EP0988048A1 - Zusammensetzungen aus ligand und lytischem peptid sowie anwendungsverfahren - Google Patents

Zusammensetzungen aus ligand und lytischem peptid sowie anwendungsverfahren

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Publication number
EP0988048A1
EP0988048A1 EP98912077A EP98912077A EP0988048A1 EP 0988048 A1 EP0988048 A1 EP 0988048A1 EP 98912077 A EP98912077 A EP 98912077A EP 98912077 A EP98912077 A EP 98912077A EP 0988048 A1 EP0988048 A1 EP 0988048A1
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EP
European Patent Office
Prior art keywords
cells
gnrh
hormone
seq
peptide
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Application number
EP98912077A
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English (en)
French (fr)
Other versions
EP0988048A4 (de
Inventor
Frederick M. Enright
Jesse M. Jaynes
William B. Hansel
Kenneth L. Koonce
Lane D. Foil
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Demeter Biotechnologies Ltd
Louisiana State University and Agricultural and Mechanical College
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Demeter Biotechnologies Ltd
Louisiana State University and Agricultural and Mechanical College
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Application filed by Demeter Biotechnologies Ltd, Louisiana State University and Agricultural and Mechanical College filed Critical Demeter Biotechnologies Ltd
Publication of EP0988048A1 publication Critical patent/EP0988048A1/de
Publication of EP0988048A4 publication Critical patent/EP0988048A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/31—Somatostatins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • A61K38/34—Melanocyte stimulating hormone [MSH], e.g. alpha- or beta-melanotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • compositions and methods for specifically inhibiting cells that are driven by or are dependent on specific ligand interactions are compositions and methods for long-term contraception or sterilization; compositions and methods for inhibiting or killing malignant and non-malignant, hormone-dependent tumors; compositions and methods for selectively killing virally infected cells; and compositions and methods for selectively destroying lymphocytes responsible for autoimmune disorders.
  • compositions that have sometimes been used for long-term contraception include those based upon natural or synthetic steroidal hormones to "trick" the female reproductive tract into a "false pregnancy.” These steroidal hormones must be administered repeatedly to prevent completion of the estrous cycle and conception. Steroids have side effects that can be potentially dangerous.
  • Antibodies can be developed to specific hormone receptors (such as the LH receptor) and then coupled to a toxin. All cells with LH receptors should then be destroyed. Although various cell types have not been characterized in dog corpora lutea, destruction of any luteal cell type could potentially result in luteolysis if cell types communicate. " (citations omitted)
  • Tissue-specific c totoxm ⁇ -Permanent contraception in females and males might be achieved by administration of a cytotoxin that is linked to gonadotropin-releasing hormone (GnRH) and that selectively destroys gonadotrop in-secreting pituitary cells.
  • GnRH gonadotropin-releasing hormone
  • a cytotoxin linked to antibodies against gonadotropin receptors could be targeted to alter gonadal function. Toxins would need to be carefully targeted to specific cells, yet be safe for all other body tissues.
  • a prodrug is then administered to the patient.
  • the prodrug is substantially less toxic than the drug that results from activation of d e drug at the tumor site by the conjugated enzyme.
  • the activated drug then selectively attacks tumor cells. See, e.g., D. Kerr et al, "Regressions and Cures of Melanoma Xenografts following Treatment with Monoclonal Antibody ⁇ -Lactamase Conjugates in Combination with Anticancer Prodrugs," Cancer Research, vol. 55, pp. 3558-3563 (1995); and H. Svensson et al.
  • EGF-Ricin Is a Potent Toxin while EGF-Diphtheria Fragment A is Nontoxic, " Cell, vol. 22, pp. 563-570 (1980).
  • E. Vitetta et al. "Redesigning Nature's Poisons to Create Anti-Tumor Reagents,"
  • Verhaert et al. "Substances Resembling Peptides of the Vertebrate Gonadotropin System Occur in the Central Nervous System of Periplaneta americana L. ,” Insect Biochem. , vol. 16, pp. 191-197 (1986).
  • U.S. Patents No. 5,378,688; 5,488,036; and 5,492,893 disclose compounds said to be useful in inducing sterility in mammals, and in treating certain sex hormone-related cancers in mammals.
  • the disclosed compounds were generically described as GnRH (or a GnRH analog) conjugated to a toxin.
  • the toxin was preferably linked to the sixth amino acid of the GnRH agonist.
  • the toxin was preferably one with a translocation domain to facilitate uptake into a cell.
  • conjugation of the GnRH agonist to the toxin is necessary because, for the most part, the above toxins, by themselves, are not capable of binding with cell membranes in general.
  • the toxins specifically mentioned appear all to have been metabolic toxins, for example ricin, abrin, modeccin, various plant-derived ribosome-inhibiting proteins, pokeweed antiviral protein, ⁇ -amanitin, diphtheria toxin, pseudomonas exotoxin, shiga toxin, melphalan, methotrexate, nitrogen mustard, doxorubicin, and daunomycin. None of these toxins is believed to be toxic due to direct interaction with the cell membrane. In the in vivo experiments reported, the most effective time course was reported to be weekly injections for 4 weeks. (E.g., Pat. 5,488,036, col.
  • the GnRH analog was preferably linked to the toxin with one of several specified heterobifunctional reagents. The specifications suggest that considerable effort was expended in conjugating the toxin to the GnRH agonist.
  • the toxins must in general be internalized into the target cells to have effect, and do not act on cell membranes; in addition, at least some of these toxins must be secondarily transported from the membrane-bound vesicle into the cytoplasm to interact with ribosomes, mitochondria, or other cellular components. M.
  • LH-RH receptors See also A. Jungwirth et al. , "Regression of rat Dunning R-3227-H prostate carcinoma by treatment with targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 containing 2-pyrrolinodoxorubicin, " Intl. J. Oncol. , vol. 10, pp. 877-884 (1997) R. Moretti et al., "Luteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145, " J. Clin. Endocrin. & Metab. , vol. 81 , pp.
  • LH- releasing hormone agonists inhibit both androgen-dependent (LNCaP) and androgen- independent (DU 145) human prostatic cancer cell lines, and suggests that the agonists may inhibit proliferation of the tumor cells by interfering with the stimulatory actions of epidermal growth factor.
  • GnRH gonadotropin-releasing hormone
  • United States patents 5,597,945 and 5,597,946 disclose plants transformed with genes encoding various lytic peptides.
  • amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to specifically inhibit abnormal or normal cells that are driven by or are dependent upon a specific ligand interaction; for example, to induce sterility or long-term contraception, or to attack tumor cells, or to selectively lyse virally-infected cells, or to attack lymphocytes responsible for autoimmune diseases.
  • the peptides act directly on cell membranes, and need not be internalized.
  • GnRH gonadotropin-releasing hormone
  • a membrane-active lytic peptide For example, administering a combination of gonadotropin-releasing hormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptide produces long-term contraception or sterilization in animals in vivo.
  • GnRH gonadotropin-releasing hormone
  • a membrane-active lytic peptide For example, administering a combination of gonadotropin-releasing hormone (GnRH) (or a GnRH agonist) and a membrane-active lytic peptide produces long-term contraception or sterilization in animals in vivo.
  • sterility results even when the combination is administered to a sexually immature animal: The combination then prevents sexual maturation.
  • Administering in vivo a combination of a ligand and a membrane-active lytic peptide kills cells with a receptor for the ligand.
  • the compounds used in the present invention are relatively small, and will not be antigenic. (Lytic peptides are known not to be very antigenic; and the ligands are not antigenic at all.)
  • the compounds may be administered in a single dose, or in two or more closely spaced doses. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes.) Lysis of tumor cells is rapid.
  • the two components - the ligand and the lytic peptide - may optionally be administered as a fusion peptide, or they may be administered separately, with the ligand administered slightly before the lytic peptide, to activate cells with receptors for the ligand, and thereby make those cells susceptible to lysis by the lytic peptide.
  • a fusion peptide it has been unexpectedly discovered that a linking moiety is not necessary to join the ligand to the lytic peptide: one may be bonded directly to the other, without the need for any intervening linkage; bonding may be performed by bonding one end of the ligand to one end of the peptide, or by bonding to the middle of either.
  • the toxin does not need a translocation domain, and need not be internalized, as it binds to and acts directly on the activated cell membrane to cause lysis.
  • the ligand may be a full native compound, or it may instead be the binding domain alone; the latter is preferred where the full ligand is relatively large.
  • the compounds of the present invention are well-suited for use in gene therapy to treat malignant or non-malignant tumors, and outer diseases caused by clones or populations of "normal" host cells bearing specific receptors (such as lymphocytes), because genes encoding a lytic peptide or encoding a lytic peptide/peptide hormone fusion may readily be inserted into hematopoietic stem cells or myeloid precursor cells.
  • cancer cells (uterine, endometrial, prostate, testicular, and ovarian) express LH or hCG receptors.
  • Tao et al. "Expression of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Gene in Benign Prostatic Hyperplasia and in Prostatic Carcinoma in Humans," Biol. Reprod. , vol. 56, pp. 67-72 (1997).
  • Conjugates of a lytic peptide and LH or a portion of the LH molecule may thus be used to destroy these cells selectively.
  • the genes encoding such hormones as FSH, TRH, and LH are known, and may be linked to a DNA sequence encoding a lytic peptide to produce a secreted fusion peptide, all under the control of a suitable promoter such as the acute-phase responsive promoters disclosed in United States patent application S.N. 08/474,678, filed June 7, 1995, and in PCT application WO 95/01095, published January 12, 1995.
  • a binding site from a hormone may be used in lieu of the entire hormone, for example the fifteen amino acid binding site of LH and hCG. See D.
  • D-amino acid form of GnRH will bind to gonadotropes in the pituitary, to GnRH neurons in the brain, and to various types of cancer cells. It is also known that the D-amino acid forms of lytic peptides have essentially the same propensity to lyse cell membranes as do the L-amino acid forms. Compounds of the present invention (whether administered as a fusion peptide or separately) may therefore be administered either in L-form or D-form. D-form peptides, although generally more expensive than L-form, have the advantage that they are not degraded by normal enzymatic processes, so that the D-form peptides may therefore be administered orally and generally have a longer biological half-life.
  • Oral administration of the D-form peptide may be enhanced by linking the peptide/hormone fusion product to a suitable carrier to facilitate uptake by the intestine, for example vitamin B 12 , following generally the B 12 -conjugation technique of G. Russell-Jones et al., "Synthesis of LHRH Antagonists Suitable for Oral Administration via the Vitamin B 12 Uptake System," Bioconjugate Chem., vol. 6, pp. 34-42 (1995).
  • GnRH or GnRH analogs may be used in the present invention. It has been reported that substitutions at the 6 and 10 positions of the GnRH decapeptide can produce "superagonists" having greater binding affinity to the GnRH receptor than does GnRH itself. These "superagonists” include goserelin, leuprolide, buserelin, and nafarelin. See U.S. Patent 5,488,036.
  • GnRH activates gonadotropic cells in the pituitary gland, as well as neuroendocrine GnRH neurons in the brain.
  • the activated cells have substantially increased susceptibility to lysis by a lytic peptide.
  • the lytic peptide then preferentially destroys (or severely damages) these activated cells.
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • the ligand and the lytic peptide may be administered separately, it is preferred to link the two in a single molecule, because such a linkage greatly increases the effective concentration of the lytic peptide in the vicinity of ligand-activated cells. Furthermore, this increase in the effective lytic peptide concentration can obviate the need for activation of the cells, allowing the peptide to be linked to a binding site of a ligand alone, without needing to include the "remainder" of a native ligand that would normally be needed for activating the target cells.
  • This linkage may be in either order: for example, GnRH/peptide or peptide/GnRH. Examples are modified GnRH/hecate (SEQ. ID NO. 3) and hecate/modified GnRH (SEQ. ID NO. 4). Note that no intermediate linker is necessary, and that the carboxy terminus of one of the two peptides may be bonded directly to the amino terminus of the other.
  • the pituitary gland of an adult female rat was harvested and divided into six sections of approximately equal size. One section was placed in each of six wells containing tissue culture medium at 37°C. A different treatment was applied to each well, as described below. Ten hours after treatment, the tissue from each well was fixed, and the histology of each was examined microscopically.
  • Treatment 1 applied tissue culture medium alone as a control. The histology of this tissue after treatment appeared normal.
  • Treatment 2 was an application of 5 nanograms of GnRH (SEQ. ID NO. 1) per mL of medium. The histology of this tissue after treatment was normal; a small degree of cellular vacuolization was noted. For comparison, the concentration of GnRH in normal, untreated rats varies from as low as 1 ng/mL to as high as 20 ng/mL during the LH surge phase of the estrous cycle.
  • Treatment 3 was an application of 50 ⁇ M of the lytic peptide hecate (SEQ. ID NO. 2) in the medium. The histology of this tissue after treatment appeared normal.
  • Treatment 4 was an initial application of 5 nanograms of GnRH per mL of medium for
  • Treatment 5 was an application of 50 ⁇ M of the fusion peptide modified GnRH/hecate (SEQ. ID NO. 3). Widespread basophilic (gonadotropic) cellular destruction was observed after the treatment.
  • Treatment 6 was an initial application of the fusion peptide GnRH/hecate (SEQ. ID NO. 3), followed by a second application of the fusion peptide GnRH/hecate two hours later.
  • Example 8 Two sexually immature female rats from the same litter as those of Example 7 (age 33 days) were given two intravenous injections of 500 ⁇ g GnRH/hecate fusion peptide in saline 24 hours apart. After the rats reached breeding age, they were examined 105 days post- inoculation. The external genitalia appeared small. Unlike the control rats, insertion of a cotton-tipped swab into the vagina was difficult. During a fourteen-day monitoring period 107 days to 121 days post-inoculation, neither of the treated rats demonstrated estrous or metestrous. The rats were then sacrificed and necropsied. The peptide-treated rats had thinned, inactive uterine and oviductal epithelia. Their ovaries contained no large follicles, and had a high number of atretic follicles (i.e., those that had failed to ovulate).
  • Eighteen sexually mature, mixed breed, female rats were randomly assigned to one of six groups containing three rats each. Each group of rats received a double treatment intravenously, as described below. Two weeks after the treatment, the rats were sacrificed and necropsied. The reproductive and endocrine organs were sectioned, weighed, and examined histologically.
  • Treatment 9 was a saline control.
  • the rats in this group exhibited normal ovarian function (e.g., normal follicles and new corpora lutea).
  • the pituitaries from this group were of normal size. Histology showed a normal number of pituitary basophilic cells.
  • Treatment 10 was injection with a total of 1.0 mg GnRH/hecate fusion peptide in saline, divided into two equal 0.5 mg injections administered 24 hours apart.
  • the rats in this group showed an arrest of normal ovarian follicular development. Few corpora lutea were present, and those that were present appeared old. Follicles were large, and had not ruptured. Uterine morphology was consistent with hormonal inactivity.
  • the pituitaries from this group were slightly smaller than the pituitaries from the saline control group. Histology revealed a 60% to 70% reduction in the number of pituitary basophilic cells compared to the controls.
  • Treatment 11 was injection of 100 ⁇ L of a 1.35 mM solution of GnRH (162 ⁇ g) in saline, followed 15 minutes later by injection with 100 ⁇ L of a 1.35 mM solution of hecate (337 ⁇ g) in saline. The same two-step treatment was repeated 24 hours later.
  • the rats in this group showed altered ovarian histology. Few corpora lutea were present, and those that were present appeared old. Follicles were large, and had not ruptured. Uterine morphology was consistent with hormonal inactivity. The pituitaries and the pituitary histology were similar to those observed in Treatment 10.
  • Treatment 12 was injection of 100 ⁇ L of a 1.35 mM solution of hecate (337 ⁇ g) in saline. The treatment was repeated after 24 hours. The rats in this group exhibited normal ovarian function (e.g., normal follicles and new corpora lutea). The pituitaries and the pituitary histology were similar to those observed in Treatment 9.
  • Treatment 13 was injection of 100 ⁇ L of a 1.35 mM solution of GnRH (162 ⁇ g) in saline. The treatment was repeated after 24 hours. The rats in this group exhibited normal ovarian function (e.g. , normal follicles and new corpora lutea). The pituitaries and the pituitary histology were similar to those observed in Treatment 9.
  • Treatment 14 was identical to Treatment 10, except that the GnRH/hecate fusion peptide was further purified by HPLC.
  • the rats in this group showed an arrest of normal ovarian follicular development. Few corpora lutea were present, and those that were present appeared old. Follicles were large, and had not ruptured. Uterine morphology was consistent with hormonal inactivity. The pituitaries and the pituitary histology were similar to those observed in Treatment 10.
  • GnRH and the lytic peptide may be administered either separately or as a fusion peptide, although the fusion peptide is preferred as it is expected to be more active at lower doses.
  • Tissue and cell specificity of cytotoxic conjugates could be further enhanced by using various hormones or hormone analogs coupled to a lytic peptide.
  • hormones or hormone analogs coupled to a lytic peptide Some examples follow.
  • GnRH -hecate conjugate Few cells in the central nervous system should be damaged by this treatment, because the chicken II GnRH and lamprey HI GnRH forms are the primary molecules affecting brain function in most mammals.
  • Fertility control may also be selectively accomplished by treating animals with a bLH-hecate conjugate; this compound should specifically affect GnRH neurons controlling reproduction and the gonads.
  • the 1-LHRH-III- hecate conjugate could be used since it binds to receptors on cancer cells, and has no significant known action on the brain. (Other lytic peptides may be used in place of hecate in these conjugates.)
  • compositions of the present invention may be administered as described, or as pharmaceutically acceptable salts.
  • the compositions may be administered intravenously, subcutaneously, intramuscularly, or orally (especially when in D-amino acid form, preferably complexed with a carrier, e.g., vitamin B 12 ).
  • Applications of the present invention include long-term contraception or sterilization in humans; and long-term contraception or sterilization in domesticated or wild mammals, birds, reptiles, amphibians, bony fish, cartilaginous fish, jawless fish, and invertebrates such as insects or molluscs.
  • domesticated mammals in which this invention may be used include, for example, dogs, cats, cattle, horses, pigs, and sheep.
  • long-term replacement hormone therapy may be needed to prevent undesirable side effects, such as premature menopause.
  • Administration of gonadotropic hormones in a sterilized individual will temporarily restore fertility if desired. The sterilization is reversible in this sense.
  • this invention may be used in the humane population control of an unwanted introduced species. Sterilization of domesticated birds such as chickens and turkeys can increase their growth rate.
  • Avian GnRH or analogs may be used in practicing this invention to sterilize birds. There are two forms of avian GnRH - Chicken I GnRH (SEQ. ID NO. 17) and Chicken II GnRH (SEQ. ID NO. 18). Either form of avian GnRH may be used in this invention.
  • position 6 of Chicken I GnRH is linked to a lytic peptide such as hecate to form a fusion peptide.
  • a GnRH agonist or antagonist may be used.
  • Expression in the plant tissue could be constitutive, or alternatively could be induced by stimuli that induce the plant's native defense mechanisms, for example by placing the peptide gene under the control of native promoters that are so induced in plants. See, e.g. , United States patent application S.N. 08/279,472, filed July 22, 1994.
  • the compounds of the present invention may be simply administered in the water, from which they will be taken up by the animals in adult, juvenile, or larval stages.
  • the peptides are encapsulated in liposomes, which are fed to the animals as spat, fry, juveniles, or adults; the animals feed on the liposomes, which then release the compounds into the animal's circulation, causing sterilization.
  • the peptides may be injected into an animal that has reached sufficient size.
  • the compounds may be used to sterilize undesirable exotic molluscs such as the zebra mussel. Sterilization of aquaculture species may also be desirable. For example, sterilization of oysters will prevent the oysters from ripening gonads in the summer (when they would otherwise do so), thereby improving their marketability.
  • Examples 15-22 Eight sexually mature, Sprague-Dawley female rats were randomly assigned to one of eight treatments. Each group of rats received a single treatment intravenously, as described below. Rats were sacrificed and necropsied either 48 or 96 hours after treatment. The ovaries, uterus, pancreas, liver, spleen, lungs, heart, thyroid, and adrenal glands were fixed in 10% buffered formalin; sectioned; and stained with H&E (hematoxylin and eosin) stain; except that the pituitary glands were stained with PAS (periodic acid-Schiff) stain with no counter-stain. The treatments were selected so that each animal received an equimolar amount of the compound with which it was treated.
  • H&E hematoxylin and eosin
  • Treatments 15 and 16 were IV-injection with 674 ⁇ g of D-hecate in 200 ⁇ L saline
  • the rat in treatment 15 was sacrificed 48 hours after injection, and the rat in treatment 16 was sacrificed 96 hours after injection. No gross lesions were noted in the organs of either animal. The pituitary glands of both rats contained a normal number of PAS-positive cells.
  • Treatments 17 and 18 were IV-injection with 334 ⁇ g of GnRH in 200 ⁇ L saline (1.35 mM).
  • the rat in treatment 17 was sacrificed 48 hours after injection, and the rat in treatment 18 was sacrificed 96 hours after injection. No gross lesions were noted in the organs of either animal.
  • the pituitary glands of both rats contained a normal number of PAS-positive cells.
  • Treatments 19-22 were IV-injection with 1 mg GnRH-hecate fusion peptide (SEQ. ID NO. 3) in 100 ⁇ L saline (2.7 mM).
  • the rats in treatments 19 and 20 were sacrificed 48 hours after injection, and the rats in treatments 21 and 22 were sacrificed 96 hours after injection. No gross lesions were noted in the organs of any of the four animals, other than the pituitary.
  • the pituitary glands of the animals from treatments 19 and 20 were slightly enlarged, hyperemic, and edematous.
  • the pituitary glands of the animals from treatments 21 and 22 were slightly hyperemic, but of expected size.
  • MSH glycopeptide hormones
  • Hecate is an amphipathic lytic peptide that acts on cell membranes without being internalized. It is a synthetic peptide analog of melittin, the primary toxin in honeybee venom. Hecate is believed to act by disrupting cell membranes.
  • the structure of the modified GnRH- hecate conjugate used in these studies was SEQ. ID NO. 3.
  • D-Lys 6 GnRH SEQ. ID NO. 13
  • Displacement by D-Lys 6 GnRH-hecate was comparable to (and actually slightly greater than) displacement by native mammalian GnRH, as measured by cpm of radioactivity.
  • Treatment 25 10 ng bLH (positive control) Treatment 26 hecate-bLH, 10 ⁇ M Treatment 27 hecate-bLH, 5 ⁇ M Treatment 28 hecate-bLH, 1 ⁇ M Treatment 29 hecate (alone), 10 ⁇ M
  • Treatment 30 hecate (alone), 5 ⁇ M Treatment 31 hecate (alone), 1 ⁇ M
  • Examples 32-33 We also studied in vitro lysis of bovine granulosa cells with GnRH-hecate conjugate and with hecate-bLH conjugate. Granulosa cells were isolated from bovine pre-ovulatory follicles.
  • the pituitaries were processed for histological analysis of PAS-stained cells and for cells stained immunocytochemically for bLH, BFSH (bovine follicle stimulating hormone), adrenocorticotropic hormone, and other proopiomelanocortin peptide products (most notably alpha-melanocyte stimulating hormone (MSH)), thyroid stimulating hormone (TSH), prolactin (PRL), vasopressin (VP), oxytocin (OXY) or growth hormone (GH).
  • MSH alpha-melanocyte stimulating hormone
  • TSH thyroid stimulating hormone
  • PRL prolactin
  • VP vasopressin
  • OXY oxytocin
  • GH growth hormone
  • Treatment 34 0.03 ⁇ g GnRH (a normal physiological dose) (two rats)
  • Treatment 35 1.62 ⁇ g GnRH (the molar equivalent to the amount of GnRH in Treatment 36) (one rat)
  • Treatment 36 0.5 mg GnRH-hecate (one rat)
  • VP expression probably in corticotropin-releasing neurons, may cause a shift in the post-translational processing of proopiomelanocortin peptide products in the pars distalis, since GnRH-hecate and GnRH + hecate treatments reduced adrenocorticotropic hormone levels and increased the number of alpha-MSH-stained cells in this subdivision of the pituitary. No pathological changes were noted in any other tissues. Since neurons in the brain do not regenerate, severe damage to these neurons could make sterilization with a GnRH/lytic peptide combination permanent (but temporarily reversible by administration of gonadotrophic hormones).
  • Examples 38-42 Sexually immature (33 day old) female rats (randomly allocated into groups of three) were injected intravenously with saline or GnRH-hecate in saline as follows:
  • Treatment 40 0.5 mg GnRH-hecate
  • Treatment 41 1.0 mg GnRH-hecate
  • Treatment 42 1.5 mg GnRH-hecate.
  • the GnRH-hecate treatment increased the number of PAS-stained pituitary cells in the pars distalis to 177% of that for control rabbits; this increase appeared to reflect increased numbers of cells staining alpha-MSH, and reduced numbers of cells staining for LH.
  • the GnRH-hecate treatment reduced the numbers of tertiary ovarian follicles, and the numbers of GnRH-induced ovulations. No effects were noticed either on peripheral tissues or on pituitary weight.
  • the effects of GnRH-hecate on CNS morphology and immunocytochemical results were similar to those described above in Examples 34-45. Again, the effects were more pronounced on GnRH neurons than on staining of pituitary gonadotropes.
  • GnRH-hecate were reduced as compared to saline controls.
  • the mean number of ovulation sites in the five rabbits given 10 mg of GnRH-hecate was 3.6 ⁇ 1.1, with
  • Late-stage Diatraea saccharalis (sugar cane borer) pupae were inoculated with 1.0 ⁇ L of saline solution containing 1.35 mM concentration of peptide as stated, or saline alone as control. The pupae were allowed to complete metamorphosis. No gross morphological defects were observed in any of the insects completing metamorphosis.
  • Adult female moths were allowed to mate with treated males, and then lay eggs. The viability of the eggs was measured by counting the number hatching into larvae.
  • Treatment 48 was the control, inoculation of 21 pupae with saline alone. Twelve of the pupae completed metamorphosis into adult moths (4 males, 8 females). The 8 females laid a total of about 900 eggs, an average of about 112 eggs per female. About 22% of these eggs hatched, or about 25 hatched larvae per female.
  • Treatment 49 was inoculation of 10 pupae with 1.35 mM GnRH.
  • Four of the pupae completed metamorphosis into adult moths (2 males, 2 females).
  • the 2 females laid a total of about 300 eggs, or an average of about 150 eggs per female.
  • About 40% of these eggs hatched, or about 60 hatched larvae per female.
  • Treatment 50 was inoculation of 10 pupae with 1.35 mM GnRH-hecate (SEQ. ID NO.
  • Treatment 51 was inoculation of 10 pupae with D-hecate. Six of the pupae completed metamorphosis into adult moths (2 males, 4 females). The 4 females laid a total of 18 eggs, or an average of 4.5 eggs per female. 100% of these eggs hatched, or 4.5 hatched larvae per female.
  • insects have a receptor that responds to mammalian GnRH.
  • GnRH alone stimulates reproductive activity in insects.
  • GnRH coupled to a lytic peptide attacks the intermediate cells in the insects, inhibiting reproductive activity.
  • Metamorphosis is a time of high cell activity. Lytic peptides generally have greater activity against active cells. The observed response to hecate alone is believed to be a generalized response by activated cells, not a specific response mediated by a receptor. The fact that the D-conformation of hecate was used in this experiment may be significant, since D- form peptides generally have a longer biological half-life. It is currently unknown whether similar results would be seen with L-hecate alone. (D-hecate was used in Treatment 26 for the simple reason that previously-synthesized D-hecate was readily available to the investigators.)
  • compositions of the present invention are useful in killing or inhibiting the growth of malignant and benign tumor cells that express receptors for GnRH, LH, hCG, 1-LHRH-III, or steroids.
  • the ligand is administered with a lytic peptide (either sequentially, or linked to one another), and the targeted tumor cells are killed or inhibited.
  • lytic peptides may be attached to the hormone for which the tumor expresses a receptor or set of receptors, e.g., an estrogen, testosterone, LH, FSH, estradiol-17 ⁇ , transforming growth factor alpha (TGF ⁇ ), epidermal growth factor (EGF), GnRH, LH, hCG, lamprey III LHRH (1-LHRH-III), and melanocyte stimulating hormone.
  • a receptor or set of receptors e.g., an estrogen, testosterone, LH, FSH, estradiol-17 ⁇ , transforming growth factor alpha (TGF ⁇ ), epidermal growth factor (EGF), GnRH, LH, hCG, lamprey III LHRH (1-LHRH-III), and melanocyte stimulating hormone.
  • an ester linkage of a lytic peptide to estradiol or testosterone can conveniently be made by condensing the carboxy terminus of the lytic peptide with the hydroxyl group at the
  • estradiol/lytic peptide combination may be used as a treatment against breast or ovarian cancer; and a testosterone/lytic peptide combination may be used to treat prostate cancer.
  • specific binding domains of the peptide hormone LH or FSH may be used in fusion peptides with a lytic peptide to selectively bind the fusion peptide to target tumor cells with cell surface receptors for these hormones.
  • the receptor binding site of the ⁇ -subunit of LH and hCG may be used (SEQ. ID NO. 11). See Morbeck et al., Mol. and Cell Endocrin., vol. 97, pp. 173-186 (1993).
  • the anterior pituitary contains different types of epithelial cells that control the complex processes of growth, reproduction, lactation, thyroid function, and adrenal functions. Due to the high functional plasticity of pituitary cells (i.e., their ability to differentiate into different cellular phenotypes in response to stimuli), these cells are particularly prone to aberrant behavior. Because many of the signals to which the pituitary responds are receptor-mediated, pathological states may be controlled by co-opting the appropriate ligand-receptor interaction.
  • the lytic peptide may be linked to dopamine, for examine, by an amide group formed by condensing the carboxy terminus of the peptide with the amino group of dopamine.
  • This therapy will be effective not only for prolactinomas, but also for other adenomas expressing dopamine receptors, such as growth hormone-secreting adenomas, thyrotropin- releasing hormone secreting adenomas, and gonadotropin-secreting adenomas.
  • GH growth hormone
  • somatostatin receptors may be homogeneously distributed, located exclusively in one portion of the tumor tissue, or in between.
  • Somatostatin receptors are also present in other types of pituitary tumors. It has been reported that the cell surfaces of a majority of GH- and thyrotropin releasing hormone (TRH)- secreting adenomas have an elevated number of somatostatin receptors. Such tumors may be treated by the present invention by a somatostatin/lytic peptide combination.
  • TRH thyrotropin releasing hormone
  • TRH TRH
  • MSH MSH
  • GnRH corticotropin-releasing hormone
  • growth hormone-releasing hormone growth hormone-releasing hormone
  • vasoactive intestinal polypeptide and pituitary adenylate cyclase activating peptide.
  • a short chain analog of ⁇ MSH that may be used in place of MSH is Ser- Tyr-Cys-Met-Glu-His-Phe-Arg-Trp-Asn-Lys-Pro-Val-NH 2 (SEQ. ID NO. 10).
  • the ligand/lytic peptide combination of the present invention may be used to treat endocrine-related diseases generally.
  • a disease is causally related to dysfunction of cells having certain hormone receptors
  • cells with such receptors may be selectively inactivated by administering a combination of the hormone and a lytic peptide.
  • LNCaP FGC and DU145 human prostate cancer cell lines were purchased from the American Type Culture Collection (ATCC, Rockville MD), ATCC accession numbers CRL 1740 and HTB-81, respectively.
  • the LNCaP FGC adenocarcinoma cell line was originally obtained from a 50 year old male Caucasian. LNCaP FGC cells are sensitive to dihydrotestosterone and to estrogens (A+).
  • the DU145 carcinoma was originally isolated from the brain of a 69 year old male Caucasian with metastatic carcinoma of the prostate; this cell line is not sensitive to steroid hormones (A-).
  • Treatment 56 50 ⁇ M GnRH-hecate
  • Treatment 57 10 ⁇ M GnRH-hecate
  • Treatment 58 FSH pre-treatment, followed by 90 ⁇ M hecate-bLH Trypan blue exclusion was used to assess viability of the cells after treatment.
  • the treatment that most consistently and effectively killed both the A+ and the A- cancer cell lines was the higher dose (50 ⁇ M) of GnRH-hecate.
  • the lower dose (10 ⁇ M) of GnRH-hecate was equally effective against the androgen-insensitive DU145 cells.
  • the DU145 cells were also killed by hecate alone.
  • treatment with a lytic peptide alone may not be selective in vivo unless specific cell types are separately stimulated, for example by hormones controlling their activity.
  • the hecate-bLH conjugate killed almost all DU145 cells, but had little effect on A+ LNCaP. This result is consistent with specific binding of LH to DU145 cells but not to LNCaP cells. LH specifically binds DU145 cells, but we have not been able to consistently measure specific binding of LH to the A + LNCaP cells.
  • the LNCaP cells pre-treated with FSH were more sensitive to the hecate-bLH conjugate than those that were not pre-treated.
  • Abnormal Cells This invention may be used wherever it is desirable to specifically inhibit abnormal (or normal) cells that are driven by or are dependent on specific ligand interactions. As another example, this invention may be used in treating autoimmune diseases for which the antigen or epitope responsible for the autoimmune disease is known.
  • autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, Addison's disease, Goodpasture's syndrome, autoimmune hemolytic anemia, Grave's disease, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, insulin-dependent diabetes mellitus, myasthenia gravis, myocardial infarction, aplastic anemia, pernicious anemia, poststreptococcal glomerulonephritis, spontaneous infertility, ankylosing spondylitis, scleroderma, and Sj ⁇ grens' syndrome.
  • autoimmune disease is characterized by lymphocytes with specific receptors for a self epitope that triggers their function -- i.e., antibody secretion, proliferation, secretion of cytotoxic factors, or secretion of inflammatory cytokines. These responses cause damage or destruction to self cells or organs.
  • the specific antigens and even epitopes that act as ligands to stimulate the lymphocytes have been identified for several autoimmune diseases, typically by the in vitro proliferative response they induce in lymphocytes.
  • thyrotropin has been implicated as the self- antigen recognized by lymphocytes in Hashimoto's Disease.
  • the autoimmune disease may be treated by administering a compound containing that epitope linked to a lytic peptide, which will selectively delete clones of the autoreactive lymphocytes.
  • Certain abnormal cells display surface receptors that are not found on normal cells. In some cases, these receptors are encoded by viral nucleic acids.
  • Ligands for these receptors such as monoclonal antibodies to those receptors, or the receptor/ligand pairs shown in Table 2 of D. Fitzgerald et al. , "Targeted Toxin Therapy for the Treatment of Cancer," J. Natl. Cancer Inst. , vol. 81, pp.
  • ligand/lytic peptide combination of the present invention may be used in the ligand/lytic peptide combination of the present invention to selectively destroy cells displaying the receptor. Destruction of such a virally-infected cell, for example, before completion of the viral maturation cycle results in the release of incomplete, non-infectious viral particles, thereby treating the viral infection. Destruction of such a cancer cell prevents further metastasis.
  • an antibody is used as the ligand, it will often be preferable to administer the antibody and the lytic peptide sequentially, rather than linked to one another. Complement and other responses to the bound antibodies make the cells more susceptible to attack by the lytic peptides.
  • Lytic Peptides Useful in the Present Invention It is believed (without wishing to be bound by this theory) that lytic peptides act by disrupting cell membranes. "Resting" eukaryotic cells protect themselves through their ability to repair the resulting membrane damage. By contrast, activated cells (e.g., cells stimulated by GnRH) are unable (or less able) to repair damaged membranes. Because GnRH-activated pituitary cells have a diminished capacity to repair membranes, they are preferentially destroyed by lytic peptides, while adjacent non-activated cells repair their membranes and survive.
  • Lytic peptides are small, basic peptides. Native lytic peptides appear to be major components of the antimicrobial defense systems of a number of animal species, including those of insects, amphibians, and mammals. They typically comprise 23-39 amino acids, although they can be smaller. They have the potential for forming amphipathic alpha-helices. See Boman et al. , "Humoral immunity in Cecropia pupae," Curr. Top. Microbiol. Immunol, vol. 94/95, pp. 75-91 (1981); Boman et al, “Cell-free immunity in insects," Annu. Rev. Microbiol , vol. 41, pp. 103-126 (1987); Zasloff, "Magainins, a class of antimicrobial peptides from
  • Xenopus skin isolation, characterization of two active forms, and partial DNA sequence of a precursor
  • Known amino acid sequences for lytic peptides may be modified to create new peptides that would also be expected to have lytic activity by substitutions of amino acid residues that preserve the amphipathic nature of the peptides (e.g., replacing a polar residue with another polar residue, or a non-polar residue with another non-polar residue, etc.); by substitutions that preserve the charge distribution (e.g., replacing an acidic residue with another acidic residue, or a basic residue with another basic residue, etc.); or by lengthening or shortening the amino acid sequence while preserving its amphipathic character or its charge distribution.
  • substitutions of amino acid residues that preserve the amphipathic nature of the peptides e.g., replacing a polar residue with another polar residue, or a non-polar residue with another non-polar residue, etc.
  • substitutions that preserve the charge distribution e.g., replacing an acidic residue with another acidic residue, or a basic residue with another basic residue, etc.
  • Lytic peptides and their sequences are disclosed in Yamada et al., "Production of recombinant sarcotoxin IA in Bombyx mori cells," Biochem. J. , vol. 272, pp. 633-666 (1990); Taniai et al, “Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori,” Biochimica Et Biophysica Acta, vol. 1132, pp. 203-206 (1992); Boman et al, "Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids," Febs Letters, vol. 259, pp.
  • Families of naturally-occurring lytic peptides include the cecropins, the defensins, the sarcotoxins, the melittins, and the magainins.
  • Boman and coworkers in Sweden performed the original work on the humoral defense system of Hyalophora cecropia, the giant silk moth, to protect itself from bacterial infection. See Hultmark et al, "Insect immunity. Purification of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia," Eur. J. Biochem. , vol. 106, pp. 7-16 (1980); and Hultmark et al, "Insect immunity.
  • cecropins capable of disrupting bacterial cell membranes, resulting in lysis and cell death.
  • cecropins those known collectively as cecropins.
  • the principal cecropins -- cecropin A, cecropin B, and cecropin D - are small, highly homologous, basic peptides.
  • Boman' s group showed that the amino-terminal half of the various cecropins contains a sequence that will form an amphipathic alpha-helix. Andrequ et al, "N-terminal analogues of cecropin A: synthesis, antibacterial activity, and conformational properties," Biochem. , vol. 24, pp. 1683-1688 (1985).
  • the carboxy-terminal half of the peptide comprises a hydrophobic tail. See also Boman et al, "Cell-free immunity in Cecropia,” Eur. J. Biochem., vol. 201, pp. 23-31 (1991).
  • a cecropin-like peptide has been isolated from porcine intestine.
  • Lee et al "Antibacterial peptides from pig intestine: isolation of a mammalian cecropin,” Proc. Natl. Acad. Sci. USA, vol. 86, pp. 9159-9162 (1989).
  • Cecropin peptides have been observed to kill a number of animal pathogens other than bacteria. See Jaynes et al, "In Vitro Cytocidal Effect of Novel Lytic Peptides on Plasmodium falciparum and Trypanosoma cruzi," FASEB, 2878-2883 (1988); Arrowood et al , "Hemolytic properties of lytic peptides active against the sporozoites of Cryptosporidium parvum," J. Protozool , vol. 38, No. 6, pp. 161S-163S (1991); and Arrowood et al, "In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum," Antimicrob. Agents Chemother.
  • Defensins originally found in mammals, are small peptides containing six to eight cysteine residues. Ganz et al, "Defensins natural peptide antibiotics of human neutrophils," J. Clin. Invest. , vol. 76, pp. 1427-1435 (1985). Extracts from normal human neutrophils contain three defensin peptides: human neutrophil peptides HNP-1, HNP-2, and HNP-3. Defensin peptides have also been described in insects and higher plants.
  • sarcotoxins Slightly larger peptides called sarcotoxins have been purified from the fleshfly Sarcophaga peregrina.
  • Okada et al "Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae," J. Biol. Chem. , vol. 260, pp. 7174-7177 (1985).
  • the sarcotoxins presumably have a similar antibiotic function.
  • Other lytic peptides have been found in amphibians.
  • Zasloff showed that the Xenopus-de ⁇ ved peptides have antimicrobial activity, and renamed them magainins. Zasloff, "Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial DNA sequence of a precursor," Proc. Natl. Acad. Sci. USA, vol. 84, pp. 3628-3632 (1987).
  • Cecropins have been shown to target pathogens or compromised cells selectively, without affecting normal host cells.
  • the synthetic lytic peptide known as S-1 (or Shiva 1) has been shown to destroy intracellular Brucella abortus-, Trypanosoma cruzi-, Cryptosporidium parvum-, and infectious bovine herpes virus I (IBR)-infected host cells, with little or no toxic effects on noninfected mammalian cells.
  • S-1 or Shiva 1
  • IBR infectious bovine herpes virus I
  • Lytic peptides such as are known generally in the art may be used in practicing the present inventions.
  • Selective toxicity to ligand-activated cells is desirable, especially when the ligand and peptide are administered separately.
  • Selective toxicity is less important when the ligand and peptide are linked to one another, because in that case the peptide is effectively concentrated in the immediate vicinity of cells having receptors for the ligand.
  • Examples of such peptides are those designated D1A21 (SEQ. ID NO. 5), D2A21 (SEQ. ID NO. 6), D5C (SEQ. ID NO. 7), and D5C1 (SEQ. ID NO. 8).
  • D1A21 SEQ. ID NO. 5
  • D2A21 SEQ. ID NO. 6
  • D5C SEQ. ID NO. 7
  • D5C1 SEQ. ID NO. 8
  • in vitro LD 50 values against human prostate cancer cell lines have ranged from about 0.57 ⁇ M to about 1.61 ⁇ M.
  • LD 50 values against human breast, bladder, colon, cervix, lung, colon, and skin cancer cell lines have ranged from about 0.28 ⁇ M to about 3.1 ⁇ M.
  • LD 50 has been measured to be greater than 100 ⁇ M for each of D2A21, D5C, and D5C1 for each of d e following types of normal, non-cancerous human cells: endothelial cells, fibroblasts, enteric cells, and keratinocytes.
  • D2A21 LD 50 has been measured to be about 100 ⁇ M for human peripheral blood monocytes, and to be greater than 100 ⁇ M for human peripheral blood T-cells.
  • GnRH analogs may be conjugated with a lytic peptide in accordance with this invention.
  • analogs that may be used as part of such a conjugate is 1-LHRH-III (or 1-
  • GnRH-III SEQ. ID NO. 16. This peptide has been reported to suppress growth of several cancer cells. See I. Mez ⁇ et al, "Synthesis of Gonadotropin-Releasing Hormone III Analogs.
  • 1-LHRH-III selectively causes the release of FSH. See W. Yu et al., "A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat," Proc. Natl. Acad. Sci USA, vol. 94, pp. 9499-9503 (1997); and United States patent application S.N. 08/869,153, filed June 4, 1997. Lytic peptide conjugates of 1-LHRH-IH will be useful as contraceptives, and in the treatment of cancers such as prostate cancers. Agonists of 1-LHRH-III, such as are disclosed in United States patent application S.N. 08/869,153, may be used as well. 5.
  • an “effective amount” of a composition is an amount sufficient to selectively kill the targeted cells in a background population of non-targeted cells. Where appropriate in context, an “effective amount” of a composition is also an amount that is 0 sufficient to induce long-term contraception or sterility in an animal. Where appropriate in context, an “effective amount” of GnRH or 1-LHRH-III is an amount sufficient to temporarily restore fertility in an animal that has been made sterile by destruction of gonadotropic cells. As used in the Claims, the term "animal” is intended to include both human and non-human metazoans. 5
  • Ala Arg Lys lie Ala Arg Leu Gly Val Ala Phe 20 25

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EP1273309A1 (de) * 2001-07-05 2003-01-08 Georg-August Universität Göttingen Gentherapeutisches Verfahren zur Behandlung von GnRH-Rezeptor-positiven Karzinomen durch GnRH induzierte tumorzellspezifische Aktivierung eines therapeutischen Gens, zugehörige Nukleinsäurekonstrukte und Vektoren
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DE69837915T2 (de) 2008-02-21
JP2000514836A (ja) 2000-11-07
CA2283630A1 (en) 1998-10-01
EP0988048A4 (de) 2003-05-28
ES2289775T3 (es) 2008-02-01
ATE364392T1 (de) 2007-07-15
WO1998042365A1 (en) 1998-10-01
AU6587998A (en) 1998-10-20
EP0975354A4 (de) 2003-07-16
EP0975354B1 (de) 2007-06-13
EP0975354A1 (de) 2000-02-02
CA2283630C (en) 2011-08-30
WO1998042364A1 (en) 1998-10-01

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