WO1999011282A9 - Compositions and methods for contraception in or sterilization of mammals - Google Patents

Abstract

Amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to induce sterility or long-term contraception in mammals. The peptides act directly on cell membranes, and need not be internalized. 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 mammals in vivo. The compounds are relatively small, and are not antigenic. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes). 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.

Description

COMPOSITIONS AND METHODS FOR CONTRACEPTION IN OR STERILIZATION OF MAMMALS

The benefit of the September 3, 1997 filing date of provisional application 60/057,456 is claimed under 35 U.S.C. § 119(e) in the United States, and is claimed under applicable treaties and conventions outside the United States.

TECHNICAL FIELD

This invention pertains to compositions and methods for long-term contraception or sterilization of mammals.

BACKGROUND ART

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.

P. Olson et al., "Endocrine Regulation of the Corpus Luteum of the Bitch as a Potential Target for Altering Fertility," J. Reprod. Fert. Suppl , vol. 39, pp. 27-40 (1989) discusses the luteal phase and its regulation in bitches. The following discussion appears at page 37: "Specific toxins can be linked to an antibody or hormone and carried to a specific target cell (or cells) which is then killed by the toxin. The idea of developing a 'magic bullet' has been discussed for decades but is now gaining renewed recognition as a potential, highly selective method for destroying specific tissues while leaving other tissues unharmed. For many years it was impossible to develop large quantities of antibodies which would react specifically with only single antigenic determinants. However, with the advent of monoclonal antibodies, this problem has been largely overcome. Antibodies can be developed to specific hormone receptors (such as die 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) P. Olson et al., "New Developments in Small Animal Population Control, " JAVMA, vol. 202, pp. 904-909 (1993) gives an overview of methods for preventing or terminating unwanted pregnancies in small animals. The following discussion appears at page 905: "Tissue-specific cytotoxins- 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 gonadotropin-secreting pituitary cells. Similarly, 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." (citation omitted).

T. Janaky et al., "Short Chain Analogs of Luteinizing Hormone-Releasing Hormone Containing Cytotoxic Moieties," Proc. Natl. Acad. Sci. USA, vol. 89, pp. 10203-10207

(1992) discloses the use of certain hexapeptide and heptapeptide analogs of GnRH as carriers for certain alkylating nitrogen mustards, certain anthraquinone derivatives, antimetabolite, and cisplatin-like platinum complex.

S. Sealfon et al., "Molecular mechanisms of ligand interaction with the gonadotropin- releasing hormone receptor," Endocrine Reviews, vol. 18, pp. 180-205 (1997) provides a review of research concerning the interaction between GnRH and its receptor.

D. Morbeck et al., "A Receptor Binding Site Identified in the Region 81-95 of the jS-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin (hCG),"

Molecular and Cellular Endocrinology, vol. 97, pp. 173-181 (1993) disclosed a fifteen amino acid region of LH and hCG that acted as a receptor binding site. (LH and hCG are homologous hormones that produce similar effects.)

S. Cho et al., "Evidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells," Mol. Brain Res., vol. 50, pp. 51-58 (1997) discloses that neuroendocrine populations of GnRH neurons have high affinity receptors for GnRH and for GnRH analogs.

N. Mores et al., "Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release," Endocrinology, vol. 137, pp. 5731-5734 (1996) discloses mat LH acts directly on neuroendocrine neurons in the brain. See also Z. Lei et al., "Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons," Mol. & Cell. Endocήnology, vol. 109, pp. 151-157 (1995).

Conventional targeted toxin therapies have several drawbacks. There is a small window for treatment with a particular targeted toxin (on the order of two weeks) before the recipient's immune system mounts an antibody response to the targeted toxin. These antibodies will neutralize the toxin; or worse, may result in the deposition of the toxin in reticuloendothelial tissues (e.g., liver, spleen, lymph nodes, lungs, bone marrow), where they may damage otherwise healthy tissue. Aside from this drawback, the toxin must be internalized by the targeted cell and translocated into the cytoplasm to have effect.

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. 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. The inventors noted that 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. That is to say that applicants have found that it is only when a GnRH analog of the type described herein is linked to a toxin of the type noted above does that toxin become capable of binding to cell membranes . . . ." (E.g., Pat. No. 5,488,036, col. 7, lines 46-52.) 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, α-amanitin, diphtiieria 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 die in vivo experiments reported, d e most effective time course was reported to be weekly injections for 4 weeks. (E.g., Pat. 5,488,036, col. 20, lines 46-47.) Because most of die conjugates cited are relatively large compounds, antigenicity could be a problem when such multiple administrations are used. 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 d e 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 widi ribosomes, mitochondria, or other cellular components. DISCLOSURE OF INVENTION

It has been unexpectedly discovered that amphipathic lytic peptides are ideally suited to use in a ligand/cytotoxin combination to specifically induce sterility or long-term contraception in mammals. The peptides act directly on cell membranes, and need not be internalized. 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 mammals in vivo. Particularly surprising, sterility results even when the combination is administered to a sexually immature animal: The combination then prevents sexual maturation. The compounds used in die 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, although they may also be given in two or more closely spaced doses. Lysis of gonadotropes has been observed to be very rapid (on the order of ten minutes.) The two components - the ligand and die lytic peptide — may optionally be administered as a fusion peptide, or they may be administered separately, wim the ligand administered slightly before the lytic peptide, to activate cells widi receptors for the ligand, and thereby make those cells susceptible to lysis by the lytic peptide. If a fusion peptide is used, it has been unexpectedly discovered that a linking moiety is not necessary to join the ligand to die lytic peptide: one may be bonded directly to the other, without the need for any intervening linkage; bonding is preferably performed by bonding one end of the ligand to one end of die peptide, not by bonding to die middle of either. The toxin, the lytic peptide, 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.

MODES FOR CARRYING OUT THE INVENTION

It is known that the D-amino acid form of GnRH will bind to gonadotropes in the pituitary and to GnRH neurons in the brain. It is also known that the D-amino acid forms of lytic peptides have essentially die same propensity to lyse cell membranes as do die L-amino acid forms. Compounds of the present invention (whedier administered as a fusion peptide or separately) may therefore be administered eidier in L-form or D-form. D-form peptides, al iough generally more expensive than L-form, have the advantage diat they are not degraded by normal enzymatic processes, so that ie 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 die peptide/hormone fusion product to a suitable carrier to facilitate uptake by the intestine, for example vitamin B₁₂, following generally the B_]2-conjugation technique of G. Russell-Jones et al., "Synthesis of LHRH Antagonists Suitable for Oral Administration via the Vitamin B_n Uptake System," Bioconjugate Chem., vol. 6, pp. 34-42 (1995).

GnRH or GnRH analogs (collectively, "GnRH agonists") may be used in the present invention. It has been reported diat substitutions at die 6 and 10 positions of the GnRH decapeptide can produce "superagonists" having greater binding affinity to die GnRH receptor than does GnRH itself. These "superagonists" include goserelin, leuprolide, buserelin, and nafarelin. See U.S. Patent 5,488,036.

Without wishing to be bound by this theory, it is believed diat die mechanism underlying the invention is as follows: 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) tiiese activated cells. When die gonadotrophic cells in the pituitary are destroyed and are deprived of GnRH from the brain, die pituitary no longer secretes follicle stimulating hormone (FSH) or luteinizing hormone (LH), rendering the mammal temporarily or permanently sterile.

Although die ligand and die lytic peptide may be administered separately, it is preferred to link die two in a single molecule, because such a linkage greatly increases the effective concentration of the lytic peptide in die vicinity of ligand-activated cells.

Furthermore, mis increase in the effective lytic peptide concentration can obviate the need for activation of die cells, allowing the peptide to be linked to a binding site of a ligand alone, witiiout needing to include the "remainder" of a native ligand diat 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 GnRH/hecate (SEQ. ID NO. 3) and hecate/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. (We have found that die initial pyro-glutamic acid residue of the GnRH or the GnRH portion of a fusion peptide may be substituted widi glutamine without substantially changing die activity of the respective peptides. See, e.g., SEQ. ID Nos. 9, 3, and 4.) Experimental Results

Examples 1-6

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 die histology of each was examined microscopically.

Treatment 1 applied tissue culture medium alone as a control. The histology of mis 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 μM 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 15 minutes. Following this incubation, die medium containing GnRH was removed, and the tissue was washed once with plain medium. This medium was men removed, and was replaced widi medium containing 50 μM of the lytic peptide hecate. Widespread basophilic

(gonadotropic) cellular destruction was observed after this treatment.

Treatment 5 was an application of 50 μM 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 die fusion peptide GnRH/hecate two hours later. After treatment the tissue was virtually destroyed, with only stromal cells remaining.

Example 7 Two sexually immature female rats from the same litter (age 33 days) were given two intravenous injections of saline control solution 24 hours apart. After the rats reached breeding age, they were examined 105 days post-inoculation. The external genitalia appeared normal. During a fourteen-day monitoring period 107 days to 121 days post-inoculation, each of die control rats completed two estrous cycles. The rats were then sacrificed and necropsied. The reproductive organs appeared histologically normal. 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 μg GnRH/hecate fusion peptide in saline 24 hours apart. After the rats reached breeding age, diey 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 unned, inactive uterine and oviductal epidielia. Their ovaries contained no large follicles, and had a high number of atretic follicles (i.e., those diat had failed to ovulate).

Examples 9-14

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 tiiat 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 die pituitaries from the saline control group. Histology revealed a 60% to 70% reduction in die number of pituitary basophilic cells compared to die controls.

Treatment 11 was injection of 100 μL of a 1.35 mM solution of GnRH (162 μg) in saline, followed 15 minutes later by injection with 100 μL of a 1.35 mM solution of hecate (337 μg) 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 diat were present appeared old. Follicles were large, and had not ruptured. Uterine morphology was consistent with hormonal inactivity. The pituitaries and die pituitary histology were similar to those observed in Treatment 10. Treatment 12 was injection of 100 μL of a 1.35 mM solution of hecate (337 μg) 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 μL of a 1.35 mM solution of GnRH (162 μg) 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 die 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 diat were present appeared old. Follicles were large, and had not ruptured. Uterine morphology was consistent with hormonal inactivity. The pituitaries and die pituitary histology were similar to those observed in Treatment 10.

These experiments demonstrate that GnRH and the lytic peptide may be administered either separately or as a fusion peptide, altiiough the fusion peptide is preferred as it is expected to be more active at lower doses.

Aldiough experiments to determine optimum dosages had not been performed by the time mis application is being filed, a person of ordinary skill in the art, who is given the teachings of die present specification, may readily ascertain optimum dosages through routine testing.

Although the experiments to date have been performed on female mammals, similar results are expected for male mammals, because GnRH signals pituitary cells to release gonadotropins in botii males and females.

Tissue and cell specificity of cytotoxic conjugates could be further enhanced by using various hormones or hormone analogs coupled to a lytic peptide. Some examples follow. For fertility control, both die pituitary and the central GnRH neuronal component of the reproductive axis are selectively damaged by GnRH-hecate conjugate. Few cells in the central nervous system should be damaged by this treatment, because the chicken π GnRH and lamprey III 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 die gonads. (Odier lytic peptides may be used in place of hecate in these conjugates.)

The compositions of the present invention may be administered as described, or as pharmaceutically acceptable salts. The compositions may be administered intravenously, subcutaneously, intramuscularly, cr (especially when in D-amino acid form and complexed widi a carrier such as vitamin B_n) orally.

Applications of the present invention include long-term contraception or sterilization in humans; and long-term contraception or sterilization in domesticated or wild mammals.

Domesticated mammals in which this invention may be used include, for example, dogs, cats, cattle, horses, pigs, and sheep. When used in humans, 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.

As one example, mis invention may be used in die humane population control of an unwanted introduced species.

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 eidier 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 diat die pituitary glands were stained widi PAS (periodic acid-Schiff) stain with no counter-stain. The treatments were selected so diat each animal received an equimolar amount of the compound widi which it was treated.

Treatments 15 and 16 were IV-injection widi 674 μg of D-hecate in 200 μL saline (1.35 mM). 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 die 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 μg of GnRH in 200 μL saline

(1.35 mM). The rat in treatment 17 was sacrificed 48 hours after injection, and die 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 μL saline (2.7 mM). The rats in treatments 19 and 20 were sacrificed 48 hours after injection, and die rats in treatments 21 and 22 were sacrificed 96 hours after injection. No gross lesions were noted in die organs of any of the four animals, other than die pituitary. The pimitary 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. The pituitary glands of all four rats showed a marked depletion of PAS-positive cells; it was estimated mat die depletion was 80 to 90% compared to tiiose of control groups. (PAS stain preferentially stains glycopeptides.

LH, FSH, and MSH are glycopeptide hormones; cells containing these hormones stored in their secretory vacuoles stain positive widi PAS.)

It was dius seen diat die GnRH-lytic peptide combination caused morphological and functional alterations in the adult female rat reproductive system, and in preventing sexual maturity in pre-pubertal female rats, but that the fusion peptide selectively eliminated a specific population of PAS-positive staining cells in die pituitary.

Example 23

Subsequent experiments were conducted on rats using treatments generally similar to treatments 9-14 above. Observations made with immunohistochemical staining found that die effective treatments selectively killed (1) gonadotropes in the pituitary, and (2) neurons in the brain bearing GnRH receptors. The selective killing of these cells was seen after the GnRH- hecate fusion peptide was administered; and after the administration of GnRH alone, followed 10 minutes later by the administration of hecate alone. In these cases, it was also observed diat pituitary cells no longer secreted either LH or FSH following die effective treatments.

Example 24

Hecate is an amphipafhic lytic peptide diat 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.

We also syn iesized D-Lys⁶GnRH (SEQ. ID NO. 13), so that hecate could be conjugated to the D-Lys⁶, a position that could minimize interference wi i binding of the GnRH domain to die GnRH receptor. These synthetic peptides specifically displaced radiolabelled monoiodinated-GnRII from rat pituitary membranes. Displacement by D- Lys⁶GnRH-hecate was comparable to (and actually slightly greater than) displacement by native mammalian GnRH, as measured by cpm of radioactivity. When GnRH and GnRH- hecate binding were compared on a molar basis over a 1000-fold concentration range (n = 6) the GnRH-hecate specifically displaced die radiolabelled peptide to an extent equal to

123 % ± 4% of the binding exhibited by equimolar concentrations of GnRH; equimolar concentrations of D-Lys⁶GnRH displaced 187% ± 8% of the cpm displaced by native GnRH.

Examples 25-32 We studied in vitro lysis of bovine luteal cells with GnRH-hecate conjugate and wid hecate-bLH conjugate (SEQ. ID NO. 12). (The bLH component of the conjugate is a 15-mer fragment of the beta chain of luteinizing hormone, SEQ. ID NO. 11). Small luteal cells were collected from cattle corpora lutea post-slaughter. Small luteal cells are rich in LH receptors, and were found to be highly susceptible to lysis by die hecate-bLH conjugate. Small luteal cells in culture were incubated with one of the following treatments for 22 hours, and were then examined for viability using Trypan Blue exclusion and release of lactic dehydrogenase.

Treatment 25 control: no additional treatment (media alone) Treatment 26 10 ng bLH (positive control) Treatment 27 hecate-bLH, 10 μM

Treatment 28 hecate-bLH, 5 μM Treatment 29 hecate-bLH, 1 μM Treatment 30 hecate (alone), 10 μM Treatment 31 hecate (alone), 5 μM Treatment 32 hecate (alone), 1 μM

Significant killing of small luteal cells was observed following 22 hr. incubation with

10 μM hecate alone, and with 5 μM hecate alone (approximately 50% killing). Cell death for

1 μM hecate alone did not differ significantly from negative control (media) or from bLH alone. All three treatment doses with hecate-bLH caused significant increases in cell deadi as compared to treatment with hecate alone. The hecate-bLH conjugate killed approximately twice the number of cells as were killed by hecate alone at die same concentrations.

Observed levels of lactic dehydrogenase activity also demonstrated diat the hecate-bLH treatment killed a significantly greater number of cells than did hecate alone.

Examples 33-34

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. (Granulosa cells are hormonally active cells with numerous LH receptors.) Our experiments with granulosa cells were otherwise generally similar to those described above for

Examples 25-32. These experiments demonstrated (1) that the granulosa cells were much more susceptible to killing by hecate alone man were the small luteal cells, and (2) that, as had been die case widi die small luteal cells, the granulosa cells were significantly more susceptible to hecate-bLH at even the lowest concentration (1 μM) than they were to hecate alone. At 1 μM, the hecate-bLH conjugate killed about twice die number of target cells as did hecate alone. Again, the levels of lactic dehydrogenase released following the hecate-bLH 1 μM treatment were significantly higher than die levels of enzyme released following treatment with 1 μM hecate alone.

Additional studies (data not shown) demonstrated that a 15-mer fragment of the bLH subunit specifically bound to LH receptors on the target granulosa cells, but did not initiate the production of steroid hormones that would be indicative of a stimulus-coupled response. We thus demonstrated at the selective killing of target cells resulted from the physical proximity of the lytic peptide to die cell, which was caused by binding of the LH subunit. Stimulation of target cell hormone production was not required for cell lysis. This result was surprising, as we had previously expected diat activation of the target cells was required for increased susceptibility to lysis. These data demonstrate mat such activation is not required. These data are, however, consistent widi our other data showing that cell activation is also a route that can lead to increased susceptibility to the lytic peptide.

Examples 35-38

Anodier set of experiments was performed to study die in vivo effects of the GnRH- hecate conjugate on female rats and rabbits. The ovaries, uterus, oviducts, adrenals, spleen, thyroids, pancreas, liver, lungs, and heart were processed for histological analysis. 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). The immunocytochemical staining procedures we used followed generally die procedures of M. Rahmanian et al., "Histological and immunocytochemical characterization of pimitary cell types in ponies," Proc. 13th Soc. Equine Nutrition & Phys. Symp. , pp. 348-349 (1993); M. Rahmanian et al., "Immunocytochemical localization of luteinizing hormone and follicle- stimulating hormone in the equine pituitary," J. Anim. Sci. , vol. 76, pp. 839-846 (1998); M. Rahmanian et al., "Immunocytochemical localization of prolactin and growth hormone in the equine pituitary, " Animal Sci. , vol. 75, pp. 3010-3018 (1997); and P. Melrose et al,

"Comparative topography of the immunoreactive alpha-melanocyte-stimulating hormone neuronal system in the brains of horses and rats," Brain Beh. & Evol. , vol. 32, pp. 226-235 (1988).

Brains were serially sectioned on a Vibrotome from the level of the diagonal band of Broca to the mammillary body. Alternate sections were consecutively divided into four to five dishes, and sections in alternate dishes were stained widi cresyl violet, or were stained immunocytochemically for GnRH or the GnRH precursor, VP, OXY, or tyrosine hydroxylase

(die rate-limiting enzyme in catecholamine synthesis). In addition to die staining procedures cited above, we also used the immunocytochemical staining procedures of P. Melrose et al., "Distribution and morphology of immunoreactive gonadotropin-releasing hormone (GnRH) neurons in the basal forebrain of ponies," J. Comp. Neurol. vol. 339, pp. 269-287 (1994); and P. Melrose et al., "Topography of oxytocin and vasopressin neurons in the forebrain of

Equus caballus: Further support of proposed evolutionary relationships for proopiomelanocortin, oxytocin and vasopressin neurons," Brain, Beh. & Evol. , vol. 33, pp. 193-204 (1989).

Thirty-three-day-old, sexually immature female rats were given intravenous administrations as follow:

Treatment 35: 0.03 μg GnRH (a normal physiological dose) (two rats)

Treatment 36: 1.62 μg GnRH (the molar equivalent to the amount of GnRH in Treatment 37) (one rat)

Treatment 37: 0.5 mg GnRH-hecate (one rat)

Treatment 38: 0.03 μg GnRH, followed 11 minutes later by 0.337 μg hecate

(two rats).

Animals were sacrificed 14 days after treatment. As compared to the two GnRH control groups, the treatment with GnRH-hecate and the treatment with GnRH followed by hecate alone reduced pituitary weights by 13 % and 14%, respectively, and reduced die numbers of bLH-specific gonadotropes by 92% and 87% , respectively. Further, following these two experimental treatments the cell bodies of GnRH-stained neurons in hypophysiotropic areas of die brain were frequently deformed; and a substantial amount of immunoreactive material leached into surrounding areas where numerous cell bodies are concentrated (the organum vasculosum of the lamina terminalis). There was histological damage to cells from the two experimental treatments in the Cl and C3 fields of die hippocampus, and increased staining of parvicellular VP neurons in the paraventricular nucleus. (The VP staining may have been caused by formation of a precipitate in certain areas of the brain. Subsequent studies widi more highly purified peptide did not show a precipitate). The change in 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 me number of alpha-MSH-stained cells in this subdivision of the pimitary. No pathological changes were noted in any o er 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 39-43

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 39 0.0 mg GnRH-hecate

Treatment 40 0.1 mg GnRH-hecate Treatment 41 0.5 mg GnRH-hecate

Treatment 42 1.0 mg GnRH-hecate Treatment 43 1.5 mg GnRH-hecate.

Animals were sacrificed at 24 hours or at 14 days after treatment. Results were similar to those reported above for Examples 35-38, except that no precipitate was found in the brain, and VP staining in the CNS was not altered. The treatments with higher levels of

GnRH-hecate produced a large number of GnRH -receptor-containing neurons having abnormal morphologies, including distortion of the somatic portion of die cells, and degeneration of neurites. In the rats sacrificed fourteen days after treatment, 66% and 87% of the GnRH- receptor-containing neurons were abnormal in the rats that had received 1.0 and 1.5 mg of GnRH-hecate, respectively. Axonal degeneration in the 1.5 mg GnRH-hecate group was accompanied by over 90% reduction in median eminence staining for GnRH.

Examples 44-46 Seven sexually mature female New Zealand rabbits were injected intravenously with saline containing GnRH-hecate as follows:

Treatment 44: 0 mg GnRH-hecate (n = 3) Treatment 45: 5 mg GnRH-hecate (n = 3) Treatment 46: 10 mg GnRH-hecate (n = 1). Forty-six days later all rabbits were injected intramuscularly with 100 μg GnRH.

Blood samples were collected at 0, 1, 4, and 24 hours, and LH and FSH levels in the blood samples were measured by radioimmunoassay. Hormone analyses revealed diat both control and experimental animals released LH in response to ie GnRH, suggesting that diere may be at least some degree of reversibility following treatment, at least for pituitary gonadotropes at lower doses of ligand/peptide. The rabbits were sacrificed die next day (day 47) for postmortem histological analysis. We found mat the numbers of tertiary follicles, corpora lutea, and GnRH-induced ovulations were reduced by GnRH-hecate treatment. Ovarian and pimitary weights were reduced by the 10 mg GnRH-hecate treatment. In tissues from the GnRH-hecate treatments, observed immunoreactive GnRH was faint and diffusely localized in CNS areas normally containing cell bodies; normal individual cell bodies were reduced in number by at least 50%; and die terminal fields, which normally contain the axons of GnRH receptor neurons, were not stained for GnRH. These observations suggest that the most pronounced effects of the GnRH-hecate treatments in these experiments on rabbits may have been on neuroendocrine neurons in the brain. The hippocampus and other areas of the brain containing high concentrations of GnRH were not discernibly affected by GnRH-hecate treatments. The GnRH-hecate treatment increased the number of PAS-stained pimitary 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.

Examples 47-48

Nine sexually mature female rabbits were injected intravenously with saline containing

0 mg (n = 4) (Treatment 47) or 10 mg GnRH-hecate (n = 5) (Treatment 48). Rabbits were injected intramuscularly with GnRH on day 6 posttreatment. Blood samples were collected for radioimmunoassay of LH and FSH as described above, and the animals were sacrificed on day 7 post-treatment. Both control and experimental animals released LH in response to the GnRH; however, the amount of LH released was lower in the treated animals than in die controls. The GnRH-hecate treatment reduced die numbers of tertiary ovarian follicles, and die numbers of GnRH-induced ovulations. No effects were noticed either on peripheral tissues or on pimitary weight. The effects of GnRH-hecate on CNS morphology and immunocytochemical results were similar to those described above in Examples 35-46.

Again, die effects were more pronounced on GnRH neurons than on staining of pimitary gonadotropes.

The number of ovulation sites in rabbits in Examples 47 and 48 treated with 10 mg GnRH-hecate were reduced as compared to saline controls. The mean number of ovulation sites in four saline controls equalled 12.2 + 5.4, with S.E.M. = 2.7. The mean number of ovulation sites in the five rabbits given 10 mg of GnRH-hecate was 3.6 ± 1.1, with S.E.M. = 0.5. This difference from control was significant (p = 0.025).

The "LH surge" (the level of LH at one hour post-GnRH challenge, minus the resting level before challenge) in the four controls was 61.2 + 16.5 ng/mL, with S.E.M. = 8.3; and in the treated group was 49.6 ± 26.1 ng/mL, with S.E.M. = 12 (p = 0.22). Thus there was a trend towards lower LH levels in the treated group.

The in vivo studies clearly demonstrated diat die GnRH-hecate conjugate selectively damaged GnRH receptor-bearing cells in me brain (neurons) and in the pimitary (gonadotrophic cells). Further, these studies demonstrated a significant alteration in the ovary, presumably a consequence of alteration in the reproductive centers of the brain- pituitary axis. Selectivity of the conjugate was demonstrated by die following observations: (1) No cytotoxic changes were seen in neurons that lacked GnRH receptors. (2) No changes were seen in pimitary cells that lacked GnRH receptors. (3) No changes were seen in other endocrine and non-endocrine tissues (except for the ovary, which presumably responded indirectly to the destruction of gonadotrophs in the pimitary).

Many of the events referred to as "ovulations" in the GnRH-hecate treated rabbits possibly were not functional ovulation sites, but may instead have represented hemorrhagic pre-ovulatory degenerative changes. Additional breeding trials will be conducted to verify that ovulation of functional ova is prevented.

Lytic Peptides Useful in the Present Invention

It is believed (widiout wishing to be bound by diis dieory) that lytic peptides act by disrupting cell membranes. "Resting" eukaryotic cells protect themselves through tiieir 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 pimitary 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.

Although the embodiments of this invention that have been tested to date have used hecate as the effector lytic peptide, this invention will work with a combination of GnRH with other lytic peptides as well. Many lytic peptides are known in the art and include, for example, those mentioned in the references cited in the following discussion.

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 tiiose of insects, amphibians, and mammals. They typically comprise 23-39 amino acids, although they can be smaller. Th y 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, " Proc. Nat!. Acad. Sci. USA, vol. 84, pp. 3628-3632 (1987); Ganz et al., "Defensins natural peptide antibiotics of human neutrophils," J. Clin. Invest. , vol. 76, pp. 1427-1435 (1985); and Lee et al., "Antibacterial peptides from pig intestine: isolation of a mammalian cecropin," Proc. Natl. Acad. Sci. USA, vol. 86, pp. 9159-9162 (1989).

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 widi anodier 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 me amino acid sequence while preserving its amphipathic character or its charge distribution. 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. 103-106 (1989); Tessier et al, "Enhanced secretion from insect cells of a foreign protein fused to die honeybee melittin signal peptide," Gene, vol. 98, pp. 177-183 (1991); Blondelle et al., "Hemolytic and antimicrobial activities of the twenty-four individual omission analogs of melittin," Biochemistry, vol. 30, pp. 4671-4678 (1991); Andreu et al., "Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity," Febs Letters, vol. 296, pp. 190-194 (1992); Macias et al.,

"Bactericidal activity of magainin 2: use of lipopolysaccharide mutants, " Can. J. MicrobioL , vol. 36, pp. 582-584 (1990); Rana et al., "Interactions between magainin-2 and Salmonella typhimurium outer membranes: effect of Lipopolysaccharide structure," Biochemistry, vol. 30, pp. 5858-5866 (1991); Diamond ei al., "Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene," Proc. Natl. Acad. Sci. USA, vol. 90, pp. 4596 ff

(1993); Selsted et al., "Purification, primary structures and antibacterial activities of β- defensins, a new family of antimicrobial peptides from bovine neutrophils," J. Biol. Chem. , vol. 268, pp. 6641 (1993); Tang et al., "Characterization of the disulfide motif in BNBD- 12, an antimicrobial -defensin peptide from bovine neutrophils," J. Biol. Chem. , vol. 268, pp. 6649 (1993); Lehrer et al., Blood, vol. 76, pp. 2169-2181 (1990); Ganz et al., Sem.

Resp. Infect. I. , pp. 107-117 (1986); Kagan et al, Proc. Natl. Acad. Sci. USA, vol. 87, pp. 210-214 (1990); Wade et al, Proc. Natl. Acad. Sci. USA, vol. 87, pp. 4761-4765 (1990); Romeo et al, J. Biol. Chem. , vol. 263, pp. 9573-9575 (1988); Jaynes et al, "Therapeutic Antimicrobial Polypeptides, Their Use and Methods for Preparation," WO 89/00199 (1989); Jaynes, "Lytic Peptides, Use for Growth, Infection and Cancer, " WO 90/12866 (1990);

Berkowitz, "Prophylaxis and Treatment of Adverse Oral Conditions with Biologically Active Peptides," WO 93/01723 (1993).

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. Isolation and structure of cecropin D. and four minor antibacterial components from cecropia pupae," Eur. J. Biochem. , vol. 127, pp. 207-217 (1982).

Infection in H. cecropia induces the syntiiesis of specialized proteins capable of disrupting bacterial cell membranes, resulting in lysis and cell death. Among diese specialized proteins are diose known collectively as cecropins. The principal cecropins - cecropin A, cecropin B, and cecropin D -- are small, highly homologous, basic peptides. In collaboration with Merrifield, Boman's group showed that the amino-terminal half of the various cecropins contains a sequence mat 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 otiier than bacteria. See Jaynes et al, "In Vitro Cytocidal Effect of Novel Lytic Peptides on

Plasmodium falciparum and Trypanosom 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 die sporozoites of Cryptosporidium parvum, " Antimicrob. Agents Chemother. , vol. 35, pp. 224-227 (1991). However, normal mammalian cells do not appear to be adversely affected by cecropins, even at high concentrations. See Jaynes et al, "In vitro effect of lytic peptides on normal and transformed mammalian cell lines, " Peptide Research, vol. 2, No. 2, pp. 1-5 (1989); and Reed et al, "Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented widi an amphipathic peptide," Mol. Reprod. DeveL , vol. 31, No. 2, pp. 106-113 (1992).

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. Dimarcq et al,

"Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranvae," EMBO J., vol. 9, pp. 2507-2515 (1990); Fisher et al, Proc. Natl. Acad. Sci. USA, vol. 84, pp. 3628-3632 (1987).

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 Sarcopfiaga peregrina (flesh fly) larvae," J. Biol. Chem. , vol. 260, pp. 7174-7177 (1985). Although highly divergent from the cecropins and defensins, me sarcotoxins presumably have a similar antibiotic function.

Other lytic peptides have been found in amphibians. Gibson and collaborators isolated two peptides from the African clawed frog, Xenopus laevis, peptides which tiαey named PGS and Gly¹⁰Lys² PGS. Gibson et al., "Novel peptide fragments originating from PGL_a and the caervlein and xenopsin precursors from Xenopus laevis," J. Biol. Chem. , vol. 261, pp. 5341- 5349 (1986); and Givannini et al, "Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones," Biochem. J., vol. 243, pp. 113-120 (1987). Zasloff showed diat the Xenopus-deήveά 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).

Synthesis of nonhomologous analogs of different classes of lytic peptides has been reported to reveal that a positively charged, amphipathic sequence containing at least 20 amino acids appeared to be a requirement for lytic activity in some classes of peptides. Shiba et al, "Structure-activity relationship of Lepidopteran, a self-defense peptide of Bombyx more," Tetrahedron, vol. 44, No. 3, pp.787-803 (1988). Other work has shown that smaller peptides can also be lytic. See McLaughlin et al, cited below. Cecropins have been shown to target pathogens or compromised cells selectively, without affecting normal host cells. The synthetic lytic peptide known as S-l (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, wi i little or no toxic effects on noninfected mammalian cells. See Jaynes et al, "In vitro effect of lytic peptides on normal and transformed mammalian cell lines," Peptide Research, vol. 2, No. 2, pp. 1-5

(1989); Wood et al, "Toxicity of a Novel Antimicrobial Agent to Cattle and Hamster cells In vitro," Proc. Ann. Amer. Soc. Anim. Sci., Utah State University, Logan, UT. J. Anim. Sci. (Suppl. 1), vol. 65, p. 380 (1987); 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); Arrowood et al, "In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum," Antimicrob. Agents Chemother. , vol. 35, pp. 224- 227 (1991); and Reed et al, "Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented widi an amphipathic peptide," Mol. Reprod. DeveL , vol. 31, No. 2, pp. 106-113 (1992). Morvan et al, "In vitro activity of the antimicrobial peptide magainin 1 against

Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis," Mol. Mar. Biol, vol. 3, pp. 327-333 (1994) reports the in vitro use of a magainin to selectively reduce the viability of the parasite Bonamia ostreae at doses that did not affect cells of the flat oyster Ostrea edulis. Also of interest are the syndietic peptides disclosed in the following pending patent applications, peptides diat have lytic activity with as few as 10-14 amino acid residues: McLaughlin et al, "Amphipathic Peptides," United States patent 5,789,542, issued August 4, 1998; and Mark L. McLaughlin et al, "Short Amphipathic Peptides widi Activity against Bacteria and Intracellular Pathogens," United States patent application serial number

08/796,123, filed February 6, 1997.

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 die 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 die 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). These peptides and other lytic peptides suitable for use in the present invention are disclosed in Jaynes, "Methods for the Design of Amphipathic Peptides Having Enhanced Biological Activities," United States provisional patent application serial number 60/027,628, filed October 4, 1996.

Miscellaneous

As used in the Claims, an "effective amount" of a composition is an amount that is sufficient to induce long-term contraception or sterility in a mammal. Where appropriate in context, an "effective amount" of GnRH is an amount sufficient to temporarily restore fertility in a mammal that has been made sterile by destruction of gonadotropic cells. As used in die

Claims, the term "mammal" is intended to include both human and non-human mammals.

The complete disclosures of all references cited in this specification are hereby incorporated by reference; as are the full disclosures of United States provisional application 60/041,009, filed March 27, 1997; United States provisional application 60/057,456, filed September 3, 1997; United States non-provisional application 08/869,153, filed June 4, 1997; and international application PCT/US98/06114, filed March 27, 1998. In the event of an otherwise irreconcilable conflict, however, the present specification shall control. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Enright, Frederick M. Jaynes, Jesse M. Hansel, William

Elzer, Philip H. Melrose, Patricia A.

(ii) TITLE OF INVENTION: Compositions and Methods for Contraception in or Sterilization of Mammals

(iii) NUMBER OF SEQUENCES: 13, 1 of which is deliberately blank

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: John H. Runnels

(B) STREET: P. O. Box 2471

(C) CITY: Baton Rouge

(D) STATE: LA

(E) COUNTRY: USA (F) ZIP: 70821-2471

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS -DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 01-SEP-1998

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Runnels, John H. (B) REGISTRATION NUMBER: 33,451

(C) REFERENCE/DOCKET NUMBER: 96A3.1-PCT

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (225) 387-3221 (B) TELEFAX: (225) 346-8049

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide (B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "Xaa in position 1 denotes pyro-glutamic acid. This sequence is GnRH."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..23 (D) OTHER INFORMATION: /note= "This sequence is hecate."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Ala Leu Lys Lys Leu Lys 1 5 10 15

Lys Ala Leu Lys Lys Ala Leu 20 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..3.3

(D) OTHER INFORMATION: /note= "This sequence is a modified GnRH/hecate fusion peptide."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gin His Trp Ser Tyr Gly Leu Arg Pro Gly Phe Ala Leu Ala Leu Lys 1 5 10 15

Ala Leu Lys Lys Ala Leu Lys Lys Leu Lys Lys Ala Leu Lys Lys Ala 20 25 30 Leu

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide (B) LOCATION: 1..33

(D) OTHER INFORMATION: /note= "This sequence is a hecate/mo ified GnRH fusion peptide." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Ala Leu Lys Lys Leu Lys 1 5 10 15

Lys Ala Leu Lys Lys Ala Leu Gin His Trp Ser Tyr Gly Leu Arg Pro 20 25 30

Gly

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..23 (D) OTHER INFORMATION: /note= "This sequence is D1A21."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Phe Ala Phe Ala Phe Lys Ala Phe Lys Lys Ala Phe Lys Lys Phe Lys

1 5 10 15

Lys Ala Phe Lys Lys Ala Phe 20

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /note= "This sequence is D2A21."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe 1 5 10 15

Ala Lys Phe Ala Phe Ala Phe 20 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide ( ix) FEATURE :

(A) NAME/KEY: Peptide

(B) LOCATION: 1..27

(D) OTHER INFORMATION: /note= "This sequence is D5C."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu 1 5 10 15

Ala Arg Lys lie Ala Arg Leu Gly Val Ala Phe 20 25 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..37

(D) OTHER INFORMATION: /note= "This sequence is D5C1. "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Leu 1 5 10 15

Ala Arg Lys lie Ala Arg Leu Gly Val Ala Lys Leu Ala Gly Leu Arg 20 25 30

Ala Val Leu Lys Phe 35

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..10 (D) OTHER INFORMATION: /note= "This sequence is a modified

GnRH . "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Gin His Trp Ser Tyr G]y Leu Arg Pro Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO: 10:

[SEQ ID NO 10 deliberately left blank] (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..15 (D) OTHER INFORMATION: /note= "This sequence is bLH."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Ser Tyr Ala Val Ala Leu Ser Cys Gin Cys Ala Leu Cys Arg Arg 1 5 10 15

(2) INFORMATION FOR SEQ ID NO : 12 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..38 (D) OTHER INFORMATION: /note= "This sequence is hecate-bLH fusion peptide."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Ala Leu Lys Lys Leu Lys 1 5 10 15

Lys Ala Leu Lys Lys Ala Leu Ser Tyr Ala Val Ala Leu Ser Cys Gin 20 25 30

Cys Ala Leu Cys Arg Arg 35 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "Xaa in position 1 denotes pyro-glutamic acid. Xaa in position 6 denotes D-lysine. This sequence is D-Lys-6 GnRH." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Xaa His Trp Ser Tyr Xaa Leu Arg Pro Gly 1 5 10

Claims

What is claimed:

1. A compound comprising: (a) a hormone domain selected from the group consisting of gonadotropin-releasing hormone, and analogues of gonadotropin-releasing hormone; and (b) a lytic peptide domain.

2. A compound as recited in Claim 1, wherein said hormone domain is bonded directly to said lytic peptide domain, without an intermediate linking domain joining said hormone domain to said lytic peptide domain.

3. A compound as recited in Claim 1, wherein said lytic peptide domain is selected from the group consisting of a cecropin peptide, a melittin peptide, a defensin peptide, a magainin peptide, a sarcotoxin peptide, and analogs of said peptides.

4. A compound as recited in Claim 1, wherein said lytic peptide domain comprises hecate.

5. A compound as recited in Claim 1, wherein said hormone domain comprises gonadotropin-releasing hormone.

6. A compound as recited in Claim 1, wherein said compound has me sequence SEQ. ID NO. 3.

7. A compound as recited in Claim 1, wherein said compound has die sequence SEQ. ID NO. 4.

8. A compound as recited in Claim 1, wherein said hormone domain, or said lytic peptide domain, or both comprise D-conformation amino acid residues.

9. A compound as recited in Claim 8, additionally comprising a carrier domain to facilitate uptake by the intestine when the compound is administered orally.

10. A compound as recited in Claim 9, wherein said carrier domain comprises a vitamin B₁₂ domain.

11. A method for producing long-term contraception or sterility in a mammal, comprising administering to the mammal an effective amount of gonadotropin-releasing hormone and an effective amount of a lytic peptide.

12. A method as recited in Claim 11, wherein the lytic peptide is administered after the gonadotropin-releasing hormone is administered.

13. A method as recited in Claim 11, wherein the gonadotropin-releasing hormone, or the lytic peptide, or both comprise D-conformation amino acid residues.

14. A method as recited in Claim 13, wherein the compound containing D-conformation amino acid residues additionally comprises a carrier domain to facilitate uptake by the intestine when the compound is administered orally.

15. A method as recited in Claim 14, wherein the carrier domain comprises a vitamin B₁₂ domain.

16. A method for producing long-term contraception or sterility in a mammal, comprising administering to the mammal a compound comprising a gonadotropin-releasing hormone domain, and a lytic peptide domain.

17. A method as recited in Claim 16, wherein the hormone domain is bonded directly to the lytic peptide domain, without an intermediate linking domain joining the hormone domain to the lytic peptide domain.

18. A method as recited in Claim 16, wherein the lytic peptide domain is selected from the group consisting of a cecropin peptide, a melittin peptide, a defensin peptide, a magainin peptide, a sarcotoxin peptide, and analogs of said peptides.

19. A method as recited in Claim 16, wherein the lytic peptide domain comprises hecate.

20. A method as recited in Claim 16, wherein the compound has me sequence SEQ. ID NO. 3.

21. A method as recited in Claim 16, wherein the compound has the sequence SEQ. ID NO. 4.

22. A method of temporarily restoring fertility in a mammal that had been made sterile by the selective destruction of gonadotropes in the pimitary, comprising administering to the mammal an effective amount of gonadotropin-releasing hormone.

23. A method as recited in Claim 22, wherein fertility is restored in a mammal that had previously been made sterile by administering to the mammal an effective amount of gonadotropin-releasing hormone and an effective amount of a lytic peptide.

24. A method as recited in Claim 22, wherein fertility is restored in a mammal that had previously been made sterile by administering to the mammal an effective amount of a compound comprising a gonadotropin-releasing hormone domain, and a lytic peptide domain.

25. A method as recited in Claim 11, wherein the mammal is a dog.

26. A method as recited in Claim 11, wherein the mammal is a cat.

27. A method as recited in Claim 11, wherein the mammal is a cow or bull.

28. A method as recited in Claim 11, wherein the mammal is a pig.

29. A method as recited in Claim 11, wherein the mammal is a horse.

30. A method as recited in Claim 11, wherein me mammal is a sheep.

31. A method as recited in Claim 11, wherein the mammal is a human.

32. A method as recited in Claim 16, wherein the mammal is a dog.

33. A method as recited in Claim 16, wherein me mammal is a cat.

34. A method as recited in Claim 16, wherein the mammal is a cow or bull.

35. A method as recited in Claim 16, wherein the mammal is a pig.

36. A method as recited in Claim 16, wherein the mammal is a horse.

37. A method as recited in Claim 16, wherein the mammal is a sheep.

38. A method as recited in Claim 16, wherein the mammal is a human.

39. A method for selectively killing gonadotrophic cells in the pimitary of a mammal, comprising administering to the mammal: (a) an effective amount of gonadotropin- releasing hormone, and (b) an effective amount of a lytic peptide.

40. A mediod for selectively killing gonadotrophic cells in the pimitary of a mammal, comprising administering to the mammal an effective amount of a compound comprising a gonadotropin-releasing hormone domain and a lytic peptide domain.

41. A method for selectively killing neurons having gonadotrophic receptors in a mammal, comprising administering to the mammal: (a) an effective amount of gonadotropin- releasing hormone, and (b) an effective amount of a lytic peptide.

42. A method for selectively killing neurons having gonadotrophic receptors in a mammal, comprising administering to the mammal an effective amount of a compound comprising a gonadotropin-releasing hormone domain and a lytic peptide domain.

43. A method as recited in Claim 11, wherein the mammal is sexually immature.

44. A method as recited in Claim 16, wherein the mammal is sexually immature.