JP2001514231A - Compositions and methods for performing contraception or infertility in animals - Google Patents

Abstract

  (57) [Summary]     Amphiphilic cytolytic peptides are typically suitable for use in a combination of a ligand and a cytotoxin to cause contraception or long-term infertility in a mammal. The peptide acts directly on the cell membrane and does not need to be taken up into the cell. Administration of a combination of gonadotropin releasing hormone (GnRH) (or an agonist of GnRH) and a lytic peptide that acts on cell membranes results in long-term contraception or sterility in vivo in a mammal. This compound is relatively small and lacks antigenicity. Gonad stimulator cells lysed very quickly (about 10 minutes). The two components, the ligand and the cytolytic peptide, may be administered as a fusion peptide, or may be administered separately, with the ligand being administered slightly earlier than the lytic peptide. This separate administration activates cells that have the receptor for the ligand, making them more susceptible to lysis by the lytic peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The filing date of provisional patent application Ser. No. 60 / 057,456 (September 3, 1997) in the United States pursuant to Section 119 (e) of the United States Patent Act and in accordance with applicable treaties and agreements outside the United States ).

TECHNICAL FIELD The present invention relates to compositions and methods for performing long-term contraception or infertility treatment in mammals. Background Art Compositions sometimes used for long-term contraception "fool" female reproductive organs
Includes those based on natural or synthetic steroid hormones that render them "pseudopregnant." Such steroid hormones must be administered repeatedly to prevent the estrous cycle and completion of conception. Steroids can have dangerous side effects.

[0003] The "Endocrine Regulation of the Corpus Lute", "Pot Olson et al."
um of the Bitch as a Potential Target for Altering Fertility ")", J. R
eprod. Fert. Suppl. 39: 27-40 (1989) discusses the luteal phase and its regulation in female dogs. The following description is on page 37. "When a specific toxin binds to an antibody or hormone and is transported to a specific target cell, the toxin can kill the target cell. This magic bullet (magi
c bullet)] has been debated for decades, but recently this idea has been highly selective, destroying only certain organizations without affecting other organizations. As a method, it is receiving new attention. For a long time, it has not been possible to obtain large quantities of antibodies that specifically react with only one antigenic determinant. But,
This problem has been greatly overcome with the advent of monoclonal antibodies. It is possible to make an antibody specific to a specific hormone receptor (such as the LH receptor) and to bind a toxin to this. This destroys all cells that have LH receptors. Not all of the different cells have been characterized in the canine luteum, but if these cells do interact, any disruption of any type of luteal cell can lead to luteal lysis . ”(Quotation omitted)

[0004] P. Olson et al., "New developments in small animal population control (
"New Developments in Small Animal Population Control") ", JAVA., No. 20
Volume 2, pages 904-909 (1993), outlines ways to prevent unwanted pregnancy or abortion in small animals. On page 905, the following description is found. "Tissue-specific cytotoxin ... a cytotoxin that binds to gonadotropin-releasing hormone (GnRH) and is persistent in female and male animals by the administration of a cytotoxin that selectively destroys gonadotropin-secreting pituitary cells. Contraception is possible, as is cytotoxicity linked to antibodies to the gonadotropin receptor, which can alter gonadal function.Toxins must be carefully targeted to specific cells, and all other Must be secure for the organization. "

[0005] "Short Chain Analogs of Luteinizing Horm", "T. Janaky" et al.
one-Releasing Hormone Containing Cytoxic Moieties ")", Proc. Natl. Acad
Sci. USA, 89, 10203-10207 (1992) describes the specific use of GnRH as a carrier for certain alkylated nitrogen mustards, certain anthraquinone derivatives, antimetabolites, and cisplatin-like platinum complexes. It discloses the use of hexapeptide and heptapeptide analogs.

[0006] S. Sealfon et al., "Molecular mechanisms for ligand interaction with gonadotropin-releasing hormone receptor"
of ligand interaction with the gonadotropin releasing hormone receptor)
", Endocrine Reviews, 18, 180-205 (1997), discusses studies on the interaction of GnRH with its receptors.

A receptor binding site identified in the 81-95 region of the beta subunit of human luteinizing hormone (LH) and human chorionic gonadotropin (hCG), such as D. Morbeck et al. Binding Site Identified in
the Region 81-95 of the b-Subunit of Human Luteinizing Hormone (LH) and
chorionic gonadotropin (hCG) ")", Molecular and Cellular Endocrinology
97, pp. 173-181 (1993) describe L-functioning as a receptor binding site.
H and 15 regions of hCG amino acids are disclosed. (LH and hCG
Is a homologous hormone that produces a similar effect. )

[0008] Evidence for autocrine inhibition of gonadotropin-releasing by gonadotropin-releasing hormone (GnRH) in hypothalamic GT1-1 neurons by S. Cho et al.
hormone (GnRH) gene transcription by GnRH in hypothalamic G T1-1 neuron
al cells. ")", Mol. Brain Res., Vol. 50, pp. 51-58 (1997) show that the neuroendocrine group of GnRH neurons has a receptor with high affinity for GnRH and GnRH analogs. It is disclosed that.

"Activation of LH receptors expressed on GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release (N. Mores, et al.). in GnRH neurons stimulat
es cyclic AMP production and inhibits pulsatile neuropeptide release ")
Endocrinology, Vol. 137, pages 573-5734 (1996), states that LH acts directly on neuroendocrine neurons in the brain. Zee Ray (Z.
Lei) et al., "Signaling and transacting factors in the transcriptional in transcriptional inhibition of the immortalized hypothalamic GT1-7 neuron gonadotropin-releasing hormone gene by human chorionic gonadotropin.
hibition of gonadotropin releasing hormone gene by human chorionic gonad
otropin in immortalized hypothalamic GT1-7 neurons ")", Mol. & Cell. E
See also ndocrinology, Vol. 109, pp. 151-157 (1995).

[0010] Conventional selective toxin therapy has several disadvantages. The time it takes for the recipient's immune system to mount an immune response that produces antibodies to the toxin is short (approximately two weeks) with treatment with certain selective toxins. These antibodies neutralize the toxin,
In some cases, toxins can accumulate in reticuloendothelial tissues (eg, liver, spleen, lymph nodes, lungs, bone marrow) and damage healthy tissues. Apart from this drawback, the toxin must be taken up by target cells and transported into the cytoplasm in order to be effective.

US Pat. Nos. 5,378,688, 5,488,036, and 5,49
No. 2,893 discloses compounds useful in inducing fertility in mammals. The disclosed compounds are collectively referred to as GnRHs (or GnRH analogs) that bind to toxins. The toxin preferably binds to the sixth amino acid of the GnRH agonist. The toxin preferably has a translocation domain that facilitates uptake into cells. We believe that binding of a GnRH agonist to a toxin is "required"
That was shown. "The reason is that, in many cases, the toxins described above are unable to bind to the cell membrane by themselves;
It has been found that the toxin can bind to the cell membrane only if the nRH analogue binds to the toxin of the type described above ... (Eg, US Pat. No. 5,488,488)
, No. 036, 7th column, lines 46-52). The toxins specifically mentioned here are ricin, abrin, modeccin, various plant-derived ribosome inhibitory proteins, anti-viral protein of American pokeweed, alpha-amanitin, diphtheria toxin, pseudomonas exotoxin, Shiga toxin, melphalan, methotrexate , Nylotogen mustard, doxorubicin, daunomycin, etc. are all metabolic toxins. None of these toxins appears to be toxic by direct interaction with the cell membrane. In vivo experiments reported here report that injection administration once a week for four weeks is most effective (eg, US Pat.
488, 036, column 20, lines 46-47). Because many of the conjugates described are relatively large compounds, antigenicity can be an issue when such multiple dose regimens are used. The GnRH analog is preferably bound to the toxin by one of several specific heterobifunctional substances. This specification suggests that significant effort was required to bind the toxin to the GnRH agonist. Normally, these toxins must be taken up into target cells in order to be effective and do not act on cell membranes. Furthermore, at least some of these toxins must be secondarily transported from membrane-bound vesicles into the cytoplasm in order to interact with ribosomes, mitochondria and other organelles.

DISCLOSURE OF THE INVENTION An amphipathic cytolytic peptide is used to produce infertility or long-term contraception in animals.
It was unexpectedly discovered to be suitable for use in combination with a ligand and a cytotoxin. The peptide acts directly on the cell membrane and does not need to be taken up by cells. By administering a combination of gonadotropin releasing hormone (GnRH) (or an agonist of GnRH) and a lytic peptide that acts on cell membranes, the animal is subjected to long-term contraception or sterility in vivo. It is particularly surprising that the combination also produces an infertile effect when administered to sexually immature animals. That is, this combination prevents sexual maturation.

This compound used in the present invention will not exhibit antigenicity because it is relatively small (it is known that the cytolytic peptide is not very antigenic,
The ligand lacks any antigenicity. ). The compounds of the present invention may be given in two or more doses, approximately in time, but may be given in a single dose. Gonad stimulator cells lysed very quickly (about 10 minutes). The two components, the ligand and the cytolytic peptide, may be administered as a fusion peptide or may be administered separately, with the ligand being administered slightly earlier than the lytic peptide. This separate administration activates cells that have receptors for the ligand, resulting in
Cells are more susceptible to lysis by lytic peptides. When using a fusion peptide,
It was unexpectedly discovered that a linking moiety for linking the ligand to the lytic peptide was unnecessary. That is, one can be directly bonded to the other without any intervening connecting portion. It is preferable that the binding is performed by binding one end of the ligand to one end of the peptide, instead of binding the ligand and the peptide to the center of both. This toxin or lytic peptide does not require a translocation domain and does not need to be taken up into cells since it acts directly on the cell membrane of activated cells to cause cell lysis.

MODES FOR CARRYING OUT THE INVENTION It is known that D-amino acid type GnRH binds to gonad stimulating cells of the pituitary gland and binds to GnRH neurons of the brain. The D-amino acid form of the cytolytic peptide has almost the same property of lysing cell membranes as the L-amino acid form. Thus, the compounds of the present invention (which may be administered as a fusion peptide or separately) may be administered in either the L- or D-form. D-type peptides are generally more expensive than L-type peptides, but have the advantage that they are not degraded by normal enzymatic reactions, so that they can be administered orally and have a longer biological half-life. Having. In oral administration of a D-type peptide, the fusion product of the peptide and the hormone can be bound to an appropriate carrier to promote absorption in the intestine and enhance its effect. Examples of suitable carriers may vitamin B _12, in this case, G Russell - Jones Synthesis of LHRH antagonists suitable for oral administration by absorbing system "vitamin B ₁₂ in (G. Russell-Jones), etc. (" Synthesis of LHR
H Antagonists Suitable for Oral Administration via the Vitamin B ₁₂ Uptak
e System ")", Bioconjugate Chem., Vol. 6, performs almost therefore administered to vitamin B ₁₂ binding technique described in 34-42 pages (1995).

GnRH or GnRH analogs (collectively referred to as “GnRH agonists”) can be used in the present invention. By substituting the 6th and 10th amino acids of the GnRH decapeptide, a "super agonist" having a higher affinity for the GnRH receptor than GnRH itself can be produced. "Superagonists" include goserelin, leuprolide, buserelin, nafarelin and the like. See U.S. Patent No. 5,488,036.

Without wishing to be bound by theory, it is believed that the mechanism underlying the present invention is as follows. That is, GnRH is a neuroendocrine GnRH in the brain.
Activates neuronal and pituitary gonadotropes. Activated cells are very susceptible to lysis by cytolytic peptides. Here, the activated cells are selectively destroyed (or severely damaged) by the cytolytic peptide. When the pituitary gland stimulating cells are destroyed and GnRH from the brain becomes deficient, the pituitary gland does not secrete follicle stimulating hormone (FSH) or luteinizing hormone (LH),
Animals become temporarily or permanently infertile.

While it is possible for the ligand and the lytic peptide to be administered separately, it is preferred to couple the two to one molecule. The reason for this is that such binding greatly increases the effective concentration of lytic peptide in the vicinity of the cells activated by the ligand. In addition, higher effective concentrations of lytic peptide reduce the need to activate cells, the peptide only needs to bind to the binding site of the ligand, and ligands typically required for activation of target cells The "rest" is not necessary. This binding is GnRH / peptide or peptide / GnR
H may be in any order. Examples include GnRH / hecate (SEQ ID NO: 3) and Hecate / GnRH (SEQ ID NO: 4). Note that an intermediate conjugate is not required and it is possible to link the carboxyl terminus of one of the two peptides directly to the amino terminus of the other (the inventors consider the fusion peptide GnRH
Alternatively, it has been found that the original pyroglutamic acid residue in the GnRH moiety can be replaced with glutamine without substantially changing the activity of each peptide. SEQ ID NOS: 9, 3 and 4).

Experimental Results Example 1-6 Pituitary glands of adult female rats were collected and divided into six sections of approximately equal size.
Pituitary sections were placed, one at each of the six wells containing tissue culture at 37 ° C. Each well was treated differently as described below. Ten hours after the treatment, the tissue in each well was fixed, and each tissue structure was examined under a microscope.

In treatment 1, only a tissue culture solution was added as a control. The tissue after treatment was normal. In treatment 2, 5 nanograms of GnRH (SEQ ID NO: 1) was added per mL of culture. The tissue after treatment was histologically normal, but some vesicle formation of cells was observed. For comparison, the concentration of GnRH in untreated normal rats varies from 1 ng / mL to 20 ng / mL during the LH-enhancing period of the estrous cycle.

In treatment 3, hecate (SEQ ID NO: 2), which is a dissolved peptide, was added to the culture solution to a concentration of 50 μM. The tissue after treatment was histologically normal. In treatment 4, 5 ng of GnRH was added per 1 mL of the culture solution, and the mixture was incubated for 15 minutes. After this incubation, the culture containing GnRH was removed and the tissue was washed once with plain culture. Then remove this culture,
The culture solution containing 50 μM of the lytic peptide, hecate, was replaced. With this treatment, destruction of basophil (gonad stimulation) cells was widely observed.

In treatment 5, 50 μM of the fusion peptide, modified GnRH / Hecate (SEQ ID NO: 3) was added. With this treatment, destruction of basophil (gonad stimulation) cells was widely observed.

In treatment 6, the fusion peptide GnRH / hecate (SEQ ID NO: 3) was first added, and then the second fusion peptide GnRH / hecate was added 2 hours later. This treatment almost completely destroyed the cells except for the stromal cells.

Example 7 Two sexually immature female rats (33 days old) of the same litter were intravenously injected with a saline control solution in two divided doses at 24 hour intervals. After the rats reached the mating age, they were observed for 105 days after inoculation. The appearance of the external genitalia was normal. 10 after inoculation
During a 14-day monitoring period from days 7 to 121, control rats each completed at least two estrous cycles. After this the rats were killed and necropsied. The genitals were histologically normal.

Example 8 Two sexually immature rats (33 days old) littermated with the rats of Example 7 were subjected to GnRH
/ Hecate fusion peptide dissolved in physiological saline was injected twice at 24 hour intervals in 500 μg increments. After the rats reached the mating age, they were observed for 105 days after inoculation. External genitalia were smaller. Unlike control rats,
It was difficult to insert a cotton swab into the vagina. None of the treated rats showed estrus or late estrus during the 14 day monitoring period from day 107 to day 121 after inoculation. After this the rats were killed and necropsied. In rats treated with peptide, uterine epithelium and fallopian tube epithelium were thin and inactivated. No large follicles were found in the ovaries, and many regressive follicles (non-ovulated follicles) were found.

Examples 9-14 Eighteen sexually mature mixed race female rats were randomly divided into six groups, three in each group. Rats in each group were treated twice by intravenous injection as described below. Two weeks after the treatment, the rats were killed and necropsied. Genital and endocrine organs were removed, weighed, and examined for histology.

In treatment 9, a saline control was used. Ovarian function was normal in this group of rats (normal follicles, new corpus luteum). Pituitary size in this group was normal. Histologically normal numbers of pituitary basophils were found.

In treatment 10, 1.0 mg of the GnRH / hecate fusion peptide dissolved in physiological saline was injected at 24 hour intervals in two equal 0.5 mg doses. In this group of rats, normal ovarian follicle development was impaired. The corpus luteum was almost nonexistent, and what was present was old. The follicle was large but not torn. Uterine morphology was consistent with hormone inactivation. The pituitary gland in this group was slightly smaller compared to the saline control group. Histologically, there was a 60-70% reduction in the number of basophil cells in the pituitary gland compared to controls.

In the treatment 11, GnRH (162 μg) was dissolved in physiological saline to prepare 1.35 m
100 μL of the solution prepared as M was injected, and 15 minutes later, 100 μL of a solution obtained by dissolving hecate (337 μg) in physiological saline to make 1.35 mM was injected. Twenty-four hours later, the same two-step treatment was performed. The ovaries of rats in this group showed histological changes. The corpus luteum was almost nonexistent, and the corpus luteum was old.
The follicle was large but not torn. Uterine morphology was consistent with hormone inactivation. The pituitary gland and pituitary histological findings were similar to those observed in treatment 10.

In treatment 12, Hecate (337 μg) was dissolved in physiological saline to give 1.35 mM
100 μL of the prepared solution was injected. This treatment was performed again after 24 hours. In this group of rats, ovarian function was normal (normal follicles and new corpus luteum). Pituitary gland and pituitary histological findings were similar to those observed in treatment 9.

In treatment 13, GnRH (162 μg) was dissolved in physiological saline to prepare 1.35 m
M solution was injected at 100 μL. This treatment was performed again after 24 hours. In this group of rats, ovarian function was normal (normal follicles and new corpus luteum). Pituitary gland and pituitary histological findings were similar to those observed in treatment 9.

In treatment 14, the same treatment as treatment 10 was performed, except that the GnRH / hecate fusion peptide was further purified by high performance liquid chromatography (HPLC). In this group of rats, normal follicular development was impaired. The corpus luteum was almost nonexistent, and the corpus luteum was old. The follicle was large but not torn.
Uterine morphology was consistent with hormone inactivation. Pituitary and pituitary histological findings were similar to those observed in treatment 10.

[0032] These experiments showed that GnRH and the lytic peptide could be administered separately or as a fusion peptide, but the fusion peptide was larger at lower doses. It is more preferable because the activity can be obtained.

Although experiments to determine the optimal dose could not be performed before the filing of this application, those skilled in the art will appreciate that the teachings of this specification will make it possible for It can be easily ascertained.

Although experiments to date have been performed on female mammals, GnRH sends a signal that causes pituitary cells to release gonadotropin in both males and females. Is expected to be obtained.

The tissue specificity and cell specificity of the cytotoxic conjugate can be further improved by utilizing various hormones or hormone analogs that bind to the lytic peptide. Here are some examples. To regulate fertility, GnRH
/ Hecate conjugate selectively damages central GnRH nerve components of the pituitary and reproductive axis. Chicken type II GnRH and lamprey type III GnRH are:
Because of the major molecules that affect brain function in many mammals, very few cells of the central nervous system should be damaged by this treatment. Regulation of fertility can also be achieved selectively by treating animals with the bLH / hecate conjugate. The bLH / hecate conjugate selectively acts on GnRH neurons that regulate reproduction and gonads. (Other lytic peptides may be used instead of Hecate in these conjugates).

The compounds of the present invention may be administered as described above, or may be administered as a pharmaceutically acceptable salt. The composition is suitable for intravenous, subcutaneous, intramuscular or oral administration (particularly, preferably if a D- amino acid form obtained by forming a carrier and complexes, such as vitamin B ₁₂₎ can be.

Uses of the present invention include long-term contraception or sterility in humans, and long-term contraception and sterility in livestock and wild mammals. Examples of livestock to which the present invention can be applied include dogs, cats, cows, horses, pigs, and sheep. When used in humans, to prevent unwanted side effects such as premature menopause,
Long-term hormone replacement therapy may be required. By administering gonadotropin hormone to patients who have undergone infertility treatment, it is possible to temporarily restore fertility as needed. In this sense, the fertility treatment of the present invention is reversible.

As an example, the present invention can be used to humanely regulate the population of unwanted alien species. Example 15-22

Eight sexually mature Sprague-Dawley female rats were randomly assigned one of eight different treatments. Rats in each group
One treatment was received intravenously as described below. 48 hours or 9 after treatment
The rats were sacrificed after 6 hours and necropsied. The ovaries, uterus, pancreas, liver, spleen, lung, heart, thyroid, and adrenal gland were fixed with 10% buffered formalin, sectioned, and stained with H & E (hematoxylin and eosin) stain. However, the pituitary gland was stained by PAS (periodic acid Schiff staining) staining method, and no counterstaining was performed. Treatments were chosen such that each animal received an equimolar amount of the compound.

In treatments 15 and 16, 674 μg of D-hecate was dissolved in 200 μl of physiological saline (1.35 mM) and injected intravenously. Treatment 15 rats were sacrificed 48 hours after injection, and treatment 16 rats were sacrificed 96 hours after injection. No gross lesion was observed in any animal organ. The pituitary gland of each rat had a normal number of PAS-positive cells.

In treatments 17 and 18, 334 μg of GnRH was dissolved in 200 μl of saline (1.35 mM) and injected intravenously. The rats from treatment 17 were killed 48 hours after injection, and the rats from treatment 18 were killed 96 hours after injection. No gross lesion was observed in any animal organ. The pituitary gland of each rat had a normal number of PAS-positive cells.

In treatments 19-22, 1 mg of the GnRH-hecate fusion peptide (SEQ ID NO: 3) was dissolved in 100 μl of physiological saline (2.7 mM) and injected intravenously. Processing 19
And 20 rats were sacrificed 48 hours after injection and treatment 21 and 22 rats were injected 9 hours after injection.
Killed 6 hours later. No gross lesions were observed in any of the four animals except for the pituitary gland. The pituitary glands of the animals of treatments 19 and 20 showed slight hypertrophy, hyperemia and edema. The pituitary gland of the animals of treatments 21 and 22 showed some hyperemia but normal size. The pituitary gland of all four rats was significantly deficient in PAS-positive cells. This deficiency was estimated to be 80-90% of the number of PAS-positive cells in the control group (PAS staining selectively stains glycopeptides. LH, FSH, TSH, MSH are glycopeptide hormones; Cells that have stored these hormones in secretory vesicles stain positive for PAS).

That is, the GnRH-lytic peptide combination causes morphological and functional changes in the reproductive system of adult female rats and also prevents sexual maturation of prepubescent female rats. It is understood that the fusion peptide selectively removes a specific population of pituitary PAS-positive cells while causing functional changes.

Example 23 The following experiment was performed on rats using a procedure generally similar to the procedures 9-14 described above. Observations made by immunohistochemical staining showed that effective treatment selectively killed (1) pituitary gonad stimulator cells and (2) GnRH receptor-bearing neurons. It was found that after administration of the GnRH-hecate fusion peptide, and after administration of only Hecate only 10 minutes after administration of GnRH alone, the cells were selectively killed. In this case, it was also observed that pituitary cells did not secrete either LH or FSH after effective treatment.

Example 24 Hecate is an amphipathic cytolytic peptide that acts on cell membranes without being taken up into cells. Hecate is a synthetic peptide analog of melittin, the major toxin of bee venom. Hecate is thought to work by disrupting cell membranes. The structure of the modified GnRH-hecate conjugate used in this study is shown in SEQ ID NO: 3.

[0046] We also, the D-Lys ⁶ is a position where interference with binding to GnRH receptors GnRH domain is minimized as Hecate is coupled, D-Ly s ⁶ GnRH (SEQ ID NO: 13) Was synthesized. These synthetic peptides specifically displaced radiolabeled monoiodinated GnRH from rat pituitary cell membranes. When measuring the radioactivity in cpm, substitution with D-Lys ⁶ GnRH-Hecate was equivalent to substitution with a natural mammalian GnRH (actually greater slightly than). The binding of GnRH and the binding of GnRH-hecate were within a 1000-fold concentration range (n =
When compared on a molar basis over 6), GnRH-Hecate specifically displaced the radiolabeled peptide to an extent equal to 123% ± 4% of binding by equal molar concentrations of GnRH. Equimolar concentrations of D-Lys ⁶ GnRH displaced 187% ± 8% of cpm replaced by native GnRH.

Examples 25-32 We examined the in vitro lysis of bovine luteal cells using the GnRH-hecate conjugate and the hecate-bLH conjugate (SEQ ID NO: 12) (conjugate b
The LH portion (SEQ ID NO: 11) is a 15 amino acid fragment of the beta chain of luteinizing hormone. ). Small luteal cells were collected from the corpus luteum of sacrificed cattle. Small luteal cells are L
It was found to be rich in H receptors and very susceptible to lysis by the hecate-bLH conjugate.

The luteal cells were incubated in the culture for 22 hours by performing one of the following treatments, and by the trypan blue exclusion test and the release of lactate dehydrogenase,
The viability was examined.

Process 25 Control. No additional treatment (culture only). 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 (single), 10 μM treatment 31 Hecate (single), 5 μM treatment 32 Hecate (single ), 1 μM

After incubation for 22 hours with 10 μM Hecate alone and 5 μM Hecate alone, significant microluteal cell death was observed (approximately 50% lethality). There was no significant difference in cell death in the case of 1 μM hecate alone compared to the negative control (culture medium only) or bLH alone. All three treatments receiving Hecate-bLH showed a significant increase in cell death as compared to Hecate alone treatment. The Hecate-bLH conjugate provides approximately 2% of the same concentration of Hecate alone.
Twice the cell count died.

The observed lactate dehydrogenase activity levels also showed that hecate-bLH treatment killed a greater number of cells compared to treatment with hecate alone.

Examples 33-34 We further investigated the in vitro cell lysis of bovine granulosa cells by GnRH-hecate and hecate-bLH conjugates. Granulosa cells were isolated from bovine preovulatory follicles (granulosa cells are hormone-active cells with many LH receptors). Experiments using granulosa cells are generally similar to the experiments described above for Examples 25-32, with the following exceptions. In other words, according to this experiment, (1) granulosa cells are much more likely to be killed by treatment with hecate alone than microluteal cells, and (2) as in the case of small luteal cells, granulosa cells have the lowest concentration ( 1
μM), it was demonstrated that hecate-bLH was significantly more susceptible than hecate alone. At 1 μM, the Hecate-bLH conjugate killed about twice as many target cells as compared to Hecate alone. Again, the level of lactate dehydrogenase released after treatment with 1 μM Hecate-bLH was 1
Significantly higher levels of the same enzyme were released after treatment with μM Hecate alone.

Further studies (data not shown) indicate that the 15-mer fragment of the bLH subunit specifically bound to the LH receptor of target granulosa cells, but that a response to stimuli was taking place. It was found that steroid hormone production was not initiated. The inventors therefore clearly show that the selective killing of target cells is due to the presence of the lytic peptide in physical proximity to the target cells, which is due to the binding of the LH subunit. did. Stimulation of target cell hormone production was not required for cell lysis. This result was surprising, as it was previously thought that activation of target cells was necessary to make them more susceptible to cell lysis. These data indicate that such activation is not required. However, these data are consistent with our other data indicating that cell activation is also a pathway to render cells susceptible to lytic peptides.

Examples 35-38 A further series of experiments was performed to determine the effect of the GnRH-hecate conjugate on female rats and rabbits in vivo. Histological analysis was performed on the ovaries, uterus, fallopian tubes, adrenal glands, spleen, thyroid, pancreas, liver, lungs, and heart. In the pituitary gland, P
AS stained cells, bLH, BFSH (bovine follicle stimulating hormone), adrenocortical stimulating hormone, other pro-opiomelanocortin peptide products (most famously alpha-melanocyte stimulating hormone), thyroid stimulation Histological analysis was performed on cells immunohistologically stained for hormone (TSH), prolactin (PRL), vasopressin (VP), oxytocin (OXY), and growth hormone (GH). The immunocytological staining method used was generally described by M. Rahmanian et al., "Histological and immunocytochemical characteriz- ing of pituitary cell types in ponies."
ation of pituitary cell types in ponies ")", Proc. 13th Soc. Equine Nut
rition & Phys. Symp., pp. 348-349 (1993), M. Rahman (M. Rah
Manian) et al., "Immunocytochemical localization of luteinizing horm in the pituitary gland of the horse."
one and follicle-stimulating hormone in the equine pituitary ")", J.
Anim. Sci., Vol. 76, pp. 839-846 (1998), M. Rahm
anian) et al., “Immunocytochemical localization of prolactin and growth hormone”.
mone in the equine pituitary ")", J. Anim. Sci., Vol. 75, pp. 3010-3018 (1997), P. Melrose, et al., "Immune-reactive alpha-melanocytes in horse and rat brain." Comparative topography of the immunoreactive alpha-melanocyte-sti
mulating hormone neuronal system in the brains of horses and rats ")"
The method of Brain Beh. & Evol., Vol. 32, pages 226-235 (1988) was generally followed.

The brain was serially sectioned on a vibrotome from the level of the diagonal band to the papillary body. Every other section is serially divided into 4-5 dishes, and sections of every other dish are stained with cresyl violet or GnRH or GnR.
Immunocytological staining for H precursor, VP, OXY, or tyrosine hydroxylase (the rate-limiting enzyme in catecholamine synthesis). In addition to the staining methods cited above, the inventors have described "Distribution and morphology of immunoreactive gonadotropin releasing hormone (GnRH) neurons in the basal forebrain of ponies." of immunoreactive gonadotropin-
releasing hormone (GnRH) neurons in the basal forebrain of ponies ")"
J. Comp. Neurol. 339, pp. 269-287 (1994), P. Melrose (PM
elrose) et al., "Topography of oxytocin and vasopressin neurons in the forebrain of Equus caballus: pro-opiomelanocortin neurons,
Further support for the proposed evolutionary relationship for oxytocin and vasopressin neurons ("Topography of oxytocin and vasopressin neurons in t
he forebrain of Equus caballus: Further support of proposed evolutionary
relationships for proopiomelanocortin, oxytocin and vasopression neuro
ns ")", Brain, Beh. & Evol., Vol. 33, pp. 193-204 (1989).

A 33-day-old sexually immature female rat was administered intravenously as follows. Treatment 35 0.03 μg GnRH (normal physiological dose) (2 rats)
Treatment 36 1.62 μg GnRH (equimolar to GnRH of treatment 37) (1 rat) Treatment 37 0.5 mg GnRH-Hecate (1 rat) Treatment 38 0.337 μg Hecate (11 min after administration of 0.03 μg GnRH)
(2 rats)

Animals were sacrificed 14 days after treatment. G compared to the two GnRH control groups
Treatment with nRH-hecate and treatment with GnRH followed by hecate alone resulted in a 13% and 14% reduction in pituitary weight, respectively, with bLH
-There was a 92% and 87% reduction in the number of specific gonadotropes, respectively. Furthermore, after these two treatments, deformities were often seen in the cell bodies of GnRH-stained neurons in the pituitary-stimulated areas of the brain. Also, a significant amount of immunoreactive material had penetrated into the peripheral area where many cell bodies were dense (end plate vascular organs). 2
The two treatments showed cell structure damage in the C1 and C3 areas of the hippocampus, and markedly stained small cell VP neurons in the paraventricular nucleus (VP staining was due to the formation of precipitates in specific areas in the brain) (A later study with a more purified peptide did not result in precipitation.) Changes in VP expression are probably in corticotropin-releasing neurons, but based on this change in VP expression, post-translational processing of the proopiomelanocortin peptide product in the terminal pituitary can be altered. Because GnRH-hecate treatment and G
This is because nRH + hecate treatment reduced the level of adrenocorticotropic hormone and increased the number of alpha-MSH stained cells in this pituitary section. No pathological changes were observed in other tissues.

Since neurons in the brain do not regenerate, if these neurons are severely damaged,
Infertility due to the GnRH / lytic peptide combination can be permanent (but can be temporarily reversed by administration of gonadotropin).

Examples 39-41 Sexually immature (33-day-old) female rats (randomly divided into groups of three) were treated with saline or GnRH-hecate dissolved in saline as follows: Iv injection.

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 24 hours or 14 days after treatment. The results were similar to those described above for Examples 35-38, except that no precipitate was seen in the brain and VP staining in the central nervous system did not change. High level GnRH
-The treatment with Hecate administered, such as deformation of the cell body part of the cells and degeneration of neurites
Many GnRH receptor-bearing neurons with abnormal morphology were generated. Processing 1
In rats sacrificed on day 4, 66% and 87% of GnRH receptor-containing neurons in rats receiving 1.0 mg and 1.5 mg of GnRH-hecate, respectively.
Was abnormal. Axonal degeneration in the group receiving 1.5 mg of GnRH-hecate reduced more than 90% of the median ridge staining for GnRH.

Examples 44-46 GnR as shown below in 7 sexually mature New Zealand rabbits
Saline containing H-hecate was injected intravenously.

Treatment 440 0 mg GnRH-Hecate (n = 3) Treatment 455 5 mg GnRH-Hecate (n = 3) Treatment 46 10 mg GnRH-Hecate (n = 1)

After 46 days, all rabbits were injected intramuscularly with 100 μg of GnRH. 0, 1,
Blood samples were taken after 4, and 24 hours, and LH and FSH levels in the samples were measured by radioimmunoassay. Hormone analysis indicated that LH was released in response to GnRH in both control and treated animals. This suggests that there is some reversibility to the treatment, at least in the pituitary gonad-stimulating cells at low doses of ligand / peptide. The rabbits were killed the next day (day 47) for histological analysis. The inventors have found that the number of tertiary follicles, corpus luteum, and GnRH-induced ovulation is reduced by GnRH-Hecate treatment. Ovarian and pituitary weights were reduced by 10 mg GnRH-hecate treatment. In tissues treated with GnRH-Hecate treatment, immunoreactive GnRH was trace and diffused and localized in areas of the central nervous system that usually contained cell bodies. The number of normal cell bodies was reduced by at least 50% and the terminal areas where axons of GnRH receptor neurons are normally found were not stained for GnRH. These observations suggest that the most pronounced effect of GnRH-Hecate treatment on rabbits in a series of experiments may have been exerted on neuroendocrine neurons in the brain. High concentration of GnR
The hippocampus and other regions of the brain, including H, were not significantly affected by GnRH-Hecate treatment. GnRH-Hecate treatment increased the number of PAS-stained pituitary cells at the terminal pituitary to 177% of that in control rabbits. This increase appears to reflect an increase in the number of alpha-MSH stained cells and a decrease in the number of LH stained cells.

Examples 47-48 Nine sexually mature female rabbits were treated with 0 mg (n = 4) (treatment 46) or 10 mg
A saline solution containing mg (n = 5) (treatment 48) of GnRH-hecate was injected intravenously. Six days after the treatment, rabbits were injected intramuscularly with GnRH. Blood samples were collected and subjected to radioimmunoassay for LH and FSH as in the above example, and rabbits were killed 7 days after treatment. G in both control and treated animals
LH was released relative to nRH, but the amount of LH released was lower in treated animals than in controls. GnRH-Hecate treatment reduced the number of tertiary follicles and the number of GnRH-induced ovulation. No effect was seen on either the surrounding tissue or the pituitary weight. The effects of GnRH-Hecate on central nervous system morphology and immunocytochemical results were similar to those described in Examples 35-46 above. Again, the effect was more pronounced on GnRH neurons than on staining of pituitary gonadotropes.

The number of ovulation sites was reduced in the rabbits of Examples 47 and 48 treated with 10 mg of GnRH-Hecate compared to the saline control. The average number of ovulation sites for the four saline controls was 12.2 ± 5.4 (SEM = 2.7). The average number of ovulation sites in five rabbits fed 10 mg of GnRH-hecate was 3.6 ± 1.1 (SEM = 0.5). The difference from the control was significant (p = 0.025).

The “increase in LH” (the level of LH 1 hour after GnRH administration minus the level before administration) was 61.2 ± 16.5 ng / mL (SE in 4 controls).
. M. = 8.3), and 49.6 ± 26.1 ng / mL (SE.
M. = 12) (p = 0.22). Therefore, the LH level tends to decrease in the treatment group.

The above in vivo studies clearly show that the GnRH-hecate conjugate selectively damages GnRH receptor-containing cells (neurons) in the brain and GnRH receptor-containing cells (gonadotropic cells) in the pituitary gland. Indicated. In addition, these studies showed significant changes in the ovaries, presumably due to changes in the reproductive center of the brain-pituitary axis. The observations listed below also showed the selectivity of the conjugate. (1) No change due to cytotoxicity was observed in neurons lacking GnRH receptor, (2) No change was observed in pituitary cells lacking GnRH receptor, (3) Other endocrine And no changes were observed in non-endocrine tissues (except for the ovaries, which are likely to respond indirectly to the destruction of pituitary gonadotropes).

Many of the phenomena called “ovulation” in GnRH-hecate-treated rabbits are not ovulatory sites that function but may also represent hemorrhagic preovulatory degenerative changes. Further mating tests are performed to ensure that ovulation of the functioning eggs is prevented.

Cytolytic peptides useful in the present invention (without being bound by this theory) It is believed that lytic peptides act by disrupting cell membranes. "Hibernating" eukaryotic cells protect themselves by their ability to repair cell membrane damage. In contrast, activated cells (Gn
RH stimulated cells) cannot repair (or have poor repair capacity) damaged cell membranes. The cell membrane repair ability of pituitary cells activated by GnRH is reduced, so that these cells are selectively destroyed by the lytic peptide, while the peripheral unactivated cells repair the cell membrane and survive.

Although hecate is used as a lytic peptide as an effector in the embodiments of the present invention that have been tested to date, other lytic peptide and ligand combinations can be used in the present invention. Many lytic peptides are known in the art, including those mentioned in the references cited in the following description.

[0072] Cytolytic peptides are small basic peptides. Naturally occurring lytic peptides are a major component of the defense system against bacteria in many animal species, such as insects, amphibians, and mammals. Lysed peptides usually consist of 23 to 29 amino acids, but may be smaller. The lytic peptide may have an amphipathic alpha-helical structure. Boman et al., "Humoral immunity in Cecropia pupae", Curr. Top. Microbiol
Immunol., 94/95, pp. 75-91 (1981), Bowman et al., "Cell-free immunity in insects", Annu. Rev.
Microbiol., Vol. 41, pp. 103-126 (1987), Zasloff, "Magainin, a class of antimicrobial peptides obtained from Xenopus skin: isolation, properties of two active forms, and precursors Partial DNA sequence ("Magainins, a class of antimic
robial peptides from Xenopus skin: isolation, characterization of two ac
tive forms, and partial DNA sequence of a precursor ")", Proc. Natl. Ac
ad. Sci. USA, 84, 3628-3632 (1987), Ganz et al., "Defensins n, a natural peptide antibiotic present in human neutrophils.
atural peptide antibiotics of human neutrophils ")", J. Clin. Invest.,
76, 1427-1435 (1985), and Lee et al., "Antibacterial peptides from pig intestine: isolation of mammalian cecropin."
ntestine: isolation of mammalian cecropin ")", Proc. Natl. Acad. Sci. U
See SA, 86, 9159-9162 (1989).

Substitution of amino acid residues such that the amphipathicity of the peptide is preserved (eg, replacing a polar residue with another polar residue, or replacing a non-polar residue with another non-polar residue), Substitutions that preserve the charge distribution (eg, replace an acidic residue with another acidic residue, or replace a basic residue with another basic residue), or preserve the amphipathic or charge distribution By lengthening or shortening the amino acid sequence as it is, it is possible to modify the known amino acid sequence of the lytic peptide and to form a new peptide also having cytolytic activity. Lytic peptides and their sequences are disclosed in the following documents.
Yamada et al., "Production of recombinant sarcotoxin IA in Bombyx mori cells,""Production of recombinant sarcotoxin IA in Bombyx mori cells", Bi.
ochem. J. Vol. 272, pp. 633-666 (1990), Taniai et al.
isolation and nucleotide sequence of a cecropin B cDNA clone from mbyx mori,
"Isolation and nucleotide sequence of cecropin B cDNA clones from the si
lkworm, Bombyx mori ", Biochimica Et Biophysica Acta., 1132, 203-2
06 (1992), Bowman et al., “Antibacterial and antimalaria properties of hybrid peptides of cecropin and melittin.”
l properties of peptides that are cecropin-melittin hybrids ", Febs Let
ters, Vol. 259, pp. 103-106 (1989), Tessier et al., "Enhanced secretion from insect cells of a foreign prote,"
tein fused to the honeybee melittin signal peptide ", Gene, Vol. 98, 17
7-183 (1991), Blondel et al., "Hemolytic and antimicrobial activity of each of the 24 truncated analogs of melittin," Hemolytic and antimicrobial activ.
ities of the twenty-four individual omission analogues of melittin ",
Biochemistry, 30, 4671-4678 (1991), Andrew, et al., "A shortened hybrid of cecropin A and melittin retains strong antibiotic activity even when the size is greatly reduced." cecropin A-melittin hybrids.
Significant size reduction retains potent antibiotic activity "", Febs L
etters, Vol. 296, pp. 190-194 (1992), Macias et al., "Bactericidal activity of magain, Bactericidal activity of magain."
in 2: use of lipopolysaccharide mutants "", Can J. Microbiol., Vol. 36,
582-584 (1990), Rana et al., “Interactions between magainin-2 and the outer membrane of Salmonella typhimurium: Influence of lipopolysaccharide structure,” Interactions between magainin-2.
and Salmonella typhimurium outer membranes: effect of Lipopolysaccharid
e structure ", Biochemistry, Vol. 30, p. 5858-5866 (1991), Diamond, etc.," Airway epithelial cells are the site of expression. of a mammali
an antimicrobial peptide gene "", Proc. Natl. Acad. Sci. USA, 90, 45
From page 96 (1993), Selsted et al., "Purification, primary structures and antibacterial activities o"
f b-defensins, a new family of antimicrobial peptides from bovine neutro
phils "", J. Biol. Chem., vol. 268, p. 6641 (1993), Tang
BNBD-12, an antimicrobial beta-defensin peptide derived from bovine neutrophils
Characterization of the disulfide motif in
motife in BNBD-12, an antimicrobial b-defensin peptide from bovi ne neu
trophils ")", J. Biol. Chem., vol. 268, pp. 6649 (1993), Lehrer et al., Blood, Vol. 76, pp. 2169-2181 (1990), Ganz (Ganz)
Sem. Resp. Infect. I., pp. 107-117 (1986); Kagan et al., Proc. Nat. Acad. Sci. USA, 87, 210-214 (1990), Wade (1990).
Wade) et al., Proc. Nat. Acad. Sci. USA, 87, 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 P,"
reparation "", International Patent Application Publication No. 89/00199 (1989), Jaynes
"Lytic Peptide. How to Use for Growth, Infection and Cancer," Lytic Peptide
s, Use for Growth, Infection and Cancer ", International Patent Application Publication No. 90/12866.
(1990), Berkowitz, "Prevention and treatment of adverse oral conditions with biologically active peptides," Prophylaxis and Treatment of Adverse Oral Con.
ditions with Biologically Active Peptides ", International Patent Application Publication No. 93/01723.
No. (1993).

A family of naturally occurring lytic peptides includes cecropins, defensins, sarcotoxins, melittins, magainins and the like. Sweden's Boman et al. Have performed an original study on a humoral defense system that allows a large silkworm, Hyalophora cecropia, to protect itself from bacterial infection. Hultmark et al., “Insect immunity Hyalophora c
Purification of three inducible bactericidal proteins from haemolymph of immunized pupae of ecropia, "Insect immunity. Purification of three inducible bactericidal proteins
s from hemolymph of immunized pupae of Hyalophora cecropia ", Eur. J. Bi
Ochem. 106, 7-16, (1980), Hultmark et al., "Isolation and structure of cecropin D and four small antibacterial components from pupae of insect immunity cecropia," Insect. i mmunity.Isolation and structure of cecropin D.
and four minor antibacterial components from cecropia pupae ", Eur. J. B
See iochem. 127, 207-217 (1982).

Infection with H. cecropia induces the synthesis of special proteins capable of disrupting bacterial cell membranes and capable of causing lysis and cell death. Among these special proteins are those known as cecropins. The major cecropins, cecropin A, cecropin B, and cecropin D, are small, highly homologous basic peptides. Bowman's group, in collaboration with Merrifield, has shown that the amino-terminal half of various cecropins contains an amphipathic alpha-helix-forming sequence. "N-Terminal Analogs of Cecropin A: Synthesis, Antibacterial Activity, and Conformational Properties," N-termi
nal analogues of cecropin A: synthesis, antibacterial activity, and con
formational properties ", Eur. J. Biochem., Vol. 24, pages 1683-1688 (1
985). The carboxyl terminal half of the same peptide has a hydrophobic tail. Boman et al., "Cell-free immunity in Cecropia"
in Cecropia ")", Eur. J. Biochem., Volume 201, pp. 23-31 (1991).

[0076] Lee et al., In which a cecropin-like peptide has been isolated from the small intestine of pigs, "Antibacterial peptide derived from pig intestine: isolation of mammalian cecropin."
s from pig intestine: isolation of a mammalian cecropin ") Proc. Natl. Ac
ad. Sci. USA, vol. 86, pp. 9159-9162 (1989)).

[0077] Cecropin peptides have been observed to kill many non-bacterial animal pathogens, such as Jaynes et al., "Effects of novel lytic peptides on the in vitro cell destruction of Plasmodium falciparum and Trypanosoma cruzi, ("In Vi
tro Cytocida Effect of Novel Lytic Peptides on Plasmodium falciparum and
Trypanosoma cruzi ")", FASEB, pages 2878-2883 (1988), Allowood (
Arrowood) et al., "Hemolytic properties of lytic peptides active against the seeds of Cryptosporidium parvum"
sporozoites of Cryptosporidium parvum ")", J. Protozool. 38, 6
No., 161S-163S (1991), Arrowood, etc., "Cryptosporidium par
activity of lytic peptides in vitro against vum spp. ("In vitro activ
ities of lytic peptides against th e sporozoites of Cryptosporidium parv
um "), Antimicrob. Agents Chemother., Vol. 35, pp. 224-227 (1991). However, even at high concentrations, normal mammalian cells are not adversely affected by cecropin. It doesn't seem like Joyness
es) et al., "In vitro effect of lytic peptides on normal and traverse mammalian cell lines."
nsformed mammalian cell lines ")", Peptide Research, Volume 2, Issue 2, 1-
5 (1989), Reed et al., "Increased in vitro growth of murine fibroblasts and preimplantation embryos cultured in media supplemented with amphipathic peptides, (" Enh.
anced in vitro growth of murine fibroblast cells and preimplantation emb
ryos cultured in medium supplemented with an amphipathic peptide ")", Mol. Reprod. Devel., Vol. 31, No. 2, pp. 106-113 (1992).

Defensins are small peptides with 6-8 cysteine residues originally found in mammals. "Defensins, natural peptide antibiotics of huma."
n neutrophils ", J. Clin. Invest., Vol. 76, pp. 1427-1435 (1985)). Extracts from normal human neutrophils include the human neutrophil peptide, HNP-1
, HNP-2, and HNP-3. Defensin peptides have also been found in insects and higher plants. Dimareq et al., "Insect immunity: Expression of two major inducible antimicrobial peptides, defensin and diptericin in Phormia terranvae, (" Insect i
mmunity: expression of the two major inducible antibacterial peptides, d
efensin and diptericin, in Phormia terranvae ", EMBO J., Vol. 9, 2507-
2515 (1990), Fisher et al., Proc. Natl. Acad. Sci. USA,
84, 3628-3632 (1987)).

A relatively large peptide, called sarcotoxin, has been isolated from the fly, Sarcophaga peregrina. Okada et al., "Sarcoxin I, Sarcophag
a Primary structure of sarcotoxin I, an antibacterial protein induc induced by hemolymph of larvae of a peregrina
ed in the hemolymph of Sarcophaga peregrina (flesh fly) larvae "", J.
Biol. Chem., Vol. 260, pp. 7174-7177 (1985)). Although sarcotoxin is quite different from cecropin and defensin, it is thought that it has a similar action as an antibiotic.

Other lytic peptides have been found in amphibians. Gibson, along with co-workers, from Xenopus laevis, PGS and Gly10Lys
Two peptides, designated 22PGS, were isolated. Gibson et al., "A novel peptide fragment derived from Xenopus laevis PGLa, carveline and xenopsin precursors," Novel peptide fragments originating from PGLa and the c.
aervlein and xenopsin precursors from Xenopus laevis "", J. Biol. Chem
, Vol. 261, pages 5341-5349 (1986), Givanini et al., "X
biosynthesis and degradation of peptides derived from prohormones of enopus laevis, "Biosynthesi
Biochem. J., Vol. 243, pp. 113-120 (1987), and degradation of peptides derived from Xenopus laevis prohormones. Sasloff (Zasloff)
The peptide from Xenopus was shown to have antibacterial activity and was named Mageinin (Zasloff), "Magainin, a class of antibacterial peptides obtained from Xenopus skin: isolation, two active properties, and The partial DNA sequence of the precursor ("Mag
ainins, a class of antimicrobial peptides from Xenopus skin: isolation,
characterization of two active forms, and partial DNA sequence of a prec
urlor ")", Proc. Natl. Acad. Sci. USA, 84, 3628-3632 (1987).
.

Due to the synthesis of heterologous analogs of different classes of lytic peptides, a positively charged amphipathic sequence containing at least 20 amino acids in one class of peptides is required for cytolytic activity (Shiba et al., "Structure-activity relationship of Lepidopteran, a self-defense pepti", "Structural-activity relationship of Lepidopteran, a self-defense pepti."
de of Bombyx more ", Tetrahedron., Volume 44, Issue 3, Pages 787-803 (1988)
). Other studies have shown that smaller peptides also show solubility (see McLaughganhlin et al., Quoted below).

Cecropin has been shown to selectively attack pathogens and susceptible cells without affecting normal host cells. Synthetic lytic peptides, known as S-1 (or Shiva1), are available from Brucella abortus, Trypanosoma cruzi, Cryptos
It has been shown to destroy host cells infected intracellularly with poridium parvum and infectious bovine herpesvirus I (IBR), but show no or little cytotoxicity to uninfected mammalian cells. Jaynes et al., "In vitro effect of lytic peptides on normal and transformed mamma cells."
lian cell lines ")", Peptide Research, Volume 2, Issue 2, pages 1-5 (1989
"Wood, et al.," Toxicity of a Novel Antimicrobial Agent to Catt. "
le and Hamster cells In Vitro "", Proc. Ann. Amer. Soc. Anim. Sci., Uta
h State University, Logan, UT. J. Anim. Sci.
Vol. 65, p. 380 (1987), Arrowood et al., "Cryptosporidium pa
Hemolytic properties of lytic peptides active against rvum spp.
of lytic peptides active against the sporozoites of Cryptosporidium parv
um ")", J. Protozool., Vol. 38, No. 6, 161S-163S (1991), Arrowood, et al., "In vitro activity of dissolved peptides against Cryptosporidium parvum spp.," of lytic peptides against the
sporozoites of Cryptosporidium parvum "), Antimicrob. Agents Chemother
35, pp. 224-227 (1991), Reed et al., "Increased in vitro growth of murine fibroblasts and preimplantation embryos cultured in media supplemented with amphipathic peptides, ( "Enhanced in vitro growth of murine fibroblast cells and p
reimplantation embryos cultured in medium supplemented with an amphipat
hic peptide ")", Mol. Reprod. Devel., Vol. 31, No. 2, pp. 106-113 (1992).

Morvan et al., “Bona, a blood cell parasite of the flat persimmon Ostrea edulis.
In vitro activity of the antimicrobial peptide magainin 1 against mia ostreae, "In
vitro activity of the antimicrobial peptide magain in 1 against Bonamia
ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis ", Mol. Mar. Biol., Vol. 3, pp. 327-333 (1994), uses magainin in vitro to produce cells of Ostrea edulis, a kind of oyster. Have been reported to selectively reduce the viability of the parasite Bonamia ostreae in non-influential amounts.

Also of interest are the synthetic peptides disclosed in the following pending patent applications. This peptide exhibits cytolytic activity with only 10 to 14 amino acid residues (McLaughlin et al., "Amphipathic peptides",
U.S. Pat. No. 5,789,542, issued Aug. 4, 1998, and Mark L. McLaughlin et al., "Short Amphipathic Peptides with Activity Against Bacteria and Intracellular Pathogens," Short Amphipathic peptides with Activ
ity a gainst Bacteria and Intracellular Pathogens ", US patent application Ser. No. 08 / 796,123, filed Feb. 6, 1997).

In practicing the present invention, it is possible to use lytic peptides as generally known in the art. Particularly when the ligand and the peptide are administered separately, it is desirable to have selective toxicity to cells activated by the ligand. Selective toxicity is less important when the ligand and peptide are linked together. This is because in such a case, the concentration of the peptide is efficiently increased in the vicinity of the cell having the receptor for the ligand.

Examples of such peptides include those designated as D1A21 (SEQ ID NO: 5), D2A21 (SEQ ID NO: 6), D5C (SEQ ID NO: 7), and D5C1 (SEQ ID NO: 8). These and other lytic peptides suitable for use in the present invention are described by Jaynes in "Methods for the Design of Amphipathic Peptides Having Enhancement."
d Biological Activities ", US Provisional Application No. 60 / 027,628, 19
It is disclosed in the application filed on October 4, 1996.

Supplemental An “effective amount” of a composition as used in the claims refers to an amount sufficient to cause long-term contraception or infertility in a mammal. Depending on the meaning, G
An “effective amount” of nRH refers to an amount sufficient to temporarily restore fertility in a mammal rendered infertile by the destruction of gonadotropes. “Mammal” as used in the claims shall include humans and non-human mammals.

The entire disclosure of all references cited herein is provided in US Provisional Application No. 6
No. 0 / 041,009 (filed on Mar. 27, 1997), US Provisional Application No. 60/05.
7,456 (filed September 3, 1997) and U.S. Patent Application Serial No. 08/869.
153, filed June 4, 1997, which is incorporated herein by reference. However, in cases of non-reconcilable disputes, the present specification will be adjusted.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 29, 2000 (2000.2.29)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 22

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0087

[Correction method] Change

[Correction contents]

Supplemental An “effective amount” of a composition as used in the claims refers to an amount sufficient to cause long-term contraception or infertility in a mammal. The meaning, the term "effective amount" Gore nad Toro pins hormone refers to an amount sufficient to restore the temporarily fertility to a mammal that is an infertile state by destruction of the gonadotropic cells. “Mammal” as used in the claims shall include humans and non-human mammals.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HU, ID, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (71) Applicant Board of Supervisors of Louisiana State University and Agricultural and Mechanical College Board of Supervisor Baton Rouge P. S. Louisiana State and University of Agricultural and Mechanic College United States 70895-5505 Louisiana, U.S.A. Oh. Box 25055 LSU Agricultural Center Building Room 104 F. Louisiana Agricultural Experiment Station (72) Inventor Enlight, Frederick United States 70808 Louisiana, Baton Rouge North Charlett Coat 5238 (72) Inventor Jane's, Jesse M. United States 27606 Raleigh High Ridge Drive, North Carola Ina 2417 (72) Inventor Hansel, William United States 70808 Louisiana, Baton Rouge Kenilworth Parkway 356 (72) Inventor Elser, Philip H. United States 70809 Baton Rouge, Azuloc Avenue, Louisiana 10231 (72) Inventor Melrose, Patricia A. United States 70803 Louisiana, Baton Rouge Louisiana Station IT University Veterinary Medical Building 2526 F-term (reference) 4C084 BA01 BA09 BA19 BA41 BA44 DA32 DB10 DC50 MA52 NA11 ZA012 ZA862 ZC612 4H045 BA10 BA15 BA19 BA41 CA45 DA47 EA26

Claims (44)

[Claims] 1. A compound comprising (a) a domain of a hormone selected from the group consisting of gonadotropin-releasing hormone and analogs of gonadotropin-releasing hormone, and (b) a domain of a cytolytic peptide. 2. The compound according to claim 1, wherein the hormone domain is directly bound to the cytolytic peptide domain without using an intermediate linking domain connecting the hormone domain and the cytolytic peptide domain. 3. The cytolytic peptide domain is selected from the group consisting of cecropin peptide, melittin peptide, defensin peptide, magainin peptide, sarcotoxin peptide, and analogs of these peptides.
The compound according to the above. 4. The compound of claim 1, wherein said cytolytic peptide domain comprises hecate. 5. The compound of claim 1, wherein said hormone domain comprises gonadotropin releasing hormone. 6. The compound according to claim 1, wherein said compound has the sequence of SEQ ID NO: 3. 7. The compound of claim 1, wherein said compound has the sequence of SEQ ID NO: 4. 8. The compound of claim 1, wherein said hormone domain, said cytolytic peptide domain, or both, comprise a D-type amino acid residue. 9. The compound of claim 8, further comprising a carrier domain to enhance intestinal absorption when the compound is administered orally. 10. A compound according to claim 9 wherein the carrier domain comprising domains of vitamin B _12. 11. A method for long-term contraception or infertility in a mammal, comprising administering to said mammal an effective amount of a gonadotropin releasing hormone and an effective amount of a cytolytic peptide. 12. The method of claim 11, wherein said cytolytic peptide is administered after said gonadotropin releasing hormone is administered. 13. The method of claim 11, wherein said gonadotropin-releasing hormone, said cytolytic peptide, or both comprise D-type amino acid residues. 14. The compound comprising a D-amino acid residue further comprises a carrier domain for promoting intestinal absorption when the compound is administered orally.
The method described in. 15. The method of claim 14 wherein the carrier domain comprising domains of vitamin B _12. 16. A method for long term contraception or infertility in a mammal, comprising administering to the mammal a compound comprising a gonadotropin releasing hormone domain and a cytolytic peptide domain. 17. The method of claim 16, wherein said hormone domain is directly linked to said cytolytic peptide domain without using an intermediate linking domain connecting said hormone domain and said cytolytic peptide domain. 18. The lytic peptide domain is a cecropin peptide,
17. The method of claim 16, wherein the method is selected from the group consisting of melittin peptide, defensin peptide, magainin peptide, sarcotoxin peptide, and analogs of these peptides. 19. The cytolytic peptide domain comprises hecate.
The method described in. 20. The method of claim 16, wherein said compound has the sequence of SEQ ID NO: 3. 21. The method of claim 16, wherein said compound has the sequence of SEQ ID NO: 4. 22. A method for temporarily restoring fertility in a mammal rendered infertile by selective destruction of pituitary gonadotropes, comprising administering to said mammal an effective amount of a gonadotropin-releasing hormone. Administering to the subject. 23. The method according to claim 22, wherein an effective amount of a gonadotropin-releasing hormone and an effective amount of a cytolytic peptide are administered to a previously sterilized mammal to restore the fertility of the mammal. Method. 24. The method according to claim 22, wherein an effective amount of a compound containing a gonadotropin-releasing hormone domain and a cytolytic peptide domain is administered to a previously sterilized mammal to restore the fertility of the animal. the method of. 25. The method of claim 11, wherein said mammal is a dog. 26. The method of claim 11, wherein said mammal is a cat. 27. The method of claim 11, wherein said mammal is a cow or a bull. 28. The method of claim 11, wherein said mammal is a pig. 29. The method according to claim 11, wherein said mammal is a horse. 30. The method of claim 11, wherein said mammal is a sheep. 31. The method of claim 11, wherein said mammal is a human. 32. The method of claim 16, wherein said mammal is a dog. 33. The method of claim 16, wherein said mammal is a cat. 34. The method according to claim 16, wherein said mammal is a cow or a bull. 35. The method of claim 16, wherein said mammal is a pig. 36. The method according to claim 16, wherein said mammal is a horse. 37. The method of claim 16, wherein said mammal is a sheep. 38. The method of claim 16, wherein said mammal is a human. 39. A method for selectively killing gonadotropic cells of the pituitary gland of a mammal, comprising: (a) an effective amount of a gonadotropin-releasing hormone;
A method comprising administering an effective amount of a cytolytic peptide. 40. A method for selectively killing gonadotropic cells of the pituitary gland of a mammal, comprising administering to the mammal an effective amount of a compound comprising a gonadotropin releasing hormone domain and a cytolytic peptide domain. A method that includes 41. A method for selectively killing a mammalian gonadotropin receptor-containing neuron, comprising: (a) an effective amount of a gonadotropin releasing hormone; and (b) an effective amount of a cytolytic peptide. A method comprising administering. 42. A method for selectively killing a mammalian gonadotropin receptor-containing neuron, comprising administering to said mammal an effective amount of a compound comprising a gonadotropin releasing hormone domain and a cytolytic peptide domain. Method. 43. The method of claim 11, wherein said mammal is sexually immature. 44. The method of claim 16, wherein said mammal is sexually immature.