter region function in cellulo. Rat and human nuclear proteins, more specifically described as novel trans-activating proteins that specifically bind to this region of the androgen receptor gene, are also identified. These proteins may be used to prepare specific antibodies to the protein, as well as to regulate the expression of the AR gene. The oligonucleotide compositions of the invention may be used as molecular probes for the androgen receptor gene, as well as to inhibit the expression of the androgen receptor gene. The compositions may also be used in the treatment of pathologies characterized by overexpression of the androgen receptor gene, including prostatic hypertrophy and androgenetic alopecia. 15 Claims, 7 Drawing Figures The government owns rights in the subject matter of the claimed invention as research was funded by NIH grants R01-AG- 63527 NIH grant P01-AG 06872, and NIH grant R 37-DK 14744. Other References: Mackellar et al. (1992) Nucleic Acids Res. 20(13), 34 11-3417. Milligan et al. (1993) J Med. Chem. 36(14), 1923-1937. Uhlmann et al. (1990) Chem. Rev. 90(4), 543-584. Wasylyk (1988) Biochem Biophys Acta 951, 17-35. Xodo et al. (1991) Nucleic. Acids Res. 19(20), 5625-5631. Agrawal, Sudhir, et al. “Cellular Uptake and Anti-HIV Activity of Oligonucleotides and Their Analogs”, Gene Regulation: Biology of Antisenses RNA and DNA, 2737-283, 1992. Agrawal, Sudhir and J.-Y. Tang, “Efficient Synthesis of Oligoribonucleotide and its Phosphorothioate Analogue Using H-Phosphonate Approach”, Tetrahedron Letters, 31(52):7541-7544, 1990. Akhtar, Saghir, et al., “Pharmaceutical Aspects of the Biological Stability and Membrane Transport Characteristics of Antisense Oligonucleotides”, Gene Regulation: Biology of Antisense RNA and DNA, 133-145, 1992. Baarends, Willy M., et al., “The rat androgen receptor gene promoter”, Molecular and Cellular Endocrinology, 74:75-84, 1990. Bankier, A. T., et al., “Random cloning and sequencing by the M13/dideoxynucleotide chaim termination method”, Methods Enzymol, 155:51-93, 1987. Beato, M., “Gene regulation by steroid hormones” Cell, 56(3): 335-44, 1989. Biggin, M.D. and Tjian, R., “Transcription factors and the control of Drosophila development”, Trends in Gent, 5(11):377-83. Biro, Sadatoshi, et al., “Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration”, Proc. Natl. Acad. Sci. USA, 90:654-658, 1993. Boutorin, A.S., et al., “Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′-terminus and their interaction with DNA within mammalian cells”, Institute of Biorganic Chemistry, Siberian Division of the USSR Academny of Sciences, 254(1,2):129-132, 1989. “Antisense Therapeutics”, Gene Regulation: Biology of Antisense RNA and DNA, 285-293, 1992. Briggs, M. R., et al., “Purification and Biochemical Characterization of the Promoter-Specific Transcription Factor”, Science, 234:47-52, 1986. Carthew, R. W., et al., “An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter”, Cell, 43(2 Pt 1):439-48, 1985. Chang, R. W., et al., “Molecular cloning of human and rat complementary DNA encoding androgen receptors”, Science, 240:324-26, (1988). Christy, B. and Nathans, “DNA binding site of the growth factor-inducible protein zif268″, Proc Natl Acad Sci U S A 86(22):8737-41, 1989. Cohen, Jack S., “Chemically modified oligodeoxynucleotide analogs as regulators of viral and cellular gene expression”, Gene Regulation: biology of Antisense RNA and DNA, 247-259, 1992. Coffey, D. S. and Pienta, K. J., “New Concepts in Stydying the Control of Normal and Cancer Growth of the Prostate”, Prog. Clin. Biol. Res., 239:1-73, (1987). Colvard, D. S., et al., “Identification of Androgen Receptors in Normal Human Osteoblast-like Cells”, Proc. Natl. Acad. Sci U S A 86(3):854-7, (1989). Curran, T. and Franza, B. R., Jr., “Fos and Jun: the AP-1 Connection”, Cell, 55(3):395-7, (1988). deWet, “Firefly Luciferase Gene: Structure and Expression in Mammalian Cells”, Mol Cel Biol, 2:1044-51, 1987. Encio, I. J. and Detera-Wadleight, S. D. “The genomic structure of the human glucocorticoid receptor”, J Biol CHem, 266:7182-88, 1991. Evans, R. M., “The steroid and thyroid hormone receptor superfamily”, Science, 240(4854):889-95, 1988. Faber, P. W., et al., “Characterization of the human androgen receptor transcription unit”, J Biol Chem, 266(17):10743-49, 1991. Hall, C. V., et al., “Expression and Regulation of Escherichia colilacZ gene gusions in mammalian cells”, J Mol Appl Genet, 2:101-0, 1983. Hanvey, Jeffery C., et al., “Antisense and Antigene Properties of Peptide Nucleic Acids”, Science, 258:1481-1485, 1992. Harris, Georgianna, et al., “Indentification and selective inhibition of an isozyme of steroid 5a-reductase in human scalp”, Proc. Natl. Acad. Sci. USA, 89:10787-10791, 1992. Huckaby, C. S., et al., “Structure of the chromosomal chicken progesterone receptor gene”, Proc Natl Acad Sci U S A 84(23):8380-4. Issemann, I. and Green, S., “Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferactors”, Nature, 347:645-50, 1990. Jenster, G., et al., “Functional domains of the human androgen receptor”, J Steroid Biochem Mol Biol, 41(3-8):671-75, 1992. Klemsz, M. J., et al., “The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene”, Cell, 61:113-24, 1990. Krieg, Arthur M., et al., “Modification of antisense phosphodiester oligodeoxynucleotides by a 5′ cholesteryl moiety increases cellular association and improves efficacy”, Proc. Natl. Acad. Sci. USA, 90:1048-1052, 1993. Leclerc, S., et al., “Purification of a human glucocorticoid receptor gene promoter-binding protein”, J Biol Chem, 266(14):8711-19, 1991. Leiter, Josef M., et al., “Inhibition of influenza virus replication by phosphorothioate oligodeoxynucleotides”, Proc. Natl. Acad. Sci. USA, 87:3430-3434, 1990. Lubahn, D. B., et al., “Cloning of human androgen receptor complementary DNA and localizing to the X chromosone”, Science, 240(4850):327-30, 1988. Marcelli, M., et al., “Androgen resistance associated with a mutation of the androgen receptor at amino acid levels and impairment of receptor function”, J Clin Endocrinol Metab, 73(2):318-25, 1991. Marshall, W. S. and M. H. Caruthers, “Phosphorodithioate DNA as a Potential Therapeutic Drug”, Science, 259:1564-1570, 1993. Martin, M. E., et al., “Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins”, Proc Natl Acad Sci U S A, 85:5839-43, 1988. McEwen, B. S., “Binding and metabolism of sex steroids by the hypothalamic-pituitary unit: physiological implications”, Annu Rev Physiol, 42:97-110, 1980. Milin, B. and Roy, A. K., “Androgen receptor in rat liver: Cytosol receptor deficiency in the pseudohermaphrodite male rate”, Nature: New Biol, 242:248-50, 1973. Newmark, J. R., et al., “Androgen receptor gene mutations in human prostate cancer”, Proc Natl Acad Sci U S A, 89:6319-23, 1992. Oberhauser, Berndt and Ernest Wagner, “Effective incorporation of 2′-O-methyl-oligoribonucleotides into liposomes and enhanced cell association through modification with thiocholesterol”, Nucleic Acids Research, 20(3):533-538, 1992. O’Malley, B., “The steriod receptor superfamily: More excitement predicted for the future”, Mol Endocrinol, 4:363-69, 1990. Pajunen, A. E. I., et al., “Androgenic regulation of ornithine decarboxylase activity in mouse kidney and its relationship to changes in cytosol and nuclear androgen receptor concentrations”, J Biol Chem, 257(14):8190-98, 1982. Pinsky, L. and Kaufman, M., “Genetics of steroid receptors and their disorders”, Adv Hum Genet, 16;299-472, 1987. Roberts, Richard W. and Donald M. Crothers, “Stability and Properties of Double and Triple Helices: Dramatic Effects of RNA or DNA Backbone Composition”, Science, 258:1463-1466, 1992. Ron, D., et al, “An inducible 50 kilodalton NFkB-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene”, Mol Cell Biol, 10:1023-32, 1990. Saunders, P. T. K., et al., “Point mutations detected in the androgen receptor gene of three men with partial androgen insensitivity syndrome”, Clin Endocrinol, 37(3):214-20, 1992. Song, Chung, S., et al., “Androgen Receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging and calorie restriction”, Endocrinology, 128(1):349-56, 1991. Song, Chung S., et al., “A Distal activation domain is critical in the regulation of the rat androgen receptor gene promoter”, Biochem. J., 294, 1993. Song, Chung S., et al., “Characterization of Distal Activation and Repressor Domains Critical for Regulation of Rat Androgen Receptor Gene Promotor”, 74 Annual Endocrine Society Prog. & Abstracts Abstract, 1505:428, 1992. Song, Chung S., et al., “Cloning and Characterization of the Upstream Regulatory Region of the Rat Androgen Receptor Gene”, 74 Annual Endocrine Society Prog. & Abstracts Abstract, 954:289, 1993. Stoner, Elizabeth, “The Clinical Development of a 5.alpha.-Reductase Inhibitor, Finasteride”, J. Steroid Biochem. Molec. Biol., 37(3):375-378, 1990. Tilley, Wayne D. et al., “Expression of the Human Androgen Receptor Gene Utilizes a Common Promoter in Diverse Human Tissues and Cell Lines”, The Journal of Biological Chemistry, 265(23):13776-13781, 1990. Trapman, J., et al., “Cloning, Structure and Expression of a cDNA Encoding the Human Androgen Receptor”, Biochemical and Biophysical Research Communications, 153(1):241-248, 1988. To, Richard Y. -L. and Paul E. Neiman, “The Potential for Effective Antisense Inhibition of Retroviral Replication Mediated by Retroviral Vectors”, Gene Regulation: Biology of Antisense RNA and DNA, 261-271, 1992. The Upjohn Company, Rogaine, “The Only Product Ever Proven to Regrow Hair”, 1992. Vinson, C. R., “Scissors-grip model for DNA recognition by a family of leucine zipper protein”, Science, 246:911-16, 1989. Yarbrough, W. G., et al., “A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat”, J Biol Chem, 265(15):8893-8900, 1990. Dialog Search Report dated Dec. 9, 1992 (8:56 a.m.). Dialog Search Report dated Dec. 9, 1992 (9:07 a.m.). Dialog Search Report dated Dec. 9, 1992 (10:33 a.m.). Dialog Search Report dated Jan. 26, 1994. Primary Examiner: Fleisher; Mindy Assistant Examiner: Schmickel; David Attorney, Agent or Firm: Akin, Gump, Strauss, Hauer & Feld, L.L.P.