U.S. patent application number 14/468713 was filed with the patent office on 2015-02-26 for compound, use and method.
The applicant listed for this patent is Medical Research Council. Invention is credited to Robert Peter Millar, Antonia Kathryn Roseweir.
Application Number | 20150057430 14/468713 |
Document ID | / |
Family ID | 38739273 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150057430 |
Kind Code |
A1 |
Millar; Robert Peter ; et
al. |
February 26, 2015 |
COMPOUND, USE AND METHOD
Abstract
The present invention relates to the use of an antagonist of
kisspeptin in the manufacture of a medicament for the treatment of
a condition induced and/or worsened by kisspeptin activity in an
individual. The invention also provides certain defined peptide
molecules, which may act as an antagonist of kisspeptin, which are
of use in treating a condition induced and/or worsened by
kisspeptin activity in an individual. In addition, the invention
provides methods of identifying and/or using antagonists of
kisspeptin and/or the defined peptides, and pharmaceutical
compositions thereof.
Inventors: |
Millar; Robert Peter; (North
Berwick, GB) ; Roseweir; Antonia Kathryn;
(Inverkeithing, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medical Research Council |
Swindon |
|
GB |
|
|
Family ID: |
38739273 |
Appl. No.: |
14/468713 |
Filed: |
August 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12681857 |
Sep 30, 2010 |
8916681 |
|
|
PCT/GB2008/003426 |
Oct 8, 2008 |
|
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14468713 |
|
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60978179 |
Oct 8, 2007 |
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Current U.S.
Class: |
530/326 ;
530/328 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61P 13/08 20180101; A61P 5/02 20180101; A61P 39/00 20180101; A61P
1/18 20180101; A61P 5/00 20180101; A61P 17/02 20180101; A61P 5/10
20180101; A61P 15/00 20180101; A61P 9/00 20180101; C07K 7/06
20130101; A61P 5/24 20180101; G01N 2500/04 20130101; A61P 9/12
20180101; A61P 37/02 20180101; A61P 15/08 20180101; A61P 43/00
20180101; A61P 25/00 20180101; A61P 35/00 20180101; C07K 7/08
20130101; A61P 5/06 20180101 |
Class at
Publication: |
530/326 ;
530/328 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 2007 |
GB |
0719592.8 |
Claims
1. Use of an antagonist of kisspeptin in the manufacture of a
medicament for the treatment of a condition induced and/or worsened
by kisspeptin activity in an individual.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/681,857, filed Sep. 30, 2010, which is the U.S. National
Stage of International Application No. PCT/GB2008/003426, filed on
Oct. 8, 2008, published in English, and claims priority under 35
U.S.C. .sctn.119 or 365 to Great Britain Application No. 0719592.8,
filed Oct. 8, 2007 and claims the benefit of U.S. Provisional
Application No. 60/978,179, filed on Oct. 8, 2007. The entire
teachings of the above applications are incorporated herein by
reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0002] This application incorporates by reference the Sequence
Listing contained in the following ASCII text file being submitted
concurrently herewith:
[0003] a) File name: 44751007001SEQLIST.txt; created Aug. 22, 2014,
31 KB in size.
BACKGROUND OF THE INVENTION
[0004] The invention relates to kisspeptin antagonists and their
use for treating a condition induced and/or worsened by kisspeptin
activity in an individual. In addition, the invention relates to a
method for identifying kisspeptin antagonists.
[0005] RFamides are a group of peptide hormones that have a common
Arg-Phe-NH2 motif at their C-terminus and all bind to G
protein-coupled receptors. RFamides have been shown to be involved
in many processes throughout the body including inflammatory
responses, control of food intake and development. One major area
where RFamides play a role is reproduction, involving two RFamides:
Gonadotrophin inhibitory Hormone (GnIH) and Kisspeptin.
[0006] The KiSS-1 gene is located on human Chromosome 1q32 and
consists of four exons, two non-translated and two partially
translated to give rise to a 145 amino acid (aa) peptide[1]. The
precursor peptide is cleaved to 54 amino acids in length, which can
be further truncated to 14, 13 or 10 amino acids; these collective
truncations are known as the Kisspeptins, and are highly conserved
within mammals. These peptides are now known to be the ligands for
the orphan G protein-coupled receptor (GPCRs) known as GPR54 in rat
and AXOR12 in humans [2-4]. The 10aa peptide, YNWNSFGLRF-NH2 (SEQ
ID NO:1) is sufficient to activate the receptor.
[0007] The GPR54 receptor has a 396aa open reading frame and is
related to the galanin receptor family, though it does not bind
galanin. Rat GPR54 is highly conserved among mammals with an 81%
homology to the human receptor and an 85% homology to the mouse [2,
4, 5]. Both the receptor and ligand have been highly characterised
within the brain and peripheral tissues, where GPR54 and KiSS-1
have been located in the hypothalamus, aorta, ovary, prostate and
placenta with GPR54 also being expressed in the pituitary [2, 4, 6,
7].
[0008] Considering the locations of receptor and ligand expression,
a role for them in reproduction was hypothesised, and confirmed
when it was shown that loss of function heterozygous and homozygous
mutations within GPR54 cause idiopathic Hypogonoadotropic
Hypogonadism (iHH), where patients exhibit delayed or failed
puberty, characterised by low or absent plasma luteinising hormone
(LH) pulsatility [8-13]. This was also observed in GPR54-/- mice,
which had small testis/ovaries, small external genitalia and
seminiferous tubules, Leydig cells and uterine horns that were
hypoplastic, very similar to that seen in iHH humans [14]. This
then eluded to the hypothesis that KiSS-1 and GPR54 played a role
in the control of puberty and in particular, its timing. Since
then, KiSS-1 mRNA level has been found to be higher in pubertal
compared to juvenile monkeys [15]. In mice, there are no KiSS-1
neurons at day 10 (d10), they begin to appear at day 25 and
increase to adult levels around day 45 in males and day 61 in
females in the Anterioventral Paraventricular nucleus (AVPV). This
coincides with the timing of puberty in these animals [16].
However, GPR54 mRNA levels remain constant throughout postnatal
development and therefore kisspeptin infusion can stimulate LH
release at all stages. Kisspeptin is therefore seen as the main
regulator of GnRH secretion in the initiation of puberty [17].
[0009] The expression of Kisspeptin and GPR54 in the hypothalamus
gave rise to the hypothesis that Kisspeptins may be involved in the
regulation of the Hypothalmic-Pituitary-Gonadal (HPG) axis. This
hypothesis was confirmed when Kisspeptin was shown to rapidly
increase plasma Luteinising Hormone (LH), Follicle Stimulating
Hormone (FSH) and Testosterone in the male rat and humans in a
dose-dependant manner, with a 10-fold more potent effect on LH over
FSH [18-20]. It has been further shown that this increase in
gonadotrophins can be abolished by administering a Gonadotrophin
Releasing Hormone Receptor (GnRHR) antagonist. This suggests that
Kisspeptin works at the level of the hypothalamus to stimulate GnRH
release[15]. This was further established when it was found that
GnRH neurons in the Median Eminence of the hypothalamus possess the
GPR54 receptor and that 90% of KiSS-1 neuron fibres are
co-localised with GnRH-immunoreactive neurons [21, 22]. Also GT1-7
hypothalamic cells express KiSS-1 and GPR54 mRNA which is increased
in response to estradiol and Kisspeptin stimulates GnRH release
from these cells after 24 hrs [23]. The above data also suggests
that Kisspeptin does not work at the level of the pituitary, though
actions directly at the pituitary level have not been ruled out.
Other groups have also shown that Kisspeptin is expressed in the
pituitary in gonadotropes and somatotropes and that Kisspeptin can
directly stimulate release of LH and growth hormone[24, 25]
[0010] KiSS-1 neurons have been localised to the Arcuate nucleus
(ARC) and AVPV of the hypothalamus in rodents and primates. However
in sheep and rats, they are only found in the ARC [16, 26, 27]. It
has now been established that the ARC is involved in negative
steroid regulation and AVPV in the positive feedback control of
gonadotrophin release. This was first noted following the
castration of male mice, when it was seen that in the ARC, KiSS-1
cell numbers and mRNA expression levels increased and that estrogen
replacement lowered this to normal levels, whereas in the AVPV,
KiSS-1 cell numbers decreased and estrogen administration increased
this to back to natural levels [26]. KiSS-1 cell bodies are now
proven to express estrogen receptor .alpha. (ER.alpha.) and the
progesterone receptor, signifying a role for KiSS-1 in steroid
feedback loops [27, 28]. This suggests that the ARC is under
negative control to regulate the LH pulses and that AVPV is under
positive control possibly to stimulate the LH surge and induction
of ovulation. This has been demonstrated by Kisspeptin antiserum
inhibition of LH surge in rats even in the presence of high
estrogen and as there are 10.times. more KiSS-1 neurons in the AVPV
of female compared to male mice. This steroid control is operative
over the estrus cycle as KiSS-1-positive cell numbers in AVPV are
highest at proestrus and lowest at diestrus with the opposite
situation in the ARC [16, 29]. In sheep, both positive and negative
feedback act at the ARC to give the same response as in other
mammals with the caudal region facilitating the LH surge. As well
as being under steroid control, KiSS-1 is also regulated by
photoperiod via melatonin, and KiSS-1 neurons possess the Mel1c
receptor on their cell bodies. Also female Siberian hamsters, held
in short day lengths had a reduced response to exogenous Kisspeptin
compared to those held in long day conditions [30-33]. Leptin also
seems to play an influential role, as severely fasted rodents have
decrease KiSS-1 mRNA, GnRH, LH and FSH as well as delayed puberty
[34, 35].
[0011] Kisspeptin is also found outside the HPG axis in peripheral
tissues such as the cardiovascular system, were Kisspeptin-10/13
have been shown to act as vasoconstrictors of aortic smooth muscle
[7, 36]. GPR54 is also highly expressed in the Central Nervous
System (CNS), in the hippocampus region of the brain, were it has
been shown to reversibly enhance the synaptic transmission in the
hippocampul dentate granule cells through mechanisms involving MAP
kinases, which appear to be regulated via calcium-activated kinases
and tyrosine kinases [37]. KiSS-1 and GPR54 mRNA have also been
located within the rat ovary where expression is again correlated
to the estrus cycle. In the ovary, KiSS-1 is located within the
ovarian surface epithelium, interstitial glands, corpus luteum and
in follicles, being present within the theca cells until proestrus
when expression shifts to the granulosa layers [6]. Recently, KiSS
mRNA was detected in the oviduct of the uterus and has been
hypothesised to be involved in the prevention of ectopic pregnancy
[38]. However, the highest levels of KiSS-1 & GPR54 are in the
placenta.
[0012] In the placenta, KiSS-1 and GPR54 are located within the
syncytiotrophoblast cells in mice [39] and humans [40]; and within
giant cells in rats [41]. GPR54 is also located in the
extravillious trophoblast cells, suggesting possible paracrine
actions [42]. Both ligand and receptor are present at high levels
in the first trimester but only KiSS-1 is present at term in humans
and both are absent by E18.5 in rats. This corresponds with
trophoblast invasion and KiSS-1 has been hypothesized as an
inhibitor of this due to its anti-metastatic properties [39,
41].
[0013] Kisspeptin is also known to be an anti-metastatic factor in
many cancer tissues, such as pancreatic, thyroid and heptocellular
carcinomas [43-45]. Different mechanisms for this function have
been hypothesised including antagonism of stromal cell-derived
factor-1 (SDF-1) to inhibit the metastatic properties of its
chemokine receptor CXCR4 and the up regulation of modulatory
calcineurin-interactin protein-1 (MCIP-1), a chemokine capable of
inhibiting the calcinuerin signalling pathway [46, 47]. Another
hypothesis is that Kisspeptin is regulated by Specificity Protein 1
(SP1) and its co-activator DRIP130 which is located on chromosome
region 6q16.3q23. When Loss of hetrozygosity (LOH) occurs at this
region KiSS-1 is frequently lost from tumours and this allows
metastasis to occur. This can be rescued by SP1 and DRIP130 which
inhibit invasion and migration[48, 49].
[0014] This inhibition has also been shown in cultured cells and
was used as an output to investigate signalling pathways used by
Kisspeptin. In chinese hamster ovary (CHO) cells, Kisspeptin-10
(Kp10) can inhibit chemotaxis, migration, colony formation and
growth and can cause cell rounding. Kp-10 can also cause focal
adhesion and stress fibre formation via the phosphorylation of
focal adhesion kinase (FAK) and paxillin [3, 50, 51]. Kisspeptin
and GPR54 have been shown to increase Inositol-3-phosphate,
intracellular calcium and pERK, showing GPR54 activates the Gq/11
signalling pathway [4]. This does not entirely concur with the
anti-metastatic properties as this pathway usually stimulates
growth and migration. It has therefore been suggested that
Kisspeptin may activate other pathways such as the Rho and
Rac/Cdc42 pathways [5, 51].
SUMMARY OF THE INVENTION
[0015] Against this background, the inventors have surprisingly
found that kisspeptin antagonists may be useful in treating a
condition induced and/or worsened by kisspeptin activity in an
individual.
[0016] Accordingly, in a first aspect the invention provides the
use of an antagonist of kisspeptin in the manufacture of a
medicament for the treatment of a condition induced and/or worsened
by kisspeptin activity in an individual. In second aspect, the
invention provides an antagonist of kisspeptin for use in the
treatment of a condition induced and/or worsened by kisspeptin
activity in an individual.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In particular, antagonists of the GPR54 receptor have been
found to be useful to manipulate gonadotropin and sex
steroid-related disorders via inhibiting GnRH release at the
hypothalamus. Kisspeptin antagonists are therefore an important
discovery as they could potentially replace GnRH agonists and
antagonists for gonadotropin inhibition and may be more rapid and
complete in their actions.
[0018] GnRH analogues are used in an inhibitory modality to inhibit
gonadotropin and sex hormones, and they have since acquired
extensive therapeutic application in treating hormone-dependent
diseases (such as prostate, breast and ovarian cancers), in in
vitro fertilisation for inducing ovulation and as new-generation
contraceptives.
[0019] Accordingly, as kisspeptin is the primary stimulator of GnRH
(for example, it is known that mutations in the kisspeptin
receptor, GPR54, cause infertility) the present inventors have
discovered that kisspeptin antagonists would have a similar range
of applications as GnRH antagonists; furthermore, because
kisspeptin operates upstream of the GnRH neurone, kisspeptin
antagonists would be expected to be more rapid and effective in its
action than GnRH-based therapies.
[0020] By "kisspeptin activity in an individual" we include any in
vitro and/or in vivo property, activity or characteristic of
kisspeptin. Thus, by "an antagonist of kisspeptin" we include the
meaning of a molecule that is capable of preventing and/or reducing
any in vitro and/or in vivo property, activity or characteristic of
kisspeptin. For example, a kisspeptin antagonist may prevent and/or
reduce the ability of kisspeptin to physically associate with, or
bind to, the kisspeptin receptor or other cell components.
[0021] Thus, the molecules and antagonists of the invention may be
capable of reversibly or irreversibly binding to the kisspeptin
receptor and/or selectively binding to the kisspeptin receptor. By
"selectively binding" we include the ability of the molecules and
antagonists of the invention to bind at least 10-fold more strongly
to the kisspeptin receptor than to another polypeptide; preferably
at least 50-fold more strongly and more preferably at least
100-fold more strongly. Preferably, the molecules and antagonists
of the invention bind to the kisspeptin receptor under
physiological conditions, for example, in vivo. Methods for
measuring the binding or association of a peptide with its ligand
or receptor (either in vivo or in vitro) are well-known to those
skilled in the arts of biochemistry and cell biology.
[0022] For example, as shown in the accompanying Examples, the
antagonists of the invention may be competitive antagonists of
kisspeptin which bind to, but do not activate, the kisspeptin
cognate receptor (such as GPR54).
[0023] A kisspeptin antagonist may also prevent and/or reduce the
ability of kisspeptin to stimulate particular cellular behaviour,
such as the kisspeptin-mediated stimulation of inositol phosphate
production in a cell (such as in transformed or primary cell lines,
like CHO, HEK, COS, the GnRH GTI neuronal cell-line, and normal
trophoblasts and cell lines such as JEG). Biochemical assays for
the cellular measurement of inositol phosphate production are well
known in the art.
[0024] In a particularly preferred aspect, the antagonist is a
peptide analogue of the kisspeptin peptide. The structure/activity
relationship (SAR) for peptide analogues of the kisspeptin peptides
has received relatively sparse attention and, until now, focused
entirely on agonism [52, 53]. The amino acid sequence of kisspeptin
(also known as kisspeptin 1-54) is:
TABLE-US-00001 (SEQ ID NO: 2)
gtslspppessgsrqqpglsaphsrqipapqgavlvqrekdlpnynwnsf
glrf.nh.sub.2
[0025] Thus, in a third aspect, the invention provides the use of a
peptide molecule comprising or consisting of the sequence:
TABLE-US-00002 (SEQ ID NO: 3) X.sup.1 - G / W - X.sup.2 - R / (D)R
- X.sup.3
wherein: X.sup.1 is F or A or any D-amino-acid residue; X.sup.2 is
L or A or any D-amino-acid residue;
X.sup.3 is F or W; and
[0026] wherein the C-terminal amino acid residue of the peptide
molecule contains group z which removes the charge on that
residue;
[0027] or a fragment or variant thereof;
[0028] and wherein the peptide sequence is not:
TABLE-US-00003 (SEQ ID NO: 4) F - G - L - R - F; (SEQ ID NO: 5) F -
G - L - R - W; or (SEQ ID NO: 6) F - G - (D)F - R - F.
for the treatment of a condition induced and/or worsened by
kisspeptin activity in a patient or in the manufacture of a
medicament for the treatment of a condition induced and/or worsened
by kisspeptin activity in a patient
[0029] Preferably, where the C-terminal amino acid residue of the
peptide molecules of the invention contains group z, the negative
charge on that C-terminal residue is removed.
[0030] It will be appreciated that the peptide molecules of the
invention described herein are defined using the conventional
one-letter code used to denote amino acids. The term "amino acid"
includes any of a group of water-soluble organic compounds that
possess both a carboxyl (--COOH) and an amino (--NH.sub.2) group
attached to the .alpha.-carbon atom. Amino acids can be represented
by the general formula R--CH(NH.sub.2)COOH; the R group is hydrogen
or an organic group and determines the properties of any particular
amino acid. The tetrahedral array of four different groups about
the .alpha.-carbon atom confers optical activity on amino acids.
The two-mirror image forms are called an L-isomer and a D-isomer.
Typically, only L-amino acids are constituents of proteins (such as
eukaryotic proteins).
[0031] Unless otherwise stated, the peptide molecules of the
invention contain L-amino acids. When present in the peptide
molecules of the invention, D-amino acids are referred to with the
prefix "(D)" prior to the usual one-letter amino acid code.
[0032] Where stated, the molecules of the invention can comprise or
consist of peptide sequences having "any D-amino acid" at a given
position. By "any D-amino acid" we include any natural or unnatural
(for example, chemically-modified) D-amino acid in that position in
the sequence. Examples of natural D-amino acids are: D-alanine;
D-aspartic acid; D-cysteine; D-glutamic acid; D-phenylalanine;
;D-glycine; D-histidine; D-isoleucine; D-lysine; D-leucine;
D-methionine; D-asparagine; D-proline; D-glutamine; D-arginine;
D-serine; D-threonine; D-valine; D-tryptophan; D-tyrosine.
[0033] Examples of unnatural D-amino acids are: napthylalanine;
D-pyridyl alanine; D-tertiarybutyl serine; D-ornithine; D-epsilon
amino lysine; D-homoarginine; D-.alpha. methyl leucine along with
halide substitutions (e.g. F) of protons in these and other
unnatural amino acids.
[0034] Through the formation of peptide bonds, amino acids join
together to form short chains (peptides) or longer chains
(polypeptides). It is well known that proteins and/or peptides are
composed of varying proportions of approximately 20
commonly-occurring amino acids, the sequence of which determines
the shape, properties and biological role of the protein and/or
peptide. Amino acid residues within such peptide or polypeptide
chains are conventionally referred to by their numbered position in
the chain, with the first position (i.e. position 1) assigned to
the amino acid at the N-terminal end of the chain.
[0035] Peptide sequences of the molecules of the invention may be
synthesised by the Fmoc-polyamide mode of solid-phase peptide
synthesis as disclosed by Lu et at (1981) J. Org. Chem. 46, 3433
and references therein. Temporary N-amino group protection is
afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group.
Repetitive cleavage of this highly base-labile protecting group is
effected using 20% piperidine in N,N-dimethylformamide. Side-chain
functionalities may be protected as their butyl ethers (in the case
of serine threonine and tyrosine), butyl esters (in the case of
glutamic acid and aspartic acid), butyloxycarbonyl derivative (in
the case of lysine and histidine), trityl derivative (in the case
of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl
derivative (in the case of arginine). Where glutamine or asparagine
are C-terminal residues, use is made of the
4,4'-dimethoxybenzhydryl group for protection of the side chain
amido functionalities. The solid-phase support is based on a
polydimethyl-acrylamide polymer constituted from the three monomers
dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine
(cross linker) and acryloylsarcosine methyl ester (functionalising
agent). The peptide-to-resin cleavable linked agent used is the
acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All
amino acid derivatives are added as their preformed symmetrical
anhydride derivatives with the exception of asparagine and
glutamine, which are added using a reversed
N,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated
coupling procedure. All coupling and deprotection reactions are
monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin
test procedures. Upon completion of synthesis, peptides are cleaved
from the resin support with concomitant removal of side-chain
protecting groups by treatment with 95% trifluoroacetic acid
containing a 50% scavenger mix. Scavengers commonly used are
ethanedithiol, phenol, anisole and water, the exact choice
depending on the constituent amino acids of the peptide being
synthesised. Trifluoroacetic acid is removed by evaporation in
vacuo, with subsequent trituration with diethyl ether affording the
crude peptide. Any scavengers present are removed by a simple
extraction procedure which on lyophilisation of the aqueous phase
affords the crude peptide free of scavengers. Reagents for peptide
synthesis are generally available from Calbiochem-Novabiochem (UK)
Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any
one, or a combination of, techniques such as size exclusion
chromatography, ion-exchange chromatography and (principally)
reverse-phase high performance liquid chromatography. Analysis of
peptides may be carried out using thin layer chromatography,
reverse-phase high performance liquid chromatography, amino-acid
analysis after acid hydrolysis and by fast atom bombardment (FAB)
mass spectrometric analysis.
[0036] The peptide sequence of the molecules of the invention may
also be synthesised using liquid phase methodology, which is well
known those skilled in the art of chemistry and biochemistry.
[0037] The peptide sequence of the molecules of the invention may
comprise or consist of peptidomimetic compounds. The term
"peptidomimetic" refers to a compound that mimics the conformation
and desirable features of a particular peptide as a therapeutic
agent, but that avoids the undesirable features. For example,
morphine is a compound which can be orally administered, and which
is a peptidomimetic of the peptides enkephalin and/or
endorphin.
[0038] In general, therapeutic applications involving peptides are
limited due to lack of oral bioavailability and to proteolytic
degradation., and are typically administered by injection.
Typically, for example, peptides are rapidly degraded in vivo by
exo- and endopeptidases, resulting in generally very short
biological half-lives. Another deficiency of peptides as potential
therapeutic agents is their lack of bioavailability via oral
administration. Degradation of the peptides by proteolytic enzymes
in the gastrointestinal tract is likely to be an important
contributing factor. The problem is, however, more complicated
because it has been recognised that even small, cyclic peptides or
peptides comprising D-amino acids which are not subject to rapid
metabolite inactivation nevertheless exhibit poor oral
bioavailability. This is likely to be due to poor transport across
the intestinal membrane and rapid clearance from the blood by
hepatic extraction and subsequent excretion into the intestine.
These observations suggest that multiple amide bonds may interfere
with oral bioavailability. It is thought that the peptide bonds
linking the amino acid residues in the peptide chain may break
apart or be cleaved when the peptide drug is orally
administered.
[0039] There are a number of different approaches to the design and
synthesis of peptidomimetics. In one approach, such as disclosed by
Sherman and Spatola, J. Am. Chem. Soc., 112: 433 (1990), one or
more amide bonds have been replaced in an essentially isoteric
manner by a variety of chemical functional groups. This stepwise
approach has met with some success in that active analogues have
been obtained. In some instances, these analogues have been shown
to possess longer biological half-lives than their
naturally-occurring counterparts. Nevertheless, this approach has
limitations. Successful replacement of more than one amide bond has
been rare. Consequently, the resulting analogues have remained
susceptible to enzymatic inactivation elsewhere in the molecule.
When replacing the peptide bond it is preferred that the new linker
moiety has substantially the same charge distribution and
substantially the same planarity as a peptide bond.
[0040] Retro-inverso peptidomimetics, in which the peptide bonds
are reversed, can be synthesised by methods known in the art, for
example such as those described in Meziere et at (1997) J. Immunol.
159 3230-3237. This approach involves making pseudopeptides
containing changes involving the backbone, and not the orientation
of side chains. Retro-inverse peptides, which contain NH--CO bonds
instead of CO--NH peptide bonds, are much more resistant to
proteolysis. Retro-inverso peptidomimetics of certain GnRH peptides
have been synthesised previously (Fromme, 2003, Endocrinology,
144:3262-9).
[0041] In another approach, a variety of un-coded or modified amino
acids such as D-amino acids and N-methyl amino acids have been used
to modify mammalian peptides. Alternatively, a presumed bioactive
conformation has been stabilised by a covalent modification, such
as cyclisation or by incorporation of .gamma.-lactam or other types
of bridges. See, for example, Veber et al, Proc. Natl. Acad. Sci.
USA, 75:2636 (1978) and Thursell et al, Biochem. Biophys. Res.
Comm., 111:166 (1983).
[0042] A common theme among many of the synthetic strategies has
been the introduction of some cyclic moiety into a peptide-based
framework. The cyclic moiety restricts the conformational space of
the peptide structure and this frequently results in an increased
affinity of the peptide for a particular biological receptor. An
added advantage of this strategy is that the introduction of a
cyclic moiety into a peptide may also result in the peptide having
a diminished sensitivity to cellular peptidases.
[0043] One approach to the synthesis of cyclic stabilised
peptidomimetics is ring closing metathesis (RCM). This method
involves steps of synthesising a peptide precursor and contacting
it with a RCM catalyst to yield a conformationally-restricted
peptide. Suitable peptide precursors may contain two or more
unsaturated C--C bonds. The method may be carried out using
solid-phase-peptide-synthesis techniques. In this embodiment, the
precursor, which is anchored to a solid support, is contacted with
a RCM catalyst and the product is then cleaved from the solid
support to yield a conformationally restricted peptide.
[0044] Another approach, disclosed by D. H. Rich in Protease
Inhibitors, Barrett and Selveson, eds., Elsevier (1986), has been
to design peptide mimics through the application of the transition
state analogue concept in enzyme inhibitor design. For example, it
is known that the secondary alcohol of staline mimics the
tetrahedral transition state of the scissile amide bond of the
pepsin substrate. However, the transition state analogue concept
has no apparent relevance to hormone agonist/antagonist design.
[0045] For the avoidance of doubt, it is not necessary that the
amino acid residues in the peptide sequence are joined by standard
peptide bonds. For example, as discussed above, the amino acid
residues may be linked by reverse peptide bonds, or they may be
joined together by other bonds which mimic the bond distance and
spatial orientation of a standard peptide bond.
[0046] Peptide sequences of the agents of the invention may be
purified following synthesis using methods known in the art, such
as HPLC and chromatography
[0047] By "a molecule" we include salts (e.g. organic or inorganic
acid addition salts), esters and solvates of the molecules
comprising or consisting of the peptide sequences of the invention.
It will be appreciated that the term further includes derivatives
that have the same biological function and/or activity as the
relevant molecule. Moreover, for the purposes of this invention,
the term also includes prodrugs of the relevant molecule (for
example, esters). The term "prodrug" includes any composition of
matter that, following oral or parenteral administration, is
metabolised in vivo to form the relevant agent in an
experimentally-detectable amount, and within a predetermined time
of dosing.
[0048] The molecules of the invention may further consist of or
comprise one or more moiety which is capable of targeting and/or
localising the molecule of the invention to a target cell (such as
a cancer cell) and/or to increase the half-life (t1/2) of the
molecule of the invention. Such moieties can therefore increase
efficacy of the molecule of the invention. Preferably, one or more
moiety may be included in a molecule of the invention when the
molecule comprises or consists of a peptide sequence comprising or
consisting of a D-amino acid as those amino acid residues are
particularly amenable to modification.
[0049] The molecules of the invention may further consist of or
comprise one or more moiety which is capable of targeting and/or
localising the molecule of the invention to a target cell (such as
a cancer cell) and/or to increase the half-life (t1/2) of the
molecule of the invention. Such moieties can therefore increase
efficacy of the molecules of the invention. Preferably, one or more
moiety may be included in a molecule of the invention when the
agent comprises or consists of a peptide sequence comprising a
D-amino acid, as those amino acid residues are particularly
amenable to modification.
[0050] Preferably, the one or more moiety is a steroid hormone
molecule (including, for example, progesterone, testosterone,
estradiol or cortisol) and is conjugated to the side chain of a
D-amino acid. Steroid hormone molecules are capable of binding to
plasma proteins and have been shown to reduce the metabolic
clearance of peptides (Ratcliffe et al., 2006, Endocrinology,
147:571-9). For example, GnRH peptides conjugated to steroid
hormones are described in WO2004/08725, incorporated herein by
reference. Alternatively, the one or more moiety is a vitamin, such
as vitamin B.sub.12 or vitamin D, and is conjugated to the NH.sub.2
terminus of the kisspeptin analogues or a suitable side chain of a
natural or D-amino acid. Vitamins have been shown to improve the
oral bioavailability of peptides (Russell-Jones et al., 1995,
Bioconjug. Chem., 6:34-42; Russell-Jones et al., 1995, Bioconjug.
Chem., 6:459-465).
[0051] Preferably, the ability of the molecule of the invention to
act as an antagonist of kisspeptin is not affected and/or
significantly affected by the one or more moiety.
[0052] Preferably, the invention provides a use wherein the peptide
sequence is not:
TABLE-US-00004 (SEQ ID NO: 7) F - G - A - R - W; (SEQ ID NO: 8) F -
G - L - (D)R - W; (SEQ ID NO9) F - G - (D)L - R - W; or (SEQ ID NO:
10) (D)F - G - L - R - W.
[0053] Conveniently, the invention provides a use wherein X.sup.1
is (D)F. Preferably, X.sup.2 is a D-amino-acid residue selected
from the group consisting of: (D)F, (D)L and (D)W.
[0054] More preferably, the peptide sequence is selected from the
group consisting of:
TABLE-US-00005 (SEQ ID NO: 11) (D)F - W - L - R - W; or (SEQ ID NO:
12) F - G - (D)W - R - F.
[0055] In one embodiment, the invention provides a use wherein the
N-terminal residue contains group y which removes the charge on
that residue. Typically, X.sup.1 contains group y which removes the
charge on that residue.
[0056] The group y may be selected from the group consisting of: an
acetyl group (represented throughout this application as "ac"); a
trifluoroacetyl group; a cyclised amino acid; or a synthetic amino
acid lacking a charge at the N-terminus (such as an amino acid with
an amino group that has been modified by the addition of a further
compound (such as pyroglutamic acid) or chemical group, for example
an alkyl group (e.g. formyl, acetyl, propyl, butyl and longer alkyl
groups).
[0057] Preferably, the invention provides a use wherein X.sup.3
contains group z which removes the charge on that residue.
[0058] In one embodiment, the invention provides a use wherein the
sequence is selected from the group consisting of:
TABLE-US-00006 (SEQ ID NO: 13) ac.F - G - (D)F - R - W.z; (SEQ ID
NO: 14) ac.F - G - (D)L - R - W.z; (SEQ ID NO: 15) ac.F - G - L -
(D)R - W.z; (SEQ ID NO: 16) ac.F - G - A - R - W.z; (SEQ ID NO: 17)
ac.A - G - L - R - W.z; (SEQ ID NO: 18) ac.(D)F - W - L - R - W.z;
or (SEQ ID NO: 19) ac.F - G - (D)W - R - F.z.
[0059] In one embodiment, the acetyl group on the N-terminal
residue of any of peptides (I) to (VI), above, (which is
represented above and throughout this application as "ac") is
replaced with a group y which removes the charge on that residue,
and is preferably selected from the group consisting of: an acetyl
group; a trifluoroacetyl group; a cyclised amino acid; or a
synthetic amino acid lacking a charge at the N-terminus.
[0060] In one embodiment, the use according to the third aspect of
the invention involves a molecule in which the peptide sequence
comprises additional amino acid residues, or peptides, at the N-
and/or C-terminus of the peptide sequence of the third aspect of
the invention, thereby incorporating the peptide sequence into a
larger polypeptide or protein molecule. Thus, the peptide sequence
of the third aspect of the invention may comprise at the N- and/or
C-terminus between 0 and 10 amino acids; or between 10 and 20 amino
acids; or between 20 and 30 amino acids; or between 30 and 40 amino
acids; or between 40 and 50 amino acids; or between 50 and 60 amino
acids; or between 60 and 70 amino acids; or between 70 and 80 amino
acids; or between 80 and 90 amino acids; or between 90 and 100
amino acids; or more than 100 amino acids.
[0061] In a preferred embodiment, the use according to the third
aspect of the invention involves a molecule comprising or
consisting of the additional peptide sequence R-R-M-K-W-K-K-Y (SEQ
ID NO:20) or ac.R-R-M-K-W-K-K-Y (SEQ ID NO:20) at the N-terminus of
the peptide sequence.
[0062] The R-R-M-K-W-K-K-Y (SEQ ID NO:20) sequence is a
heptapeptide sequence from Antennapedia, which facilitates and/or
improves intracellular delivery of peptide sequences, thereby
improving the therapeutic use of the peptides of the invention. The
Antennapedia heptapeptide is described in Fisher et al., 2003, J.
Peptide Res., 55:163-72; and Wang et al., 2006, Bioorganic and
Medicinal Chemistry Letters, 16:2628-2631.
[0063] Accordingly, the peptide of the invention may comprise or
consist of the sequence:
TABLE-US-00007 (SEQ ID NO: 21) R - R - M - K - W - K - K - Y - F -
G - (D)F - R - W.z; (SEQ ID NO: 22) R - R - M - K - W - K - K - Y -
F - G - (D)L - R - W.z; (SEQ ID NO: 23) R - R - M - K - W - K - K -
Y - F - G - L - (D)R - W.z; (SEQ ID NO: 24) R - R - M - K - W - K -
K - Y - F - G - A - R - W.z; (SEQ ID NO: 25) R - R - M - K - W - K
- K - Y - A - G - L - R - W.z; (SEQ ID NO: 26) R - R - M - K - W -
K - K - Y - (D)F - W - L - R - W.z; or (SEQ ID NO: 27) R - R - M -
K - W - K - K - Y - F - G - (D)W - R - F.z.
[0064] Optionally, the N-terminal residue may comprise group y at
the N-terminus, as defined above, and is preferably acetyl
(ac).
[0065] Preferably, the peptide sequence of the third aspect of the
invention is incorporated within some or all of the sequence of the
full-length kisspeptin peptide. More preferably, the N-terminus of
the peptide sequence of the third aspect of the invention can be
extended to any length incorporating the peptide sequence of
kisspeptin 1-54 (i.e. amino acid residues 1 to 54 of the kisspeptin
sequence) and/or the peptide sequence of kisspeptin 1-45 (i.e.
amino acid residues 1 to 45 of the kisspeptin sequence). For
example, the peptide sequence of the third aspect of the invention
may have the following sequence at its N-terminus:
TABLE-US-00008 (SEQ ID NO: 28)
gtslspppessgsrqqpglsaphsrqipapqgavlvqrekdlpnynwns
[0066] Alternatively, the peptide sequence of the third aspect of
the invention can be incorporated within a protein, such as an
albumin and/or immunoglobulin protein. Incorporation into such
molecules is thought to increase half-life and decrease the
metabolic clearance within an individual of the peptide molecules
of the invention.
[0067] In a fourth aspect, the invention provides the use of a
peptide molecule comprising or consisting of the sequence:
TABLE-US-00009 (SEQ ID NO: 39) X.sup.A - X.sup.B - X.sup.C - N -
X.sup.D - X.sup.E - G - X.sup.F - R - F
[0068] wherein:
[0069] X.sup.A is Y or any D-amino-acid residue;
[0070] X.sup.B is N or any D-amino acid residue;
[0071] X.sup.C is W or any D-amino acid residue;
[0072] X.sup.D is G or S or any D-amino acid residue;
[0073] X.sup.E is F or (D)W or (D)L;
[0074] X.sup.F is W or L or any D-amino acid residue; and
[0075] wherein the C-terminal amino acid residue of the peptide
molecule contains group z which removes the charge on that
residue;
[0076] or a fragment or variant thereof;
[0077] and wherein the peptide sequence is not:
TABLE-US-00010 (SEQ ID NO: 30) Y - N - W - N - S - F - G - L - R -
F; (SEQ ID NO: 31) (D)Y - (D)N - W - N - S - F - G - W - R - F;
(SEQ ID NO: 32) (D)Y - (D)N - W - N - G - F - G - W - R - F; (SEQ
ID NO: 33) (D)Y - (D)N - W - N - S - F - G - (D)W - R - F; or (SEQ
ID NO: 34) (D)Y - (D)N - W - N - G - F - G - (D)W - R - F.
[0078] for the treatment of a condition induced and/or worsened by
kisspeptin activity in a patient or in the manufacture of a
medicament for the treatment of a condition induced and/or worsened
by kisspeptin activity in a patient.
[0079] Preferably, X.sup.A is a D-amino-acid residue selected from
the group consisting of: (D)F and (D)Y and (D)A. Typically, X.sup.B
is a D-amino-acid residue selected from the group consisting of:
(D)A and (D)N. In one aspect, when one of X.sup.A or X.sup.B is
(D)Y, the other is not (D)N. In another aspect, X.sup.A and X.sup.B
are not both a D-amino acid residue. Preferably, when X.sup.F is
(D)W, X.sup.A is (D)F.
[0080] Preferably, the invention provides a use wherein X.sup.C is
a D-amino-acid residue selected from the group consisting of: (D)A
and (D)W.
[0081] In one embodiment, X.sup.D is a D-amino-acid residue
selected from the group consisting of: (D)A and (D)W. Preferably,
when X.sup.D is S, X.sup.F is (D)W and/or X.sup.A is not (D)Y and
more preferably, when X.sup.D is S, X.sup.F is (D)W and/or X.sup.A
is (D)A.
[0082] Preferably, X.sup.F is a D-amino-acid residue selected from
the group consisting of: (D)L and (D)W.
[0083] In one embodiment, when X.sup.E and X.sup.F are both (D)W,
X.sup.A is not (D)Y. Preferably, when X.sup.E and X.sup.F are both
(D)W, X.sup.A is (D)A.
[0084] Typically, the invention provides a use wherein the
N-terminal residue contains group y which removes the charge on
that residue. Preferably, X.sup.1 contains group y which removes
the charge on that residue. The group y is selected from the group
consisting of: an acetyl group; a trifluoroacetyl group; a cyclised
amino acid; or a synthetic amino acid lacking a charge at the
N-terminus.
[0085] In one preferred embodiment, the invention provides a use
wherein the C-terminal F residue of the peptide molecule contains
group z which removes the charge on that residue.
[0086] In one embodiment, the peptide sequence is selected from the
group consisting of:
TABLE-US-00011 (SEQ ID NO: 35) Y - N - W - N - G - F - G - L - R -
F.z; (SEQ ID NO: 36) Y - N - W - N - G - F - G - (D)L - R - F.z;
(SEQ ID NO: 37) Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID
NO: 38) Y - N - W - N - G - (D)W - G - L - R - F.z; (SEQ ID NO: 39)
ac.Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 40) ac.Y
- N - W - N - (D)W - F - G - (D)W - R - F.z; (SEQ ID NO: 41)
ac.(D)Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 42)
ac.Y - N - (D)W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 43)
ac.Y - (D)N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 44)
ac.Y - N - W - N - (D)A - F - G - (D)W - R - F.z; (SEQ ID NO: 45)
ac.(D)A - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 46)
ac.Y - N - (D)A - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 47)
ac.Y - (D)A - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 48)
ac.(D)W - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 49)
ac.(D)F - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 50)
ac.(D)Y - N - W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID NO:
51) ac.(D)A - N - W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID
NO: 52) ac.(D)A - N - W - N - S - F - G - (D)W - R - F.z; (SEQ ID
NO: 53) ac.D - A - N - W - N - G - F - G - W - R - F.z; (SEQ ID NO:
54) ac.(D)A - N - W - N - (D)S - F - G - (D)W - R - F.z; or (SEQ ID
NO: 55) ac.(D)A - N - W - N - G - F - G - (D)L - R - F.z.
[0087] In one embodiment, the use according to the fourth aspect of
the invention involves a molecule in which the peptide sequence
comprises additional amino acid residues, or peptides, at the N-
and/or C-terminus of the peptide sequence of the fourth aspect of
the invention, thereby incorporating the peptide sequence into a
larger polypeptide or protein molecule. Thus, the peptide sequence
of the fourth aspect of the invention may comprise at the N- and/or
C-terminus between 0 and 10 amino acids; or between 10 and 20 amino
acids; or between 20 and 30 amino acids; or between 30 and 40 amino
acids; or between 40 and 50 amino acids; or between 50 and 60 amino
acids; or between 60 and 70 amino acids; or between 70 and 80 amino
acids; or between 80 and 90 amino acids; or between 90 and 100
amino acids; or more than 100 amino acids.
[0088] In a preferred embodiment, the use according to the fourth
aspect of the invention involves a molecule comprising or
consisting of the additional peptide sequence R-R-M-K-W-K-K-Y (SEQ
ID NO:20) or ac.R-R-M-K-W-K-K-Y (SEQ ID NO:20) at the N-terminus of
the peptide sequence.
[0089] The R-R-M-K-W-K-K-Y (SEQ ID NO:20) sequence is a
heptapeptide sequence from Antennapedia, as discussed above.
Accordingly, the peptide of the fourth aspect of the invention may
comprise or consist of the sequence:
TABLE-US-00012 (SEQ ID NO: 56) R - R - M - K - W - K - K - Y - Y -
N - W - N - G - F - G - L - R - F.z; (SEQ ID NO: 57) R - R - M - K
- W - K - K - Y - Y - N - W - N - G - F - G - (D)L - R - F.z; (SEQ
ID NO: 58) R - R - M - K - W - K - K - Y - Y - N - W - N - G - F -
G - (D)W - R - F.z; (SEQ ID NO: 59) R - R - M - K - W - K - K - Y -
Y - N - W - N - G - (D)W - G - L - R - F.z; (SEQ ID NO: 60) R - R -
M - K - W - K - K - Y - Y - N - W - N - G - F - G - (D)W - R - F.z;
(SEQ ID NO: 61) R - R - M - K - W - K - K - Y - Y - N - W - N -
(D)W - F - G - (D)W - R - F.z; (SEQ ID NO: 62) R - R - M - K - W -
K - K - Y - (D)Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID
NO: 63) R - R - M - K - W - K - K - Y - Y - N - (D)W - N - G - F -
G - (D)W - R - F.z; (SEQ ID NO: 64) R - R - M - K - W - K - K - Y -
Y - (D)N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 65) R -
R - M - K - W - K - K - Y - Y - N - W - N - (D)A - F - G - (D)W - R
- F.z; (SEQ ID NO: 66) R - R - M - K - W - K - K - Y - (D)A - N - W
- N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 67) R - R - M - K - W
- K - K - Y - Y - N - (D)A - N - G - F - G - (D)W - R - F.z; (SEQ
ID NO: 68) R - R - M - K - W - K - K - Y - Y - (D)A - W - N - G - F
- G - (D)W - R - F.z; (SEQ ID NO: 69) R - R - M - K - W - K - K - Y
- (D)W - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 70) R
- R - M - K - W - K - K - Y - (D)F - N - W - N - G - F - G - (D)W -
R - F.z; (SEQ ID NO: 71) R - R - M - K - W - K - K - Y - (D)Y - N -
W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID NO: 72) R - R - M -
K - W - K - K - Y - (D)A - N - W - N - G - (D)W - G - (D)W - R -
F.z; (SEQ ID NO: 73) R - R - M - K - W - K - K - Y - (D)A - N - W -
N - S - F - G - (D)W - R - F.z; (SEQ ID NO: 74) R - R - M - K - W -
K - K - Y - (D)A - N - W - N - G - F - G - W - R - F.z; (SEQ ID NO:
75) R - R - M - K - W - K - K - Y - (D)A - N - W - N - (D)S - F - G
- (D)W - R - F.z; or (SEQ ID NO: 76) R - R - M - K - W - K - K - Y
- (D)A - N - W - N - G - F - G - (D)L - R - F.z.
[0090] Optionally, the N-terminal residue may comprise group y at
the N-terminus, as defined above, and is preferably acetyl
(ac).
[0091] In a particularly preferred embodiment, the peptide sequence
of the fourth aspect of the invention is selected from the group
consisting of:
TABLE-US-00013 (SEQ ID NO: 77) R - R - M - K - W - K - K - Y - (D)A
- N - W - N - G - F - G - (D)W - R - F.z; or (SEQ ID NO: 77) ac.R -
R - M - K - W - K - K - Y - (D)A - N - W - N - G - F - G - (D)W - R
- F.z.
[0092] As demonstrated in the accompanying examples, peptides of
the invention comprising the N-terminal Antennapedia heptapeptide
retain kisspeptin antagonist function.
[0093] Preferably, the peptide sequence of the fourth aspect of the
invention is incorporated within some or all of the sequence of the
full-length kisspeptin peptide. More preferably, the N-terminus of
the peptide sequence of the fourth aspect of the invention can be
extended to any length incorporating the peptide sequence of
kisspeptin 1-54 (i.e. amino acid residues 1 to 54 of the kisspeptin
sequence) and/or the peptide sequence of kisspeptin 1-45 (i.e.
amino acid residues 1 to 45 of the kisspeptin sequence). For
example, the peptide sequence of the fourth aspect of the invention
may have the following sequence at its N-terminus:
TABLE-US-00014 (SEQ ID NO: 78)
gtslspppessgsrqqpglsaphsrqipapqgavlvqrekdlp
[0094] Alternatively, the peptide sequence of the fourth aspect of
the invention can be incorporated within a protein, such as an
albumin and/or immunoglobulin protein. Incorporation into such
molecules is thought to increase half-life and decrease the
metabolic clearance within an individual of the peptide molecules
of the invention.
[0095] By "a condition induced and/or worsened by kisspeptin
activity in an individual" we include conditions in which the
symptoms and/or progression and/or outcome of that condition in an
individual are initiated, induced, elevated, increased and/or made
more severe as a consequence of the activity of kisspeptin.
[0096] Preferably, the condition induced and/or worsened by
kisspeptin activity in an individual is selected from the group
comprising: a proliferative disorder; endometriosis; uterine
fibroids; precocious puberty; pre-eclampsia; inter-uterine growth
retardation (IUGR); ectopic pregnancy; menorrhagia; hypertension;
coronary heart disease; pathologies of the central nervous system
(CNS), pancreas and/or immune system; suppressed or inhibited
ovulation; fertility; chemotherapy-induced and/or
radiotherapy-induced damage to reproductive tissues; suppressed
and/or inhibited wound healing; suppressed and/or inhibited growth
hormone production.
[0097] For example, in reproductive tissues stimulated by
gonadotropins and sex steroid hormones, kisspeptin antagonists will
inhibit GnRH secretion, thereby inhibiting stimulation of
reproductive tissues and reproductive cancers. Accordingly,
antagonists of kisspeptin can be used to inhibit endogenous
gonadotropin so that exogenous gonadotropin can be administered in
a controlled manner to treat individuals having suppressed or
inhibited ovulation, thereby inducing or elevating ovulation in
that individual. Antagonists of kisspeptin may therefore also be
used to reduce or inhibit fertility, resulting in a means of
contraception in both males and females. Furthermore, the
inhibition of stimulation of reproductive tissues by kisspeptin
antagonists will permit their use in preventing
chemotherapy-induced or radiotherapy-induced damage to reproductive
tissues, thereby protecting reproductive tissues in an individual
treated with chemotherapy and/or radiation therapy of cancer.
[0098] As demonstrated in the accompanying examples, the
antagonists of the invention are capable of inhibiting and/or
reducing the inhibitory effect of kisspeptin on cell migration.
Since cell migration occurs, and is required, in wound healing,
kisspeptin therefore plays a role in inhibiting wound healing.
Accordingly, in one embodiment, the antagonists of the invention
may be used to induce and/or promote wound healing in an individual
in need thereof.
[0099] As demonstrated in the accompanying examples, the
antagonists of the invention surprisingly elevated growth hormone
levels in test individuals. Accordingly, the antagonists of the
invention may be used to promote and/or induce growth hormone
production in an individual in need thereof. For example, the
antagonists of the invention could be used alongside existing
treatments in which growth hormone therapy is used, such as in the
treatment of renal failure.
[0100] Kisspeptin independently inhibits trophoblast invasion into
the uterine wall (i.e. implantation), as occurs in pre-eclampsia or
IUGR. In relation to pre-eclampsia, it has been shown that KiSS-1
mRNA is increased, preventing proper trophoblast invasion of the
maternal blood supply [54]. Thus kisspeptin antagonists may be used
to relieve that inhibition and, for example, allow trophoblast
invasiveness during pregnancy in disorders such as
pre-eclampsia.
[0101] It is known that kisspeptin independently induces
vasoconstriction, and kisspeptin antagonists could therefore be
used to treat hypertension, for example in peripheral tissues as a
hypertensive agent to reduce vasoconstriction of blood vessels.
GPR54 is expressed in the CNS and kisspeptin antagonists may be
employed in modulating CNS function (such as in the hypocampul
dentate granule cells).
[0102] In one embodiment, the acetyl group on the N-terminal
residue of any of peptides (e) to (s), above, is replaced with a
group y which removes the charge on that residue, and is preferably
selected from the group consisting of: an acetyl group; a
trifluoroacetyl group; a cyclised amino acid; or a synthetic amino
acid lacking a charge at the N-terminus.
[0103] Kisspeptin is known to be expressed at high levels in the
placenta and has been implicated in implantation. Poor implantation
can cause pre-eclampsia and intra-uterine growth retardation, for
which (until the present invention) there is no effective
treatment.
[0104] Kisspeptin and its receptor (GPR54) is also expressed in
trophoblast and in uterine cells (Hiden et al., 2007, Rev. Endocr.
Metab. Disord., 8:31-39) and, as shown in the accompanying
Examples, the inventors have shown that kisspeptin causes foetal
growth retardation. Accordingly, modulation of kisspeptin input
will have application in pre-eclampsia, inter-uterine growth
retardation (IUGR) and ectopic pregnancy.
[0105] Furthermore, kisspeptin and GPR54 induce increased
vasoconstriction (Mead et al., 2007, Endocrinology, 148:140-147)
and are present in atherosclerotic plaque, such that kisspeptin
antagonists could be used to treat hypertension and coronary heart
disease.
[0106] Kisspeptin and its receptor (GPR54) are also expressed
widely in the central nervous system, pancreas and immune system
(Muir et al., 2001, J. Biol. Chem., 276:28969-28975; Kotani et
al.., 2001, J. Biol. Chem., 276:34631-34636) such that antagonists
may have application in pathologies of those tissues.
[0107] Preferably, the condition induced and/or worsened by
kisspeptin activity is a condition of an animal. The animal may be
a human, or may be any mammal such as a domesticated mammal
(preferably of agricultural or commercial significance including a
chicken; cat; dog; pig; sheep; cow; horse).
[0108] More preferably, the proliferative disorder is selected from
the group consisting or comprising: benign prostatic hyperplasia;
cancer; cancer of reproductive tissues; gynaecological cancer;
prostate cancer; breast cancer; ovarian cancer; uterine cancer;
cervical cancer; endometrial cancer; melanoma; pancreatic cancer;
gastric cancer.
[0109] All cancers which express the kisspeptin receptor could
potentially be treated using the molecules of the invention.
Preferably, the cancer is a reproductive cancer (including
prostate, endometrial, cervical, ovarian and breast cancers), all
of which express the kisspeptin receptor.
[0110] Methods for detecting expression of cellular proteins are
well known in the art. Methods suitable for detecting expression of
the kisspeptin receptor include: in situ hybridisation and/or PCR
for detecting the presence of mRNA encoding the kisspeptin
receptor; radio-ligand binding for detecting the presence of the
kisspeptin receptor protein; and methods involving antibodies
capable of specifically binding to the kisspeptin receptor (for
example, immunoblotting, immunohistochemistry, immunofluorescence,
and ELISA).
[0111] It is preferred that the peptide molecule as defined in the
third and/or fourth aspects of the invention is an antagonist of
kisspeptin, or that the fragment or variant thereof is an
antagonist of kisspeptin.
[0112] Preferably, the invention provides an antagonist according
to the first and/or second aspect of the invention or the peptide
molecule as defined in the third or fourth aspect of the invention
for use in medicine. More preferably, the peptide molecule is an
antagonist of kisspeptin.
[0113] In a fifth embodiment, the invention provides a peptide
molecule comprising or consisting of the sequence:
TABLE-US-00015 (SEQ ID NO: 3) X.sup.1 - G / W - X.sup.2 - R / (D)R
- X.sup.3
[0114] wherein:
[0115] X.sup.1 is F or A or any D-amino-acid residue;
[0116] X.sup.2 is L or A or any D-amino-acid residue;
[0117] X.sup.3 is F or W; and
[0118] wherein the C-terminal amino acid residue of the peptide
molecule contains group z which removes the charge on that
residue;
[0119] or a fragment or variant thereof;
[0120] and wherein the peptide sequence is not:
TABLE-US-00016 (SEQ ID NO: 4) F - G - L - R - F; (SEQ ID NO: 5) F -
G - L - R - W; (SEQ ID NO: 6) F - G - (D)F - R - F; (SEQ ID NO: 7)
F - G - A - R - W; (SEQ ID NO: 8) F - G - L - (D)R - W; (SEQ ID NO:
9) F - G - (D)L - R - W; (SEQ ID NO: 17) A - G - L - R - W; or (SEQ
ID NO: 10) (D)F - G - L - R - W.
[0121] Preferably, the invention provides a peptide molecule
wherein X.sup.1 is (D)F. Typically, X.sup.2 is a D-amino-acid
residue selected from the group consisting of: (D)F, (D)L and
(D)W.
[0122] Conveniently, the peptide molecule according to the fifth
aspect of the invention is selected from the group consisting
of:
TABLE-US-00017 (SEQ ID NO: 11) (D)F - W - L - R - W; or (SEQ ID NO:
12) F - G - (D)W - R - F.
[0123] In a preferred embodiment, the N-terminal residue contains
group y which removes the charge on that residue. Preferably,
X.sup.1 contains group y which removes the charge on that residue.
It is particularly preferred that group y is selected from the
group consisting of: an acetyl group; a trifluoroacetyl group; a
cyclised amino acid; or a synthetic amino acid lacking a charge at
the N-terminus.
[0124] In one embodiment, X.sup.3 contains group z which removes
the charge on that residue.
[0125] Preferably, the peptide sequence is selected from the group
consisting of:
TABLE-US-00018 (SEQ ID NO: 13) ac.F - G - (D)F - R - W.z; (SEQ ID
NO: 14) ac.F - G - (D)L - R - W.z; (SEQ ID NO: 15) ac.F - G - L -
(D)R - W.z; (SEQ ID NO: 16) ac.F - G - A - R - W.z; (SEQ ID NO: 17)
ac.A - G - L - R - W.z; (SEQ ID NO: 18) ac.(D)F - W - L - R - W.z;
or (SEQ ID NO: 19) ac.F - G - (D)W - R - F.z.
[0126] In one embodiment, the acetyl group on the N-terminal
residue of any of peptides (I) to (VI), above, is replaced with a
group y which removes the charge on that residue, and is preferably
selected from the group consisting of: an acetyl group; a
trifluoroacetyl group; a cyclised amino acid; or a synthetic amino
acid lacking a charge at the N-terminus.
[0127] In one embodiment, the peptide molecule according to the
fifth aspect of the invention involves a molecule comprising or
consisting of additional amino acid residues, or peptides, at the
N- and/or C-terminus of the peptide sequence of the fifth aspect of
the invention, thereby incorporating the peptide sequence into a
larger polypeptide or protein molecule. Thus, the peptide sequence
of the fifth aspect of the invention may comprise at the N- and/or
C-terminus between 0 and 10 amino acids; or between 10 and 20 amino
acids; or between 20 and 30 amino acids; or between 30 and 40 amino
acids; or between 40 and 50 amino acids; or between 50 and 60 amino
acids; or between 60 and 70 amino acids; or between 70 and 80 amino
acids; or between 80 and 90 amino acids; or between 90 and 100
amino acids; or more than 100 amino acids.
[0128] In a preferred embodiment, the peptide molecule according to
the fifth aspect of the invention comprises or consists of the
additional peptide sequence R-R-M-K-W-K-K-Y (SEQ ID NO:20) or
ac.R-R-M-K-W-K-K-Y (SEQ ID NO:20) at the N-terminus.
[0129] The R-R-M-K-W-K-K-Y (SEQ ID NO:20) sequence is a
heptapeptide sequence from Antennapedia, as described above.
Accordingly, the peptide molecule of the fifth aspect of the
invention may comprise or consist of the sequence:
TABLE-US-00019 (SEQ ID NO: 26) R - R - M - K - W - K - K - Y - (D)F
- W - L - R - W; (SEQ ID NO: 27) R - R - M - K - W - K - K - Y - F
- G - (D)W - R - F; (SEQ ID NO: 21) R - R - M - K - W - K - K - Y -
F - G - (D)F - R - W.z; (SEQ ID NO: 22) R - R - M - K - W - K - K -
Y - F - G - (D)L - R - W.z; (SEQ ID NO: 23) R - R - M - K - W - K -
K - Y - F - G - L - (D)R - W.z; (SEQ ID NO: 24) R - R - M - K - W -
K - K - Y - F - G - A - R - W.z; (SEQ ID NO: 25) R - R - M - K - W
- K - K - Y - A - G - L - R - W.z; (SEQ ID NO: 26) R - R - M - K -
W - K - K - Y - (D)F - W - L - R - W.z; or (SEQ ID NO: 27) R - R -
M - K - W - K - K - Y - F - G - (D)W - R - F.z.
[0130] Optionally, the N-terminal residue may comprise group y at
the N-terminus, as defined above, and is preferably acetyl
(ac).
[0131] Preferably, the peptide sequence of the fifth aspect of the
invention is incorporated within some or all of the sequence of the
full-length kisspeptin peptide. More preferably, the N-terminus of
the peptide sequence of the fifth aspect of the invention can be
extended to any length incorporating the peptide sequence of
kisspeptin 1-54 (i.e. amino acid residues 1 to 54 of the kisspeptin
sequence) and/or the peptide sequence of kisspeptin 1-45 (i.e.
amino acid residues 1 to 45 of the kisspeptin sequence). For
example, the peptide sequence of the fifth aspect of the invention
may have the following sequence at its N-terminus:
TABLE-US-00020 (SEQ ID NO: 28)
gtslspppessgsrqqpglsaphsrqipapqgavlvqrekdlpnynwns
[0132] Alternatively, the peptide sequence of the fifth aspect of
the invention can be incorporated within a protein, such as an
albumin and/or immunoglobulin protein. Incorporation into such
molecules is thought to increase half-life and decrease the
metabolic clearance within an individual of the peptide molecules
of the invention.
[0133] In a sixth aspect, the invention provides a peptide molecule
comprising or consisting of the sequence:
TABLE-US-00021 (SEQ ID NO: 29) X.sup.A - X.sup.B - X.sup.C - N -
X.sup.D - X.sup.E - G - X.sup.F - R - F
[0134] wherein:
[0135] X.sup.A is Y or any D-amino-acid residue;
[0136] X.sup.B is N or any D-amino acid residue;
[0137] X.sup.C is W or any D-amino acid residue;
[0138] X.sup.D is G or S or any D-amino acid residue;
[0139] X.sup.E is F or (D)W or (D)L;
[0140] X.sup.F is W or L or any D-amino acid residue; and
[0141] wherein the C-terminal amino acid residue of the peptide
molecule contains group z which removes the charge on that
residue;
[0142] or a fragment or variant thereof;
[0143] and wherein the peptide sequence is not:
TABLE-US-00022 (SEQ ID NO: 30) Y - N - W - N - S - F - G - L - R -
F; (SEQ ID NO: 31) (D)Y - (D)N - W - N - S - F - G - W - R - F;
(SEQ ID NO: 32) (D)Y - (D)N - W - N - G - F - G - W - R - F; (SEQ
ID NO: 33) (D)Y - (D)N - W - N - S - F - G - (D)W - R - F; or (SEQ
ID NO: 34) (D)Y - (D)N - W - N - G - F - G - (D)W - R - F.
[0144] Preferably, the peptide molecule of the sixth aspect of the
invention provides that X.sup.A is a D-amino-acid residue selected
from the group consisting of: (D)F and (D)Y and (D)A. It is
preferred that X.sup.B is a D-amino-acid residue selected from the
group consisting of: (D)A and (D)N.
[0145] In one embodiment, the invention provides a peptide molecule
wherein when one of X.sup.A or X.sup.B is (D)Y, the other is not
(D)N. One particularly preferred embodiment is wherein X.sup.A and
X.sup.B are not both a D-amino acid residue. In one embodiment,
when X.sup.F is (D)W, X.sup.A is (D)F.
[0146] Preferably, X.sup.C is a D-amino-acid residue selected from
the group consisting of: (D)A and (D)W. Conveniently, X.sup.D is a
D-amino-acid residue selected from the group consisting of: (D)A
and (D)W. Typically, X.sup.F is a D-amino-acid residue selected
from the group consisting of: (D)L and (D)W.
[0147] In one embodiment, when X.sup.D is S, X.sup.F is (D)W and/or
X.sup.A is not (D)Y. Preferably, when X.sup.D is S, X.sup.F is (D)W
and/or X.sup.A is (D)A.
[0148] In one embodiment, when X.sup.E and X.sup.F are both (D)W,
X.sup.A is not (D)Y. Preferably, when X.sup.E and X.sup.F are both
(D)W, X.sup.A is (D)A.
[0149] Conveniently, the invention provides a peptide molecule
wherein the N-terminal residue contains group y which removes the
charge on that residue. In one embodiment, X.sup.1 contains group y
which removes the charge on that residue. Typically, group y is
selected from the group consisting of: an acetyl group; a
trifluoroacetyl group; a cyclised amino acid; or a synthetic amino
acid lacking a charge at the N-terminus.
[0150] In a preferred embodiment, the C-terminal F residue of the
peptide molecule contains group z which removes the charge on that
residue.
[0151] It is most preferred that the peptide molecule according to
the sixth aspect of the invention comprises or consists of a
peptide sequence selected from the group consisting of:
TABLE-US-00023 (SEQ ID NO: 35) Y - N - W - N - G - F - G - L - R -
F.z; (SEQ ID NO: 36) Y - N - W - N - G - F - G - (D)L - R - F.z;
(SEQ ID NO: 37) Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID
NO: 38) Y - N - W - N - G - (D)W - G - L - R - F.z; (SEQ ID NO: 39)
ac.Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 40) ac.Y
- N - W - N - (D)W - F - G - (D)W - R - F.z; (SEQ ID NO: 41)
ac.(D)Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 42)
ac.Y - N - (D)W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 43)
ac.Y - (D)N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 44)
ac.Y - N - W - N - (D)A - F - G - (D)W - R - F.z; (SEQ ID NO: 45)
ac.(D)A - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 46)
ac.Y - N - (D)A - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 47)
ac.Y - (D)A - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 48)
ac.(D)W - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 49)
ac.(D)F - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 50)
ac.(D)Y - N - W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID NO:
51) ac.(D)A - N - W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID
NO: 52) ac.(D)A - N - W - N - S - F - G - (D)W - R - F.z; (SEQ ID
NO: 53) ac.(D) - A - N - W - N - G - F - G - W - R - F.z; (SEQ ID
NO: 54) ac.(D)A - N - W - N - (D)S - F - G - (D)W - R - F.z; or
(SEQ ID NO: 55) ac.(D)A - N - W - N - G - F - G - (D)L - R -
F.z.
[0152] In one embodiment, the acetyl group on the N-terminal
residue of any of peptides (e) to (s), above, is replaced with a
group y which removes the charge on that residue, and is preferably
selected from the group consisting of: an acetyl group; a
trifluoroacetyl group; a cyclised amino acid; or a synthetic amino
acid lacking a charge at the N-terminus.
[0153] In one embodiment, the peptide molecule according to the
sixth aspect of the invention involves a molecule comprising or
consisting of additional amino acid residues, or peptides, at the
N- and/or C-terminus of the peptide sequence of the sixth aspect of
the invention, thereby incorporating the peptide sequence into a
larger polypeptide or protein molecule. Thus, the peptide sequence
of the sixth aspect of the invention may comprise at the N- and/or
C-terminus between 0 and 10 amino acids; or between 10 and 20 amino
acids; or between 20 and 30 amino acids; or between 30 and 40 amino
acids; or between 40 and 50 amino acids; or between 50 and 60 amino
acids; or between 60 and 70 amino acids; or between 70 and 80 amino
acids; or between 80 and 90 amino acids; or between 90 and 100
amino acids; or more than 100 amino acids.
[0154] In a preferred embodiment, the peptide molecule according to
the sixth aspect of the invention involves a molecule comprising or
consisting of the additional peptide sequence R-R-M-K-W-K-K-Y (SEQ
ID NO:20) orac.R-R-M-K-W-K-K-Y (SEQ ID NO:20) at the N-terminus of
the peptide sequence.
[0155] The R-R-M-K-W-K-K-Y (SEQ ID NO:20) sequence is a
heptapeptide sequence from Antennapedia, as discussed above.
Accordingly, the peptide of the invention may comprise or consist
of the sequence:
TABLE-US-00024 (SEQ ID NO: 56) R - R - M - K - W - K - K - Y - Y -
N - W - N - G - F - G - L - R - F.z; (SEQ ID NO: 57) R - R - M - K
- W - K - K - Y - Y - N - W - N - G - F - G - (D)L - R - F.z; (SEQ
ID NO: 58) R - R - M - K - W - K - K - Y - Y - N - W - N - G - F -
G - (D)W - R - F.z; (SEQ ID NO: 59) R - R - M - K - W - K - K - Y -
Y - N - W - N - G - (D)W - G - L - R - F.z; (SEQ ID NO: 60) R - R -
M - K - W - K - K - Y - Y - N - W - N - G - F - G - (D)W - R - F.z;
(SEQ ID NO: 61) R - R - M - K - W - K - K - Y - Y - N - W - N -
(D)W - F - G - (D)W - R - F.z; (SEQ ID NO: 62) R - R - M - K - W -
K - K - Y - (D)Y - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID
NO: 63) R - R - M - K - W - K - K - Y - Y - N - (D)W - N - G - F -
G - (D)W - R - F.z; (SEQ ID NO: 64) R - R - M - K - W - K - K - Y -
Y - (D)N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 65) R -
R - M - K - W - K - K - Y - Y - N - W - N - (D)A - F - G - (D)W - R
- F.z; (SEQ ID NO: 66) R - R - M - K - W - K - K - Y - (D)A - N - W
- N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 67) R - R - M - K - W
- K - K - Y - Y - N - (D)A - N - G - F - G - (D)W - R - F.z; (SEQ
ID NO: 68) R - R - M - K - W - K - K - Y - Y - (D)A - W - N - G - F
- G - (D)W - R - F.z; (SEQ ID NO: 69) R - R - M - K - W - K - K - Y
- (D)W - N - W - N - G - F - G - (D)W - R - F.z; (SEQ ID NO: 70) R
- R - M - K - W - K - K - Y - (D)F - N - W - N - G - F - G - (D)W -
R - F.z; (SEQ ID NO: 71) R - R - M - K - W - K - K - Y - (D)Y - N -
W - N - G - (D)W - G - (D)W - R - F.z; (SEQ ID NO: 72) R - R - M -
K - W - K - K - Y - (D)A - N - W - N - G - (D)W - G - (D)W - R -
F.z; (SEQ ID NO: 73) R - R - M - K - W - K - K - Y - (D)A - N - W -
N - S - F - G - (D)W - R - F.z; (SEQ ID NO: 74) R - R - M - K - W -
K - K - Y - (D)A - N - W - N - G - F - G - W - R - F.z; (SEQ ID NO:
75) R - R - M - K - W - K - K - Y - (D)A - N - W - N - (D)S - F - G
- (D)W - R - F.z; or (SEQ ID NO: 76) R - R - M - K - W - K - K - Y
- (D)A - N - W - N - G - F - G - (D)L - R - F.z.
[0156] Optionally, the N-terminal residue may comprise group y at
the N-terminus, as defined above, and is preferably acetyl
(ac).
[0157] In a particularly preferred embodiment, the peptide sequence
is selected from the group consisting of:
TABLE-US-00025 (SEQ ID NO: 77) R - R - M - K - W - K - K - Y - (D)A
- N - W - N - G - F - G - (D)W - R - F.z; or (SEQ ID NO: 77) ac.R -
R - M - K - W - K - K - Y - (D)A - N - W - N - G - F - G - (D)W - R
- F.z.
[0158] As demonstrated in the accompanying examples, peptides of
the invention comprising the N-terminal Antennapedia heptapeptide
retain kisspeptin antagonist function.
[0159] Preferably, the peptide sequence of the sixth aspect of the
invention is incorporated within some or all of the sequence of the
full-length kisspeptin peptide. More preferably, the N-terminus of
the peptide sequence of the sixth aspect of the invention can be
extended to any length incorporating the peptide sequence of
kisspeptin 1-54 (i.e. amino acid residues 1 to 54 of the kisspeptin
sequence) and/or the peptide sequence of kisspeptin 1-45 (i.e.
amino acid residues 1 to 45 of the kisspeptin sequence). For
example, the peptide sequence of the sixth aspect of the invention
may have the following sequence at its N-terminus:
TABLE-US-00026 (SEQ ID NO: 78)
gtslspppessgsrqqpglsaphsrqipapqgavlvqrekdlp
[0160] Alternatively, the peptide sequence of the sixth aspect of
the invention can be incorporated within a protein, such as an
albumin and/or immunoglobulin protein. Incorporation into such
molecules is thought to increase half-life and decrease the
metabolic clearance within an individual of the peptide molecules
of the invention.
[0161] In one embodiment, the invention provides a peptide molecule
wherein z is NH.sub.2 or N-propylamide or N-ethylamide (NHEt) or
N-methylamide or N-butylamide.
[0162] Preferably, z has a molecular weight of less than 200,
preferably less than 150, preferably less than 100. Preferably, z
is NHR' wherein R' is H or C.sub.1 to C.sub.4 alkyl or z is OR''
wherein R'' is C.sub.1 to C.sub.4 alkyl. Preferably, z is an amide.
Preferably, z is NH.sub.2 or N-propylamide or N-ethylamide (NHEt)
or N-methylamide or N-butylamide.
[0163] In a seventh embodiment, the invention provides a
pharmaceutical composition comprising or consisting of an effective
amount of an antagonist according to the invention or a peptide
molecule according to the invention, and a
pharmaceutically-acceptable excipient or diluent.
[0164] By "effective amount" we include an amount of the molecule
of the invention that is sufficient to reduce and/or alleviate
and/or prevent symptoms associated with a condition induced and/or
worsened by kisspeptin activity in an individual. An "effective
amount" may, for example, be determined by undertaking dose studies
in animals (and, if possible, humans) for inhibition of
gonadotrophin and/or steroid hormones; doses would likely be in the
region of 1-100 mg per day delivered subcutaneously or
intraperitoneally or orally.
[0165] An effective amount could also be determined in vitro by
using the methods described in the Examples (for example, the
methods used to monitor antagonism of kisspeptin binding to the
kisspeptin receptor and/or antagonism of kisspeptin-mediated
stimulation of inositol phosphate production in a cell) or in vivo
by monitoring the reduction and/or alleviation and/or prevention of
symptoms in an individual (such as an animal) associated with a
condition induced and/or worsened by kisspeptin activity in an
individual, which symptoms will be know to those skilled in the
relevant medical field.
[0166] In relation to in vitro methods of monitoring kisspeptin
antagonism, those skilled in the art will understand that other or
additional cellular and enzymatic activities could be monitored to
measure kisspeptin-mediated stimulation. For example, it is known
that the Gq G protein activates phospholipase (which generates IP
and diacylglycerol that in turn mobilise Ca.sup.2+ and activate
protein kinase C, respectively) and that protein kinase C
phosphorylates and activates extracellular-regulated kinase (ERK)
and/or the mitogen-activated protein kinase (MAPK) cascade and
members thereof; accordingly, a skilled person could monitor the
activity and/or presence of any or all of those cellular components
to determine kisspeptin-mediated stimulation.
[0167] In an eighth embodiment, the invention provides a method of
treating a condition induced and/or worsened by kisspeptin activity
in an individual comprising or consisting of the step of
administering to the individual a pharmaceutical composition
according to the seventh aspect of the invention, or an effective
amount of an antagonist of kisspeptin according to the first or
second aspect of the invention, or an effective amount of a peptide
molecule according to the second, third, fourth and/or fifth aspect
of the invention.
[0168] The molecules, medicaments and pharmaceutical compositions
of the present invention may be delivered using an injectable
sustained-release drug delivery system. These are designed
specifically to reduce the frequency of injections. An example of
such a system is Nutropin Depot which encapsulates recombinant
human growth hormone (rhGH) in biodegradable microspheres that,
once injected, release rhGH slowly over a sustained period.
Preferably, delivery is performed intra-muscularly (i.m.) and/or
sub-cutaneously (s.c.) and/or intravenously (i.v.).
[0169] The molecules, medicaments and pharmaceutical compositions
of the present invention can be administered by a surgically
implanted device that releases the drug directly to the required
site. For example, Vitrasert releases ganciclovir directly into the
eye to treat CMV retinitis. The direct application of this toxic
agent to the site of disease achieves effective therapy without the
drug's significant systemic side-effects.
[0170] Electroporation therapy (EPT) systems can also be employed
for the administration of the agents, medicaments and
pharmaceutical compositions of the invention. A device which
delivers a pulsed electric field to cells increases the
permeability of the cell membranes to the drug, resulting in a
significant enhancement of intracellular drug delivery.
[0171] The molecules, medicaments and pharmaceutical compositions
of the invention can also be delivered by electroincorporation
(EI). EI occurs when small particles of up to 30 microns in
diameter on the surface of the skin experience electrical pulses
identical or similar to those used in electroporation. In EI, these
particles are driven through the stratum corneum and into deeper
layers of the skin. The particles can be loaded or coated with
drugs or genes or can simply act as "bullets" that generate pores
in the skin through which the drugs can enter.
[0172] An alternative method of delivery of the molecules,
medicaments and pharmaceutical compositions of the invention is the
ReGel injectable system that is thermo-sensitive. Below body
temperature, ReGel is an injectable liquid while at body
temperature it immediately forms a gel reservoir that slowly erodes
and dissolves into known, safe, biodegradable polymers. The active
substance is delivered over time as the biopolymers dissolve.
[0173] The molecules, medicaments and pharmaceutical compositions
of the invention can also be delivered orally. The process employs
a natural process for oral uptake of vitamin B.sub.12 and/or
vitamin D in the body to co-deliver proteins and peptides. By
riding the vitamin B.sub.12 and/or vitamin D uptake system, the
nucleic acids, molecules and pharmaceutical formulations of the
invention can move through the intestinal wall. Complexes are
synthesised between vitamin B.sub.12 analogues and/or vitamin D
analogues and the drug that retain both significant affinity for
intrinsic factor (IF) in the vitamin B.sub.12 portion/vitamin D
portion of the complex and significant bioactivity of the active
substance of the complex.
[0174] The molecules, medicaments and pharmaceutical compositions
of the invention can be introduced to cells by "Trojan peptides".
These are a class of polypeptides called penetratins which have
translocating properties and are capable of carrying hydrophilic
compounds across the plasma membrane. This system allows direct
targeting of oligopeptides to the cytoplasm and nucleus, and may be
non-cell type specific and highly efficient. See Derossi et al.
(1998), Trends Cell Biol 8, 84-87.
[0175] Preferably, the medicament and/or pharmaceutical composition
of the present invention is a unit dosage containing a daily dose
or unit, daily sub-dose or an appropriate fraction thereof, of the
active ingredient.
[0176] The molecules, medicaments and pharmaceutical compositions
of the invention will normally be administered orally or by any
parenteral route, in the form of a pharmaceutical composition
comprising the active ingredient, optionally in the form of a
non-toxic organic, or inorganic, acid, or base, addition salt, in a
pharmaceutically acceptable dosage form. Depending upon the
disorder and patient to be treated, as well as the route of
administration, the compositions may be administered at varying
doses.
[0177] In human therapy, the molecules, medicaments and
pharmaceutical compositions of the invention can be administered
alone but will generally be administered in admixture with a
suitable pharmaceutical excipient, diluent or carrier selected with
regard to the intended route of administration and standard
pharmaceutical practice.
[0178] For example, the molecules, medicaments and pharmaceutical
compositions of the invention can be administered orally, buccally
or sublingually in the form of tablets, capsules, ovules, elixirs,
solutions or suspensions, which may contain flavouring or colouring
agents, for immediate-, delayed- or controlled-release
applications. The molecules, medicaments and pharmaceutical
compositions of the invention may also be administered via
intracavernosal injection.
[0179] Such tablets may contain excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dibasic
calcium phosphate and glycine, disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch
glycollate, croscarmellose sodium and certain complex silicates,
and granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC),
sucrose, gelatin and acacia. Additionally, lubricating agents such
as magnesium stearate, stearic acid, glyceryl behenate and talc may
be included.
[0180] Solid compositions of a similar type may also be employed as
fillers in gelatin capsules. Preferred excipients in this regard
include lactose, starch, cellulose, milk sugar or high molecular
weight polyethylene glycols. For aqueous suspensions and/or
elixirs, the agents of the invention may be combined with various
sweetening or flavouring agents, colouring matter or dyes, with
emulsifying and/or suspending agents and with diluents such as
water, ethanol, propylene glycol and glycerin, and combinations
thereof.
[0181] The molecules, medicaments and pharmaceutical compositions
of the invention can also be administered parenterally, for
example, intravenously, intra-arterially, intraperitoneally,
intra-thecally, intraventricularly, intrasternally, intracranially,
intra-muscularly or subcutaneously, or they may be administered by
infusion techniques. They are best used in the form of a sterile
aqueous solution which may contain other substances, for example,
enough salts or glucose to make the solution isotonic with blood.
The aqueous solutions should be suitably buffered (preferably to a
pH of from 3 to 9), if necessary. The preparation of suitable
parenteral formulations under sterile conditions is readily
accomplished by standard pharmaceutical techniques well-known to
those skilled in the art.
[0182] Medicaments and pharmaceutical compositions suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents. The medicaments and compositions may be
presented in unit-dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets of the kind
previously described.
[0183] For oral and parenteral administration to human patients,
the daily dosage level of the molecules, medicaments and
pharmaceutical compositions of the invention will usually be from
0.1 to 100 mg per adult per day administered in single or divided
doses.
[0184] Thus, for example, the tablets or capsules of the molecules
of the invention may contain from 0.1 mg to 100 mg of active agent
for administration singly or two or more at a time, as appropriate.
The physician in any event will determine the actual dosage which
will be most suitable for any individual patient and it will vary
with the age, weight and response of the particular patient. The
above dosages are exemplary of the average case. There can, of
course, be individual instances where higher or lower dosage ranges
are merited and such are within the scope of this invention.
[0185] The molecules, medicaments and pharmaceutical compositions
of the invention can also be administered intranasally or by
inhalation and are conveniently delivered in the form of a dry
powder inhaler or an aerosol spray presentation from a pressurised
container, pump, spray or nebuliser with the use of a suitable
propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoro-ethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134A3 or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or
other suitable gas. In the case of a pressurised aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurised container, pump, spray or nebuliser
may contain a solution or suspension of the active agent, e.g.
using a mixture of ethanol and the propellant as the solvent, which
may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use
in an inhaler or insufflator may be formulated to contain a powder
mix of a agent of the invention and a suitable powder base such as
lactose or starch.
[0186] Aerosol or dry powder formulations are preferably arranged
so that each metered dose or "puff" contains at least 0.1 mg of a
molecule of the invention for delivery to the patient. It will be
appreciated that he overall daily dose with an aerosol will vary
from patient to patient, and may be administered in a single dose
or, more usually, in divided doses throughout the day.
[0187] Alternatively, the molecules, medicaments and pharmaceutical
compositions of the invention can be administered in the form of a
suppository or pessary, or they may be applied topically in the
form of a lotion, solution, cream, gel, ointment or dusting powder.
The molecules, medicaments and pharmaceutical compositions of the
invention may also be transdermally administered, for example, by
the use of a skin patch. They may also be administered by the
ocular route, particularly for treating diseases of the eye.
[0188] For ophthalmic use, the molecules, medicaments and
pharmaceutical compositions of the invention can be formulated as
micronised suspensions in isotonic, pH adjusted, sterile saline,
or, preferably, as solutions in isotonic, pH adjusted, sterile
saline, optionally in combination with a preservative such as a
benzylalkonium chloride. Alternatively, they may be formulated in
an ointment such as petrolatum.
[0189] For application topically to the skin, the molecules,
medicaments and pharmaceutical compositions of the invention can be
formulated as a suitable ointment containing the active agent
suspended or dissolved in, for example, a mixture with one or more
of the following: mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene polyoxypropylene agent,
emulsifying wax and water. Alternatively, they can be formulated as
a suitable lotion or cream, suspended or dissolved in, for example,
a mixture of one or more of the following: mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and water.
[0190] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavoured basis, usually sucrose and acacia or tragacanth;
pastilles comprising the active ingredient in an inert basis such
as gelatin and glycerin, or sucrose and acacia; and mouth-washes
comprising the active ingredient in a suitable liquid carrier.
[0191] Generally, in humans, oral or parenteral administration of
the molecules, medicaments and pharmaceutical compositions of the
invention agents of the invention is the preferred route, being the
most convenient.
[0192] For veterinary use, the molecules, medicaments and
pharmaceutical compositions of the invention is administered as a
suitably acceptable formulation in accordance with normal
veterinary practice and the veterinary surgeon will determine the
dosing regimen and route of administration which will be most
appropriate for a particular animal.
[0193] Conveniently, the formulation is a pharmaceutical
formulation.
[0194] Advantageously, the formulation is a veterinary
formulation.
[0195] It is particularly preferred that the molecules of the
invention are formulated using peptide formulations already known
in the arts of medicine and pharmacy, such as those used in
formulations of GnRH peptide agonists (for example, formulations
comprising or consisting of polyglycyl lactide copolymer and/or
polyethylene glycol and/or oil and/or microcrystalline suspensions
may be useful for injecting the molecules of the invention).
[0196] In a ninth aspect of the invention, the invention provides a
method for identifying an antagonist of kisspeptin comprising or
consisting of the step(s) of:
[0197] providing a compound to be tested;
[0198] determining the ability of the compound in (i) to bind to
the kisspeptin receptor;
[0199] determining the ability of the compound in (i) to antagonise
kisspeptin-mediated stimulation of inositol phosphate production in
a cell; and
[0200] identifying the compound as an antagonist of kisspeptin in
the event that it is capable of binding to the kisspeptin receptor
and capable of antagonising kisspeptin-mediated stimulation of
inositol phosphate production in a cell.
[0201] Thus, the method of the ninth aspect of the invention may be
performed to determine whether a particular test compound or test
molecule (for example, a test compound or test molecule comprising
or consisting of a peptide sequence that is an analogue of part or
all of the kisspeptin peptide sequence) has the ability to bind to
the kisspeptin receptor and functionally antagonise
kisspeptin-mediated stimulation of inositol phosphate production in
a cell (such as cell lines, like CHO, HEK or COS cell lines,
transiently or stably transfected with the human kisspeptin
receptor, GPR54). The accompanying Examples describe experimental
methods suitable for determining the binding ability of a compound
to the kisspeptin receptor and for measuring kisspeptin-mediated
stimulation of inositol phosphate production in a cell. Other
appropriate methods will be known to those skilled in the art.
[0202] For example, as discussed above, those skilled in the art
will understand that other or additional cellular and enzymatic
activities could be monitored to measure kisspeptin-mediated
stimulation. For example, it is known that the Gq G protein
activates phospholipase (which generates IP and diacylglycerol that
in turn mobilise Ca.sup.2+ and activate protein kinase C,
respectively) and that protein kinase C phosphorylates and
activates extracellular-regulated kinase (ERK) and/or the
mitogen-activated protein kinase (MAPK) cascade and members
thereof; accordingly, a skilled person could monitor the activity
and/or presence of any or all of those cellular components to
determine kisspeptin-mediated stimulation.
[0203] Preferably, the test compound or molecule will be identified
as capable of binding to the kisspeptin receptor if it has a
binding affinity (that is, a Kd) of <100 nM.
[0204] Preferably, the test compound or molecule will be identified
as capable of antagonising the ability of 10 nM of kisspeptin to
stimulate inositol phosphate production in a cell, as described in
the accompanying examples. Preferably, the test compound or
molecule will be identified as capable of achieving at least 40%
inhibition of the stimulation by 10 nM kisspeptin; more preferably,
at least 50% or 60% or 70% or 80% or 90% or 100% inhibition will be
achieved.
[0205] For example, a compound capable of achieving 40% or less
inhibition of stimulation by 10 mM kisspeptin may not be regarded
as an antagonist. A compound capable of achieving between around
40% to 80% inhibition of stimulation by 10 mM kisspeptin may be
regarded as a "poor" antagonist. A compound capable of achieving
80% or more inhibition of stimulation by 10 mM kisspeptin may be
regarded as a "good" antagonist.
[0206] Preferably, the method of the ninth aspect of the invention
further comprises or consisting of the step of making or
synthesising the compound or molecule identified as an antagonist
of kisspeptin in step (iv), according to the methods described
herein or known to those skilled in the art.
[0207] More preferably, the method further comprises or consisting
of the step of formulating the compound or molecule identified in
the ninth aspect of the invention (or the synthesised compound or
molecule) into a pharmaceutical composition or medicament,
according to the methods described herein or known to those skilled
in the art.
[0208] The listing or discussion of a prior-published document in
this specification should not necessarily be taken as an
acknowledgement that the document is part of the state of the art
or is common general knowledge.
[0209] Preferred, non-limiting examples which embody certain
aspects of the invention will now be described, with reference to
the following figures:
Table 1: The stimulation of inositol phosphate (IP) and inhibition
of 10 nM Kisspeptin stimulation of IP by Kisspeptin analogues.
[0210] FIG. 1: A--Binding dose response for kisspeptin in CHO cells
stably transfected with GPR54.
[0211] B--IP dose response for kisspeptin in CHO cells stably
transfected with GPR54.
[0212] FIG. 2: A--Ability of repeated injections of compound 210
(0, 60, 120 min) to interfere with the potent LH- and testosterone
releasing effect of kisspeptin-10 (100 pmol)(120 min) in intact
male rats.
[0213] B--Ability of repeated injections of compound 234 to
interfere with the potent LH and testosterone-releasing effect of
kisspeptin-10 (100 pmol) in intact male rats.
[0214] C--Ability of repeated injections (0, 60, 120 min) of
compound 210 to attenuate the elevated LH levels in orchidectomised
male rats.
[0215] FIG. 3: Central infusion of kisspeptin antagonist inhibits
the secretory pulses of LH in OVX ewes. Concentrations of LH are
shown in ewes treated with kisspeptin antagonist (closed bars) or
control (opened bars). Arrows indicate LH pulses as defined in the
Materials and Methods.
[0216] FIG. 4: Dose response binding curve and dose response IP
curve and IP competition assays for peptide antagonists.
[0217] FIG. 5: Inhibition of kiss stimulation of GnRH neurone
firing by antagonist 234.
[0218] FIG. 6: Fetal weight is decreased by kisspeptin treatment
which causes poor implantation. Therefore kisspeptin plays a role
in fetal growth retardation suggesting that antagonists could be
used to treat females with a history of poor implantation (which
leads to, for example, miscarriage and pre-eclampsia).
[0219] FIG. 7: N-terminal substitutions and C-terminal truncations
create partial agonists. Amino acid substitutions were made at the
N-terminus and truncations were made at the c-terminus of
kisspeptin-10. Truncation to 7 amino acids (a) or 5 amino acids (b)
in length caused partial agonism. When truncation to 5 amino acids
was combined with (D)-Leu at Leu.sup.8 this further enhanced the
partial agonism (c).
[0220] FIG. 8: Amino acid substitutions create antagonists at the
human gpr-54 receptor with Gly5.sup.5 and D-Trp.sup.8 being
critical. (a, b) Amino acid substitutions at serine.sup.5 with
glycine or D-Ala combined with substitutions at Leu.sup.8 with
D-amino acids created partial agonist. (c, d) Further substitutions
at Tyr.sup.1 with D-Ala or D-Tyr enhance this antagonism further to
create full antagonists at the gpr-54 receptor. However, removal of
Gly.sup.5 and D-Trp.sup.8 substitutions from antagonist with a
D-amino acid at Tyr.sup.1 decreases or abolishes antagonistic
properties of analogues. (e) This is most pronounced when Tyr.sup.1
is replaced with a D-Tyr as removal of one or both substitutions
abolishes antagonism. (f) The effect is less obvious when Tyr.sup.1
is replaced with D-Ala but antagonism is still reduced
(*=p<0.05, **=p<0.01, ***=p<0.001).
[0221] FIG. 9: Peptide 234 binds with the same affinity as
Kisspeptin-10. When I.sup.125-labelled kisspeptin-10 was competed
with either kisspeptin-10 (a) or peptide 234 (b) they both bound
with an affinity of 2 nM.
[0222] FIG. 10: Peptide 234 antagonizes kisspeptin-10 excitation of
GnRH neurons. Representative traces of GnRH neuronal firing rate
over time. (a) Increased GnRH firing rate after 1 nM kisspeptin-10
(bar). Downward spikes are individual action currents. (b)
Inhibition of kisspeptin-10 (1 nM) stimulation by peptide 234 (1
nM, bar). (c) Summary bar graph showing mean.+-.SEM fold change in
firing rate during baseline (white bars) and kisspeptin-10 (black
bars) kisspeptin-10 significantly increased firing activity of GnRH
neurons (n=7, * p<0.002). Response to kisspeptin-10 was
significantly reduced with the presence of 1, 10, 100 nM peptide
234 (1 nM n=5, 10 nM n=6, 100 nM n=7, # p<0.001 all groups).
[0223] FIG. 11: Peptide 234 suppresses GnRH release in vivo.
Representative cases from the effects of peptide 234 on GnRH
release and group mean (.+-.sem, n=6) are shown. (a) Pulsatile GnRH
release in the hypothalamus was suppressed by 10 nM peptide 234
infusion to the stalk-median eminence regions (dark shaded bar).
Short arrows indicate GnRH peaks identified by PULSAR. (b) In
contrast, vehicle infusion as a control did not cause any
significant changes in GnRH release (light shaded bar). (c) Data
analysis indicated that peptide 234 significantly (p<0.05)
suppressed GnRH release as compare to levels prior to peptide 234
as well as to the vehicle control. (d) Vehicle infusion did not
cause any significant changes. The estimated concentration of
peptide 234 in the stalk-median eminence region was 1 nM, based on
our previous assessment that the dialysis membrane passes
.about.10% of peptides with a similar size. * p<0.05 vs. before
peptide 234; a p<0.05 vs. control at corresponding time
period.
[0224] FIG. 12: Peptide 234 effects on basal and
kisspeptin-10-stimulated plasma LH in intact and castrated male
rats. Peptide 234 (a) inhibits kisspeptin-10 induced LH secretion
in intact male rats. The animals were given two boluses of peptide
234 at 0 and 60 min, followed by one infusion of kisspeptin-10 at
120 mins. The peptide significantly inhibited LH production over
the following 2 hours, calculated as area under the curve (AUC;
p<0.05). Testosterone levels at 60 min after kisspeptin infusion
(i.e., at180-min of experiment) are also shown as bars. In
addition, castrated male rats were given three infusions of peptide
234, which inhibited LH secretion (b).
[0225] FIG. 13: Central infusion of peptide 234 inhibits the
secretory pulses of LH in OVX ewes. Concentrations of LH are shown
in ewes treated with peptide 234 (closed bars) or control (opened
bars). Arrows indicate LH pulses as defined in the Materials and
Methods. Analysis revealed a significant reduction in the mean LH
concentration and pulse amplitude after peptide 234 infusion.
[0226] Table 2: Mean LH pulse amplitude, LH concentration, and GH
concentration (ng/ml) before, during and after infusion with
peptide 234. Data are the mean.+-.SEM for vehicle and peptide-234
treated ewes before (0-180 min), during (180-240 min), and after
(240-360 min) infusion. Repeated measures ANOVAs revealed a
significant interaction between treatment and time (P<0.05).
Individual P values for each time period are shown.
[0227] FIG. 14: Effect of central infusion of antagonist 234 on GH
in OVX ewes. Concentrations of GH are shown in ewes treated with
peptide 234 (closed bars) or control (opened bars). Analysis
revealed a significant increase in the mean GH concentration during
peptide 234 infusion.
[0228] FIG. 15: Effect of central infusion of antagonist 234 on
prolactin in OVX ewes. Concentrations of prolactin are shown in
ewes treated with peptide 234 (closed bars) or control (opened
bars). Analysis showed no effect of peptide 234 infusion on
prolactin.
[0229] FIG. 16: Effect of central infusion of antagonist 234 on
cortisol in OVX ewes. Concentrations of cortisol are shown in ewes
treated with peptide 234 (closed bars) or control (opened bars).
Analysis showed no effect of peptide 234 infusion on cortisol
[0230] FIG. 17: Effect of antagonists on cell migration
[0231] Peptides 233, 234 and 273 act as antagonist in HUVEC and CHO
cells at the human gpr-54, as they almost completely inhibit the
effect of kisspeptin on migration. (A) In CHO (Chinese hamster
ovary) cells, 100 nM Kisspeptin inhibits cell migration by 50%.
Peptide 234 completely abolishes this inhibition. (B) In HUVECs
(Human Umbilical Vein Endothelial Cells), 1 nM Kisspeptin inhibits
migration in to the wound by 50%. However, in the presence of
peptides 233, 234 or 273, inhibition is reduced to only 10%.
EXAMPLE 1
Experimental Data Relating to Kisspeptin Analogues
Materials and Methods
Method Used for Dose Response Bind (IC50) Column in Table 1
[0232] Whole Cell Receptor Binding Assay--
[0233] Kisspeptin-10 and analogues were prepared at 1:1000
dilutions in HEPES modified Dulbecco's modified Eagle's medium
(DMEM) supplemented with .sup.125I-labelled Kisspeptin to produce
100,000 counts per minute (cpm). Cell monolayers were placed on ice
and exposed to 0.5 mls peptide (10 pM-1 uM); the cells were then
incubated at 4.degree. C. for 4 hr. After 4 hrs, cells were washed
twice with ice cold Dulbecco's phosphate buffered saline (DPBS;
with Calcium and Magnesium) and then 0.5 mls 0.1M Sodium Hydroxide
(NaOH) were added to cells for 20 minutes, shaking to lyse cells.
Lysates were transferred to plastic tubes and bound radioactivity
counted on the gamma counter for 60 seconds. IC50s were measured as
the concentration at which 50% of receptor binding was competed for
by the analogue.
Method Used for Dose Response IP (EC50) Column in Table 1
[0234] Inositol-3-Phoshate Stimulation Assay
[0235] Cell monolayers had 0.5 mls HEPES modified DMEM supplemented
with 1% Penicillin/Streptomycin for 30 mins at 37.degree. C. to
block IP.sub.3 breakdown. Cells were then stimulated with 0.5 mls
Kisspeptin-10 and analogues (10 pM-1 uM) diluted at 1:1000 in the
above media for 1 hr at 37.degree. C., then in 10 mM Formic acid at
4.degree. C. for 1 hr to lyse cells. Lysate was then transferred to
plastic tubes containing 0.5 mls Dowex resin to bind the
radioactive IP3 and the resin was then washed with 1 ml H.sub.2O.
The resin was then washed with 60 mM NH4 formate/5 mM Sodium
tetraborate followed by 1M NH4 formate/0.1M Formic acid to release
the bound radiation. Then 800 .mu.ls of the radioactive solution
were transferred to scintillation vials containing 2.5 mls
scintillation fluid and radioactivity counted on a Beta counter for
60 seconds. EC50s were measured as the concentration that
stimulated a response at 50% of the maximum stimulation.
Method Used for Antagonist IP Inhibition (IC50) Column in Table
1
[0236] Inositol-3-Phoshate Antagonistic Assay
[0237] Cell monolayers had 0.5 mls HEPES modified DMEM supplemented
with 1% Penicillin/Streptomycin for 30 mins at 37.degree. C. to
block IP.sub.3 breakdown. Cells were then stimulated with 0.25 mls
10 nM Kisspeptin-10 plus 0.25 mls of 10 nM Kisspeptin-10 or
analogues (10 pM-10 uM) diluted at 1:1000 in the above media for 1
hr at 37.degree. C., then in 10 mM Formic acid at 4.degree. C. for
1 hr to lyse cells. Lysate was then transferred to plastic tubes
containing 0.5 mls Dowex resin to bind the radioactive IP3 and the
resin was then washed with 1 ml H.sub.2O. The resin was then washed
with 60 mM NH4 formate/5 mM Sodium tetraborate followed by 1M NH4
formate/0.1M Formic acid to release the bound radiation. Then 800
.mu.ls of the radioactive solution were transferred to
scintillation vials containing 2.5 mls scintillation fluid and
radioactivity counted on a Beta counter for 60 seconds. IC50s were
calculated as the concentration required to inhibit 10 nM
Kisspeptin-10 stimulation by 50%.
[0238] P-values were calculated using student's T-tests and the
most significant value for each analogue is quoted.
[0239] IP stimulation refers to intrinsic residual agonist activity
where estimateable.
[0240] IC50 is dose of antagonist required to inhibit to inhibit IP
stimulation by 10 nM Kisspeptin-10 by 50%.
Central Kisspeptin Antagonist Treatment in OVX Ewes
[0241] All experimental procedures were conducted under a protocol
approved by the Monash School of Biomedical Sciences "A" Animal
Ethics Committee.
[0242] Adult Corriedale ewes were housed under natural lighting and
were bilaterally ovariectomized (OVX) at least 1 month before any
experimental manipulations. Permanent indwelling third cerebral
ventricular (3V) cannulae were implanted in a subsequent surgical
procedure as previously described (Barker-Gibb et al., 1995, J.
Endocrinol., 147:565-79). Approximately two weeks after 3V surgery,
one external jugular vein was cannulated for blood sampling and
animals housed in single pens; cannulae were kept patent with
heparinized saline.
[0243] Ewes were assigned to two treatment groups (n=4 per group);
kisspeptin antagonist (diluted in artificial cerebrospinal fluid,
aCSF; 150 mM NaCl, 1.2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 2.8 mM KCl)
or control (aCSF only). The following day, infusion lines were
connected to 3V cannulae and blood sampling commenced at 07.00 h.
Samples were collected every 10 min. After 3 h of sampling,
kisspeptin antagonist (or control) was infused into the 3V at a
dose of 40 .mu.g/h for 1 h, with an initial loading dose of 10
.mu.g. Both kisspeptin antagonist and vehicle were infused at 200
.mu.l/h for 1 h using Graseby.RTM. MS 16A infusion pumps (Smiths
Medical Australasia Pty. Ltd., Gold Coast, Qld, Australia). After
infusion, 3V lines remained in place and blood sampling continued
for a further 2 h (total of 6 h). Plasma was harvested immediately
from samples, and frozen at -20 C until assayed.
[0244] Plasma LH concentrations were measured in duplicate, using
the method of Lee et al. (1976, J. Reprod. Fertil., 46:1-6) with
NIH-oLH-S18 as standard. Assay results were calculated using the
program of Burger et al. (1972, J. Lab. Clin. Med., 80:302-12).
Assay sensitivity was 0.08 ng/ml and the intra-assay coefficient of
variation was less than 10% over the range of 0.3-12.8 ng/ml. A
pulse analysis of the LH data was performed based on the method
described for GnRH (Clarke, 1993, Endocrinology, 133:1624-32).
Results
Central Kisspeptin Antagonist Treatment in OVX Ewes
[0245] Central infusion of kisspeptin antagonist appeared to
inhibit the secretory pulses of LH in OVX ewes (FIG. 3). LH pulse
analysis revealed in control treated OVX ewes--and in OVX ewes
prior to kisspeptin antagonist treatment--the major secretory
episodes of LH were clearly distinguishable. The number of
secretory pulses was reduced after ventricular administration of
kisspeptin antagonist. Where LH pulses were identified, these
appeared to be of diminished amplitude.
EXAMPLE 2
Further Experimental Data Relating to Kisspeptin Analogues
Overview
[0246] Gonadotropin releasing hormone (GnRH) is the central
regulator of the reproductive system and GnRH analogues are
extensively employed for treating infertility and hormone-dependent
diseases such as prostatic cancer and endometriosis. GnRH neuron
activity is modulated by numerous factors, including photoperiod,
nutrition, stress and steroid hormones. The discovery that
mutations in gpr-54 cause hypogonadotropic hypogonadism led to the
recognition that its cognate ligand (kisspeptin) mediates these
effects on GnRH neurons. We report on the development of kisspeptin
antagonists and their application in elucidating the role of
kisspeptin in puberty and steroid hormone feedback. Substitution of
amino acids in kisspeptin-10 identified a series of potent
antagonists. A selected antagonist inhibited GnRH neuron firing in
mouse brain and ablated GnRH pulses in pubertal female rhesus
monkeys; the latter supporting the key role of kisspeptin in
puberty onset. Moreover, the antagonist blunted the luteinizing
hormone (LH) responses to exogenous kisspeptin in rats, and
inhibited the LH increase after gonadectomy in ewes, rats and mice,
which indirectly evidences that kisspeptin-neurons are targets for
the feedback action of sex steroids. The development of kisspeptin
antagonists thus provides novel tools for interrogating the
physiological and pathophysiological roles of kisspeptin in the
regulation of reproduction and therapeutics for intervention in
hormone-dependent diseases.
Introduction
[0247] Gonadotropin-releasing hormone (GnRH) is the primary
effector of gonadotropin secretion, which is critical for
downstream regulation of gamete and steroid hormone production by
the testes and ovaries. Neurons that produce GnRH are modulated by
a host of factors, including photoperiod, metabolic signals, stress
and gonadal steroids, which control GnRH secretion by activating or
inhibiting neural circuits in the hypothalamus but the mechanisms
transducing these effects into changes in GnRH secretion have
remained elusive (1-3). These mechanisms are now emerging through
the discovery of a unique type of infertility termed
hypogonadotropic hypogonadism which was found to be associated with
a mutation in a G protein-coupled receptor (gpr-54) (4, 5). Gpr-54
is the cognate receptor for a family of neuropeptides, called
kisspeptins (6, 7), and kisspeptin/gpr-54 signaling has emerged as
a linchpin in the neuroendocrine regulation of reproduction(8).
Indeed, it has been revealed that kisspeptins (encoded by the Kissl
gene) are potent secretagogues for GnRH and that GnRH neurons
express the kisspeptin receptor(9, 10). Moreover, kisspeptin
neurons have been implicated as conduits that relay environmental
and hormonal inputs to GnRH neurons (11-16).
[0248] Through their modulation of the
hypothalamic-pituitary-gonadal (HPG) axis, GnRH analogues are
extensively employed to treat hormone-dependent diseases including
prostatic, breast and ovarian cancers, endometriosis, uterine
fibroids and precocious puberty, as well as in the induction of
ovulation for infertility and IVF (17, 18). Since kisspeptin exerts
such a powerful stimulatory effect on GnRH and gonadotropin
secretion, intervention at the level of the kisspeptin/gpr-54
system may have potential for treatment of these conditions, and
possibly with greater and more effective control than achieved with
GnRH analogues.
[0249] Kisspeptin is markedly elevated in pregnancy (19) and is
produced by the trophoblast (20) and inhibits trophoblast cell
invasion (implantation) through gpr-54 receptors in trophoblast
cells and in the uterine epithelium by inhibiting matrix
metalloproteinases (MMPs)(21). Thus kisspeptin/gpr-54 dysregulation
may play a role in placental and fetal pathologies such as
pre-eclampsia, placenta acretia, ectopic pregnancy and
inter-uterine growth retardation but direct evidence for this is
lacking Kisspeptin is also a potent vasoconstrictor through gpr-54
in selective regions of the human vasculature (22).
[0250] The development of kisspeptin antagonists would therefore
provide the wherewithal to elucidate the role of kisspeptin in the
positive and negative gonadal feedback on GnRH cells and in
mediating metabolic and stress effects on the reproductive system.
Kisspeptin antagonists also offer potential therapeutic
intervention in a wide range of hormone-dependent diseases and
possibly also in placental and vasculature dysfunction.
[0251] We report on a systematic structure-activity study of
kisspeptin analogues and the development of potent and specific
antagonists that are active in vitro and in vivo in laboratory
rodents, sheep and a non-human primate. The studies indicate a
pivotal role for kisspeptin in puberty and steroid hormone feedback
underlining the wide utility of these antagonists
Results
[0252] Effects of Substitutions of Amino Acids within Kisspeptin-10
on Stimulation of Inositol Phosphate (IP) Release in CHO Cells
Stably-Expressing Human Gpr-54
[0253] As kisspeptin-10 is the minimal sequence required for full
receptor binding and activation, we explored the effect of
systematic substitution of amino acids in this region monitoring IP
production in cells stably-expressing the human gpr-54. Because the
C-terminal sequence RF.NH.sub.2 is conserved in this large and
ancient peptide family, we reasoned that this is essential for
receptor binding and focused our attention on the preceding eight
amino acids. We truncated the N-terminal five amino acids and
introduced various substitutions with D-amino acids. This resulted
in a reduced efficacy in IP production and a weaker ability to
inhibit IP stimulation by 10 nM-kisspeptin-10 (FIG. 7; Table 1).
This indicated the first five amino acids are involved in receptor
activation. We then substituted residues alone or in combination in
the full ten amino acid peptide, and monitored the effect this had
on intrinsic IP stimulation and any inhibitory effects these
substitutions had on IP stimulation by 10 nM kisspeptin-10. This
systematic substitution of amino acids allowed us to develop a
range of analogues with partial agonistic and antagonistic
properties at this receptor (Table 1). Many of these analogues
incorporated a glycine substitution for Ser.sup.6 in combination
with a D-amino acid (tryptophan or leucine) at Leu.sup.8. Although
these substitutions alone did significantly (P<0.01) inhibit 10
nM kisspeptin-10 stimulation of IP, they generally did not reduce
this stimulation to basal levels (FIG. 8a,b; Table 1).
[0254] To develop full antagonists, further D-amino acid
substitutions were made at Tyr.sup.1, Asn.sup.2 and Trp.sup.3. The
most effective antagonists with substitutions in these positions
were peptides 230, 232, 233, 234, 235 and 236 which alone had
little or no stimulation of IP, but had IC.sub.50s of
<10.sup.-8M and a maximal inhibition of 66-93% of kisspeptin
stimulation of IP (FIG. 8b-d; Table 1). The most complete
inhibition was achieved with D-Ala' substitution (234 in Table 1;
FIG. 8d). This combination of substitutions inhibited 10 nM
kisspeptin-10 stimulation of IP by 93%, with an IC.sub.50 of 7 nM
and had no intrinsic IP stimulation, signifying high antagonist
activity. These studies have also highlighted the significance of
the glycine substitution at Ser.sup.6 and the D-Trp at Leu.sup.8,
as removal of one or both of these substitutions from the above
analogues either reduces the antagonist activity when D-Tyr is
substituted at Tyr.sup.1 (FIG. 8e) or reduces the antagonism when
D-Ala is substituted (FIG. 8f). This suggests that these residues
may be important for receptor activation. The active analogues
displayed high binding affinities using .sup.125I-kisspeptin-10,
for example peptide 234 had a binding affinity of 2.7 nM (FIG.
9).
Peptide 234 Inhibits Kisspeptin-10 Stimulation of GnRH Neuron
Firing
[0255] As has been demonstrated previously (23) (J.
Pieleka-Fortuna, Z Chu, S M Meonter, Endocrine Society Meeting
2006, Abstract P1-8), 1 nM kisspeptin-10 markedly increased GnRH
neuron firing activity (FIG. 10a,c). Under these experimental
conditions, there was no effect on GnRH neuron firing activity of
peptide 234 alone (1 nM pre 0.18.+-.0.12 Hz, post 0.27.+-.0.08 Hz,
n=5, P>0.05; 10 nM pre 0.34.+-.0.15 Hz, post 0.29.+-.0.17 Hz,
n=6, P>0.05; 100 nM pre 0.45.+-.0.12 Hz, 0.62.+-.0.14 Hz, n=7,
P>0.05, paired t test). In contrast to the lack of effect of
peptide 234 on basal firing, pretreatment with this peptide blocked
the response to 1 nM kisspeptin-10 (P<0.001 for all doses)
compared to cells treated with kisspeptin-10 alone (FIG. 10b,
c).
Peptide 234 Inhibits Pulsatile GnRH Release in Female Rhesus
Monkeys
[0256] Since peptide 234 inhibited GnRH neuronal firing we examined
whether this resulted in an inhibition of GnRH release in pubertal
female rhesus monkeys using methods previously described (24).
Infusion of 10 nM peptide 234 over 30 min through a microdialysis
probe located in the stalk-median eminence region promptly and
consistently suppressed GnRH pulses as well as mean GnRH levels
(FIG. 11a,c). In contrast, vehicle infusion through the probe did
not cause any significant changes in GnRH release (FIG. 11b,d). The
peptide 234-induced GnRH suppression was significantly different
from values prior to peptide 234 infusion as well as those from the
vehicle control (for both p<0.05). Based on our previous
assessment that the dialysis membrane passes .about.10% of peptides
with a similar size (25), it is estimated that the concentration of
peptide 234 in the stalk-median eminence region was 1 nM.
Peptide 234 Inhibits Kisspeptin-10 Stimulated LH in Intact Male
Rats and the Increase in LH after Castration
[0257] The effects of the putative kisspeptin antagonist activity
of peptide 234 were tested in vivo using adult male rats.
Pharmacological tests involved repeated (.times.3) intracerebral
injections of the compounds and serial blood sampling, in order to
assess potential effects of antagonists on basal LH levels. The
third injection was accompanied by co-administration of a
submaximal dose (100 pmol) of kisspeptin-10 and a 1 nmol dose of
peptide 234, in order to monitor the ability to inhibit the
kisspeptin-10 stimulation of LH and testosterone via its
stimulation of GnRH. Administration of 1 nmol peptide 234 icy did
not significantly modify basal LH levels at 15, 60, 75 and 120 min
after injection, Central injection of 1 pmol kisspeptin-10 to
vehicle-treated animals (at 120-min) evoked the expected rise in
serum LH levels (FIG. 12a). In spite of its lack of effect on basal
levels, co-administration of peptide 234 significantly blunted the
LH secretory responses induced by central injection of
kisspeptin-10, with a significant (P<0.01) reduction in the net
LH secretory mass (AUC) during the 120-min period following
co-injection of peptide 234 and kisspeptin-10. In good agreement,
testosterone levels at 60-min after combined injection of peptide
234 and kisspeptin-10 were significantly (P<0.01) lower than in
animals injected with kisspetin-10 alone (FIG. 12a). LH levels were
elevated in castrated rats over the 240 min monitoring period.
Administration of 1 nmol peptide 234 at 0-, 60 and 120 min tended
to reduce serum LH levels in castrated males; a reduction that
reached statistical significance at 240 min (FIG. 12b).
Peptide 234 Inhibits LH Pulse Frequency and Amplitude in
Ovariectomized Ewes
[0258] Major secretory episodes of LH were clearly distinguishable
in control ovariectomized ewes and in treated ewes prior to peptide
234 administration (FIG. 13). The number of secretory pulses was
reduced after ventricular administration of peptide 234. Where LH
pulses were identified, these were of diminished amplitude
following peptide 234 infusion (P<0.05, (Table 2). Mean LH
levels were similar before and during the infusion, but reduced
following peptide 234 infusion (P<0.05)
[0259] Infusion of peptide 234 significantly (P<0.05) increased
the mean concentration of GH in OVX ewes (FIG. 14 and Table 2).
This effect was only seen during the infusion, with mean GH levels
similar between control and peptide 234 treated ewes before and
after the infusion. Interestingly, this stimulatory effect appeared
to be more immediate than the inhibitory effect on LH. Peptide 234
had no effect on the concentrations of prolactin or cortisol
before, during or after infusion (FIGS. 15 & 16).
[0260] As can be seen in FIG. 15, antagonist administration
surprisingly elevated growth hormone levels in sheep. Accordingly,
the antagonists of the invention may be used to promote and/or
induce growth hormone production in an individual. For example, the
antagonists of the invention could be used alongside existing
treatments in which growth hormone therapy is used, such as in the
treatment of renal failure.
Peptide 234 Inhibits LH Pulse Frequency and Amplitude in
Ovariectomized Ewes
[0261] Human Umbilical Vein Endothelial Cells (HUVECs) isolated
from neonatal umbilical cord were used as they endogenously express
gpr-54, which is though to be involved in cell migration in the
placenta. Chinese hamster ovary (CHO) cells which stably express
the human gpr-54 receptor were used as a model cell line for wound
healing assays.
[0262] Cell monolayers of WT HUVECs or CHO cells stably expressing
human gpr-54 had a scratch made down the middle of each well in a
12-well plate with a pipette tip, and then plates were washed with
PBS to remove any loose cells. Complete media was added along with
1 nM Kisspeptin-10 for HUVECs and 100 nM Kisspeptin-10 for CHO
cells, the optimal concentrations to inhibit cell migration (wound
healing) in each cell type, in the presence and absence of
antagonist 233, 234, 273 and 276 for HUVECs and 234 for CHOs. Cells
were then incubated at 37.degree. C. with 5% CO.sub.2 in air for 22
hrs. Cells were photographed on the axiovert 2000 at 20.times.
magnification in phase contrast (Hori et al., 2001. Metastin
suppresses the motility and growth of CHO cells transfected with
its receptor. Biochem Biophys Res Commun 286, 958-963; Stafford et
al., 2002, Identification and characterization of mouse
metastasis-suppressor KiSS1 and its G-protein-coupled receptor.
Cancer Res 62, 5399-5404).
[0263] In FIG. 17(A), the results show that in the CHO cells, 100
nM Kisspeptin inhibits cell migration by 50% but that peptide 234
completely abolishes this inhibition. In FIG. 17(B), the results
show that in the HUVECs, 1 nM Kisspeptin inhibits migration in to
the wound by 50%. However, when this is done in the presence of
peptides 233, 234 or 273 this inhibition is reduced to only
10%.
[0264] From this we can conclude that peptides 233, 234 and 273 act
as antagonists in these cells at the human gpr-54 as they almost
completely inhibit the effect of kisspeptin on migration.
Accordingly, the antagonists of the invention can be used to induce
and/or promote wound healing.
Addition of the Antennapedia Heptapeptide to the N-Terminus of
Peptide 234
[0265] As discussed above, the R-R-M-K-W-K-K-Y (SEQ ID NO:20)
sequence is a heptapeptide sequence from Antennapedia. The peptide
sequence R-R-M-K-W-K-K-Y (SEQ ID NO:20) was added to the N-terminus
of Peptide 234, and the resulting peptide designated Peptide
271.
[0266] Accordingly, Peptide 271 has the sequence:
TABLE-US-00027 (SEQ ID NO: 77) ac.R - R - M - K - W - K - K - Y -
(D)A - N - W - N - G - F - G - (D)W - R - F.z
[0267] As shown in Table 1, Peptide 271, which comprises the
N-terminal Antennapedia heptapeptide, retains kisspeptin antagonist
function.
Discussion
[0268] The pleiotropic effects of kisspeptin as an antimetastatic
agent, and in regulating the HPG axis, and possibly implantation,
vasoconstriction and CNS neurons makes the gpr-54 receptor an
attractive therapeutic target. Until now the major emphasis has
been on developing kisspeptin agonists as antimetastatic agents
(26-28). However, the majority of therapeutic interventions in
kisspeptin roles in the HPG axis, in implantation and in
vasoconstriction would require development of antagonists. To
realise this objective we have systematically substituted amino
acids in the minimal kisspeptin structure required for biological
activity (kisspeptin-10) and developed antagonists with high
binding affinity and efficacy in vitro and in vivo. Studies in
pursuit of kisspeptin agonists have established that Phe.sup.6,
Arg.sup.9 and Phe.sup.10.NH.sub.2 constitute a binding
pharmacophore (26). The evolutionary conservation of the
RF.NH.sub.2 moiety amongst a large and ancient family of peptides
predicts that these C-terminal residues are essential for receptor
engagement in agreement with their identification in the
pharmacophore. Our pilot studies on kisspeptin amino acid
substitutions confirmed that changes in the RF.NH.sub.2 moiety may
reduce binding. We therefore focused on other residues in seeking
antagonist structures. We first truncated the N-terminal five amino
acids in the rodent and human kisspeptin-10 sequences and found
that this reduced agonist activity and antagonist properties.
Substitution of Phe.sup.6 or Leu.sup.8 in these truncated peptides
revealed that D-Trp substitution of Leu.sup.8 produced the most
promising antagonist. Incorporation of this substitution in the
full decapeptide sequences produced better antagonists especially
when accompanied by the substitution of Ser.sup.5 with the achiral
amino acid, Gly, which permits greater flexibility peptides and can
obviate some of the steric hindrances of the other amino acid
substitutions which have bulky side chains (e.g. D-Trp).
[0269] Further exploration revealed that substitution of the
N-terminal Tyr.sup.1 with D-amino acids was also tolerable for
antagonistic activity but not if D-Tyr.sup.1 or D-Trp' accompanied
D-Trp.sup.8 substitution. As expected substitution of the
pharmacophore residue Phe.sup.6 resulted in a decrease in agonist
activity and generation of some antagonist activity (e.g. 211 and
212). However, substitution of Phe.sup.6 with D-Trp.sup.6 combined
with Leu.sup.8 substitution by D-Trp.sup.8 reduced antagonism
compared with D-Trp.sup.8 alone (compare 213, 245, and 246 with
210). Certain substitutions of Asn.sup.2 (e.g. in 232 and 236) and
Trp.sup.3 (e.g. in 231 and 235) were also acceptable for antagonism
when combined with D-Trp.sup.8 substitution of Leu.sup.8. Overall a
consensus sequence for antagonism was
X.sup.1-X.sup.2-X.sup.3-N-G-F-G-X.sup.8-R-F.NH2 (SEQ ID NO:79)
where X.sup.1 is D-Tyr or D-Ala, X.sup.2 is Asn or D-Ala or D-Trp,
X.sup.3 is Trp or D-Trp or D-Ala, and X.sup.8 is D-Leu, D-Phe or
D-Trp.
[0270] Our studies have also identified amino acids that appear to
be involved in receptor activation as substitution of Ser.sup.6,
Leu.sup.8 and to some extent Tyr.sup.1, Asn.sup.2 and Trp.sup.3
contributed to antagonism. This is interesting because research to
date suggests that the N-terminus contains the activation domain
and the C-terminus the binding domain. Our findings suggest,
however, that the two sites overlap, as it has been shown that
Phe.sup.6, Arg.sup.9 and Phe.sup.10 form a pharmacophore for
binding (26), thus placing the Leu.sup.8 activating residue within
the binding site.
[0271] Of the analogues exhibiting good antagonist activity in
vitro we selected peptide 234 for ex vivo and in vivo studies.
Since kisspeptin action in the HPG axis is thought to be
predominantly through stimulating GnRH secretion (10, 29) we first
investigated the ability of peptide 234 to inhibit kisspeptin-10
stimulation of firing in GnRH neurons recorded in acutely prepared
mouse brain slices. Peptide 234 was found to block kisspeptin-10
stimulation of GnRH neuron firing at 100 nM, 10 nM and even at 1 nM
which is the same concentration as that of kisspeptin-10 used for
stimulation of the GnRH neuron.
[0272] The inhibition of kisspeptin action on GnRH neuronal firing
by peptide 234 suggests that the antagonist would reduce
hypothalamic GnRH release in vivo. We therefore determined whether
kisspeptin modified pulsatile GnRH release by administering peptide
234 directly to the stalk-median eminence region in pubertal female
rhesus monkeys. The suppression of GnRH release during peptide 234
infusion provides the first direct evidence that input from
kisspeptin neurons in the hypothalamus is a significantly important
signal for GnRH release. The importance of kisspeptin in GnRH
pulsatility is further supported by previous observations
indicating that kisspeptin-54 release is pulsatile and
kisspeptin-54 pulses coincide with GnRH pulses 75% of the time
(24). However, because clinical studies suggest that patients with
gpr-54 mutations exhibited attenuated LH pulses with an
approximately normal pulse frequency (4, 5)'(30), the issue of
whether kisspeptin neurons are critical for GnRH pulse-generation
or if it is just a modulator of GnRH pulse amplitude, remains to be
investigated. The complete ablation of GnRH pulses in the monkey
model and LH pulses in the ovariectomised sheep may therefore
simply reflect a total inhibition of amplitude rather than
frequency.
[0273] Peptide 234 also blocked kisspeptin-10 stimulation of LH and
testosterone in adult male rats over 2 hours (FIG. 12a), presumably
by blocking kisspeptin-10 effects on GnRH secretion. The inhibition
of GnRH secretion in intact female rhesus monkeys by peptide 234
suggests that the former explanation is most likely. In castrated
male rats peptide 234 blocked the increase in LH that ensues
following castration and the removal of negative feedback. The
findings suggest that removal of gonadal steroids leads to a rise
in kisspeptin activity that is translated into increased GnRH and
LH secretion. Previous demonstrations of increased KiSS-1 gene
expression in the arcuate nucleus in gonadectomised male and female
rats (10, 29) and the ability to ablate the resulting LH rise with
GnRH antagonist (10) is consistent with the presumption that
kisspeptin-producing neurons are targets for the feedback actions
of sex steroids (11). Levels of mRNA may not, however, reflect the
biosynthesis and secretion of kisspeptin to engage gpr-54 in GnRH
neurons. Our studies with a novel kisspeptin antagonist now provide
direct evidence for kisspeptin modulation of the GnRH neuron in
negative feedback by the gonads and emphasize the value of a
kisspeptin antagonist for elucidating physiological mechanisms.
[0274] Frequent blood sampling (every 10 min) and antagonist
treatment in ovariectomised ewes allowed us to interrogate the role
of endogenous kisspeptin on LH pulse frequency and amplitude. LH
pulse frequency and amplitude was markedly reduced by icy
administration of peptide 234 suggesting a decrease in the
amplitude and possibly frequency of GnRH secretion. This accords
with our demonstration of peptide 234 inhibition of GnRH neuron
firing rate in mice and its inhibition of GnRH pulses in rhesus
monkeys. These findings imply that the endogenous secretion of
kisspeptin is pulsatile in nature and drives the ultradian
secretion of GnRH. The ability to increase GnRH neuron firing with
kisspeptin-10 and to reduce it with kisspeptin antagonist suggests
therapeutic potential of kisspeptin analogues in pathological
conditions associated with dysfunctional LH pulse frequency such as
the increased LH pulse frequency characteristic of polycystic ovary
syndrome (31) and decreased frequency found in psychological
disorders, stress (32) and in under nutrition (e.g., anorexia
nervosa) (33).
[0275] Peptide 234 appears to be specific in its actions as it had
no effect on prolactin and cortisol secretion in ovariectomised
ewes (FIGS. 15 & 16) and did not bind to GnRH or LH receptors
(data not shown). Growth hormone appeared to increase during
infusion of peptide 234 in all 4 ewes suggesting that endogenous
kisspeptin inhibits growth hormone (FIG. 14). We have not as yet
examined this phenomenon in depth but have observed that the
kisspeptin receptor (gpr-54) is exclusively expressed in growth
hormone cells in the pituitary. Positive feedback by estrogen in
inducing the LH surge of ovulation has been shown in rodents and
sheep to be accompanied by an increase in Kiss-1 gene expression in
specific hypothalamic areas (34-36).
[0276] Studies in female rats(37) and women (38) also show a
greater LH sensitivity to exogenous kisspeptin at the time of the
ovulatory LH surge suggesting that positive feedback by estrogen is
mediated by increased kisspeptin input (37). This concurs with the
demonstration of increased KiSS-1 gene expression after estrogen
treatment (34, 35) and the LH surge, which is ablated by
administration of kisspeptin antiserum (35) in female rats. We are
currently utilizing peptide 234 to determine the role of kisspeptin
in positive feedback on GnRH and LH secretion.
[0277] The majority of our studies administered antagonist icy in
order to have a more direct intervention of kisspeptin input on
GnRH neurons and more clearly delineate the role of kisspeptin in
the physiological regulation of the HPG axis. Systemic
administration is, however, a prerequisite for therapeutic
application of kisspeptin analogues. Since intravenous and
subcutaneous kisspeptin administration stimulate LH release in men
(39) and women (38) the peptide evidently enters the brain or acts
at sites that are outside the blood-brain barrier such as the
arcuate nucleus or median eminence and acts on GnRH neurons. It
therefore seems plausible that systemically delivered kisspeptin
antagonist would also reach GnRH neurons or their axons in the
median eminence, thus providing an effective and feasible
therapeutic target.
[0278] A wide spectrum of pathologies is associated with
dysfunction of the HPG axis and many conditions are exacerbated by
sex steroid stimulation. These include infertility, polycystic
ovary syndrome, endometriosis, uterine fibroids, excessive
menstrual bleeding, delayed and precocious puberty, and breast,
prostatic and ovarian cancers. The availability of potent, specific
and efficacious kisspeptin antagonists will provide the potential
for novel treatments of these conditions and for interrogating the
physiological regulation of the HPG axis. In addition to these
primary indications, kisspeptin antagonists may have potential in
modulating vasoconstriction (22), CNS function (40) and trophoblast
invasion and the developments of adequate maternal blood supply
(19, 21, 41).
Methods
Peptides
Materials
[0279] Human kisspeptin-10 and peptide analogues 186-191, 200-203,
206-213 and 228-248 (10 .mu.g) were custom synthesized by
EZBiolabs. The source of all other reagents is indicated in the
text.
In Vitro Assays for Antagonistic Activity
Cell Culture
[0280] Chinese hamster ovary (CHO) cells stably-expressing the
human gpr-54 receptor (CHO/gpr-54) were obtained from Prof G.
Vassart, Univ. Brussels. The cells were maintained in Dulbecco's
modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal
calf serum, 2% glutamine and 1% penicillin (10,000
units/ml)/streptomycin (10,000 mg/ml) at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere.
Inositol Phosphate (IP) Stimulation Assay
[0281] Prior to stimulation CHO/gpr-54 cells were washed twice with
Dulbecco's phosphate-buffered saline (DPBS; without calcium or
magnesium) then incubated overnight with .sup.3H-myoinositol,
labeled HEPES-modified DMEM with 1% penicillin/streptomycin at
37.degree. C. HEPES-modified DMEM supplemented with 1%
penicillin/streptomycin and 1% lithium chloride (0.5 ml) was added
to cells for 30 min at 37.degree. C. to block IP hydrolysis. Cells
were then stimulated with 0.5 ml kisspeptin-10 and analogues (10
pM-1 .mu.M) diluted at 1:100 in the above media for 1 h at
37.degree. C., then with 10 mM formic acid at 4.degree. C. for 1 h
to lyse cells. Lysates were transferred to plastic tubes containing
0.5 ml Dowex resin to bind the radioactive IP and the resin was
then washed with 1 ml water. The resin was next washed with 60 mM
ammonium formate/5 mM sodium tetraborate. followed by 1M ammonium
formate/0.1M formic acid to release the bound radiation. Then 800
.mu.l of the radioactive solution were transferred to scintillation
vials containing 2.5 ml scintillation fluid and radioactivity
counted on a Beta counter for 60 sec. Experiments were repeated 3-5
times. IP production was plotted as mean values.+-.SEM and analyzed
by using a Student's t-Test (p.gtoreq.0.05).
Inositol Phosphate (IP) Antagonism Assay
[0282] CHO/gpr-54 cell monolayers were stimulated with 0.25 ml
kisspeptin (10 nM) alone or in combination with 0.25 ml peptide
analogues (100 pM-1 .mu.M), to investigate the inhibition of
kisspeptin stimulation of IP production. Experiments were repeated
3-5 times. IP production was plotted as mean values.+-.SEM and
analyzed using a Student's t-Test (p.gtoreq.0.05).
GnRH Neuron Firing
Animals
[0283] Firing of GnRH neurons were recorded in brain slices from
transgenic female mice in which GFP is genetically targeted to GnRH
neurons (42). Mice were housed on a 14 h light, 10 h dark cycle,
with lights off at 1630 h, and were maintained on Harlan 2916
rodent chow (Harlan) and water ad libitum. All procedures were
approved by the Animal Care and Use Committee of University of
Virginia and were conducted within the guidelines of the National
Research Council's Guide for the Care and Use of Laboratory
Animals. Adult female GnRH-GFP mice were ovariectomized under
isoflurane (Abbott Laboratories) anesthesia. Postoperative
analgesia was provided by a long-acting local anesthetic (0.25%
bupivicaine; 7.5 .mu.l/site; Abbott Laboratories). At the time of
surgery, mice received Silastic (Dow Corning) capsules containing
0.625 .mu.g estradiol in sesame oil. Recordings were done 2-4 days
after surgery in the AM, when estradiol has been demonstrated to
have a negative feedback effect (43). No more than four cells from
a single animal were recorded, all in different slices.
Brain Slice Preparation
[0284] Brain slices were prepared using previously described method
(44). Briefly, all solutions were bubbled with a 95% O.sub.2/5%
CO.sub.2 mixture throughout the experiments and for at least 15 min
before exposure to the tissue. The brain was rapidly removed and
placed in ice-cold, high-sucrose saline solution containing 250 mM
sucrose, 3.5 mM KCl, 26 mM NaHCO.sub.3, 10 mM glucose, 1.25 mM
Na.sub.2HPO.sub.4, 1.2 mM MgSO.sub.4, and 2.5 mM MgCl.sub.2.
Coronal 300-.mu.m brain slices were cut with a Vibratome 3000
(Technical Products, International, Inc., St. Louis, Mo.). Slices
were incubated for 30 min at 30-32C in a solution of 50%
high-sucrose saline and 50% normal saline (NS) containing 135 mM
NaCl, 3.5 mM KCl, 10 mM glucose, 1.3 mM Na.sub.2HPO.sub.4, 1.2 mM
MgSO.sub.4, and 2.5 mM CaCl.sub.2 and then were transferred to a
solution of 100% NS at room temperature and kept at least 30 min
and no more than 6 h before recording.
Electrophysiological Recordings
[0285] Targeted extracellular recordings (also known as
loose-patch) were used to study effects of kisspeptin-10 and
peptide 234 on GnRH neuron firing activity (45). Because low
resistance seals (<50 M.OMEGA.) do not influence the cell
membrane this approach is a minimally invasive method for
monitoring the endogenous electrical activity of a single cell.
Although these events are not action potentials per se, they
accurately reflect changes in the action potential firing rate. For
simplicity, we have used the phrases firing rate, firing pattern,
and/or firing activity to refer to these events.
[0286] Individual brain slices were placed in a recording chamber
continuously superfused with oxygenated NS solution and kept at
29-31 C. Cells were visualized with an Olympus BX50WI upright
fluorescent microscope with infrared differential interference
contrast (Opelco). GnRH neurons were identified by brief
illumination at 470 nm to visualize the GFP signal. Patch
borosilicate pipettes (World Precision Instruments), which ranged
from 1.5-3.OMEGA. M were filled with normal HEPES-buffered solution
containing 150 mM NaCl, 10 mM HEPES, 10 mM glucose, 2.5 mM
CaCl.sub.2, 1.3 mM MgCl.sub.2, and 3.5 mM KCl. Pipettes were placed
in contact with the GnRH neurons using an MP-285 micromanipulator
(Sutter Instruments). Seal resistances ranged from 8 M.OMEGA. and
either remained stable or increased during recording up to as high
as 50 M.OMEGA.. Current traces were obtained using an EPC-8
amplifier (HEKA) with the PulseControl XOP (Instrutech) running in
Igor Pro (Wavemetrics) on the G4 Macintosh computer (Apple
Computer) to acquire data. A voltage-clamp mode with a pipette
holding potential of 0 mV, filtering at 10 kHz, digitized with an
ITC-18 acquisition interface (Instrutech) was used for the
recordings.
Experimental Design
[0287] Human kisspeptin-10, 1 nM, (Phoenix Pharmaceuticals) was
applied via the incubation bath. Different doses (1 nM, 10 nM, 100
nM) of peptide 234 were used to examine its antagonistic action on
kisspeptin-10 activation of gpr-54. Positive control cells were
recorded for a 10-min stable baseline, and then treated with
kisspeptin-10 for 5 min, followed by a 15-min washout. Experimental
cells were recorded for a 5-min stable baseline in NS, followed by
10 min in peptide 234 (1, 10 or 100 nM), then 5 min in
kisspeptin-10 plus the same dose of antagonist, followed by a wash
in NS. Washing with a solution containing antagonist yielded
similar results (1 nM peptide 234, n=5 cells, not shown). One cell
in the 10 nM peptide 234 group showed no activity; addition 15 mM
KCl confirmed cell viability and recording integrity.
Data Analysis
[0288] Using programs written for Igor Pro,
extracellularly-recorded events were counted and binned at 1-min
intervals to identify changes in firing rate of GnRH neurons.
Binned event data were analyzed using Microsoft Excel (Microsoft
Corp) for the mean firing rate before kisspeptin-10 treatment
(baseline, note this is during peptide 234 treatment in cells in
the antagonist treatment groups) during kisspeptin-10 application
(treatment), and during washout. For cells in the antagonist
treatment group firing rates in NS vs. antagonist before
kisspeptin-10 application were also compared. Mean firing rate was
determined by dividing the total number of events detected by the
duration of recording in each condition; two minutes were skipped
after drug changes to eliminate transition periods. Since GnRH
neuron firing activity changes with time (44, 45) the fold change
for each treatment group was calculated. Groups were compared using
ANOVA followed by Bonferroni post hoc test.
Effects of Peptide 234 on GnRH Release in Female Rhesus Monkeys
Animals
[0289] Four female rhesus monkeys, born and raised in the Wisconsin
National Primate Research Center, were used in this study. They
were housed in pairs (cages 172.times.86.times.86 cm) in rooms with
12 h of light 12 h of darkness and controlled temperature
(22.degree. C.). The animals were fed a standard diet of Harlan 20%
Protein Primate Diet twice each day, supplemented with fresh fruit
several times per week. Water was available ad libitum. The
protocol for this study was reviewed and approved by the Animal
Care and Use Committee, University of Wisconsin, and all
experiments were conducted under the guidelines established by the
NIH and USDA.
Experimental Design
[0290] The effects of peptide 234 on GnRH release were examined
using the microdialysis method. This method allows collection of
dialysate samples for GnRH measurement while peptide 234 was
infused through the semi-permeable membrane, as described
previously (25)'(24). Peptide 234 dissolved in CNS perfusion fluid
was infused in the stalk median eminence region through the
microdialysis probe for 30 min while dialysates were continuously
collected. For a control, vehicle was similarly infused. Each
animal was examined with peptide 234 (10 nM concentration) or
vehicle at a random order in the same experiment a 2 hour-minimum
interval between two challenges. Two of the 4 animals were examined
twice in separate experiments totaling 6 experiments. During the
entire experiment monkeys were placed in proximity to a companion
monkey, given constant access to food and water, and were provided
frequently with fruit, cereal, raisins and other snacks. The mean
age of sampling was 33.1.+-.0.4 months.
Cranial Pedestal Implantation
[0291] For collection of perfusates in the stalk-median eminence
region, we used the microdialysis method, as previously described
(24). Prior to experiments, monkeys at 29.1.+-.1.2 months of age
(body weight: 3.6.+-.0.1 kg) were well-adapted to the primate
chair, experimental environment, and investigator. They were
implanted with a cranial pedestal under isoflurane anesthesia,
similar to those described previously for push-pull perfusion
method (46). Animals were allowed to recover for at least 1 month
prior to experimentation.
Microdialysis Methods
[0292] On the day of experiment the monkey was placed in the
stereotaxic apparatus under ketamine (15 mg/kg b.w.) and
medetomidine (0.03-0.05 mg/kg b.w.) anesthesia. The custom-made
guide cannula (CMA 12) consisted of a stainless steel shaft (76.0
mm in length, 0.91 mm o.d.) and a removable stainless steel stylet
(96.0 mm in length, 0.6 mm o.d.), which extruded 20 mm from the
guide cannula tip. It was inserted into the skull 5 mm above the
S-ME with a hydraulic microdrive unit (M095-B). The microdrive unit
allowed for accurate three-dimensional adjustment of the tip
location. The x, y, and z coordinates for the S-ME were calculated
using ventriculographs and the final radiographs taken during
cranial pedestal implantation surgery. Cannula placement was
confirmed with radiographic visualization. Following placement of
the guide cannula, the monkey was removed from the stereotaxic
apparatus and placed into a primate chair. Upon proper placement in
the chair, the inner stylet was removed from the guide cannula and
a custom-made microdialysis probe (a stainless steel shaft 96.0 mm
in length, 0.6 mm o.d.), fitted with a membrane (5 mm in length,
0.5 mm o.d.), was inserted into the S-ME through the guide cannula
as described previously (25)'(24). To reverse the effects of
medetomidine, atipamazole (0.15-0.25 mg/kg) was injected (i.v.)
into the animal. The animal was fully awake within 1 h after probe
insertion.
[0293] A CNS perfusion fluid consisting of NaCl 147 mM, KCl 2.7 mM,
CaCl2 1.2 mM, MgCl2 0.85 mM, purchased from CMA/Microdialysis with
bacitracin (4 U/ml) added, was infused through the inflow tubing at
2 .mu.l/min with the CMA/102 microdialysis pump outfitted with a 1
or 2.5 ml Hamilton gas tight syringe (Reno). Perfusates were
continuously collected at 10 min intervals for up to 12 h through
the outflow tubing into 12.times.75 mm borosilicate tube, on ice
with a fraction collector (Model FC203B). The perfusate samples
were immediately frozen on dry ice and stored at -80.degree. C.
GnRH RIA
[0294] RIA was conducted with the antiserum R42 kindly provided by
Dr. Terry Nett (Colorado State University), as described previously
(46). Assay sensitivity was 0.02 pg/tube and intra and interassay
coefficients of variation were 8.1% and 11.3%, respectively.
Data Analysis
[0295] Peaks of GnRH release were identified using the PULSAR
algorithm as described previously (47). The mean value of a 30 min
period of GnRH collection before and after peptide 234 (or vehicle)
challenge was calculated in each experiment. Subsequently, the
means (.+-.sem) of all experiments during each time period were
calculated for statistical comparison. The difference between
before, during and after the treatment (within treatment) as well
as between treatments (peptide 234 vs. vehicle) was examined using
2-way analysis of variance followed by Tukey's multiple comparison
test. Differences were considered significant at P<0.05.
Dynamic Studies on Peptide 234 Inhibition of LH in Male Rats
Experimental Design
[0296] Adult male rats (BW: 280-310 g) bred in the vivarium of the
University of Cordoba were used. The animals were maintained under
constant conditions of light (14 h of light, from 0700 h) and
temperature (22 C), and, before cannula implantation, were kept in
groups of four rats per cage with free access to pelleted food and
tap water. Experimental procedures were approved by the Cordoba
University Ethical Committee for animal experimentation and were
conducted in accordance with the European Union normative for care
and use of experimental animals.
[0297] The animals were implanted with icy cannulae under light
ether anaesthesia and caged individually thereafter. To allow
delivery of compounds into the lateral cerebral ventricle, the
cannulae were lowered to a depth of 4 mm beneath the surface of the
skull; the insert point was 1 mm posterior and 1.2 mm lateral to
bregma. Functional tests were conducted at least 24-48 h after
cannula implantation.
[0298] Pharmacological tests in intact males involved 5 .mu.l
injections at 60 min intervals (icy) of 1 nmol doses of the peptide
234. The last injection was accompanied by the injection of an
effective (but sub-maximal) dose of 100 pmol kisspeptin-10 (Phoenix
Pharmaceuticals Ltd). Animals injected with vehicle (physiological
saline) served as controls. Blood samples (250 .mu.L) were taken by
jugular venipuncture at 15- and 60-min following each icy
injection. In addition, blood samples were taken immediately before
initiation of the experiment (time: 0-min) and at 120-min after
last injection (time: 240-min). Experimental procedures and
kisspeptin doses were selected on the basis of previous studies
(16, 34, 48). For each time-point, 10-12 samples were taken per
group.
[0299] In addition, repeated icy injection of the peptide 234 was
also conducted in orchidectomized (ORX) rats. The animals were
subjected to bilateral ORX via the scrotal route, and after 1-wk
after surgery were subjected to cannula implantation as described
above. The test involved three intracerebral injections of the
antagonist (1 nmol; at 0-, 60- and 120-min of the procedure) and
blood sampling by jugular venipuncture in basal conditions (0-min)
and 15-min after each icy injection. Additional blood samples were
obtained at 180- and 240-min after central administration of
peptide 234. For each time-point, 10-12 samples were taken per
group.
Hormone Measurements
[0300] Serum LH levels were measured in a volume of 50 .mu.l using
a double-antibody method and radioimmunoassay kits supplied by the
NIH (Dr. A F Parlow, NIDDK). Rat LH-I-10 was labeled with .sup.125I
using Iodo-Gen precoated tubes, following the instructions of the
manufacturer (Pierce), and hormone concentrations were expressed
using reference preparation LH-RP-3 as standard. Intra- and
inter-assay coefficients of variation were below 8 and 10%,
respectively. The sensitivity of the assay was 5 pg/tube. In
addition, serum testosterone (T) measurements were selectively
conducted in blood samples taken 60-min after co-administration of
kp-10 and peptide 234 (Time: 180-min). To this end, a commercial
kit from MP Biomedicals was used, following the instructions of the
manufacturer. The sensitivity of the assay was 0.1 ng/tube and the
intra-assay coefficient of variation was 4.5%. For each hormone,
all samples were measured in the same assay. Accuracy of hormone
determinations was confirmed by assessment of rat serum samples of
known hormone concentrations used as external controls.
Data Analysis
[0301] Hormonal determinations were conducted in duplicate, with a
minimal total number of 10 samples per group. When appropriate, in
addition to individual time-point measurements, integrated LH
secretory responses were calculated as the area under the curve
(AUC), following the trapezoidal rule. Hormonal data are presented
as mean.+-.SEM. Results were analyzed for statistically significant
differences using Student t-test or ANOVA followed by
Student-Newman-Keuls multiple range test (SigmaStat 2.0).
P.ltoreq.0.05 was considered significant.
Effect of Peptide 234 on LH in Ovariectomised Ewes
Experimental Procedure
[0302] All experimental procedures were conducted under a protocol
approved by the Monash School of Biomedical Sciences Animal Ethics
Committee. Adult Corriedale ewes were housed under natural lighting
and were bilaterally ovariectomized (OVX) at least 1 month before
any experimental manipulations. Permanent indwelling third cerebral
ventricular (3V) cannulae were implanted in a subsequent surgical
procedure as previously described (49). Approximately 2 weeks after
3V surgery, one external jugular vein was cannulated for blood
sampling and animals housed in single pens; cannulae were kept
patent with heparinized saline. Ewes were assigned to two treatment
groups (4/group); peptide 234 (diluted in artificial cerebrospinal
fluid, aCSF; 150 mM NaCl, 1.2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 2.8
mM KCl) or control (aCSF only). The following day, infusion lines
were connected to 3V cannulae and blood sampling commenced at 07.00
h. Samples were collected every 10 min. After 3 h of sampling,
peptide 234 (or control) was infused into the 3V at a dose of 40
.mu.g/h for 1 h, with an initial loading dose of 10 Both peptide
234 and vehicle were infused at 200 .mu.l/h using Graseby.RTM. MS
16A infusion pumps (Smith Medical Australasia Pty. Ltd.). After
infusion, 3V lines remained in place and blood sampling continued
for a further 2 h (total of 6 h). Plasma was harvested immediately
from samples, and frozen at -20 C until assayed.
[0303] Plasma LH concentrations were measured in duplicate, with
NIH-oLH-S 18 as standard (49). Assay sensitivity was 0.1 ng/ml and
the intra-assay coefficient of variation was less than 10% over the
range of 0.3-12.8 ng/ml. Pulse analysis of the plasma LH data was
as previously described (36). Plasma samples were assayed for GH in
duplicate using the standard NIDDK-oGH-I-4 and NIDDK-anti-oGH-2
antiserum (50). The assay sensitivity was 1 ng/ml, the intra-assay
CV was less than 10% between 4 and 51 ng/ml and the inter-assay CV
was 20%.
[0304] Plasma prolactin concentrations were measured in duplicate
using Sigma, Lot 114F-0558, NOL-7135 as standard (50). The
sensitivity of the assay was 1 ng/ml. Intra-assay CV was less than
10% between 1 and 22 ng/ml.
[0305] Plasma cortisol concentrations were assayed in duplicate
using antiserum no. 3368 (Bioquest Ltd) and .sup.125I-labeled
cortisol (Amersham Pharmacia Biotech Ltd). The sensitivity of the
assay was 3 ng/ml. Intra-assay CV was less than 10% between 3 and
18 ng/ml, and the inter-assay CV was 15%.
Data Analysis
[0306] For data analysis of LH, GH, prolactin and cortisol, the
mean plasma value in each ewe was calculated for the period before
(0-180 min), during (180-240 min), and after (240-360 min) the
infusion. In addition, the mean LH pulse amplitude for each ewe was
also calculated before, during, and after the infusion. Repeated
Measures ANOVAs were used to determine the effect of peptide 234
treatment on hormone levels over each period, and where appropriate
one-way ANOVAs were employed to assess specific difference within
each period.
EXAMPLE 3
Exemplary Pharmaceutical Formulations
[0307] Whilst it is possible for a molecule of the invention to be
administered alone, it is preferable to present it as a
pharmaceutical formulation, together with one or more acceptable
carriers. The carrier(s) must be "acceptable" in the sense of being
compatible with the agent of the invention and not deleterious to
the recipients thereof. Typically, the carriers will be water or
saline which will be sterile and pyrogen-free.
[0308] The following examples illustrate medicaments and
pharmaceutical compositions according to the invention in which the
active ingredient is a molecule of the invention.
[0309] Preferably, the molecule of the invention is provided in an
amount from 10 .mu.g to 500 mg. It will be appreciated that the
following exemplary medicaments and pharmaceutical compositions may
be prepared containing an amount of the molecule of the invention
from 10 .mu.g to 500 mg. For example, the agent of the invention
may be present in a 10.sup.th or 100.sup.th or 200.sup.th or
500.sup.th of the amount shown in the following exemplary
medicaments and pharmaceutical compositions with the amounts of the
remaining ingredients changed accordingly.
Example A
Tablet
TABLE-US-00028 [0310] Active ingredient 1 mg Lactose 200 mg Starch
50 mg Polyvinylpyrrolidone 5 mg Magnesium stearate 4 mg
[0311] Tablets are prepared from the foregoing ingredients by wet
granulation followed by compression.
Example B
Ophthalmic Solution
TABLE-US-00029 [0312] Active ingredient 1 mg Sodium chloride,
analytical grade 0.9 g Thiomersal 0.001 g Purified water to 100 ml
pH adjusted to 7.5
Example C
Tablet Formulations
[0313] The following formulations A and B are prepared by wet
granulation of the ingredients with a solution of povidone,
followed by addition of magnesium stearate and compression.
Formulation A
TABLE-US-00030 [0314] mg/tablet mg/tablet (a) Active ingredient 1 1
(b) Lactose B.P. 210 26 (c) Povidone B.P. 15 9 (d) Sodium Starch
Glycolate 20 12 (e) Magnesium Stearate 5 3 251 51
Formulation B
TABLE-US-00031 [0315] mg/tablet mg/tablet (a) Active ingredient 1 1
(b) Lactose 150 -- (c) Avicel PH 101 .RTM. 60 26 (d) Povidone B.P.
15 9 (e) Sodium Starch Glycolate 20 12 (f) Magnesium Stearate 5 3
251 51
Formulation C
TABLE-US-00032 [0316] mg/tablet Active ingredient 1 Lactose 200
Starch 50 Povidone 5 Magnesium stearate 4 260
[0317] The following formulations, D and E, are prepared by direct
compression of the admixed ingredients. The lactose used in
formulation E is of the direction compression type.
Formulation D
TABLE-US-00033 [0318] mg/capsule Active Ingredient 1 Pregelatinised
Starch NF15 150 151
Formulation E
TABLE-US-00034 [0319] mg/capsule Active Ingredient 1 Lactose 150
Avicel .RTM. 100 251
Formulation F (Controlled Release Formulation)
[0320] The formulation is prepared by wet granulation of the
ingredients (below) with a solution of povidone followed by the
addition of magnesium stearate and compression.
TABLE-US-00035 mg/tablet (a) Active Ingredient 1 (b)
Hydroxypropylmethylcellulose 112 (Methocel K4M Premium) .RTM. (c)
Lactose B.P. 53 (d) Povidone B.P.C. 28 (e) Magnesium Stearate 7
201
[0321] Drug release takes place over a period of about 6-8 hours
and was complete after 12 hours.
Example D
Capsule Formulations
Formulation A
[0322] A capsule formulation is prepared by admixing the
ingredients of Formulation D in Example C above and filling into a
two-part hard gelatin capsule. Formulation B (infra) is prepared in
a similar manner.
Formulation B
TABLE-US-00036 [0323] mg/capsule (a) Active ingredient 1 (b)
Lactose B.P. 143 (c) Sodium Starch Glycolate 25 (d) Magnesium
Stearate 2 171
Formulation C
TABLE-US-00037 [0324] mg/capsule (a) Active ingredient 1 (b)
Macrogol 4000 BP 350 351
[0325] Capsules are prepared by melting the Macrogol 4000 BP,
dispersing the active ingredient in the melt and filling the melt
into a two-part hard gelatin capsule.
Formulation D
TABLE-US-00038 [0326] mg/capsule Active ingredient 1 Lecithin 100
Arachis Oil 100 201
[0327] Capsules are prepared by dispersing the active ingredient in
the lecithin and arachis oil and filling the dispersion into soft,
elastic gelatin capsules.
Formulation E (Controlled Release Capsule)
[0328] The following controlled release capsule formulation is
prepared by extruding ingredients a, b, and c using an extruder,
followed by spheronisation of the extrudate and drying. The dried
pellets are then coated with release-controlling membrane (d) and
filled into a two-piece, hard gelatin capsule.
TABLE-US-00039 mg/capsule (a) Active ingredient 1 (b)
Microcrystalline Cellulose 125 (c) Lactose BP 125 (d) Ethyl
Cellulose 13 264
Example E
Injectable Formulation
TABLE-US-00040 [0329] Active ingredient 1 mg Sterile, pyrogen free
phosphate buffer (pH7.0) to 10 ml
[0330] The active ingredient is dissolved in most of the phosphate
buffer (35-40.degree. C.), then made up to volume and filtered
through a sterile micropore filter into a sterile 10 ml amber glass
vial (type 1) and sealed with sterile closures and overseals.
Example F
Intramuscular Injection
TABLE-US-00041 [0331] Active ingredient 1 mg Benzyl Alcohol 0.10 g
Glucofurol 75 .RTM. 1.45 g Water for Injection q.s. to 3.00 ml
[0332] The active ingredient is dissolved in the glycofurol. The
benzyl alcohol is then added and dissolved, and water added to 3
ml. The mixture is then filtered through a sterile micropore filter
and sealed in sterile 3 ml glass vials (type 1).
Example G
Syrup Suspension
TABLE-US-00042 [0333] Active ingredient 1 mg Sorbitol Solution
1.5000 g Glycerol 2.0000 g Dispersible Cellulose 0.0750 g Sodium
Benzoate 0.0050 g Flavour, Peach 17.42.3169 0.0125 ml Purified
Water q.s. to 5.0000 ml
[0334] The sodium benzoate is dissolved in a portion of the
purified water and the sorbitol solution added. The active
ingredient is added and dispersed. In the glycerol is dispersed the
thickener (dispersible cellulose). The two dispersions are mixed
and made up to the required volume with the purified water. Further
thickening is achieved as required by extra shearing of the
suspension.
Example H
Suppository
TABLE-US-00043 [0335] mg/suppository Active ingredient (63 .mu.m)*
1 Hard Fat, BP (Witepsol H15-Dynamit Nobel) 1770 1771 *The active
ingredient is used as a powder wherein at least 90% of the
particles are of 63 .mu.m diameter or less.
[0336] One fifth of the Witepsol H15 is melted in a steam-jacketed
pan at 45.degree. C. maximum. The active ingredient is sifted
through a 200 .mu.m sieve and added to the molten base with mixing,
using a silverson fitted with a cutting head, until a smooth
dispersion is achieved. Maintaining the mixture at 45.degree. C.,
the remaining Witepsol H15 is added to the suspension and stirred
to ensure a homogenous mix. The entire suspension is passed through
a 250 .mu.m stainless steel screen and, with continuous stirring,
is allowed to cool to 40.degree. C. At a temperature of 38.degree.
C. to 40.degree. C. 2.02 g of the mixture is filled into suitable
plastic moulds. The suppositories are allowed to cool to room
temperature.
Example I
Pessaries
TABLE-US-00044 [0337] mg/pessary Active ingredient 1 Anhydrate
Dextrose 380 Potato Starch 363 Magnesium Stearate 7 751
[0338] The above ingredients are mixed directly and pessaries
prepared by direct compression of the resulting mixture.
[0339] The agents of the invention may also be formulated as for
Zoladex, Leuprolide, Teverelix, Abarelix, Ganarelix, Goserelin
etc.
EXAMPLE 4
Treatment of a Proliferative Disorder Using an Agent of the
Invention
[0340] A patient with prostatic cancer who has not responded to
anti-androgen therapy is administered 1 mg of an agent of the
invention per day intramuscularly or a depot preparation delivering
this dose according to the methods of the invention. The antagonist
will decrease androgen.
EXAMPLE 5
Treatment of a Proliferative Disorder Using an Agent of the
Invention
[0341] A patient with endometriosis or uterine fibroids or breast
cancer is administered 1 mg of an agent of the invention per day
intramuscularly or a depot preparation delivering this dose
according to the methods of the invention.
EXAMPLE 6
Treatment of a Pre-Eclampsia Using an Agent of the Invention
[0342] A patient with pre-eclampsia is administered 1 mg of an
agent of the invention per day intramuscularly or a depot
preparation delivering this dose according to the methods of the
invention.
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Numbered References Cited in Description
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Sequence CWU 1
1
93110PRTHomo sapiensSITE(1)...(10)Kisspeptin 1-10 sequence 1Tyr Asn
Trp Asn Ser Phe Gly Leu Arg Phe1 5 10 254PRTHomo
sapiensSITE(1)...(54)Kisspeptin 1-54 sequence 2Gly Thr Ser Leu Ser
Pro Pro Pro Glu Ser Ser Gly Ser Arg Gln Gln1 5 10 15 Pro Gly Leu
Ser Ala Pro His Ser Arg Gln Ile Pro Ala Pro Gln Gly 20 25 30 Ala
Val Leu Val Gln Arg Glu Lys Asp Leu Pro Asn Tyr Asn Trp Asn 35 40
45 Ser Phe Gly Leu Arg Phe 50 35PRTArtificial SequenceKisspeptin
variant sequence 3Xaa Gly Xaa Arg Xaa1 5 45PRTArtificial
SequenceKisspeptin variant sequence 4Phe Gly Leu Arg Phe1 5
55PRTArtificial SequenceKisspeptin variant sequence 5Phe Gly Leu
Arg Trp1 5 65PRTArtificial SequenceKisspeptin variant sequence 6Phe
Gly Phe Arg Phe1 5 75PRTArtificial SequenceKisspeptin variant
sequence 7Phe Gly Ala Arg Trp1 5 85PRTArtificial SequenceKisspeptin
variant sequence 8Phe Gly Leu Arg Trp1 5 95PRTArtificial
SequenceKisspeptin variant sequence 9Phe Gly Leu Arg Trp1 5
105PRTArtificial SequenceKisspeptin variant sequence 10Phe Gly Leu
Arg Trp1 5 115PRTArtificial SequenceKisspeptiin Variant Sequence
11Phe Trp Leu Arg Trp1 5 125PRTArtificial SequenceKisspeptiin
Variant Sequence 12Phe Gly Trp Arg Phe1 5 135PRTArtificial
SequenceKisspeptiin Variant Sequence 13Phe Gly Phe Arg Trp1 5
145PRTArtificial SequenceKisspeptiin Variant Sequence 14Phe Gly Leu
Arg Trp1 5 155PRTArtificial SequenceKisspeptiin Variant Sequence
15Phe Gly Leu Arg Trp1 5 165PRTArtificial SequenceKisspeptiin
Variant Sequence 16Phe Gly Ala Arg Trp1 5 175PRTArtificial
SequenceKisspeptiin Variant Sequence 17Ala Gly Leu Arg Trp1 5
185PRTArtificial SequenceKisspeptiin Variant Sequence 18Phe Trp Leu
Arg Trp1 5 195PRTArtificial SequenceKisspeptiin Variant Sequence
19Phe Gly Trp Arg Phe1 5 208PRTArtificial SequenceDrosophila
melanogaster 20Arg Arg Met Lys Trp Lys Lys Tyr1 5 2113PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 21Arg Arg Met Lys Trp Lys Lys Tyr Phe Gly Phe Arg Trp1 5
10 2213PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 22Arg Arg Met Lys Trp Lys Lys Tyr
Phe Gly Leu Arg Trp1 5 10 2313PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 23Arg Arg
Met Lys Trp Lys Lys Tyr Phe Gly Leu Arg Trp1 5 10 2413PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 24Arg Arg Met Lys Trp Lys Lys Tyr Phe Gly Ala Arg Trp1 5
10 2513PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 25Arg Arg Met Lys Trp Lys Lys Tyr
Ala Gly Leu Arg Trp1 5 10 2613PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 26Arg Arg
Met Lys Trp Lys Lys Tyr Phe Trp Leu Arg Trp1 5 10 2713PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 27Arg Arg Met Lys Trp Lys Lys Tyr Phe Gly Trp Arg Phe1 5
10 2849PRTHomo sapiensSITE(1)...(49)Kisspeptin 1-45 sequence 28Gly
Thr Ser Leu Ser Pro Pro Pro Glu Ser Ser Gly Ser Arg Gln Gln1 5 10
15 Pro Gly Leu Ser Ala Pro His Ser Arg Gln Ile Pro Ala Pro Gln Gly
20 25 30 Ala Val Leu Val Gln Arg Glu Lys Asp Leu Pro Asn Tyr Asn
Trp Asn 35 40 45 Ser2910PRTArtificial SequenceKisspeptin variant
sequence 29Xaa Xaa Xaa Asn Xaa Xaa Gly Xaa Arg Phe1 5 10
3010PRTArtificial SequenceKisspeptin variant sequence 30Tyr Asn Trp
Asn Ser Phe Gly Leu Arg Phe1 5 10 3110PRTArtificial
SequenceKisspeptin variant sequence 31Tyr Asn Trp Asn Ser Phe Gly
Trp Arg Phe1 5 10 3210PRTArtificial SequenceKisspeptin variant
sequence 32Tyr Asn Trp Asn Gly Phe Gly Trp Arg Phe1 5 10
3310PRTArtificial SequenceKisspeptin variant sequence 33Tyr Asn Trp
Asn Ser Phe Gly Trp Arg Phe1 5 10 3410PRTArtificial
SequenceKisspeptin variant sequence 34Tyr Asn Trp Asn Gly Phe Gly
Trp Arg Phe1 5 10 3510PRTArtificial SequenceKisspeptin variant
sequence 35Tyr Asn Trp Asn Gly Phe Gly Leu Arg Phe1 5 10
3610PRTArtificial SequenceKisspeptin variant sequence 36Tyr Asn Trp
Asn Gly Phe Gly Leu Arg Phe1 5 10 3710PRTArtificial
SequenceKisspeptin variant sequence 37Tyr Asn Trp Asn Gly Phe Gly
Trp Arg Phe1 5 10 3810PRTArtificial SequenceKisspeptin variant
sequence 38Tyr Asn Trp Asn Gly Trp Gly Leu Arg Phe1 5 10
3910PRTArtificial SequenceKisspeptin variant sequence 39Tyr Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 4010PRTArtificial
SequenceKisspeptin variant sequence 40Tyr Asn Trp Asn Trp Phe Gly
Trp Arg Phe1 5 10 4110PRTArtificial SequenceKisspeptin variant
sequence 41Tyr Asn Trp Asn Gly Phe Gly Trp Arg Phe1 5 10
4210PRTArtificial SequenceKisspeptin variant sequence 42Tyr Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 4310PRTArtificial
SequenceKisspeptin variant sequence 43Tyr Asn Trp Asn Gly Phe Gly
Trp Arg Phe1 5 10 4410PRTArtificial SequenceKisspeptin variant
sequence 44Tyr Asn Trp Asn Ala Phe Gly Trp Arg Phe1 5 10
4510PRTArtificial SequenceKisspeptin variant sequence 45Ala Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 4610PRTArtificial
SequenceKisspeptin variant sequence 46Tyr Asn Ala Asn Gly Phe Gly
Trp Arg Phe1 5 10 4710PRTArtificial SequenceKisspeptin variant
sequence 47Tyr Ala Trp Asn Gly Phe Gly Trp Arg Phe1 5 10
4810PRTArtificial SequenceKisspeptin variant sequence 48Trp Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 4910PRTArtificial
SequenceKisspeptin variant sequence 49Phe Asn Trp Asn Gly Phe Gly
Trp Arg Phe1 5 10 5010PRTArtificial SequenceKisspeptin variant
sequence 50Tyr Asn Trp Asn Gly Trp Gly Trp Arg Phe1 5 10
5110PRTArtificial SequenceKisspeptin variant sequence 51Ala Asn Trp
Asn Gly Trp Gly Trp Arg Phe1 5 10 5210PRTArtificial
SequenceKisspeptin variant sequence 52Ala Asn Trp Asn Ser Phe Gly
Trp Arg Phe1 5 10 5311PRTArtificial SequenceKisspeptin variant
sequence 53Asp Ala Asn Trp Asn Gly Phe Gly Trp Arg Phe1 5 10
5410PRTArtificial SequenceKisspeptin variant sequence 54Ala Asn Trp
Asn Ser Phe Gly Trp Arg Phe1 5 10 5510PRTArtificial
SequenceKisspeptin variant sequence 55Ala Asn Trp Asn Gly Phe Gly
Leu Arg Phe1 5 10 5618PRTArtificial SequenceKisspeptin variant
sequence with N terminal antennapedia sequence 56Arg Arg Met Lys
Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Phe Gly Leu1 5 10 15 Arg
Phe5718PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 57Arg Arg Met Lys Trp Lys Lys Tyr
Tyr Asn Trp Asn Gly Phe Gly Leu1 5 10 15 Arg Phe5818PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 58Arg Arg Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Phe
Gly Trp1 5 10 15 Arg Phe5918PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 59Arg Arg
Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Trp Gly Leu1 5 10 15
Arg Phe6018PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 60Arg Arg Met Lys Trp Lys Lys Tyr
Tyr Asn Trp Asn Gly Phe Gly Trp1 5 10 15 Arg Phe6118PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 61Arg Arg Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Trp Phe
Gly Trp1 5 10 15 Arg Phe6218PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 62Arg Arg
Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Phe Gly Trp1 5 10 15
Arg Phe6318PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 63Arg Arg Met Lys Trp Lys Lys Tyr
Tyr Asn Trp Asn Gly Phe Gly Trp1 5 10 15 Arg Phe6418PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 64Arg Arg Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Phe
Gly Trp1 5 10 15 Arg Phe6518PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 65Arg Arg
Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Ala Phe Gly Trp1 5 10 15
Arg Phe6618PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 66Arg Arg Met Lys Trp Lys Lys Tyr
Ala Asn Trp Asn Gly Phe Gly Trp1 5 10 15 Arg Phe6718PRTArtificial
SequenceAntennapedia N terminal sequence 67Arg Arg Met Lys Trp Lys
Lys Tyr Tyr Asn Ala Asn Gly Phe Gly Trp1 5 10 15 Arg
Phe6818PRTArtificial SequenceAntennapedia N terminal sequence 68Arg
Arg Met Lys Trp Lys Lys Tyr Tyr Ala Trp Asn Gly Phe Gly Trp1 5 10
15 Arg Phe6918PRTArtificial SequenceKisspeptin variant sequence
with N terminal antennapedia sequence 69Arg Arg Met Lys Trp Lys Lys
Tyr Trp Asn Trp Asn Gly Phe Gly Trp1 5 10 15 Arg
Phe7018PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 70Arg Arg Met Lys Trp Lys Lys Tyr
Phe Asn Trp Asn Gly Phe Gly Trp1 5 10 15 Arg Phe7118PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 71Arg Arg Met Lys Trp Lys Lys Tyr Tyr Asn Trp Asn Gly Trp
Gly Trp1 5 10 15 Arg Phe7218PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 72Arg Arg
Met Lys Trp Lys Lys Tyr Ala Asn Trp Asn Gly Trp Gly Trp1 5 10 15
Arg Phe7318PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 73Arg Arg Met Lys Trp Lys Lys Tyr
Ala Asn Trp Asn Ser Phe Gly Trp1 5 10 15 Arg Phe7418PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 74Arg Arg Met Lys Trp Lys Lys Tyr Ala Asn Trp Asn Gly Phe
Gly Trp1 5 10 15 Arg Phe7518PRTArtificial SequenceKisspeptin
variant sequence with N terminal antennapedia sequence 75Arg Arg
Met Lys Trp Lys Lys Tyr Ala Asn Trp Asn Ser Phe Gly Trp1 5 10 15
Arg Phe7618PRTArtificial SequenceKisspeptin variant sequence with N
terminal antennapedia sequence 76Arg Arg Met Lys Trp Lys Lys Tyr
Ala Asn Trp Asn Gly Phe Gly Leu1 5 10 15 Arg Phe7718PRTArtificial
SequenceKisspeptin variant sequence with N terminal antennapedia
sequence 77Arg Arg Met Lys Trp Lys Lys Tyr Ala Asn Trp Asn Gly Phe
Gly Trp1 5 10 15 Arg Phe7843PRTArtificial SequenceKisspeptin
sequence 78Gly Thr Ser Leu Ser Pro Pro Pro Glu Ser Ser Gly Ser Arg
Gln Gln1 5 10 15 Pro Gly Leu Ser Ala Pro His Ser Arg Gln Ile Pro
Ala Pro Gln Gly 20 25 30 Ala Val Leu Val Gln Arg Glu Lys Asp Leu
Pro 35 40 7910PRTArtificial SequenceConsensus peptide sequence
79Xaa Xaa Xaa Asn Gly Phe Gly Xaa Arg Phe1 5 10 806PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 80Ser Phe Gly
Leu Arg Phe1 5 816PRTArtificial SequenceArtificially synthesized
Kisspeptin analog 81Ser Phe Gly Leu Arg Trp1 5 8210PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 82Tyr Asn Trp
Asn Gly Phe Gly Leu Arg Phe1 5 10 8310PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 83Tyr Asn Trp
Asn Gly Leu Gly Leu Arg Phe1 5 10 8410PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 84Tyr Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 8510PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 85Tyr Asn Trp
Asn Ser Phe Gly Trp Arg Phe1 5 10 8610PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 86Tyr Asn Trp
Asn Ser Phe Gly Trp Arg Phe1 5 10 8710PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 87Tyr Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 8810PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 88Tyr Asn Trp
Asn Ser Phe Gly Trp Arg Phe1 5 10 8910PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 89Tyr Asn Trp
Asn Gly Phe Gly Trp Arg Phe1 5 10 9010PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 90Ala Asn Trp
Asn Pro Phe Gly Trp Arg Phe1 5 10 9110PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 91Ala Asn Trp
Asn Pro Phe Gly Trp Arg Phe1 5 10 9210PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 92Ala Asn Trp
Asn Ala Phe Gly Leu Arg Phe1 5 10 9310PRTArtificial
SequenceArtificially synthesized Kisspeptin analog 93Ala Asn Trp
Asn Gly Phe Gly Leu Arg Phe1 5 10
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