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United States Patent (19) 11 Patent Number: 6,103,881 Nett Et Al

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United States Patent (19) 11 Patent Number: 6,103,881 Nett Et Al USOO6103881A United States Patent (19) 11 Patent Number: 6,103,881 Nett et al. (45) Date of Patent: Aug. 15, 2000 54) GNRH ANALOGS FOR DESTROYING Chang et al., “Artificial Hybrid Protein Containing a Toxin GONADOTROPHS Protein Fragment and a Cell Membrane Receptor-Binding Moiety in a Disulfide Conjugate”, pp. 1515-1522, 1977, J. 75 Inventors: Torrance M. Nett, Ft. Collins; Biol. Chem., vol. 252, Feb. Leonard Michael Glode, Aurora; Chaudhary et al., “Activity of a Recombinant Fusion Protein Marat Karpeisky, Boulder, all of Colo. Between Transforming Growth Factor Type C. and Pseudomonas Toxin'", pp. 4538-4542, 1987, Proc. Natl. 73 Assignee: Colorado State University Research Acad. Sci. USA, Vol. 84, Jul. Foundation, Ft. Collins, Colo. Colombatti et al., “Cloned Fragment of Diphtheria Toxin Linked to T Cell-specific Antibody Identifies Regions of B 21 Appl. No.: 09/015,729 Chain Active in Cell Entry”, pp. 3030-3035, 1986 J. Biol. 22 Filed: Apr. 7, 1998 Chem., vol. 261, Mar. Greenfield et al., “Mutations in Diphtheria Toxin Separate Related U.S. Application Data Binding from Entry and Amplify Immunotoxin Selectivity”, pp. 536-539, 1987, Science, vol. 238, Oct. 63 Continuation-in-part of application No. 07/314,653, Feb. 23, Meyers et al., “Specific Chemical Cleavage of Diphtheria 1989, abandoned, and a division of application No. 08/481, Toxin With Hydroxylamine”, pp. 17122–17127, 1988, J. 128, Jun. 7, 1995, Pat. No. 5,786,457. Biol. Chem., vol. 263, No. 32, Nov. 51 Int. Cl." ............................. A61K38/00; CO7K 1/00; Murphy et al., “Genetic Construction, Expression, and C07K5/00; CO7K 7/00 Melanoma-Selective Cytotoxicity of a Diphtheria Toxin 52 U.S. Cl. .......................... 530/402; 530/324; 530/326; Related C-Melanocyte-Stimulating Hormone Fusion Pro 530/313; 530/328; 514/12; 514/14: 514/15; tein", pp. 8258–8262, 1986 Proc. Natl. Acad. Sci. USA, vol. 514/16 83, Nov. 58 Field of Search .................................. 514/15, 12, 14, Myers, “Hybrid Toxins: An Approach to Cell Specific Tox 514/16; 530/313, 324, 328,326, 402 icity”, 1987, Dissertation submitted to the Division of Ani mal Science and the Graduate School of the University of 56) References Cited Wyoming, Laramie, Wyoming. U.S. PATENT DOCUMENTS Myers D.A., et al., “Protein-Peptide Conjugation by a Two-Phase Reaction”, p. 343, 1985, Biochem J., 227:1. 4,081,533 3/1978 Cheesman ............................... 424/177 4,201,770 5/1980 Stevens ..... ... 424/177 Oeltmann et al., “A Hybrid Protein Containing the Toxic 4,302,386 11/1981 Stevens ..... ... 260/112 Subunit of Ricin and the Cell-Specific Subunit of Human 4,400,376 8/1983 Sanderson ................................. 424/88 Chorionic Gonadotrophin', pp. 1028-1032, 1979, J. Biol. 4,444,759 4/1984 Rivier et al. .. ... 424/177 Chem., vol. 4, Feb. 4,526,716 7/1985 Stevens ..... 260/112.5 Oeltmann, “Synthesis and In Vitro Activity of a Hormone 4,691,006 9/1987 Stevens ..... ... 530/324 Diphtheria Toxin Fragment of a Hybrid”, pp. 430-435, 4,767,842 8/1988 Stevens ................................... 530/324 1985, Biochem Biophys. Res. Commun., vol. 133, Dec. 4,863,857 9/1989 Blalock et al. ........................... 435/68 Pastan et al., “Immunotoxins”, pp. 641–648, 1986, Cell, vol. 5,378,688 1/1995 Nett et al. ................................. 514/17 47, Dec. 5,488,036 1/1996 Nett et al. ................................. 514/15 5,492.893 2/1996 Nett et al. ................................. 514/15 Pineda M.H. et al., “Atrophy of Rabbit Testes Associated 5,631,229 5/1997 Nett et al. ................................. 514/15 with Production of Antiserum to Bovine Luteinizing Hor 5,786,457 7/1998 Nett et al. ............................... 530/402 mone”, pp. 665-668, 1967, Proc. Soc. Exp. Bio. Med., vol. OTHER PUBLICATIONS 125, No. 3, Jul. Bacha et al., “Organ-Specific Binding of a Thyrotropin-Re (List continued on next page.) leasing Hormone-Diphtheria Toxin Complex after Intrave nous Administration to Rats', pp. 1072-1076, 1983, Endo Primary Examiner Avis M. Davenport crinology, Vol. 113. Attorney, Agent, or Firm-Sheridan Ross P.C. Bacha et al., Systemic Toxicity of Diphtheria Toxin-Related 57 ABSTRACT Fragments (CRM26, CRM45), a Hormone-Toxin Hybrid Protein (TRH-CRM45), and Ricin A' (42234), pp. Certain toxic compounds (T) Such as, for example, com 131-138, 1986, Proc. Soc. Exp. Biol. Med., vol. 181. pounds based upon diphtheria toxin, ricintoxin, pseudomo Bacha et al., “Thryotropin-Releasing Hormone-Diphtheria nas exotoxin, C.-amanitin, pokeweed antiviral protein (PAP), Toxin-Related Polypeptide Conjugates; Potential Role of ribosome inhibiting proteins, especially the ribosome inhib the Hydrophobic Domain in Toxin Entry”. pp. 1565-1570, iting proteins of barley, wheat, corn, rye, gelonin and abrin, J. Biol. Chem., vol. 258, Feb. as well as certain cytotoxic chemicals. Such as, for example, Bourdon et al., “The Potential of Monoclonal Antibodies as melphalan and daunomycin can be conjugated to certain Carriers of Radiation and Drugs for Immunodetection and analogs of gonadotropin-releasing hormone to form a class Therapy of Brain Tumors”, pp. 79-101, 1984, Prog. Exp. of compounds which, when injected into an animal, destroy Tumor Res., vol. 28. the gonadotrophs of the animals anterior pituitary gland. Cawley et al., “Epidermal Growth Factor-Toxin A Chain Hence Such compounds may be used to Sterilize Such Conjugates: EGF-Ricin A is a Potent Toxin While EGF animals and/or to treat certain SeX hormone related diseases. Diphtheria Fragment A is Nontoxic', pp. 563-570, 1980, Cell, vol. 22, Nov. 5 Claims, 8 Drawing Sheets 6,103,881 Page 2 OTHER PUBLICATIONS Vitetta et al., “Redsigning Nature's Poisons to Create Anti Tumor Reagents”, pp. 1098-1104, 1987, Science, vol. 238, Quadri S.K. et al., “Inhibition of Spermatogenesis and Nov. Ovulation in Rabbits with Antiovine LH Rabbit Serum', pp. Youle, et al., “Immunotoxins Show Rapid Entry of Diph 809–814, 1966, Proc. Soc. Exp. Biol. Med., vol. 123. theria Toxin but not Ricin via the T3 Antigen”, pp. 93-98, Schwartz et al., “A New Cytotoxin Specific for the Target 1986, J. Immunol, vol. 136, Jan. Cells of Corticotrophin-Releasing Factor”, pp. 1454-1460, CRC of Chemistry of Protein Conjugation and Cross-Link 1987, Endocrinology, vol. 121. ing, pp. 58, 152-160, 189, 267-285, 288-293. Singh et al., “Controlled Release of LHRH-DT From Bio Youle, et al., “Cytotoxix Ribonucleases and Chimeras in erodible Hydrogel Microsphers', pp. R5-R8, 1976 Interna Cancer Therapy”, Crit. Rev. in Therapeutic Drug Carrier tional Journal of Pharmacology. Systems, 10(1), pp. 1-28 (1993). Tallgat M. et al., “Impairment of Spermatogenesis and Conn et al., “Gonadotrophin-Releasing Hormone and its Libido Through Antibodies to Luteinizing Hormone”, pp. Analogues”, The New England J. of Med., pp. 93-103, Jan., 113–118, 1971, Fertility and Sterility, vol. 22, No. 2, Feb. 1991. U.S. Patent Aug. 15, 2000 Sheet 1 of 8 6,103,881 1 OO GnRH - A GnRH - A - Toxin 50 U.S. Patent Aug. 15, 2000 Sheet 2 of 8 6,103,881 O PAP - O - Lys SALNE CONTROL X GnRH CHALLENGE X8 X 8O & k: X aaaaaaa 8/20 8/27 9/3 9/11 9/17 U.S. Patent Aug. 15, 2000 Sheet 3 of 8 6,103,881 WHEAT HEMTOXN 120 Molar ratioS of SPDP Hemitoxin 20:1 1 OO A. C 8 O 4.6OO 2 O 100 O 102 103 104 Hemitoxin added to Protein Synthesis Reaction Fig. 2A U.S. Patent Aug. 15, 2000 Sheet 4 of 8 6,103,881 BARLEY HEMTOXN 120 Molar ratioS Of SPDP Hemitoxin 20:1 1 OO TV O 8 O 6 O 4 O 2 O 100 101 102 103 104 Hemitoxin added to Protein Fig. 2B U.S. Patent Aug. 15, 2000 Sheet 5 of 8 6,103,881 ||60 8/ 9 61·9 #7G 2€ | 08 U.S. Patent Aug. 15, 2000 Sheet 6 of 8 6,103,881 Effect of 2 - minothiolane Conjugation on Barley Toxin Activity 20,000 18,000 16,000 14,000 12,000 10,000 8,000 6,000 4,000 O- - Unconjugated \ o y A--- i.44 Molar Ratio U.S. Patent Aug. 15, 2000 Sheet 7 of 8 6,103,881 U.S. Patent Aug. 15, 2000 Sheet 8 of 8 6,103,881 Toxin Conjugate Receptor Binding 102 10 100 10 102 103 ng D-Lys 6 Equivalents in Conjugate 1 -- D - Lys - 6 - GnRH 4 - - - - Geionin - C - D - Lys - 6 - GnRH 4.5 - A - Barley - 21T - D - Lys - 6. GnRH 45 - O - Barley - C - D. Lys - 6 - G 15a -0- PAP. SPDP - D - Lys. 6. GnRH 16ac -a - Barley - EACA - C - D. Lys - 6 - 4 ---g)- Wheat - C - D - Lys - 6 - GnRH GnRH 4 - - -O-- Rye - C - D - Lys - 6 - GnRH Fig. 5 6,103,881 1 2 GNRH ANALOGS FOR DESTROYING levels. On the other hand, in a prepubertal calf, or an adult GONADOTROPHS bull, discharges of luteinizing hormone from the anterior pituitary occur more frequently and the testeS produce RELATED APPLICATIONS considerably larger amounts of testosterone and other Ste roids. It is thought that these conditions result from the This patent application is a continuation-in-part of U.S. following factors: (1) decreases in the concentration of patent application Ser. No. 07/314,653 filed Feb. 23, 1989, estradiol receptors in the hypothalamus, (2) concomitant now abandoned and also entitled “GnRH Analogs For increases in the concentration of estradiol receptors in the Destroying Gonadotrophs, and a division of Ser. No. 08/481, anterior pituitary, and (3) increases the number of 128, filed Jun. 7, 1995, now U.S. Pat. No. 5,786,457. gonadotropin-releasing hormone GnRH) receptors in the anterior pituitary. This increase in GnRH receptorS is gen FIELD OF THE INVENTION erally regarded as a prerequisite for an animal to pass from The present invention generally relates to methods for the infantile Stage to the prepubertal and mature Stages of Sterilizing animals and to methods for medically treating endocrine development.
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