U.S. patent application number 14/896449 was filed with the patent office on 2016-05-05 for ghrelin splice variant for treating neuronal damage,neurodegenerative disease, parkinsons disease, alzheimers disease, and/or depression.
The applicant listed for this patent is Liat MINTZ. Invention is credited to Liat Mintz.
Application Number | 20160122403 14/896449 |
Document ID | / |
Family ID | 52432330 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160122403 |
Kind Code |
A1 |
Mintz; Liat |
May 5, 2016 |
GHRELIN SPLICE VARIANT FOR TREATING NEURONAL
DAMAGE,NEURODEGENERATIVE DISEASE, PARKINSONS DISEASE, ALZHEIMERS
DISEASE, AND/OR DEPRESSION
Abstract
The present disclosure relates, in one aspect, to use of ghrelin
splice variant or an analogue thereof, or a ghrelin splice
variant-like compound for the preparation of a medicament for one
or more of: treatment and/or prevention of neuronal damage and/or
neurodegeneration and, prophylaxis or treatment of neuronal damage
and/or neurodegenerative disease, Parkinson's disease, Alzheimer's
disease, depression stimulation of neuronal activity. A further
aspect relates to a number of new ghrelin splice variant-like
compounds and uses thereof, as well as to pharmaceutical
compositions and medical packaging comprising the new ghrelin
splice variant-like compounds.
Inventors: |
Mintz; Liat; (East
Brunswick, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MINTZ; Liat |
East Brunswick |
NJ |
US |
|
|
Family ID: |
52432330 |
Appl. No.: |
14/896449 |
Filed: |
July 15, 2014 |
PCT Filed: |
July 15, 2014 |
PCT NO: |
PCT/US14/46681 |
371 Date: |
December 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61846920 |
Jul 16, 2013 |
|
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Current U.S.
Class: |
514/17.8 ;
514/17.7; 525/54.1; 530/324; 530/325; 530/326 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/25 20130101; A61P 25/28 20180101; A61P 25/16 20180101; A61P
25/24 20180101; A61P 25/00 20180101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Claims
1. Use of an isolated ghrelin splice variant-like compound having
the formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally
present protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1 in the manufacture of a medicament for
the induction of neuron function, protection of neuron function,
alteration neuron function, or a combination thereof.
2. The use of claim 1, wherein the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 has at least 98% homology to SEQ ID
NO:1.
3. The use of claim 1, wherein the compound is 22-29 amino acids in
length.
4. The use of claim 1, wherein the bulky hydrophobic group is an
acyl group or a fatty acid group.
5. The use of claim 4, wherein the acyl group is a C.sub.1-C.sub.35
acyl group.
6. The use of claim 5, wherein the acyl group is a C.sub.7-C.sub.12
acyl group.
7. The use of claim 1, wherein the compound has the formula
Z1-Gly-(X1)m-(X2)-(X3)n-Z2, Z1-Gly-Ser-(X2)-(X3)n-Z2, or
Z1-Gly-(X2)-(X3)n-Z2.
8. The use of claim 1, wherein (X1)m is Gly, Gly-Ser, Gly-Cys,
Gly-Lys, Gly-Asp, Gly-Glu, Gly-Arg, Gly-His, Gly-Asn, Gly-Gln,
Gly-Thr, or Gly-Tyr.
9. The use of claim 1, wherein (X2) is modified Ser, modified Cys,
modified Asp, modified Lys, modified Trp, modified Phe, modified
Ile, or modified Leu.
10. The use of claim 1, wherein (X3)n comprises a fragment of SEQ
ID NO:6.
11. The use of claim 1, wherein Z1 is selected from the group
consisting of C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 substituted
alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 substituted
alkenyl, aryl, C.sub.1-C.sub.6 alkyl aryl,
C(O)--(CH.sub.2)--(C.sub.1-C.sub.6 alkyl)-COOH,
C(O)--(C.sub.1-C.sub.6 alkyl), C(O)-aryl,
C(O)--O--(C.sub.1-C.sub.6-alkyl), and C(O)--O-aryl.
12. The use of claim 1, wherein Z2 is selected from the group
consisting of amide, methylamide, and ethylamide.
13. The use of claim 1 which binds to the growth hormone
secretagogue (GHS) receptor GHS-R 1a.
14. The use of claim 13, wherein the compound has an EC50 potency
on the GHS-R1a of less than 500 nM.
15. The use of claim 13, wherein the compound has a dissociation
constant of less than 500 nM.
16. The use of claim 1, wherein the compound has at least about 50%
of the functional activity of ghrelin.
17. The use of claim 16, wherein functional activity is activation
of Gq/G11, accumulation of inositol phosphate, mobilization of
calcium from intracellular stores, activation or deactivation of
MAP kinases, NF.kappa.B translocation, CRE driven gene
transcription, binding of arrestin to ghrelin receptor, or a
combination thereof.
18. The use of claim 1, wherein the compound is conjugated to a
polymer molecule.
19. The use of claim 18, wherein the polymer molecule is selected
from the group consisting of polyalkylene oxide, polyalkylene
glycol, poly-vinyl alcohol, poly-carboxylate,
poly-(vinylpyrolidone), polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, and dextran.
20. The use of claim 1, wherein the compound is modified with a
chemically reactive group.
21. The use of claim 20, wherein the chemically reactive group is
selected from the group consisting of N-hydroxysuccinimide,
N-hydroxy-sulfosuccinimide, maleimide-benzoyl-succinimide,
gamma-maleimido-butyryloxy succinimide ester, and
maleimidopropionic acid.
22. A method for treating neuronal damage comprising administering
to a mammal in need thereof a pharmaceutically acceptable amount
of: (a) ghrelin splice variant; (b) a ghrelin splice variant-like
compound having the formula Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein
Z1 is an optionally present protecting group; each X1 is
independently selected from a naturally occurring amino acid and a
synthetic amino acid; X2 is selected from a naturally occurring
amino acid and a synthetic amino acid, said amino acid being
modified with a bulky hydrophobic group; each X3 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid, wherein one or more of X1 and X3 optionally may be
modified with a bulky hydrophobic group; Z2 is an optionally
present protecting group; m is an integer in the range of from
1-10; n is an integer in the range of from 4-92; provided that the
compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino
acids in length, has at least 95% homology to SEQ ID NO:1, and
induce neuron function, protects neuron function, alters neuron
function, or a combination thereof of a subject when administered
thereto; or (c) a mixture thereof.
23. A method for treating neurodegenerative disease comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of: (a) ghrelin splice variant; (b) a ghrelin
splice variant-like compound having the formula
Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
24. A method for preventing neuronal damage and/or
neurodegenerative disease comprising administering to a mammal in
need thereof a pharmaceutically acceptable amount of: (a) ghrelin
splice variant; (b) a ghrelin splice variant-like compound having
the formula Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an
optionally present protecting group; each X1 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid; X2 is selected from a naturally occurring amino acid
and a synthetic amino acid, said amino acid being modified with a
bulky hydrophobic group; each X3 is independently selected from a
naturally occurring amino acid and a synthetic amino acid, wherein
one or more of X1 and X3 optionally may be modified with a bulky
hydrophobic group; Z2 is an optionally present protecting group; m
is an integer in the range of from 1-10; n is an integer in the
range of from 4-92; provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
25. A method for treating Parkinson's disease comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of: (a) ghrelin splice variant; (b) a ghrelin
splice variant-like compound having the formula
Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
26. A method for preventing Parkinson's disease comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of: (a) ghrelin splice variant; (b) a ghrelin
splice variant-like compound having the formula
Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
27. A method for treating Alzheimer's disease comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of (a) ghrelin splice variant; (b) a ghrelin
splice variant-like compound having the formula
Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
28. A method for preventing Alzheimer's disease comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of: (a) ghrelin splice variant; (b) a ghrelin
splice variant-like compound having the formula
Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
29. A method for treating depression comprising administering to a
mammal in need thereof a pharmaceutically acceptable amount of: (a)
ghrelin splice variant; (b) a ghrelin splice variant-like compound
having the formula Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein Z1 is an
optionally present protecting group; each X1 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid; X2 is selected from a naturally occurring amino acid
and a synthetic amino acid, said amino acid being modified with a
bulky hydrophobic group; each X3 is independently selected from a
naturally occurring amino acid and a synthetic amino acid, wherein
one or more of X1 and X3 optionally may be modified with a bulky
hydrophobic group Z2 is an optionally present protecting group; m
is an integer in the range of from 1-10; n is an integer in the
range of from 4-92; provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1, and induce neuron function, protects
neuron function, alters neuron function, or a combination thereof
of a subject when administered thereto; or (c) a mixture
thereof.
30. A method for preventing depression comprising administering to
a mammal in need thereof a pharmaceutically acceptable amount of:
(a) ghrelin splice variant; (b) a ghrelin splice variant-like
compound having the formula Z1-(X1).sub.m--(X2)-(X3)n-Z2, wherein
Z1 is an optionally present protecting group; each X1 is
independently selected from a naturally occurring amino acid and a
synthetic amino acid; X2 is selected from a naturally occurring
amino acid and a synthetic amino acid, said amino acid being
modified with a bulky hydrophobic group; each X3 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid, wherein one or more of X1 and X3 optionally may be
modified with a bulky hydrophobic group; Z2 is an optionally
present protecting group; m is an integer in the range of from
1-10; n is an integer in the range of from 4-92; provided that the
compound according to formula Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino
acids in length, has at least 95% homology to SEQ ID NO:1, and
induce neuron function, protects neuron function, alters neuron
function, or a combination thereof of a subject when administered
thereto; or (c) a mixture thereof.
Description
FIELD OF INVENTION
[0001] The present disclosure relates to compounds for treating or
preventing neurodegeneration and/or Parkinson's disease and/or
Alzheimer's disease and/or depression and conditions related
thereto.
BACKGROUND OF INVENTION
[0002] Alzheimer's disease (AD) and Parkinson's disease (PD) are
the two most common neurodegenerative disorders. Parkinson's
disease (PD) is characterized by the progressive degeneration of
dopamine (DA) neurons projecting from the substantia nigra pars
compacta (SNpc) to the dorsal striatum. The resulting loss of
dopamine in the striatum leads to debilitating motor dysfunction,
including rigidity, resting tremor, postural instability, and
bradykinesia. Familial or genetic causes of PD only account for 10%
of all cases, whereas 90% are considered sporadic and may manifest
as a result of a variety of factors. Currently available therapies
for Parkinson's disease (PD) provide relief of motor symptoms, but
fall short of exhibiting the neuroprotective effect required to
prevent progressive degeneration of dopamine (DA) neurons.
Furthermore, no therapies simultaneously target the multiple,
debilitating, non-motor symptoms of PD.
[0003] Alzheimer's disease (AD) is a multifactorial progressive
neurodegenerative disorder characterized by loss of memory and
cognitive deficits, strongly influenced by the metabolic status, in
which the impairment of neuropeptides/neurotransmitters systems has
been previously observed. Currently available therapies for
Alzheimer's disease (AD) have shown limited efficacy, with no true
cure to this day being present. Recent work, both clinical and
experimental, indicates that many neurodegenerative disorders often
display a coexisting metabolic dysfunction which may exacerbate
neurological symptoms. It stands to reason therefore that metabolic
pathways may themselves contain promising therapeutic targets for
major neurodegenerative diseases.
[0004] Ghrelin is a bioactive peptide that induces food intake,
body weight gain, and adiposity in rodents (Tschop M. et al.,
Nature 407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47
(2001)). Acute administration of ghrelin induces food intake in
healthy men and women (Wren A. M. et al., J. Clin. Endocrinol.
Metab. 86:5992-95 (2001); Druce M. R. et al., Int. J. Obes. Relat.
Metab. Disord. 29:1130-36 (2005)) as well as in cancer patients
with anorexia (Neary N. M. et al., J. Clin. Endocrinol. Metab.
89:2832-36 (2004)). Repeated administration of ghrelin increases
lean body mass, body weight and food intake in cachectic patients
with Chronic Obstructive Pulmonary Disease (COPD) (Nagaya N. et
al., Chest 128:1187-93 (2005)) and improved muscle wasting in
patients with chronic heart failure (Nagaya N. et al., Circulation
110:3674-79 (2004)). A similar effect was also shown in a mouse
model (Hanada T. et al., Biochem. Biophys. Res. Commun. 301:275-79
(2003)).
[0005] Ghrelin has been associated with the progression of obesity
and metabolic syndrome, but has been also linked to
neuromodulation, neuroprotection and memory and learning processes.
In addition, ghrelin system also acts in an autocrine/paracrine
fashion where the majority of its components ghrelin variants,
acylation enzyme (GOAT) and receptors (GHS-Rs)] are expressed in
the different regions of central nervous system. Gahete et al.
analyzed the mRNA expression of the ghrelin system in three
different regions of the temporal gyrus (inferior, medial and
superior) of control and AD human brains, since the temporal lobe
is considered as one of the most important cortical structures in
memory and cognition, and is one of the most affected regions in AD
(Gahete et al., Peptides 32: 2225-2228 (2011)). This report showed,
for the first time, that AD patients have a reduction in local
brain ghrelin production, as compared with age-matched controls. In
addition, they analyzed the expression of a newly described
In1-ghrelin splice variant (In1-ghrelin is identical to applicants
SEQ ID 32 from U.S. Pat. No. 7,176,292), which could also be
acylated by the ghrelin-O-acyltransferase (GOAT) enzyme. Results
revealed that, similar to ghrelin, In1-ghrelin variant (In1-ghrelin
is applicants SEQ ID 32 from U.S. Pat. No. 7,176,292) is also
expressed in the temporal lobe, and is down-regulated in AD,
thereby showing, together with ghrelin, a region-dependent
alteration with AD. Consistent with a similar distribution between
ghrelin and GOAT in other tissues, GOAT was also expressed in the
temporal lobe of the brain and was impaired in AD, suggesting that
a functional autocrine and/or paracrine pathway might be in place
within the temporal lobe, and that changes in locally produced
acylated/non-acylated ghrelin and In1-ghrelin variant may be of
(patho)-physiologically relevance.
[0006] The first evidence showing a direct effect of ghrelin on
AD-like alterations was reported in a mouse model widely used to
examine the pathophysiology of early defects seen in AD. The
senescence-accelerated mouse prone8 or SAMP8 mice develop early
abnormalities in learning and memory related to abnormalities in
septo-hippocampal function, which are due to overproduction of
.beta.-amyloid. In this mouse model, ghrelin was able to improve
retention of T-maze foot shock avoidance in 12 and 14 month-old
mice, compared to their controls (Diano et al. Nat Neurosci.
9:381-8 (2006)). More recently, a different mouse model has been
used to analyze in more detail the role that ghrelin plays in
AD-related endpoints. This model was generated by intrahippocampal
injection of oligomeric forms of the AP peptide (A130), which have
been directly related with AD-associated damage (Moon et al. J.
Alzheimers Dis.; 23:147-59 (2011)). Results of this study revealed
that systemic injection of ghrelin rescues memory deficits observed
following intrahippocampal A.beta.O injection, using two
independent behavioral paradigms (Y-maze and passive avoidance
tasks). In addition, the AD-associated neuropathological
abnormalities observed in these A.beta.O mice were also attenuated
by ghrelin. Indeed, ghrelin inhibited the reactive microgliosis
originated by A.beta.O, thus preventing the inflammatory response.
Ghrelin also prevented A.beta.O-induced neuronal cell loss in the
dentate gyrus and increased the density of hippocampal synaptic and
cholinergic nerve fibers. Collectively, these data show that
systemic injection of ghrelin rescue cognitive impairments induced
by A.beta.O, possibly through inhibition of both, microgliosis and
impairment of neuronal integrity.
[0007] Recent data (Andrews et al. The Journal of Neuroscience
29(45):14057-14065 (2009)) demonstrate that ghrelin administration
decreases substantia nigra pars compacta DA cell loss and limits
striatal DA loss in a PD mouse model. Ghrelin has also been shown
to defend against depression (Lutter et al. Nature Neuroscience
11(7):752-3 (2008)) and to stimulate appetite (Tschop M. et al.,
Nature 407:908-13 (2000); Wren A. M. et al., Diabetes 50:2540-47
(2001)) in animal models, suggesting additional benefit to these
distressing non-motor symptoms of PD (Uc et al. Movement Disorders
21(7):930-6 (2006); Chen et al. Annual Neurology 53(5):676-9
(2003); Delinkanaki-Skaribas et al. Movement Disorders 15;
24(5):667-71 (2009)). It was also shown that subjects at putative
preclinical (iRBD) and clinical stages of PD suffer from an
impaired ghrelin excretion. Lately it was shown that the GHSR1 and
DRD2 receptors form heteromers in hypothalamic neurons and that you
can inhibit dopamine signaling using GHSR1 antagonists (Kern A. et
al., Neuron 73:317-332 (2012)). From the studies conducted, ghrelin
has an acute effect on the firing rate of SNpc DA neurons, which
enhances dopamine availability during the course of degeneration
and lowers the loss of dopamine levels in the dorsal striatum. In
addition, ghrelin-induced enhancement of UCP2-dependent
mitochondrial respiration and proliferation provides a bioenergetic
status that makes these neurons more resistant to cellular stress.
UCP2 is a mitochondrial protein known to protect SNpc DA cells from
MPTP intoxication5. Ghrelin also regulates UCP2 mRNA in the brain,
consistent with the role of UCP2 as a component of ghrelin-induced
neuroprotection. Ghrelin-/- and ghsr-/- mice were more susceptible
to DA cell loss in the SNpc and DA loss in the striatum after MPTP
than their wt littermates. This suggests that any factor leading to
reduced ghrelin production or secretion, whether genetic or
environmental, may predispose individuals to nigrostriatal
dopaminergic dysfunction. Taken together, these data provide a
strong biological rationale for ghrelin peptide family as a novel
class of neurodegenerative diseases therapeutics that promotes
neuroprotection and relieves non-motor symptoms.
[0008] The GHRL (ghrelin) gene encodes a variety of products
resulting from alternatively spliced transcripts, various types of
cleavage of the prepropeptide, and various post-translational
modifications (Kojima M. & Kangawa M., Physiol. Rev. 85:495-522
(2005); Zhang J. V. et al., Science 310:996-99 (2005)). In
addition, different degradation products are produced by various
tissues (De Vriese C. et al., Endocrinology 145:4997-5005 (2004)).
Some of these GHRL products are described herein.
[0009] Ghrelin is a 28 amino acid peptide bearing an n-octanoyl
side chain on the third serine, resulting from the cleavage of
signal and propeptide from the 117 amino acid preproghrelin and an
acylation event. The acylated N-terminus of ghrelin is essential
for the endocrine functions (Kojima M. et al., Nature 402:656-60
(1999); Bednarek M. A. et al., J. Med. Chem. 43:4370-76 (2000)).
Des-acyl ghrelin, which lacks the endocrine functions, was shown to
have an antagonistic effect to that of ghrelin on glucose output in
vitro (Gauna C. et al., J. Clin. Endocrinol. Metab. 89:5035-42
(2004)). An alternatively-spliced ghrelin mRNA encodes a 116 amino
acid prepropeptide that is further processed to a Des-Gln14-ghrelin
and a 27 amino acid processed peptide (Hosoda H. et al., J Biol.
Chem. 275:21995-22000 (2000)). Another peptide, Obestatin, is
cleaved from the preproghrelin and has no sequence overlap with
processed ghrelin peptide. This peptide was shown to have some
antagonistic effect to acylated ghrelin, inhibiting food intake and
body weight gain (Zhang J. V. et al., Science 310:996-99 (2005)).
Yet another peptide, the 66 amino acid C-terminus of the
preproghrelin, may also be functional (Pemberton C. et al.,
Biochem. Biophys. Res. Comm. 310:567-73 (2003)). A variety of
isoforms, including isoforms encoded by different splice variants,
are known for other proteins, e.g. for vascular endothelial growth
factor (VEGF), where different isoforms share roles as
angiogenesis, while differing in some other characteristics, such
as binding affinity (Neufeld G. et al., FASEB J. 13:9-22 1999).
Thus, the variety of products of the GHRL gene may reflect a
similarly complex control of the endocrine and paracrine action of
the ghrelin isoforms.
[0010] Previously, administration of ghrelin by continuous
infusions of 5 pmol/kg/min doses for 270 minutes was shown to
increase food intake in healthy humans (Wren A. M. et al., J. Clin.
Endocrinol. Metab. 86:5992-95 (2001)). It was also shown that
infusion of ghrelin for 90 minutes could increase food intake by
30% in cancer cachexia patients (Abstract P09, Digestive Hormones,
Appetite and Energy Balance, Baylis and Starling meeting, London,
June 2003). Recently, it was shown that subcutaneous injection of
3.6 nmol/kg acylated ghrelin prior to a meal, thereby ensuring a
close mimic of the natural pre-meal situation, increased energy
intake by 27%. Ghrelin also appeared to enhance the perceived
palatability of the food offered (Druce M. R. et al., Int. J. Obes.
Relat. Metab. Disord. 29:1130-36 (2005)).
[0011] These studies demonstrate that parenteral administration of
ghrelin can increase appetite in both normal subjects and in
patients with loss of appetite. Furthermore, Applicant has found
that it is possible to obtain a significant effect of body weight
gain and a significant increase in food consumption with a novel
ghrelin splice variant (see co-owned, published U.S. Pat. No.
7,981,860 and/or U.S. Pat. No. 7,763,707, incorporated herein by
reference) when administered to a subject, in particular when
administered subcutaneously prior to a meal, thereby ensuring a
close mimic of the natural pre-meal situation. Applicant's novel
ghrelin splice variant effect of weight gain is mainly on lean mass
while ghrelin's effect on weight gain is mainly on fat mass.
[0012] In addition applicant has found that it is possible to
decrease substantia nigra pars compacta DA cell loss and limits
striatal DA loss with a novel ghrelin splice variant (see co-owned,
published U.S. Pat. No. 7,981,860 and/or U.S. Pat. No. 7,763,707,
incorporated herein by reference) when administered to a subject in
a PD animal model.
SUMMARY OF THE INVENTION
[0013] One aspect is a ghrelin splice variant-like compound having
the formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally
present protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1.
[0014] Another aspect is a pharmaceutical composition comprising
the ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1.
[0015] A further aspect is a method of treating Parkinson's Disease
comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0016] An additional aspect is a method for preventing Parkinson's
Disease comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0017] A further aspect is a method of treating Alzheimer's Disease
comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0018] An additional aspect is a method for preventing Alzheimer's
Disease comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0019] A further aspect is a method for the stimulation of neuronal
activity comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0020] Another aspect is a method for treating depression
comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0021] A further aspect is a method for preventing depression
comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0022] Another aspect is a method for treating neuronal
degeneration comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0023] A further aspect is a method for preventing neuronal
degeneration comprising administering to a mammal in need thereof a
pharmaceutically acceptable amount of (a) ghrelin splice variant;
(b) a ghrelin splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof.
[0024] An additional aspect is a method of treating Parkinson's
Disease and/or depression and/or neuronal degeneration comprising
administering to a mammal in need thereof a pharmaceutically
acceptable amount of a secretagogue comprising (a) ghrelin splice
variant; (b) a ghrelin splice variant-like compound having the
formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (c) a mixture thereof;
wherein treatment is selected from the group consisting of
prophylaxis or treatment of Parkinson's disease, prophylaxis or
treatment of Alzheimer's disease, prophylaxis or treatment of
depression, stimulation of neuronal activity, stimulation of
neuronal regeneration, stimulation of appetite, stimulation of
weight gain, increasing body fat mass, increasing lean body mass,
or a combination thereof.
[0025] A further aspect is a kit for administering ghrelin splice
variant or a ghrelin splice variant-like compound comprising (a) a
dosage form comprising a pharmaceutically acceptable amount of (1)
ghrelin splice variant; (2) a ghrelin splice variant-like compound
having the formula Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an
optionally present protecting group; each X1 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid; X2 is selected from a naturally occurring amino acid
and a synthetic amino acid, said amino acid being modified with a
bulky hydrophobic group; each X3 is independently selected from a
naturally occurring amino acid and a synthetic amino acid, wherein
one or more of X1 and X3 optionally may be modified with a bulky
hydrophobic group; Z2 is an optionally present protecting group; m
is an integer in the range of from 1-10; n is an integer in the
range of from 4-92; provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% homology to SEQ ID NO:1; or (3) a mixture thereof; and
(b) optionally, instructions for administering (a).
[0026] Another aspect is for a method of producing a ghrelin splice
variant-like compound, said method comprising: (a) providing a cDNA
comprising a polynucleotide sequence encoding a ghrelin splice
variant-like compound as described above; (b) inserting said cDNA
in an expression vector such that the cDNA is operably linked to a
promoter; (c) introducing said expression vector into a host cell
whereby said host cell produces said ghrelin splice variant-like
compound; and (d) optionally recovering the ghrelin splice
variant-like compound produced in step (c).
[0027] An additional aspect is for use of an isolated ghrelin
splice variant-like compound having the formula
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length, has at least
95% homology to SEQ ID NO:1 in the manufacture of a medicament for
the induction of neuron function, protection of neuron function,
alteration neuron function, or a combination thereof.
[0028] Other objects and advantages will become apparent to those
skilled in the art upon reference to the detailed description that
hereinafter follows.
BRIEF DESCRIPTION OF THE SEQUENCES
[0029] SEQ ID NO:1 represents human prepro ghrelin splice variant
after the signaling sequence. SEQ ID NO:2 represents 22 amino acid
human ghrelin splice variant. SEQ ID NO:3 represents 24 amino acid
human ghrelin splice variant. SEQ ID NO:4 represents 24 amino acid
modified human ghrelin splice variant. SEQ ID NO:5 represents 29
amino acid human ghrelin splice variant. SEQ ID NO:6 represents a
fragment of full-length human ghrelin splice variant. SEQ ID NO:7
represents mouse prepro ghrelin splice variant after the signaling
sequence. SEQ ID NO:8 represents rat prepro ghrelin splice variant
after the signaling sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0030] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0031] FIG. 1. In FIG. 1 it is clearly shown that the toxicity of
H2O2 leads to the survival of only 50% of the cells. It is also
clear that treatment with Ghrelin splice variant alone or in
combination with Ghrelin exerts an effective protective action
against the damages produced by H2O2. Specifically H2O2+Ghrelin
splice variant & H2O2+Ghrelin & H2O2+Ghrelin+Ghrelin splice
variant treatments are statistically significant in terms of cell
viability compared to the negative control H2O2. Furthermore, the
efficacy of Ghrelin splice variant seems to be greater than that of
Ghrelin.
[0032] FIG. 2. Ghrelin Splice Variant Increases Action Potential
Generation in SNpc DA Neurons in Mice. Ghrelin Splice Variant (1
.mu.M) was applied to the recording chamber via bath application.
Ghrelin Splice Variant induced a significant depolarization (about
10 mV) and increment in AP firing in this DA neuron. The effect of
Ghrelin Splice Variant lasted for at 20 minutes after washout. A.
Raw traces recorded before (Control), during (Ghrelin Splice
Variant), and after (Washout) application of Ghrelin Splice
Variant. B. Time course of Ghrelin Splice Variant-induced increase
in frequency of action potential (FAP).
[0033] FIG. 3. Ghrelin Splice Variant Elevates the Frequency of
mESPCs in WT Mice. A. Significant elevation in the frequency of
mEPSCs compared with saline control after treatment with Ghrelin
Splice Variant (67.2+3.1 (n=6) versus Saline 30.35+2.5 (n=8). B.
Conversely, a significant decrease in the frequency of mIPSCs was
triggered by Ghrelin Splice Variant (Ghrelin Splice Variant:
60.5+8.9 (n=6) versus Saline 72.5+11.2 (n=5)).
[0034] FIG. 4. of Ghrelin Splice Variant on Firing Rate of SNpc DA
Neurons in GHSR1a KO mice and their WT Littermates. In WT animals
Ghrelin Splice Variant (1 .mu.M)-induced mEPSC frequency of
98.1+/-24.7 per min (n=31) and 27.3+/-2.8 per min (n=9) in GHSR-KO
mice indicating the involvement of GHSR-mediated effects of Ghrelin
Splice Variant on SN neurons. The raw traces of mEPSCs recorded in
the presence of TTX (0.5 .mu.M) and bicuculline (30 .mu.M) from WT
and KO mice are shown in A. The mean frequency of mEPSC for WT and
KO mice are shown in B. H current was induced with voltage steps
from -60 mV to -120 mV to identify DA neurons in the SNC as shown
in the box.
[0035] FIG. 5. Effect of Ghrelin Splice Variant on Cumulative
Weight Gain of MPTP and Vehicle Treated Mice. Ghrelin Splice
Variant or vehicle was be administered once-a-day immediately
before the dark phase, and food was removed overnight starting
seven days prior to MPTP challenge and continued until end-point
day 10 after MPTP. Body weight was monitored daily in all mice
administered Ghrelin Splice Variant or Vehicle. Ghrelin Splice
Variant treated group managed to recover after the MP IP treatment
while most of the animals in the Vehicle treated group died.
[0036] FIG. 6. Ghrelin Splice Variant attenuates MPTP-induced
nigrostriatal DA damage. Ghrelin Splice Variant or vehicle were
administered once-a-day immediately before the dark phase, and food
was removed overnight starting seven days prior to MPTP challenge
and continued until end-point day 10 after MPTP. Note the
significant loss of DA neurons in wt mice treated with MPTP
compared with Ghrelin Splice Variant MPTP-treated mice and the un
treated control group.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Applicants specifically incorporate the entire content of
all cited references in this disclosure. Further, when an amount,
concentration, or other value or parameter is given as either a
range, preferred range, or a list of upper preferable values and
lower preferable values, this is to be understood as specifically
disclosing all ranges formed from any pair of any upper range limit
or preferred value and any lower range limit or preferred value,
regardless of whether ranges are separately disclosed. Where a
range of numerical values is recited herein, unless otherwise
stated, the range is intended to include the endpoints thereof, and
all integers and fractions within the range. It is not intended
that the scope of the invention be limited to the specific values
recited when defining a range.
[0038] In the context of this disclosure, a number of terms shall
be utilized.
[0039] "Affinity" as used herein means the strength of binding
between receptors and their ligands, for example between an
antibody and its antigen.
[0040] "Amino acid residue" as used herein means an amino acid
formed upon chemical digestion (hydrolysis) of a polypeptide at its
peptide linkages. The amino acid residues described herein are
preferably in the "L" isomeric form. However, the amino acid
encompasses every amino acid such as L-amino acid, D-amino acid,
alpha-amino acid, beta-amino acid, gamma-amino acid, natural amino
acid and synthetic amino acid or the like as long as the desired
functional property is retained by the polypeptide. NH.sub.2 refers
to the free amino group present at the amino terminus of a
polypeptide. COOH refers to the free carboxy group present at the
carboxy terminus of a polypeptide. Standard polypeptide
abbreviations for amino acid residues are shown in Table 1.
TABLE-US-00001 TABLE 1 1-Letter Code 3-Letter Code Amino Acid A Ala
Alanine B Asx, Asn, and/or Asp Aspartic Acid and/or Asparagine C
Cys Cysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe
Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys
Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro
Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine
V Val Valine W Trp Tryptophan X Xaa Unknown or Other Y Tyr Tyrosine
Z Glx, Gln, and/or Glu Glutamic Acid and/or glutamine -- Dpr
2,3-diaminopropionic acid
It should be noted that all amino acid residue sequences
represented herein by formula have a left-to-right orientation in
the conventional direction of amino terminus to carboxy terminus.
In addition, the phrase "amino acid residue" is broadly defined to
include the amino acids listed in the Table 1 and modified and
non-naturally occurring amino acids. Furthermore, it should be
noted that a dash at the beginning or end of an amino acid residue
sequence indicates a peptide bond to a further sequence of one or
more amino acid residues or a covalent bond to an amino-terminal
group such as NH.sub.2 or acetyl or to a carboxy-terminal group
such as COOH.
[0041] "Parkinson's disease" as used herein means a slowly
progressive neurologic disease characterized by a fixed
inexpressive face, a tremor at rest, slowing of voluntary
movements, a gait with short accelerating steps, peculiar posture
and muscle weakness, caused by degeneration of an area of the brain
called the basal ganglia, and by low production of the
neurotransmitter dopamine.
[0042] "Parkinson's disease specific assessments" as used herein
means tests used to assess the severity of Parkinson's disease in a
mammal. For example: Parts I, II, and III of the revised Movement
Disorders Society Unified Parkinson Disease Rating Scale
(MDS-UPDRS) (Goetz et al. Mov Disord 15; 23(15):2129-70 (2008))
and/or Geriatric Depression Scale (GDS) (Yesavage et al. J
Psychiatr Res 17(1):37-49 (1982)) and/or The Montreal Cognitive
Assessment (MoCA) (Nasreddine et al. J Am Geriatr Soc; 53(4):695-9
(2005))
[0043] "Activating a neuron" as used herein refers to increasing
the action potential frequency (i.e., firing rate) above baseline.
Baseline frequency is the frequency in the absence of a ghrelin or
ghrelin mimetic. A dopamine neuron releases dopamine when it is
activated.
[0044] "Alzheimer's disease" as used herein means a multifactorial
progressive neurodegenerative disorder characterized by loss of
memory and cognitive deficits, strongly influenced by the metabolic
status, in which the impairment of neuropeptides/neurotransmitters
systems has been previously observed. Alzheimer's disease is the
most common form of both senile and presenile dementia in the world
and is recognized clinically as relentlessly progressive dementia
that presents with increasing loss of memory, intellectual function
and disturbances in speech (Merritt, 1979, A Textbook of Neurology,
6th edition, pp. 484-489 Lea & Febiger, Philadelphia). The
disease itself usually has a slow and insidious progress that
affects both sexes equally, worldwide. It begins with mildly
inappropriate behavior, uncritical statements, irritability, a
tendency towards grandiosity, euphoria and deteriorating
performance at work; it progresses through deterioration in
operational judgement, loss of insight, depression and loss of
recent memory; it ends in severe disorientation and confusion,
apraxia of gait, generalized rigidity and incontinence (Gilroy
& Meyer, 1979, Medical Neurology, pp. 175-179 MacMillan
Publishing Co) Alzheimer's disease afflicts an estimated 4 million
human beings in the United States alone at a cost of 35 billion
dollars a year (Hay & Ernst, 1987, Am. J. Public Health,
77:1169-1175). It is found in 10% of the population over the age of
65 and 47% of the population over the age of 85 (Evans et al.,
1989, JAMA, 262:2551-2556). In addition, the disease is found at
much lower levels in the younger age groups, usually beginning at
about 30 years of age and even rarely in late childhood (Adams
& Victor, 1977, Principles of Neurology, pp. 401-407). The
etiology of Alzheimer's disease is unknown.
[0045] "Body Mass Index" or "BMI" is a measure of an individual's
height to weight ratio. BMI is determined by calculating weight in
kilograms divided by the square of height in meters. The BMI
"normal" range is 18.5-25.
[0046] "Body fat mass" can be measured, e.g., by the fat fold
technique. In the fat fold technique, a pincer-type caliper is used
to measure subcutaneous fat by determining skin fold thickness at
representative sites on the body. These skin fold measurements are
then used to compute body fat by either adding the scores from the
various measurements, and using this value as an indication of the
relative degree of fatness among individuals, or by using the
measurements in mathematical equations that have been developed to
predict percent body fat (Fogelholm M. & van Marken Lichtenbelt
W., Eur. J. Clin. Nutr. 51:495-503 (1997)).
[0047] "Concentration equivalent" as used herein means an
equivalent dosage of a ghrelin splice variant-like compound having
in vitro and/or in vivo the same response as evaluated from a
dosage-response curve of the ghrelin splice variant.
[0048] "Dissociation constant" or "Kd" is a measure describing the
strength of binding (or affinity or avidity) between receptors and
their ligands, for example an antibody and its antigen. The smaller
the Kd, the stronger the binding.
[0049] A "fusion polypeptide" is a polypeptide comprised of at
least two polypeptides and a linking sequence to operatively link
the two polypeptides into one continuous polypeptide. The two
polypeptides linked in a fusion polypeptide are typically derived
from two independent sources, and therefore a fusion polypeptide
comprises two linked polypeptides not normally found linked in
nature.
[0050] "Human ghrelin" as used herein is a polypeptide having the
amino acid sequence as set forth in GenBank.RTM. Accession No.
NP_057446 or Swiss-Prot Identifier GHRL_HUMAN. Human ghrelin
preprotein has 117 amino acids. This preprotein undergoes the
following post-translational processing. The signal peptide (amino
acids 1-23) is removed and the remaining 94 amino acids are cleaved
by a protease to provide a mature 28 amino acid ghrelin (amino
acids 24-51) or a mature 27 amino acid ghrelin (amino acids 24-50)
and a mature 23 amino acid obestatin (amino acids 76-98). The 27 or
28 amino acid mature ghrelin peptides can be further modified at
the serine at position 26 in the preprotein by either an 0-octanoyl
group or an O-decanoyl group. The obestatin mature peptide can be
further modified at the lysine at position 98 of the preprotein by
an amide group. An additional ghrelin preprotein is known, which
lacks the glutamine at position 37 of the preprotein.
[0051] "Ghrelin splice variant" is a polypeptide having the amino
acid sequence as set forth in SEQ ID NO:1 or any peptide of 15
amino acids or more from SEQ ID NO:1 with or without post
translational modification, or any SEQ ID NO:1 homologs as set
forth in SEQ ID NO:7 or SEQ ID NO:8, and/or any peptide of 15 amino
acids or more from SEQ ID NO:7 or SEQ ID NO:8 with or without post
translational modification.
[0052] "Ghrelin splice variant-like compound" as used herein refers
to any compound which mimics the function of ghrelin splice
variant, in particular human ghrelin splice variant, particularly
in terms of the ghrelin splice variant functions leading to the
desired therapeutic effects described herein, such as stimulation
of appetite and/or treatment and/or prophylaxis of
neurodegeneration and is defined by the Formula I:
Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1 is an optionally present
protecting group; each X1 is independently selected from a
naturally occurring amino acid and a synthetic amino acid; X2 is
selected from a naturally occurring amino acid and a synthetic
amino acid, said amino acid being modified with a bulky hydrophobic
group; each X3 is independently selected from a naturally occurring
amino acid and a synthetic amino acid, wherein one or more of X1
and X3 optionally may be modified with a bulky hydrophobic group;
Z2 is an optionally present protecting group; m is an integer in
the range of from 1-10; n is an integer in the range of from 4-92;
provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% (or, in alternative embodiments, 85%, 90%, 93%, 95%, 97%,
98%, 99%, 100%) homology to SEQ ID NO:1. In a preferred embodiment,
the ghrelin splice variant-like compound is 22-29 amino acids in
length.
[0053] "Immunologically distinct" refers to the ability to
distinguish between two polypeptides on the ability of an antibody
to specifically bind one of the polypeptides and not specifically
bind the other polypeptide.
[0054] An "individual" is an animal or human susceptible to a
condition, in particular a cachectic condition as defined herein.
In preferred embodiments, the individual is a mammal, including
human, and non-human mammals such as dogs, cats, pigs, cows, sheep,
goats, horses, rats, and mice. In the most preferred embodiment,
the individual is a human.
[0055] "Isolated" is used to describe the various ghrelin splice
variant-like compounds, i.e. polypeptides and nucleotides disclosed
herein, that have been identified and separated and/or recovered
from a component of its natural environment. Contaminant components
of a natural environment are materials that would typically
interfere with diagnostic or therapeutic uses for the polypeptide,
and may include enzymes, hormones, and other proteinaceous or
non-proteinaceous solutes. In preferred embodiments, the ghrelin
splice variant-like compounds will be purified.
[0056] "Modified amino acid" as used herein is an amino acid
wherein an arbitrary group thereof is chemically modified.
[0057] "Non-standard amino acid" as used herein is an amino acid
that does not belong to the standard 20 amino acids. Non-standard
amino acids are usually formed through chemical modifications to
standard amino acids. They can also be formed naturally as an
intermediate component of a metabolic pathway or by microorganisms
and/or plants.
[0058] "Monoclonal Antibody", in its various grammatical forms,
refers to a population of antibody molecules that contains only one
species of antibody combining site capable of immunoreacting with a
particular antigen.
[0059] A "non-acylated ghrelin splice variant-like compound" is a
ghrelin splice variant-like compound, as defined herein, which does
not contain an acyl group attached to any of its constituent amino
acids.
[0060] "Palliative treatment" is a treatment which relieves or
sooths the symptoms of a disease or disorder but without a curing
effect.
[0061] "Polyclonal antibodies" are a mixture of antibody molecules
recognizing a specific given antigen; hence, polyclonal antibodies
may recognize different epitopes within said antigen.
[0062] "Polypeptide" refers to a molecule comprising amino acid
residues which do not contain linkages other than amide linkages
between adjacent amino acid residues.
[0063] "Processed ghrelin" means the 27 or 28 amino acid residue,
acylated ghrelin (the post-translational product of cleavage and
acylation of 116 and 117 amino acid residues long preproghrelin,
respectively (e.g., SWISS-PROT Q9UBU3 GHRL_HUMAN)).
[0064] A "receptor" is a molecule, such as a protein, glycoprotein
and the like, that can specifically (non-randomly) bind to another
molecule.
[0065] A "secretagogue" is a substance stimulating growth hormone
release, such as ghrelin or a ghrelin-like compound. A secretagogue
according to the present disclosure may for example be selected
from L-692-429 and L-692-585 (benzoelactam compounds; available
from Merck & Co, Inc., Whitehouse Station, N.J.), MK677
(spiroindaner; available from Merck) G-7203, G-7039, G-7502
(isonipecotic acid peptidomimetic; available from Genentech, Inc.,
South San Francisco, Calif.), NN703 (Novo Nordisk Inc., Princeton,
N.J.), or ipamorelin. In particular, the secretagogue is a ghrelin
splice variant-like compound, including a 29 amino acid human
ghrelin splice variant, a 24 amino acid human ghrelin splice
variant, or a 22 amino acid human ghrelin splice variant (e.g., SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5). The growth
hormone secretagogue may in one embodiment be non-acylated, for
instance a non-acylated form of ghrelin splice variant or a
non-acylated ghrelin splice variant-like compound.
[0066] A "surfactant molecule" is a molecule comprising a
hydrophobic part and a hydrophilic part; i.e. a molecule capable of
being present in the interphase between a lipophilic phase and a
hydrophilic phase.
Indications
[0067] The present disclosure relates to the use of a secretagogue,
such as a ghrelin splice variant-like compound, in the treatment or
prophylaxis of conditions, e.g., relating to neuronal damage and/or
neurodegenerative disease and/or depression including a)
prophylaxis or treatment of parkinson's disease, and/or b)
prophylaxis or treatment of Alzheimer's disease, and/or c)
prophylaxis or treatment of neuron degeneration, and/or d)
prophylaxis or treatment of depression, and/or e) stimulation of
neuron activity, fg. In particular, the present disclosure relates
to the treatment or prophylaxis of parkinson's disease and/or
prophylaxis of alzheimer's disease and/or the stimulation of neuron
activity, prevention of neuron degeneration and/or the treatment or
prophylaxis of depression, increasing quality of life, most
preferably the prophylaxis or treatment of parkinson's disease
and/or alzheimer's disease.
[0068] Neurodegenerative Disease
[0069] In accordance with the above, the compounds disclosed herein
are particularly suitable for treating or preventing neuronal
damage and/or neurodegenerative diseases in an individual suffering
from the following Alzheimer's disease, Parkinson's disease,
Huntington's disease, Amyotrophic lateral sclerosis (ALS), Aging.
In another preferred embodiment, a secretagogue, such as ghrelin
splice variant or a ghrelin splice variant-like compound, is used
in the treatment or prevention of neuronal damage and/or
neurodegenerative disease, Alzheimer's disease, Parkinson's
disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS),
Aging. In one preferred embodiment, the individual treated for
neuronal damage and/or neurodegenerative disease is elderly, such
as 60-120 years old, such as 70-120 years old, such as 80-120 years
old, such as 90-120 years old. Equally preferable are embodiments
where said individual is a child, such as from 0-20 years old, such
as 0-15 years old, such as 0-10 years old, such as 0-5 years old,
such as 0-1 years old, such as a newborn child less than 2 months
old.
[0070] In one embodiment, it is preferred that the secretagogue,
such as a ghrelin splice variant-like compound, is administered
prophylactically for preventing a neurodegenerative state. In this
embodiment, the treatment may be started before any
anti-neurodegenerative treatment initiates. The secretagogue may be
administered continuously during the anti-neurodegenerative
treatment, or it may be administered at intervals, for example
between periods with anti-neurodegenerative therapy. By
administering during and in particular between the periods of
anti-neurodegenerative therapy, the risk that the treated
individual acquires infections and/or other complications may be
reduced due to better health conditions of the individual.
[0071] Treatment of neuronal damage and/or neurodegenerative
disease using a secretagogue, such as ghrelin splice variant or a
ghrelin splice variant-like compound, may be achieved using any
administration method known in the art. Preferably, treatment may
be achieved using any of the administration methods described
herein, more preferably using intravenous or subcutaneous
administration, most preferably using subcutaneous administration
methods.
Subcutaneous Administration
[0072] It is important to note that ghrelin and ghrelin splice
variant activate the GHS receptors and additional yet to be
identified receptors. These receptors are found on GH producing
cells, in the hypothalamic centers for appetite control and in a
number of additional places in the organism. In the CNS, these
receptors are tuned to receiving signals from local ghrelin splice
variant-containing neurons. Peripherally-secreted or
artificially-administered ghrelin splice variants will reach such
sites and will pass the blood brain barrier specifically activating
the appropriate receptors and triggering a specific pathway.
However, currently available "so-called" GH secretagogues, which
are small organic compounds such as MK-0677 (Merck), generally
targeted to bind the GHS receptor will pass the blood brain barrier
and also reach these sites, activating various GHS receptor related
pathways and consequently having the danger of causing unwanted
side effects such as dizziness, nausea, falling, elevated fasting
serum glucose and insulin, and blurred vision. Thus, such compounds
which do have the advantage of being, for example, orally active
will not be optimal for mimicking the natural pre-meal,
appetite-inducing surge of ghrelin splice variant, since they will
activate non-specifically all GHS receptor related pathways in the
body. In contrast, by using the natural peptide, ghrelin splice
variant itself, or homologues thereof, and administering it
peripherally--as in a preferred embodiment--it is ensured that only
the relevant, appetite-regulating ghrelin splice variant receptors
and pathways are reached and stimulated.
[0073] Any parenteral administration form that will ensure that the
ghrelin splice variant receptors which normally are the target for
peripherally-produced ghrelin splice variant in the pre-meal
situation will be exposed to sufficient levels of the bioactive
form of ghrelin splice variant to ensure robust and appropriate
appetite stimulation, without causing desensitization of the
system, may be part of an embodiment of the present disclosure.
However, taking into consideration that the individuals to be
treated possibly will have to receive treatment for a longer
period, such as weeks or months, it is preferred that the
administration form is well-suited thereto. Accordingly, it is
preferred that the secretagogue, such as a ghrelin splice
variant-like compound, is administered subcutaneously in an amount
allowing sufficient levels of the bioactive form of ghrelin splice
variant, i.e. the acylated form, to reach the receptors prior to
the forthcoming meal.
[0074] The present disclosure preferably deals with methods for
administering a secretagogue, such as ghrelin splice variant, in a
way which mimics the physiologically pre-meal situation as closely
as possible yet provides patients in need of increased food intake,
for example fragile elderly, post-operative patients, and/or
patients with lost appetite as part of neuronal damage and/or
neurodegenerative disease for example precipitated by Parkinson's
disease, alzheimer's disease, etc., with a sufficient extra
stimulatory input to their appetite-regulating ghrelin splice
variant receptors, which normally are reached by ghrelin splice
variant in the pre-meal situation.
[0075] Bolus Administration
[0076] Furthermore, from a molecular pharmacological point-of-view,
it is important to note that it has been found that the ghrelin
receptor, and therefore ghrelin splice variant receptor, normally
is exposed to short-lived surges in ghrelin concentration. The
GHS-R 1a receptor (growth hormone secretagogue receptor 1a) belongs
to the class of G protein coupled receptors or 7TM receptors, which
upon continued exposure to an agonist will be desensitized,
internalized, and down-regulated. These mechanisms, which are
inherent to the overall signal transduction system, involve
processes such as receptor phosphorylation (which, in itself,
decreases the affinity of the receptor for the agonist) and binding
of inhibitory proteins such as arrestin (which sterically block the
binding of signal transduction molecules such as G proteins).
Another part of the agonist-mediated desensitization process is
receptor internalization (i.e. physical removal of the receptor
from the cell surface where it could bind the agonist) as well as
receptor down regulation (i.e. decreased production/expression of
the receptor). Receptor internalization could, after short-lived
exposure of the receptor to agonist, be followed by a
re-sensitization process, where the receptor is dephosphorylated
and recycled to the cell surface to be used again. Without being
bound by theory, it is believed that, upon prolonged stimulation
which would occur for example during a long-lasting continuous
infusion of the agonist, the receptor down-regulation process
ensures that the target cell is adjusted in its signal transduction
system to this situation.
Optimal Administration
[0077] The present disclosure also provides a procedure for an
optimal administration of ghrelin splice variant to patients in
order to obtain a maximal response and to avoid, for example,
desensitization mechanisms.
[0078] Accordingly, one aspect relates to administration of a
secretagogue, such as a ghrelin splice variant-like compound, in
boluses, preferably a bolus prior to each main meal. It has been
found, in contrary to the prolonged administration processes in the
prior art, that a bolus administration leads not only to
stimulation of appetite, but also to stimulation of feed intake and
more important to stimulation of weight gain. Without being bound
by theory, it is believed that pre-meal subcutaneous injection,
intravenous injection, or short-term infusions of appropriate doses
of a secretagogue, such as ghrelin splice variant or a ghrelin
splice variant-like compound, will ensure that a robust stimulation
of appetite-inducing ghrelin splice variant receptors will be
obtained with minimal constraint to, e.g., the mobility of the
patient. Thus, for example, patients with hip fractures can, in the
post-operative situation, be treated in the pre-meal period and, if
required during the meal as such, be free to move around and
participate in the important post-operative physiotherapeutic
regimens. In one preferred embodiment, a secretagogue such as
ghrelin splice variant or a ghrelin splice variant-like compound is
administered as a bolus in an amount equivalent to 10 .mu.g per kg
body weight.
Ghrelin Splice Variant-Like Compound
[0079] Any secretagogue, such as ghrelin splice variant or a
ghrelin splice variant-like compound, may be used in the presently
disclosed methods. One preferred type of ghrelin splice
variant-like compound described herein is a compound comprising a
structure defined by Formula I: Z1-(X1)m-(X2)-(X3)n-Z2, wherein Z1
is an optionally present protecting group; each X1 is independently
selected from a naturally occurring amino acid and a synthetic
amino acid; X2 is selected from a naturally occurring amino acid
and a synthetic amino acid, said amino acid being modified with a
bulky hydrophobic group; each X3 is independently selected from a
naturally occurring amino acid and a synthetic amino acid, wherein
one or more of X1 and X3 optionally may be modified with a bulky
hydrophobic group; Z2 is an optionally present protecting group; m
is an integer in the range of from 1-10; n is an integer in the
range of from 4-92; provided that the compound according to formula
Z1-(X1)m-(X2)-(X3)n-Z2 is 15-94 amino acids in length and has at
least 80% (or, in alternative embodiments, 85%, 90%, 93%, 95%, 97%,
98%, 99%, 100%) homology to SEQ ID NO:1. In a preferred embodiment,
the ghrelin splice variant-like compound is 22-29 amino acids in
length.
[0080] Accordingly, the term "secretagogue" includes the naturally
occurring 29 amino acid human ghrelin splice variant, the amino
acid sequence of which is shown in SEQ ID NO:5, as well as the
naturally occurring 22 amino acid human ghrelin splice variant, the
amino acid sequence of which is shown in SEQ ID NO:2, and the
naturally occurring 24 amino acid human ghrelin splice variant, the
sequence of which is shown in SEQ ID NO:3.
[0081] The present disclosure includes diastereomers as well as
their racemic and resolved enantiomerically pure forms.
Secretagogues can contain D-amino acids, L-amino acids, alpha-amino
acid, beta-amino acid, gamma-amino acid, natural amino acid and/or
synthetic amino acid or the like or a combination thereof.
Preferably, amino acids present in a ghrelin splice variant-like
compound are the L-enantiomers.
[0082] The ghrelin splice variant-like compound preferably
comprises an amino acid modified with a bulky hydrophobic group.
The number of amino acids N-terminal to the modified amino acid is
preferably within the range of from 1-9. Accordingly, m is
preferably an integer in the range of from 1-9, such as of from
1-8, such as of from 1-7, such as of from 1-6, such as of from 1-5,
such as of from 1-4, such as of from 1-3, such as of from 1-2, such
as 2.
[0083] It is more preferred that the number of amino acids
N-terminally to the modified amino acid is low, such as of from
1-3, such as of from 1-2. Most preferably 2 amino acids are
positioned N-terminal to the modified amino acid.
[0084] In a preferred embodiment, (X1)m has a Gly residue in the
N-terminal part of the sequence. Accordingly, in preferred
embodiment, (X1)m is selected from the sequences: Gly, Gly-Ser,
Gly-Cys, Gly-Lys, Gly-Asp, Gly-Glu, Gly-Arg, Gly-His, Gly-Asn,
Gly-Gln, Gly-Thr, and Gly-Tyr. More preferably (X1)m is selected
from Gly-Ser, and Gly-Cys; most preferably (X1)m is Gly-Ser.
[0085] In other words, in a preferred embodiment the ghrelin splice
variant-like compound is selected from Z1-Gly-(X1)m-(X2)-(X3)n-Z2
(Formula II), Z1-Gly-Ser-(X2)-(X3)n-Z2 (Formula III), and
Z1-Gly-(X2)-(X3)n-Z2 (Formula IV). More preferably, the ghrelin
splice variant-like compound has Formula III.
[0086] As described above, X2 may be any amino acid modified with a
bulky hydrophobic group. In particular, X2 is selected from the
group consisting of modified Ser, modified Cys, modified Asp,
modified Lys, modified Trp, modified Phe, modified Ile, and
modified Leu. More preferably, X2 is selected from the group
consisting of modified Ser, modified Cys, and modified Lys; most
preferably X2 is modified Ser.
[0087] Furthermore, (X1)m-(X2) is preferably Gly-Xaa-Ser* or
Gly-Xaa-Cys*, wherein Xaa is any amino acid, more preferably
(X1)m-(X2) is Gly-Ser-Ser* or Gly-Ser-Cys*, wherein * indicates
that the amino acid residue is modified with a bulky hydrophobic
group.
[0088] (X3)n preferably comprises a sequence which is a fragment of
ghrelin splice variant, such as human ghrelin splice variant.
Accordingly, (X3)n preferably comprises a fragment of the following
sequence (SEQ ID NO:6): Phe Leu Ser Pro Glu His Gln Arg Val Gln Val
Arg Pro Pro His Lys Ala Pro His Val Val Pro Ala Leu Pro Leu Ser Asn
Gln Leu Cys Asp Leu Glu Gln Gln Arg His Leu Trp Ala Ser Val Phe Ser
Gln Ser Thr Lys Asp Ser Gly Ser Asp Leu Thr Val Ser Gly Arg Thr Trp
Gly Leu Arg Val Leu Asn Arg Leu Phe Pro Pro Ser Ser Arg Glu Arg Ser
Arg Arg Ser His Gln Pro Ser Cys Ser Pro Glu Leu.
[0089] In a preferred embodiment, the length of the ghrelin splice
variant-like compound is substantially similar to the length of
processed human ghrelin, i.e. 29 amino acids. Accordingly, n is
preferably an integer in the range of from 4 to 25, such as of from
4 to 24, such as of from 4 to 22, such as of from 4 to 15, such as
of from 4 to 10, such as of from 10 to 25, such as of from 10 to
24, such as of from 15 to 25, such as of from 15 to 24. Most
preferably, a ghrelin splice variant-like compound is the 29 amino
acid human ghrelin splice variant, the amino acid sequence of which
is shown in SEQ ID NO:5; is the 22 amino acid human ghrelin splice
variant, the amino acid sequence of which is shown in SEQ ID NO:2;
is the 24 amino acid human ghrelin splice variant, the amino acid
sequence of which is shown in SEQ ID NO:3; or is the 24 amino acid
human ghrelin splice variant having a 2,3-diaminopropionic acid
(Dpr) residue in the third position, the amino acid sequence of
which is shown in SEQ ID NO:4.
Functionality
[0090] The secretagogues described herein are active at the
receptor for GHS as described above, i.e. the receptor GHS-R 1a.
The compounds can bind to GHS-R 1a, and preferably, stimulate
receptor activity. In some embodiments, the compounds can bind
other receptors and, optionally, stimulate their activity.
[0091] Receptor activity can be measured using different techniques
such as detecting a change in the intracellular conformation of the
receptor, in the G-protein coupled activities, and/or in the
intracellular messengers. One simple measure of the ability of a
ghrelin splice variant-like compound to activate the ghrelin splice
variant receptor is to measure its EC50, i.e. the dose at which the
compound is able to activate the signaling of the receptor to half
of the maximal effect of the compound. When measuring, e.g., EC50,
the receptor can either be expressed endogenously on primary cell
cultures, for example pituitary cells, or heterologously expressed
on cells transfected with the ghrelin receptor. Whole cell assays
or assays using membranes prepared from either of these cell types
can be used depending on the type of assay.
[0092] As the receptor is generally believed to be primarily
coupled to the Gq signaling pathway, any suitable assay which
monitors activity in the Gq/G11 signaling pathway can be used, for
example: [0093] 1) An assay measuring the activation of Gq/G11
performed, for example, by measurement of GTPgS binding combined
with, e.g., anti-G-alpha-q or -11 antibody precipitation in order
to increase the signal to noise ratio. This assay may also detect
coupling to other G-proteins than Gq/11. [0094] 2) An assay which
measures the activity of phospholipase C (PLC), one of the first
down-stream effector molecules in the pathway, for example by
measuring the accumulation of inositol phosphate which is one of
the products of PLC. [0095] 3) More down stream in the signaling
cascade is the mobilization of calcium from the intracellular
stores, which can be measured by any method known to one of
ordinary skill in the art. [0096] 4) Even more down stream,
signaling molecules such as the activity of different kinds of MAP
kinases (p38, jun, etc.), NF.kappa.B translocation and CRE driven
gene transcription may also be measured. [0097] 5) Binding of
fluorescently-tagged arrestin to the activated ghrelin
receptor.
[0098] Examples of suitable protocols for use in determining
secretagogue functionality are given in Example 4, infra.
[0099] In one embodiment, the binding of a compound to the receptor
GHS-R 1a can be measured by the use of the assay described herein
above.
[0100] A ghrelin splice variant-like compound preferably has at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, or at least about 90%, functional activity relative to
the 28 amino acid human wild-type ghrelin as determined using an
assay described herein above, and/or an EC50 greater than about
1,000, greater than about 100, or greater than about 50, or greater
than about 10. Greater refers to potency and thus indicates a
lesser amount is needed to achieve binding inhibition.
[0101] In one embodiment, the compound has potency (EC50) on the
GHS-R 1a of less than 500 nM. In another embodiment the compound
has a potency (EC50) on the GHS-R 1a of less than 100 nM, such as
less than 80 nM, such as less than 60 nM, such as less than 40 nM,
such as less than 20 nM, such as less than 10 nM, such as less than
5 nM, such as less than 1 nM, such as less than 0.5 nM, such as
less than 0.1 nM, such as less than 0.05 nM, such as less than 0.01
nM.
[0102] In a further embodiment, the dissociation constant (Kd) of
the compound is less than 500 nM. In a still further embodiment the
dissociation constant (Kd) of the ligand is less than 100 nM, such
as less than 80 nM, such as less than 60 nM, such as less than 40
nM, such as less than 20 nM, such as less than 10 nM, such as less
than 5 nM, such as less than 1 nM, such as less than 0.5 nM, such
as less than 0.1 nM, such as less than 0.05 nM, such as less than
0.01 nM.
[0103] Binding assays can be performed using recombinantly-produced
receptor polypeptides present in different environments. Such
environments include, for example, cell extracts and purified cell
extracts containing the receptor polypeptide expressed from
recombinant nucleic acid or naturally occurring nucleic acid, and
also include, for example, the use of a purified GHS receptor
polypeptide produced by recombinant means or from naturally
occurring nucleic acid which is introduced into a different
environment. Using a recombinantly expressed GHS receptor offers
several advantages, such as the ability to express the receptor in
a defined cell system, so that a response to a compound at the
receptor can more readily be differentiated from responses at other
receptors. For example, the receptor can be expressed in a cell
line such as HEK 293, COS 7, and CHO not normally expressing the
receptor by an expression vector, wherein the same cell line
without the expression vector can act as a control.
Identity and Homology
[0104] The term "identity" or "homology" shall be construed to mean
the percentage of amino acid residues in the candidate sequence
that are identical with the residue of a corresponding sequence to
which it is compared, after aligning the sequences and introducing
gaps, if necessary to achieve the maximum percent identity for the
entire sequence, and not considering any conservative substitutions
as part of the sequence identity. Neither N- or C-terminal
extensions nor insertions shall be construed as reducing identity
or homology. Methods and computer programs for the alignment are
well known in the art. Sequence identity may be measured using
sequence analysis software (e.g., Sequence Analysis Software
Package, Genetics Computer Group, University of Wisconsin
Biotechnology Center, Madison, Wis.). This software matches similar
sequences by assigning degrees of homology to various
substitutions, deletions, and other modifications.
[0105] A ghrelin splice variant homologue of one or more of the
sequences specified herein may vary in one or more amino acids as
compared to the sequences defined, but is capable of performing the
same function, i.e. a homologue may be envisaged as a functional
equivalent of a predetermined sequence.
[0106] A ghrelin splice variant homologue is preferably a ghrelin
splice variant-like compound as defined above.
[0107] As described above, a homologue of any of the predetermined
sequences herein may be defined as i) homologues comprising an
amino acid sequence capable of being recognized by an antibody,
said antibody also recognizing the 22 amino acid and/or the 24
amino acid human ghrelin splice variant and/or the 29 amino acid
human ghrelin splice variant, preferably the acylated 22 amino acid
and/or the 24 amino acid human ghrelin splice variant and/or the 29
amino acid human ghrelin splice variant, and/or ii) homologues
comprising an amino acid sequence capable of binding selectively to
GHS-R 1a, and/or iii) homologues having a substantially similar or
higher binding affinity to GHS-R 1a than the 22 amino acid and/or
the 24 amino acid human ghrelin splice variant and/or the 29 amino
acid human ghrelin splice variant, preferably the acylated 22 amino
acid and/or the 24 amino acid human ghrelin splice variant and/or
the 29 amino acid human ghrelin splice variant. (The 22 amino acid
and/or the 24 amino acid and/or the 29 amino acid human ghrelin
splice variant can have the sequence shown in SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, or SEQ ID NO:5, and, when acylated, is acylated
at position 3.)
[0108] The antibodies used herein may be antibodies binding the
N-terminal region of ghrelin splice variant or the C-terminal
region of ghrelin splice variant, preferably the N-terminal region.
The antibodies may be antibodies as described in Ariyasu H. et al.,
Endocrinology 143:3341-50 (2002).
[0109] Exemplary homologues comprise one or more conservative amino
acid substitutions including one or more conservative amino acid
substitutions within the same group of predetermined amino acids,
or a plurality of conservative amino acid substitutions, wherein
each conservative substitution is generated by substitution within
a different group of predetermined amino acids. Homologues may thus
comprise conservative substitutions independent of one another,
wherein at least one glycine (Gly) of said homologue is substituted
with an amino acid selected from the group of amino acids
consisting of Ala, Val, Leu, and lie, and independently thereof,
homologues, wherein at least one of said alanines (Ala) of said
homologue thereof is substituted with an amino acid selected from
the group of amino acids consisting of Gly, Val, Leu, and Ile; and,
independently thereof, homologues wherein at least one valine (Val)
of said homologue thereof is substituted with an amino acid
selected from the group of amino acids consisting of Gly, Ala, Leu,
and Ile; and, independently thereof, homologues wherein at least
one of said leucines (Leu) of said homologue thereof is substituted
with an amino acid selected from the group of amino acids
consisting of Gly, Ala, Val, and Ile; and, independently thereof,
homologues wherein at least one isoleucine (Ile) of said homologues
thereof is substituted with an amino acid selected from the group
of amino acids consisting of Gly, Ala, Val and Leu; and,
independently thereof, homologues wherein at least one of said
aspartic acids (Asp) of said homologue thereof is substituted with
an amino acid selected from the group of amino acids consisting of
Glu, Asn, and Gln; and, independently thereof, homologues wherein
at least one of said phenylalanines (Phe) of said homologues
thereof is substituted with an amino acid selected from the group
of amino acids consisting of Tyr, Trp, His, and Pro, and preferably
selected from the group of amino acids consisting of Tyr and Trp;
and, independently thereof, homologues wherein at least one of said
tyrosines (Tyr) of said homologues thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Phe, Trp, His, and Pro, preferably an amino acid selected from the
group of amino acids consisting of Phe and Trp; and, independently
thereof, homologues wherein at least one of said arginines (Arg) of
said fragment is substituted with an amino acid selected from the
group of amino acids consisting of Lys and His; and, independently
thereof, homologues wherein at least one lysine (Lys) of said
homologues thereof is substituted with an amino acid selected from
the group of amino acids consisting of Arg and His; and,
independently thereof, homologues wherein at least one of said
asparagines (Asn) of said homologues thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Asp, Glu, and Gln; and, independently thereof, homologues wherein
at least one glutamine (Gln) of said homologues thereof is
substituted with an amino acid selected from the group of amino
acids consisting of Asp, Glu, and Asn; and, independently thereof,
homologues wherein at least one proline (Pro) of said homologues
thereof is substituted with an amino acid selected from the group
of amino acids consisting of Phe, Tyr, Trp, and His; and,
independently thereof, homologues wherein at least one of said
cysteines (Cys) of said homologues thereof is substituted with an
amino acid selected from the group of amino acids consisting of
Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, and Tyr.
[0110] Conservative substitutions may be introduced in any position
of a preferred predetermined sequence. It may however also be
desirable to introduce non-conservative substitutions,
particularly, but not limited to, a non-conservative substitution
in any one or more positions.
[0111] A non-conservative substitution leading to the formation of
a functionally equivalent homologue of the sequences herein would
for example i) differ substantially in polarity, for example a
residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile,
Gly, Leu, Phe or Met) substituted for a residue with a polar side
chain such as Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino
acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a
polar residue for a non-polar one; and/or ii) differ substantially
in its effect on polypeptide backbone orientation such as
substitution of or for Pro or Gly by another residue; and/or iii)
differ substantially in electric charge, for example substitution
of a negatively charged residue such as Glu or Asp for a positively
charged residue such as Lys, His or Arg (and vice versa); and/or
iv) differ substantially in steric bulk, for example substitution
of a bulky residue such as His, Trp, Phe or Tyr for one having a
minor side chain, e.g. Ala, Gly or Ser (and vice versa).
[0112] Substitution of amino acids may in one embodiment be made
based upon their hydrophobicity and hydrophilicity values and the
relative similarity of the amino acid side-chain substituents,
including charge, size, and the like. Exemplary amino acid
substitutions which take various of the foregoing characteristics
into consideration are well known to those of skill in the art and
include, for example, arginine and lysine; glutamate and aspartate;
serine and threonine; glutamine and asparagine; and valine,
leucine, and isoleucine.
[0113] In a preferred embodiment, the binding domain comprises a
homologue having an amino acid sequence at least 60% homologous to
SEQ ID NO:1. More preferably the homology is at least 65%, such as
at least 70% homologous, such as at least 75% homologous, such as
at least 80% homologous, such as at least 85% homologous, such as
at least 90% homologous, such as at least 95% homologous, such as
at least 98% homologous to SEQ ID NO:1. In a more preferred
embodiment, the percentages mentioned above relate to the identity
of the sequence of a homologue as compared to SEQ ID NO:1.
Homologues to SEQ ID NO:1 may be 22 amino acid human ghrelin splice
variant (SEQ ID NO:2), 24 amino acid human ghrelin splice variant
(SEQ ID NO:3), or 29 amino acid human ghrelin splice variant (SEQ
ID NO:5). Other homologues are the variants described in EP 1 197
496 (Kangawa), incorporated herein by reference.
Bulky Hydrophobic Group
[0114] The bulky hydrophobic group of secretagogues disclosed
herein is any bulky hydrophobic group capable of providing the
des-acylated 28 amino acid human wild-type ghrelin, or an analogue
thereof, with binding affinity to GHS-R 1a and or GS-R-lb. Any
suitable amino acid may be modified with any suitable bulky
hydrophobic group. In a preferred embodiment, a Ser residue
(preferably, amino acid number 3 in the ghrelin splice variant
amino acid chain) is modified with the bulky hydrophobic group.
When the amino acid being modified contains, e.g., --OH, --SH, --NH
or --NH.sub.2 as a substituent group in a side chain thereof, a
group formed by acylating such a substituent group is preferred.
The mode of linkage may thus be selected from the group consisting
of ester, ether, thioester, thioether, amide and carbamide. For
example, if the modified amino acid is serine, threonine, tyrosine
or oxyproline, the amino acid has a hydroxyl group in the side
chain. If the modified amino acid is cysteine, the amino acid has a
mercapto group in the side chain. If the modified amino acid is
lysine, arginine, histidine, tryptophan, proline or oxyproline, it
has an amino group or imino group in the side chain.
[0115] The hydroxyl group, mercapto group, amino group and imino
group described above may thus be chemically modified. That is, the
hydroxyl group or mercapto group may be, for example, etherized,
esterified, thioetherified or thioesterified. The imino group may
be, for example, iminoetherified, iminothioetherified or alkylated.
The amino group may be, for example, amidated, thioamidated or
carbamidated. Further, the mercapto group may be, for example,
disulfidated; the imino group may be, for example, amidated or
thioamidated; and the amino group may be, for example, alkylated or
thiocarbamidated.
[0116] In a preferred embodiment, the modified amino acid is Ser
coupled through an ester linkage to the hydrophobic group or, in
another preferred embodiment, the modified amino acid is Dpr
coupled through an amide linkage to the hydrophobic group.
[0117] The hydrophobic group may be any group with a saturated or
unsaturated alkyl or acyl group containing one or more carbon
atoms. In one embodiment, the bulky hydrophobic group is an acyl
group, including groups formed by removing a hydroxyl group from an
organic carboxylic acid, organic sulfonic acid or organic
phosphoric acid. The organic carboxylic acid includes, e.g., fatty
acids, and the number of carbon atoms thereof is preferably 1 to
35. In the organic sulfonic acid or organic phosphoric acid, the
number of carbon atoms thereof is preferably 1 to 35.
[0118] Accordingly, the acyl group is preferably selected from a
C.sub.1-C.sub.35 acyl group, such as a C.sub.1-C.sub.20 acyl group,
such as a C.sub.1-C.sub.15 acyl group, such as a C.sub.6-C.sub.15
acyl group, such as a C.sub.6-C.sub.12 acyl group, such as a
C.sub.8-C.sub.12 acyl group. More preferably, the acyl group is
selected from the group consisting of C.sub.7 acyl group, C.sub.8
acyl group, C.sub.9 acyl group, C.sub.10 acyl group, C.sub.ii acyl
group, and C.sub.12 acyl group. Such acyl group may be formed from
octanoic acid (preferably caprylic acid), decanoic acid (preferably
capric acid), or dodecanoic acid (preferably lauric acid), as well
as monoene or polyene fatty acids thereof.
[0119] Furthermore, the modified amino acid may be any amino acid
wherein a group is modified as described in EP 1 197 496 (Kangawa),
which is hereby incorporated by reference.
Protecting Group
[0120] The ghrelin splice variant-like compound according to the
present disclosure may comprise a protecting group at the
N-terminus or the C-terminus or at both. A protecting group
covalently joined to the N-terminal amino group reduces the
reactivity of the amino terminus under in vivo conditions. Amino
protecting groups include, for example, C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.10 substituted alkyl, C.sub.2-C.sub.10 alkenyl,
C.sub.2-C.sub.10 substituted alkenyl, aryl, C.sub.1-C.sub.6 alkyl
aryl, C(O)--(CH.sub.2)--(C.sub.1-C.sub.6 alkyl)-COOH,
C(O)--(C.sub.1-C.sub.6 alkyl), C(O)-aryl,
C(O)--O--(C.sub.1-C.sub.6-alkyl), or C(O)--O-aryl. Preferably, the
amino terminus protecting group is acetyl, propyl, succinyl,
benzyl, benzyloxycarbonyl or t-butyloxycarbonyl.
[0121] A protecting group covalently joined to the C-terminal
carboxy group reduces the reactivity of the carboxy terminus under
in vivo conditions. The carboxy terminus protecting group is
preferably attached to the a-carbonyl group of the last amino acid.
Carboxy terminus protecting groups include, for example, amide,
methylamide, and ethylamide.
Conjugates
[0122] The secretagogue, such as a ghrelin splice variant-like
compound, may be provided in the form of a secretagogue conjugate,
i.e. a molecule comprising the secretagogue conjugated to another
entity. The other entity may be any substance that is capable of
conferring improved properties to the secretagogue, e.g. in terms
of improved stability, half-life, etc. Examples of suitable
entities are described in the following. For example, the
secretagogue may be conjugated to a peptide, such as a peptide
having effect on nociceptin receptor ORL1. In one embodiment, the
conjugate is a conjugate of ghrelin splice variant or a derivative
or homologue thereof and a peptide having effect on ORL1, e.g. the
peptide Ac--RYY (RK) (WI) RK)--NH.sub.2, where the brackets show
allowable variation of amino acid residues. Examples of other
suitable peptides are found in Published U.S. Patent Application
Nos. 2003/040472 and 2002/004483, and U.S. Pat. No. 5,869,046, each
of which is incorporated herein by reference.
[0123] In another embodiment, a secretagogue, such as ghrelin
splice variant or a ghrelin splice variant-like compound, is
conjugated to a polymer molecule. The polymer molecule may be any
suitable polymer molecule, such as a natural or synthetic polymer,
typically with a molecular weight in the range of about 1-100 kDa,
such as about 3-20 kDa, such as 5-10 kDa. The polymer is attached
to a reactive group present on the secretagogue, e.g. an amine
group or a thiol group. Examples of suitable polymer molecules
include polymer molecules selected from the group consisting of
polyalkylene oxide (PAO), including polyalkylene glycol (PAG), such
as linear or branched polyethylene glycol (PEG) and polypropylene
glycol (PPG); poly-vinyl alcohol (PVA); poly-carboxylate;
poly-(vinylpyrolidone); polyethylene-co-maleic acid anhydride;
polystyrene-co-maleic acid anhydride; and dextran, including
carboxymethyl-dextran. Preferably, the polymer molecule is a PEG
molecule, in particular a monofunctional PEG, such as
methoxypolyethylene glycol (mPEG). Suitable activated PEG molecules
are available from, for example, Nektar Therapeutics Inc.
(Huntsville, Ala.) or Valentis, Inc. (Burlingame, Calif.).
Alternatively, the polymer molecules can be activated by
conventional methods known in the art, e.g., as disclosed in WO
90/13540, incorporated herein by reference. Specific examples of
activated PEG polymers include the following linear PEGs: NHS-PEG
(e.g., SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG,
and SCM-PEG), NOR-PEG, BTC-PEG, EPDX-PEG, NCO-PEG, NPC-PEG,
CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and
branched PEGs such as PEG2-NHS and those disclosed in U.S. Pat.
Nos. 5,932,462 and 5,643,575, both of which are incorporated herein
by reference.
[0124] The PEGylation (i.e., conjugation of the secretagogue
polypeptide and the activated polymer molecule) is conducted in
accordance with established procedures, e.g., as described in the
following references (which also describe suitable methods for
activation of polymer molecules): R. F. Taylor, (1991), "Protein
Immobilization. Fundamentals and Applications", Marcel Dekker, N.
Y.; S. S. Wong, (1992), "Chemistry of Protein Conjugation and
Crosslinking", CRC Press, Boca Raton; G. T. Hermanson et al.,
(1993), "Immobilized Affinity Ligand Techniques", Academic Press,
N. Y.).
[0125] It is also contemplated according to the present disclosure
to couple the polymer molecules to the secretagogue through a
linker. Suitable linkers are well known to the skilled person. A
preferred example is cyanuric chloride (Abuchowski A. et al., J.
Biol. Chem. 252:3578-81 (1977); U. S. Pat. No. 4,179,337; Shafer et
al., J. Polym. Sci. Polym. Chem. Ed. 24:375-78 (1986)).
[0126] In yet another embodiment, the secretagogue is conjugated to
an oligosaccharide molecule, such as dextran, glycan, transferrin,
etc. Such conjugation may be achieved in accordance with
established technologies, e.g. those available from Neose
Technologies, Inc. (Horsham, Pa.).
[0127] In yet another embodiment, the secretagogue is conjugated to
an Fc region of an IgG molecule, typically in the form of a fusion
protein. For instance, a salvage receptor binding epitope of the Fc
region of an IgG (i.e. the Fc portion of an immunoglobulin of the
isotype IgG) is incorporated into the secretagogue so as to
increase its circulatory half-life, but so as not to lose its
biological activity. This can take place by any means, such as by
mutation of the appropriate region in the secretagogue to mimic the
Fc region or by incorporating the epitope into a peptide tag that
is then fused to the secretagogue at either end or in the middle or
by DNA or peptide synthesis.
[0128] The salvage receptor binding epitope is any suitable such
epitope as known to the person skilled in the art, and its nature
will depend, e.g., on the type of secretagogue being modified. The
epitope is introduced into the secretagogue such that the
biological activity of the secretagogue is maintained, i.e., the
epitope does not adversely affect the conformation of the
secretagogue or affect its binding to ligands that confers its
biological activity.
[0129] Alternatively to providing the secretagogue in the form of a
conjugate, the secretagogue may be modified to include suitable
reactive groups, whereby the thus modified secretagogue is capable
of forming a conjugate in vivo (after having been administered to
an individual) through covalent bonding with available reactive
functionalities on blood components. The present disclosure also
relates to such modified secretagogues, and methods for their use.
Also, the present disclosure relates to conjugates formed in vitro
between a modified secretagogue as described above and a blood
component. The conjugates formed in accordance with this embodiment
are contemplated to have an increased in vivo half life as compared
to the corresponding non-modified secretagogue.
[0130] In accordance with this embodiment, the secretagogue is
modified with a chemically reactive group (reactive entity). The
reactive entity may, e.g., be selected from the wide variety of
active carboxyl groups, particularly esters, where the hydroxyl
moiety is physiologically acceptable. Such groups may be selected
from the group consisting of N-hydroxysuccinimide (NHS),
N-hydroxy-sulfosuccinimide (sulfo-NHS),
maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy
succinimide ester (GMBS) and maleimidopropionic acid (MPA). The
principal targets for this group of entities are primary amines on
the blood component. Another group of active entities is
constituted by a maleimido-containing group such as MPA and
gamma-maleimide-butrylamide (GMBA). Such groups react with thiol
groups present on the blood component (Lee V. H. L. in "Peptide and
Protein Drug Delivery", New York, N. Y., M. Dekker, 1990).
[0131] The blood component with which the modified secretagogue is
designed to react may be any blood component having an available
target group, e.g. an amine or a thiol group, and which is suitable
as a carrier for binding the modified secretagogue in vivo and
thereby extend the circulating half-life thereof. Examples of such
blood components are serum albumin and IgG.
[0132] As mentioned above, the covalent bonding of a modified
secretagogue to a blood component may be achieved in vivo by
administration of the modified secretagogue directly to the
patient. The administration may be done in any suitable form, such
as in the form of a bolus or introduced slowly over time by
infusion using metered flow or the like. Alternatively, the
secretagogue/blood component conjugate may also be prepared ex vivo
by combining blood with the modified secretagogue, allowing
covalent bonding of the modified secretagogue to reactive
functionalities on blood components and then returning or
administering the conjugated blood to the individual.
[0133] Moreover, the above may also be accomplished by first
purifying an individual blood component or limited number of
components, such as red blood cells, immunoglobulins, serum
albumin, or the like, and combining the component or components ex
vivo with the chemically reactive secretagogues.
Production of Ghrelin Splice Variant-Like Compounds
[0134] Ghrelin splice variant-like compounds can be produced using
techniques well known in the art. For example, a polypeptide region
of a ghrelin splice variant-like compound can be chemically or
biochemical synthesized and modified. Techniques for chemical
synthesis of polypeptides are well known in the art (see, e.g., Lee
V. H. L. in "Peptide and Protein Drug Delivery", New York, N. Y.,
M. Dekker, 1990). Examples of techniques for biochemical synthesis
involving the introduction of a nucleic acid into a cell and
expression of nucleic acids are provided in Ausubel F. M. et al.,
"Current Protocols in Molecular Biology", John Wiley, 1987-1998,
and Sambrook J. et al., "Molecular Cloning, A Laboratory Manual",
2d Edition, Cold Spring Harbor Laboratory Press, 1989, each of
which is incorporated herein by reference. Another exemplary
technique, described in U.S. Pat. No. 5,304,489, incorporated
herein by reference, is the use of a transgenic mammals having
mammary gland-targeted mutations which result in the production and
secretion of synthesized ghrelin splice variant-like compound in
the milk of the transgenic mammal.
[0135] The ghrelin splice variant-like compounds can also be
recombinantly produced using routine expression methods known in
the art. The polynucleotide encoding the desired ghrelin splice
variant-like compound is operably linked to a promoter into an
expression vector suitable for any convenient host. Both eukaryotic
and prokaryotic host systems are used in forming recombinant
ghrelin splice variant-like compounds. The ghrelin splice
variant-like compound is then isolated from lysed cells or from the
culture medium and purified to the extent needed for its intended
use.
[0136] Consequently, a further embodiment is for a method of
producing a ghrelin splice variant-like compound, said method
comprising the steps of: (a) providing a cDNA comprising a
polynucleotide sequence encoding a ghrelin splice variant-like
compound; (b) inserting said cDNA in an expression vector such that
the cDNA is operably linked to a promoter; and (c) introducing said
expression vector into a host cell whereby said host cell produces
said ghrelin splice variant-like compound.
[0137] In one aspect of this embodiment, the method further
comprises the step of recovering the ghrelin splice variant-like
compound produced in step (c). Another embodiment is a ghrelin
splice variant-like compound obtainable by the method described in
the preceding paragraph. The expression vector is any of the
mammalian, yeast, insect, or bacterial expression systems known in
the art. Commercially available vectors and expression systems are
available from a variety of suppliers including Genetics Institute
(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega
(Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to
enhance expression and facilitate proper protein folding, the codon
context and codon pairing of the sequence is optimized for the
particular expression organism in which the expression vector is
introduced, as explained in U.S. Pat. No. 5,082,767, which
disclosure is hereby incorporated by reference in its entirety.
[0138] In another embodiment, it is often advantageous to add to
the recombinant polynucleotide additional nucleotide sequence(s)
which codes for secretory or leader sequences, pro-sequences,
sequences which aid in purification, such as multiple histidine
residues, or an additional sequence for stability during
recombinant production.
[0139] Introduction of a polynucleotide encoding a ghrelin splice
variant-like compound into a host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation,
transduction, infection, or other methods. Such methods are
described in many standard laboratory manuals, such as Davis et
al., (1986) Basic Methods in Molecular Biology, ed., Elsevier
Press, NY, which disclosure is hereby incorporated by reference in
its entirety. It is specifically contemplated that the ghrelin
splice variant-like compounds disclosed herein may in fact be
expressed by a host cell lacking a recombinant vector or naturally
produced by a cell.
[0140] Ghrelin splice variant-like compounds can be recovered and
purified from recombinant cell cultures by well-known methods
including differential extraction, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography (see, for
example, "Methods in Enzymology: Aqueous Two-Phase Systems", Walter
H et al. (eds.), Academic Press (1993), incorporated herein by
reference, for a variety of methods for purifying proteins). In one
embodiment, high performance liquid chromatography ("HPLC") is
employed for purification. A recombinantly produced version of a
ghrelin splice variant-like compound can be substantially purified
using techniques described herein or otherwise known in the art,
such as, for example, by the one-step method described in Smith
& Johnson, Gene 67:31 40 (1988), which disclosure is hereby
incorporated by reference in its entirety. Ghrelin splice
variant-like compounds also can be purified from recombinant
sources using antibodies directed against the ghrelin splice
variant-like compounds, such as those described herein, in methods
which are well known in the art of protein purification.
[0141] In one embodiment, the recombinantly expressed ghrelin
splice variant-like compound is purified using standard
immunochromatography techniques. In such procedures, a solution
containing the ghrelin splice variant-like compound of interest,
such as the culture medium or a cell extract, is applied to a
column having antibodies against the ghrelin splice variant-like
compound attached to the chromatography matrix. The recombinant
ghrelin splice variant-like compound is allowed to bind the
immunochromatography column. Thereafter, the column is washed to
remove non-specifically bound proteins. The specifically bound
secreted ghrelin splice variant-like compound is then released from
the column and recovered using standard techniques.
[0142] Depending upon the host employed in a recombinant production
procedure, the ghrelin splice variant-like compounds may be
glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
Thus, it is well known in the art that the N-terminal methionine
encoded by the translation initiation codon generally is removed
with high efficiency from any protein after translation in all
eukaryotic cells. While the N-terminal methionine on most proteins
also is efficiently removed in most prokaryotes, for some proteins,
this prokaryotic removal process is inefficient, depending on the
nature of the amino acid to which the N-terminal methionine is
covalently linked.
Pharmaceutical Compositions
[0143] While it is possible for the compounds or salts of the
present disclosure to be administered as the raw chemical, it is
preferred to present them in the form of a pharmaceutical
composition. Accordingly, one aspect relates to a pharmaceutical
composition comprising a ghrelin splice variant-like compound as
defined in Formula I.
[0144] Another embodiment relates to a pharmaceutical composition
comprising a mixture of at least two different ghrelin splice
variant-like compounds, such as a mixture of a ghrelin splice
variant-like compound acylated with a C.sub.8 acyl and a ghrelin
splice variant-like compound acylated with a C.sub.10 acyl. Without
being bound by theory, it is believed that such a mixture will have
a longer half-life in plasma.
[0145] In yet another embodiment, the pharmaceutical composition
comprises acylated ghrelin splice variant-like compounds,
optionally compounds having different acyl chain lengths preferably
selected from the group consisting of C.sub.7 acyl group, C.sub.9
acyl group, and C.sub.11 acyl group, optionally in combination with
a desacylated ghrelin splice variant-like compound.
[0146] Another aspect relates to a pharmaceutical composition
comprising any secretagogue, such as any ghrelin splice
variant-like compound as defined above or a pharmaceutically
acceptable salt thereof and pharmaceutical acceptable carriers,
vehicles and/or excipients; said composition further comprising
transport molecules. The transport molecules are primarily added in
order to increase the half-life of the acylated compound,
preventing premature des-acylation, since the des-acylated ghrelin
splice variant might not be active at the GHS-R 1a.
[0147] Transport molecules act by having incorporated into or
anchored to it a compound disclosed herein. Any suitable transport
molecule known to the skilled person may be used. Examples of
transport molecules are those described in the conjugate section,
supra. Other preferred examples are liposomes, micelles, and/or
microspheres.
[0148] Conventional liposomes are typically composed of
phospholipids (neutral or negatively charged) and/or cholesterol.
The liposomes are vesicular structures based on lipid bilayer
surrounding aqueous compartments. They can vary in their
physio-chemical properties such as size, lipid composition, surface
charge and number, and fluidity of the phospholipids bilayer. The
most frequently used lipids for liposome formation are:
1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC),
1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC),
1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC),
1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC),
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC),
1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE),
1,2-Dipaimitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE),
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),
1,2-Dimyristoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DMPA),
1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DPPA),
1,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DOPA),
1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium
Salt) (DMPG),
1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)) (Sodium
Salt) (DPPG), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]
(Sodium Salt) (DOPG),
1,2-Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt)
(DMPS), 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium
Salt) (DPPS), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium
Salt) (DOPS),
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(glutaryl) (Sodium
Salt) and 1,1',2,2'-Tetramyristoyl Cardiolipin (Ammonium Salt).
Formulations composed of DPPC in combination with other lipid or
modifiers of liposomes are preferred, e.g., in combination with
cholesterol and/or phosphatidylcholine.
[0149] Long-circulating liposomes are characterized by their
ability to extravasate at body sites where the permeability of the
vascular wall is increased. A preferred way to produce long
circulating liposomes is to attach hydrophilic polymer polyethylene
glycol (PEG) covalently to the outer surface of the liposome. Some
of the preferred lipids are:
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy
(Polyethylene glycol)-2000] (Ammonium Salt),
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy
(Polyethylene glycol)-5000] (Ammonium Salt),
1,2-Dioleoyl-3-Trimethylammonium-Propane (Chloride Salt)
(DOTAP).
[0150] Possible lipids applicable for liposomes are supplied by
Avanti Polar Lipids, Inc., (Alabaster, Ala.). Additionally, the
liposome suspension may include lipid-protective agents which
protect lipids against free-radical and lipid-peroxidative damages
on storage. Lipophilic free-radical quenchers, such as
alpha-tocopherol and water-soluble iron-specific chelators, such as
ferrioxianine, are preferred.
[0151] A variety of methods are available for preparing liposomes,
as described in, e.g., Szoka F. & Papahadjopolous D., Ann. Rev.
Biophys. Bioeng. 9:467-508 (1980); U. S. Pat. Nos. 4,235,871,
4,501,728 and 4,837,028; all of which are incorporated herein by
reference. Another method produces multilamellar vesicles of
heterogeneous sizes. In this method, the vesicle-forming lipids are
dissolved in a suitable organic solvent or solvent system and dried
under vacuum or an inert gas to form a thin lipid film. If desired,
the film may be redissolved in a suitable solvent, such as tertiary
butanol, and then lyophilized to form a more homogeneous lipid
mixture which is in a more easily hydrated powder-like foam. This
film is covered with an aqueous solution of the targeted drug and
the targeting component and allowed to hydrate, typically over a
15-60 minute period with agitation. The size distribution of the
resulting multilamellar vesicles can be shifted toward smaller
sizes by hydrating the lipids under more vigorous agitation
conditions or by adding solubilizing detergents such as
deoxycholate.
[0152] Micelles are formed by surfactants (molecules that contain a
hydrophobic portion and one or more ionic or otherwise strongly
hydrophilic groups) in aqueous solution. As the concentration of a
solid surfactant increases, its monolayers adsorbed at the
air/water or glass/water interface become so tightly packed that
further occupancy requires excessive compression of the surfactant
molecules already in the two monolayers. Further increments in the
amount of dissolved surfactant beyond that concentration cause
amounts equivalent to the new molecules to aggregate into micelles.
This process begins at a characteristic concentration called
"critical micelle concentration".
[0153] The shape of micelles formed in dilute surfactant solutions
is approximately spherical. The polar head groups of the surfactant
molecules are arranged in an outer spherical shell whereas their
hydrocarbon chains are oriented toward the center, forming a
spherical core for the micelle. The hydrocarbon chains are randomly
coiled and entangled and the micellar interior has a nonpolar,
liquid-like character. In the micelles of polyoxyethylated nonionic
detergents, the polyoxyethlene moieties are oriented outward and
permeated by water. This arrangement is energetically favorable
since the hydrophilic head groups are in contact with water and the
hydrocarbon moieties are removed from the aqueous medium and partly
shielded from contact with water by the polar head groups. The
hydrocarbon tails of the surfactant molecules, located in the
interior of the micelle, interact with one another by weak van der
Waals forces.
[0154] The size of a micelle or its aggregation number is governed
largely by geometric factors. The radius of the hydrocarbon core
cannot exceed the length of the extended hydrocarbon chain of the
surfactant molecule. Therefore, increasing the chain length or
ascending homologous series increases the aggregation number of
spherical micelles. If the surfactant concentration is increased
beyond a few percent and if electrolytes are added (in the case of
ionic surfactants) or the temperature is raised (in the case of
nonionic surfactants), the micelles increase in size. Under these
conditions, the micelles are too large to remain spherical and
become ellipsoidal, cylindrical or finally lamellar in shape.
[0155] Common surfactants well known to one of skill in the art can
be used in the micelles of the present disclosure. Suitable
surfactants include sodium laureate, sodium oleate, sodium lauryl
sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and
PLURONIC.RTM. F-127 (BASF Corp., Florham Park, N.J.). Preferred
surfactants are nonionic polyoxyethylene and polyoxypropylene
detergents compatible with intravenous injection such as,
TWEEN.RTM.-80, PLURONIC.RTM. F-68, n-octyl-beta-D-glucopyranoside,
and the like. In addition, phospholipids, such as those described
for use in the production of liposomes, may also be used for
micelle formation.
[0156] In another preferred embodiment, the compounds disclosed
herein are formulated as described in the literature for an
administration route selected from: buccal delivery, sublingual
delivery, transdermal delivery, inhalation and needle-free
injection, such as using the methods developed by Powderjet.
[0157] For inhalation, the compounds disclosed herein can be
formulated using methods known to those skilled in the art, for
example an aerosol, dry powder or solubilized such as in
microdroplets, preferably in a device intended for such delivery
(such as commercially available from Aradigm Corp. (Hayward,
Calif.), Alkermes, Inc. (Cambridge, Mass.), or Nektar Therapeutics
(San Carlos, Calif.)).
Administration
[0158] Suitable dosing regimens for the various compounds and
methods of the present disclosure are preferably determined taking
into account factors well known in the art including, e.g., type of
subject being dosed; age, weight, sex and medical condition of the
subject; the route of administration; the renal and hepatic
function of the subject; the desired effect; and the particular
compound employed. Preferably, the composition will comprise about
0.5% to 75% by weight of a secretagogue disclosed herein, with the
remainder consisting of suitable pharmaceutical excipients.
[0159] Optimal precision in achieving concentrations of drug within
the range that yields efficacy without toxicity requires a regimen
based on the kinetics of the drug's availability to target sites.
This involves a consideration of the distribution, equilibrium, and
elimination of a drug.
[0160] As described above, in one aspect, the secretagogue, such as
ghrelin splice variant or a ghrelin splice variant-like compound,
is administered subcutaneously.
[0161] In another aspect, the secretagogue, such as ghrelin splice
variant or a ghrelin splice variant-like compound, is administered
as a premeal bolus, wherein the administration form may be any
suitable parenteral form. In a preferred embodiment, the
secretagogue, such as ghrelin splice variant or a ghrelin splice
variant-like compound, is administered subcutaneously in a premeal
bolus.
[0162] The secretagogue, such as ghrelin splice variant or a
ghrelin splice variant-like compound, can also be administered
during a meal as a bolus. The mode of administration during a meal
includes subcutaneous administration, such as a subcutaneously
administered bolus.
[0163] Pharmaceutical compositions for parenteral administration
include sterile aqueous and non-aqueous injectable solutions,
dispersions, suspensions or emulsions, as well as sterile powders
to be reconstituted in sterile injectable solutions or dispersions
prior to use. Other suitable administration forms include
suppositories, sprays, ointments, creams, gels, inhalants, dermal
patches, implants, pills, tablets, lozenges and capsules.
[0164] A typical dosage is in a concentration equivalent to from 10
ng to 10 mg ghrelin splice variant per kg bodyweight. The
concentrations and amounts herein are given in equivalents of
amount ghrelin splice variant, wherein the ghrelin splice variant
is a 22 amino acid human ghrelin splice variant (SEQ ID NO:2)
and/or a 29 amino acid human ghrelin splice variant (SEQ ID NO:5)
and/or a 24 amino acid human ghrelin splice variant (SEQ ID NO:3)
and/or a 24 amino acid human ghrelin splice variant having a Dpr
residue at the third position (SEQ ID NO:4). Equivalents may be
tested as described in the section entitled "Functionality",
above.
[0165] In a preferred embodiment, the medicament is administered in
a concentration equivalent to from 0.1 .mu.g to 1 mg ghrelin splice
variant per kg bodyweight, such as from 0.5 .mu.g to 0.5 mg ghrelin
splice variant per kg bodyweight, such as from 1.0 .mu.g to 0.1 mg
ghrelin splice variant per kg bodyweight, such as from 1.0 .mu.g to
50 .mu.g ghrelin splice variant per kg bodyweight, such as from 1.0
.mu.g to 10 .mu.g ghrelin splice variant per kg bodyweight.
[0166] As described above, the secretagogue, such as ghrelin splice
variant or a ghrelin splice variant-like compound, is preferably
administered as a bolus. Accordingly, in one embodiment the
medicament is administered as a bolus prior to a meal, said bolus
comprising an amount of the secretagogue or a salt thereof
equivalent to from 0.3 .mu.g to 600 mg ghrelin splice variant. More
preferably, the medicament is administered as a bolus prior to a
meal, said bolus comprising an amount of the secretagogue or a salt
thereof equivalent to from 2.0 .mu.g to 200 mg ghrelin splice
variant, such as from 5.0 .mu.g to 100 mg ghrelin splice variant,
such as from 10 .mu.g to 50 mg ghrelin splice variant, such as from
10 .mu.g to 5 mg ghrelin splice variant, such as from 10 .mu.g to
1.0 mg ghrelin splice variant.
[0167] It should be noted that the normal ghrelin splice
variant-like response which occurs before a meal is a short-lived
surge in plasma concentrations of ghrelin splice variant and that,
due to the relatively short half life of the peptide, an
intravenous injection of ghrelin splice variant will ensure that a
similar short-lived peak on ghrelin splice variant concentrations
can be obtained. The administration route must ensure that the
non-degraded, bioactive form of the peptide will be the dominating
form in the circulation, which will reach and stimulate the ghrelin
splice variant receptors.
[0168] Thus, in order to obtain the maximum effect of the
medicament, it is preferably administered from one to three times
daily, each administration being within 45 minutes of a meal, such
as within 30 minutes of a meal, such as within 25 minutes of a
meal, such as within 20 minutes of a meal, such as within 15
minutes of a meal, such as within 10 minutes of a meal, such as
within 5 minutes of a meal. More preferably, the medicament is
administered prior to each main meal, such as administered three
times daily.
[0169] Compounds disclosed herein may also be formulated for nasal
administration. The solutions or suspensions are applied directly
to the nasal cavity by conventional means, for example with a
dropper, pipette or spray. The compositions may be provided in a
single or multidose form. In the latter case of a dropper or
pipette, this may be achieved by the patient administering an
appropriate, predetermined volume of the solution or suspension. In
the case of a spray, this may be achieved for example by means of a
metering atomizing spray pump.
[0170] The compounds disclosed herein may be formulated for aerosol
administration, particularly to the respiratory tract and including
intranasal administration. The compound will generally have a small
particle size, for example of the order of 5 microns or less. Such
a particle size may be obtained by means known in the art, for
example by micronization. The active ingredient is provided in a
pressurized pack with a suitable propellant such as a
hydrofluoroalkane (HFA) for example hydrofluoroalkane-134a and
hydrofluoroalkane-227, carbon dioxide or other suitable gas. The
aerosol may conveniently also contain a surfactant such as
lecithin. The dose of drug may be controlled by a metered valve.
Alternatively, the active ingredients may be provided in a form of
a dry powder, for example a powder mix of the compound in a
suitable powder base such as lactose, starch, starch derivatives
such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine
(PVP). The powder carrier will form a gel in the nasal cavity. The
powder composition may be presented in unit dose form for example
in capsules or cartridges of, e.g., gelatin or blister packs from
which the powder may be administered by means of an inhaler.
[0171] Compositions administered by aerosols may be prepared, for
example, as solutions in saline, employing benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, employing fluorocarbons, and/or employing other
solubilizing or dispersing agents.
[0172] Compounds disclosed herein may also be formulated for
administration by injection pen in a similar way as for cartridged
growth hormone (GH) or Insulin. The cartridge contains compounds
disclosed herein in solvents. The pen, which is basically a needle,
syringe and vial in one piece, is operated by a turning movement
and allows different doses to be administrated. This device offers
simplicity, convenience, and enhanced safety features for compounds
delivery. It provides a simple device design, few administration
steps and one-step dial-back dose knob. Such injection pen can be
obtained by means known in art. For example, several manufacturers
offer drug developers injection pens to be used with the drug
developers compounds (BD--Medical--Pharmaceutical Systems, Inc.;
Owen Mumford Inc. etc.).
Compositions for Oral Administration
[0173] Those secretagogue types capable of remaining biologically
active in an individual after oral administration (such as, e.g.,
small molecules and short peptides) can be formulated in a wide
range of oral administration dosage forms. The pharmaceutical
compositions and dosage forms may comprise the compounds disclosed
herein or their pharmaceutically acceptable salt or crystal forms
thereof as the active component.
[0174] The pharmaceutical acceptable carriers can be either solid
or liquid. Solid form preparations include powders, tablets, pills,
capsules, cachets, suppositories, and dispersible granules. A solid
carrier can be one or more substances which may also act as
diluents, flavoring agents, solubilizers, lubricants, suspending
agents, binders, preservatives, wetting agents, tablet
disintegrating agents, or an encapsulating material.
[0175] For oral administration, such excipients include, e.g.,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, gelatin,
sucrose, magnesium carbonate, and the like.
[0176] In powders, the carrier is a finely divided solid which is a
mixture with the finely divided active component. In tablets, the
active component is mixed with the carrier having the necessary
binding capacity in suitable proportions and compacted in the shape
and size desired. The powders and tablets preferably containing
from one to about seventy percent of the active compound. Suitable
carriers are magnesium carbonate, magnesium stearate, talc, sugar,
lactose, pectin, dextrin, starch, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose, a low melting wax,
cocoa butter, and the like. The term "preparation" is intended to
include a composition comprising an active compound disclosed
herein with encapsulating material as carrier providing a capsule
in which the active component, with or without carriers, is
surrounded by a carrier, which is in association with it.
Similarly, cachets and lozenges are included.
[0177] Tablets, powders, capsules, pills, cachets, and lozenges can
be as solid forms suitable for oral administration.
[0178] Drops may comprise sterile or nonsterile aqueous or oil
solutions or suspensions, and may be prepared by dissolving the
active ingredient in a suitable aqueous solution, optionally
including a bactericidal and/or fungicidal agent and/or any other
suitable preservative, and optionally including a surface active
agent. The resulting solution may then be clarified by filtration,
transferred to a suitable container which is then sealed and
sterilized by autoclaving or maintaining at 98-100.degree. C. for
half an hour. Alternatively, the solution may be sterilized by
filtration and transferred to the container aseptically. Examples
of bactericidal and fungicidal agents suitable for inclusion in the
drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium
chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable
solvents for the preparation of an oily solution include glycerol,
diluted alcohol and propylene glycol.
[0179] Also included are solid form preparations which are intended
to be converted, shortly before use, to liquid form preparations
for oral administration. Such liquid forms include solutions,
suspensions, and emulsions. These preparations may contain, in
addition to the active component, colorants, flavors, stabilizers,
buffers, artificial and natural sweeteners, dispersants,
thickeners, solubilizing agents, and the like.
[0180] Other forms suitable for oral administration include liquid
form preparations including emulsions, syrups, elixirs, aqueous
solutions, aqueous suspensions, toothpaste, gel dentifrice, chewing
gum, or solid form preparations which are intended to be converted
shortly before use to liquid form preparations. Emulsions may be
prepared in solutions in aqueous propylene glycol solutions or may
contain emulsifying agents such as lecithin, sorbitan monooleate,
or acacia. Aqueous solutions can be prepared by dissolving the
active component in water and adding suitable colorants, flavors,
stabilizing and thickening agents. Aqueous suspensions can be
prepared by dispersing the finely divided active component in water
with viscous material, such as natural or synthetic gums, resins,
methylcellulose, sodium carboxymethylcellulose, and other well
known suspending agents. Solid form preparations include solutions,
suspensions, and emulsions, and may contain, in addition to the
active component, colorants, flavors, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like.
Compositions for Parenteral Administration
[0181] The compounds disclosed herein may be formulated for
parenteral administration (e.g., by injection, for example bolus
injection or continuous infusion) and may be presented in unit dose
form in ampules, pre-filled syringes, small volume infusion or in
multi-dose containers with an added preservative. The compositions
may take such forms as suspensions, solutions, or emulsions in oily
or aqueous vehicles, for example solutions in aqueous polyethylene
glycol. Examples of oily or nonaqueous carriers, diluents, solvents
or vehicles include propylene glycol, polyethylene glycol,
vegetable oils (e.g., olive oil), and injectable organic esters
(e.g., ethyl oleate), and may contain formulatory agents such as
preserving, wetting, emulsifying or suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilization from solution for constitution before use with a
suitable vehicle, e.g., sterile, pyrogen-free water. Aqueous
solutions should be suitably buffered if necessary, and the liquid
diluent first rendered isotonic with sufficient saline or glucose.
The aqueous solutions are particularly suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration. The
sterile aqueous media employed are all readily available by
standard techniques known to those skilled in the art.
[0182] Solutions of ghrelin splice variant or a ghrelin splice
variant-like compound or pharmaceutical acceptable salt thereof
(and for example antigenic epitopes and protease inhibitors) can be
prepared in water or saline, and optionally mixed with a nontoxic
surfactant. Compositions for intravenous or intra-arterial
administration may include sterile aqueous solutions that may also
contain buffers, liposomes, diluents and other suitable
additives.
[0183] Oils useful in parenteral compositions include petroleum,
animal, vegetable, or synthetic oils. Specific examples of oils
useful in such compositions include peanut, soybean, sesame,
cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty
acids for use in parenteral compositions include oleic acid,
stearic acid, and isostearic acid. Ethyl oleate and isopropyl
myristate are examples of suitable fatty acid esters.
[0184] Suitable soaps for use in parenteral compositions include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides; (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates; (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers; (d) amphoteric detergents
such as, for example, alkyl-beta-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures
thereof.
[0185] The parenteral compositions typically will contain from
about 0.5 to about 25% by weight of the active ingredient in
solution. Preservatives and buffers may be used. In order to
minimize or eliminate irritation at the site of injection, such
compositions may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such compositions will typically
range from about 5 to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral compositions can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0186] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions
comprising the active ingredient that are adapted for
administration by encapsulation in liposomes. In all cases, the
ultimate dosage form must be sterile, fluid and stable under the
conditions of manufacture and storage.
[0187] Sterile injectable solutions are prepared by incorporating
ghrelin splice variant or a ghrelin splice variant-like compound or
pharmaceutical acceptable salt thereof in the required amount in
the appropriate solvent with various of the other ingredients
enumerated above, as required, followed by, e.g., filter
sterilization.
Compositions for Topical Administration
[0188] The compounds disclosed herein can also be delivered
topically. Regions for topical administration include the skin
surface and also mucous membrane tissues of the rectum, nose,
mouth, and throat. Compositions for topical administration via the
skin and mucous membranes should not give rise to signs of
irritation, such as swelling or redness.
[0189] The topical composition may include a pharmaceutical
acceptable carrier adapted for topical administration. Thus, the
composition may take the form of, for example, a suspension,
solution, ointment, lotion, cream, foam, aerosol, spray,
suppository, implant, inhalant, tablet, capsule, dry powder, syrup,
balm or lozenge. Methods for preparing such compositions are well
known in the pharmaceutical industry.
[0190] The compounds disclosed herein may be formulated for topical
administration to the epidermis as ointments, creams or lotions, or
as a transdermal patch. Ointments and creams may, for example, be
formulated with an aqueous or oily base with the addition of
suitable thickening and/or gelling agents. Lotions may be
formulated with an aqueous or oily base and will in general also
containing one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents. Compositions suitable for topical administration
in the mouth include lozenges comprising active agents in a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatin
and glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0191] Creams, ointments or pastes according to the present
disclosure are semi-solid compositions for external application
comprising the active ingredient. They may be made by mixing the
active ingredient in finely-divided or powdered form, alone or in
solution or suspension in an aqueous or non-aqueous fluid, with the
aid of suitable machinery, with a greasy or non-greasy base. The
base may comprise hydrocarbons such as hard, soft or liquid
paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of
natural origin such as almond, corn, arachis, castor or olive oil;
wool fat or its derivatives; or a fatty acid such as steric or
oleic acid together with an alcohol such as propylene glycol or a
macrogel. The composition may incorporate any suitable surface
active agent such as an anionic, cationic or non-ionic surfactant
such as a sorbitan ester or a polyoxyethylene derivative thereof.
Suspending agents such as natural gums, cellulose derivatives or
inorganic materials such as silicaceous silicas, and other
ingredients such as lanolin, may also be included.
[0192] Lotions according to the present disclosure include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0193] The compounds described herein can be administered
transdermally. Transdermal administration typically involves the
delivery of a pharmaceutical agent for percutaneous passage of the
drug into the systemic circulation of the patient. The skin sites
include anatomic regions for transdermally administering the drug
and include the forearm, abdomen, chest, back, buttock, mastoidal
area, and the like.
[0194] Transdermal delivery is accomplished by exposing a source of
the active compound to a patient's skin for an extended period of
time. Transdermal patches have the added advantage of providing
controlled delivery of a compound complex to the body (see
Transdermal Drug Delivery: Developmental Issues and Research
Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);
Controlled Drug Delivery: Fundamentals and Applications, Robinson
and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal
Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC
Press, (1987)). Such dosage forms can be made by dissolving,
dispersing, or otherwise incorporating a compound disclosed herein
in a proper medium, such as an elastomeric matrix material.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate of such flux can be controlled
by either providing a rate-controlling membrane or dispersing the
compound in a polymer matrix or gel.
[0195] A variety of types of transdermal patches will find use in
the methods described herein. For example, a simple adhesive patch
can be prepared from a backing material and an acrylate adhesive.
The active compound and any enhancer are formulated into the
adhesive casting solution and allowed to mix thoroughly. The
solution is cast directly onto the backing material and the casting
solvent is evaporated in an oven, leaving an adhesive film. The
release liner can be attached to complete the system.
[0196] Alternatively, a polyurethane matrix patch can be employed
to deliver a compound disclosed herein. The layers of this patch
comprise a backing, a polyurethane drug/enhancer matrix, a
membrane, an adhesive, and a release liner. The polyurethane matrix
is prepared using a room temperature curing polyurethane
prepolymer. Addition of water, alcohol, and complex to the
prepolymer results in the formation of a tacky firm elastomer that
can be directly cast only the backing material.
[0197] A further embodiment will utilize a hydrogel matrix patch.
Typically, the hydrogel matrix will comprise alcohol, water, drug,
and several hydrophilic polymers. This hydrogel matrix can be
incorporated into a transdermal patch between the backing and the
adhesive layer.
[0198] A liquid reservoir patch will also find use in the methods
described herein. This patch comprises an impermeable or
semipermeable, heat sealable backing material, a heat sealable
membrane, an acrylate based pressure sensitive skin adhesive, and a
siliconized release liner. The backing is heat sealed to the
membrane to form a reservoir which can then be filled with a
solution of the complex, enhancers, gelling agent, and other
excipients.
[0199] Foam matrix patches are similar in design and components to
the liquid reservoir system, except that the gelled pharmaceutical
agent-chemical modifier solution is constrained in a thin foam
layer, typically a polyurethane. This foam layer is situated
between the backing and the membrane which have been heat sealed at
the periphery of the patch.
[0200] For passive delivery systems, the rate of release is
typically controlled by a membrane placed between the reservoir and
the skin, by diffusion from a monolithic device, or by the skin
itself serving as a rate-controlling barrier in the delivery system
(See U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580,
4,788,062; and the like, all of which are incorporated herein by
reference). The rate of drug delivery will be dependent, in part,
upon the nature of the membrane. For example, the rate of drug
delivery across membranes within the body is generally higher than
across dermal barriers. The rate at which the active compound is
delivered from the device to the membrane is most advantageously
controlled by the use of rate-limiting membranes which are placed
between the reservoir and the skin. Assuming that the skin is
sufficiently permeable to the active compound (i.e., absorption
through the skin is greater than the rate of passage through the
membrane), the membrane will serve to control the dosage rate
experienced by the patient.
[0201] Suitable permeable membrane materials may be selected based
on the desired degree of permeability, the nature of the active
compound, and the mechanical considerations related to constructing
the device. Exemplary permeable membrane materials include a wide
variety of natural and synthetic polymers, such as
polydiniethylsiloxanes (silicone rubbers), ethylenevinylacetate
copolymer (EVA), polyurethanes, polyurethane-polyether copolymers,
polyethylenes, polyamides, polyvinylchlorides (PVC),
polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE),
cellulosic materials, e.g., cellulose triacetate and cellulose
nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate
(HEMA).
[0202] Other items may be contained in the device, such as other
conventional components of therapeutic products, depending upon the
desired device characteristics. For example, the compositions
disclosed herein may also include one or more preservatives or
bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl
hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the
like. These pharmaceutical compositions also can contain other
active ingredients such as antimicrobial agents, particularly
antibiotics, anesthetics, analgesics, and antipruritic agents.
Compositions for Administration as Suppositories
[0203] The compounds disclosed herein may be formulated for
administration as suppositories. A typical suppository is produced
by providing a low melting wax, such as a mixture of fatty acid
glycerides or cocoa butter, that is first melted and the active
component is dispersed homogeneously therein, for example, by
stirring. The molten homogeneous mixture is then poured into
convenient sized molds, allowed to cool, and to solidify.
[0204] The active compound may be formulated into a suppository
comprising, for example, about 0.5% to about 50% of a compound
disclosed herein, disposed in a polyethylene glycol (PEG) carrier
(e.g., PEG 1000 [96%] and PEG 4000 [4%]).
Formulation
[0205] A preferred aspect contemplates pharmaceutical compositions
useful for practicing the therapeutic methods described herein.
Pharmaceutical compositions can contain a physiologically tolerable
carrier together with at least one species of a secretagogue, such
as ghrelin splice variant or a ghrelin splice variant-like compound
as described herein, dissolved or dispersed therein as an active
ingredient. In a preferred embodiment, the pharmaceutical
composition is not immunogenic when administered to a human
individual for therapeutic purposes, unless that purpose is to
induce an immune response.
[0206] One aspect relates to a pharmaceutical composition
comprising at least one secretagogue, such as ghrelin splice
variant or a ghrelin splice variant-like compound as defined above
in Formula I. In a preferred embodiment, the pharmaceutical
composition comprises at least two different ghrelin splice
variant-like compounds as defined above in Formula I in order to
increase the effect of the treatment. The difference may for
example be compounds having different acylations as discussed
above.
[0207] As used herein, the terms "pharmaceutically acceptable",
"physiologically tolerable" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to or upon a human without the production of
undesirable physiological effects such as nausea, dizziness,
gastric upset and the like.
[0208] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art. Typically, such compositions are prepared as
sterile injectables either as liquid solutions or suspensions,
aqueous or non-aqueous; however, solid forms suitable for solution,
or suspensions, in liquid prior to use can also be prepared. The
preparation can also be emulsified.
[0209] The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient and in amounts suitable for use in the therapeutic
methods described herein. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol or the like and
combinations thereof. In addition, if desired, the composition can
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like which enhance
the effectiveness of the active ingredient. It is preferred that
the formulation has a pH within the range of 3.5-8, such as in the
range 4.5-7.5, such as in the range 5.5-7, such as in the range
6-7.5, most preferably around 7.3. However, as is understood by one
skilled in the art, the pH range may be adjusted according to the
individual treated and the administration procedure. For example,
certain secretagogues, such as ghrelin splice variant and ghrelin
splice variant homologs, may be easily stabilized at a lower pH;
so, in another preferred embodiment, the formulation has a pH
within the range 3.5-7, such as 4-6, such as 5-6, such as 5.3-5.7,
such as 5.5.
[0210] Pharmaceutical compositions disclosed herein can include
pharmaceutically acceptable salts of the compounds therein. These
salts will be ones which are acceptable in their application to a
pharmaceutical use, meaning that the salt will retain the
biological activity of the parent compound and the salt will not
have untoward or deleterious effects in its application and use in
treating diseases.
[0211] Pharmaceutically acceptable salts are prepared in a standard
manner. If the parent compound is a base, it is treated with an
excess of an organic or inorganic acid in a suitable solvent. If
the parent compound is an acid, it is treated with an inorganic or
organic base in a suitable solvent.
[0212] The compounds disclosed herein may be administered in the
form of an alkali metal or earth alkali metal salt thereof,
concurrently, simultaneously, or together with a pharmaceutically
acceptable carrier or diluent, especially and preferably in the
form of a pharmaceutical composition thereof, whether by, e.g.,
oral, rectal, or parenteral (including subcutaneous) route, in an
effective amount.
[0213] Examples of pharmaceutical acceptable acid addition salts
for use in the present inventive pharmaceutical composition include
those derived from mineral acids, such as, e.g., hydrochloric,
hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids,
and organic acids, such as, e.g., tartaric, acetic, citric, malic,
lactic, fumaric, benzoic, glycolic, gluconic, succinic,
p-toluenesulphonic, and arylsulphonic acids.
[0214] Other suitable pharmaceutically acceptable salts include the
acid addition salts (formed with the free amino groups of the
polypeptide). Other examples of salts include pharmaceutically
acceptable acid addition salts, pharmaceutically acceptable metal
salts, ammonium salts and alkylated ammonium salts. Acid addition
salts include salts of inorganic acids as well as organic acids.
Representative examples of suitable inorganic acids include
hydrochloric, hydrobromic, hydriodic, phosphoric, sulfuric and
nitric acids and the like. Representative examples of suitable
organic acids include formic, acetic, trichloroacetic,
trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,
glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,
pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic,
tartaric, ascorbic, pamoic, bismethylene salicylic,
ethanedisulfonic, gluconic, citraconic, aspartic, stearic,
palmitic, ethylenediaminetetraacetic (EDTA), p-aminobenzoic,
glutamic, benzenesulfonic, and p-toluenesulfonic acids and the
like. Further examples of pharmaceutically acceptable inorganic or
organic acid addition salts include the pharmaceutical acceptable
salts listed in Berge S. M. et al., J. Pharm. Sci. 66:1-19 (1977),
which is incorporated herein by reference. Examples of metal salts
include lithium, sodium, potassium and magnesium salts and the
like.
[0215] Examples of ammonium and alkylated ammonium salts include
ammonium, methylammonium, dimethylammonium, trimethylammonium,
ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium
and tetramethylammonium salts and the like.
[0216] Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine and the like.
[0217] Also included within the scope of compounds or
pharmaceutical acceptable acid addition salts thereof in the
context of the present disclosure are any hydrates (hydrated forms)
thereof.
[0218] For parenteral administration, solutions of the present
compounds in sterile aqueous solution, aqueous propylene glycol or
sesame or peanut oil may be employed. Such aqueous solutions should
be suitably buffered if necessary, and the liquid diluent first
rendered isotonic with sufficient saline or glucose. The aqueous
solutions are particularly suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. The sterile
aqueous media employed are all readily available by standard
techniques known to those skilled in the art.
[0219] Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as
cottonseed oil, organic esters such as ethyl oleate, and water-oil
emulsions.
[0220] Suitable pharmaceutical carriers include inert solid
diluents or fillers, sterile aqueous solution and various organic
solvents. Examples of solid carriers are lactose, terra alba,
sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia,
magnesium stearate, stearic acid or lower alkyl ethers of
cellulose. Examples of liquid carriers are syrup, peanut oil, olive
oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene
or water. Nasal aerosol or inhalation formulations may be prepared,
for example, as solutions in saline, employing benzyl alcohol or
other suitable preservatives, absorption promoters to enhance
bioavailability, employing fluorocarbons, and/or employing other
solubilizing or dispersing agents.
[0221] The pharmaceutical compositions formed by combining the
compounds disclosed herein and the pharmaceutical acceptable
carriers are then readily administered in a variety of dosage forms
suitable for the disclosed routes of administration. The
formulations may conveniently be presented in unit dosage form by
methods known in the art of pharmacy.
[0222] In a preferred embodiment, the formulation comprises the
secretagogue or a salt thereof as a lyophilisate, and the
formulation further comprises a solvent, said lyophilisate and said
solvent being in separate compartments until administration. In
another embodiment, the formulation is a solution of the
secretagogue or a salt thereof. In either embodiment, the solvent
may be any suitable solvent, such as those described herein, and
preferably the solvent is saline.
[0223] Another aspect relates to a method for preparing a
medicament or pharmaceutical composition comprising a compound
disclosed herein, the method comprising admixing at least one
ghrelin splice variant-like compound, as defined above in Formula
I, with a physiologically acceptable carrier. A further aspect
relates to a pharmaceutical composition comprising, as an active
ingredient, a compound as defined above in Formula I or a
pharmaceutically acceptable salt thereof together with a
pharmaceutically-acceptable carrier. Accordingly, the formulation
may further include the transport molecules as described above.
Combination Treatments
[0224] In a further aspect, the present compounds may be
administered in combination with additional
pharmacologically-active substances or other
pharmacologically-active material and/or may be administered in
combination with another therapeutic method. By the phrase "in
combination with another substance(s) and/or therapeutic method(s)"
is meant herein that said another substance(s) and/or therapeutic
method(s) is administered to the individual thus treated before,
during (including concurrently with) and/or after treatment of an
individual with a secretagogue. In all cases of combination
treatment described herein, the combination may be in the form of
kit-in-part systems, wherein the combined active substances may be
used for simultaneous, sequential or separate administration. In
all cases, it is preferred that any of the herein-mentioned
medicaments are administered in pharmaceutically effective amounts,
i.e. an administration involving a total amount of each active
component of the medicament or pharmaceutical composition or method
that is sufficient to show a meaningful patient benefit.
[0225] In the following sections, combination therapies for use in
preferred embodiments are grouped as follows:
1) Combinations wherein all active ingredients are
appetite-regulating agents or in other ways useful for treating
neuronal damage and/or neurodegenerative disease.
[0226] The secretagogue(s) according to the present disclosure can
be administered in combination with other appetite-regulating
agents, including more than one type of growth hormone
secretagogue, such as another ghrelin splice variant-like compound,
such as a ghrelin splice variant-like compound comprising a
structure defined by Formula I, described herein. Other
secretagogues suitable for combination administration with another
secretagogue compound are any of the secretagogue compounds
described herein. In one preferred embodiment, ghrelin splice
variant (most preferably human ghrelin splice variant) is
administered in combination with a different, ghrelin splice
variant-like compound--this combination is envisaged to enhance
and/or prolong the effect of the secretagogues on the ghrelin
receptor. In a similar way, several different secretagogues may be
administered to an individual to increase efficacy on the ghrelin
receptor, such as greater than 2 different secretagogue types, such
as 3, such as 4, such as 5, such as 6, such as 7, such as greater
than 8 different secretagogue types. The secretagogue according to
the present disclosure, such as ghrelin splice variant or a ghrelin
splice variant-like compound(s) can also be administered in
combination with a pharmaceutically effective amount of a growth
hormone, including hGH.
[0227] In one preferred embodiment, the secretagogue, such as
ghrelin splice variant or a ghrelin splice variant-like compound,
may be administered in combination with IGF-1, IGFBP-3, or ALP,
preferably with IGF-1. The rationale behind this combination
treatment is to increase the level of IGF-1, IGFBP-3, and/or ALP
found to be low in cachectic individuals.
[0228] In a further embodiment, the secretagogues, such as ghrelin
splice variant or a ghrelin splice variant-like compound, may be
administered in combination with compounds known to stimulate
appetite, such as ghrelin, melanocortin receptor antagonists,
neuropeptide Y receptor agonists including agonists selective for
individual subtypes of the neuropeptide Y receptors, leptin or
leptin receptor agonists, cannabinoids including marijuana and
marijuana derivatives, antipsychotics, especially atypical
antipsychotics such as sertindole, Sufpirid, Clozapine,
Risperidone, Quetiapin, Amisulpride, Ziprasidon, and
Olanzapine.
2) Combinations of the secretagogue, such as ghrelin splice variant
or a ghrelin splice variant-like compound, with an ingredient or
therapy active against a disease causing or being associated with
the disease or condition treated with the secretagogue, such as
ghrelin splice variant or a ghrelin splice variant-like
compound.
[0229] Particularly in relation to neuronal damage and/or
neurodegenerative disease, administration of a secretagogue, such
as a ghrelin splice variant-like compound, may be performed in
combination with any anti-neurodegenerative or supportive therapy,
including levodopa and/or dopamine agonist therapy and surgical
treatment. In particular, it is used in combination with levodopa
and dopamine agonist therapy. Thus, one embodiment relates to a
method of treating cancer comprising administering an effective
amount of dopamine agonist therapy and an effective amount of a
secretagogue, such as a ghrelin splice variant-like compound
according to the present disclosure. The treatment with the
secretagogue, such as a ghrelin splice variant-like compound, may
be started before the levodopa and/or dopamine agonist therapy
treatment initiates. It may be administered continuously during the
levodopa and/or dopamine agonist therapy or it may be administered
at intervals, for example between periods with radiotherapy
therapy.
[0230] Another embodiment relates to a method of treating
neurodegenerative disease comprising administering an effective
amount of levodopa and/or dopamine agonist therapy and an effective
amount of a secretagogue, such as a ghrelin splice variant-like
compound according to the present disclosure. The treatment with
the secretagogue, such as a ghrelin splice variant-like compound,
may be started before the levodopa and/or dopamine agonist therapy
treatment initiates. It may be administered continuously during the
levodopa and/or dopamine agonist therapy, or it may be administered
at intervals, for example between periods with levodopa and/or
dopamine agonist therapy.
[0231] Furthermore, the combination treatment may be
co-formulations of the secretagogue, such as a ghrelin splice
variant-like compound, and the anti neurodegenerative therapy.
[0232] A secretagogue according to the present disclosure, such as
ghrelin splice variant or a ghrelin splice variant-like compound,
may also be administered in combination with a pharmaceutically
effective amount of glucocorticoid steroids and prokinetic
treatment as well as other treatment used in cancer therapy. Thus,
in another preferred embodiment, a secretagogue according to the
present disclosure, such as ghrelin splice variant or a ghrelin
splice variant-like compound, is administered in combination with a
pharmaceutical effective amount of one or more of: progestational
drugs, such as megastrol and/or cyproheptadines (and/or other 5-HT
receptor antagonists); and/or branched chain amino acids; and/or
oxandralin; and/or anti-TNF-.alpha. agents, such as infliximab,
etanercept, or adalimumab; and/or testosterone; and/or a "cocktail"
comprising immunonutrition drugs, antioxidants and COX2 inhibitors;
and/or cannabinoids; and/or eicosapentaenoic acid; and/or
melatonin; and/or thalidomide; and/or a f32 adrenergic drug; most
preferably for the treatment of neurodegenerative disease, such as
parkinson's disease or alzheimers disease.
[0233] In yet another embodiment, the secretagogue, such as a
ghrelin splice variant-like compound, is administered in
combination with anti-inflammatory compounds, preferably an NSAID,
such as indomethacin, and COX1 inhibitors or COX2 inhibitors;
and/or anti-TNF-.alpha. compounds such as infliximab, etanercept,
or adalimumab. Another combination may be with erythropoietin/EPO.
Another combination can be with angiotensin II lowering agents,
such as Vitor. Another combination can be with selective androgen
receptor modulator(s). Another combination may be with one or more
of leptin, agonists of the renin-angiotensin system, opioid
receptor agonists or peroxisome proliferator-activated receptor
gamma agonists.
[0234] In relation to treatment of lipodystrophy, another
embodiment relates to a treatment wherein a secretagogue, such as
ghrelin splice variant, more preferably a ghrelin splice
variant-like compound, is administered in combination with a
lipodystrophy treatment, such as one or more of the treatments or
compounds described herein suitable for treating a lipodystrophic
syndrome.
[0235] Thus, other pharmacologically active substances that may be
administered in combination with said secretagogue, such as a
ghrelin splice variant-like compound, in the methods of the present
disclosure comprise: [0236] (a) Leptin: Leptin has been shown to
have a positive effect on the metabolic abnormalities associated
with lipodystrophy (Oral E. A. et al., J. Clin. Endocrinol. Metab.
91:621-28 (2006)). This treatment has proven to be beneficial both
to those patients that suffer from a low plasma level of leptin and
to those that have a normal level. [0237] (b) Peroxisome
proliferator-activated receptor (PPAR-.gamma.) agonists:
PPAR-.gamma. has in several studies been demonstrated to be
important for adipocyte metabolism and metabolic syndrome, and it
is proposed that PPAR-.gamma. agonists will decrease the symptoms
of lipodystrophy (Semple R. K. et al., J. Clin. Invest. 116:581-89
(2006)). [0238] (c) Agonists of the renin-angiotensin system: It
has been shown that treatment with HAART increases the activity of
ACE in the T-cells, which means that agonists of the
renin-angiotensin system may improve HAART induced lipodystrophy
(Hegele R. A. & Leff T., J. Clin. Invest. 114:163-65 (2004)).
[0239] (d) Opioid receptor antagonists: Opioid receptor
antagonists, such as Naloxone and Naltrexone, have been shown to
prolong the period of time from protease inhibitor treatment to
development of the first symptoms of lipodystrophy (AIDS Patient
Care STDS 14:283 (2000)). [0240] (e) Des-acyl ghrelin splice
variant: Ghrelin splice variant in combination with des-acyl
ghrelin splice variant has been found to decrease insulin
resistance, which is an important feature of the lipodystrophy
syndrome (Koutkia P. et al., Am. J. Physiol. Endocrinol. Metab.
286:E296-303 (2004)). [0241] (f) Adiponectin and anti-diabetic
treatment including other compounds for the treatment and/or
prevention of insulin resistance and diseases wherein insulin
resistance is the pathophysiological mechanism. [0242] (g) Therapy
with rhGH has been reported to cause reduction in the size of
"buffalo hump", truncal fat and to increase the lean body mass in a
small number of patients (Lo J. C. et al., J. Clin. Endocrinol.
Metab. 86:3480-87 (2001)). However, fat loss and lipid
abnormalities did not improve and blood glucose control worsened.
Examples of syndromes treated with hGH include HIV, AIDS and
cancer. Without being bound by theory, it is believed that
treatment with ghrelin splice variant or a analog thereof would
maintain and/or increase body fat in patients being treated with
hGH, thereby effectively counteracting or at least reducing
lipodystrophy caused by hGH. Thus, one preferred embodiment relates
to use of ghrelin splice variant or ghrelin splice variant-like
compound in combination with a growth hormone, preferably in
individuals suffering from neurodegenerative disease. Said
treatment with ghrelin splice variant or an analog thereof may be
prior to, and/or during and/or after the individual is subjected to
treatment with a growth hormone. Said growth hormone is preferably
hGH. [0243] (h) Treatment with combinations of different
secretagogues as described above under group 1), supra. 3)
Combinations of the secretagogue, such as ghrelin splice variant or
a ghrelin splice variant-like compound, with an ingredient active
or therapy against symptoms associated with the disease or
condition treated with the secretagogue, such as ghrelin splice
variant or a ghrelin splice variant-like compound.
[0244] One aspect relates to combination treatment, wherein one of
the ingredients in the combination is used for treating symptoms or
conditions that may be encountered in individuals suffering from
neuronal damage and/or neurodegenerative disease. Thus, uses and
combination treatments involving administration of a secretagogue,
such as the ghrelin splice variant-like compound according to the
present disclosure, can also involve treatment in combination with
one or more of [0245] a) prophylaxis and/or alleviation and/or
treatment of a clinical depression, which combination treatment
further comprises administering an antidepressant, a prodrug
thereof, or a pharmaceutical acceptable salt of said antidepressant
or said prodrug. In the above combination treatment, the
antidepressant is preferably a norepinephrine reuptake inhibitor
(NERD, a selective serotonin reuptake inhibitor (SSRI), a monoamine
oxidase inhibitor (MAO), a combined NERI/SSRI, or an atypical
antidepressant, a prodrug of said antidepressant or a
pharmaceutically acceptable salt of said antidepressant or said
prodrug. Preferred antidepressants are S SRI, a prodrug thereof or
a pharmaceutical acceptable salt of said SSRI or said prodrug. The
SSRI is preferably citalopram, escitalopram, femoxetine,
fluoxetine, fluvoxamine, indalpine, indeloxazine, milnacipran,
paroxetine, sertraline, sibutramine or zimeldine, a prodrug of said
SSRI or a pharmaceutical acceptable salt of said SSRI or said
prodrug. Of the above, citalopram and escitalopram, a prodrug or a
pharmaceutical acceptable salt thereof, are preferred in certain
embodiments of combination treatments. [0246] b) prophylaxis and/or
alleviation and/or treatment of an emetic condition, including
nausea and vomiting, which combination treatment further comprises
administering an antiemetic agent, a prodrug thereof, or a
pharmaceutically acceptable salt of said antiemetic agent or said
prodrug. Preferred antiemetic agents used in combination treatments
according to the present disclosure include meclizine
hydrochloride, prochlorperazine, promethazine, trimethobenzamide
hydrochloride and ondansetron hydrochloride. In particular, emesis
may be caused by cancer, either due to the anti-cancer treatment or
due to the cancer disease as such. [0247] c) prophylaxis and/or
alleviation and/or treatment of a psychotic condition, which
combination treatment further comprises administering an
antipsychotic agent, a prodrug thereof or a pharmaceutical
acceptable salt of said antipsychotic agent or said prodrug.
Preferred antipsychotic agents used in combination treatments in
accordance with the present disclosure include chlorpromazine,
haloperidol, clozapine, loxapine, molindone hydrochloride,
thiothixene, olanzapine, ziprasidone, ziprasidone hydrochloride,
prochlorperazine, perphenazine, trifluoperazine hydrochloride and
risperidone. [0248] d) prophylaxis and/or alleviation and/or
treatment of anxiety, which combination treatment further comprises
administering an antianxiety agent, a prodrug thereof or a
pharmaceutically acceptable salt of said antianxiety agent or said
prodrug. Preferred antianxiety agents used in combination
treatments in accordance with the present disclosure include
alprazolam, clonazepam, lorazepam, oxazepam, chlordiazepoxide
hydrochloride, diazepam, buspirone hydrochloride, doxepin
hydrochloride, hydroxyzine pamoate and clonazepam.
[0249] Of course, combinations of the above groups (1-3) are also
within the scope of this disclosure.
Medical Packaging
[0250] The compounds disclosed herein may be administered alone or
in combination with pharmaceutically acceptable carriers or
excipients, in either single or multiple doses.
[0251] The formulations may conveniently be presented in unit
dosage form by methods known to those skilled in the art.
[0252] It is preferred that the compounds according to the present
disclosure are provided in a kit. Such a kit typically contains an
active compound in dosage forms for administration. A dosage form
contains a sufficient amount of active compound such that a
desirable effect can be obtained when administered to a subject,
preferably prior to at least one meal a day, more preferably prior
to each main meal, such as three times a day, during the course of
1 or more days. Thus, it is preferred that the medical packaging
comprises an amount of dosage units corresponding to the relevant
dosage regimen. Accordingly, in one embodiment, the medical
packaging comprises a pharmaceutical composition comprising a
compound as defined above or a pharmaceutically acceptable salt
thereof and pharmaceutically acceptable carriers, vehicles and/or
excipients, said packaging having from 7 to 21 dosage units, or
multiple thereof, thereby having dosage units for one week of
administration or several weeks of administration.
[0253] In one embodiment, the medical packaging is for
administration once daily in a week, and comprises 7 dosage units,
in another embodiment the medical packaging is for administration
twice daily, and comprises 14 dosage units. In yet another more
preferred embodiment, the medical packaging is for administration
three times daily, and comprises 21 dosage units.
[0254] The dosage units are as defined above, i.e. a dosage unit
preferably comprises an amount of the ghrelin splice variant-like
compound or a salt thereof equivalent to from 0.3 .mu.g to 600 mg
ghrelin splice variant, such as of from 2.0 .mu.g to 200 mg ghrelin
splice variant, such as from 5.0 .mu.g to 100 mg ghrelin splice
variant, such as from 10 .mu.g to 50 mg ghrelin splice variant,
such as from 10 .mu.g to 5 mg ghrelin splice variant, such as from
10 .mu.g to 1.0 mg ghrelin splice variant.
[0255] The medical packaging may be in any suitable form for
parenteral, in particular subcutaneous, administration. In a
preferred embodiment, the packaging is in the form of a cartridge,
such as a cartridge for an injection pen, the injection pen being
such as an injection pen known from insulin treatment or from hGH
treatment.
[0256] When the medical packaging comprises more than one dosage
unit, it is preferred that the medical packaging is provided with a
mechanism to adjust each administration to one dosage unit
only.
[0257] Preferably, a kit contains instructions indicating the use
of the dosage form to achieve a desirable affect and the amount of
dosage form to be taken over a specified time period. Accordingly,
in one embodiment the medical packaging comprises instructions for
administering the pharmaceutical composition. In particular said
instructions may include instructions referring to administration
of said pharmaceutical composition either during a meal, or
preferably at the most 45 minutes prior to a meal, such as at the
most 30 minutes prior to a meal, such as at the most 25 minutes
prior to a meal, such as at the most 20 minutes prior to a meal,
such as at the most 15 minutes prior to a meal, such as at the most
10 minutes prior to a meal, such as at the most 5 minutes prior to
a meal.
Method for Monitoring the Effect of Treatment with Ghrelin Splice
Variant and/or a Ghrelin Splice Variant-Like Compound
[0258] Another aspect relates to a method for monitoring the effect
of the administration of a secretagogue, such as the ghrelin splice
variant-like compounds disclosed herein, in a method of the present
disclosure, comprising measuring one or more markers, in particular
markers, selected from GH, IGF-1, IGFBP-3, ALP (acidic labeled),
thyroid hormones, sex hormones, and albumin; more preferably
selected from IGF-I, IGFBP-3, ALP (acidic labeled); even more
preferably IGF-1. These markers are all low in cachetic patients
and are expected to increase after treatment with ghrelin splice
variant. Other markers that are expected to increase after
treatment with ghrelin splice variant are the blood GH level and
the body weight. In addition, the body composition is expected to
change, and the lean body mass is expected to increase. The body
composition changes can be assessed by using MRI or NMR.
[0259] Thus, one embodiment relates to a method for monitoring the
effect of any of the treatments of an individual with a
secretagogue described herein, said method comprising measuring the
blood level of said individual of one or more of: (i) IGF-1 and/or
(ii) IGFBP-3 and/or (iii) ALP and/or (iv) one or more thyroid
hormones and/or (v) one or more sex hormones and/or (vi) albumin
or, more preferably, one or more of: (i) IGF-1 and/or (ii) IGFBP-3
and/or (iii) ALP and/or (iv) GH and/or (v) body weight and/or (vi)
body composition.
[0260] Methods for measuring substances in the blood level of an
individual are well known in the art. As an example, an isolated
blood sample may be tested by methods such as Western blot or by
enzyme-linked assay (ELISA).
EXAMPLES
[0261] The present disclosure is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the preferred features of this disclosure,
and without departing from the spirit and scope thereof, can make
various changes and modifications to adapt it to various uses and
conditions.
[0262] Examples 2, 5, 6, 7, 8, and 9 are working examples. Examples
1, 3, 4, 10, and 11 are prophetic examples.
Example 1
Competition Binding Assays
[0263] Transfected COS-7 cells are transferred to culture plates
one day after transfection at a density of 1.times.10.sup.5 cells
per well aiming at 5-8% binding of the radioactive ligand. Two days
after transfection, competition binding experiments are performed
for 3 hours at 4.degree. C. using 25 pM of [.sup.125I]Ghrelin (GE
Healthcare, Piscataway, N.J., USA). Binding assays are performed in
0.5 ml of a 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM
CaCl.sub.2, 5 mM MgCl.sub.2, and 0.1% (w/v) bovine serum albumin,
40 .mu.g/ml bacitracin. Non-specific binding is determined as the
binding in the presence of 1 micromole of unlabeled ghrelin splice
variant. Cells are washed twice in 0.5 ml of ice-cold buffer and
0.5-1 ml of lysis buffer (8 M Urea, 2% NP40 in 3 M acetic acid) is
added and the bound radioactivity is counted. Determinations are
made in duplicate.
Example 2
Synthetic Production of Ghrelin Splice Variant-Like Compound
[0264] Amino acid derivatives and synthesis reagents can be
obtained from commercial sources. Peptide chain extension can be
performed using Applied Biosystem 433A synthesizer produced by
Perkin Elmer, and a protected peptide derivative-resin can be
constructed by the Boc or Fmoc method. The protected peptide resin
obtained by the Boc method is deprotected with anhydrous hydrogen
fluoride (HF) in the presence of p-cresol thereby releasing the
peptide, which is then purified. The protected peptide resin
obtained by the Fmoc method is deprotected with trifluoroacetic
acid (TFA) or dilute WA containing various scavengers, and the
released peptide is purified. Purification is performed in reversed
phase HPLC on a C4 or C18 column. The purity of the purified
product can be confirmed by reverse phase HPLC, and its structure
can be confirmed by amino acid composition analysis and mass
spectrometry.
[0265] Peptides disclosed herein can be produced by a conventional
peptide synthesis method. Specifically, synthesis of acylated or
alkylated peptides is exemplified below.
[0266] Abbreviations: "HMP resin" means
4-hydroxymethyl-phenoxymethyl resin; "Fmoc amide resin" means
4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxyacetamido-ethyl
resin; "PAM resin" means phenylacetoamidomethyl resin; "HBTU" means
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; "TBTU" means
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; "HOBt" means 1-hydroxybenzotriazole; "DCC" means
dicyclohexylcarbodiimide; "DIPCI" means diisopropylcarbodiimide;
"TFA" means trifluoroacetic acid; "DIPEA" means
diisopropylethylamine; "TIPS" means triisopropylsilane; "Fmoc"
means fluorenylmethoxycarbonyl; "Boc" means t-butyloxycarbonyl;
"Tit" means trityl; "Bu" means t-butyl; "Pmc" means
2,2,5,7,8-pentamethylchroman-6-sulfonyl; "Prl" means propionyl;
"PhPrl" means phenylpropionyl; "Bzl" means benzyl; "Bom" means
benzyloxymethyl; "Tos" means toluenesulfonyl; "CI-Z" means
2-chloro-benzyloxycarbonyl; "Pis" means 2-phenylisopropyl; "Mtt"
means 4-methyltrityl; "DMF" means N,N-dimethylformamide; "NMP"
means N-methylpyrrolidone; "DMAP" means 4-dimethylaminopyridine;
"HOSu" means N-hydroxysucciniimide; "Adod" means 2-aminododecanoic
acid; "Aib" means 2-aminoisobutylic acid; "Ape" means
5-aminopentanoic acid; "Cha" means cyclohexylalanine; "Dap" means
2,3-diaminopropionic acid; "Nal" means naphtylalanine; "Nie" means
norleucine.
[0267] Protecting amino acids which can be used in synthesis Fmoc
method: Boc-Gly, Fmoc-Gly, Fmoc-Ser (Bu), Fmoc-Ser (Trt), Fmoc-Glu
(OBu), Fmoc-His (Boc), Fmoc-Gln (Trt), Fmoc-Arg (Pmc), Fmoc-Lys
(Boc), Fmoc-Pro, Fmoc-Leu, Fmoc-Ala, Fmoc-Val, Fmoc-Phe, Fmoc-Phe,
Fmoc-Ser (n-C.sub.8H.sub.17), Fmoc-Ser (n-C.sub.8H.sub.17),
Fmoc-Cys (n-C.sub.8H.sub.17), Fmoc-Asp (OPis), Fmoc-Ser (Bzl),
Fmoc-Cys (Trt), Fmoc-Dap (Octanoyl), Fmoc-2-Nal, Fmoc-2-Nal,
Fmoc-Nle, Fmoc-Lys (Mtt), Fmoc-Aib-OH, Fmoc-Asp
(O--C.sub.7H.sub.15). Boc method: Boc-Gly, Boc-Ser (Bzl), Boc-Ser
(Ac), Boc-Ser (Prl), Boc-Glu (OBzl), Boc-His (Born), Boc-Gln,
Boc-Arg (Tos), Boc-Lys (CI-Z), Boc-Pro, Boc-Leu, Boc-Ala, Boc-Val,
Boc-Phe, Boc-Cys (n-C.sub.8H.sub.17), Boc-Ape, Boc-Ser
(n-C.sub.8H.sub.17)
[0268] Units used: [0269] (a) Analytical HPLC system Unit: Shimadzu
LC-10A System; Column: YMC PROTEIN-RP (4.6 mm.times.150 mm); Column
temperature: 40.degree. C.; Eluent: A linear gradient of from 0 to
50% acetonitrile for 20 minutes in 0.1% trifluoroacetic acid; Flow
rate: 1 mL/min; Detection: UV (210 nm); Injection volume: 10 to 100
mu I. [0270] (b) Preparative HPLC system Unit: Waters 600
Multisolvent Delivery System; Columns: YMC-Pack-ODS-A (5 mu m, 20
mm.times.250 mm) YMC-Pack-PROTEIN-RP (5 mu m, C4, 10 mm.times.250
mm) YMC-Pack PROTEIN-RP (5 mu m, C4, 20 mm.times.250 mm) YMC
PROTEIN-RP (4.6 mm.times.150 mm); Eluent: A suitable linear
gradient of acetonitrile concentration in 0.1% trifluoroacetic
acid; Flow rate: 10 mL/min. (for columns of an inner diameter of 20
mm), 3 mL/min. (for the column of an inner diameter of 10 mm), 1
mL/min. (for the column of an inner diameter of 4.6 mm); Detection:
210 nm, 260 nm; Injection: 10 to 2000 mu I (2000 mu I or more was
injected via a pump) [0271] (c) Mass spectrometer Unit: Finnigan
MAT TSQ700; Ion source: ESI; Detection ion mode: Positive Spray;
Voltage: 4.5 kV; Capillary temperature: 250.degree. C.; Mobile
phase: A mixture of 0.2% acetic acid and methanol (1:1); Flow rate:
0.2 mL/min; Scan range: m/z 300 to 1,500 [0272] (d) Analysis of
amino acid sequence Unit: Applied Biosystem 477A, 492 model
sequencer manufactured by Perkin Elmer [0273] (e) Analysis of amino
acid composition Unit: L-8500 model amino acid analyzer
manufactured by Hitachi, Co., Ltd.; Sample: Unless otherwise
specified, the sample is hydrolyzed with 6 M HCl at 110.degree. C.
for 24 hours in a sealed tube.
Example of Synthesis of a Derivative Having Acyl Serine (Fmoc
Method, Carboxyl-Terminal Amide Derivatives) Ghrelin Splice
Variant
[0274] GSS (CO--C.sub.7H.sub.15) FLSPEHQRVQVRPPHKAPH Fmoc-His
(Pmc)-HMP-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) is treated with
20% piperazine for 20 minutes and subjected repeatedly to
introduction of Fmoc-amino acid by HBTU/HOBt and elimination of
Fmoc by piperazine sequentially to construct Fmoc-Ser (Bu)-Ser
(Trt)-Phe-Leu-Ser (tBu)-Pro-Glu (OBu)-His (Boc)-Gln (Trt)-Arg
(Pmc)-Val-Gln-Val (Trt)-Arg (Pmc)-Pro-Pro-His (Boc)-Lys (Boc)-Ala
(Boc)-Pro (Boc)-Pro-His (Pmc)-resin. After Boc-Gly is finally
introduced by DCC/HOBt, the resulting protected peptide resin (1.3
g) is treated with 1% TFA-5% TIPS-methylene chloride solution (15
mL) for 30 minutes.
[0275] The peptide resin is filtrated, washed several times with
methylene chloride (30 mL), and washed with 5% DI EA (10 mL) and
then with methylene chloride (30 mL). The resulting de-Trt peptide
resin (about 1.3 g) is swollen with NMP (10 mL), and octanoic acid
(144.2 mg, 1.0 mmol) and DIPCI (126.2 mg, 1.0 mmol) are added
thereto in the presence of DMAP (61.1 mg, 0.5 mmol) and allowed to
react for 8 hours. The resin is recovered by filtration and washed
with NMP and then with methylene chloride, followed by drying under
vacuum to give about 1.2 g protected peptide resin wherein the side
chain of third serine is octanoylated. To this product is added a
de-protecting reagent (10 mL) consisting of 88% TFA-5% phenol-2%
TIPS-5% H.sub.2O, and the mixture is stirred at room temperature
for 2 hours. The resin is removed by filtration, and the filtrate
is concentrated followed by adding ether to the resulting residues
to form precipitates. The precipitates are recovered by filtration
and dried to give about 550 mg crude peptide. 200 mg of this
product is dissolved in 10 mL water and applied to YMC-Pack
PROTEIN-RP (C4, 20 mm.times.250 mm) and eluted with a linear
gradient (flow rate: 10 mL/min) for 60 minutes of from 0 to 54%
acetonitrile in 0.1% trifluoroacetic acid. The desired fractions
are collected and lyophilized to give about 120 mg of the desired
product.
Example of Synthesis of a Derivative Having Acyl Serine (Fmoc
Method, Carboxyl-Terminal Amide Compounds) Ghrelin Splice Variant
(1-22)-NH.sub.2
[0276] GSS (CO--C.sub.7H.sub.15) FLSPEHQRVQVRPPHKAPH-NH.sub.2
Fmoc-amide-resin (403 mg, 0.25 mmol, ABI Co., Ltd.) is treated with
20% piperazine for 20 minutes and subjected repeatedly to
introduction of Fmoc-amino acid by HBTU/HOBt and elimination of
Fmoc by piperazine sequentially to construct Fmoc-Ser (Bu)-Ser
(Trt)-Phe-Leu-Ser (Bu)-Pro-Glu (OBu)-His (Boc)-Gln (Trt)-Arg
(Pmc)-Val-Gln-Val (Trt)-Arg (Pmc)-Pro-Pro-His (Boc)-Lys (Boc)-Ala
(Boc)-Pro (Boc)-Pro-His (Boc)-resin. After Boc-Gly is finally
introduced by DCC/HOBt, the resulting protected peptide resin
(about 550 mg) is treated with 1% TFA-5% TIPS-methylene chloride
solution (10 mL) for 30 minutes. The peptide resin is recovered by
filtration, washed several times with methylene chloride (30 mL),
and washed with 5% DIEA (10 mL) and then with methylene chloride
(30 mL). The resulting de-Trt peptide resin (about 750 mg) is
swollen with NMP (10 mL), and octanoic acid (144.2 mg, 1.0 mmol)
and DIPCI (126.2 mg, 1 mmol) are added thereto in the presence of
DMAP (61.1 mg, 0.5 mmol) and allowed to react for 4 hours. The
resin is recovered by filtration and washed with NMP and then with
methylene chloride, followed by drying under vacuum to give about
800 mg protected peptide resin wherein the side chain of third
serine is octanoylated. TFA (10 mL) is added to this product and
stirred at room temperature for 30 minutes. The resin is removed by
filtration, and the filtrate is then concentrated followed by
adding ether to the resulting residues to form precipitates. The
precipitates are recovered by filtration and dried to give about
250 mg crude peptide. About 200 mg of this product is dissolved in
10 mL of 30% aqueous acetic acid and applied to YMC-Pack PROTEIN-RP
(C4, 20 mm.times.250 mm) and eluted with a linear gradient (flow
rate: 10 mL/min.) for 60 minutes of from 0 to 54% acetonitrile in
0.1% trifluoroacetic acid. The desired fractions are collected and
then lyophilized to give about 150 mg of the desired product.
Example of Synthesis of a Derivative Having Acyl Serine (Boc
Method) [Ser3 (Propionyl)]-Ghrelin Splice Variant (1-22)
[0277] GSS (CO--CH.sub.2CH.sub.3) FLSPEHQRVQVRPPHKAPH protected
ghrelin splice variant resin (4-22) is constructed from Boc-His
(Tos)-Pam resin (0.75 g, 0.5 mmol) by Boc chemistry, and Boc-Ser
(CO--CH.sub.2CH.sub.3)--OH, Boc-Ser (Bzl)-OH and Boc-Gly-OH are
condensed with a half (1.4 g) of the resin. The resulting resin,
1.5 g, is then treated with a mixture of HF and p-cresol (8.5
mL:1.5 mL) at 0.degree. C. for 1 hour, and the HF is evaporated.
Ether is added to the residues, whereby 671 mg crude peptide is
obtained. This sample is then dissolved in 50% acetic acid (AcOH)
and applied to a preparative column YMC-Pack-ODS-A (5 mu m, 20
mm.times.250 mm) and eluted at a rate of 10 mL/min by a gradient of
from 0 to 95% acetonitrile concentration in 0.1% TFA solution for
75 minutes. Those fractions containing the desired product are
lyophilized to give approximately 135.8 mg crude peptide. A part
(0.5 mg) of this product is applied to YMC-A-302 column (C18, 4.6
mm.times.150 mm) and eluted at a flow rate of 1 mL/min. by a
gradient of from 15 to 19% concentration acetonitrile. This
purification procedure is then repeated and the desired fractions
are combined to give approximately 0.41 mg of the desired
product.
[0278] Other compounds according to the present disclosure can be
produced likewise.
[0279] The above method was used to synthesize acylated and
non-acylated SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5.
Example 3
A Randomized, Single Center, Four-Period Crossover Trial to
Investigate the Absolute Bioavailability of Intravenously
Administered Ghrelin Splice Variant and Subcutaneously Administered
Ghrelin Splice Variant at Three Different Single Doses in Healthy
Subjects
Objectives:
[0280] Primary: To investigate the absolute bioavailability of
three different doses of ghrelin splice variant administered as
single intravenous and subcutaneous doses. [0281] Secondary: [0282]
1) To investigate the dose linearity (dose proportionality) of the
ascending doses. [0283] 2) To investigate and compare the
pharmacodynamic profiles between the treatments. [0284] 3) To
assess the safety and local tolerability. Trial Design: A
randomized, single center, unbalanced block design, four-period
crossover trial to investigate the absolute bioavailability between
intravenously administered ghrelin splice variant and
subcutaneously administered ghrelin splice variant at three
different single doses in healthy subjects. Three doses will be
used for each way of administration: low, medium and high. To
reduce the number of dosings to each individual and hence reduce
the length of the trial, each subject will only receive four doses
of the total of six doses, i.e. two dose levels administered as
intravenous and subcutaneous, respectively. The unbalanced block
design will ensure that all three-dose levels will be covered in
this way although not all subjects will receive all dose levels. A
sufficient washout period will be placed between the individual
dosing periods.
Endpoints:
[0284] [0285] Pharmacokinetics of ghrelin splice variant:
AUC.sub.0-t, AUC, C.sub.max, t.sub.max, t, C.sub.1/f, V.sub.z/f,
C.sub.1, V.sub.z, t.sub.1/z [0286] MRT Pharmacodynamics: GH: AUC,
C.sub.max and t.sub.max Cardiac output, assessment of hunger,
food/energy intake, degree of pleasure related to food intake, body
mass, energy expenditure, DEXA. Safety: Safety and local
tolerability will be assessed throughout the study by clinical
evaluations (physical examination and vital signs),
electrocardiography and laboratory tests (hematology and clinical
chemistry). Trial population and power calculation: Healthy male
subjects, aged 18-45 years with a body mass index (BMI) of 19-26
kg/m.sup.2 (both inclusive).
[0287] The primary objective of this study is to investigate the
absolute bioavailability of ghrelin splice variant administered via
intravenous and subcutaneous administration. An unbalanced block
design will be applied to reduce the trial period time and reduce
the number of dosings per subject. The number of subjects needed to
perform a statistical analysis of absolute bioavailability per dose
levels as well as an analysis of dose linearity between doses will
be calculated based on existing literature data.
Trial products: ghrelin splice variant for intravenous and
subcutaneous administration.
Example 4
Functional Tests on the Ghrelin Receptor
[0288] Transfections and tissue culture-COS-7 cells are grown in
Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal
calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin. Cells are
transfected using calcium phosphate precipitation method with
chloroquine addition as previously described (Hoist B. et al., Mol.
Pharmacol. 53:166-175 (1998)). For gene dose experiments, variable
amounts of DNA are used. The amount of cDNA (20 .mu.g/75 cm.sup.2)
resulting in maximal signaling is used for dose response curves.
HEK-293 cells are grown in D-MEM, Dulbecco's modified Eagle's
medium 31966 with high glucose supplemented with 10% fetal calf
serum, 2 mM glutamine and 0.01 mg/ml gentamicin. Cells are
transfected with Lipofectamine.TM. 2000 (Invitrogen Corp.,
Carlsbad, Calif.).
Phosphatidylinositol turnover: One day after transfection, COS-7
cells are incubated for 24 hours with 5 .mu.Ci of [3H]-myo-inositol
(GE Healthcare, Piscataway, N.J.) in 1 ml medium supplemented with
10% fetal calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin per
well. Cells are washed twice in buffer, 20 mM HEPES, pH 7.4,
supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO.sub.4, 1 mM
CaCl.sub.2, 10 mM glucose, 0.05% (w/v) bovine serum; and are
incubated in 0.5 ml buffer supplemented with 10 mM LiCl at
37.degree. C. for 30 min. After stimulation with various
concentrations of peptide for 45 min at 37.degree. C., cells are
extracted with 10% ice-cold perchloric acid followed by incubation
on ice for 30 min. The resulting supernatants are neutralized with
KOH in HEPES buffer, and the generated [3H]-inositol phosphate is
purified on Bio-Rad AG 1-X8 anion-exchange resin (Bio-Rad
Laboratories, Hercules, Calif.) as per manufacturer's instructions.
Determinations are made in duplicates. CRE, SRE and NF-.kappa.-B
reporter assay: HEK293 cells (30,000 cells/well) seeded in 96-well
plates are transiently transfected. In case of the CRE reporter
assay, the cells are transfected with a mixture of pFA2-CREB and
pFR-Luc reporter plasmid (PathDetect CREB trans-Reporting System,
Stratagene, La Jolla, Calif.) or SRE-Luc (PathDetect SRE
Cis-Reporting System, Stratagene, La Jolla, Calif.) and the
indicated amounts of receptor DNA. Following transfection, cells
are maintained in low serum (2.5%) throughout the experiments and
are treated with the respective inhibitor of intracellular
signaling pathways. One day after transfection, cells are treated
with the respective ligands in an assay volume of 100 .mu.l medium
for 5 hrs. The assay is terminated by washing the cells twice with
PBS and addition of 100 .mu.l Luciferase.RTM. assay reagent
(LucLite.RTM., PerkinElmer, Inc., Wellesley, Mass.). Luminescence
is measured in a TopCounter (Top Count NETT, Packard Instrument
Co., Meriden, Conn.) for 5 sec. Luminescence values are given as
relative light units (RLU). MAP Kinase assay: COS 7 cells (seeding
density 150,000 cells/well) are transfected in the assay plates.
Two days after transfection, the indicated concentration of ligand
are added to assay medium without any serum and incubated for 10
min at 37.degree. C. The reaction is stopped by removing the medium
and two washing steps with ice cold PBS. The cells are lysed in
sample buffer and separated on 10% SDS-PAGE according to Laemmli U.
K., Nature 227:680-85 (1970). Proteins are transferred onto
nitrocellulose and Western blot analysis carried out using a 1:5000
dilution of mouse monoclonal antiphospho-ERK1/2 antibody (Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif.). Total ERK protein is
determined using a 1:10000 dilution of anti-ERK antibody (Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif.). Blots are probed
with anti-mouse horseradish peroxidase-conjugated secondary
antibodies, visualized using enhanced chemiluminescence reagent (GE
Healthcare, Piscataway, N.J.) and quantified by densiometric
analysis. ERK1/2 phosphorylation is normalized according to the
loading of protein by expressing the data as a ratio of
phosphoERK1/2 over total ERK1/2. Results are expressed as
percentage of the value obtained in non-stimulated mock transfected
cells.
Example 5
Neuroprotective Effect of Acylated Ghrelin Splice Variant
[0289] The protective effects of ghrelin splice variant against
induced cell death, cell viability, of C57B1/6 mice primary cell
culture, was analysed by trypan blue assay, an indirect method
based on the dye exclusion of live cells with intact cell membrane.
Cells were treated with 50 .mu.M H.sub.2O.sub.2 to induce cell
damages and death. The neuroprotective effect of ghrelin splice
variant was tested by treating the cells with either H.sub.2O.sub.2
plus 1 .mu.M ghrelin splice variant, H.sub.2O.sub.2 plus 1 .mu.M
ghrelin or H.sub.2O.sub.2 plus both peptides. Controls samples were
untreated cells. In FIG. 1, results clearly show that
H.sub.2O.sub.2 treatment induced a massive cell death, the toxicity
of the compound leads to 50% of the cell survival, moreover,
ghrelin splice variant and ghrelin treatments, alone or in
combination, seem to exert a statistically significant effective
protective action against the damages produced by H.sub.2O.sub.2 in
terms of cell viability compared to the negative control
(H.sub.2O.sub.2). Furthermore, the efficacy of ghrelin splice
variant peptide seems to be greater than ghrelin treatment, and the
presence of ghrelin splice variant and ghrelin together results in
a synergic activity of the two compounds.
Example 6
Patch Clamp Recordings from SN DA Neurons. Determining the Effect
of Ghrelin Splice Variant on Synaptic Input Organization and Firing
Rate of Nigral DA Neurons in GHSR1 Knockout Animals and their Wild
Type Littermates
[0290] Brain slices preparation: Brain slices (300 .quadrature.m)
containing the SN, will be cut on a vibratome from 4-6 week old
males (n=10 per group). Briefly, animals will be anesthetized with
Nembutal (80 mg/kg) and then decapitated. The brains will be
rapidly removed and immersed in cold (40 C) oxygenated bath
solution (containing (mM): NaCl 150, KCl 2.5, CaCl2 2, MgCl2 2
Hepes 10, and glucose 10, pH 7.3 with NaOH). After being trimmed to
contain only the SN, slices will be transferred to a recording
chamber where they are constantly perfused with bath solution at 2
ml/min. DA neurons in the SN are identified by presence of a large
Ih current (>100 pA) evoked by hyperpolarizing voltage steps
from -50 to -120 mV for 2 s (22). This approach identifies
dopaminergic cells with >90% accuracy. In brain slices,
whole-cell current clamp will be used to observe spontaneous action
potentials. Slices will be maintained at 330 C and perfused
continuously with ASCF (bubbled with 5% CO2 and 95% 02) containing
(in mM): NaCl, 124; KCl, 3; CaCl2, 2; MgCl2, 2; NaH2PO4, 1.23;
NaHCO3, 26; glucose, 10; pH 7.4 with NaOH. Ghrelin Splice Variant
will be applied to the recording chamber via bath application. The
pipette solution container (mM): Gluconic Acid 140, CaCl2, MgCl2 2,
EGTA 1, HEPES 10, Mg-ATP 4, and Na2-GTP 0.5, pH 7.3 with KOH. To
further explore the mechanism of Ghrelin Splice Variant-mediated
effect on action potentials in DA neurons, we will analyze the
effect of Ghrelin Splice Variant on action potential frequency
either in the presence of ionotropic glutamate receptor antagonists
CNQX (10 .quadrature.M) and AP5 (50 .quadrature.M) in all
solutions, or in the presence of bicuculine (30 .quadrature.M; to
block diminish GABA effects) in all solutions. Analysis: All data
will be sampled at 3-10 kHz and filtered at 1-3 kHz with an Apple
Macintosh computer using Axograph 4.9 (Axon Instruments).
Electrophysiological data will be analyzed with Axograph 4.9 (Axon
Instruments) and plotted with Igor Pro software (WaveMetrics, Lake
Oswego, Oreg., USA). To complement, the electron microscopic
measurements of synapses, we will also analyze mEPSC and mIPSC of
SN DA neurons. Mice (C57BL/J6; n=10) between the ages of 4-6 weeks
of age will be injected with Ghrelin Splice Variant (30 .mu.g in
0.3 ml) or vehicle ip at 09:00 hrs. Two hours later, animals will
be sacrificed under nembutal anesthesia and their brains collected
as described above. For the recording of mEPSCs and IPSCs, SN DA
cells will be patched with pipettes with a tip resistance of 4-6
M.OMEGA. and partially compensated by the amplifier. The pipette
will be filled with a solution containing the following (mM):
KMeSO, 145 (mEPSC) or KCL, 145 (mIPSC); MgCl2, 2; HEPES, 10;
Mg-ATP, 2; Nat-GTP, 0.3; and KOH to adjust pH to 7.3. mEPSC
recordings will be achieved under the presence of TTX and
bicucullin, whereas mIPSC will be monitored under the presence of
TTX, AP5 and CNQX. The frequency and amplitude of mEPSC and IPSC
will be recorded as described previously. Differences in the
frequency of mEPSCs and mIPSCs of DA cells from Ghrelin Splice
Variant and saline treated animals will be compared using unpaired
t-tests with the critical level set at .alpha.=0.05). In FIG. 2 it
is clear that Ghrelin splice variant induced a significant
depolarization (about 10 mV) and an increment in action potential
firing in this dopaminergic neuron. The effect of the Ghrelin
splice variant lasted for at least 20 minutes after washout
indicating that the Ghrelin splice variant induces plasticity. In
FIG. 3. As can be seen in FIG. 3 there was a significant elevation
in the frequency of mEPSCs compared with saline control after
treatment with Ghrelin splice variant in WT mice: Ghrelin splice
variant (67.2+3.1 (n=6) versus Saline 30.35+2.5 (n=8)). Conversely,
a significant decrease in the frequency of mIPSCs was triggered by
Ghrelin splice variant: (Ghrelin splice variant: 60.5+8.9 (n=6)
versus Saline 72.5+11.2 (n=5)). N=number of cells recorded. In FIG.
4. mEPSC recordings were taken in the presence of tetrodotoxin
(TTX) and bicucullin in GHSR1a knockout mice and their WT
littermates'. The frequency and amplitude of mEPSCs was determined.
Ghrelin splice variant induced mEPSC frequency in WT animals was
98.1+/-24.7 per min (n=31) and Ghrelin splice variant induced mEPSC
frequency in GHSR1a knockout mice was 27.3+/-2.8 per min (n=9)
(N=number of cells recorded). These results indicate the
involvement of GHSR1a-mediated effects of Ghrelin splice variant on
SN neurons.
Example 7
Altering MPTP-Induced DA Cell Degeneration and Motor Dysfunctions
by Ghrelin Splice Variant in Mice (n=10 Per Group, Ghrelin Splice
Variant Treatment in a Mouse Model of Parkinson's Disease
[0291] In the MPTP model, the bilateral dopaminergic neuronal death
of the substantia nigra and dopamine depletion of the striatum is
created by i.p. injection of MPTP. Experimental groups: [0292]
Group 1: 10 Sham-mice treated with vehicle (10 ml/kg) once-a-day
starting day-7 and continuing until the end of the study day 10
[0293] Group 2: 10 MPTP-mice treated with vehicle (10 ml/kg)
once-a-day starting day-7 and continuing until the end of the study
day 10 [0294] Group 3: 10 MPTP-mice treated with Ghrelin
SplicVariant (10 nmol) once-a-day starting day-7 and continuing
until the end of the study day 10 Ghrelin Splice Variant or vehicle
was administered once-a-day immediately before the dark phase, and
food and food were removed overnight starting seven days prior to
MPTP challenge and continued until end-point day 10 after MPTP.
Striatal levels of dopamine (DA), 3,2-dihydroxyphenylacetic acid
(DOPAC) and homovanillic acid (HVA) are evaluated 10 days after the
injection of MPTP with HPLC. In addition, the selective damage to
dopaminergic neurons in the SNc is evaluated with tyrosine
hydroxylase (TH) immunoreactivity. Food intake and body weight were
monitored daily in all mice administered Ghrelin Splice
Variant.
Example 8
Effect of Subcutaneous Administration of Acylated Ghrelin Spice
Variant on GH Release
[0295] Acylated ghrelin splice variants (20 .mu.g) or the Vehicle
(1.6% mannitol) were administered by a subcutaneous bolus injection
(Corresponding to 0.3 .mu.mol/kg) to 5 mice each. Blood was sampled
10 and 20 min after injection. Serum samples were stored at
-70.degree. C. and analyzed by the RAT/MOUSE GROWTH HORMONE ELISA
KIT 96-Well Plate (Cat. EZRMGH-45K) (Millipore Corporation,
Billerica, Mass., USA). Results: The serum and brain growth hormone
concentration 10 minutes after subcutaneous administration of
acylated ghrelin splice variant or the Vehicle was 2-14-fold higher
in the ghrelin splice variant group in comparison to the vehicle
group.
Example 9
Pharmacokinetics of Acylated Ghrelin Splice Variant Range Finder
into Mice
[0296] Subcutaneous administration of ghrelin splice variants was
performed at three dose levels of 0.5, 2.5 and 10 mg/kg
corresponding to concentrations of 0.1, 0.5 and 2 mg/ml,
respectively, and at a constant volume dosage of 5 ml/kg.
Intravenous injection was performed at one dose level of 0.5 mg/kg
corresponding to a concentration of 0.1 mg/ml and a constant volume
dosage of 5 ml/kg as well. The study comprised 9 male and 9 female
Sprague-Dawley.TM. (SD.TM.) rats per dose level and route of
administration.
[0297] Bleeding sampling design was confined to 9 bleeding time
points for each dose level: pre-dosing, 5, 15, 30, 60 and 90 min,
3, 5 and 24 hrs, post-dosing. Each group was divided into 3
sub-groups, each being assigned 3 specific bleeding time points, in
order to receive 3 individual samples/time point/group (total of 27
individual samples/group). Mean group body weight values at
initiation of the study were similar among all groups and did not
exceed .+-.20% of the mean weight per gender. Whole blood samples
were kept on ice from the time of blood collection until the time
of centrifugation. The obtained plasma samples were flash frozen in
liquid nitrogen and kept on dry ice until removal to -70.degree.
C.
[0298] Concentrations of the Test Item in plasma were determined by
LC/MS/MS (liquid chromatography/mass spectrometry/mass
spectrometry). Pharmacokinetic analysis of Ghrelin Splice Variant
was based on mean plasma concentration time profiles for each dose
group as obtained by Non-compartmental Pharmacokinetic analysis,
generated by the use of the computer software: "PK Solutions 2.0"
(Summit Research Services, CO, USA).
[0299] Pharmacokinetic analysis for the SC route showed that
AUC.sub.0-.infin. values were similar for males and females
administered the low dose (6.1 and 5.2 mcgmin/ml, respectively) and
mid dose (18.8 and 20.8 mcgmin/ml, respectively). At the high dose,
AUC values of the females were substantially lower (49.1 mcgmin/ml)
than that of males (79.2 mcgmin/ml). T.sub.max occurred at 5
minutes post-dosing for all dose groups. T.sub.1/2 values ranged
from 17.4 to 26.4 minutes for male rats and 10.7 to 28.9 minutes
for female rats.
Example 10
Examples of Diaries/Questionnaires Assessing Patient Quality of
Life
[0300] A) EORTC QLQ-C30 (Aaronson et al., J. Natl. Cancer Inst. 85:
365-76 (1993)), see the National Institutes of Health website and
see, for example, a specimen of EORTC QLQ-C30 (version 3.0),
available on the EORTC website and incorporated herein by
reference. We are interested in information regarding you and your
health. Please answer the follow questions by ticking off the
number that applies best to you. There are no "right" or "wrong"
answers. This information will be treated with confidentiality.
Questions: See specimen of EORTC QLQ-C30 (version 3.0), available
on the EORTC website and incorporated herein by reference).
Example 11
Examples of Suitable Formulations for Preparing Pharmaceutical
Compositions for Use in the Present Disclosure
[0301] 4% Mannitol Solution is prepared by dissolving D-Mannitol in
Water for Injection to achieve final concentration of 40 mg/ml.
Ghrelin Splice Variant Stock Solution is prepared by dissolving
ghrelin splice variant in TFA salt or in Acetate (5 mg) in 10 ml of
4% Mannitol solution, divided into aliquots and kept frozen
(-70.degree. C.) until the time of use.
Ghrelin Splice Variant Dosing Solution: On each day of dosing, the
required amount of each test item is thawed and diluted with
Physiological Saline to a concentration of 0.2 mg/ml. Control Item
Dosing Solution: Prepared on each day of dosing, by diluting 4%
Mannitol Solution with Physiological Saline at the same ratio as
the Ghrelin Splice Variant Dosing Solution. Patients: Patients with
documented neuronal damage and/or neurodegenerative disease and
documented parkinson's disease or alzheimer's disease. Efficacy of
ghrelin splice variant action will be assessed according to
clinical assessments: [0302] (1) Effect of Ghrelin Splice Variant
on change in motor, mood, and cognitive function [0303] (2)
Parkinson's disease-specific assessments: Parts I, II, and III of
the revised Movement Disorders Society Unified Parkinson Disease
Rating Scale (MDS-UPDRS), Geriatric Depression Scale (GDS), The
Montreal Cognitive Assessment (MoCA) [0304] (3) Acute Food intake:
Dietician-assessed food intake during the infusion. [0305] (4)
Chronic Food intake: A daily report of the amount of food consumed
during the day, and assessment of the pleasantness related to the
food intake. This will be validated by urine nitrogen excretion,
based on 4 day diet diary. [0306] (5) Body-weight: Standard and
calibrated scale will be used at the clinic. [0307] (6) Resting
energy expenditure (REE) is a very important measurement, since it
is affected both by the state of the disease and the body size.
[0308] (7) Exercise test: Actigraph is used according to standard
protocol described on the Actigraph website. [0309] (8)
Health-related QOL using standard forms as described supra. [0310]
(9) Para-clinical assessments: [0311] (i) Nitrogen excretion in the
urine: 24 h urine collection should be used as validation of the
reported food intake. [0312] (ii) Plasma glucose, plasma FFA,
plasma triglycerides, plasma glycerol and plasma amino acids:
Plasma substrates measured to ensure the reported food intake is in
accordance with the absorbed amount of food intake. [0313] (iii)
Lean body mass and fat mass assessed by TSF thickness and mid-arm
circumference as a measurement of body composition. [0314] (iv)
Total body fat (and fat free mass) will be assessed DEXA scan,
using software 1.31 for the lunar DPX-L (Scanexport Medical,
Helsingborg, Sweden). [0315] (v) Plasma Leptin: Leptin is produced
by and secreted from the fat cell. The plasma level of leptin gives
an estimate of the total fat cell burden. [0316] (vi) Plasma
Ghrelin: The basal ghrelin level tends to be increased in cachectic
patients. [0317] (vii) Plasma-GH: In previous studies, GH has been
measured as a control for the effect of ghrelin administration
(Enomoto M. et al., Clin. Sci. (Lond). 105:431-35 (2003)). [0318]
(viii) IGF-I: A single determination of IGF-I summarizes 24 h of GH
secretion. This has been demonstrated in healthy volunteers where
levels of circulating IGF-I have been shown to correlate with
spontaneous GH secretion (Rose S. R. et al., N. Engl. J. Med.
319:201-07 (1988)). IGF-I may also increase independently of GH
increase by improved nutritional status. [0319] (ix) IGFBP-3: One
of the carrier proteins for IGF-I. It increases in parallel with
IGF-I but with a slower response rate. [0320] (x) Albumin: Is an
indicator of nutritional status. [0321] (xi) Prealbumin: Indicator
of nutritional status, with quicker response to alterations than
albumin. [0322] (xii) Cortisol: Ghrelin administration has been
shown to increase the serum cortisol level (Broglio F. et al., J.
Clin. Endocrinol. Metab. 88:1537-42 (2003)). Corticosteroids have
been shown to have a significant anti-nausea effect and to improve
asthenia and pain control, which may be beneficial for the
cachectic cancer patient. However, cortisol has never been shown to
increase weight in cachectic cancer patients. [0323] (xiii) CRP and
ESR: Acute phase proteins and ESR are often good indicators of
systemic inflammation related to the cancer process (Inui A., CA
Cancer J. Clin. 52:72-91 (2002)).
Example 12
Treatment of Patients Parkinson's Disease
[0324] Thirty-two Parkinson's Disease (PD) patients will be
enrolled in four, 8-person dose-escalating cohorts. Subjects in
each cohort will be randomized 6:2 (active:placebo) to receive
subcutaneous injections of Dln101 or placebo once daily for 7
consecutive days. Safety parameters will be assessed on Day 1 to
Day 8 and Day 15. The next cohort will begin dosing approximately 7
days after the Day 15 safety parameters have been reviewed for the
previous cohort by the study investigators and medical monitor.
Blood will be drawn for determination of anti-Dln101 antibodies at
Day 22. Subjects will have a diagnosis of probable PD, based on
Gelb criteria, within the last 5 years with Hoehn and Yahr stage
1-3 (not severely disabled). Use of levodopa and dopamine agonist
therapy will be permitted. Patients will receive subcutaneous
administration of a single dose of 10, 50, 100, or 150 .mu.g/kg of
Ghreline Splice Variant or placebo. The protocol will start at
08.00 hours after an overnight fast. A 22-gauge catheter will be
inserted into an antecubital vein for blood sampling. After an
equilibration period of 30 min, ghrelin splice variant (10
.mu.g/kg) or placebo (0.9% saline) will be administered
subcutaneously. Investigational treatment: Ghrelin splice variant
will be available in GMP-quality in prepared vials of 10 .mu.g/kg
from BACHEM AG, Switzerland or NeoMPS Inc., USA. Placebo consists
of normal saline (or the vehicle used to dissolve study substance),
which will be provided by a hospital pharmacy. Ghrelin splice
variant is dissolved in saline, and a dose of 10 .mu.g/kg ghrelin
splice variant will be administered to the patient. Assessments of
efficacy: [0325] (1) Effect of Ghrelin Splice Variant on change in
motor, mood, and cognitive function [0326] (2) Quality of life:
will be assessed using the EORTC-QLQ-C30 questionnaire (see example
9). [0327] (3) Nutritional intake and food preferences: food intake
measurement will be by percentage calculation of food products
consumed at each meal by the patient, the clinical dietician will
assess the food preferences as part of their routine assessments.
[0328] (4) Food pleasantness: will be assessed after lunch using
visual analogue scales, following established anchors. [0329] (5)
Perceived appetite, hunger, nausea and satiety: will be assessed in
the morning, before infusion, and before and after lunch using
visual analogue scale, following established anchors. Applicant
will also apply a shortened ad hoc taste questionnaire. [0330] (6)
Growth hormone (GH): since GH reflects directly the biological
function of ghrelin, with a rapid increase of GH after ghrelin
injections, Applicant will also monitor GH levels at the same time
points as ghrelin. A standard ghrelin assay will be used. [0331]
(7) Body composition: body compositions will be assessed by BMI,
bioimpedance analysis and dual photon absorptiometry/dual energy
x-ray absorptiometry (DEXA). [0332] (8) Albumin and transferrin
levels will be determined as parameters for nutritional status.
[0333] (9) Cardiovascular autonomic function: for the screening of
autonomic disorders, a 20 minute Holter EKG will be performed, and
the SDNN value determined. [0334] (10) Parkinson's disease-specific
assessments: Parts I, II, and III of the revised Movement Disorders
Society Unified Parkinson Disease Rating Scale (MDS-UPDRS),
Geriatric Depression Scale (GDS), The Montreal Cognitive Assessment
(MoCA)
Sequence CWU 1
1
8194PRTHomo sapiens 1Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg
Val Gln Val Arg Pro 1 5 10 15 Pro His Lys Ala Pro His Val Val Pro
Ala Leu Pro Leu Ser Asn Gln 20 25 30 Leu Cys Asp Leu Glu Gln Gln
Arg His Leu Trp Ala Ser Val Phe Ser 35 40 45 Gln Ser Thr Lys Asp
Ser Gly Ser Asp Leu Thr Val Ser Gly Arg Thr 50 55 60 Trp Gly Leu
Arg Val Leu Asn Gln Leu Phe Pro Pro Ser Ser Arg Glu 65 70 75 80 Arg
Ser Arg Arg Ser His Gln Pro Ser Cys Ser Pro Glu Leu 85 90
222PRTHomo sapiens 2Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val
Gln Val Arg Pro 1 5 10 15 Pro His Lys Ala Pro His 20 324PRTHomo
sapiens 3Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg Val Gln Val
Arg Pro 1 5 10 15 Pro His Lys Ala Pro His Val Val 20 424PRTHomo
sapiensMISC_FEATURE(3)..(3)Xaa is Dpr 4Gly Ser Xaa Phe Leu Ser Pro
Glu His Gln Arg Val Gln Val Arg Pro 1 5 10 15 Pro His Lys Ala Pro
His Val Val 20 529PRTHomo sapiens 5Gly Ser Ser Phe Leu Ser Pro Glu
His Gln Arg Val Gln Val Arg Pro 1 5 10 15 Pro His Lys Ala Pro His
Val Val Pro Ala Leu Pro Leu 20 25 691PRTHomo sapiens 6Phe Leu Ser
Pro Glu His Gln Arg Val Gln Val Arg Pro Pro His Lys 1 5 10 15 Ala
Pro His Val Val Pro Ala Leu Pro Leu Ser Asn Gln Leu Cys Asp 20 25
30 Leu Glu Gln Gln Arg His Leu Trp Ala Ser Val Phe Ser Gln Ser Thr
35 40 45 Lys Asp Ser Gly Ser Asp Leu Thr Val Ser Gly Arg Thr Trp
Gly Leu 50 55 60 Arg Val Leu Asn Gln Leu Phe Pro Pro Ser Ser Arg
Glu Arg Ser Arg 65 70 75 80 Arg Ser His Gln Pro Ser Cys Ser Pro Glu
Leu 85 90 7181PRTMus musculus 7Gly Ser Ser Phe Leu Ser Pro Glu His
Gln Lys Ala Gln Val Ser Gln 1 5 10 15 Ser Val Ser Leu Ser Pro His
Ile Tyr Pro Asp Leu Cys Val Cys Val 20 25 30 Arg Glu Arg Glu Arg
Glu Pro Ser Phe Pro Phe Gln Gln Arg Lys Glu 35 40 45 Ser Lys Lys
Pro Pro Ala Lys Leu Gln Pro Arg Ala Leu Glu Gly Trp 50 55 60 Leu
His Pro Glu Asp Arg Gly Gln Ala Glu Glu Thr Glu Glu Glu Leu 65 70
75 80 Glu Ile Arg Val Cys Thr Gln Ala Pro Ala Cys Ser Tyr Asn Ser
Lys 85 90 95 Gly Val Gly Val Trp Arg Val Ser His Met Leu Ala Phe
Gln Ala Thr 100 105 110 Gln Gly Leu Glu Ser Ser Thr Asn Ser Ser Thr
Arg Gly Ser Glu Ser 115 120 125 Pro Ser Gln Glu Val Thr Val Ser Arg
Val Ala Arg Glu Gln Gln Thr 130 135 140 Cys Ala Gln Lys Thr Lys Gln
Ile Glu Gly Ser Gln Glu Pro Gly Ser 145 150 155 160 Thr Asp Gly Tyr
Arg Asn Arg Arg Lys Pro Cys Leu Ser Gln Asp Leu 165 170 175 Ser Gly
Leu Pro Trp 180 890PRTRattus norvegicus 8Gly Ser Ser Phe Leu Ser
Pro Glu His Gln Lys Ala Gln Val Ser Leu 1 5 10 15 Ser Pro Gln Val
Pro His Leu Ser Trp Ser Val Val Cys Ser Phe Pro 20 25 30 Phe Gln
Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro 35 40 45
Arg Ala Leu Glu Gly Trp Leu His Pro Glu Asp Arg Gly Gln Ala Glu 50
55 60 Glu Ala Glu Glu Glu Leu Glu Ile Arg Val Gly Pro Arg Ala Pro
Ala 65 70 75 80 Tyr Ser Cys Asn Ser Lys Gly Phe Gly Val 85 90
* * * * *