Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR
CONTRACEPTION IN OR STERILIZATION OF MAMMALS
Frederick M. Enright, Jesse M. Jaynes, William Hansel,
Philip H. Elzer, Patricia A. Melrose
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
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hormone receptors (such as the LH receptor) and then coupled to a toxin. All
cells with LH
receptors should then be destroyed. Although various cell types have not been
characterized
in dog corpora lutea, destruction of any luteal cell type could potentially
result in luteolysis if
cell types communicate." (citations omitted)
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:
"7lssue-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
/3-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 GTI-1 neuronal cells," Mot.
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 that LH acts directly on neuroendocrine neurons
in the brain.
See also Z. Lei et al., "Signaling and transacting factors in the
transcriptional inhibition of
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3
gonadotropin releasing hormone gene by human chorionic gonadotropin in
immortalized
hypothalamic GTI-7 neurons," Mol. & Cell. Endocrinology, 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, a-amanitin,
diphtheria toxin,
pseudomonas exotoxin, shiga toxin, melphalan, methotrexate, nitrogen mustard,
doxorubicin,
and daunomycin. None of these toxins is believed to be toxic due to direct
interaction with
the cell membrane. In the in vivo experiments reported, the most effective
time course was
reported to be weekly injections for 4 weeks. (E.g., Pat. 5,488,036, col. 20,
lines 46-47.)
Because most of the 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
the toxin to the
GnRH agonist. The toxins must in general be internalized into the target cells
to have effect,
and do not act on cell membranes; in addition, at least some of these toxins
must be
secondarily transported from the membrane-bound vesicle into the cytoplasm to
interact with
ribosomes, mitochondria, or other cellular components.
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4
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
internalised. Administering a combination of gonadotripin-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.
One embodiment of the present invention provides for the use of an
effective amount of gonadotropin-releasing hormone and an effective amount of
an
amphipathic lytic peptide that acts on cell membranes for producing long-term
contraception or sterility in a mammal.
Yet another embodiment of the present invention provides for the use of a
compound comprising a first domain and a second domain wherein (a) the first
domain
comprises a hormone selected from the group consisting of gonadotropin-
releasing
hormone, the beta subunit of luteinizing hormone, the beta subunit of
chorionic
gonadotropin and analogues of these hormones and (b) the second domain
comprises an
amphipathic lytic peptide that acts on cell membranes for producing long-term
contraception or sterility in a mammal.
The compounds used in the present invention are relatively small, and will not
be
antigenic (Lytic peptides are known not to be very antigenic; and the ligands
are not
antigenic at all.) The compounds may be administered in a single dose,
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 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. If a fusion peptide is used, it has been
unexpectedly discovered
that a linking moiety is not necessary to join the ligand to the lytic
peptide: one may be
bonded directly to the other, without the need for any intervening linkage;
bonding is
preferably performed by bonding one end of the ligand to one end of the
peptide, not by
bonding to the middle of either. The toxin, the lytic peptide, does not need a
translocation
domain, and need not be internalised, as it binds to and acts directly on the
activated cell
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4a
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 the same propensity to lyse cell membranes
as do the L-
amino acid forms. Compounds of the present invention (whether administered as
a fusion
peptide or separately) may therefore be administered either in L-form or D-
form. D-form
peptides, although generally more expensive than L-form, have the advantage
that they are
not degraded by normal enzymatic processes, so that the D-form peptides may
therefore
be administered orally and generally have a longer biological half-life. Oral
administration of the D-form peptide may be enhanced by linking the peptide/
hormone
fusion product to a suitable carrier to facilitate uptake by the intestine,
for example vitamin
B12, following generally the
25
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B,2-conjugation technique of G. Russell-Jones et al., "Synthesis of LHRH
Antagonists Suitable
for Oral Administration via the Vitamin B12 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 that substitutions at the 6 and 10 positions
of the GnRH
decapeptide can produce "superagonists" having greater binding affinity to the
GnRH receptor
than does GnRH itself. These "superagonists" include goserelin, leuprolide,
buserelin, and
nafarelin. See U.S. Patent 5,488,036.
Without wishing to be bound by this theory, it is believed that the 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) these activated cells. When the
gonadotrophic
cells in the pituitary are destroyed and are deprived of GnRH from the brain,
the pituitary no
longer secretes follicle stimulating hormone (FSH) or luteinizing hormone
(LH), rendering the
mammal temporarily or permanently sterile.
Although the ligand and the lytic peptide may be administered separately, it
is
preferred to link the two in a single molecule, because such a linkage greatly
increases the
effective concentration of the lytic peptide in the vicinity of ligand-
activated cells.
Furthermore, this increase in the effective lytic peptide concentration can
obviate the need for
activation of the cells, allowing the peptide to be linked to a binding site
of a ligand alone,
without needing to include the "remainder" of a native ligand that would
normally be needed
for activating the target cells. This linkage may be in either order: for
example,
GnRH/peptide or peptide/GnRH. Examples are 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 the initial pyro-glutamic acid residue of the
GnRH or the
GnRH portion of a fusion peptide may be substituted with glutamine without
substantially
changing the activity of the respective peptides. See, e.g., SEQ. ID Nos. 9,
3, and 4.)
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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 the
histology of each was
examined microscopically.
Treatment I applied tissue culture medium alone as a control. The histology of
this
tissue after treatment appeared normal.
Treatment 2 was an application of 5 nanograms of GnRH (SEQ. ID NO. 1) per mL
of medium. The histology of this tissue after treatment was normal; a small
degree of cellular
vacuolization was noted. For comparison, the concentration of GnRH in normal,
untreated
rats varies from as low as 1 ng/mL to as high as 20 ng/mL during the LH surge
phase of the
estrous cycle.
Treatment 3 was an application of 50 M of the lytic peptide hecate (SEQ. ID
NO.
2) in the medium. The histology of this tissue after treatment appeared
normal.
Treatment 4 was an initial application of 5 nanograms of GnRH per mL of medium
for 15 minutes. Following this incubation, the medium containing GnRH was
removed, and
the tissue was washed once with plain medium. This medium was then removed,
and was
replaced with medium containing 50 M of the lytic peptide hecate. Widespread
basophilic
(gonadotropic) cellular destruction was observed after this treatment.
Treatment 5 was an application of 50 AM of the fusion peptide modified
GnRH/hecate (SEQ. ID NO. 3). Widespread basophilic (gonadotropic) cellular
destruction
was observed after the treatment.
Treatment 6 was an initial application of the fusion peptide GnRH/hecate (SEQ.
ID
NO. 3), followed by a second application of the fusion peptide GnRH/hecate two
hours later.
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 the control rats completed two estrous cycles. The rats were then
sacrificed and
necropsied. The reproductive organs appeared histologically normal.
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Example 8
Two sexually immature female rats from the same litter as those of Example 7
(age 33
days) were given two intravenous injections of 500 g GnRH/hecate fusion
peptide in saline
24 hours apart. After the rats reached breeding age, they were examined 105
days post-
inoculation. The external genitalia appeared small. Unlike the control rats,
insertion of a
cotton-tipped swab into the vagina was difficult. During a fourteen-day
monitoring period 107
days to 121 days post-inoculation, neither of the treated rats demonstrated
estrous or
metestrous. The rats were then sacrificed and necropsied. The peptide-treated
rats had
thinned, inactive uterine and oviductal epithelia. Their ovaries contained no
large follicles,
and had a high number of atretic follicles (i.e., those that had failed to
ovulate).
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 that were present appeared old. Follicles were large, and
had not ruptured.
Uterine morphology was consistent with hormonal inactivity. The pituitaries
from this group
were slightly smaller than the pituitaries from the saline control group.
Histology revealed a
60% to 70% reduction in the number of pituitary basophilic cells compared to
the controls.
Treatment 11 was injection of 100 L of a 1.35 mM solution of GnRH (162 g) in
saline, followed 15 minutes later by injection with 100 L of a 1.35 mM
solution of hecate
(337 g) in saline. The same two-step treatment was repeated 24 hours later.
The rats in this
group showed altered ovarian histology. Few corpora lutea were present, and
those that were
present appeared old. Follicles were large, and had not ruptured. Uterine
morphology was
consistent with hormonal inactivity. The pituitaries and the pituitary
histology were similar to
those observed in Treatment 10.
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Treatment 12 was injection of 100 L of a 1.35 mM solution of hecate (337 g)
in
saline. The treatment was repeated after 24 hours. The rats in this group
exhibited normal
ovarian function (e.g., normal follicles and new corpora lutea). The
pituitaries and the
pituitary histology were similar to those observed in Treatment 9.
Treatment 13 was injection of 100 L of a 1.35 mM solution of GnRH (162 g) in
saline. The treatment was repeated after 24 hours. The rats in this group
exhibited normal
ovarian function (e.g., normal follicles and new corpora lutea). The
pituitaries and the
pituitary histology were similar to those observed in Treatment 9.
Treatment 14 was identical to Treatment 10, except that the GnRH/hecate fusion
peptide was further purified by HPLC. The rats in this group showed an arrest
of normal
ovarian follicular development. Few corpora lutes were present, and those that
were present
appeared old. Follicles were large, and had not ruptured. Uterine morphology
was consistent
with hormonal inactivity. The pituitaries and the pituitary histology were
similar to those
observed in Treatment 10.
These experiments demonstrate that GnRH and the lytic peptide may be
administered
either separately or as a fusion peptide, although the fusion peptide is
preferred as it is
expected to be more active at lower doses.
Although experiments to determine optimum dosages had not been performed by
the
time this application is being filed, a person of ordinary skill in the art,
who is given the
teachings of the 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 both 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 the 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 II
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
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conjugate; this compound should specifically affect GnRH neurons controlling
reproduction
and the gonads. (Other 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
with a carrier such as vitamin B12) 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, this invention may be used in the 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 either 48 or 96 hours after
treatment. The
ovaries, uterus, pancreas, liver, spleen, lungs, heart, thyroid, and adrenal
glands were fixed in
10% buffered formalin; sectioned; and stained with H&E (hematoxylin and eosin)
stain;
except that the pituitary glands were stained with PAS (periodic acid-Schiff)
stain with no
counter-stain. The treatments were selected so that each animal received an
equimolar amount
of the compound with which it was treated.
Treatments 15 and 16 were IV-injection with 674 g of D-hecate in 200 jiL
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 the
organs of either animal. The pituitary glands of both rats contained a normal
number of PAS-
positive cells.
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Treatments 17 and 18 were IV-injection with 334 g of GnRH in 200 ;cL saline
(1.35 mM). The rat in treatment 17 was sacrificed 48 hours after injection,
and the rat in
treatment 18 was sacrificed 96 hours after injection. No gross lesions were
noted in the
organs of either animal. The pituitary glands of both rats contained a normal
number of PAS-
positive cells.
Treatments 19-22 were IV-injection with 1 mg GnRH-hecate fusion peptide (SEQ.
ID
NO. 3) in 100 L saline (2.7 mM). The rats in treatments 19 and 20 were
sacrificed 48
hours after injection, and the rats in treatments 21 and 22 were sacrificed 96
hours after
injection. No gross lesions were noted in the organs of any of the four
animals, other than
the pituitary. The pituitary glands of the animals from treatments 19 and 20
were slightly
enlarged, hyperemic, and edematous. The pituitary glands of the animals from
treatments 21
and 22 were slightly hyperemic, but of expected size. The pituitary glands of
all four rats
showed a marked depletion of PAS-positive cells; it was estimated that the
depletion was 80 to
90% compared to those 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 with PAS.)
It was thus seen that the 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 the 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 the
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
that pituitary cells no longer secreted either LH or FSH following the
effective treatments.
Example 24
Hecate is an amphipathic lytic peptide that acts on cell membranes without
being
internalized. It is a synthetic peptide analog of melittin, the primary toxin
in honeybee
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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 synthesized D-Lys6GnRH (SEQ. ID NO. 13), so that hecate could be
conjugated to the D-Lys6, a position that could minimize interference with
binding of the
GnRH domain to the 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 the 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 with
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 the 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 M
Treatment 28 hecate-bLH, 5 M
Treatment 29 hecate-bLH, 1 M
Treatment 30 hecate (alone), 10 M
Treatment 31 hecate (alone), 5 M
Treatment 32 hecate (alone), 1 M
Significant killing of small luteal cells was observed following 22 hr.
incubation with
M hecate alone, and with 5 AM hecate alone (approximately 50% killing). Cell
death for
1 M 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 death as
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compared to treatment with hecate alone. The hecate-bLH conjugate killed
approximately
twice the number of cells as were killed by hecate alone at the same
concentrations.
Observed levels of lactic dehydrogenase activity also demonstrated that 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 than were the small luteal cells,
and (2) that, as
had been the case with the small luteal cells, the granulosa cells were
significantly more
susceptible to hecate-bLH at even the lowest concentration (1 &M) than they
were to hecate
alone. At I 1M, the hecate-bLH conjugate killed about twice the number of
target cells as did
hecate alone. Again, the levels of lactic dehydrogenase released following the
hecate-bLH 1
M treatment were significantly higher than the levels of enzyme released
following treatment
with 1 M 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 that the selective killing of target cells resulted from
the physical
proximity of the lytic peptide to the 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 that activation of the target cells
was required for
increased susceptibility to lysis. These data demonstrate that such activation
is not required.
These data are, however, consistent with our other data showing that cell
activation is also a
route that can lead to increased susceptibility to the lytic peptide.
Examples 35-38
Another set of experiments was performed to study the 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),
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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 the
procedures of M.
Rahmanian et at., "Histological and immunocytochemical characterization of
pituitary cell
types in ponies," Proc. 13th Soc. Equine Nutrition & Phys. Symp., pp. 348-349
(1993); M.
Rahmanian et at., "Immunocytochemical localization of luteinizing hormone and
follicle-
stimulating hormone in the equine pituitary," J. Anirn. Sci., vol. 76, pp. 839-
846 (1998); M.
Rahmanian et at., "Immunocytochemical localization of prolactin and growth
hormone in the
equine pituitary," Animal Sci., vol. 75, pp. 3010-3018 (1997); and P. Melrose
et at.,
"Comparative topography of the immunoreactive alpha-melanocyte-stimulating
hormone
neuronal system in the brains of horses and rats," Brain Belt. & Evol., vol.
32, pp. 226-235
(1988).
Brains were serially sectioned on a Vibratome m 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 with cresyl violet, or
were stained
i mmunocyto chemically for GnRH or the GnRH precursor, VP, OXY, or tyrosine
hydroxylase
(the rate-limiting enzyme in catecholamine synthesis). In addition to the
staining procedures
cited above, we also used the immunocytochemical staining procedures of P.
Melrose er at.,
"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, Be/z. & 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 g GnRH (a normal physiological dose) (two rats)
Treatment 36: 1.62 g 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 g GnRH, followed 11 minutes later by 0.337 g 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
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WO 99/11282 PCT/US98/18117
14
hecate alone reduced pituitary weights by 13% and 14%, respectively, and
reduced the
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 the 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 the
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 with 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 the number of alpha-MSH-
stained cells in
this subdivision of the pituitary. No pathological changes were noted in any
other tissues.
Since neurons in the brain do not regenerate, severe damage to these neurons
could
make sterilization with a GnRH/lytic peptide combination permanent (but
temporarily
reversible by administration of gonadotrophic hormones).
Examples 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 the 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
CA 02302392 2000-02-29
WO 99/11282 PCTIUS98/18117
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 g
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 that both
control
and experimental animals released LH in response to the GnRH, suggesting that
there 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 the next day (day
47) for
postmortem histological analysis. We found that the numbers of tertiary
follicles, corpora
lutes, and GnRH-induced ovulations were reduced by GnRH-hecate treatment.
Ovarian and
pituitary weights were reduced by the 10 mg GnRH-hecate treatment. In tissues
from the
GnRH-hecate treatments, observed irimunoreactive 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 the 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
pituitary cells
in the pars distalis to 177% of that for control rabbits; this increase
appeared to reflect
increased numbers of cells staining alpha-MSH, and reduced numbers of cells
staining for LH.
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
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16
GnRH; however, the amount of LH released was lower in the treated animals than
in the
controls. The GnRH-hecate treatment reduced the numbers of tertiary ovarian
follicles, and
the numbers of GnRH-induced ovulations. No effects were noticed either on
peripheral
tissues or on pituitary weight. The effects of GnRH-hecate on CNS morphology
and
immunocytochemical results were similar to those described above in Examples
35-46.
Again, the effects were more pronounced on GnRH neurons than on staining of
pituitary
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 that the GnRH-hecate conjugate
selectively
damaged GnRH receptor-bearing cells in the brain (neurons) and in the
pituitary
(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 the following
observations:
(1) No cytotoxic changes were seen in neurons that lacked GnRH receptors. (2)
No changes
were seen in pituitary 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 pituitary).
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.
Lyric Peptides Useful in the Present Invention
It is believed (without wishing to be bound by this theory) that lytic
peptides act by
disrupting cell membranes. "Resting" eukaryotic cells protect themselves
through their ability
to repair the resulting membrane damage. By contrast, activated cells (e.g.,
cells stimulated
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WO 99/11282 PCT/US98/18117
17
by GnRH) are unable (or less able) to repair damaged membranes. Because GnRH-
activated
pituitary cells have a diminished capacity to repair membranes, they are
preferentially
destroyed by lytic peptides, while adjacent non-activated cells repair their
membranes and
survive.
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
those of insects, amphibians, and mammals. They typically comprise 23-39 amino
acids,
although they can be smaller. Thcy 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 at., "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. Natl.. Acad. Sci. USA, vol. 84, pp. 3628-3632
(1987); Ganz
et at., "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 with another non-polar
residue, etc.); by
substitutions that preserve the charge distribution (e.g., replacing an acidic
residue with
another acidic residue, or a basic residue with another basic residue, etc.);
or by lengthening
or shortening the amino acid sequence while preserving its amphipathic
character or its charge
distribution. Lytic peptides and their sequences are disclosed in Yamada et
at., "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 at., "Antibacterial and antimalarial properties of peptides that are
cecropin-melittin
hybrids," Febs Letters, vol. 259, pp. 103-106 (1989); Tessier et al.,
"Enhanced secretion
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18
from insect cells of a foreign protein fused to the 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 at., "Shortened cecropin A-melittin hybrids. Significant
size reduction
retains potent antibiotic activity," Febs Letters, vol. 296, pp. 190-194
(1992); Macias et at.,
"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 et at., "Airway epithelial cells are the site of
expression of a
mammalian antimicrobial peptide gene," Proc. Natl. Acad. Sci. USA, vol. 90,
pp. 4596 if
(1993); Selsted et al., "Purification, primary structures and antibacterial
activities of 3-
defensins, a new family of antimicrobial peptides from bovine neutrophils," J.
Biol. Chem.,
vol. 268, pp. 6641, (1993); Tang et at., "Characterization of the disulfide
motif in BNBD-
12, an antimicrobial 3-defensin peptide from bovine neutrophils," J. Biol.
Chem., vol. 268,
pp. 6649 f (1993); Lehrer et al., Blood, vol. 76, pp. 2169-2181 (1990); Ganz
et at., Sem.
Resp. Infect. I., pp. 107-117 (1986); Kagan et at., 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 at., J. Biol. Chem., vol. 263, pp. 9573-9575 (1988); Jaynes et at.,
"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 at., "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 at.,
"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 synthesis of specialized proteins capable
of
disrupting bacterial cell membranes, resulting in lysis and cell death. Among
these specialized
proteins are those known collectively as cecropiris. 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
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19
contains a sequence that will form an amphipathic alpha-helix. Andrequ et al.,
"N-terminal
analogues of cecropin A: synthesis, antibacterial activity, and conformational
properties,"
Biochem., vol. 24, pp. 1683-1688 (1985). The carboxy-terminal half of the
peptide comprises
a hydrophobic tail. See also Boman et al., "Cell-free immunity in Cecropia,"
Eur. J.
Biochem., vol. 201, pp. 23-31 (1991).
A cecropin-like peptide has been isolated from porcine intestine. Lee et al.,
"Antibacterial peptides from pig intestine: isolation of a mammalian
cecropin," Proc. Natl.
Acad. Sci. USA, vol. 86, pp. 9159-9162 (1989).
Cecropin peptides have been observed to kill a number of animal pathogens
other than
bacteria. See Jaynes et al., "In Vitro Cytocidal Effect of Novel Lytic
Peptides on
Plasmodium falciparum and Trypanosoma cruzi," FASEB, 2878-2883 (1988);
Arrowood et
al., "Hemolytic properties of lytic peptides active against the sporozoites of
Cryptosporidium
parvwn," J. Protozool., vol. 38, No. 6, pp. 161S-163S (1991); and Arrowood et
al., "In vitro
activities of lytic peptides against the sporozoites of Cryptosporidium
parvwn," 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 with 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 at., "Primary structure of sarcotoxin I, an
antibacterial
protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae,"
J. Biol.
Chem., vol. 260, pp. 7174-7177 (1985). Although highly divergent from the
cecropins and
defensins, the 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 they
named PGS
CA 02302392 2000-02-29
WO 99/11282 PCT/US98/18117
and Gly10Lys?2PGS. Gibson et al., "Novel peptide fragments originating from
PGL, 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
that the Xenopus-derived peptides have antimicrobial activity, and renamed
them magainins.
Zasloff, "Magainins, a class or 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-1
(or Shiva 1) has
been shown to destroy intracellular Brucella abortus-, Trypanosoma cruzi-,
Cryptosporidium
parvum-, and infectious bovine herpes virus I (IBR)-infected host cells, with
little or no toxic
effects on noninfected mammalian cells. 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 at., "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 with 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 Mulls,"
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.
CA 02302392 2006-09-25
21
Also of interest are the synthetic peptides disclosed in the following
patents,
peptides that 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 with Activity against
Bacteria
and Intracellular Pathogens", United States Patent 6,566,334 of May 20, 2003.
Lytic peptides such as are known generally in the art may be used in
practicing the
present invention. Selective toxicity to ligand-activated cells is desirable,
especially when
the ligand and peptide are administered separately. Selective toxicity is less
important
when the ligand and peptide are linked to one another, because in that case
the peptide is
effectively concentrated in the immediate vicinity of cells having receptors
for the ligand.
Examples of such peptides are those designed DIA21 (SEQ. ID NO. 5) D2A21
(SEQ. ID NO. 6), D5C (SEQ. ID NO. 7), and D5CI (SEQ. ID NO. 8).
Miscellaneous
As used in the claims, an "effective amount" of a composition is an amount
that it
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 the claims, the term "mammal" is intended to include both human and
non-human
mammals.
Reference is also made herein to WO 98/42365 published October 1, 1998, United
States provisional Application 60/057,456, filed September 3, 1997; United
States Patent
6,300,471 and WO 98/42365 published October 1, 1998. In the event of an
otherwise
irreconcilable conflict, however, the present specification shall control.
CA 02302392 2000-07-27
22
SEQUENCE LISTING
(1) GENERAL INFORMATIONS
(i) APPLICANT: Board of Supervisors of Louisiana State
University and Agricultural and
Mechanical College
(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: McFadden, Fincham
(B) STREET: 606-225 Metcalfe Street
(C) CITY: Ottawa
(D) STATE: ON
(E) COUNTRY: Canada
(F) POSTAL CODE: K2P 1P9
(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: 2,302,392
(B) FILING DATE: O1-SEP-1998
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McFadden, Fincham
(B) REGISTRATION NUMBER: 3083
(C) REFERENCE/DOCKET NUMBER: 5318-6
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 234-1907
(B) TELEFAX: (613) 234-5233
(2) INFORMATION FOR SEQ ID NO:1:
(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
CA 02302392 2000-07-27
23
(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 N0:2:
(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
Lye Ala Lou Lys Lys Ala Leu
(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..33
(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 Lou Ala Leu Lys
1 5 10 15
Ala Leu Lys Lys Ala Leu Lys Lys Leu Lye Lys Ala Leu Lys Lye Ala
20 25 30
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Leu
(2) INFORMATION FOR SEQ ID NO:4:
(L) 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..33
(D) OTHER INFORMATION: /note- "This sequence is a
hecate/modified 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 Gln 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 DiA21."
(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
Lye Ala Phe Lys Lys Ala Phe
(2) INFORMATION FOR SEQ ID NO:6:
CA 02302392 2000-07-27
(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: l..23
(D) OTHER INFORMATION: /note- "This sequence is D2A21."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Ala Lys Lys Phe Ala Lys Lye Phe Lys Lye Phe Ala Lys Lys Phe
1 5 10 15
Ala Lys Phe Ala Phe Ala Phe
(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 Lye Leu
1 5 10 15
Ala Arg Lye Ile 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:
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26
(A) NAME/KEY: Peptide
(B) LOCATION: 1..37
(D) OTHER INFORMATION: /note- "This sequence is D5C1."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Lys Arg Lys Arg Ala Val Lys Arg Val Gly Arg Arg Leu Lys Lys Lou
1 5 10 15
Ala Arg Lys Ile Ala Arg Lou Gly Val Ala Lys Lou Ala Gly Lou Arg
20 25 30
Ala Val Lou Lys Phe
(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:
Gln His Trp Ser Tyr Gly Lou 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."
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg
1 5 10 15
(2) INFORMATION FOR SEQ ID NOsl2:
(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 a
hecate-bLH fusion peptide."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Phe Ala Leu Ala Leu Lys Ala Leu Lys Lye 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 Gln
20 25 30
Cys Ala Leu Cys Arg Arg
(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
