Title of Invention

PEPTIDIC GROWTH HORMONE SECRETAGOGUES ANALOG COMPOUND AND PREPARATION THEREOF

Abstract Chemical peptide compounds obtained by means of in silico molecular modelling whose structure enables them to perform the same functions as growth hormone peptide secretagogues. The invention also comprises the compositions that contain said compounds and use thereof in the preparation of medicinal products, nutritional supplements or other formulations for human or animal use.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“PEPTIDIC GROWTH HORMONE SECRETAGOGUES ANALOG
COMPOUNDS AND PREPARATIONS THEREOF”
CENTRO DE INGENIERIA GENÉTICA Y
BIOTECNOLOGÍA., of Ave. 31 entre 158 y 190, Cubanacán,
Playa, C. Habana 10600, Cuba
The following specification particularly describes the invention and the manner
in which it is to be performed.
1
PEPTIDIC GROWTH HORMONE SECRETAGOGUES ANALOG COMPOUNDS
AND PREPARATIONS THEREOF.
Technical Field
The present invention can be described in the field of rational design of biologically 5
active molecular entities regulating the metabolic activity and cytoprotection of the
organisms. More specifically on compounds analog to peptidic growth hormone
secretagogues whose activity includes but it is not restricted to: the controlled
release of growth hormone, cardioprotection, the increase of the functional response
of the cardiovascular system, neuroprotection, appetite regulation and control, fat 10
intake and energetic metabolism.
Previous art
The synthetic growth hormone (GH) secretagogues consist of a family of ligands
including peptidic and non peptidic molecules, being first described by Momany and 15
Bowers before the isolation of GH releasing hormone (GHRH) synthetic peptides of
6 and 7 amino acids resulting into potent GH releasing peptides (GHRPs); such
peptides were described prior to the knowledge of its function in the organism or
their way of action. Mutational studies and in vivo and in vitro experiments revealed
that the two amino acid arrangement L-D and D-L separated by one amino acid 20
acting as spacer, was considered optimal for the GH releasing activity, and
peptide(His-D-Trp-Ala-D-Trp-Phe-NH2) was conformed releasing GH at a
concentration of 10 to 30 ng/mL reaching to a peptide known as GHRP-6 (His-D-Trp-
Ala-Trp-D-Phe-Lys-NH2) where the Lys residue was needed only to improve the in
vivo activity because it was not deemed as functional in vitro (Momany F.A., Bowers 25
C.Y., et. al. (1981) Design, synthesis and biological activity of peptides which release
growth hormone, in vitro. Endocrinology, 108:31-39).
Other analog peptides were discovered; in 1993 Bowers et al. discovered two
GHRP-6 analog peptides, el GHRP-2 (D-Ala-D-ß-Nal-Ala-Trp-D-Phe-Lys-NH2) y el
GHRP-1 (Ala-His-D-ß-Nal-Ala-Trp-D-Phe-Lys-NH2). This three secretagogues 30
showed and increased GH release in vitro from incubated hypothalamus-pituitary
that from the pituitary gland alone, demonstrating that the hypothalamic impulse was
important in such action, also it was demonstrated, even in humans that the
synergistic action of GHRP and GHRH released more GH than any of the two by
2
itself (Bowers C.Y. (1993) GH-releasing peptides: structure and kinetics. J Pediatr
Endocrinol, 6(1):21-31).
From the peptide known as GHRP-2 new cyclic peptides were, by the change of Nterminal
D-Ala by an amino acid having the side chain linked to another amino acid
inserted between D-Phe and Lys. One of such peptides (D-Lys-D-ß-Nal-Ala-Trp-D- 5
Phe-Glu-Lys-NH2) resulted in to a 10 fold increased activity in vitro and a
comparable efficacy in vivo to GHRP-6 (McDowell R.S., et al. (1995) Growth
hormone secretagogues: characterization, efficacy, and minimal bioactive
conformation. PNAS USA, 92(24):11165-11169). The experiments were completed
with structural studies in solution of the DL cyclic peptides reaching to the conclusion 10
that the introduction of D amino acids in the peptidic compounds was essentially
needed to elicit the desired activity. Other investigations were directed to find active
molecules with an increased oral bioavailability and longer clearance times, yielding
the discoveries of new GHRPs and other non peptidic molecules. In 1993, the first
non peptidic GH secretagogue was described (Smith R.G., et al. (1993) A 15
nonpeptidyl growth hormone secretagogue. Science, 260:1640-43), and later is
referred the synthesis of a non peptidic and more potent GHS, MK-0677, having a
high bioavailability and able to stimulate GH secretion 24 h after a single dose oral
administration (Patchett A.A., Nargund R.P., et al. (1995) Design and biological
activities of L-163,191 (MK-0677): a potent, orally active growth hormone 20
secretagogue. PNAS USA, 92:7001-7005; Smith R.G., Van der Ploeg L.H., et al.
(1997) Peptidomimetic regulation of growth hormone secretion. Endocr. Rev,
18:621-645). More recently another peptidomimetic GHS was designed with a
selective and potent GH releasing activity (EP1572) showing a GH secretagogue
receptor (GHS-R) binding potency in human and animal tissues similar to that of 25
ghrelin and peptidic GHS inducing a marked increase in GH after the subcutaneous
administration to newborn rats (Broglio F., Boutignon F., et al. (2002) EP1572: a
novel peptido-mimetic GH secretagogue with potent and selective GH-releasing
activity in man. J Endocrinol Invest, 25:RC26-RC28).
In 1999 ghrelin was discovered as a 28 amino acid peptide produced mainly in the 30
stomach, however finding also its mARN in several other tissues. It is produced in
the stomach by the X/A cells which are the major population of endocrine cells in the
oxintic mucosa. Ghrelin is also found in the hypothalamic arcuate nucleus where its
RNA is present in NPY and AGRP neurons, involved in to the appetite control and
3
the energetic balance (Kojima M., Hosoda H., et al. (1999) Ghrelin is a growthhormone-
releasing acylated peptide from stomach. Nature, 402:656-60; Nakazato
M., Murakami N., et al. (2001) A role for ghrelin in the central regulation of feeding.
Nature, 409:194-198). Its RNA has been also localized in pancreas and intestine. It
circulates in the bloodstream of adult humans on a concentration of 100-120 fmol/ml, 5
suggesting that is secreted by the stomach cells and it may act by an endocrine
pathway. The production of ghrelin has been also reported in neoplastic tissues
(Takaya K., Ariyasu H., et al. (2000) Ghrelin strongly stimulates growth hormone
release in humans. J. Clin. Endocrinol. Metab, 85:4908-11; Papotti M., et al. (2001)
Substantial production of ghrelin by a human medullary thyroid carcinoma cell line. J 10
Clin Endoc. Metab, 86:4984–4990).
Other animal studies showed that the secretion of ghrelin is pulsatile and more
associated to appetite to the GH pulses (Tolle V., Bassant M.H., et al. (2002)
Ultradian rhythmicity of ghrelin secretion in relation with GH, feeding behaviour, and
sleep wake patterns in rats. Endocrinology, 143:1353-1361). 15
Ghrelin is the first natural hormone being found with a hydroxyl group of one of the
serines acylated with octanoic acid. This modification has being described as
essential for the binding to GHS-R1a, as well as for the GH releasing capacity, and
probably to other endocrine actions.
Non acylated ghrelin circulates in major amounts tan the acylated, although it was 20
not described a direct endocrine action it is regarded as acting in other nonendocrine
functions like cardiovascular effects, cardioprotective, antiproliferatives,
and cytoprotecting in general, probably mediated by the binding to other subtypes of
GHS-R (Matsumoto M., Hosoda H., et al. (2001) Structure-activity relationship of
ghrelin: pharmacological study of ghrelin peptides. Biochem Biophys Res Commun, 25
287:142-146; Hosoda H., Kojima M., et al. (2000) Ghrelin and des-acyl ghrelin: two
major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res
Commun, 279:909-913; Cassoni P., Papotti M., et al. (2001) Identification,
characterization, and biological activity of specific receptors for natural (ghrelin) and
synthetic growth hormone secretagogues and analogs in human breast carcinomas 30
and cell lines. J Clin Endocrinol Metab, 86:1738-1745).
There is another endogenous Ligand for GHS-R1a that can be isolated from the
stomach endocrine mucosa, des-Gln14-ghrelin as the result of an alternative
4
processing of the ghrelin gene losing Gln14 and as ghrelin it does experiment the
same acylation process on Ser3.
Studies made with several ghrelin analogs having the third residue modified with
several aliphatic or aromatic groups and several short peptides derived from the
ghrelin side chain showed that the hydrophobic groups in residue 3 are essential to 5
the activity. Also has been observed the short segments containing the first five
residues of ghrelin are capable of activating the receptor with a comparable
efficiency to the whole peptide. Tetra peptides were shown to be less potent and
fragments lacking the N-Terminal were unable to activate the receptor (Bednarek
M.A., Feighner S.D., et al. (2000) Structure-Function Studies on the New Growth 10
Hormone-Releasing Peptide, Ghrelin: Minimal Sequence of Ghrelin Necessary for
Activation of Growth Hormone Secretagogue Receptor 1a. J Med Chem, 43: 4370-
4376; Silva Elipe M.V., Bednarek M.A., et al. (2001) 1H NMR structural analysis of
human ghrelin and its six truncated analogs. Biopolymers, 59:489-501). Such
studies suggested that the complete ghrelin sequence is not essential for the activity 15
and Gly-Ser-Ser(n-octanoyl)-Phe is the active fragment in the activity as agonist of
GHS-R1a.
Before and after the discovery of ghrelin a great effort was made to find small
molecules and derivatives that can be ligands of the GHS-R an important number of
patents described molecules of such type (US Pats: US 3,239,345; 4,036,979; 20
4,411,890; 5,492,916; 5,494,919; 5,559,128; 5,663,171; 5,721,250; 5,721,251;
5,723,616; 5,726,319; 5,767,124; 5,798,337; 5,830,433; 5,919,777; 6,034,216;
6,548,501; 6,559,150; 6,576,686; 6,686,359; Intl Pats: WO 89/07110; 89/07111;
92/07578; 93/04081; 94/11012; 94/13696; 94/19367; 95/11029; 95/13069;
95/14666; 95/17422; 95/17423; 95/34311; 96/02530; 96/15148; 96/22996; 25
96/22997; 96/24580; 96/24587; 96/32943; 96/33189; 96/35713; 96/38471;
97/00894; 97/06803; 97/07117; 97/09060; 97/11697; 97/15191; 97/15573;
97/21730; 97/22004; 97/22367; 97/22620; 97/23508; 97/24369; 97/34604;
97/36873; 97/38709; 97/40023; 97/40071; 97/41878; 97/41879; 97/43278;
97/44042; 97/46252; 98/03473; 98/10653; 98/18815; 98/22124; 98/46569; 30
98/51687; 98/58947; 98/58948; 98/58949; 98/58950; 99/08697; 99/09991;
99/36431; 99/39730; 99/45029; 99/58501; 99/64456; 99/65486, 99/65488;
00/01726; 00/10975; 01/47558; 01/92292; 01/96300; 01/97831) (Carpino, P.
(2002) Recent developments in ghrelin receptor (GHS-. R1a) agonists and
5
antagonists Exp. Opin. Ther. Patents 12:1599-1618) After such extensive revision
other compounds has been described as antagonists of the GHS-R (US2005288316
y WO2005048916) and others described to also bind to GHS-R and used with
various purposes. (WO2005046682; WO2005039625; JP2003335752;
US2004009984; US2003130284; WO03004518) More recently a new series of 5
macro cyclic compounds was added to the set with the main purpose of being
agonists of GHS-R without eliciting the release of GH (US2006025566)
GHS-R is a class A G-coupled protein receptor, expressed by a single gene in the
chromosomal 3q26.2 locus in humans. Two types of cDNA were identified result of
the alternative processing of the pre-mRNA (McKee K.K., Tan C.P., et al. (1997) 10
Cloning and characterization of two human G protein-coupled receptor genes
(GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin
receptors. Genomics, 46:426-434; McKee K.K., Palyha O.C., et al. (1997) Molecular
analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors.
Mol Endocrinol, 11:415-423; US 6,242,199; WO 97/21730). cDNA 1a encode a 366 15
amino acid receptor with seven transmembrane segments (GHS-R1a). cDNA 1b
encodes a shorter protein (GHS-R1b) having 289 amino acids and five
transmembrane segments. Although the role of GHS-R1b is yet unknown, it has
been proved the expression in several endocrine and non-endocrine tissue (Howard
A.D., Feighner S.D., et al. (1996) A receptor in pituitary and hypothalamus that 20
functions in growth hormone release. Science, 273:974-977; Gnanapavan S., Kola
B., et al. (2002) The tissue distribution of the mRNA of ghrelin and subtypes of its
receptor, GHS-R, in humans. J Clin Endocrinol Metab. 87:2988; Smith R.G., Leonard
R., et al. (2001) Growth hormone secretagogue receptor family members and
ligands. Endocrine, 14:9-14). 25
The human GHS-R1a has a 96 and 93% identity with those of the rat and swine
respectively, and a close relation has been shown between the sequence of human
GHS-R1a and those of telosteous fish. Such findings suggest that GHS-R1a is highly
conserved between species and probably exert an essential biological function.
(Palyha O.C., Feighner S.D., et al. (2000) Ligand activation domain of human orphan 30
growth hormone (GH) secretagogue receptor (GHS-R) conserved from pufferfish to
humans. Mol Endocrinol. 14:160-169).
The binding of ghrelin and synthetic GHS to GHS-R1a activates the phospholipase C
signalling pathway, increasing the concentration of inositol-1,4,5 triphosphate (IP3),
6
and protein kinase C (PKS) activation, followed by the release of Ca 2+ from the
intracellular stores. The activation of GHS-R also inhibits the K+, channels allowing
the intake of Ca 2+ through L type voltage gated channels but not of type T.
Differently to GHS-R1a, GHS-R1b does not bind or responds to GHS and its function
is yet unknown. (Chen C., Wu D., et al. (1996) Signal transduction systems 5
employed by synthetic GH-releasing peptides in somatotrophs. J Endocrinol.
148:381-386; Casanueva F.F., Dieguez C. (1999) Neuroendocrine regulation and
actions of leptin. Front Neuroendocrinol, 20:317-363; Howard A.D., Feighner S.D., et
al. (1996) A receptor in pituitary and hypothalamus that functions in growth hormone
release. Science, 273:974-977). 10
Synthetic GHS, ghrelin and its natural isoform (des-Gln14-ghrelin) bind with high
affinity to GHS-R1a, and the efficiency on displacing membrane bound [35S] MK-
0677 or [125I] [Tyr4]ghrelin is correlated to the concentration required to stimulate GH
release (Muccioli G., Papotti M., et al. (2001) Binding of 125I-labeled ghrelin to
membranes from human hypothalamus and pituitary gland. J Endocrinol Invest. 15
24:RC7-RC9; Hosoda H., Kojima M., et al. (2000) Purification and characterization of
rat des-Gln14-ghrelin, a second endogenous ligand for the growth hormone
secretagogue receptor. J Biol Chem, 275:21995–22000).
To determine the essential structural characteristics of ghrelin for the binding and
activation of GHS-R1a, short ghrelin peptides were studies in HEK-293 cells, 20
expressing human GHS-R1a observing that 4 and 5 amino acid ghrelin N-terminal
peptides were able to activate the receptor. Based on this in vitro results it is
postulated that Gly-Ser-Ser(n-octanoyl)-Phe is essentially required for the activation
of the receptor (Van der Lely A.J., Tschop M., et al. (2004) Biological, Physiological,
Pathophysiological, and Pharmacological Aspects of Ghrelin. Endocrine Reviews, 25
25(3):426-457). The first 7 amino acid of ghrelin are conserved among all studied
species, however the ability of ghrelin derivatives to activate GHS-R1a in transfected
cells does not seems an indication for the capacity to stimulate GH release in
somatotroph cells, recently it was shown that (1-4) and (1-8) octanoyl ghrelin are not
able to stimulate the release of GH in rats and were not effective displacing [125I] 30
[Tyr4] ghrelin from the binding site in preparations of human pituitary or hypothalamic
membranes (Torsello A., Ghe C., et. al. (2002) Short ghrelin peptides neither
displace ghrelin binding in vitro nor stimulate GH release in vivo. Endocrinology,
143:1968–1971). Other study on the same cells expressing human or swine GHS7
R1a it was found that adenosine also activates the receptor, but like the short ghrelin
analogs can not stimulate the GH secretion, suggesting that adenosine is a partial
agonist of GHS-R1a bound to a different site in the receptor that MK-0677 or GHRP-
6 (Smith R.G., Griffin P.R., et. al. (2000) Adenosine: a partial agonist of the growth
hormone secretagogue receptor. Biochem Biophys Res Commun, 276:1306–1313). 5
More recently has been reported that GHS-R1a can also bind cortistatin (CST), a
somatostatin (SS) homolog neuropeptide not being able by itself to recognize GHSR1a
(Deghenghi R., Papotti M., et. al. (2001) Cortistatin, but not somatostatin, binds
to growth hormone secretagogue (GHS) receptors of human pituitary gland. J
Endocrinol Invest, 24:RC1–RC3). GHS-R1a is expressed in the arcuate nucleus and 10
pituitary somatotroph cells, crucial zones for neuroendocrine and appetite stimulation
activities of ghrelin and synthetic GHS. (Willesen M.G., Kristensen P., Romer J.
(1999) Co-localization of growth hormone secretagogue receptor and NPY mRNA in
the arcuate nucleus of the rat. Neuroendocrinology, 70:306–316; Bluet-Pajot M.T.,
Tolle V., et. al. (2001) Growth hormone secretagogues and hypothalamic networks. 15
Endocrine, 14:1–8; Shintani M., Ogawa Y., et. al. (2001) Ghrelin, an endogenous
growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin
action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway.
Diabetes, 50:227–232). Ghrelin and synthetic GHS, stimulate the expression of
neuronal activity markers (c-fos and EGR-1) in neurons of the arcuate nucleus an 20
GHS-R1a mRNA has been detected in extra hypothalamic areas like the dented
twist and regions CA2 and CA3 on the hippocampus, substance nigra pars
compacta, and ventral tegmental area, dorsal and medial Raphe nuclei, Edinger-
Westphal nucleus, bridge and spinal bulb, indicating possible extra hypothalamic
actions. mRNA has been also found on several peripheral organs like stomach, 25
intestine, pancreas, kidney, heart, aorta, several human adenomas and some human
lung, stomach and pancreas neoplasm. (Hewson A.K., Dickson S.L. (2000) Systemic
administration of ghrelin induces Fos and Egr-1 proteins in the hypothalamic arcuate
nucleus of fasted and fed rats. J Neuroendocrinol, 12:1047–1049; Muccioli G., Ghe
et. al. (1998) Specific receptors for synthetic GH secretagogues in the human brain 30
and pituitary gland. J Endocrinol, 157:99–106; Guan X.M., Yu H., et. al. (1997)
Distribution of mRNA encoding the growth hormone secretagogue receptor in brain
and peripheral tissues. Brain Res Mol Brain Res, 48:23–29;; Mori K., Yoshimoto et.
al. (2000) Kidney produces a novel acylated peptide, ghrelin. FEBS Lett, 486:213–
8
216; Nagaya N., Miyatake K., et. al. (2001) Hemodynamic, renal, and hormonal
effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol
Metab, 86:5854–5859;; Korbonits M., Bustin S.A., et. al. (2001) The expression of
the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal
human pituitary and other neuroendocrine tumours. J Clin Endocrinol Metab, 5
86:881–887; Papotti M., Cassoni P., et. al. (2001) Ghrelin-producing endocrine
tumors of the stomach and intestine. J Clin Endocrinol Metab, 86:5052–5059).
Ghrelin ad GHS have a high affinity to GHS-R1a. However there are evidences of
other additional sites for GHS. Specific sites for Tyr-Ala-hexarelin and other GHS
with a similar receptor density at least equal to the density found in pituitary has 10
been found in human and rat heart and many other non endocrine peripheral tissues
like: lungs, arteries, skeletal muscles, kidney, and liver (Muccioli G., Ghe C., et. al.
(1998) Specific receptors for synthetic GH secretagogues in the human brain and
pituitary gland. J Endocrinol, 157:99–106; Muccioli G., Broglio F., et. al. (2000)
Growth hormone-releasing peptides and the cardiovascular system. Ann Endocrinol 15
(Paris), 61:27–31; Bodart V., Bouchard J.F., et. al. (1999) Identification and
characterization of a new growth hormone-releasing peptide receptor in the heart.
Circ Res, 85:796–802; Katugampola S., Davenport A. (2003) Emerging roles for
orphan G protein-coupled receptors in the cardiovascular system. Trends Pharmacol
Sci, 24:30–35; Ghigo E., Arvat E., et. al. (2001) Biologic activities of growth hormone 20
secretagogues in humans. Endocrine, 14:87–93; Papotti M., Ghe C., Cassoni P., et.
al. (2000) Growth hormone secretagogue binding sites in peripheral human tissues.
J Clin Endocrinol Metab, 85:3803–3807). Such binding sites showed low affinity for
ghrelin and are probably not ghrelin receptors but peptide ghrelin analog receptors.
Heart GHS-R has a higher (84 kDa) molecular weight than GHS-R1a and no 25
sequence homology, the predicted amino acid sequence for the receptor in the heart
is similar to CD36 (Papotti M., Ghe C., et. al. (2000) Growth hormone secretagogue
binding sites in peripheral human tissues. J Clin Endocrinol Metab, 85:3803–3807;
Bodart V., Febbraio M., et. al. (2002) CD36 mediates the cardiovascular action of
growth hormone-releasing peptides in the heart. Circ Res, 90:844–849). The 30
functional meaning of peripheral tissue GHS receptors and findings in the
cardiovascular system suggest that such binding sites modulate the cardioprotective
activities of peptidic GHS.
9
Ghrelin an other synthetic secretagogues stimulate the release of GH by the
somatotroph cells in vitro probably by membrane depolarization and by the
increment of the GH secreted per cell, reporting also a stimulatory effect of GHS on
the GH synthesis.(Kojima M., Hosoda H., et. al. (1999) Ghrelin is a growth-hormonereleasing
acylated peptide from stomach. Nature, 402:656–660; Sartor O., Bowers 5
C.Y., Chang D. (1985) Parallel studies of His-DTrp-Ala-Trp-DPhe-Lys-NH2 and
human pancreatic growth hormone releasing factor-44-NH2 in rat primary pituitary
cell monolayer culture. Endocrinology, 116:952–957; Bowers C.Y., Sartor A.O., et.
al. (1991) On the actions of the growth hormone-releasing hexapeptide, GHRP.
Endocrinology, 128:2027-2035; Wu D., Chen C., et al. (1994) The effect of GH- 10
releasing peptide-2 (GHRP-2 or KP 102) on GH secretion from primary cultured
ovine pituitary cells can be abolished by a specific GH-releasing factor (GRF)
receptor antagonist. J Endocrinol, 140:R9-R13;).
Early studies showed GHS stimulating the GH secretion using a different receptor
and pathway the GHRH: An antagonist of the GHRH receptor inhibits the GHRH 15
elicited GH secretion, but not the release of GHRH stimulated by secretagogues and
an alleged GHS-R antagonist does not affect the GH release in response to GHRH,
GHRP-6 does not compete with GHRH in receptor binding assays for GHRH binding
sites, there is an additive effect on the GH release upon the co administration of
GHS and GHRH, and there is no crossed desensitation between GHRH and GHS in 20
terms of GH release. (Wu D., Chen C., et al. (1994) The effect of GH-releasing
peptide-2 (GHRP-2 or KP102) on GH secretion from primary cultured ovine pituitary
cells can be abolished by a specific GH-releasing factor (GRF) receptor antagonist. J
Endocrinol, 140:R9-13; Thorner M.O., Hartman M.L., et al. (1994) Current status of
therapy with growth hormone-releasing neuropeptides. Savage MO, Bourguignon J, 25
Grossman AB (eds). Frontiers in Paediatric Neuroendocrinology, 161-167).
The GH releasing activity of GHS is larger in pituitary-hypothalamus preparations tan
in isolated pituitary, in agreement with the evidence of the larger in vivo GH
stimulating effects. (Mazza E., Ghigo E., et. al. (1989) Effect of the potentiation of
cholinergic activity on the variability in individual GH response to GH-releasing 30
hormone. J Endocrinol Invest, 12:795–798; Bowers C.Y., Sartor A.O., et. al. (1991)
On the actions of the growth hormone-releasing hexapeptide, GHRP. Endocrinology,
128:2027–2035; Clark R.G., Carlsson M.S., et. al. (1989) The effects of a growth
10
hormone-releasing peptide and growth hormone releasing factor in conscious and
anaesthetized rats. J Neuroendocrinol, 1:249–255).
On the hypothalamic level, ghrelin and GHS act upon the GHRH secretor neurons
and incremented levels of GHRH has been observed in the pituitary portal circulation
after the administration of GHS in sheep. (Conley L.K., Teik J.A., et. al. (1995) 5
Mechanism of action of hexarelin and GHRP-6: analysis of the involvement of GHRH
and somatostatin in the rat. Neuroendocrinology, 61:44–50; Guillaume V., Magnan
E., et. al. (1994) Growth hormone (GH)-releasing hormone secretion is stimulated by
a new GH-releasing hexapeptide in sheep. Endocrinology, 135:1073–1076).
GHS requires of GHRH to fully express its GH releasing effect, in humans the GH 10
response is inhibited by GHRH receptor antagonists, and by pituitary-hypothalamic
disconnection. (Bluet-Pajot M.T., Tolle V., et al. (2001) Growth hormone
secretagogues and hypothalamic networks. Endocrine, 14:1–8; 148:371–380;
Popovic V., Miljic D., et al. (2003) Ghrelin main action on the regulation of growth
hormone release is exerted at hypothalamic level. J Clin Endocrinol Metab, 88:3450- 15
3453). Patients with a deficiency on the GHRH receptor do not show an increase in
GH secretion as a response to GHS stimulation but keep the capacity to increase the
cortisol, ACTH and PRL after the GHS stimulation. (Maheshwari H.G., Pezzoli S.S.,
et al. (2002) Pulsatile growth hormone secretion persists in genetic growth hormonereleasing
hormone resistance. Am J Physiol Endocrinol Metab, 282:E943-E951; 20
Maheshwari H.G., Rahim A., et al. (1999) Selective lack of growth hormone (GH)
response to the GH-releasing peptide hexarelin in patients with GH-releasing
hormone receptor deficiency. J Clin Endocrinol Metab, 84:956-959; Gondo R.G.,
Aguiar-Oliveira M.H., Hayashida C.Y., et al. (2001) Growth hormone-releasing
peptide-2 stimulates GH secretion in GH-deficient patients with mutated GH- 25
releasing hormone receptor. J Clin Endocrinol Metab, 86:3279-3283), In animals
and humans there is evidence of GHS and GHRH induced homologous but not
heterologous desensitation, GHS activity homologous desensitation has been shown
during GHS infusion, but not on the intermittent daily oral or nasal administration of
the peptide for more than 15 days. (Ghigo E., Arvat E., et al. (1994) Growth 30
hormone-releasing activity of hexarelin, a new synthetic hexapeptide, after
intravenous, subcutaneous, intranasal, and oral administration in man. J Clin
Endocrinol Metab, 78:693-698; Ghigo E., Arvat E., et al. (1996) Short-term
administration of intranasal or oral hexarelin, a synthetic hexapeptide, does not
11
desensitize the growth hormone responsiveness in human aging. Eur J Endocrinol,
135:407–412). On the other hand the parenteral, intranasal or oral administration of
GHS increases the GH spontaneous pulse and raises the IGF-1 levels in young
healthy adults, like in children and elderly subjects. (Chapman I.M., Bach M.A., et al.
(1996) Stimulation of the growth hormone (GH)-insulin-like growth factor I axis by 5
daily oral administration of a GH secretagogue (MK-677) in healthy elderly subjects.
J Clin Endocrinol Metab, 81:4249–4257; Copinschi G., Van Onderbergen A., et al.
(1996) Effects of a 7-day treatment with a novel, orally active, growth hormone (GH)
secretagogue, MK-0677, on 24-hour GH profiles, insulin-like growth factor I, and
adrenocortical function in normal young men. J Clin Endocrinol Metab, 81:2776– 10
2782; Laron Z., Frenkel J., et al. (1995) Intranasal administration of the GHRP
hexarelin accelerates growth in short children. Clin Endocrinol (Oxf), 43:631–635).
Ghrelin is capable of stimulating the appetite in rats and this property could be
mediates by the syntheses of NPY and AGRP. Intraventricular ghrelin is also
capable of nullifying the anorexigenic effects of leptin, and is postulated that there is 15
a competitive interaction between this two peptides on appetite and energy
homeostasis control. The circulating concentrations of ghrelin in the rat are
augmented upon fasting and are smaller after feeding or glucose ingestion (Shintani
M., Ogawa Y., et al. (2001) Ghrelin, an endogenous growth hormone secretagogue,
is a novel orexigenic peptide that antagonizes leptin action through the activation of 20
hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes, 50:227-32; Nakazato
M., Murakami N., et al. (2001) A role for ghrelin in the central regulation of feeding.
Nature, 409(6817):194-198; Tschöp M., Smiley D.L., Heiman M.L. (2000) Ghrelin
induces adiposity in rodents. Nature, 407:908-13).
GHS also stimulate appetite and weight gain. Chronic treatment with GHRP-2 25
stimulate the accumulation of adipose tissue in NPY deficient mice and increase the
hypothalamic expression of AGRP mRNA in the controls (Torsello, A., Luoni, M., et
al. (1998) Novel hexarelin analogs stimulate feeding in the rat through a mechanism
not involving growth hormone release. Eur. J. Pharmacol, 360:123-129; Ghigo, E.,
Arvat, E., et al. (1999) Endocrine and non-endocrine activities of growth hormone 30
secretagogues in humans. Horm. Res, 51:9-15; Tschop, M., Statnick, et al. (2002)
GH-releasing peptide-2 increases fat mass in mice lacking NPY: indication for a
crucial mediating role of hypothalamic agouti-related protein. Endocrinology,
143:558–568).
12
Ghrelin administration to rats yield gain in appetite and weight by a significative
increment on the fat tissue without observing changes in the lean mass, bone tissue
or growth stimulation. The lipogenic effect of ghrelin is independent of the GH action,
and it can be found in a genetically GH deficient rat. GH elicits an increment in the
energy expenditure and causes fat elimination, allowing for a balance with ghrelin, 5
ghrelin increases the fat tissue and GH does not allow a decrease in the lean tissue.
(Nakazato M., Murakami N., et al. (2001) A role for ghrelin in the central regulation of
feeding. Nature, 409(6817):194-198; Wren A.M., Small C.J., et al. (2000) The novel
hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion.
Endocrinology, 141(11):4325-4328; Tschop M., Smiley D.L., Heiman M.L. (2000) 10
Ghrelin induces adiposity in rodents. Nature, 407:980-913).
In obese individuals ghrelin levels are depleted and does not decrease after feeding,
this is a reversible condition, because weight loss and ghrelin mean plasma levels
are incremented. Plasma levels of ghrelin negatively correlate to the body weight
index, the body fat weight, the size of the adiposities and the plasma levels of insulin, 15
glucose and leptin (English P.J., Ghatei M.A., et. al. (2002) Food fails to suppress
ghrelin levels in obese humans. J Clin Endocrinol Metab, 87(6):2984; Tschop M.,
Weyer C., et. al. (2001) Circulating ghrelin levels are decreased in human obesity.
Diabetes, 50(4):707-9).
GH insufficiency in obese patients has been reported as reversible after a prolonged 20
diet and a marked weight loss. The chronic increase on the free fatty acids and
hyperinsulinism associated with the low ghrelin levels may have an important role
causing GH insufficiency in obesity. (Maccario M., Tassone F., Grottoli S., Rossetto
R., Gauna C., Ghigo E. (2002) Neuroendocrine and metabolic determinants of the
adaptation of GH/IGF-I axis to obesity. Ann Endocrinol (Paris), 63(2 Pt 1):140-144). 25
As ghrelin is being found adipogenic and orexigenic it can be thought on
antagonizing it for obesity treatment, however the consequences of such antagonism
lower the GH secretion and are associated with the fat mass increase. (Jorgensen
J.O., Vahl N., (1996) Influence of growth hormone and androgens on body
composition in adults. Horm Res, 45:94-98). Long term administration of agonists or 30
antagonists of ghrelin will reveal which of the two effects dominates and determine
its influence on the energy balance.
In the obese man the circulating concentrations of ghrelin are diminished and
negatively correlated to the body fat tissue and the circulating levels of insulin and
13
leptin (Tschöp M., Weyer C., et. al. (2001) Circulating ghrelin levels are decreased in
human obesity. Diabetes, 50:707-9).
GH/IGF-I axis has a very important role during cardiac development and for the
maintenance of the structure and function or the heart; deterioration on the
cardiovascular performance is one of the symptoms of GH deficiency that can be 5
reverted with a GH therapy. (Sacca L, Cittadini A, Fazio S (1994) Growth hormone
and the heart. Endocr Rev 15:555–573; Caidahl K, Eden S, Bengtsson BÅ 1994
Cardiovascular and renal effects of growth hormone. Clin Endocrinol (Oxf) 40:393–
400).
There are experimental data showing an improvement on the performance of the 10
cardiac muscle due to GH, among them many studies using a myocardial infarction
(MI) model in rats, GH treatment after MI resulted in an increment on the systolic
ejection volume, cardiac output and other systolic variables, along with a pronounced
vasodilation and a lower total peripheral resistance due to GH/IGF-I, probably
contributing to improve the myocardial contractility. (Timsit J, Riou B, et al. 1990 15
Effects of chronic growth hormone hypersecretion on intrinsic contractility,
energetics, isomyosin pattern and myosin adenosine triphosphate activity of rat left
ventricle. J Clin Invest 86:507–515; Tajima M, et al. (1999) Treatment with growth
hormone enhances contractile reserve and intracellular calcium transients in
myocytes from rats with post infarction heart failure. Circulation 99:127-134). 20
On the other hand, animal models with an excess of GH display a shift to a myosin
isoform with a low adenosine triphosphatase activity, they might lower the energy
demand on the contraction process. (Timsit J, Riou B, et al. (1990) Effects of chronic
growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin
pattern and myosin adenosine triphosphate activity of rat left ventricle. J Clin Invest 25
86:507–515).
There are several studies on the cardiac and peripheral effects of GH and/or IGF-I,
among them good clinical data pointing to a future role of GH/IGF-I on the
cardiovascular therapy. (Fazio S., Sabatini D., et al. (1996) A preliminary study of
growth hormone in the treatment of dilated cardiomyopathy. N Engl J Med, 334:809- 30
814).
Several synthetic GHS and ghrelin have cardioprotective properties in several in vivo
studies improving several cardiac function variables, having a comparable effect with
those of GH. The likelihood of hexarelin haemodinamic profile with those of GH
14
could suggest that GHS action is mediated by GH, recent studies however support a
direct action on the heart. (Locatelli V., Rossoni G., (1999) Growth Hormone
independent cardioprotective effects of hexarelin in the rat. Endocrinology,
140:4024-4031; Tivesten Å., Bollano E., (2000) The growth hormone secretagogue
hexarelin improves cardiac function in rats after experimental myocardial infarction. 5
Endocrinology, 141:60-66).
GHS-R1a mRNA has been found in aorta and heart, and it is also increased in
cardiomyocyte cultures after preincubation with hexarelin (Gnanapavan S., Kola B.,
et al. (2002) The tissue distribution of the mRNA of ghrelin and subtypes of its
receptor, GHS-R, in humans. J Clin Endocrinol Metab, 87: 2988-2991; Nagoya N., 10
Kojima M., et al. (2001) Hemodynamic and hormonal effects of human ghrelin in
healthy volunteers. Am J Physiol Regul Integr Comp Physiol, 280: R1483-R1487;
Pang J.-J., Xu R.-K., et al. (2004) Hexarelin protects rat cardiomyocytes from
angiotensin II-induced apoptosis in vitro. Am J Physiol Heart Circ Physiol, 286(3):
H1063-1069). 15
Specific ghrelin binding sites have been identified in rat heart and human arteries,
where the receptor density is increased by atherosclerosis and radioactively labelled
peptidic GHS were found specifically bound to rat myocardial cells and several
human cardiovascular tissues (ventricle, auricle, aorta, coronaries, carotid,
endocardium and vena cava), in higher amount than to the pituitary (Katugampola 20
S.D. (2001) [125I-His(9)]-ghrelin, a novel radioligand for localising GHS orphan
receptors in human and rat tissue: up-regulation of receptors with atherosclerosis. Br
J Pharmacol, 134:143-149; Ong H., McNicoll N., et al. (1998) Identification of a
pituitary growth hormone-releasing peptide (GHRP) receptor subtype by photo
affinity labeling. Endocrinology, 139:432– 435; Bodart V., McNicoll N., et al. (1999) 25
Identification and characterization of a new GHRP receptor in the heart. Circ Res,
85:796-808; Papotti M., Ghe C., et al. (2000) Growth hormone secretagogue binding
site in periferical human tissues. J Clin Endocrinol Metab, 85: 3803-3807).
Even though the administration of high pharmacological doses of peptidic GHS
induce a clear but transient vasoconstriction in perfunded rat heart using young rats 30
with induced GH deficiency by immunization with GHRH, has been also found that
hexarelin can protect against the myocardial damage induced by ischemia ad
reperfusion, such protective activity has been associated to prostacyclin release and
the recovery of Angiotensin II vasopressor activity. (Bodart V., Febbario M., et al.
15
(2000) CD36 mediates the cardiovascular action of growth hormone-releasing
peptides in the heart. Circ Res, 90:844-849; de Gennaro Colonna V., Rossoni G., et
al. (1997) Hexarelin, a growth hormone-releasing peptide, discloses protectant
activity against cardiovascular damage in rats with isolated growth hormone
deficiency. Cardiologia, 42:1165-1172; de Gennaro Colonna V., et al. (1997) Cardiac 5
ischemia and impairment of vascular endothelium function in hearts from growth
hormone-deficient rats: protection by hexarelin. Eur J Pharmacol, 334:201-207).
Similar results are obtained in aged rats in which treatment with hexarelin resulted in
a strong protection against the post ischemic ventricular dysfunction. Complete
recovery of the cardiac function was observed in the reperfusion and the 10
simultaneous reduction in the creatine kinase levels corroborated the integrity of the
heart membranes and the preservation of the contractile weakness following the
oxygen readmission. The hexarelin protective effect was also shown by the
production of 6-keto-PGF1a and the restoration of the coronary vascular reactivity to
Angiotensin II (Rossoni G., de Gennaro Colonna V., et al. (1998) Protectant activity 15
of hexarelin or growth hormone against post ischemic ventricular dysfunction in
hearts from aged rats. J Cardiovasc Pharmacol, 32:260-265; Rossoni G., de
Gennaro Colonna V., et al. (1998) Protectant activity of hexarelin or growth hormone
against post ischemic ventricular dysfunction in hearts from aged rats. J Cardiovasc
Pharmacol, 32:260-265; Locatelli V., Rossoni G., et al. (1999) Growth hormone- 20
independent cardioprotective effects of hexarelin in the rat. Endocrinology,
140:4024-4031). Studies in hypophysectomised rats showed the GHS
cardioprotective effects independents of GH and mediated by specific myocardial
receptors (Locatelli V., Rossoni G., et. al. (1999) Growth hormone-independent
cardioprotective effects of hexarelin in the rat. Endocrinology, 140:4024-4031; Bodart 25
V., McNicoll N., et al. (1999) Identification and characterization of a new GHRP
receptor in the heart. Circ Res, 85:796-808).
Hexarelin increases the systolic ejection volume and cardiac output, and reduces the
total peripheral resistance in a 4 week rat model after the myocardial infarction
induction. Although the mechanism of the synthetic GHS inotropic activity is not 30
clear, there are evidence of the increase in the papillary muscle contractility by action
on the endothelial cells or in the nerve endings (Tivesten A., Bollano et al. (2000)
.The growth hormone secretagogue Hexarelin improve cardiac function in rats after
experimental myocardial infarction. Endocrinology, 141:60-66; Bedendi I., Gallo
16
M.P., et al. (2001) Role of endothelial cells in modulation of contractility induced by
hexarelin in rat ventricle. Life Sci, 69:2189-2201)
Ghrelin does not share all the cardiovascular actions of the synthetic GHS, ghrelin
gives a poor protection to the heart suggesting that the synthetic GHS effects are
due to the binding and activation of GHS specific sites studies with [125I]Tyr-Ala- 5
hexarelin revealed many binding sites in rat myocardium and in human
cardiovascular tissues distinct of GHSR-1a, suggesting the existence of another
receptor, with a similar sequence to CD36 mediating the coronary actions of
synthetic GHS (Torsello A., Bresciani E., et al. (2003) Ghrelin plays a minor role in
the physiological control of cardiac function in the rat. Endocrinology, 144:1787– 10
1792; Muccioli G., Broglio F., et al. (2000) Growth hormone-releasing peptides and
the cardiovascular system. Ann Endocrinol (Paris) 61:27–31; Bodart V., Febbraio M.,
et al. (2002) CD36 mediates the cardiovascular action of growth hormone-releasing
peptides in the heart. Circ Res, 90:844–849). Although ghrelin is mostly inactive at a
coronary level, it does present other cardiovascular effects, Ghrelin has a very potent 15
in vivo and in vitro vasodilating effect, such ghrelin action is directed towards the non
striated muscles with potency comparable to the natriuretic peptides. In human
atherosclerosis patients ghrelin receptors are augmented suggesting that it plays a
role on the compensation for the vasoconstriction increment observed in such
condition. (Okumura H., Nagaya N., et al. (2002) Vasodilatory effect of ghrelin, an 20
endogenous peptide from the stomach. J Cardiovasc Pharmacol, 39:779-783; Wiley
K.E., Davenport A.P. (2002) Comparison of vasodilators in human internal mammary
artery: ghrelin is a potent physiological antagonist of endothelin-1. Br. J. Pharmacol,
136:1146-1152; Katugampola S.D. (2001) [125I-His(9)]-ghrelin, a novel radioligand
for localising GHS orphan receptors in human and rat tissue: up-regulation of 25
receptors with atherosclerosis. Br J Pharmacol, 134:143-149).
Other studies showed that hexarelin, acylated ghrelin and even ghrelin can prevent
doxorubicin induced cell death of H9c2 cardiomyocytes and endothelial cells,
probably stimulating intracellular signalling like the activation of
ERK1/2 and PI 3-kinase/AKT (Baldanzi G., Filigheddu N., et. al. (2002) Ghrelin and 30
des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through
ERK1/2 and PI 3-kinase/AKT. J Cell Biol, 159:1029-1037; Filigheddu N., Fubini A., et
al. (2001) Hexarelin protects H9c2 cardiomyocytes from doxorubicin-induced cell
death. Endocrine, 14:113–119).
17
In vivo studies on cardiomyocytes and endothelial cells suggests that the
antiapoptotic effects of GHS are mediated by the activation of ERK and AKT and by
the inhibition of the activation of caspase 3 and BAX expression increasing the
expression of BCL-2 (Pang J.J., Xu R.K., et al. (2004) Hexarelin protects rat
cardiomyocytes from angiotensin II-induced apoptosis in vitro. Am J Physiol Heart 5
Circ Physiol, 286:H1063-H1069). Such data enforces the hypothesis of the existence
of another GHS-R subtype, because non acylated ghrelin does not activate GHSR1a.
Ghrelin and GHS have indeed cardiovascular activity in humans, its administration to
healthy volunteers and patients with chronic cardiac failure reduced the systemic 10
vascular resistance and increase the cardiac output and the systolic ejection volume,
with a reduction of the mean arterial pressure but not showing any change in the
heart rate, pressure on the medial pulmonary artery or capillary pulmonary pressure.
(Nagaya N., Kojima M., et al. (2001) Hemodynamic and hormonal effects of human
ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol, 280:R1483– 15
R1487; Enomoto M., Nagaya N., et al. (2003) Cardiovascular and hormonal effects
of subcutaneous administration of ghrelin, a novel growth hormone-releasing
peptide, in healthy humans. Clin Sci (Lond), 105:431–435)
It has also been observed that several trophic factors, including GH and IGF-I have
neuroprotecting properties during the second phase of in vivo hypoxic ischemia (HI) 20
and has been shown that the activation of the PI3K pathway with AKT
phosphorylation is the mediator of the neuronal survival rate in vitro induced by
growth factors, phosphorylated AKT promoted cell survival and can inhibit apoptosis
by inactivation of several antiapoptotic targets like Bad, glycogen synthase 3 beta
(GSK3ß), caspase 9 or transcriptional factor modification. (Kulik G., Klippel A., 25
Weber M.J. (1997). Antiapoptotic signalling by the insulin-like growth factor I
receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol, 17:1595-1606)
Another pathway activated by growth factors is MAPK p42/44 ERK. ERK activation
has been found to inhibit the hypoxia induced apoptosis, besides the neuroprotection
BDNF in neonatal rats has shown to be mediated by the activation of MAPK/ERK 30
and treatment IGF-I after HI activates Akt and EKR (Buckley S., Driscoll B., et al.
(1999) ERK activation protects against DNA damage and apoptosis in hyperoxic rat
AEC2. Am J Physiol, 277:159-166; Han B.H., Holtzman D.M. (2000) BDNF protects
18
the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J
Neurosci, 20:5775-5781)
Hexarelin reduces the brain damage on an in vivo model of HI. This protection is
related to AKT and GSK3ß phosphorylation indicating the possibility of the
involvement of PI3K pathway, observing its protective effect to cortex, hippocampus 5
thalamus, but not in the striatum, spatial distribution of the protection is correlated to
the localization of GH receptor and hexarelin (Brywe K.G., Leverin A.-L., et al. (2005)
Growth Hormone Releasing Peptide Hexarelin reduces neonatal brain injury and
alters Akt/Glycogen Synthase Kinase-3ß phosphorylation. Endocrinology, 146: 4665-
4672; Lobie P.E., García-Aragón J., et al. (1993) Localization and ontogeny of 10
growth hormone receptor gene expression in the central nervous system. Dev Brain
Res, 74:225-233; Scheepens A., Sirimanne E.S., et al. (2001) Growth hormone as a
neuronal rescue factor during recovery from CNS injury. Neuroscience, 104:677-
687). Such findings suggest that the hexarelin protective effect could be GH
mediated or GH and hexarelin share common pathways for the cell protection 15
because GHS-R mRNA has been found in several of the brain structures.
Administration of GHRP-6 to adult rats under physiological conditions showed an
increase on the levels of IGF-I in hypothalamus, cerebellum, hippocampus but not in
cortex, although this could be to the increase of the IGF-I expression, the same
effect has not been found in hexarelin treated rats 24 hours after HI, on the other 20
hand if IGF-I was an important mediator of hexarelin effects it could be also expected
a reduction of the brain damage in the striatum, as IGF-I receptors are present there.
(Frago L.M., Paneda C., Dickson S.L., et al. (2002) Growth hormone (GH) and GHreleasing
peptide-6 increase brain insulin-like growth factor-I expression and activate
intracellular signalling pathways involved in neuroprotection. Endocrinology, 25
143:4113-4122; Guan J., Williams C., et al. (1993) The effects of IGF-1 treatment
after hypoxic-ischemic brain injury in adult rats. J Cereb Blood Flow Metab, 13:609-
616). Hexarelin also activates PI3K pathway in the Central Nervous System (CNS)
after HI but it does not affects ERK phosphorylation, IGF-I in contrast activates both
ERK and PI3K pathways. 30
Hexarelin increase the phosphorylation of the IGF-I receptor in the absence of an
obvious induction of IGF-I, the phosphorylation increase could be due to a receptor
transactivation by hexarelin or an endogenous ligand. Previously has been reported
that GPCR agonists like angiotensin-II, thrombin and endothelin can stimulate the
19
IGF-I and/or AKT (Sumitomo M., Milowsky M.I., et al. (2001) Neutral endopeptidase
inhibits neuropeptide-mediated transactivation of the insulin-like growth factor
receptor-Akt cell survival pathway. Cancer Res, 61:3294-3298; Zahradka P., Litchie
B., et al. (2004) Transactivation of the insulin-like growth factor-I receptor by
angiotensin II mediates downstream signalling from the angiotensin II type 1 receptor 5
to phosphatidylinositol 3-kinase. Endocrinology, 145:2978-2987).
The neuroprotective effect of hexarelin does not seem to be mediated primarily by an
induction of the GH/IGF-I axis, though a increased signalling on the IGF-I receptor
could contribute to the reduction of the brain damage.
10
Detailed description of the invention
In spite of the vast work on this field, described in the state of the art, it is evident
however that all ghrelin mimetic compounds and those of non peptidic nature are not
capable of exerting all possible functions attributed to ghrelin in the organism, being
preferred the usage of compounds of peptidic nature, having a larger structural 15
similarity, the description of such peptidic analogs is constrained however to the use
of non natural D stereochemistry amino acids as part of the compositions.
Taking into account the importance of the peptidic secretagogues in the previously
described functions and the capacity of such compounds on the endocrine and non
endocrine functions in a large variety of organisms, systems and cells, the present 20
invention describe, in effect for the first time chemical molecules of a peptidic nature,
with internal cycles and composed solely of amino acids with an L stereochemistry
for the chiral carbon, capable of exerting due to their chemical structure, similar
functions of those attributed to ghrelin, des-acyl ghrelin and other peptidic GHS,
including but not restricted to the GH releasing capacity, cardioprotection and in 25
general functional improving of the cardiac muscle and the reticuloendothelial
system, neuroprotection that does not only includes the brain but all the nervous
system cells, and the control and regulation of appetite including the regulation of fat
and energy metabolism.
The peptidic chemical compounds described in the invention have an structure 30
allowing them to fulfil the requirements to bind the ghrelin specific receptors and at
the same time the receptors described for the binding of other secretagogues
performing all of the aforementioned functions
20
In a particular realization, the invention refers to chemical molecules having the
following structure:
I. [Aa1...Aan] X1 [Ab1...Abn ] X2 [Ac1 … Acn ] Adn
Were Aa are L-amino acids selected from the set of [Cys, Gly, Ser, His, Ala, Leu,
Met o Thr], varying in combinations of 1 to 4 residues, Ab are L-amino acids, 5
selected from the set of [Pro, Ile, Ala, Phe, Trp, Lys, Asp, Asn, Glu, Gln, Gly, Leu,
Met, Tyr o Thr], varying in combinations of 1 to 4 residues, Ac are L-amino acids
selected from the set of [Arg, Leu, Pro, Val, Thr, Glu, His, Gln, Asn, Asp, Trp, Tyr,
Phe, Ser, Ala , Gly o Ile], varying in combination of 1 to 5 and Ad are L-amino acids,
natural or not without limit in number, X1 y X2 are L-amino acids, natural o not, with 10
the side chains covalently bound forming an internal cycle, using any chemical
reaction for the direct link or using a binding compound as a bridge.
Compounds belonging to the structural classes are shown as follows:
A221 GSKFDSPEHQ (SEC. ID NO: 1)
A222 HGSKFDLEFG (SEC. ID NO: 2) 15
A223 HCKFDLDWH (SEC. ID NO: 3)
A224 SSDFKLYWG (SEC. ID NO: 4)
A225 ALDFKPNIP (SEC. ID NO: 5)
A226 STDFKPFAI (SEC. ID NO: 6)
A227 HSKGYDLDH (SEC. ID NO: 7) 20
A228 GKFGDLSPEHQ (SEC. ID NO: 8)
A229 HAKPGGIDPEQ (SEC. ID NO: 9)
A230 GKFDSPEHQ (SEC. ID NO: 10)
A231 GGGKFWDIPHH (SEC. ID NO: 11)
A232 HKGIDSPEQH (SEC. ID NO: 12) 25
A233 GKFDLSPEHQ (SEC. ID NO: 13)
A234 GDAGAKLLSSR (SEC. ID NO: 14)
A235 GMEAGIKLCHRQ (SEC. ID NO: 15)
A236 GEGYKLDERSQ (SEC. ID NO: 16)
A237 GGEAGKLCPPRY (SEC. ID NO: 17) 30
A238 GLEFKLLHQ (SEC. ID NO: 18)
Were the underlined amino acids are linked by the side chains.
21
The aforementioned molecules were described for the function by the exhaustive
molecular modelling of the human ghrelin receptor using combined techniques of
homology modelling, molecular dynamics and exhaustive conformational search
techniques.
Once the receptor was modelled binding models were built based in the modelling of 5
ghrelin and other secretagogues, based upon the receptor-ligand interactions a
virtual library was built with several thousand of structures having such
characteristics to perform a conformational analysis, and a massive docking
experiment was performed against the receptor model.
Based on this analysis a series of compounds were selected representing several 10
structural families that were chemically synthesized and tested with several in vivo
and in vitro systems, after the biological assays the compounds were reoptimized
and new libraries were generated and the structural analysis was repeated to seek
for a larger action on the biological systems, having more specific structural
regularities. 15
The invention also includes any homolog variant of the aforementioned compounds.
Being understood as “homolog variant” any molecule of chemical nature similar in
70% or more of the amino acid sequence to those described in this invention (page
21), including non-natural amino acids, with a structure allowing it to perform the
same effect of the hereby described compounds. 20
In another preferred realization of the invention, the pharmaceutical composition
contains one or more of the described compounds or its allowed salts, along with
acceptable additive or vehicles for the application purpose. Also it is part of the
present invention, the use of the compounds for the manufacturing of medicines,
nutritional supplements, or other formulations of human or animal use in aquaculture 25
or other breeding or animal improvement activities, in vivo, in vitro, in body
associated devices or in devices for controlled release to the medium, associated to
the action similar to other GHS, directly related or not to their endocrine action.
The molecules described herein were defined by the capacity of interacting to the
human ghrelin receptor, but we can not rule out another proteins not having similar 30
structure or amino acid sequence but have the capacity to bind this type of
compound and affect in any way their biological action being by activation,
potentiation, repression, competition or synergism with other substrates, or by any
mechanism, described or not but experimentally documented.
22
For the definition of the chemical compounds described in the invention, the
molecular modelling of the human ghrelin receptor was performed, using combined
techniques of homology modelling, molecular dynamics and exhaustive
conformational search techniques. Once the receptor was modelled binding models
were built based in the modelling of ghrelin and other secretagogues, based upon 5
the receptor-ligand interactions a virtual library was built with several thousand of
structures having such characteristics to perform a conformational analysis, and a
massive docking experiment was performed against the receptor model.
Based on this analysis a series of compounds were selected representing several
structural families that were chemically synthesized and tested with several in vivo 10
and in vitro systems, after the biological assays the compounds were reoptimized
and new libraries were generated and the structural analysis was repeated with
another round of molecular docking with the receptor to extract structural regularities,
the chemical nature of the second round was optimized to reach higher values of
calculated binding energy, ranging between -58 and -32 KJ/mol and analyzed again 15
to look for a larger action on the biological systems, having more specific structural
regularities. A representative selection of 18 such compounds with binding energies
better than -40 KJ/mol, were synthesized, purified using High Performance Liquid
Chromatography, analyzed by Mass Spectrometry and evaluated for the in vivo and
in vitro effectiveness. 20
Description of the figures
Figure 1: Effects of the treatment with compounds A221(a), A228(b) y A233(c) in
the prevention of Doxorubicin (Dx) induced myocardial failure.
Figure 2: Protective effect of compounds A221(a), A228(b) y A233(c) on forced 25
stress in Dx treated rats.
Figure 3: Effect of the treatment with compounds A221(a), A228(b) y A233(c) in time
and reversion of Doxorubicin induced dilated cardiomyopathy in treated groups with
doses ranging from 100 to 500 µg/kg of animal weight.
Figure 4: Effect of the treatment with compounds A221(a), A228(b) y A233(c) in the 30
survival of animals with doxorubicin (Dx) induced dilated cardiomyopathy.
23
Examples
The present invention is explained in the following examples:
Example 1: Selection of the compounds by in silico molecular modelling.
The compounds obtained in the second cycle of the computational evaluation as
described above were optimized to obtain better energy values and more specific 5
regularities upon receptor binding, 18 representative compounds with energies better
than -40 KJ/mol were selected as shown in table 1.
Table 1. Calculated interaction energy of the interaction with the Growth
Hormone Secretagogue Receptor model after molecular docking.
Compound Energy (KJ/mol) Compound Energy(KJ/mol)
A221 -52.54 A230 -56.27
A222 -49.80 A231 -42.32
A223 -43.76 A232 -50.30
A224 -42.93 A233 -58.06
A225 -54.99 A234 -53.14
A226 -40.00 A235 -45.94
A227 -41.01 A236 -45.20
A228 -40.93 A237 -50.01
A229 -52.25 A238 -51.11
10
Example 2: Prevention of NGF deprivation induced death on PC12 cells.
PC12 cells were stored in 75 cm2 culture flasks on DMEM containing 5% bovine
foetal serum and 10% horse serum, with 50 µg/ml gentamicin. Cells were incubated
at 37°C in 5% CO2 .To induce differentiation cells were transferred at a density of
1X104 to polylysine covered 96 well plates in NGF supplemented DMEM media for 7 15
days, with medium replacement every 2-3 days. After differentiation cells were
incubated with peptidic GHS analog compounds, at different concentrations for 72 h.
Cell survival and proliferation was determined using the Promega non-radioactive
cytotoxicity proliferation assay, Cell Titer 96, based in the conversion of
3-(4,5-dimethyltiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in to a 20
spectrophotometrically detectable product. After deprivation of NGF medium is
removed and 15µl of the DMEM dissolved dye is added, after 4 hours of 37°C
24
incubation 100 µl of the stopping solution is added and absorbance is measured at
570 nm
The compounds showed a concentration dependent neuroprotection effect, IC50 for
each of the compounds is shown in Table 2.
Table 2. IC50 values of each compound during the NGF deprivation induced 5
neuronal death.
Compound IC50 uM Compound IC50 uM
A221 2.02 A230 4.06
A222 2.03 A231 4.00
A223 3.12 A232 4.89
A224 2.37 A233 5.00
A225 4.07 A234 5.86
A226 4.87 A235 2.05
A227 3.06 A236 3.00
A228 3.99 A237 3.33
A229 3.41 A238 2.04
Example 3: Prevention of the induced neuronal damage by Hydrogen Peroxide
addition to a primary culture of neurons.
Primary cultures of granular cerebellum cells were obtained from 7-9 days Wistar 10
rats. After a rapid dissection, rat cerebellums were submerged in a cold solution and
meningeal membranes were removed, each organ was transferred to a 2-3 ml fresh
medium solution and finely sliced. Cells were dissociated using a Pasteur pipette
and filtered through a nylon 40 mu.M membrane (Falcon, Franklin Lakes, N.J.). The
number of viable cells was determined by cell counting in a hematocytometer with 15
tripan blue as a marker. Cells were cultured on polylysine covered 96 well plates at a
density of 6250 cells in 200 ml final volume. Cultures were kept at 37°C in 5% . CO2
after 24h, 10 µM of cytosine arabinofuranose (AraC; Sigma) was added to inhibit the
proliferation of non-neuronal cells.
The capacity of neural damage prevention was tested adding 500 µM hydrogen 20
peroxide in different concentrations of peptidic GHS analog compounds, Cell
survival was determined using the Promega non-radioactive cytotoxicity proliferation
assay, Cell Titer 96 (Promega).
25
The compounds showed a concentration dependent neuroprotection effect, IC50 for
each of the compounds is shown in Table 3.
Table 3. IC50 values of each compound during the induced neuronal damage
by Hydrogen Peroxide addition to a primary culture of neurons.
Compound IC50 uM Compound IC50 uM
A221 1.80 A230 3.81
A222 1.30 A231 3.46
A223 2.47 A232 3.28
A224 3.20 A233 3.56
A225 3.99 A234 3.72
A226 3.58 A235 1.01
A227 2.26 A236 3.33
A228 1.77 A237 2.51
A229 1.33 A238 1.00
5
Example 4: Demonstration of the biological activity of the peptidic GHS analog
compounds in fish.
IGF-I mRNa was determined in the liver of intraperitoneally injected tilapias
monitoring also the GH level time course, showing the peptidic GHS analog
compounds as able to stimulate in fish the GH levels in the bloodstream and at 10
incrementing the IGF-I mRNA levels after the injection of the compounds as shown
in table 4.
Table 4. Normalized IGF-I mRNA levels to a non related synthetic peptide
control group.
Compound IGF1 Compound IGF1
A221 1.32 A230 1.48
A222 1.115 A231 1.39
A223 1.40 A232 1.23
A224 1.41 A233 1.69
A225 1.38 A234 1.17
A226 1.13 A235 0.9
A227 1.28 A236 1.13
26
A228 1.18 A237 1.201
A229 1.09 A238 1.24
Example 5: Experiment on juvenile tilapia treated with peptidic GHS analog
compounds:
5.1 Growth acceleration on tilapias treated intraperitoneally (ip) with peptidic
GHS analog compounds. 5
The compounds were dissolved in a sodium phosphate (PBS) buffer solution and
injected twice a week, during three weeks at 0.1 µg/g of humid fish weight
(gbw).Compounds were applied individually to a group of 10 male tilapias with an
average weight of 60.41 ± 10.36 g and a control group with an average weight of
60.58 ± 19.67 g was receiving PBS only as a control, measuring the average weight 10
every week, all animals in the experiment were labelled with microchips (Stoelting
Co. Wood Dale, USA.). for proper identification. A weight increase was obtained in
the treated group with 165% peak relative to the control group as shown in table 5.
Table 5. Weight increment in % for the treated group taking as 100% the 15
growing of the control group.
In the same experiment we have studied the presence of monogeneous Trichodinics
and Helmints on the animals used in the assay to observe and compare the
extension of the invasion of pathogenic agents in the treated group. Table 6 shows 20
the comparison with the non treated animals that showed six crosses as average.
Compound Weight inc. (%) Compound Weight Inc. (%)
A221 98.0 A230 158.0
A222 96.2 A231 150.2
A223 105.0 A232 160.1
A224 132.7 A233 165.0
A225 120.0 A234 110.6
A226 122.4 A235 89.9
A227 139.9 A236 99.0
A228 130.6 A237 100.0
A229 126.5 A238 129.4
27
Table 6. Intensity of the pathogenic infection with Trichodinics and Helmints in
treated animals.
5
5.2 Stimulation by immersion, of the growth of tilapia (Oreochromis sp) larvae
with the peptidic GHS analogs.
Growth stimulation experiments on tilapia Oreochromis sp. larvae were performed
evaluating groups of 100 larvae with 0.01g average, using the peptidic GHS analogs,
in a 100 µg/L concentration, twice a week using an immersion time of one hour. On a 10
three weeks course a top growth stimulation of 155% of average weight was
obtained as shown in table 7, relative to the control group that was receiving PBS
immersions.
Compound Pathogens Compound Pathogens
A221 +++++ A230 ++
A222 ++++ A231 ++
A223 ++++ A232 ++
A224 ++++ A233 ++
A225 ++++ A234 +++
A226 +++ A235 +++
A227 ++++ A236 ++++
A228 +++ A237 +++
A229 +++ A238 +++
28
Table 7. Weight increment in % for the treated group taking as 100% the
growing of the control group.
5
10
During this experiment lysozyme levels were also monitored and an increase of this
immunity marker was obtained in the treated animals as shown in table 8.
Table 8. Lysozyme levels of the treated animals relative to the control group. 15
Compound Weight Inc. (%) Compound Weight Inc. (%)
A221 97.0 A230 150.0
A222 96.0 A231 151.0
A223 102.0 A232 148.3
A224 130.0 A233 155.0
A225 98.0 A234 120.6
A226 120.4 A235 90.0
A227 140.6 A236 105.0
A228 132.0 A237 109.9
A229 125.0 A238 112.6
Compound Lysozyme Compound Lysozyme
A221 1.01 A230 1.33
A222 1.43 A231 1.89
A223 1.52 A232 2.41
A224 1.37 A233 2.68
A225 1.43 A234 2.77
A226 1.17 A235 1.90
A227 1.52 A236 1.42
A228 1.08 A237 1.01
A229 1.15 A238 1.33
29
Example 6: Growing of shrimps v Litopenaeus vanamei by dipping in a
solution of the peptidic GHS analogs.
Shrimp larvae were subjected two four dips, for one hour every three days with
different peptidic GHS analogs at 0.1g/L. The control group was subjected to the
same frequency of dipping with 1 mg/L BSA. 5
As a result it was observed that in the treated group the quality of the larvae was
improved with a 120-150% weight gain and 10-25% of size increase as shown in
table 9, showing also a larger number of gill ramifications and rostral modifications.
Besides it was found that n general in the treated group the animals had a lower
muscular water content and better values of RNA/DNA, Protein/DNA, showing the 10
higher activation of the metabolism in the treated larvae.
Table 9. Weight and size increment in % for the treated group taking as 100%
the growing of the control group.
15
This experiment was also performed in production conditions for compounds A221,
A228 y A233, with a 20% of higher survival compared with the controls, keeping a
stimulation of 110% on the weight and 30% in the size, showing on the treated
animals a better homogeneity on the size distribution with only a 30% and a 8% of
variation coefficient in weight and size respectively, on contras with a 77% and 30% 20
in the non treated group.
Compound
Weight Inc. (%)/
Size Inc. (%)
Compound
Weight Inc. (%)/
Size Inc. (%)
A221 120.1/112.0 A230 150.0/123.6
A222 121.0/112.2 A231 130.0/123.0
A223 120.0/110.9 A232 132.8/123.0
A224 127.0/116.0 A233 143.0/124.9
A225 121.0/112.6 A234 123.6/114.2
A226 120.1/112.2 A235 121.0/112.5
A227 128.6/118.5 A236 121.0/112.0
A228 128.2/118.9 A237 127.0/116.2
A229 126.1/115.9 A238 129.2/117.9
30
Example 7: Growth stimulation in shrimps by the dietary supplementation with
the peptidic GHS analogs.
The peptidic GHS analogs were included at 1% in a post-larvae crustacean diet.
Post-larvae of Litopenaeus vanamei were fed with the aforementioned diet in parallel
with a control group with 1% BSA addition. The effect was measured with an optical 5
micrometer and weighing the animals in a 0.1 mg precision scale.
The added compound produced a size increase of 30-40 % compared with the
control group as shown in table 10.
Table 10. Size increment in % for the treated group taking as 100% the growing 10
of the control group.
7.1: Artemia salina encapsulation
The peptidic GHS analogs were bioencapsulated in Artemia to be fed to
Litopenaeus vanamei post-larvae. For the encapsulation the compounds were added 15
in a 10mh/L left for an hour, harvested and washed. The animals were fed four times
a day for one month while the control group was fed with BSA encapsulated Artemia
The effect was measured with an optical micrometer and weighing the animals in a
0.1 mg precision scale. The encapsulated compounds increased the growth of the
animals in a 30 to 40% respect to the control group with a highly significant 20
difference (p Compound Size Inc. (%) Compound Size Inc. (%)
A221 130.0 A230 140.0
A222 131.0 A231 140.1
A223 131.6 A232 139.7
A224 131.2 A233 140.1
A225 130.0 A234 138.6
A226 130.4 A235 137.0
A227 139.0 A236 137.0
A228 140.0 A237 132.0
A229 140.0 A238 130.1
31
Table 11. Size increment in % for the treated group taking as 100% the growing
of the control group.
Compound Size Inc. (%) Compound Size Inc. (%)
A221 130.2 A230 140.0
A222 130.3 A231 140.2
A223 132.0 A232 139.6
A224 130.0 A233 140.0
A225 130.0 A234 135.0
A226 132.0 A235 134.2
A227 140.0 A236 138.0
A228 140.0 A237 136.0
A229 140.0 A238 140.0
5
Example 8: Cardioprotective effect in rats of the peptidic GHS analogs.
To reproduce the physiopathogenic effects of a Dilated Cardiomyopathy (DCM)
female Wistar rats of 160 g were treated with 2mg/kg Doxorubicin (Dx) during 8
weeks. A group of this rats was also treated in parallel with compounds A221, A228
or A233 intraperitoneally at 500µg/Kg during the 8 weeks of Dx treatment, another 10
Dx treated group was also receiving saline solution as a placebo, and as a healthy
control for the experiment another group of untreated Wistar rats of the same age
was used. After the 8 week treatment all the rats were tested with an
echocardiogram, to test the ventricular functionality and assess the ventricular
ejection fraction (VEF). As seen in fig.1 rats receiving the parallel Dx-compound 15
A221(1a), A228(1b) or A233(1c) slightly modified VEF (p>0.05) with respect to the
healthy control, in contrast the group receiving placebo suffers a drop in VEF of
about a 40% (p functional implications for the stress response of the drop in VEF, the rats were
subjected to forced swimming in 4°C water foe 30 minutes, as shown in fig. 2 20
animals receiving the treatment with Dx-compound A221(2a), A228(2b) or A233(2c)
have a survival of 100% and the Dx-saline solution survived to the 45 % (p=0.0043).
32
This results suggested that the protection by compounds A221, A228 ad A233, does
not only maintains the VEF but also yields the heart resistant to forced stress.
Example 9: Cardioprotective effect and reversion of the Dilated
Cardiomyopathy (DCM) in rats of the peptidic GHS analogs.
To assess if there is any dose-response effect and reversion of DCM, Wistar rats 5
were subjected to a treatment with 2mg/kg of Doxorubicin (Dx) for 8 weeks, after the
treatment all rats with a VEF drop higher than 40% were selected, divided in groups
of n=8 and treated with different doses of compounds A221, A228 or A233 as
follows:
• 500 µg/kg, 10
• 250 µg/kg,
• 100 µg/kg,
• 50 µg/kg,
• 25 µg/kg,
• 10 µg/kg 15
• Saline Solution.
Defining the groups based in the A221 doses.
As shown in fig 3, two weeks after the treatment with compounds A221(3a),
A228(3b) or A233(3c) partially reverts DCM in the concentration range of 50 µg/kg to
500 µg/kg but at 4 weeks of treatment the DCM reversion is complete in the groups 20
receiving the compounds A221, A228 or A233 in the 100 to 500 µg/kg range, 50
µg/kg dosage is not effective for the total VEF recovery but somehow effective to
reduce mortality in the group, respect to the animals receiving placebo or groups
treated with lower concentrations, that do not recovered VEF and have a lower
survival days after the treatment is finished. (Fig. 4, a A221, b A228 and c A233). 25
30
WE CLAIM:
1) Chemical molecules of a peptide nature, with internal cycles and L- amino
acids, and their homolog variants, able of exerting, due to their chemical
structure, similar functions of those attributed to ghrelin, des-acyl ghrelin and
other peptidic Growth Hormone Secretagogues, where the chemical structure
is defined by the following amino acid sequence, including a cyclation using
the amino acid side chain or a binding compound as a bridge, and can be
selected using the following structural regularities:
[Aa1...Aan] X1 [Ab1...Abn ] X2 [Ac1 … Acn ] Adn
Where Aa are L-amino acids selected from the set of [Cys, Gly, Ser, His, Ala,
Leu, Met o Thr], varying in combinations of 1 to 4 residues, Ab are L-amino
acids, selected from the set of [Pro, Ile, Ala, Phe, Trp, Lys, Asp, Asn, Glu,
Gln, Gly, Leu, Met, Tyr o Thr], varying in combinations of 1 to 4 residues, Ac
are L-amino acids selected from the set of [Arg, Leu, Pro, Val, Thr, Glu, His,
Gln, Asn, Asp, Trp, Tyr, Phe, Ser, Ala , Gly o Ile], varying in combination of 1
to 5 and Ad are L-amino acids, natural or not without limit in number, X1 and
X2 are L-amino acids, natural o not, with the side chains covalently bound
forming an internal cycle, using any chemical reaction for the direct link or
using a binding compound as a bridge.
2) Chemical molecules of a peptide nature, according to Claim 1 where the
chemical structure is defined by the following amino acid sequence: Seq ID
No. 1-Seq ID No. 18, corresponding to compounds A221-A238.
3) Pharmaceutical composition comprising one or more of the chemical
compounds described in claims 1 and 2, any of its pharmaceutically
acceptable salts, and excipients or vehicles.
4) Pharmaceutical composition according to claim 3, characterized by having the
peptidic chemical compounds in a range from 2 to 100 µg of compound per
ml, if prepared as a solution or further used as a lyophilized powder.
5) Veterinary composition for aquaculture or other animal production, or
improvement, comprising one or more of the chemical compounds described
in claims 1 and 2, any of its veterinary acceptable salts, and other excipients
or vehicles.
6) Veterinary composition, according to claim 5, characterized by the
administration of the compounds as foodstuff, in nutritional supplement, in
periodical injections or in immersion baths, for the purposes of growth
stimulation and/or eliciting disease resistance in fish or crustaceans.
7) Use of the chemical compounds of claims 1 and 2, or any of its
pharmaceutically accepted salts, for producing a pharmaceutical composition
for the induction of growth hormone in a patient requiring such treatment, by
administering one or more of such compounds.
8) Use of the chemical compounds of claims 1 and 2, or any of its
pharmaceutically accepted salts, for producing a pharmaceutical composition
for the induction of cardioprotection, and/or neuroprotection, and/or appetite
control including the fat and energy metabolism in a patient requiring such
treatment, by administering one or more of such compounds.
9) Use of the chemical compounds of claims 1 and 2, or any of its
pharmaceutically accepted salts, for producing a veterinary composition for
growth stimulation and/or eliciting disease resistance in fish or crustaceans,
by using one or more of such compounds.
10) Use of the chemical compounds of claims 1 and 2, or any of its
pharmaceutically accepted salts, according to claim 9, wherein the peptidic
chemical compounds are in the range from 0.01 to 1%, if administered as
foodstuff; in the range from 0.05 to 10 µg of compound per gram of animal
wet weight, if administered in periodic injections; or in the range from 10 to
500 µg of compound per liter, if administered in immersion baths.
ABSTRACT
PEPTIDIC GROWTH HORMONE SECRETAGOGUES ANALOG
COMPOUNDS AND PREPARATIONS THEREOF
Chemical peptide compounds obtained by means of in silico molecular modelling
whose structure enables them to perform the same functions as growth hormone
peptide secretagogues. The invention also comprises the compositions that
contain said compounds and use thereof in the preparation of medicinal
products, nutritional supplements or other formulations for human or animal
use.

Documents:

1865-MUMNP-2008-AUSTRALIAN DOCUMENT(31-12-2012).pdf

1865-MUMNP-2008-CLAIMS(AMENDED)-(22-5-2013).pdf

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1865-MUMNP-2008-CORRESPONDENCE(1-9-2008).pdf

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1865-MUMNP-2008-CORRESPONDENCE(17-3-2009).pdf

1865-MUMNP-2008-CORRESPONDENCE(18-3-2009).pdf

1865-MUMNP-2008-CORRESPONDENCE(4-2-2010).pdf

1865-MUMNP-2008-CORRESPONDENCE(8-10-2012).pdf

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1865-MUMNP-2008-DECLARATION OF TRANSLATION(18-3-2009).pdf

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1865-MUMNP-2008-EP DOCUMENT(31-12-2012).pdf

1865-MUMNP-2008-FORM 1(17-3-2009).pdf

1865-MUMNP-2008-FORM 1(28-8-2008).pdf

1865-MUMNP-2008-FORM 1(31-12-2012).pdf

1865-MUMNP-2008-FORM 1(8-10-2012).pdf

1865-MUMNP-2008-FORM 1(8-7-2013).pdf

1865-MUMNP-2008-FORM 13(8-10-2012).pdf

1865-MUMNP-2008-FORM 18(4-2-2010).pdf

1865-MUMNP-2008-FORM 2(TITLE PAGE)-(31-12-2012).pdf

1865-MUMNP-2008-FORM 26(17-3-2009).pdf

1865-MUMNP-2008-FORM 26(8-10-2012).pdf

1865-MUMNP-2008-FORM 3(10-9-2009).pdf

1865-MUMNP-2008-FORM 3(28-8-2008).pdf

1865-MUMNP-2008-FORM 3(31-12-2012).pdf

1865-MUMNP-2008-FORM 5(28-8-2008).pdf

1865-MUMNP-2008-FORM PCT-IPEA-409(31-12-2012).pdf

1865-MUMNP-2008-JAPANESE DOCUMENT(31-12-2012).pdf

1865-MUMNP-2008-PCT-RO-101(18-3-2009).pdf

1865-MUMNP-2008-PETITION UNDER RULE 137(31-12-2012).pdf

1865-MUMNP-2008-PETITION UNDER RULE 137-(31-12-2012).pdf

1865-MUMNP-2008-REPLY TO EXAMINATION REPORT (31-12-2012).pdf

1865-MUMNP-2008-REPLY TO EXAMINATION REPORT(31-12-2012).pdf

1865-MUMNP-2008-REPLY TO HEARING(22-5-2013).pdf

1865-MUMNP-2008-SEQUENCE LISTING(18-3-2009).pdf

1865-MUMNP-2008-US DOCUMENT(31-12-2012).pdf

1865-MUMNP-2008-WO INTERNATIONAL PUBLICATION REPORT A1(4-2-2010).pdf

Drawings.pdf

Form-1.pdf

Form-3.pdf

Form-5.pdf


Patent Number 256881
Indian Patent Application Number 1865/MUMNP/2008
PG Journal Number 32/2013
Publication Date 09-Aug-2013
Grant Date 06-Aug-2013
Date of Filing 28-Aug-2008
Name of Patentee CENTRO DE INGENIERIA GENÉTICA Y BIOTECNOLOGÍA
Applicant Address AVE. 31 ENTRE 158 Y 190, CUBANACÁN, PLAYA, C. HABANA 10600,
Inventors:
# Inventor's Name Inventor's Address
1 RODRÍGUEZ FERNÁNDEZ, ROLANDO EDUARDO AVENIDA 31 ENTRE 182 Y 184, # 18207 APTO 41, CUBANACÁN, PLAYA, CIUDAD DE LA HABANA 10 600,
2 DE LA NUEZ VEULENS, ANIA Calle M entre 11 y 13 # 64 apto 8D bajos Vedado Ciudad de La Habana 10 400 Cuba
3 ESTRADA GARCÍA, MARIO PABLO Calle: 186 entre 31 y 33 #3112 Apto.10 Cubanacán Playa Ciudad de La Habana 12 100 Cuba
4 MARTÍNEZ RODRÍGUEZ, REBECA Calle: 186 entre 31 y 33 #3115 Apto.8B Cubanacán Playa Ciudad de La Habana 12 100 Cuba
5 CHINEA SANTIAGO, GLAY Calle 186 #3115 entre 31 y 33 Apto 4C Cubanacán Playa Ciudad de La Habana 12 100 Cuba
6 REYES ACOSTA, OSVALDO Avenida 31 #18207 entre 182 y 184 apto 20 Cubanacán Playa Ciudad de La Habana 12 100 Cuba
7 FERNÁNDEZ MASSÓ, JULIO RAÚL Calle 186 # 3115 Apto 8A entre 31 y 33 Cubanacán Playa Ciudad de La Habana 12 100 Cuba
8 GARCÍA DEL BARCO HERRERA, DIANA Calle: 37 #3614 apto13 entre 42 y 36 Playa Ciudad de La Habana 11 600 Cuba
9 BERLANGA ACOSTA, JORGE AMADOR Avenida 31 # 18207 apto 32 entre 182 y 184 Cubanacán Playa Ciudad de La Habana 11 600 Cuba
10 MUSACCHIO LASA, ALEXIS Calle 128 No. 7117 entre 71 y 73 Mariel La Habana 32 100 Cuba
PCT International Classification Number C07K7/06
PCT International Application Number PCT/CU2007/000007
PCT International Filing date 2007-02-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2006-0050 2006-02-28 Cuba