Note: Descriptions are shown in the official language in which they were submitted.
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LONG-ACTING GONADOTROPIN-RELEASING HORMONE
ANALOGS AND METHODS OF USE THEREOF
FIELD OF INVENTION
The present invention relates to the design, synthesis and biological
evaluation of
potent long-acting gonadotropin-releasing hormone (GnRH) analogs, and to their
therapeutic use in fertility regulation or as contraceptives, and in treating
and/or
preventing sex hormone-related diseases or conditions.
BACKGROUND OF THE INVENTION
Gonadotropin-releasing hormone (GnRH; pGlu-His-Trp-Ser-Tyr-Gly-Leu-
Arg-Pro-Gly-NHZ) is a key integrator between the neural and the endocrine
systems
and plays a pivotal role in the regulation of the reproductive system. This
neurohormone is synthesized in hypothalamic neurosecretory cells and is
released in
a pulsatile pattern into the hypothalamo-hypophyseal portal circulation. This
pattern
of GnRH secretion provokes the release of the gonadotropins, luteinizing
hormone
(LH) and follicle-stimulating hormone (FSH), from the anterior pituitary,
which, in
turn, stimulate gonadal steroidogenesis and gametogenesisl~2. Chronic
administration
of GnR_H_ or its super active agonists results in down-regulation of GnR_H_
receptors
and desensitization of the pituitary gonadotrophs and thus causes the
suppression of
gonadotropin secretion3~4. Synthetic GnRH analogs, agonists as well as
antagonists,
have attracted remarkable interest because of their potential applications for
the
treatment of reproductive diseases, such as prostate and breast cancer, and
their
possible use as contraceptives5~6.The mechanism of action of GnRH analogs in
these
diseases is believed to be at least partly related to gonadal steroids
deprivation, which
results from down-regulation and desensitization of the pituitary
gonadotrophs. In
cancer therapy however, GnR_H_ analogs have been demonstrated to exert direct
inhibitory effects on the growth of cancer cells through GnR_H_ receptors that
are
present in prostate, breast and ovarian cancer5~7.
The relatively short half life time of GnR_H_ in the general circulation (2-4
min)5 is advantageous for the establishment of a pulsatile secretion pattern.
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However, potent agonists or antagonists having a prolonged bioactivity are
certainly
needed in the clinic for the induction of desensitization or contraception.
Since the
discovery of GnRH, more than 3000 analogs of the peptide have been synthesized
and evaluated for their bioactivity. Most of the super agonists usually
incorporate a
D-amino acid, substituting for Gly in position 6, and an N-ethylamide instead
of the
terminal Gly-NH2 in position 10. These chemical modifications enhance the
bioactive (3-turn confornzation of GnRH at the Gly6-Leu7 bond and decrease the
susceptibility of the peptide to proteolytic degradation8,9. In addition,
increasing the
hydrophobicity of the peptide, by incorporation of appropriate amino acid
residues,
usually results in increased biological potency, probably due to a decrease in
the rate
of clearance from the general circulation and an increase in its apparent
binding
constant to GnR_H_ receptors10,11
Conjugation of bulky moieties, such as tetramethylrhodamine, to the E-amino
group of [D-Lys6]GnR_H_ do not significantly affect the bioactivity of GnR_H-
analogsl2. Thus, in an attempt to produce effective targeted chemotherapy
against
cancer, several chemotherapeutic agents have been covalently attached to the E-
amino group of [D-Lys6]GnRHl3-15. For example, attachment of the cytotoxic
compound 2-(hydroxymethyl)anthraquinone hemiglutaratel6 (Fig. 1) to [D-
Lys6]GnR_H_ has generated a powerful agonist, T-98, which bound to GnRH
receptors
on human breast cancer cells with a binding affinity similar to that of [D-
Lys6]GnRH. This analog, however, exhibited a five-fold higher biological
activity
than the parent peptide and inhibited the growth of human breast and prostate
cancerl4.
Long acting antagonists of GnR_H_ produced by modification of positions 5 or 6
in the peptide sequence are disclosed for example in US 6,214,798, and
references
therein. The present inventors have published their initial findings
concerning the
longer duration of action of a particular GnRH agonist [D-Lys6]GnR_H_
conjugated to
emodic acidlaa
Development of new potent GnRH agonists and antagonists is of a major
interest to the medical community, largely because of their clinical
application in
modulating the reproductive system as well as in therapy of cancer and various
other
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sex-hormone related conditions. Thus, there is an urgent need in the art for
potent
GnRH agonists and antagonists that do not exhibit any toxic effects, and that
can be
safely administered as contraceptives, and as therapeutic agents in the
treatment of
sex-hormone related conditions and diseases.
SUMMARY OF THE INVENTION
The present invention relates to the design, synthesis and biological
evaluation
of potent long-acting gonadotropin-releasing hormone (GnRH) agonists or
antagonists, comprising GnRH analogs conjugated to emodic acid or an emodic
acid
derivative. These agonists and antagonists have prolonged duration of action
and are
thus useful in the control of fertility or as a contraceptive, and in treating
andlor
preventing sex-hormone related diseases or conditions.
According to a currently preferred embodiment, the present invention relates
to
the design, synthesis and biological evaluation of the potent long-acting GnRH
agonist, [D-Lys6(Emo)]GnRH. [D-Lys6(Emo)]GnRH binds GnRH receptors with
high affinity to induce LH release, is devoid of toxicity, and is thus useful
in the
control of fertility or as a contraceptive, and in treating andlor preventing
sex-
hormone related diseases or conditions.
According to another currently preferred embodiment, the present invention
relates to the design, synthesis and biological evaluation of the potent long-
acting
GnRH antagonist, [D-Pyre, D-Phe2, D-Trp3, D-Lys6(Emo)]GnRH (denoted herein as
[D-Lys6(Emo)]GnRH-Antg). [D-Lys6(Emo)]GnRH-Antg binds GnR_H_ receptors to
prevent LH release, is devoid of toxicity, and is thus useful in the control
of fertility
or as a contraceptive, and in treating and/or preventing sex-hormone related
diseases
or conditions.
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Additional GnRH agonists according to the invention are represented by [SEQ
ID NO:1 ] peptides of the general formula:
Pyr-His-Trp-Y-Tyr-X-Leu-Arg-Pro-Z
wherein X is selected from Ser(Emo), D-Ser(Emo), Lys(Emo), D-
Lys(Emo), D-Dab(Emo), D-Orn(Emo), D-hSer(Emo); Y is selected from D-
Lys(Emo), D-Dab(Emo), D-Orn(Emo), D-Ser(Emo), D-hSer(Emo); Z is
selected from Gly, Ethylamine, D-Ala; Pyr denotes pyroglutamic acid, Dab
denotes diaminobutyric acid; and pharmaceutically acceptable salts, amides,
esters and hydrates thereof.
Additional GnR_H_ antagonists according to the invention are represented by
[SEQ ID N0:2,] peptides of the general formula:
Ac-D-Nal-4-chloro-D-Phe-[3(3-Pyridyl)-D-Ala-X-Y-Z-Leu-W-Pro-D-Ala
wherein, X is selected from Ser(Emo), hSer(Emo); Y is selected from
Lys(Emo), Dab(Emo), Z is selectcd from D-Lys(Emo), D-Dab (Emo), D-
Orn(Emo), D-Ser(Emo) D-hSer(Emo); W is selected from Lys(Emo),
Dab(Emo), Orn(Emo), Ser(Emo), hSer(Emo); and pharmaceutically acceptable
salts, esters, amides and hydrates thereof.
Additional GnRH antagonists according to the invention are represented by
[SEQ ID N0:3] peptides of the general formula:
Ac-Nal-4-chloro-D-Phe-D-Pal-X-Tyr-Y-Leu-Arg-Pro-D-Ala
wherein X is selected from Ser(Emo), hSer(Emo); Y is selected from D-
Cit(Emo), D-Lys(Emo), D-Dab(Emo), D-Orn(Emo), D-Ser(Emo) D-
hSer(Emo); D-Nal denotes D-3-(2'-naphtyl)-alanine; D-Pal denotes 3-(3'-
pyridyl)-alanine; and pharmaceutically acceptable salts, amides, esters and
hydrates thereof.
In one embodiment, the present invention provides a method of controlling
fertility in a subject, comprising the step of administering to the subject a
gonadotropin releasing hormone (GnRH) agonist or antagonist peptide having the
formula [D-Lys6(Erno)]GnRH, or a pharmaceutically acceptable salt or hydrate
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thereof, in an amount effective to control fertility in the subject. In one
embodiment,
fertility is controlled to prevent conception in a female mammalian subject.
In yet another embodiment, the present invention provides a method of
contraception in a subject, comprising the step of administering to the
subject a
gonadotropin releasing hormone (GnRH) agonist or antagonist peptide having the
formula [D-Lys6(Emo)]GnRH, or a pharmaceutically acceptable salt or hydrate
thereof.
Furthermore, in another embodiment, the present invention provides a method
of treating a sex hormone-related disease or condition in a subject, the
method
comprising the step of administering to the subject a gonadotropin releasing
hormone
(GnRH) agonist or antagonist peptide having the fornmla [D-Lys6(Emo)]GnRH, or
a
pharmaceutically acceptable salt or hydrate thereof, in an amount effective to
treat the
disease or condition in said subject.
Furthermore, in another embodiment, the present invention provides a method
of preventing a sex hormone-related disease or condition in a subject, the
method
comprising the step of administering to the subject a gonadotropin releasing
hormone
(GnRH) agonist or antagonist peptide having the formula [D-Lys6(Emo)]GnRH, or
a
pharmaceutically acceptable salt or hydrate thereof, in an amount effective to
prevent
the disease or condition in the subject.
Furthermore, in another embodiment, the present invention provides a method
of promoting the release of LH or FSH in a subject, the method comprising the
step of
administering to the subject a gonadotropin releasing hormone (GnRH) agonist
or
antagonist peptide having the formula [D-Lys6(Emo)]GnRH, or a pharmaceutically
acceptable salt or hydrate thereof, in an amount effective to promote the
release of LH
or FSH in the subject.
Furthermore, in another embodiment, the present invention provides a method
of suppressing or preventing the release of LH or FSH in a subject, the method
comprising the step of administering to the subject a long-acting gonadotropin
releasing hormone (GnRH) agonist or antagonist peptide having the formula [D-
Lys6(Emo)]GnRH, or a pharmaceutically acceptable salt or hydrate thereof, in
an
amount effective to prevent or suppress the release of LH or FSH in the
subject.
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In one embodiment, the subject is a mammal. In another embodiment, the
subject is a human. In another embodiment the subject is a non-human mammal.
In
another embodiment the subject is a non-mammalian vertebrate. In another
embodiment, the subject is a male subject. In another embodiment, the subject
is a
female subject.
In one embodiment, the sex hormone-related disease or condition is a cancer,
for example prostate cancer, breast cancer, ovarian cancer, cervical cancer, a
tumor of
the pituitary, testicular cancer or uterine cancer. In another embodiment, the
sex
hormone-related disease or condition is a benign disease or condition, for
example
benign prostatic hyperplasia, precocious puberty, aberrant sexual behavior,
late luteal
phase dysphoric disorder (premenstrual syndrome), fibroids, endometriosis,
myoma,
hirsutism, cyclic auditory dysfunction, porphyria, or polycystic ovarian
syndrome.
In one embodiment, the methods of the present invention comprise
administering a pharmaceutical preparation comprising the GnRH agonist
peptide,
and a pharmaceutically acceptable carrier. In another embodiment, the
pharmaceutical preparation is orally administered in solid or liquid dosage
form. In
another embodiment, the pharmaceutical preparation is intravenously,
intraarterially,
subcutaneously, intradermally, intraperitoneally, intramuscularly,
intranasally or
intralesionally injected in liquid form. In another embodiment, the
pharmaceutical
preparation is administered as an intravaginal device or ring. In another
embodiment,
the pharmaceutical preparation is formulated as a topical formulation for
topical
application. In another embodiment, the pharmaceutical preparation is
formulated as a
pellet, a tablet, a capsule, a solution, a suspension, an emulsion, a gel, a
cream, a
suppository, an infra-vaginal ring, or a parenteral formulation. In a
particular
embodiment the pharmaceutical preparation is formulated as a depot for
providing
sustained release of the active ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be understood and appreciated more fully from the
following detailed description taken in conjunction with the appended drawings
which depict:
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Figure 1. Chemical structure of quinonic moieties: (1) 1,3,8 trihydroxy-6-
methyl-9,10-anthraquinone (Emodin); (2) 1,3,8-trihydroxy-6-oxy-9,10-
anthraquinone (Emodic acid, Emo); (3) 2-hydroxymethyl anthraquinone-
hemiglutarate (AntrQ); and (4) 2-(3-alanyl-3-chloro-1,4-naphthoquinone (NQ).
Figure 2. Solid phase synthesis of [D-Lys6(Emo)]GnRH.
Figure 3. Displacement (%) of specific binding of Izsl[D-Lys6]GnRH from
pituitary membranes of proestrous rats by increasing concentrations of
unlabeled
GnRH analogs: [D-Lys6(AntrQ)]GnRH (o); [D-Lys6(NQ)]GnRH (1); [D-
Lys6(Emo)]GnRH (v); and [D-Lys6]GnIRH (~).
Figure 4. LH releasing potency of [D-Lys6]GnRH conjugates in primary cultures
of rat pituitary cells containing the indicated concentrations of [D-
Lysb]GnR_H_
(0); [D-Lys6(AntrQ)]GnR_H_ (0); [D-Lys6(NQ)]GnR_H_ (~); or [D-
Lys6(Emo)]GnR_H_ (v).
Figure 5. Effect of a GnRH antagonist ([D-pyre, D-Phe2, D-Trp3°6]GnRH)
on the
induction of LH secretion from primary cultures of rat pituitary cells
stimulated
by [D-Lys6(Emo)]GnRH.
Figure 6. Induction of LH release in rats by intraperitoneal administration of
[D-
Lys6(Emo)]GnR_H_ (v) or of the parent peptide [D-Lys6]GnR_H_ (~). The ~'-axis
is
presented as a logarithmic scale.
Figure 7. Effects of long-term administration of GnR_H_ analogs on the weight
of
testes and prostate glands. Adult male rats were injected daily for 7 days
either
with [D-Lys6(Emo)]GnRH (0.1 or 1 nmol/rat, gray), [D-Lys6]GnRH (1 nmol/rat,
white), or PBS (control, black). Rats Were sacrificed 24 h after the last
injection,
and testes (a) and prostate glands (b) were dissected and weighed.
Figure 8. Binding of Emodic acid and GnR_H_ analogs to human serum albumin
(HSA).
Figure 9. Phototoxicity of [D-Lysb(Emo)]GnRTi and emodic acid to aT3-1 cells.
Cells were incubated ; with [D-Lys6(Emo)]GnRH (black) or emodic acid (white),
washed and illuminated. Cell survival was determined by the XTT method.
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Values are expressed as % survival. 100% survival (gray) refers to the
survival of
cells in the control group that were incubated without any emodic acid
derivatives.
Figure 10. DNA cleavage in cells treated with [D-Lys6(Emo)]GnRH and emodic
acid. ocT3-1 cells were incubated ; in darkness with 10 ~M of the tested
compounds, washed and illuminated. After 24 h of incubation DNA was isolated
and analyzed by gel electrophoresis. Lanes 1, 3 and 5 represent DNA of cells
treated with 1% DMSO in PBS, emodic acid or [D-Lys6(Emo)]GnRH,
respectively, followed by illumination. Lane 2, 4 and 6 represent cells
treated with
1% DMSO in PBS, emodic acid, or [D-Lys6(Emo)]GnRH, respectively, in
darkness.
Figure 11. Displacement of lasl[D-Lys6]-GnRH from proestrous rat pituitary
membrane receptors by increasing concentrations of unlabeled GnRH analogs.
Figure 12. The effect of a GnRH antagonist conjugate on the inhibition of LH
secretion from primary cultures of rat pituitary cells that were stimulated by
GnRH. Cells were incubated with GnRH (1 nM) in the absence or presence of
increasing concentrations of [D-Lys6]Antg or [D-Lys6(Emo)]Antg. Following the
incubation period (4 h at 37°C) media were collected and LH
concentration was
determined by RIA. Results are the mean~SEM of two independent experiments
(4 wells / experimental group, each).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the design, synthesis and biological
evaluation
of potent long-acting gonadotropin-releasing hormone (GnRH) agonists and
antagonists, which comprise a GnR_H_ analog conjugated to emodic acid or an
emodic
acid derivative.
According to the principles of the present invention a GnR_H_ analog is
conjugated to a molecule having the general formula:
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OH O OH
R ~I I % R
HO ~ ~ ~ ~(CH2)nCOOH
R O R
wherein n is an integer of 0-5 and R is independently at each occurrence
selected
from hydrogen, hydroxy, alkoxy, halogen, a straight chain, branched or cyclic
alkyl
group, lower alkenyl group, lower alkynyl group, carboxyl, carboxyalkyl,
amino,
aminoalkyl, diaminoalkyl, thin, thioalkyl, amido, alkylamido, dialkylamido or
any
other suitable substituent that yields a long acting non-toxic derivative of
said GnRH
analog.
An "alkyl" group refers to a saturated aliphatic hydrocarbon, including
straight-
chain, branched-chain and cyclic alkyl groups. In one embodiment, the alkyl
group
has 1-12 carbons. In another embodiment, the alkyl group has 1-7 carbons. In
another embodiment, the alkyl group has 1-6 carbons. In another embodiment,
the
alkyl group has 1-4 carbons. The alkyl group may be unsubstituted or
substituted by
one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido,
alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl,
thio and
thioalkyl.
A "hydroxy" group refers to an OH group. An "alkoxy" group refers to an -0-
alkyl group wherein alkyl is as defined above. A "thio" group refers to an -SH
group. A "thioalkyl" group refers to an -SR group wherein R is alkyl as
defined
above. An "amino" group refers to an -NHZ group. An "alkylamino" group refers
to
an NHR group wherein R is alkyl is as defined above. A "dialkylamino" group
refers to an NRR' group wherein R and R' are alkyl as defined above. An
"amido"
group refers to an -CONHZ group. An "alkylamido" group refers to an -CONHR
group wherein R is alkyl is as defined above. A "dialkylamido" group refers to
an -
CONRR' group wherein R and R' are alkyl as defined above. A "nitro" group
refers
to an N02 group. A "carboxyl" group refers to a COOH group. A "carboxyalkyl"
refers to a COOR group wherein R is an alkyl as defined above.
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In one embodiment, the present invention relates to the design, synthesis and
biological evaluation of a potent long-acting gonadotropin-releasing hormone
(GnRH) agonist, ([D-Lys6(Emo)]GnRH), which binds GnRH receptors with high
affinity to induce LH release, and which is devoid of any toxicity or
antiproliferative
effects. The present invention further relates to therapeutic uses of ([D-
Lys6(Emo)]GnRH) as a contraceptive, and in treating and/or preventing sex-
hormone
dependent diseases or conditions.
In another embodiment, the present invention further relates to the design,
synthesis and biological evaluation of a potent long-acting gonadotropin-
releasing
hormone (GnRH) antagonist, ([D-Lys6(Emo)]GnRH-Antg), which binds GnRH
receptors with high affinity to prevent LH release, and which useful as a
contraceptive, and in treating and/or preventing sex-hormone dependent
diseases or
conditions.
As defined herein a receptor agonist is a substance which binds receptors and
activates them. A receptor antagonist is a substance which binds receptors and
inactivates them. Assays to determine whether the compounds of the present
invention are agonists or antagonists are well known to a person skilled in
the art.
As discussed hereinabove, conjugation of bulky moieties to the s-amino group
of [D-Lys6]GnR_H_ do not significantly affect the bioactivity of GnRH analogs.
For
example, incorporation of an anthraquinone moiety such as 2,-hydroxymethyl
anthraquinone hemiglutarate to [D-Lys6]GnRH, generates an agonist ([D-
Lys6(AntrQ)]GnRH) with superior bioactivity to that of the parent peptidel4,
but
which is cytotoxic to cells, and inhibits the growth of human breast and
prostate
cancerl4.
In an attempt to develop a GnRH agonist possessing similar or superior
activity
to [D-Lys6]GnRH, but which is devoid of any toxic or antiproliferative
effects,
Applicants have unexpectedly found that incorporation of the anthraquinone-
based
emodin moiety (1,3,8-trihydroxy-6-methyl-9,10-anthraquinone) to [D-Lys6]GnRH,
generates a nontoxic, potent agonist ([D-Lys6(Emo)]GnRH), which binds with
high
affinity izz-vitz~o and izz-vivo to GnR_H_ receptors to induce LH release.
Emodin (Fig 1)
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is a naturally occurnng polyhydroxylated anthraquinone that is widely used for
preparation of laxatives. Applicants have previously shown24 that
incorporation of
emodic acid to [D-Lys6]GnRH diminished its ability to generate reactive
radical
species (ROS)25.
Moreover, Applicants have unexpectedly found that ([D-Lys6(Emo)]GnRH)
possesses long-term bioactivityl4a. Development of long acting GnRTi analogs
is of
particular interest, since in the clinic GnRH analogs are frequently
administered in
slow-release depot preparations in order to desensitize the pituitary gland.
Without
wishing to be bound by any particular concept or mechanism of action, the long
term
bioactivity may be attributed at least in part, as demonstrated herein, to the
ability of
emodin to bind with high affinity to serum proteins such as human serum
albumin
(HSA)17, which may protect the peptide from proteolytic degradation.
Accordingly, the present invention provides a potent long-acting GnRH super-
active agonist - ([D-Lys6(Emo)]GnRH), or a long-acting GnRH antagonist, - [D-
Lys6(Emo)]GnRH-Antg which is useful in the prevention and/or treatment of a
variety of sex-hormone related conditions (specifically conditions involving
cells that
carry GnRH receptors), and as a contraceptive.
In one embodiment, [D-Lys6(Emo)]GnRH is useful as a contraceptive agent to
control fertility in a subject, for example to prevent conception in a female
subject. As
described hereinabove, chronic administration of GnR_H_ agonists results in
down-
regulation of GnRH receptors and desensitization of the pituitary gonadotrophs
and
thus causes the suppression of gonadotropin secretion3~4. Thus, in one
embodiment,
[D-Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg may be administered in an
amount and dosage effective to suppress the release the gonadotropins LH and
FSH,
and thus may act as a contraceptive agent and prevent conception. In one
embodiment, [D-Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg is a potent
contraceptive in males. In another embodiment, [D-Lys6(Emo)]GnRH or [D-
Lys6(Emo)]GnRH-Antg is a potent contraceptive in females, i.e. is effective in
preventing pregnancy in females. In another embodiment, [D-Lys6(Emo)]GnRH or
[D-Lys6(Emo)]GnRH-Antg is a potent contraceptive in humans.
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Thus, in one embodiment, the present invention provides a method of
controlling fertility in a mammalian subject, comprising the step of
administering to
the subject a gonadotropin releasing hormone (GnRH) agonist or antagonist
peptide
having the formula [D-Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg , or a
pharmaceutically acceptable salt or hydrate thereof, in an amount effective to
control
fertility in the subject. In one embodiment, fertility is controlled to
prevent
conception in a female subject.
In yet another embodiment, the present invention provides a method of
contraception in a mammalian subject, comprising the step of administering to
the
subject a gonadotropin releasing hormone (GnRH) agonist or antagonist peptide
having the formula [D-Lys6(Emo)]GnR_H_ or [D-Lys6(Emo)]GnRH-Antg or a
pharmaceutically acceptable salt or hydrate thereof.
As defined herein, the term "analog" means any variant and includes both
agonists and antagonists
As defined herein, the term "conception" means the onset of pregnancy, marked
by the formation of a viable zygote and subsequent implantation of the
blastocyst. As
defined herein, the term "contraceptive" means an agent that diminishes the
likelihood of or that prevents conception.
As used herein, the term "administering" refers to bringing a subject in
contact
with a pharmaceutical composition comprising a GnRH peptide of the present
invention. As used herein, administration can be accomplished iyz vitro, i.e.
in a test
tube, or iya vivo, i.e. in cells or tissues of living organisms, for example
humans.
Furthermore, in accordance with another embodiment of the present invention,
[D-Lys6(Emo)]GnR_H_ or [D-Lysb(Emo)]GnRH-Antg is useful in the prevention
and/or treatment of a sex-hormone related diseases or conditions. Thus, in one
embodiment, the present invention provides a method of treating a sex hormone-
dependent disease in a subject, the method comprising the step of
administering to the
subject [D-Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg, or a pharmaceutically
acceptable salt or hydrate thereof, in an amount effective to treat the
disease or
condition in the subject. In another embodiment, the present invention
provides a
method of preventing a sex hormone-dependent disease in a mammalian subject,
the
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method comprising the step of administering to the subject [D-Lys6(Emo)]GnRH,
or a
pharmaceutically acceptable salt or hydrate thereof, in an amount effective to
prevent
the disease or condition in the subject.
As defined herein, the term "sex-hormone related disease or condition"
encompasses diseases or conditions involving the reproductive system, and/or
which
are dependent upon a sex hormone, i.e. a male hormone or female hormone. In
one
embodiment, these include diseases or conditions occurring due to an excess of
such
hormones in mammals or non-mammalian vertebrates (e.g. human, monkey, bovine,
horse, dog, cat, sheep, rabbit, rat, mouse, Esh etc.). In another embodiment,
the
diseases/conditions involve cells that carry GnRH receptors.
Thus, in one embodiment, [D-Lys6(Emo)]GnRH, or [D-Lys6(Emo)]GnRH-Antg
is useful for the prevention or treatment of sex hormone-dependent malignant
diseases, such as cancer. Non-limiting examples include prostate cancer,
breast
cancer, ovarian cancer, cervical cancer, tumor of the pituitary, testicular
cancer, and
uterine cancer.
In another embodiment, [D-Lys6(Emo)]GnR_H_ or [D-Lys6(Emo)]GnRH-Antg is
useful for the prevention or treatment of sex hormone-dependent non-malignant
(benign) diseases or conditions. Non-limiting examples include benign
prostatic
hyperplasia, precocious puberty, aberrant sexual behavior (treatment by
chemical
castration), late luteal phase dysphoric disorder (premenstrual syndrome),
fibroids,
endometriosis, myoma, hirsutism, cyclic auditory dysfunction, porphyria, or
polycystic ovarian syndrome.
As used herein, the term "treating" means remedial treatment, and encompasses
the terms "reducing", "suppressing" "ameliorating" and "inhibiting", which
have their
commonly understood meaning of lessening or decreasing.. The term "preventing"
means inhibiting the disease or condition, so that the disease or condition
does not
develop or progress.
Furthermore, in another embodiment, the present invention provides a method
of promoting the release of LH and FSH in a subject, the method comprising the
step
of administering to the subject a gonadotropin releasing hormone (GnRH)
agonist
peptide having the formula [D-Lys6(Emo)]GnRH, or a pharmaceutically acceptable
13
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salt or hydrate thereof, in an amount effective to promote the release of LH
and FSH
in the subject.
Furthermore, in another embodiment, the present invention provides a method
of preventing suppressing the release of LH and FSH in a subject, the method
comprising the step of administering to the subject a long-acting gonadotropin
releasing hormone (GnRH) agonist or antagonist peptide having the formula [D-
Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg. or a pharmaceutically acceptable
salt or hydrate thereof, in an amount effective to prevent the release of LH
and FSH in
the subject.
In one embodiment, the present invention comprises administering a
pharmaceutically acceptable salt of the [D-Lys6(Emo)]GnIRH or [D-
Lys6(Emo)]GnRH-Antg peptide. The term "pharmaceutically acceptable salt"
includes acid addition salts which are formed with inorganic acids such as,
for
example, hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic,
tartaric, mandelic, and the like. The term also includes base addition salts
which are
formed from inorganic bases such as, for example, sodium, potassium, ammonium,
and calcium, and from organic bases such as isopropylamine, trimethylamine,
histidine, and the like.
In another embodiment, the present invention comprises administering a
~0 hydrate of the [D-Lys6(Emo)]GnR_H_ or [D-Lys6(Emo)]GnRH-Antg peptide. The
term
"hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate,
trihydrate and the like.
In another embodiment, the methods of the present invention comprise
administering a pharmaceutical preparation comprising the GnRH agonist or
antagonist peptide, and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutical preparation" or "pharmaceutical compositions", used herein
interchangeably, means a "therapeutically effective amount" of the [D-
Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRHAntg. A "therapeutically effective
amount" as used herein refers to that amount which provides a therapeutic
effect for a
given condition and administration regimen.
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In one embodiment, pharmaceutical preparation is orally administered in solid
or liquid dosage form. In another embodiment, the pharmaceutical preparation
is
intravenously, intraarterially, subcutaneously, intradermally,
intraperitoneally,
intramuscularly or intralesionally injected in liquid form. In another
embodiment, the
S pharmaceutical preparation is administered as an intravaginal ring. In
another
embodiment, the pharmaceutical preparation is administered intranasally. In
another
embodiment, the pharmaceutical preparation is formulated as a topical
formulation
for topical application. In another embodiment, the pharmaceutical preparation
is a
pellet, a tablet, a capsule, a solution, a suspension, an emulsion, a gel, a
cream, a
suppository, an infra-vaginal ring, or a parenteral formulation. In a
particular
embodiment the pharmaceutical preparation is formulated as a depot for
providing
sustained release of the active ingredient. In another particular embodiment
the
pharmaceutical preparation is formulated for intranasal administration or
inhalation.
In one embodiment, the pharmaceutical preparations are administered orally,
and are thus formulated in a form suitable for oral administration, i.e, as a
solid or a
liquid preparation. Suitable solid oral formulations include tablets,
capsules, pellets
and the like. Suitable liquid oral formulations include solutions,
suspensions,
emulsions, and the like.
Further, in another embodiment, the pharmaceutical preparations are
administered by intravenous, intraarterial, intraperitoneal, subcutaneous,
intradermal,
intramuscular or injection of a liquid preparation. Suitable liquid
formulations
include solutions, suspensions, emulsions, and the like. Alternative
embodiments
include depots providing sustained release or prolonged duration of activity
of the
active ingredient in the subject, as are well known in the art.
Further, in another embodiment, the pharmaceutical compositions are
administered topically to body surfaces, and are thus formulated in a form
suitable for
topical administration. Suitable topical formulations include gels, ointments,
creams,
lotions, and the like. Further, in another embodiment, the pharmaceutical
compositions are administered as a suppository, for example a rectal
suppository or a
vaginal or urethral suppository. Further, in another embodiment, the
pharmaceutical
compositions can be applied on conventional intravaginal rings or other
intravaginal
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devices. Further, in another embodiment, the pharmaceutical compositions can
be are
administered intranasally or by inhalation.
As used herein a "pharmaceutically acceptable carrier" may be a solid carrier
for solid formulations, a liquid carrier for liquid formulations, or mixtures
thereof.
Solid carriers include, but are not limited to, a gum, a starch (e.g. corn
starch,
pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose),
a cellulosic
material (e.g. microcrystalline cellulose), an acrylate (e.g.
polymethylacrylate),
calcium carbonate, magnesium oxide, talc, or mixtures thereof. For liquid
formulations, pharmaceutically acceptable carriers may be aqueous or non-
aqueous
solutions, suspensions, or emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, and injectable organic esters such as
ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions
or
suspensions, including saline and buffered media.
Parenteral vehicles (for subcutaneous, intravenous, intraarterial,
intraperitoneal
or intramuscular injection) include sodium chloride solution, Ringer's
dextrose,
dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous
vehicles
include fluid and nutrient replenishers, electrolyte replenishers such as
those based on
Ringer's dextrose, and the like. Examples are sterile liquids such as water
and oils,
with or without the addition of a surfactant and other pharmaceutically
acceptable
adjuvants. In general, water, saline, aqueous dextrose and related sugar
solutions, and
glycols such as propylene glycols or polyethylene glycol are preferred liquid
carriers,
particularly for injectable solutions.
In addition, the compositions may further comprise binders (e.g. cornstarch,
gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose,
hydroxypropyl
methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato
starch,
alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum,
sodium
starch glycolate), buffers (e.g., Tris-HCI, acetate, phosphate) of various pH
and ionic
strength, additives such as gelatin to prevent absorption to surfaces,
detergents (e.g.,
Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,
surfactants
(e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,
glycerol,
polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite,
butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,
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hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer,
colloidal
silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame,
citric acid),
preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g.
stearic
acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-
aids (e.g.
colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl
citrate),
emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate),
polymer
coatings (e.g., poloxamers or poloxamines), coating and film forming agents
(e.g.
ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
In one embodiment, the pharmaceutical compositions provided herein are
controlled release compositions, i.e. compositions in which the [D-
Lys6(Emo)]GnR_H_
or [D-Lys6(Emo)]GnRH-Antg peptide is released over a period of time after
administration. In another embodiment, the composition is an immediate release
composition, i.e. a composition in which all of the [D-Lys6(Emo)]GnRH or [D-
Lysb(Emo)]GnRH-Antg. peptide is released immediately after administration.
The preparation of pharmaceutical compositions which contain an active
component is well understood in the art, for example by mixing, granulating,
or
tablet-forming processes. The active therapeutic ingredient is often mixed
with
excipients which are pharmaceutically acceptable and compatible with the
active
ingredient. For oral administration, the [D-Lys6(Emo)]GnR_H_ or [D-
Lys6(Emo)]GnRH-Antg peptide is mixed with additives customary for this
purpose,
such as vehicles, stabilizers, or inert diluents, and converted by customary
methods
into suitable forms for administration, such as tablets, coated tablets, hard
or soft
gelatin capsules, aqueous, alcoholic or oily solutions. For parenteral
administration,
the (D-Lys6(Emo)]GnRH or [D-Lys6(Emo)]GnRH-Antg peptide is converted into a
solution, suspension, or emulsion, if desired with the substances customary
and
suitable for this purpose, for example, solubilizers or other.
The following examples are presented in order to more fully illustrate certain
embodiments of the invention. They should in no way, however, be construed as
limiting the broad scope of the invention. One skilled in the art can readily
devise
many variations and modifications of the principles disclosed herein without
departing
from the scope of the invention.
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EXAMPLE 1
SYNTHESIS OF fD-Lys6IGnRH CONJUGATES
The synthesis of [D-Lys6]GnRH conjugates that are modified at the s-amino
group of [D-Lys6]GnRH was carried out by two different routes. The Brst route
involved the reaction of the free s-amino group of solid-phase synthesized [D-
Lys6]GnRH, in a homogeneous solution, with the carboxylic functional group of
the
respective quinone moieties. This method employed benzotriazole-1-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate (PyBOP) as a coupling reagent and
4-
methylmorpholine (NMM) as a base. Such a method is a 'one-pot synthesis',
which
results in better yield and purity than other methodologies that use active
esters, such
as N-hydroxysuccinimide, as coupling reagentsl9.
In the second route, [D-Lys6]GnRH derivatives were prepared by employing an
automatic multiple peptide synthesizer, using Rink amide resin as the
polymeric
support, and standard Fmoc-protected amino acids and corresponding reagents
(Fig.
2). The routinely used Fmoc-D-Lys(Boc)-OH was replaced by Fmoc-D-Lys(Mtt)-OH
and the protected peptide was not cleaved from the resin. The Mtt group was
removed
selectively from the NE-amino group of D-Lys6 by mild acidolysis (2% TFA in
CH2Clz) without effecting other protecting groups20. Consequently, direct
incorporation of emodic acid (Emo), 2-hydroxymethyl-anthraquinone
hemiglutarate
(AntrQ), and N-(2-chloro-1,4-naphthoquinonyl)-(3-alanine (NQ) (Fig. 1) to the
free
NE-amino function of D-Lys6 occurs by employing the standard coupling reagent
and
procedure. This reaction leads to the synthesis of [D-Lys6(1,3,8-trihydroxy-6-
carboxy-anthraquinone)]GnR_H_ ([D-Lys6(Emo)]GnRH', [D-Lysb(2-hydroxyrnethyl-
anthra-quinone hemiglutarate)] GnRH ([D-Lys6(AntrQ)]GnRH) and [D-Lys6(N-(2-
chloro-1,4-naphthoquinonyl)-(3-alanyl)]GnRH ([D-Lys6(NQ)]GnRH).
The naphthoquinone and anthraquinone derivatives were synthesized in order to
evaluate the generality of the synthetic manipulations and as a reference for
the
biological activity of other conjugates of [D-Lys&]GnRH. The corresponding
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molecular mass and the relative hydrophobicity of each of the conjugates are
summarized in Table 1.
Table 1. GnRH analogs: molecular mass and relative hydrophobicity and binding
affinity (ICso) to rat pituitary receptors.
Peptide analog Hydrophobicity
(%B) ~ obsd (calcd) b
[D-Lys ]GnRH 37 1254.6
(1254.4)
[D-Lys6(NQ)]GnR_H_ 59 1516
(1516.07)
[D- 84 1589,7
Lys6(AntrQ)]GnR_H_ (1588.7)
[D- 66 1537.6
Lys6(Emo)]GnR_H_ (1536.7)
a The percent of buffer B in which the analog was eluted from RP-18 column.
bObserved (obsd) and calculated (calcd) m/z values of MIT'- monoisotopes. In
most cases, an additional peak, corresponding to MNa+ ,was observed. Purities
of
the synthetic peptides were usually >98%, according to two different
analytical
HPLC solvent systems as detailed in the Experimental Section. For more details
and abbreviations see Figures 1 and 2.
EXAMPLE 2
IN VITRO BINDING OF fD-Lys6IGnRH TO GnRH RECEPTORS AND
LH-RELEASING ACTIVITY
The ability of the [D-Lys6]GnRH conjugates to bind to rat pituitary GnRH
receptors was evaluated in vitro by displacement assays, using l2sI[D-
Lys6]GnRTi as
the radioligand. Figure 3 shows that incorporation of the quinone moiety in
position 6
of the GnRH analog does not change the high binding affinity of the parent
peptide to
the GnRH receptors. Moreover, the binding affinity of [D-Lys6(NQ)]GnRH and of
[D-Lys6(AntrQ)]GnRH to GnRH receptors increased by 2 and 5 folds,
respectively,
whereas the affinity of [D-Lysb(Emo)]GnR_H_ to the GnRH receptors was reduced
by
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about 3 fold compared to the parent peptide. The binding affinities (ICso) of
the
GnRH conjugates are summarized in Table 2.
Table 2. GnRH analogs: binding affinity (ICso) to rat pituitary receptors.
Peptide analog GnRH
receptor
binding
ICso (x10-ioM)
[D-Lys ]GnRH 0.8 ~ 0.01
[D-Lysb(NQ)]GnR_H_ 0.9 ~ 0.01
D-Lys6(AntrQ)]GnRH 0.2 ~ 0.02
[D-Lys6(Emo)]GnRH 2.5 ~ 0.1
ICso= concentration of unlabeled ligand that displaces 50% of bound tracer.
Each
point is the mean~SEM of triplicates of one experiment out of three. For more
details and abbreviations see Figures 1 and 3.
In order to correlate the binding affinity of the newly synthesized GnRH
conjugates to their bioactivity, these compounds were compared to [D-
Lys6]GnRH for the LH-releasing capacity, using primary rat pituitary cell
cultures. As shown in Figure 4, all conjugates exhibited enhanced LH releasing
activity compared to the parent peptide, ira vitro. Notably, [D-
Lys6(Emo)]GnR_H_
demonstrated the highest bioactivity, despite its lowest binding affinity to
the
GnRH receptors (Fig. 3). This bioactivity was completely inhibited by the
antagonist [D-pGlul, D-Phe2, D-Trp3°6]GnRH, which reduced the [D-
Lysb(Emo)]GnRH-induced LH-secretion to basal levels (Fig. 5).
In conclusion, the in-vitro studies demonstrated a correlation between the
binding affinities to GnRH receptors and the iya vitro LH releasing potencies
of the
[D-Lys6(NQ)]GnR_H_ and [D-Lysb(AntrQ)]GnRH conjugates. [D-Lysg(Emo)]GnRH,
however, exhibited high LH-releasing potency despite its relatively low
binding
affinity. The gonadotropin-releasing activity of this conjugate could be
completely
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inhibited by the antagonist, [D-Pyre, D-Phe2, D-Trp3'6]GnRH, indicating that
this
activity is receptor mediated.
Furthermore, as demonstrated herein, the in vitro LH-releasing potency of [D-
Lys6(Emo)]GnRH was superior to that of other conjugates despite its
substantial
lower affinity to rat pituitary GnRH receptors (Figs. 3 and 4). This
discrepancy might,
perhaps, stem from the fact that the nature of its peptide-receptor binding
may be
different from that of the other derivatives, due to a combination of enhanced
hydrophobicity (Table 1) and high capacity to form hydrogen bonds through the
hydroxylic groups of emodic acid moiety. Such interactions may reduce the
binding
affinity but enhance receptor activation.
EXAMPLE 3
IN T~IT~O AGONISTIC ACTIVITY OF fD-Lys~IGnRH
The ability of [D-Lys6(Emo)]GnRH to bind GnRH receptors and promote LH
release in vitro, prompted the evaluation of its iya vivo activity. Following
intraperitoneal administration to intact rats, this conjugate proved to be a
very potent
agonist which induced a similar increase in serum LH levels as that of [D-
Lys6]GnRH, although the dose was reduced to 10°l0 of that of the parent
peptide (0.04
vs. 0.4 nmol) (Fig. 6). Moreover, the duration of the stimulation was also
longer; six h
after [D-Lys6(Emo)]GnIRH administration, LH levels were about 6 folds higher
than
in the group of rats treated with [D-Lys6]GnRH. Administration of a higher
dose of
[D-Lys6]GnR_H_ (20 nmol/rat) induced a laxger increase in serum LH levels.
Furthermore, chronic administration of [D-Lys6(Emo)]GnRH (0.1 nmol/rat) to
intact
adult male rats for 7 days resulted in a greater decrease in the testicular
and ventral
prostate gland weights as compared to rats that were treated with 10-fold
higher dose
of the parent peptide (1 nmol/rat) (Fig. 7).
In conclusion, the ira-vivo studies demonstrated that [D-Lys6(Emo)]GnRH is a
powerful long acting agonist, which preserves its activity significantly
longer than the
parent peptide [D-Lys6]GnRH. Moreover, chronic treatment of adult males rats
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showed that [D-Lys6(Emo)]GnRH is much more active in reducing the weights of
testes and prostate gland than the parent peptide.
EXAMPLE 4
BINDING OF ~D-Lys611GnRH TO HSA
Binding of peptides to serum proteins, such as human serum albumin (HSA), has
been proposed to prolong activity of the peptideslo,ls, Albumin, the most
abundant
protein in human serum (with the concentration of about 0.6 mM), possesses a
half
life in circulation of 19 days. Its role in the circulatory system is probably
to aid in the
transport, metabolism, and distribution of exogenous and endogenous ligands26.
As indicated earlier, emodin is known to bind to (HSA). In order to determine
whether the prolonged ih vivo activity of [D-Lys6(Emo)]GnRH might be related
to its
binding to plasma proteins, its association with HSA was measured.
As shown in Figure 8, emodic acid binds to HSA at the range of the
concentrations that were examined. Consequently, the conjugation of emodic
acid to
[D- Lys6]GnRH generates a superagonist, [D-(Emo)]GnRH, which binds
significantly
to HSA, and thus may explain its prolonged in vivo activity.
Development of potent, long acting GnR_H_ analogs is of particular interest,
since in the clinic GnRTi analogs are frequently administered in slow-release
depot
preparations in order to desensitize the pituitary gland. The HSA-binding
studies
demonstrate that the long-term bioactivity of [D-Lys6(Emo)]GnRH may be
attributed,
at least partly, to the high binding affinity of the emodin moiety to serum
proteins,
which may protect the peptide from proteolytic degradation.
EXAMPLE 5
IlV VITRO TOXICITY MEASUREMENT
Emodin is known to be a photoactive compound that generates reactive oxygen
species upon illuminationzi,zz, therefore its cytotoxicity as well as that of
[D-
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(Emo)]GnRH to a mouse gonadotroph cell line (aT3-1) that expresses high
affinity
binding sites for GnRH23, was measured.
As shown in Figure 9, although emodic acid demonstrated significant
phototoxicity to ocT3-1 cells at a concentration of 10 ~.M, its conjugate, [D-
Lys6
(Emo)]GnRH, did not exhibit any toxicity. Both compounds did not show any
toxicity to these cells while incubated in the dark.
To further ensure that [D-Lys6(Emo)]GnRH is not toxic to pituitary cells,
emodic acid and [D- Lys6(Emo)]GnRH were tested for their ability to induce an
apoptotic-like process in ocT3-1 cells. These experiments were carried out in
dark
conditions as well as under illumination.
As shown in Figure 10 emodic acid and its GnR_H_ analog did not cause any
DNA fragmentation in the dark (lanes 4 and 6), while upon illumination, only
emodic
acid (lane 3) but not [D-Lys6(Emo)]GnRH (lane 5) induced DNA fragmentation.
In conclusion, the assumption that the high LH releasing activity of [D-
Lys6(Emo)]GnR_H_ may result from its cytotoxicity and cellular damage,
eventually
leading to leakage of LH from the gonadotropic cells, was ruled out by
evaluation of
the toxicity as well as the apoptotic potency of the conjugate. These results
clearly
revealed that the peptide was devoid of any toxic activity.
Since emodic acid is considered to be a photoactive anthraquinone, these
studies were carried out in darkness as well as under illumination. Indeed,
the results
showed that in the dark neither emodic acid nor [D-Lys6(Emo)]GnRH were toxic
and
could not induce apoptosis to pituitary cell line, while upon illumination
emodic acid,
but not [D-Lys6(Emo)]GnRH, was cytotoxic and induced apoptosis. These results
are
supported by Applicants' previous studies which revealed that whereas emodic
acid
generated ROS upon irradiation, [D-Lys6(Emo)]GnRH was much less active in this
respect24
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EXAMPLE 6
MATERIALS AND METHODS
Abbreviations
Abbreviations of common amino acids are in accordance with the
recommendations of IUPAC. Additional abbreviations: AntrQ, 2-(Hydroxymethyl)
anthraquinone; BSA, bovine serum albumin; DMF, N,N'-dimethylformamide;
DMSO, dimethylsulfoxide; EDSO, concentration of the ligand which indicates 50%
of
maximal effect. Emo (Emodic acid), 1,3,8-trihydroxy-6-oxy-9,10-anthraquinone;
GnRH, gonadotropin-releasing hormone; HPLC, high performance liquid
chromatography; ICSO, concentration of ligand which displaces 50% of bound
tracer;
LH, luteinizing hormone; MB, maximal binding; NQ, 2-(3-alanyl-1,4-
naphthoquinone; PBS, phosphate buffered saline; pGlu, or Pyr, pyroglutamic
acid;
RIA, radioimmunoassay; SEM, standard error of the mean.
Reagents
All chemicals and reagents were of analytical grade. Rink amide resin, 9-
fluorenylmethoxycarbonyl (Fmoc) protected amino acid derivatives, and all the
reagents for solid-phase peptide synthesis were purchased from Novabiochem
(Laufelfingen, Switzerland). Side-chain protecting groups employed for peptide
synthesis were as follows: Arg, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl
(Pbf); His, trityl (Trt); D-Lys, 4-methyltrityl (Mtt) or tert-butyloxycarbonyl
(Boc);
Trp, tert-butyloxycarbonyl (Boc); Ser and Tyr, tent-butyl (tBu). Reversed
phase
HPLC was performed on a Spectra-Physics SP-8800 liquid chromatography system
equipped with an Applied Biosystems 757 variable wavelength absorbance
detector.
HPLC prepacked columns were: Lichrocart, containing Lichrosorb RP-18 (250 x 10
mm; 7 ~,m, Merck, Darmstadt, Germany) for semi-preparative purification and
Lichrospher 100 RP-18 (250 x 4 mm; 5 Vim, Merck, Darmstadt, Germany) and wide
pore butyl C4 (250 x 4.6 mm; 5 pm, J. T. Baker Inc., Phillipsburg, NJ) for
analytical
purposes. HPLC purification and analysis were achieved by using a linear
gradient
established between 0.1 % trifluoroacetic acid (TFA) in water as buffer A and
0.1
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TFA in 75% aqueous acetonitrile as buffer B. Eluent composition was 10-100% B
over 40 min, using RP-18 column, and a gradient of 0-100% B over 40 min
employing a wide pore butyl (C4) column. N-(2-chloro-1,4-naphtoquinonyl)-(3-
alanine (NQ), 1,6,8-Trihydroxy-3-carboxylic acid-anthraquinone (Emo) and 2-
hydroxymethyl-anthraquinone hemiglutarate (AntrQ) were synthesized as
described14,24.,27 (for chemical structure see Fig. 1).
Animals
Wistar-derived rats were obtained from the Weizmann Institute Animal
Resource Center. Experiments were carried out in compliance with the
regulations of
the Weizmann Institute of Science.
Cells and culture conditions
All tissue culture components were purchased from Biological Industries (Befit
Haemek, Israel). Mouse pituitary gonadotrope carcinoma cell line (aT3-1) was
obtained from Dr. M. Liscovitch, (Dept. of Biological Regulation, Weizmann
Inst. of
Science) and maintained routinely in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum (FCS) and antibiotics (complete
medium).
Cells were kept at 5% C02 atmosphere and 37° C in a humidified
incubator. Primary
pituitary cell cultures were prepared from 21 day-old Wistar-derived female
rats as
described3~ and maintained in M-199 containing 10% horse serum and antibiotics
and
incubated as described above. Irradiations were carned out with a 100 W
halogen
lamp (Philips, Germany) with an appropriate filter using a band-pass of 320-
510 nm
and with ~,m~ = 400 nm. The fluence rate used for the all irradiations was
14.3 mW
cm 2 with a total fluence of 17 J cm z.
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Synthesis of D-Lys6IGnRHI conjugates
Peptide synthesis
Automated solid phase peptide synthesis was performed using a multiple
peptide synthesizer (AMS-422, Abimed Analysen-Technik GmbH, Langenfeld,
Germany) with Rink amide resin (25 ~,mol scale) as a polymeric support,
following
the company's protocol for Fmoc strategy19~2g~29. For the synthesis of [D-
Lysb]GnR_H_
conjugates on solid phase support, the Mtt protecting group was used as the
side chain
protecting group for D-Lys6 residue. The completed peptide chain was cleaved
from
the resin, along with side-chain deprotection, using 3 mL of the mixture
TFA:H20ariethylsilane; (95:2.5:2.5, v:v), for 2 h at room temperature. The
crude
products were precipitated with ice-cold tert-butyl methyl ether. Precipitated
peptides
were washed with cold dry tert-butyl methyl ether, dissolved in water or
water/acetonitrile solution, and lyophilized. Peptide purification to
homogeneity
(usually >96%) was achieved with semi-preparative HPLC and tested by
analytical
HPLC using the above solvent systems. Samples of each of the peptides were
hydrolyzed (6N HCI, 110°C, 22 h, in vacuum) and analyzed with a Dionex
automatic
amino acid analyzer. The results were also used for quantification of the
peptide
content in each preparation. The peptides were also analyzed by a Micromass
Platform LCZ 4000 (Manchester, UK) using an electron spray ionization
technique
(ESI). For biological evaluations, pure peptides were dissolved in
dimethylsulfoxide
(DMSO) or in double distilled water to obtain 1 mM concentration as a stock
solution. The DMSO contents in the preparations used for bioassays were always
1
or lower. Identical concentrations of DMSO were tested and found to have no
significant effects on receptor binding assays or hormone secretion.
(D-Lys6IGnRH coniu~ates
Solution synthesis
[D-Lys6]GnRTi was automatically synthesized on a multiple peptide synthesizer
and lyophilized (purity of crude product was >90%) as described above. To the
DMF
solution (1 mL) of dry crude peptide (31 mg, 25 pmol) and corresponding
quinone
26
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WO 2004/030687 PCT/IL2002/000801
(27.5 pmol) in the presence of 4-methylmorpholine (NMM) (8.2 p,L, 75 ~,mol), a
DMF solution (0.5 mL) of PyBOP (13 mg, 27.5 pmol) was added. The mixture was
stirred for 2 h at room temperature. The progress of the reaction was followed
by the
disappearance of [D-Lys6]GnRH as revealed by analytical HPLC. Upon completion
of the reaction the crude peptide was precipitated with ice cold tent-butyl
methyl ether
(10 mL) and dried. Purification to homogeneity was achieved by semi-
preparative
HPLC to yield 22.7 mg (15 ~mol; 60%) of [D-Lysg(N-(2-chloro-1,4-
naphtoquinonyl)-(3-alanine)]GnRH. Mass spectrometry: found m/z [M + H]+ 1516,
calcd for C72 H92 C1N19 Ois [M + H]+ 1516.07. Amino acid analysis after
hydrolysis
with 6 M HCl at 110 °C for 22 h: Glu 1.00, His 1.00, Ser 0.87, Tyr
0.98, Lys l, Leu
0.98, Arg 1.05, Pro 1.01, Gly 0.98. Trp was destroyed under the acidic
conditions of
hydrolysis. In this manner, [D-Lys6(AntrQ)]GnRH and [D-Lys6(Emo)]GnRH were
also synthesized in solution (yield: 65% and 57%, respectively) and
characterized.
Mass spectrometry: [D-Lys6(AntrQ)]GnRH found rnlz [M + H]+ 1589.7, calcd for
C79H9gN1801$ [M + H]+ 1588.7. [D-Lys6(Emo)]GnRH found nz/z [M + H]+ 1537.6,
calcd for C74H9oN18O19 jM + H]~ 1536.7. Amino acid analysis after hydrolysis
with 6
M HCl at 110 °C for 22 h: [D-Lys6(AntrQ)]GnRH, Glu 0.99, His 1.01, Ser
0.87, Tyr
0.98, Lys 1.02, Leu 1.02, Arg 1.05, Pro 0.61, Gly 1.06. [D-Lys6(Emo)]GnRH, Glu
1.01, His 0.95, Ser 0.88, Tyr 0.97, Lys 1.02, Leu 1.04, Arg 1.04, Pro 0.42,
Gly 1.1.
fib) Solid phase synthesis
pGlu-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Lys(Mtt)-Leu-Arg(Pbf)-Pro-Gly-
[Rink amide resin] (25 ~,mol), synthesized by a multiple peptide synthesizer,
was
treated with 2% TFA in CH2C12 (2 mL x5, 5 min) to cleave the Mtt group from D-
Lys2~. The removal of the Mtt group was revealed by the ninhydrin test. The
resin
was then neutralized by 10% N,N'-diisopropylethylamine in CH2C12 (2 mL ~e3, 2
min)
and washed with CH2C12 (2 mL x3). Emodic acid (30 mg, 100 ~.mol) in DMF (0.1
mL) was then coupled to the free NE-amino group of D-Lysb following the above
mentioned company's protoco129. Cleavage of the resulting derivatives,
precipitation
and purification were carried out as described above. Yield: 31 mg (20 Eunol,
80%,
based on the initial amino group-resin loading). Mass spectrometry: found nalz
[M +
27
CA 02500897 2005-04-O1
WO 2004/030687 PCT/IL2002/000801
H]+ 1537.4, calcd for C7zH9zC1N190i6 [M + H]+ 1536.7. Similar to this
procedure, [D-
Lys6(AntrQ)]GnRH and [D-Lys6(NQ)]GnR_H_ were synthesized on polymer support to
yield 85% and 83%, respectively. Mass spectroscopy and amino acid analysis
yielded
the expected results, similar to those obtained from the synthesis in
solution.
In vitro GnRH receptor binding assay and LH releasing potency of fD-
LVs6IGnRH derivatives
Displacement binding assays were carried out using rat pituitary membrane
preparations and lasl[D-Lys6]GnRH as radioligand as described3~. Briefly,
membranes were incubated fox 90 min at 4°C with lasljD-Lys6]GnR_u_ and
with the
unlabeled peptides. Non-specific binding was defined as the residual binding
in the
presence of excess [D-Lys6]GnR_H_ (1 ~,M). Specific binding was calculated by
subtracting the non-specific binding from the maximal binding, determined in
the
absence of any competing peptide. Results are the mean of two experiments
carried
out in triplicates. SEM values are omitted for clarity (Figure 3).
For evaluating the LH releasing potencies of the [D-Lys6]GnRH derivatives, rat
pituitary cells were incubated in M-199 (without serum and antibiotics)
containing the
desired concentrations of the tested peptides and incubated in the dark at
37°C for 4 h
as described3l. The media were then collected and LH concentration were
analyzed
by double-antibody radioimmunoassay (RIA)32 using kits kindly supplied by the
National Institute of Arthritis, Metabolism and Digestive Diseases (hIIMDD).
Results
(Figure 4) are expressed in terms of LH-RP-3 rat reference preparation, and
are the
mean~SEM of LH concentration of two experiments (4 wells / experimental group,
each). The basal release after 4 h was 9.41~2 ng/mL.
To determine the effect of a GnRH antagonist ([D-pyre, D-Phe2, D-
Trp3'6]GnRH) on the induction of LH secretion from primary cultures of rat
pituitary
cells stimulated by [D-Lysb(Emo)]GnRH, cells were incubated for 4 h at
37°C with
jD-Lys6(Emo)]GnRTi (20 pM) in the absence or presence of the GnRH antagonist
(100 nM). The media were collected and LH concentration was determined by RIA
(Figure 5). Results are the mean~SEM of LH concentrations obtained from two
28
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experiments (4 wells / experimental group, each). The LH concentration in each
well ,
was determined using three different aliquots of the medium. *LH release was
significantly higher (p<0.01) than that of the other groups.
Ih vivo LH Release
Female rats (~ 250 gr) were intraperitoneally injected with the desired
concentration of [D-Lys6(Emo)]GnRH (0.04 nmol/rat) or [D-Lysb]GnRH (0.4
nmolJrat) in 0.5 mL PBS. Blood samples were withdrawn by cardiac puncture
under
light ether anesthesia at the indicated time intervals, and serum LH levels
were
determined by RIA as described above (Figure 6). Results are the mean~SEM of
LH
concentrations in the serum of five animals/experimental group. The LH
concentration was determined using three different dilutions of each serum
sample.
Similar results were obtained in two other experiments. LH concentration in
unstimulated rats was 0.53 ng/mL serum. *LH release was significantly higher
(p<0.01) than in the group treated with [D-Lys6]GnRH.
Long-term treatment of animals with GnRH analogs
Intact adult males rats (260-280 g) were injected daily intraperitoneally with
the
GnRH analogs [D-Lys6(Emo)]GnRH (0.1 or 1 nmol/rat) or with [D-Lys6]GnRH (1
nmollrat) in PBS for 7 days. Control rats were injected daily with 0.5 mL of
PBS.
Rats were sacrificed 24 h after the last injection, and the testes and
prostate glands
were immediately dissected and weighed (Figure 7). Results are the mean~SEM of
the weight of the organs (six animals/experimental group). The weight of the
testes
and prostate gland of the control group were 3.367~0.07 and 0.267~0.02 g,
respectively. * Weights are significantly different (P<0.01) from the control.
**
Significantly different (P<0.001) from control group or from rats treated with
[D-
Lys6]GnRH (1 nmol/rat).
29
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Binding to HSA
The binding capacity of [D-Lys6(Emo)]GnRH, [D-Lys6]GnR_H_ and emodic acid
to HSA (Sigma, St. Louis, MO) was evaluated by incubating various
concentrations
of the tested compounds with HSA (22.5 nmol, 1.5 mg in 0.5 mL of PBS) at 37
°C for
3 h. The concentration of HSA in the solution was determined using a molar
extinction coefficient of 39,000 M-1 cm 1 at 277.5 nm33. The unbound compound
was
then separated from the HSA solution by applying it on to a Centricon°
concentrator
column (Amicon Inc., Beverly, MA) with a band pass membrane of 30,000 kD
according to the manufacture's protocol. The HSA bound compound (~ 20 ~,L) was
then precipitated by adding it to acetonitrile (1 mL). The supernatant was
kept and the
precipitate was dissolved in PBS (100 ~L) and re-precipitated. The combined
supernatants were evaporated, the residue was dissolved in water containing
0.1
TFA (buffer A) and analyzed by HPLC to determine the amount of the tested
compounds. Results represent the mean of two experiments (Figure 8).
hz Vitro toxicity Measurements
aT3-1 cells (50,000 cells/well) were plated in 96-well tissue culture plates
in
0.1 mL of complete medium. After 24 h the medium was changed and cells were
incubated in the same medium (without serum and phenol red) containing
different
concentrations of the tested compounds for 4 h at 37 °C. The cells were
then washed
(x3) with PBS and illuminated (~,",~ = 400 nm) in PBS. The fluence rate was
14.3
mW cm 2 with a total fluence of 17 J cm 2. Following illumination the media
were
replaced by complete medium and the plates were incubated for additional 24 h
at
37°C. Cell survival was determined using the XTT (Biological
Industries, Beit-
Haemek, Israel) kit following the manufacture's protocol. Values are expressed
as
survival. 100% survival refers to the survival of cells in the control group
that were
incubated without any emodic acid derivatives (Figure 9). *Survival is
significantly
lower (p<0.001) than in the control group.
30
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DNA Fragmentation Assay
aT3-1 cells (Sx 106/dish) were incubated in phenol red and serum free complete
medium (37°C, 5% COZ) with the tested compounds for 5 h in the dark.
Cells were
then washed with PBS (x3) and illuminated or remained in darkness as described
earlier. The media were then replaced by complete medium and cells were
incubated
for additional 24 h. DNA was then isolated, using Wizard~ genomic DNA
purification kit (Promega, Madison, WI) following the company's protocol and
analyzed by gel electrophoresis (0.4% cross linked agarose, ethidium bromide
staining).
EYAMPLE 7
EMODIC ACID DERIVATIVES OF GNRH ANTAGONIST
Synthesis of fD-Pyrl, D-Phe2, D-Trp3, D-Lys6(Emo)IGnRH (denoted herein as
f D-Lys6(Emo)1 Anti)
For the synthesis of [D-Pyrl, D-Phe2, D-Trp3, D-Lys6(Emo)]GnRH, the crude
antagonist, [(D-Pyre, D-Phe2, D-Trp3, D-Lys6)GnRH, (D-Lys6)Antg] of GnRH were
automatically synthesized on a multiple peptide synthesizer. The crude
peptides were
cleaved from the resin, precipitated, dissolved in H20 and lyophilized. The
conjugation of Emodic acid to the peptides were carried out in a one pot
reaction,
using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBOP) as a coupling agent, 4-methylmorpholine (NMM) as a base and DMF as a
solvent for lh. The crude conjugates were then purified to homogeneity by semi-
preparative HPLC and characterized by LTV, MS and amino acid analysis.
Synthesis of [D-Lys6(Emo)]Antg was also carried out employing solid phase
peptide synthesis, similar to that of [D-Lys6(Emo)]GnRH.
Binding of the ([D-Lys6(Emo)]Antg)to the pituitary GnRH receptor.
The binding affinities of different GnRH conjugates to the pituitary GnRH
receptors
were compared by competitive binding experiments as described for [D-
Lys6(Emo)]GnRH, using l2sI- [D-Lys6]GnR_H_ as a tracer. [D-Lys6(Emo)]Antg
31
CA 02500897 2005-04-O1
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showed lower binding affinity to GnRH receptors as compared to the parent
peptide
or to the [D-Lys6]GnRH conjugates (Figure 11). This seems to be mainly due to
the
similar, low binding affinity of the parent antagonist to GnR_H_ receptors
(Figure 11).
It should be noted that the antagonist, [D-Pyre, D-Phe2, D-Trp3, D-Lys6]GnRH,
has a
lower binding affinity to GnR_H_ receptor as compared to [D-Lys6]GnRH and the
insertion of emodic acid to either peptides causes a similar loss of binding
affinity.
Figure 11 compares the binding affinities of agonistic and antagonistic
derivatives of
emodin and Table 3 summarizes the estimated ICSO (concentration of the
competitor
which displace 50% of the bound tracer) of different GnR_H_ analogs to the
GnR_H_
receptor.
These results indicate that conjugated analogs of GnRH, agonists as well as
antagonists, preserved the binding capacity of their carriers
Table 3. The ICSO of GnRH analogs and their emodic acid conjugates.
Peptide analog ICSo (M)
[D-Lys ]-GnRH 0.7-1.4x10-9
[D-Lys6(Emo)]GnRH 2.6x10'9
[D-Lys6JAntg* 4.Sx 10'$
[D-Lys6(Emo)]Antg l.Sx 10-~
*[D-Lyss]-Antg correspond to the antagonist, [D-Pyre, D-Phez, D-Trp3, D-
Lys6]GnRH.
The biological potencies of the GnRH conjugates.
To correlate the binding affinity with the bioactivity, we compared the effect
of each
of the GnRH conjugates with its parent peptide on LH secretion, using primary
rat
pituitary cell cultures.
Figure 12 demonstrates the inhibition of LH secretion from rat pituitary
cultures by
the twa antagonists. Incorporation of emodic acid to the antagonist decreases
its
32
CA 02500897 2005-04-O1
WO 2004/030687 PCT/IL2002/000801
binding affinity (Figure 11) however its bioactivity is superior as compared
to the
parent antagonist (Figure 12).
EXAMPLE 8
LONG ACTING GNRH ANALOGS IN ANIMAL HUSBANDRY
In animal husbandry, the management of fertility can be both difficult and
extremely important for the success of agricultural or other businesses.
Stimulation
of ovulation at appropriate times, as well as the induction of cyclicity in
some species
of domesticated animals that can become seasonally nonovulatory would result
in
increased management efEciency for these species.
For example, in the husbandry of horses, the development of an accurate,
economical method for the precise control of ovulation in the mare would
greatly
benefit reproductive management of mares and stallions. The mares' extended
estrus
period, with ovulation at any time from 1 to 10 days after the beginning of
estrus, has
made reproductive management of mares time-consuming, expensive and most
importantly, inefficient. In the mare, GnRH or its analogs are beginning to be
used as
alternative non-antigenic substitutes to replace hCG to hasten ovulation in
preovulatory mares. This is because repeated use of hCG has been associated
with
decreased response [Sullivan, J., J. Am. Vet. Med. Assoc. 63:895(1973)] and
anti-
hCG antibody formation [Roser, J. , J. Reprod. Fert. Suppl. 173-179(1974)].
Current
data suggest that ovulation induction with potent GnR_H_ analogs requires
multiple
injections of very low doses [Harrison, L., et al., J. Eq. Vet Sci. 11:163-
166(1991)] or
a very high dose given as a slow releasing implant [Jochle, W. et al., J. Eq.
Vet. Sci.
44:632(1994)].
Similar concerns and needs occur in other areas involving the raising and
breeding of mammals, in particular livestock mammals, including, for example,
the
swine and cattle husbandry industries, where control of the estrus of sows and
gifts
and of cows and heifers, respectively, also would benefit their reproductive
management.
33
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WO 2004/030687 PCT/IL2002/000801
In particular, in commercial swine production, maximizing reproductive
efficiency offers producers substantial opportunities to reduce production
costs and
enhance profitability. Currently, a precise method of determining the time of
ovulation in spontaneously cycling gifts is not available. In gifts and sows,
GnR_H_
affects the synchronization of ovulation. However, at present the variation in
time of
the onset of ovulation is large enough that two inseminations are required for
maximal fertilization. Therefore, use of a long acting GnT2H that could
stimulate an
LH surge capable of reducing the time span of ovulations so that a single
timed
insemination could be used would greatly benefit reproductive management of
gifts,
sows, and consequently, boars.
In cattle production, maximizing reproductive efficiency offers producers
substantial opportunities to reduce production costs and enhance
profitability. This is
particularly true in the heifer, due to difficulties in synchronizing estrus
compared
with cows, a factor that reduces overall herd performance. Therefore, methods
to
synchronize estrus which increase the level of response and reduce variability
would
allow management to bring replacement heifers into the herd at lower cost and
significantly impact the efftciency of beef and dairy production.
Progress toward reducing reliance on estrus detection for managing
reproduction in dairy heifers and cows is being realized by combining timed
artificial
insemination (AI) with a protocol for synchronization of ovulation that can be
initiated at a random stage of the estrous cycle. [Pursley, J.R., et al.,
Theriogenology
44:915-923 (1995); Pursley, J.R., et al., J. I?airy Sci. 80:295-300 (1997)].
This
protocol, commonly called OVSYNCH, synchronizes follicular development, luteal
regression and time of ovulation, thereby allowing for timed insemination 12
to 24
hours after the completion of the GnR_H_ / PGF / GnRH treatment protocol.
However,
hormone cost per treated cow can be significant. [Frick, P.M., et al.,
Theriogenology
50:1275-1284 (1998)]. Since retail cost of GnR_H_ constitutes the majority of
the cost
in using OVSYNCH, availability of a cost effective long acting GnRU that could
be
used in the OVSYNCH protocol would greatly benefit reproductive management of
heifers and cows.
Lastly, aquaculture is the fastest growing section of the agricultural
industry
in many countries. However, many fish do not spawn when raised in captivity &
34
CA 02500897 2005-04-O1
WO 2004/030687 PCT/IL2002/000801
techniques to induce spawning in fish are inefficient due to the lack of
methods for
controlled administration. , availability of an effective long acting GnRH
that could
be used to induce spawning in fish would greatly benefit reproductive
management in
aquaculture.
It will be appreciated by a person skilled in the art that the present
invention is
not limited by what has been particularly shown and described hereinabove.
Rather,
the scope of the invention is defined by the claims which follow:
CA 02500897 2005-04-O1
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