U.S. patent application number 12/600407 was filed with the patent office on 2010-07-01 for unacylated ghrelin as therapeutic agent in the treatment of metabolic disorders.
This patent application is currently assigned to ALIZE PHARMA SAS. Invention is credited to Ezio Ghigo, Riccarda Granata, Aart Jan Van Der Lely.
Application Number | 20100168033 12/600407 |
Document ID | / |
Family ID | 39677677 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100168033 |
Kind Code |
A1 |
Ghigo; Ezio ; et
al. |
July 1, 2010 |
UNACYLATED GHRELIN AS THERAPEUTIC AGENT IN THE TREATMENT OF
METABOLIC DISORDERS
Abstract
An isolated polypeptide comprising any amino acid fragment of
unacylated ghrelin or any analog thereof, wherein the polypeptide
has an activity selected from the group consisting of a) decreasing
blood glucose levels; b) increasing insulin secretion an/or
sensitivity; c) binding to insulin-secreting cells; and d)
promoting survival of insulin-secreting cells. As well as the use
of the polypeptide in the treatment of a disorder associated with
impaired glucose metabolism.
Inventors: |
Ghigo; Ezio; (Torino,
IT) ; Van Der Lely; Aart Jan; (Bergschenhoek, NL)
; Granata; Riccarda; (Torino, IT) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
ALIZE PHARMA SAS
ECULLY
FR
|
Family ID: |
39677677 |
Appl. No.: |
12/600407 |
Filed: |
May 30, 2008 |
PCT Filed: |
May 30, 2008 |
PCT NO: |
PCT/EP08/56727 |
371 Date: |
January 28, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60941186 |
May 31, 2007 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
435/375; 530/324; 530/325; 530/326; 530/327; 530/328; 530/329;
530/330 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 3/10 20180101; A61P 3/06 20180101; A61K 38/25 20130101; A61P
3/04 20180101; C07K 14/60 20130101 |
Class at
Publication: |
514/14 ; 435/375;
514/16; 530/324; 530/325; 530/326; 530/327; 530/328; 530/329;
530/330 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C12N 5/02 20060101 C12N005/02; A61K 38/08 20060101
A61K038/08; C07K 7/08 20060101 C07K007/08; C07K 7/06 20060101
C07K007/06 |
Claims
1. An isolated polypeptide comprising any amino acid fragment of
the amino acid sequence shown in SEQ ID NO: 9 or an analog thereof,
wherein said polypeptide has at least one activity selected from
the group consisting of a) decreasing blood glucose levels; b)
increasing insulin secretion and/or sensitivity; c) binding to
insulin-secreting cells; and d) promoting survival of
insulin-secreting cells.
2. The isolated polypeptide of claim 1, wherein said polypeptide
comprises any amino acid fragment of the amino acid sequence shown
in SEQ ID NO: 9 or an analog thereof, said fragment including at
least the amino acid residues shown in SEQ ID NO: 8 or an analog
thereof.
3. The isolated polypeptide of claim 1, wherein said polypeptide
comprises any amino acid fragment of the amino acid sequence shown
in SEQ ID NO: 9 or an analog thereof, said fragment including at
least the amino acid residues shown in SEQ ID NO: 6 or an analog
thereof.
4. The isolated polypeptide of claim 1, wherein said polypeptide
comprises any amino acid fragment of at least 6 amino acid residues
of the amino acid sequence shown in SEQ ID NO: 9 or an analog
thereof, said fragment including at least the amino acid residues
shown in SEQ ID NO: 8 or an analog thereof.
5. The isolated polypeptide of claim 1, wherein said polypeptide
comprises any amino acid fragment of at least 8 amino acid residues
of the amino acid sequence shown in SEQ ID NO: 9 or an analog
thereof, said fragment including at least the amino acid residues
shown in SEQ ID NO: 8 or an analog thereof.
6. The isolated polypeptide of claim 1, consisting of any
consecutive 5 amino acid residues of the amino acid sequence shown
in SEQ ID NO: 9.
7. The isolated polypeptide of claim 1, wherein said fragment has
an amino acid sequence selected from the group consisting of SEQ ID
NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
8. An isolated polypeptide 5 to 27 amino acid residues in length,
said polypeptide comprising the amino acid sequence
Glu-His-Gln-Arg-Val.
9. The isolated polypeptide of claim 8, wherein said amino acid
sequence optionally contains up to two conservative amino acid
substitutions at a position selected from the group consisting of
amino acid residues Glu, His and Val.
10. A method for treating a disorder associated with impaired
glucose metabolism in a patient, comprising administering to the
patient a therapeutically effective amount of the polypeptide of
claim 1.
11. The method of claim 10, wherein the disorder is diabetes.
12. The method of claim 11, wherein the diabetes is type 1
diabetes.
13. The method of claim 11, wherein the diabetes is type 2
diabetes.
14. The method of claim 10, wherein the disorder is a medical
condition associated with insulin deficiencies.
15. The method of claim 10, wherein the disorder is a medical
condition associated with insulin resistance.
16. The method of claim 10, wherein the disorder is a medical
condition associated with dyslipidemia.
17. The method of claim 10, wherein the disorder is a medical
condition associated with obesity.
18. The method of claim 10, wherein the disorder is a medical
condition related to metabolic syndrome.
19. The method of claim 10, for the treatment of pancreatic
.beta.-cells.
20. The method of claim 19, wherein the treatment is through
enhancement of proliferation or of survival of the pancreatic
.beta.-cells.
21. The method of claim 20, wherein the enhancement of
proliferation or of survival is achieved ex vivo by subjecting the
pancreatic O-cells to the polypeptide prior to administering said
cells to the patient as a graft.
22. A method for enhancing survival and/or proliferation of
insulin-secreting cells comprising culturing said cells in the
presence of a therapeutically effective amount of the polypeptide
of claim 1.
23. The method of claim 10, wherein the polypeptide is administered
through a route selected from the group consisting of intravenous,
subcutaneous, transdermal, oral, buccal, sublingual, nasal delivery
and inhalation.
24. The method of claim 10, wherein the polypeptide is administered
in a dose varying from about 0.01 .mu.g/kg to about 10 mg/kg.
25.-40. (canceled)
41. An isolated polypeptide selected from the group consisting of
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27 and SEQ ID NO: 28.
42. (canceled)
43. The isolated polypeptide of claim 41, for treating a disorder
associated with impaired glucose metabolism in a patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
provisional patent application No. 60/941,186 filed May 31, 2007,
the content of which is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to unacylated ghrelin fragments and
analogs thereof as well as to their therapeutic uses.
BACKGROUND
[0003] Ghrelin is a peptide which was isolated from the stomach but
is expressed also in many other tissues, including the endocrine
pancreas. It was discovered as a natural ligand of the
growth-hormone secretagogue receptor type 1a (GHS-R) (Refs. 1, 2).
Ghrelin acylation at serine 3 is essential for binding to GHS-R1a,
which mediates GH-releasing activity and also the orexigenic action
of acylated ghrelin. Besides stimulating GH secretion and
modulating other pituitary functions, acylated ghrelin (AG) exerts
a broad range of biological actions such as central regulation of
food intake and energy balance and control of insulin secretion and
glucose metabolism. GHS-R1a expression has been detected in a
variety of endocrine and non-endocrine, central and peripheral
animal and human tissues, including the pancreas. Notably, the link
between ghrelin and insulin seems of major relevance. AG has been
shown to possess hyperglycemic diabetogenic effects; ghrelin
knock-out mice display enhanced glucose-induced insulin release
while blockade of pancreatic islet-derived ghrelin has been shown
to enhance insulin secretion and to prevent high-fat diet-induced
glucose intolerance in rats.
[0004] In the endocrine pancreas, ghrelin has been shown to
localize to .alpha.- and .beta.-cells and to the newly identified
ghrelin-producing islet .epsilon.-cells, suggesting a role in the
regulation of .beta.-cell fate and function (Refs. 9, 22, 19).
Survival of .beta.-cells is of major importance for maintaining
normal glucose metabolism and ft-cell apoptosis is a critical event
in both type 1 and 2 diabetes (Refs. 16, 21).
[0005] Unacylated ghrelin (UAG) is the major circulating form of
ghrelin and has long been believed to be biologically inactive
since it does not bind GHS-R1a at physiological concentrations and
is thus devoid of GH-releasing activity. It is now known that UAG
is a biologically active peptide, particularly at the metabolic
level, having notably been shown to exert anti-diabetogenic effects
as described in U.S. patent application Ser. No. 10/499,376,
published on Apr. 14, 2005, under publication number US
2005-0080007. Indeed UAG is able to: a) counteract the
hyperglycemic effect of AG in humans (Ref. 6); b) directly modulate
glucose metabolism at the hepatic level by blocking basal,
glucagon-induced and acylated ghrelin-stimulated glucose output
from hepatocytes (Ref. 3); c) decrease fat deposition, food
consumption, and glucose levels in UAG transgenic animals (Ref. 7);
d) stimulate proliferation and prevent cell death and apoptosis in
.beta.-cells and human pancreatic islets (Ref. 4).
[0006] It has recently been demonstrated that UAG is able to
stimulate proliferation and to prevent cell death and apoptosis
induced by (IFN)-.gamma./tumor necrosis (TNF)-.alpha., synergism in
.beta.-cells and human pancreatic islets (Ref. 4). Noteworthy,
cytokine synergism is considered to be a major cause for
.beta.-cell destruction in type I diabetes as well as of
.beta.-cell loss in type 2 diabetes. Moreover, this work also
showed that UAG stimulated glucose-induced insulin secretion from
.beta.-cells that do not express GHS-R1a.
[0007] Together, these results reinforce the concept that UAG has a
therapeutic potential in medical conditions associated with
metabolic disorder such as conditions characterized by insulin
deficiencies or by insulin resistance, including, but not limited
to diabetes, and the effect of UAG on the .beta.-cells is one of
the mechanisms of action of UAG in these potential
applications.
[0008] Recently, the therapeutic potential of UAG was clinically
demonstrated, as a continuous infusion of UAG in healthy volunteers
resulted in a lowering of blood glucose, an improvement in insulin
sensitivity, a reduction in blood free fatty acids, and decreased
cortisol levels.
[0009] UAG is a 28 amino-acid peptide and would preferably be
administered to patients by intravenous or subcutaneous injection
in order to produce its effects, which is not a convenient way to
administer a drug to a patient. Also, peptides of this size are
usually rapidly degraded following administration and their in vivo
efficacy is often weak following intravenous, subcutaneous or
intramuscular bolus administration.
[0010] In addition, manufacturing a 28 amino-acid peptide is a long
and expensive process, whether it is manufactured by solid-phase
peptide synthesis or by recombinant technology. Finally,
chronically treating patients with a long peptide such as UAG might
represent safety risks for the patients in the form of
immunogenicity. Raising neutralizing antibodies against a natural
peptide is a potential major health risk for the patients.
[0011] Therefore, it would be highly desirable to identify smaller
size peptides that would possess a comparable biological activity
to UAG, but would be easier and less costly to manufacture.
[0012] It would be even more desirable that these smaller size
peptides would have increased biological potency when compared with
UAG.
[0013] Another advantage of these smaller size peptides would be
that they would bear fewer immunogenicity risks for patients upon
chronic and repeated administrations, and hence exhibit a better
safety profile. They may have a better bioavailability than UAG,
whatever the route of administration, and be suitable for more
convenient routes of administration, such as, but not limited to,
transdermal, pulmonary, intranasal or oral delivery, or may
constitute a starting material for the design of peptide analogs or
peptidomimetic molecules with a better oral bioavailability.
Smaller size peptides may also be compatible with drug delivery
system such as, but not limited to, polymer-based depot
formulations.
SUMMARY
[0014] In one aspect of the present invention, there is provided an
isolated polypeptide comprising any amino acid fragment of the
amino acid sequence shown in SEQ ID NO: 9 or an analog thereof,
wherein said polypeptide has at least one activity selected from
the group consisting of a) decreasing blood glucose levels; b)
increasing insulin secretion and/or sensitivity; c) binding to
insulin-secreting cells; and d) promoting survival of
insulin-secreting cells.
[0015] In one aspect of the present invention, there is provided an
isolated polypeptide 5 to 27 amino acid residues in length, said
polypeptide comprising the amino acid sequence
Glu-His-Gln-Arg-Val.
[0016] In another aspect of the present invention, there is
provided a method for treating a disorder associated with impaired
glucose metabolism in a patient, comprising administering to the
patient a therapeutically effective amount of the polypeptide as
defined herein.
[0017] In another aspect of the present invention, there is
provided a method for enhancing survival and/or proliferation of
insulin-secreting cells comprising culturing said cells in the
presence of a therapeutically effective amount of the polypeptide
as defined herein.
[0018] In another aspect of the present invention, there is
provided use of a therapeutically effective amount of the
polypeptide as defined herein, in the preparation of a medicament
for treating a disorder associated with impaired glucose metabolism
in a patient.
[0019] In a further aspect of the present invention, there is
provided use of a therapeutically effective amount of the
polypeptide as defined herein, for treating a disorder associated
with impaired glucose metabolism in a patient.
[0020] In a further aspect of the present invention, there is
provided a pharmaceutical composition for treating a metabolic
disorder associated with impaired glucose metabolism comprising a
therapeutically effective amount of the polypeptide as defined
herein.
[0021] In yet a further aspect of the present invention, there is
provided an isolated polypeptide selected from the group consisting
of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ
ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO:
22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
ID NO: 27 and SEQ ID NO: 28.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 illustrates survival of INS-1E .beta.-cells in
serum-free medium in the presence of unacylated ghrelin or the
indicated fragments of unacylated ghrelin.
[0023] FIG. 2 illustrates survival of INS-1E .beta.-cells in the
presence of TNF-.alpha./IFN-.gamma./IL-.beta. and in the presence
of unacylated ghrelin or the indicated fragments of unacylated
ghrelin.
[0024] FIGS. 3A and 3B illustrate survival of HIT-T15 .beta.-cells
in serum free medium with or without cytokines and either
unacylated ghrelin UAG (1-28) or its fragment UAG (1-14) (FIG. 3A)
or UAG (1-18) (FIG. 3B).
[0025] FIGS. 4A and 4B illustrate survival of HIT-T15 .beta.-cells
in serum free medium with or without cytokines and either
unacylated ghrelin UAG (1-28) or its fragments UAG (1-5) (FIG. 4A)
or UAG (17-28) (FIG. 4B).
[0026] FIGS. 5A and 5B illustrate survival of cytokine-treated
HIT-T15 .beta.-cell in the presence of unacylated ghrelin fragments
UAG (6-13), UAG (8-13), UAG (8-12), UAG (1-14), UAG (1-18), UAG
(1-28) (FIG. 5A) and UAG (8-11), UAG (9-12) and UAG (9-11) (FIG.
5B).
[0027] FIGS. 6A to 6C illustrate the antiapoptotic effects of
unacylated ghrelin fragments UAG (6-13) (FIG. 6A), UAG (8-13) (FIG.
6B) and UAG (8-12) (FIG. 6C) on cytokine treated HIT-T15
.beta.-cells
[0028] FIGS. 7A and 7B illustrate the survival effect on human
pancreatic islets of unacylated ghrelin (1-28) and its fragments
UAG (1-14), UAG (1-18) (FIG. 7A) and UAG (1-5) and UAG (17-28)
(FIG. 7B).
[0029] FIGS. 8A to 8D illustrate the effect of UAG (1-14) (FIG.
8A), UAG (1-18) (FIG. 8B), UAG (1-28) (FIG. 8C) and Exendin-4 (FIG.
8D) on insulin secretion in human pancreatic islets.
[0030] FIGS. 9A to 9D illustrate the in vivo effect of unacylated
ghrelin fragment UAG (6-13) on animal survival (FIG. 9A), on plasma
glucose levels (FIG. 9B) and plasma (FIG. 9C) and pancreatic (FIG.
9D) insulin levels, in Streptozotocin (STZ)-treated animals.
[0031] FIGS. 10A and 10B illustrate the binding of unacylated
ghrelin and unacylated ghrelin fragment UAG (6-13) to pancreatic
HIT-T15 (FIG. 10A) and INS-1E (FIG. 10B) .beta.-cell receptors.
[0032] FIGS. 11A and 11B illustrate the survival effects of UAG
(6-13) with alanine (Ala) substitutions at positions 6 to 13 in
HIT-T15 .beta.-cells in both the absence of serum (FIG. 11A) and in
the presence of cytokines (FIG. 11B).
[0033] FIGS. 12A and 12B illustrate the survival effects of UAG
(6-13) with conservative substitutions and N-terminal modifications
in HIT-T15 .beta.-cells, in both the absence of serum (FIG. 12A)
and in the presence of cytokines (FIG. 12B).
[0034] FIGS. 13A and 13B illustrate the survival effects of UAG
(6-13) with cyclization in HIT-T15 .beta.-cells in both the absence
of serum (FIG. 13A) and in the presence of cytokines (FIG.
13B).
[0035] FIGS. 14A and 14B illustrate the in vivo effects of UAG
(6-13) on plasma glucose levels after 2 and 4 weeks of treatment in
ob/ob mice, an animal model of diabetes associated with obesity.
FIG. 14A illustrates fed plasma glucose levels and FIG. 14B
illustrates fasting plasma glucose.
[0036] FIG. 15 illustrates fasting insulin levels after 2 and 4
weeks of treatment with UAG and UAG (6-13) in ob/ob mice, an animal
model of diabetes associated with obesity.
[0037] FIG. 16 illustrates the effect of UAG and UAG (6-13) on
gonadal fat as percent body weight in ob/ob mice, an animal model
of diabetes associated with obesity.
DETAILED DESCRIPTION
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which the invention belongs.
UAG Fragments and Analogs Thereof
[0039] For the purpose of the present invention the following terms
are defined below.
[0040] In the present application, the terms "ghrelin" and
"acylated ghrelin" or "AG" are used interchangeably and have the
same meaning.
[0041] The term "unacylated ghrelin" or "UAG" is intended to mean
peptides that contain the amino acid sequence specified in SEQ ID
NO: 1
(1-NH.sub.2Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Ly-
s-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-28; SEQ ID NO:
1). UAG may also be referred to as UAG (1-28).
[0042] Naturally-occurring variations of unacylated ghrelin include
peptides that contain substitutions, additions or deletions of one
or more amino acids which result due to discrete changes in the
nucleotide sequence of the encoding ghrelin gene or alleles thereof
or due to alternative splicing of the transcribed RNA. It is
understood that the said changes do not substantially affect the
properties, pharmacological and biological characteristics of
unacylated ghrelin variants. Those peptides may be in the form of
salts. Particularly the acidic functions of the molecule may be
replaced by a salt derivative thereof such as, but not limited to,
a trifluoroacetate salt.
[0043] As used herein, SEQ ID NO: 9 refers to the amino acid
sequence consisting of residues 6 to 18 of UAG (SEQ ID NO: 1),
namely:
6-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-18.
[0044] By "peptide", "polypeptide" or "protein" is meant any chain
of amino acids, regardless of length or post-translational
modification (e.g., glycosylation or phosphorylation), or chemical
modification, or those containing unnatural or unusual amino acids
such as D-Tyr, ornithine, amino-adipic acid. The terms are used
interchangeably in the present application.
[0045] The term "fragments" or "fragments thereof" refers to amino
acid fragments of a peptide such as unacylated ghrelin. Fragments
of unacylated ghrelin are shorter than 28 amino acid residues.
Fragments of unacylated ghrelin may therefore be 27, 26, 25, 24,
23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5 or 4 amino acid residues in length.
[0046] In some aspects of the invention, the polypeptides are used
in a form that is "purified", "isolated" or "substantially pure".
The polypeptides are "purified", "isolated" or "substantially pure"
when they are separated from the components that naturally
accompany them. Typically, a compound is substantially pure when it
is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99%, by weight, of the total material in a sample.
[0047] The term "analog of unacylated ghrelin", "analog of
fragments of unacylated ghrelin" or "analogs thereof" refers to
both structural and functional analogs of unacylated ghrelin or
fragments thereof which are, inter alia, capable of replacing
unacylated ghrelin in antagonizing the peripheral actions or
functions of ghrelin or are capable of replacing other biological
actions of unacylated ghrelin, such as, but not limited to,
stimulate proliferation and/or inhibit apoptosis in .beta.-cell
lines, lower blood glucose levels, improved insulin sensitivity
and/or secretion, decrease cortisol levels, improve lipid profile
in human beings, and thus, have the potential use to treat
metabolic disorders such as those associated with for example,
insulin resistance, insulin deficiency, dyslipidemia or cortisol
excess.
[0048] Simple structural analogs comprise peptides showing homology
with unacylated ghrelin as set forth in SEQ ID NO: 1 or homology
with any fragments thereof. For example, an isoform of ghrelin-28
(SEQ ID NO: 1), des Gln-14 Ghrelin (a 27 amino acid peptide
possessing serine 3 modification by n-octanoic acid) is shown to be
present in stomach. It is functionally identical to ghrelin in that
it binds to GHSR-1a with similar binding affinity, elicits
Ca.sup.2+ fluxes in cloned cells and induces GH secretion with
similar potency as Ghrelin-28. It is expected that UAG also has a
des Gln-14 UAG that is functionally identical to UAG.
[0049] Preferred analogs of UAG and preferred analogs of fragments
of UAG are those that vary from the native UAG sequence or from the
native UAG fragment sequence by conservative amino acid
substitutions; i.e., those that substitute a residue with another
of like characteristics. Typical substitutions include those among
Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues
Asp and Glu; among Asn and Gln; among the basic residues Lys and
Arg; and among the aromatic residues Phe and Tyr. Particularly
preferred are analogs in which several, for example, but not
limited to, 5-10, 1-5, or 1-2 amino acids are substituted, deleted,
or added in any combination. For example, the analogs of UAG may
differ in sequence from UAG by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
amino acid substitutions (preferably conservative substitutions),
deletions, or additions, or combinations thereof.
[0050] There are provided herein, analogs of the peptides of the
invention that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the
amino acid sequences described herein over its full length, and
sharing at least one of the metabolic effects or biological
activity of UAG. A person skilled in the art would readily identify
an analog sequence of unacylated ghrelin or an analog sequence of a
fragment of unacylated ghrelin.
[0051] In a further aspect, analogs of UAG or fragments thereof
are, for example, analogs obtained by alanine scans, by
substitution with D-amino acids or with synthetic amino acids or by
cyclization of the peptide. Analogs of UAG or fragments thereof may
comprise a non-naturally encoded amino acid, wherein the
non-naturally encoding amino acid refers to an amino acid that is
not one of the common amino acids or pyrrolysine or selenocysteine,
or an amino acid that occur by modification (e.g.
post-translational modification) of naturally encoded amino acid
(including, but not limited to, the 20 common amino acids or
pyrrolysine and selenocysteine) but are not themselves incorporated
into a growing polypeptide chain by the translation complex.
Examples of such non-naturally-occurring amino acids include, but
are not limited to, N-acetylglucosaminyl-L-serine,
N-acetylglucosaminyl-L-threonine and O-phosphotyrosine.
[0052] As used herein, the term "modified" refers to any changes
made to a given polypeptide, such as changes to the length of the
polypeptide, the amino acid sequence, chemical structure,
co-translational modification, or post-translational modification
of a polypeptide.
[0053] The term "post-translational modification" refers to any
modification of a natural or non-natural amino acid that occurs to
such an amino acid after it has been incorporated into a
polypeptide chain. The term encompasses, by way of example only,
co-translational in vivo modifications, co-translational in vitro
modifications (such as in cell-free translation system),
post-translational in vivo modifications, and post-translational in
vitro modifications. Examples of post-translational modifications
are, but are not limited to, glycosylation, acetylation, acylation,
amidation, carboxylation, phosphorylation, addition of salts,
amides or esters, in particular C-terminal esters, and N-acyl
derivatives of the peptides of the invention. The types of
post-translational modifications are well known.
[0054] Certain peptides according to the present invention may also
be in cyclized form, such that the N- or C-termini are linked
head-to-tail either directly, or through the insertion of a linker
moiety, such moiety itself generally comprises one or more amino
acid residues as required to join the backbone in such a manner as
to avoid altering the three-dimensional structure of the peptide
with respect to the non-cyclized form. Such peptide derivatives may
have improved stability and bioavailability relative to the
non-cyclized peptides. Methods for cyclizing peptides are well
known in the art.
[0055] Cyclisation may be accomplished by disulfide bond formation
between two side chain functional groups, amide or ester bond
formation between one side chain functional group and the backbone
.alpha.-amino or carboxyl function, amide or ester bond formation
between two side chain functional groups, or amide bond formation
between the backbone .alpha.-amino and carboxyl functions. These
cyclisation reactions have been traditionally carried out at high
dilution in solution. Cyclisation is commonly accomplished while
the peptide is attached to the resin. One of the most common ways
of synthesising cyclic peptides on a solid support is by attaching
the side chain of an amino acid to the resin. Using appropriate
protection strategies, the C- and N-termini can be selectively
deprotected and cyclised on the resin after chain assembly. This
strategy is widely used, and is compatible with either
tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc)
protocols. However, it is restricted to peptides that contain
appropriate side chain functionality to attach to the solid
support. A number of approaches may be used to achieve efficient
synthesis of cyclic peptides. One procedure for synthesising cyclic
peptides is based on cyclisation with simultaneous cleavage from
the resin. After an appropriate peptide sequence is assembled by
solid phase synthesis on the resin or a linear sequence is appended
to resin, the deprotected amino group can react with its anchoring
active linkage to produce protected cyclic peptides. In general, a
final deprotection step is required to yield the target cyclic
peptide. The procedure for synthesising cyclic peptides are well
known in the art.
[0056] For example, lactamazation, a form of cyclisation, may be
performed to form a lactam bridge using Fmoc synthesis, amino acids
with different protecting groups at the lateral chains may be
introduced, such as, but not limited to, aspartic acid (or
glutamic) protected with allyl ester at the beta ester (or gamma
ester for glutamic acid) and lysine protected with allyloxy
carbamate at the N-.epsilon.. At the end of the synthesis, with the
N-terminus of the peptide protected with Fmoc, Boc or other
protecting group different from Alloc, the allyl and alloc
protecting groups of aspartic acid and lysine may be deprotected
with, for example, palladium (0) followed by cyclization using
PyAOP (7-Azabenzotriazol-1-yloxytris(pyrrolidino)
phosphonium-hexafluorophosphate) to produce the lactam bridge.
[0057] Unless otherwise indicated, an amino acid named herein
refers to the L-form. Well recognised abbreviations in the art will
be used to describe amino acids, including levoratory amino acids
(L-amino acids or L or L-form) and dextrorotary amino acids
(D-amino acids or D or D-form), Alanine (Ala or A), Arginine (Arg
or R), Asparagine (Asn or N), Aspartic acid (Asp or D), Cysteine
(Cys or C), Glutamic acid (Glu or E), Glutamine (Gln or Q), Glycine
(Gly or G), Histidine (H is or H), Isoleucine (Ile or I), Leucine
(Leu or L), Lysine (Lys or K), Methionine (Met or M), Phenylalanine
(Phe or F), Proline (Pro or P), Serine (Ser or S), Threonine (Thr
or T), Tryptophan (Trp or W), Tyrosine (Tyr or Y) and Valine (Val
or V). An L-amino acid residue within the native peptide sequence
may be altered to any one of the 20 L-amino acids commonly found in
proteins or any one of the corresponding D-amino acids, rare amino
acids, such as, but not limited to, 4-hydroxyproline or
hydroxylysine, or a non-protein amino acid, such as P-alanine or
homoserine.
[0058] Any other analogs of UAG or fragments thereof or any other
modified UAG or fragments thereof that preserve the biological
activity of UAG are encompassed by the present invention.
[0059] General methods and synthetic strategies used in providing
functional and structural analogs of UAG or fragments thereof are
commonly used and well known in the art and are described in
publications such as "Peptide synthesis protocols" ed, M. W.
Pennigton & B. M. Dunn. Methods in Molecular Biology. Vol 35.
Humana Press, NJ., 1994.
[0060] The term "homology" refers to sequence similarity between
two peptides while retaining an equivalent biological activity.
Homology can be determined by comparing each position in the
aligned sequences. A degree of homology between amino acid
sequences is a function of the number of identical or matching
amino acids at positions shared by the sequences so that a
"homologous sequence" refers to a sequence sharing homology and an
equivalent function or biological activity. Assessment of percent
homology is known by those of skill in the art.
[0061] Methods to determine identity and similarity of peptides are
codified in publicly available computer programs. Preferred
computer program methods to determine identity and similarity
between two sequences include, but are not limited to, the GCG
program package, BLASTP, BLASTN, and FASTA. The BLAST X program is
publicly available from NCBI and other sources. The well known
Smith Waterman algorithm may also be used to determine
identity.
[0062] Preferred parameters for polypeptide sequence comparison
include the following:
[0063] Algorithm: Needleman and Wunsch, J. MoI. Biol. 48: 443-453
(1970);
[0064] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992);
[0065] Gap Penalty: 12; Gap Length Penalty: 4.
[0066] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison, Wis.
The aforementioned parameters are the default parameters for amino
acid sequence comparisons (along with no penalty for end gaps).
[0067] The polypeptides of the invention may be prepared in any
suitable manner as known in the art. Such polypeptides include
isolated naturally occurring polypeptides, recombinantly produced
polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means and methods for
preparing such polypeptides are well known in the art.
[0068] Certain aspects of the invention use UAG polynucleotides.
These include isolated polynucleotides which encode the UAG
polypeptides, fragments and analogs defined in the application.
[0069] As used herein, the term "polynucleotide" refers to a
molecule comprised of a plurality of deoxyribonucleotides or
nucleoside subunits. The linkage between the nucleoside subunits
can be provided by phosphates, phosphonates, phosphoramidates,
phosphorothioates, or the like, or by nonphosphate groups as are
known in the art, such as peptoid-type linkages utilized in peptide
nucleic acids (PNAs). The linking groups can be chiral or achiral.
The oligonucleotides or polynucleotides can range in length from 2
nucleoside subunits to hundreds or thousands of nucleoside
subunits. While oligonucleotides are preferably 5 to 100 subunits
in length, and more preferably, 5 to 60 subunits in length, the
length of polynucleotides can be much greater (e.g., up to 100).
The polynucleotide may be any of DNA and RNA. The DNA may be in any
form of genomic DNA, a genomic DNA library, cDNA derived from a
cell or tissue, and synthetic DNA. Moreover, the present invention
may, in certain aspects, use vectors which include bacteriophage,
plasmid, cosmid, or phagemid.
Survival Effect of UAG Fragments and Analogs Thereof
[0070] In one aspect of the invention, the proliferative and
antiapoptotic effects of UAG fragments and analogs thereof vs. UAG
in INS-1E .beta.-cell line, HIT-T15 .beta.-cell line as well as in
human pancreatic islets were investigated.
[0071] UAG fragments and analogs thereof which stimulate
proliferation and/or inhibit apoptosis in these cell lines will
also bear other metabolic properties of UAG including, but not
limited to, lowering blood glucose levels, improving insulin
sensitivity, decreasing cortisol levels, improving lipid profile in
human beings, and thus, have the potential use to treat metabolic
disorders associated, for example, with insulin resistance, insulin
deficiency, dyslipidemia or cortisol excess.
[0072] In one aspect of the invention, the survival effects of some
human UAG fragments listed in Table 1 below were analyzed:
TABLE-US-00001 TABLE 1 SEQ ID NAME NO: SEQUENCE UAG (1-14) 2
Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu- His-Gln-Arg-Val-Gln-Gln UAG (1-18)
3 Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu- His-Gln-Arg-Val-Gln-Gln-Arg-Lys-
Glu-Ser UAG (1-5) 4 Gly-Ser-Ser-Phe-Leu UAG (17-28) 5
Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys- Leu-Gln-Pro-Arg UAG (6-13) 6
Ser-Pro-Glu-His-Gln-Arg-val-Gln UAG (8-13) 7
Glu-His-Gln-Arg-Val-Gln UAG (8-12) 8 Glu-His-Gln-Arg-Val UAG (8-11)
10 Glu-His-Gln-Arg UAG (9-12) 11 His-Gln-Arg-Val UAG (9-11) --
His-Gln-Arg
[0073] The UAG fragments listed in Table 2 below were also
analysed:
TABLE-US-00002 TABLE 2 SEQUENCE SEQ (amino acid residues ID 6 to 13
of NAME NO: SEQ ID NO: 1) (Asp)8 UAG (6-13)NH.sub.2 12
Ser-Pro-Asp-His-Gln- Arg-Val-Gln-NH.sub.2 (Lys)11 UAG
(6-13)NH.sub.2 13 Ser-Pro-Glu-His-Gln- Lys-Val-Gln-NH.sub.2 (Gly)6
UAG (6-13)NH.sub.2 14 Gly-Pro-Glu-His-Gln- Arg-Val-Gln-NH.sub.2
(Ala)6 UAG (6-13)NH.sub.2 15 Ala-Pro-Glu-His-Gln-
Arg-Val-Gln-NH.sub.2 (Ala)7 UAG (6-13)NH.sub.2 16
Ser-Ala-Glu-His-Gln- Arg-Val-Gln-NH.sub.2 (Ala)8 UAG (6-13)NH.sub.2
17 Ser-Pro-Ala-His-Gln- Arg-Val-Gln-NH.sub.2 (Ala)9 UAG
(6-13)NH.sub.2 18 Ser-Pro-Glu-Ala-Gln- Arg-Val-Gln-NH.sub.2 (Ala)10
UAG (6-13)NH.sub.2 19 Ser-Pro-Glu-His-Ala- Arg-Val-Gln-NH.sub.2
(Ala)11 UAG (6-13)NH.sub.2 20 Ser-Pro-Glu-His-Gln-
Ala-Val-Gln-NH.sub.2 (Ala)12 UAG (6-13)NH.sub.2 21
Ser-Pro-Glu-His-Gln- Arg-Ala-Gln-NH.sub.2 (Ala)13 UAG
(6-13)NH.sub.2 22 Ser-Pro-Glu-His-Gln- Arg-Val-Ala-NH.sub.2
(Acetyl-Ser)6 UAG 23 Ac-Ser-Pro-Glu-His- (6-13)NH.sub.2
Gln-Arg-Val-Gln-NH.sub.2 (Acetyl-Ser)6, (DPro)7 UAG 24
Ac-Ser-pro-Glu-His- (6-13 )NH.sub.2 Gln-Arg-Val-Gln-NH.sub.2 Cyclo
(6-13) UAG 25 Ser-Pro-Glu-His-Gln- Arg-Val-Gln (cycl) Cyclo (8,
11), Lys 11, UAG 26 Ser-Pro-Glu-His-Gln- (6-13)amide
Lys-Val-Gln-amide Cyclo (8, 11), Acetyl- 27 Ac-Ser-Pro-Glu-His-
Ser6, Lys 11, UAG (6-13)- Gln-Lys-Val-Gln amide (cycl) Acetyl-Ser6,
Lys 11, UAG 28 Ac-Ser-Pro-Glu-His- (6-13) NH.sub.2
Gln-Lys-Val-Gln-NH.sub.2
[0074] UAG (1-14) and UAG (1-18) potently increased cell survival
of both INS-1E .beta.-cells and HIT-T15 .beta.-cells in either
serum-free conditions and after treatment with cytokines (FIGS. 1-2
for INS-1E cells, FIGS. 3A, 3B, 4A and 4B for HIT-T15
.beta.-cells). These effects were similar to that displayed by the
full-length molecule UAG (1-28). UAG (1-14) appeared even stronger
than native UAG as a protection against cytokine-induced apoptosis
in INS-1E cells. UAG (1-5) and UAG (17-28) exerted only a trivial
effect in INS-1E cells (FIGS. 1-2) and very little effect in
HIT-T15 cells (FIGS. 4A and 4B). Surprisingly, the short fragments
UAG (6-13), UAG (8-13) and UAG (8-12) were all strongly effective
in increasing survival in cytokine-induced apoptosis in HIT-T15
cells (FIG. 5A). Actually, peptides UAG (8-12) and UAG (8-13) were
at least as potent as UAG (1-14), whereas peptide UAG (6-13) was
clearly superior. UAG (1-5) and UAG (17-28) were only minimally
effective.
[0075] UAG (6-13), UAG (8-12) and UAG (8-13) were shown to exert
the strongest antiapoptotic effect in HIT-T15 .beta.-cells treated
with cytokines (FIGS. 6A, 6B and 6C).
[0076] The data presented herein demonstrate that UAG fragments
potently increase cell survival and prevent cell death in p cell
lines with potencies very comparable to that of the full-length UAG
itself or better. UAG (1-14) exhibited a potency equivalent to, if
not better than full-length UAG itself, whereas the (8-12)
fragment, a 5 amino-acid peptide, retained all the biological
activity and UAG (6-13) was even more potent.
[0077] In another aspect of the invention, the data presented
herein also demonstrate the survival effect of UAG fragments in
human pancreatic islets (FIGS. 7A and 7B). UAG (1-14) and UAG
(1-18) exert protective effects in serum-free conditions that are
similar to those displayed by UAG (1-28). On the other hand, the
protective effect of UAG (1-5) and (17-28) in human islets is
reduced or even absent in the experimental conditions tested.
Effect of UAG Fragments or Analogs Thereof on Insulin Secretion
[0078] The effects of UAG (1-14) and UAG (1-18) on insulin
secretion in human islets was also investigated. UAG (1-14),
similarly to UAG (1-28), and to exendin-4, significantly increased
glucose-induced insulin secretion in both HIT-T15 .beta.-cells
(data not shown) and in human islets (FIGS. 8A to 8D).
UAG Fragment and Analogs Thereof Reduce Diabetes In Vivo
[0079] In a further aspect, the data presented herein also show
that UAG fragments, for example UAG (6-13), increase survival of
Streptozotocin (STZ)-treated animals (FIG. 9A). UAG fragments also
reduce STZ-induced plasma glucose (FIG. 9B) and improve both plasma
and pancreatic insulin levels (FIGS. 9C and 9D) in STZ-induced
diabetic rats. The data presented herein also demonstrate that UAG
fragments, for example UAG (6-13), suppress plasma glucose levels,
enhance insulin sensitivity and modulate diabetes in vivo (FIGS.
14A, 14B and 15) and reduces body fat weight (FIG. 16).
Binding of UAG Fragments and Analogs Thereof to .beta.-Cells
[0080] In a further aspect, the data presented herein demonstrate
that UAG (6-13), UAG (1-14) and UAG (1-13) recognized and bound to
the UAG receptor on HIT-T15 and INS-1E pancreatic p cells. Among
these, UAG (6-13) displayed the highest binding activity and
possessed a binding affinity very close to that of the naturally
occurring UAG. This finding, in conjunction with the functional in
vitro studies showing that UAG (6-13) exerts, similarly to native
UAG, prosurvival effects on HIT-T15 cells, indicate that UAG (6-13)
is a potent UAG agonist with potential anti-diabetic activity.
[0081] Thus it appears that the active sequence of UAG to obtain
its metabolic effects resides in the region containing residues
8-12. This observation clearly differentiates the
structure-activity relationship of UAG to that of acylated ghrelin,
for which the minimally active sequence is ghrelin (1-5), the
serine residue in position 3 being octanoylated. This further
reinforces the hypothesis that UAG exerts its metabolic effects
through one or several receptors other than GHS-R1a, the receptor
mediating the effects of acylated ghrelin on growth hormone
secretion.
[0082] Therefore, and very surprisingly, these results show that
the full-length UAG sequence is not necessary for UAG to produce
its biological effects on .beta.-cells and on human islet. UAG
(1-14) and UAG (1-18) are at least as potent as native UAG. Even
more surprisingly, UAG (8-12) and UAG (8-13) retained all the
biological activity of full-length UAG, and UAG (6-13) was even
more potent than UAG (1-14).
[0083] The results indicate that UAG (8-12) or any peptide
comprising this 5 amino acid sequence, whether amidated or not, or
any peptide comprising, for example, any analogs of UAG (6-13), UAG
(8-12) or UAG (8-13) will share the same metabolic or biological
effects as UAG itself. Any peptide comprising a fragment of at
least 5, or at least 6, or at least 7, or at least 8 amino acid
residues of the amino acid sequence containing residues 6 to 18 of
UAG and including at least the amino acid sequence UAG (8-12) are
also preferred.
[0084] In a further aspect, the present invention provides for
peptides comprising UAG (8-12) or UAG (8-13) or UAG (6-13) or any
analogs thereof having the property to stimulate the proliferation
of .beta.-cells, to improve survival and/or inhibit death of
.beta.-cells, to decrease plasma glucose level, to increase insulin
secretion and/or sensitivity, to decrease blood lipids, such as
free fatty acids and triglycerides, to reduce cortisol secretion,
to bind to .beta.-cells, which make them useful, for example, for
the treatment of disorders associated with impaired glucose
metabolism, impaired insulin metabolism, type I diabetes, type II
diabetes and/or to improve the engraftment of pancreatic islets,
whether by ex vivo treatment of the graft or by administration in
the patient. The peptides are also useful to treat medical
conditions associated in insulin resistance, insulin deficiency,
lower blood glucose, useful for the treatment of diabetes, obesity
and dyslipidemia. Assays for measuring the properties of the
polypeptides of the invention and the procedures for carrying out
these assays are well known in the art.
[0085] In a further aspect, the present invention provides for
analogs of UAG fragments which retain the biological activity of
UAG. Examples of such analogs are, but are not limited to, (Asp) 8
UAG (6-13) where E (Glu) is substituted by D (Asp), which is as
active as UAG (6-13). The activity of this analog illustrates that
a substitution of an acidic amino-acid by another acidic residue
preserves the biological activity of UAG (6-13). (Lys)11 UAG (6-13)
where R (Arg) is substituted by K (Lys), is also as active as UAG
(6-13), illustrating the fact that a substitution of a basic
amino-acid by another basic residue preserves the biological
activity of UAG (6-13). (Gly) 6 UAG (6-13) where S (Ser) is
substituted by G (Gly), is also as active as UAG (6-13),
illustrating that a substitution based on size preserves the
biological activity of UAG (6-13). Overall, these analogs of UAG
(6-13) demonstrate that conservative substitutions preserve the
biological activity of UAG (6-13).
[0086] Further, acetylation of Ser in position 6 (N-terminus) of
UAG (6-13) preserves the biological activity of UAG (6-13) and a
combination of N-terminus acetylation and substitution of, for
example, Pro7 by D-Pro (its D form) results in an analog that also
exhibits biological activity. Therefore, strategies aiming at
stabilizing the N-terminus of UAG (6-13) to improve its resistance
to degradation by for example, exopeptidases and endopeptidases
(such as, but not limited to, DPP IV) result in peptides that still
exhibit biological activity of UAG (6-13), making them useful for
in vivo uses.
[0087] The peptides of the present invention, including analogs
thereof, can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed at least
by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY., 1987.
[0088] In general, a DNA sequence encoding the polypeptide of the
present invention is operably linked to other genetic elements
required for its expression, generally including a transcription
promoter and terminator within an expression vector. The vector
will also commonly contain one or more selectable markers and one
or more origins of replication, although those skilled in the art
will recognize that within certain systems selectable markers may
be provided on separate vectors, and replication of the exogenous
DNA may be provided by integration into the host cell genome.
Selection of promoters, terminators, selectable markers, vectors
and other elements is a matter of routine design within the level
of ordinary skill in the art. Many such elements are described in
the literature and are available through commercial suppliers.
[0089] To direct a polypeptide into the secretory pathway of a host
cell, a secretory signal sequence (also known as a leader sequence,
prepro sequence or pre sequence) may be provided in the expression
vector. The secretory signal sequence is joined to the DNA sequence
in the correct reading frame. Secretory signal sequences are
commonly positioned 5' to the DNA sequence encoding the propeptide
of interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743;
[0090] Holland et al., U.S. Pat. No. 5,143,830). The methods to
produce and/or manufacture the polypeptide of the invention are
well known and well practiced in the art.
[0091] The peptides of the invention may be synthesized by
solid-phase synthesis. Solid-phase synthesis is a common method for
synthesizing peptides. Basically, in this technique, molecules are
bound on a bead and synthesized step-by-step in a reactant
solution; compared with normal synthesis in a liquid state, it is
easier to remove excess reactant or by-product from the product. In
this method, building blocks are protected at all reactive
functional groups. The two functional groups that are able to
participate in the desired reaction between building blocks in the
solution and on the bead can be controlled by the order of
deprotection.
[0092] In the basic method of solid-phase synthesis, building
blocks that have two function groups are used. One of the
functional groups of the building block is usually protected by a
protective group. The starting material is a bead which binds to
the building block. At first, this bead is added into the solution
of the protected building block and stirred. After the reaction
between the bead and the protected building block is completed, the
solution is removed and the bead is washed. Then the protecting
group is removed and the above steps are repeated. After all steps
are finished, the synthesized compound is cleaved from the
bead.
[0093] If a compound containing more than two kinds of building
blocks is synthesized, a step is added before the deprotection of
the building block bound to the bead; a functional group which is
on the bead and did not react with an added building block has to
be protected by another protecting group which is not removed at
the deprotective condition of the building block. By-products which
lack the building block of this step only are prevented by this
step. In addition, this step makes it easy to purify the
synthesized compound after cleavage from the bead.
[0094] Usually, peptides are synthesized from the chain in this
method, although peptides are synthesized in the opposite direction
in cells. An amino-protected amino acid is bound to a bead (a
resin), forming a covalent bond between the carbonyl group and the
resin. Then the amino group is deprotected and reacted with the
carbonyl group of the next amino-protected amino acid. The bead now
bears two amino acids. This cycle is repeated to form the desired
peptide chain. After all reactions are complete, the synthesized
peptide is cleaved from the bead.
[0095] The protecting groups for the amino groups mostly used in
this peptide synthesis are, but not limited to
9-fluorenylmethyloxycarbonyl group (Fmoc) and t-butyloxycarbonyl
(Boc). The Fmoc group is removed from the amino terminus with base
while the Boc group is removed with acid. Any one of skill in the
art to which this invention pertains will be familiar with the
technique of solid-phase synthesis of peptides.
[0096] Other techniques may be used to synthesize the peptides of
the invention. The techniques to produce and obtain the peptides of
the invention are well known in the art.
[0097] The peptides of the invention can be purified using
fractionation and/or conventional purification methods and media.
For example, ammonium sulfate precipitation and acid or chaotrope
extraction may be used for fractionation of samples. Exemplary
purification steps may include hydroxyapatite, size exclusion, FPLC
and reverse-phase high performance liquid chromatography. Suitable
anion exchange media include derivatized dextrans, agarose,
cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE, QAE and Q derivatives may be used (Pharmacia, Piscataway,
N.J.). Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like. These supports may be modified with reactive groups that
allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers.
[0098] Although UAG fragments containing amino acid residues 1-5,
1-14, 1-18, 6-13, 8-12, 8-13, 8-11, 9-11, 9-12, 17-28 and analogs
of UAG fragments, have been synthesized, the present invention also
provides for any other fragments of SEQ ID NO: 1 and analogs
thereof retaining at least one of the biological activities of the
full-length UAG. A skilled person in the art, with knowledge of the
instant invention, would readily determine if a particular UAG
fragment or analog thereof has the expected biological
activities.
Therapeutic Uses and Treatments
[0099] The expression "treating a disease or a disorder" refers to
administering a therapeutic substance effective to ameliorate
symptoms associated with a disease, to lessen the severity or cure
the disease, or to prevent the disease from occurring.
[0100] As used herein, the term "treatment" refers to both
therapeutic treatment as well as to prophylactic and preventative
measures. Those in need of treatment include those already with the
disease or disorder, condition or medical condition as well as
those in which the disease, disorder, condition or medical
condition is to be prevented. Those in need of treatment are also
those in which the disorder, disease, condition or medical
condition has occurred and left after-effects or scars. Treatment
also refers to administering a therapeutic substance effective to
improve or ameliorate symptoms associated with a disease, a
disorder, condition or medical condition to lessen the severity of
or cure the disease, disorder, condition or medical condition, or
to prevent the disease, disorder or condition from occurring.
[0101] The term "metabolic disorders" refers to, but is not limited
to, disorders of carbohydrate metabolism, disorders of amino acid
metabolism, disorders of organic acid metabolism (organic
acidurias), disorders of fatty acid oxidation and mitochondrial
metabolism, disorders of porphyrin metabolism, disorders of purine
or pyrimidine metabolism, disorders of steroid metabolism,
disorders of mitochondrial function, disorders of peroxisomal
function and lysosomal storage disorders.
[0102] The term "metabolic syndrome" refers to a combination of
medical disorders that increase one's risk for cardiovascular
disease and/or diabetes.
[0103] It is thus an aspect of the invention that fragments of
unacylated ghrelin and analogs thereof and peptides comprising them
have a glucose lowering effect since unacylated ghrelin prevents
the hyperglycemic effects of acylated ghrelin, an insulin
sensitizing effect, an insulin secretion enhancement effect, a body
fat weight lowering effect, a free fatty acids (FFA) and cortisol
lowering effect, indicating an effect of fragments of unacylated
ghrelin on dyslipidemia. In addition to these properties, fragments
of unacylated ghrelin and analogs thereof are capable of
stimulating the proliferation and the survival, as well as
inhibiting death, of insulin-secreting cells such as, pancreatic
.beta.-cells.
[0104] The invention thus provides for a therapeutic potential of
fragments of unacylated ghrelin and analogs thereof in the
treatment of, for example, diabetes, other medical conditions
related to impaired glucose or insulin metabolism, insulin
deficiencies or resistance, dyslipidemia, obesity, the metabolic
syndrome and the treatment of insulin secreting cells such as
pancreatic .beta.-cells.
[0105] It is a further aspect, the invention provides for any
pharmaceutical compositions incorporating at least one of the
peptides of the invention, which share the same potential
therapeutic indication as UAG itself.
[0106] The peptides of the present invention can be used for and
can be incorporated in pharmaceutical formulations to be used in
the prevention, reduction and/or treatment of for example, but not
limited to, disorders or medical conditions associated with
impaired glucose metabolism, impaired insulin metabolism, impaired
lipid metabolism, type I diabetes, type II diabetes, obesity,
dyslipidemia, atherosclerosis, cardiovascular diseases, metabolic
syndrome disorders associated with impaired proliferation of
insulin-secreting cells or with insulin resistance.
[0107] For therapeutic and/or pharmaceutical uses, the peptides of
the invention may be formulated for, but not limited to,
intravenous, subcutaneous, transdermal, oral, buccal, sublingual,
nasal, inhalation, pulmonary, or parenteral delivery according to
conventional methods. Intravenous injection may be by bolus or
infusion over a conventional period of time. The peptides of the
invention may also be compatible with drug delivery system such as,
but not limited to, polymer-based depot formulations.
[0108] Active ingredients to be administered orally as a suspension
can be prepared according to techniques well known in the art of
pharmaceutical formulation and may contain, but are not limited to,
microcrystalline cellulose for imparting bulk, alginic acid or
sodium alginate as a suspending agent, methylcellulose as a
viscosity enhancer, and sweeteners/flavoring agents. As immediate
release tablets, these compositions may contain, but not limited to
microcrystalline cellulose, dicalcium phosphate, starch, magnesium
stearate and lactose and/or other excipients, binders, extenders,
disintegrants, diluents and lubricants.
[0109] Administered by 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.
[0110] The peptides of the invention may be administered in
intravenous (both bolus and infusion), intraperitoneal,
subcutaneous, topical with or without occlusion, or intramuscular
form. When administered by injection, the injectable solution or
suspension may be formulated using suitable non-toxic,
parenterally-acceptable diluents or solvents, well-known in the
art.
[0111] In general, pharmaceutical compositions will comprise at
least one of the peptides of the invention together with a
pharmaceutically acceptable carrier which will be well known to
those skilled in the art. The compositions may further comprise for
example, one or more suitable excipients, diluents, fillers,
solubilizers, preservatives, salts, buffering agents and other
materials well known in the art depending upon the dosage form
utilised. Methods of composition are well known in the art.
[0112] In the present context, the term "pharmaceutically
acceptable carrier" is intended to denote any material, which is
inert in the sense that it substantially does not have any
therapeutic and/or prophylactic effect per se. A pharmaceutically
acceptable carrier may be added to the peptides of the invention
with the purpose of making it possible to obtain a pharmaceutical
composition, which has acceptable technical properties.
[0113] Therapeutic dose ranges of the invention will generally vary
from about 0.01 .mu.g/kg to about 10 mg/kg. Therapeutic doses that
are outside this range but that have the desired therapeutic
effects are also encompassed by the present invention.
[0114] Suitable dosage regimens are preferably determined taking
into account factors well known in the art including, but not
limited to, 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.
[0115] For example, a therapeutically effective amount of the
peptides of the invention (also referred to herein as "active
compound") is an amount sufficient to produce a clinically
significant change in lowering blood glucose levels, improving
insulin sensitivity and/or secretion, reducing blood free fatty
acids levels, lowering body fat weight, decreasing cortisol levels
and/or increasing survival of insulin-secreting cells, amongst
other changes. The tests for measuring such parameters are known to
those of ordinary skill in the art.
[0116] The peptides of the invention can be provided in a kit. Such
a kit typically comprises an active compound in dosage form for
administration. A dosage form comprises a sufficient amount of
active compound such that a desirable effect can be obtained.
Preferably, a kit comprises instructions indicating the use of the
dosage form to achieve the desired effect and the amount of dosage
form to be taken over a specified time period.
Experiments and Data Analysis
UAG Fragments Promote INS-1E .beta.-Cell Survival
[0117] Cell survival was assessed by MTT assay in INS-1E rat
incubated with either full length human UAG (1-28) or UAG (1-14),
UAG (1-18), UAG (1-5) and UAG (17-28) in serum deprived medium,
either alone or with IFN-.gamma./TNF-.alpha./IL-1.beta., whose
synergism has been shown to be involved in .beta.-cell death in
both type 1 and type 2 diabetes (Ref. 16). The peptides were tested
at increasing concentrations, ranging from 0.1 nM to 100 nM. In
serum-free conditions, UAG (1-14) and (1-18) showed significant
survival effect, comparable to that of UAG (1-28). Under the same
conditions, UAG (1-5) and UAG (17-28) displayed reduced, although
significant, survival action (FIG. 1). In the presence of
cytokines, all the peptides significantly increased cell survival
at every concentration tested (FIG. 2). However, similarly to
serum-free condition, UAG (1-5) and UAG (17-28) displayed reduced
effect. Interestingly, UAG (1-14) and also UAG (1-18) showed to be
more potent than full length UAG (1-28) (FIG. 2). These results
indicate that UAG fragments particularly UAG (1-14) and (1-18),
similarly to full length UAG (1-28), are able to counteract
.beta.-cell death induced by either serum starvation or treatment
with cytokines.
UAG Fragments Promote HIT-T15 .beta.-Cell Survival
[0118] MTT experiments were also performed in hamster HIT-T15
.beta.-cells, to test the survival effect of UAG (1-28) or its
fragments UAG (1-14), UAG (1-18), UAG (1-5) and UAG (17-28) in
serum deprived medium, either alone or with
IFN-.gamma./TNF-.alpha./IL-1.beta.. As for the experiments
performed on INS-1E .beta.-cells, the peptides were tested at
increasing concentrations, ranging from 0.1 nM to 100 nM. With
respect to INS-1E, in HIT-T15 cells the peptides displayed
different protective effects against both serum starvation- and
cytokine-induced cell death. Indeed, whereas UAG (1-14) and UAG
(1-18) significantly increased cell viability under both
experimental conditions (FIGS. 3A and 3B), UAG (1-5) slightly
increased cell survival only in cytokine-treated cells, whereas UAG
(17-28) had no significant effect, at any condition examined (FIGS.
4A and 4B).
[0119] The survival effect of UAG (6-13), UAG (8-13), UAG (8-12),
UAG (8-11), UAG (9-12) and UAG (9-11) was assessed in
cytokine-treated HIT-T15 .beta.-cells. As expected, the cytokines
(IFN-.gamma./TNF-.alpha./IL-1.beta.) strongly reduced cell survival
with respect to normal culture conditions (serum containing
medium). UAG (6-13), at all the concentrations tested (1 nM to 100
nM) and particularly at 100 nM, potently inhibited cytokine-induced
cell death by increasing cell survival up to values similar to or
even greater than those observed in the presence of serum.
Interestingly, the survival effect of UAG (6-13) was comparable to
that of full length UAG (1-28) (FIG. 5A).
[0120] Under the same experimental condition, UAG (8-13), although
less than UAG (6-13), showed significant protective effect at all
the concentrations examined, whereas UAG (8-12) displayed
significant, although reduced protection, only at 10 nM and 100 nM.
The protective effects of peptides UAG (8-13) and UAG (8-12) were
found similar to those of UAG (1-14) and UAG (1-18) A peptide made
of the inverse sequence of UAG (1-14) and named UAG (14-1), was
used as negative control for these experiments (FIG. 5A). With
regard to UAG (8-11), UAG (9-12) and UAG (9-11) (FIG. 5B), MTT
results indicated that UAG (8-11) exerted significant survival
effect only at 100 nM and UAG (9-12) significantly increased cell
survival at both the concentrations tested (1 and 100 nM). These
effects were however lower than those of UAG (6-13) (FIG. 5B). UAG
(9-11) had no significant effect at both concentrations tested
(FIG. 5B).
UAG Fragments Exert Antiapoptotic Effects in HIT-T15
.beta.-Cells
[0121] HIT-T15 .beta.-cells were cultured for 24 h in serum-free
medium, either alone or with IFN-.gamma./TNF-.alpha./IL-1.beta.. In
both cell lines, apoptosis increased under cytokine treatment, with
respect to serum starvation alone. UAG (6-13) increased the number
of cells, induced cell enlargement and small islets formation, with
respect to cytokine condition (data not shown). Moreover, it
significantly reduced cytokine-induced apoptosis at the
concentration of 1 nM, 10 nM and, particularly, at 100 nM where the
antiapoptotic effect was even stronger than that displayed by UAG
(1-28) (FIG. 6A). UAG (8-13), although less than UAG (6-13),
significantly inhibited apoptosis at 10 and 100 nM, whereas UAG
(8-12) showed some protective effect only at 100 nM (FIGS. 6B and
6C respectively). UAG (14-1), the inverse sequence of UAG (1-14),
was used as negative control, whereas UAG (1-28), was used as
positive control in each experiment. These results indicate that,
similarly to the results obtained for cell survival, with respect
to UAG (8-13) and UAG (8-12), UAG (6-13) exerts the strongest
antiapoptotic effect in HIT-T15 .beta.-cells treated with
cytokines.
Survival Effect of UAG Fragments in Human Pancreatic Islets
[0122] The survival effect of UAG (1-14), UAG (1-18), UAG (1-5) and
UAG (17-28), with respect to that of full length UAG (1-28), was
assessed in human pancreatic islets by MTT. The peptides were
tested in islet cells cultured in serum deprived medium, either
alone or with IFN-.gamma./TNF-.alpha./IL-1.beta. (5 ng/ml each).
UAG (1-14) significantly increased cell survival in serum deprived
medium at 10 nM and 100 nM, whereas in the presence of cytokines it
prevented cell death at 100 nM (FIG. 7A). UAG (1-18) significantly
increased cell survival at 1 nM and 10 nM (FIG. 7A). UAG (1-5)
displayed little, although significant survival action at 10 nM in
serum deprived medium but showed no cell protection after addition
of cytokines, at any concentration tested (1 nM to 100 nM) (FIG.
7B). UAG (17-28) significantly increased survival of islet cells
cultured in serum deprived conditions, at 10 nM and 100 nM, but had
no effect in the presence of cytokines (FIG. 7B). In all, these
results indicate that in human islets, UAG (1-14) and UAG (1-18)
exert protective effects in serum-free conditions that are similar
to those displayed by UAG (1-28), whereas their survival capacity
is at least partly lost in cytokine-treated cells where the effect
of UAG (1-28) is still evident.
Effect of UAG Fragments on Insulin Secretion in Human Pancreatic
Islets
[0123] The effects of UAG (1-14) and UAG (1-18), both used at 100
nM, were investigated on insulin secretion in human islets. FIG. 8A
shows that UAG (1-14), similarly to UAG (1-28) (FIG. 8C) and to
exendin-4 (FIG. 8D), significantly increased insulin secretion both
in the absence and presence of glucose (2 to 25 mM), whereas UAG
(1-18) showed significant effect with 7.5 mM glucose (FIG. 8B). UAG
(1-28) and Exendin-4 were used as positive controls (FIGS. 8C and
8D). These results indicate that in human pancreatic islets UAG
(1-14) and UAG (1-18) stimulate glucose-induced insulin
secretion.
In Vivo Effect of UAG Fragment on Streptozotocin (STZ)-Treated
Animals
[0124] It is well known that Streptozotocin (STZ) treatment in
neonatal rats causes diabetes (Refs. 24, 25, 26). Herein, the
long-term effects of UAG (6-13) (one week of treatment following
STZ administration, assessment at 70 days following STZ
administration vs. those of UAG in neonatal rats treated with STZ
at day 1 of birth) was investigated. UAG (6-13) was tested at a
concentration that was equal (30 nmol/l) or higher (100 nmol/l)
than that of UAG. Interestingly, at day 9 after injection with STZ,
the animal survival rate, that was decreased by STZ with respect to
the Control group (.apprxeq.52%), was strongly increased by UAG
(.apprxeq.72%), and by both UAG (6-13) concentrations (.apprxeq.71%
and 89% for 30 nmol/l and 100 nmol/l, respectively) (FIG. 9A). At
day 70, plasma glucose was significantly increased by 150%
(P<0.01) in STZ group with respect to Control. UAG, as expected,
counteracted STZ effect by reducing glucose levels (by
.apprxeq.21%). A similar effect was obtained with both 30 nmol/l
and 100 nmol/l UAG (6-13) (reduction of 31% and 14%, respectively
vs. STZ group). Interestingly, UAG (6-13) at equal concentration
showed an effect that was stronger than that of UAG (FIG. 9B).
STZ-treated animals showed significant reduction of plasma insulin
levels; UAG, as well as UAG (6-13), at both concentrations,
significantly reduced this effect by increasing insulin levels in
STZ-treated rats (FIG. 9C). Similar results were obtained with
regard to pancreatic insulin secretion (FIG. 9D). These results
indicate that at day 70 after treatment with STZ, UAG (6-13),
similarly or even more than UAG, is able to reduce STZ-induced
plasma glucose increase and to improve both plasma and pancreatic
insulin levels.
[0125] UAG fragments modulate plasma glucose levels, insulin
sensitivity as well as gonadal fat weight in vivo in a genetic
model of diabetes associated with obesity and insulin resistance,
the ob/ob mice
[0126] Baseline tail vein plasma samples were collected from
free-fed and 16 h fasted ob/ob mice 7 and 6 days before pump
implantation into K.sub.2EDTA coated capillary tubes (Microvette
CB300 K2E; Sarstedt, Germany). The animals were then separated into
three groups with approximately equivalent weight ranges. Ten week
old mice were anesthetized, and a filled Alzet 1004 pump was
inserted, delivery portal first, into the peritoneal cavity. The
musculoperitoneal and skin layers were then closed using
interrupted sutures (Vicryl 5.0 FS-2 absorbable suture). Animals
received pumps containing either saline, 10 mg/ml UAG, or 3.5 mg/mL
UAG (6-13) (n=8 per group). Alzet 1004 pumps deliver 12 .mu.l/day,
and infused 30 .mu.g of hUAG/animal/day (.about.600 .mu.g/kg/day)
and 10 .mu.g of UAG (6-13)/animal/day (.about.200
.mu.g/kg/day).
[0127] Blood samples (at 0900-1000) were obtained from fed and
fasted animals at weeks 2 and 4 via the tail vein into EDTA
Microvette tubes. Glucose levels in tail vein blood were measured
directly using a glucometer. On the last day of treatment baseline
(fasted) blood samples were taken.
[0128] Although no statistically significant effects (RM-ANOVA)
were observed on fed plasma glucose levels during the period of
treatment, UAG (6-13) showed a consistent suppressive effect
relative to saline controls, and by week 4, UAG also suppressed
glucose levels relative to controls (FIG. 14A). In contrast,
fasting glucose concentrations were significantly suppressed by
25-30% from saline treated controls by UAG and UAG (6-13) treatment
at week 2 (FIG. 14B). This effect remained at week 4 (FIG. 14B). As
expected, both fasting and fed glucose levels in the controls
increased during the period of treatment, since ob/ob mice reach
peak hyperglycemia at approximately 12 weeks (Ref. 27).
[0129] Fasting plasma insulin levels were significantly suppressed
by UAG at 2 weeks relative to saline controls (FIG. 15). By 4 weeks
of treatment, though, fasting levels of insulin were significantly
increased above baseline levels, and relative to saline
controls.
[0130] During the period of treatment, in UAG (6-13) treated ob/ob
animals, gonadal fat pad weight was decreased by approximately 7%
relative to saline treated controls (trend p<0.06) (FIG. 16).
UAG and UAG (6-13) did not cause an increase in gonadal fat weight
over the period of treatment, as is observed with ghrelin
treatment. The trend towards a decrease in fat weight suggests that
longer exposure to UAG and UAG (6-13) will exert a lipolytic effect
translating into a reduction in fat mass, and thus might constitute
a promising treatment for obesity, with accompanying beneficial
effects on insulin sensitivity (e.g., Refs. 28, 29).
[0131] The findings from this long-term treatment protocol were
that both UAG and UAG (6-13) suppressed plasma glucose levels in
fasted animals after 2 and 4 weeks of treatment, relative to saline
control animals. UAG (6-13) also appeared to have a 30-40%
suppressive effect on plasma glucose levels in fed animals.
[0132] The effect of UAG on fasting glucose observed following 2
weeks of treatment corresponded with significantly lowered insulin
levels, indicating improved insulin sensitivity.
Binding of UAG Fragments to Pancreatic .beta.-Cell Receptors
[0133] The ability of the fragment UAG (6-13) to compete in a
concentration-dependent manner with [.sup.125I-Tyr.sup.4]-UAG for
HIT-T15 (FIG. 10A) and INS-1E (FIG. 10B) binding sites was assayed.
As shown in FIGS. 10A and 10B, unlabelled UAG (1-28) and UAG (6-13)
competed with a similar efficacy and in a concentration-dependent
fashion with [.sup.125I-Tyr.sup.4]-UAG for such binding sites in
both cell lines. The IC.sub.50 values calculated from competition
binding curves, all expressed as nM concentration, were 2.6.+-.0.5
and 2.0.+-.0.2 for UAG (1-28) and 3.8.+-.0.3 and 2.4.+-.0.3 for UAG
(6-13) in HIT-T15 and INS-1E, respectively.
Survival Effects of UAG Fragments with Alanine Substitutions on
HIT-T15 .beta.-Cells
[0134] UAG fragments with alanine (Ala) substitutions at different
amino acid positions (6 to 13) were tested with regards to their
survival effects in HIT-T15 hamster .beta.-cells. The cells were
cultured in serum deprived medium, either alone or with
IFN-.gamma./TNF-.alpha./IL-1.beta.. The peptides were tested at the
concentrations of 1 nM to 100 nM. In serum-free conditions, where
the survival rate was reduced by .apprxeq.40% with respect to the
presence of serum, UAG (6-13) significantly increased cell
survival, as expected (.apprxeq.18% and .apprxeq.30% at 1 and 100
nM, respectively). Ala 6-UAG (6-13), Ala 7-UAG (6-13), Ala 8-UAG
(6-13), Ala 9-UAG (6-13) and particularly, Ala 12-UAG (6-13) and
Ala 13-UAG (6-13), showed similar effects at both concentrations.
By contrast, very low survival effects were displayed by Ala
substitution at positions 10 and 11 (FIG. 11A). Under treatment
with cytokines, where cell survival was reduced by .apprxeq.18%
with respect to serum starved conditions, all Ala substitutions,
except those at positions 10 and 11, completely reversed cell death
and brought the survival rate to levels that were even higher than
those under serum-free conditions, at both 1 nM and 100 nM
concentrations. These effects, were similar to those elicited by
the original peptide UAG (6-13) (FIG. 11B). Ala substitutions at
positions 6 to 9 and 12 to 13 of UAG (6-13) do not affect the
peptide survival effect, whereas the side chains of amino acids at
position 10 (Q) and 11 (R) seem to play an essential role.
Survival Effects of UAG Fragments with Conservative Substitutions
and N-Terminal Modifications on HIT-T15 .beta.-Cells
[0135] In serum-free conditions, where the survival rate was
reduced by .apprxeq.35% with respect to the presence of serum, UAG
(6-13) significantly increased cell survival, as expected
(.apprxeq.18% and .apprxeq.30% at 1 and 100 nM, respectively). Asp
8-UAG (6-13), Lys 11-UAG (6-13), Gly 6-UAG (6-13), as well as AcSer
6-UAG (6-13) and AcSer 6-(D)Pro 7-UAG (6-13) showed similar effects
at both concentrations (FIG. 12A). Under the treatment with
cytokines, where cell survival was reduced by .apprxeq.20%, all the
peptides significantly increased cell survival. Particularly, the
best effect was exerted by Gly 6-UAG (6-13), whereas the lowest was
seen using AcSer 6-UAG (6-13), AcSer 6-(D)Pro 7-UAG (6-13) (FIG.
12B).
Survival Effects of Cyclized UAG Fragments on HIT-T15
.beta.-Cells
[0136] In serum-free conditions, where the survival rate was
reduced by .apprxeq.58% with respect to the presence of serum, UAG
(6-13) significantly increased cell survival, as expected
(.apprxeq.16% and .apprxeq.60% at 1 nM and 100 nM, respectively).
Cyclo 6,13 UAG (6-13), Cyclo (8,11), Acetyl-Ser6, Lys11, UAG
(6-13)amide and Acetyl-Ser6, Lys11, UAG (6-13)NH.sub.2 showed
similar effects (FIG. 13A). Similar results were found under the
treatment with cytokines (FIG. 13B).
Materials and Technical Protocols
[0137] Human UAG and UAG fragments (1-14), (1-18), (1-5) and
(17-28) as well as exendin-4 were from Phoenix Pharmaceuticals
(Belmont, Calif.). The other fragments (6-13), (8-13), (8-12),
(8-11), (9-12), (9-11) were from Tib MolBiol (Genova, Italy). Cell
culture reagents were from Invitrogen (Milano, Italy). Human UAG
(6-13) with alanine (Ala), Ala 6-UAG (6-13), Ala 7-UAG (6-13), Ala
8-UAG (6-13), Ala 9-UAG (6-13), Ala 10-UAG (6-13), Ala 11-UAG
(6-13), Ala 12-UAG (6-13) and Ala 13-UAG (6-13) were synthesized by
Tib MolBiol (Genova, Italy).
[0138] Most of the peptides defined herein were synthesised by
means of the simultaneous multiple peptide synthesis on the
following instrument: PSSM-8, SHIMADZU, Japan, using the Fmoc/But
(G. Schnorrenberg et al. Tetrahedron, 45:7759, 1989) strategy by
SHEPPARD (W. C. Chan et al., Fmoc solid phase peptide synthesis--A
practical approach, IRL Press, Oxford, 1989). Couplings were
performed using 3-6 equiv. Fmoc-amino acid/HOBt/TBTU and 6-12
equiv. N-Methylmorpholine on Tentagel HL RAM resin. The peptides
were purified by HPLC instrument SHIMADZU LC-8A. The peptides were
deprotected and cleaved from the resin by TFA/water and were
characterized by MALDI-TOF by means of a MALDI 2 DE instrument.
Finally the peptides were lyophilized in form of the TFA salt.
[0139] Cell culture--Hamster HIT-T15 insulin-secreting .beta.-cells
were obtained and cultured as described (Refs. 14, 4). INS-1E rat
.beta.-cells were kindly provided by Prof. Claes B. Wollheim
(University Medical Center, Geneva, Switzerland) and cultured as
described (Refs. 14, 4). Cell culture reagents were from Invitrogen
(Milano, Italy). Cytokines were from Biosource (Invitrogen,
Italy).
[0140] Human islet isolation--Human islets were obtained from
pancreases of multiorgan donors as described (Ref. 4). Islet
preparations with purity >70%, not suitable for transplantation,
were provided by European Consortium for Islet Transplantation
(ECIT) "Islets for Research Distribution Program," Transplant Unit,
Scientific Institute San Raffaele, Vita-Salute University, Milan.
Islets (10,000) were cultured in CMRL (Invitrogen) with 10%
FBS.
[0141] Cell survival assay--Cell survival was assessed by
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT)
as described previously (Ref. 4). Cells were seeded on 96-well
plates at a density of 5.times.10.sup.3 cells/well. After
treatments, cells were incubated with 1 mg/ml MTT for .apprxeq.1 h.
The medium was aspirated, and the formazan product solubilized with
100 .mu.l DMSO. Viability was assessed by spectrophotometry at 570
nm absorbance using a 96-well plate reader.
[0142] Insulin secretion--HIT-T15 cells were plated at density of
5.times.10.sup.5 cells into 100-mm dishes and serum starved for 24
h before incubation for 1 h at 37.degree. C. in HEPES-buffered
Krebs-Ringer bicarbonate buffer (KRBH), containing 0.5% BSA with
1.25 mM glucose. The medium was changed and the cells were
incubated again for 1 h in KRBH/0.5% BSA containing 1.25, 7.5 or 15
mM glucose. Following acid ethanol extraction of the hormone,
secreted insulin was quantitated by a radioimmunoassay kit (Linco
Research, Labodia, Yens, Switzerland) which recognizes human
insulin and cross reacts with rat insulin.
[0143] Animals--Pregnant female Sprague-Dawley rats (n=10, day
14th-15th of pregnancy) were purchased from Harlan Sri (Italy),
caged allowing free access to water and fed with a standard pellet
rat diet. Natural birth occurred 6-7 days later. Five experimental
groups were studied: 1) Control group, in which new-born rats
received a single i.p. injection of citrate buffer (0.05 mmol/l, pH
4.5); 2) STZ group, which received a single i.p. injection of STZ
(100 mg/Kg body weight), freshly dissolved in citrate buffer at day
1 of birth; 3) STZ+UAG group, which received a single i.p.
injection of STZ followed by injections of UAG, (30 nmol/kg s.c.,
twice daily) for 7 days (from day 2 to 8) after birth; 4) STZ+UAG
(6-13) group, which received a single i.p. injection of STZ
followed by injections of UAG (6-13) (30 nmol/kg s.c., twice daily)
for 7 days (from day 2 to 8) after birth; 5) STZ+UAG (6-13) group,
which received a single i.p. injection of STZ followed by
injections of UAG (6-13), (100 nmol/kg s.c., twice daily) for 7
days (from day 2 to 8) after birth. Dams were randomly assigned to
the five groups and pups from the same litter were assigned to the
same group. The numbers of dams in each of the four groups 11
(Control), 11 (STZ), 16 (STZ+UAG), and 21 (STZ+UAG (6-13), 30
nmol/kg) and 15 (STZ+UAG (6-13), 100 nmol/kg). Pups were left with
their mothers. All neonates were tested on day 2 for glycosuria
using Accu-chek compact plus (Roche). Only those animals that were
glycosuric at day 2 after birth were included in the STZ model
group. Treatments with UAG and UAG (6-13) were started after
glycosuria was confirmed. Animals were killed at day 70 after birth
by decapitation. Blood samples were collected after decapitation
and immediately centrifuged at 20,000.times.g for 2 min at
4.degree. C., and stored at -20.degree. C. until assayed.
[0144] For the experimental data illustrated in FIGS. 14A, 14B, 15
and 16, the animals were obtained from Charles River Laboratories
(Maastricht, The Netherlands). Animals (B6.V-Lep.sup.ob/J, Charles
River Laboratories, Belgian colony) were received in our animal
facilities at 8 weeks of age, and acclimatized in individual cages
for 2 weeks before treatments began. They were maintained under
standard 12:12 h light:dark conditions, 21.degree. C., and were
allowed free access to food and water. The animals were also
handled daily to accustom them to the method used for blood
collection. The peptides were dissolved in sterile, nonpyrogenic,
0.9% saline (Baxter BV, Utrecht, The Netherlands). D-glucose was
obtained from Sigma-Aldrich Chemie BV (Zwijndrecht, The
Netherlands), and was dissolved at 400 mg/ml in 0.9% saline. Alzet
pumps (model 1004) were obtained from Charles River Laboratories
(Maastricht, The Netherlands). Pumps were filled with 0.9% saline,
UAG or UAG (6-13) solution under sterile conditions, and
pre-incubated in 0.9% saline for at least 48 hours at 37.degree. C.
to initiate flow. Blood glucose levels were measured directly from
tail vein incisions using a Freestyle mini glucometer and test
strips (ART05214 Rev.A; Abbot, Amersfoort, The Netherlands). Plasma
insulin levels were assayed by Ultra-sensitive mouse insulin ELISA
(Cat. #10-1150-10; Mercodia, Sweden).
[0145] Pancreas removal and treatment--After excision, pancreases
were removed and weighed. For insulin content determination,
pancreases (35-50 mg) were homogenized and centrifuged in 5 ml
acid-ethanol (0.15 mol/l HCl in 75% [vol/vol]ethanol) at 1,000 g
for 20 min; the supernatants were stored at -80.degree. C. For
immunohistochemistry, additional pancreases were fixed in 4%
paraformaldehyde fixative for 24 h and embedded in paraffin.
[0146] Analytical techniques--Plasma glucose levels were determined
using a glucose analyzer. Insulin was measured from pancreases or
from plasma by RIA as previously described (Ref. 15).
[0147] Binding assay--Membranes from hamster HIT-T15 and rat INS-1E
to pancreatic .beta.-cells were prepared and assayed for the
presence of [.sup.125I-Tyr.sup.4]-UAG binding. The ability of UAG
fragments to compete with the radioligand for such binding sites
has been evaluated as previously described (Ref. 4). Data are
presented as mean.+-.S.E.M. of three independent experiments.
[0148] Statistical analysis--Results are expressed as means.+-.SE.
Statistical analysis were performed using Student's t test or
one-way ANOVA. Significance was established when P<0.05.
[0149] It is understood that the data reported in the present
specification are only given to illustrate the invention and may
not be regarded as constituting a limitation thereof.
[0150] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
[0151] All published documents mentioned in the above specification
are herein incorporated by reference.
REFERENCES
[0152] 1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa
K; (1999) Ghrelin is a growth-hormone-releasing acylated peptide
from stomach. Nature 402:656-660. [0153] 2. van der Lely A J,
Tschop M, Heiman M L, Ghigo E; (2004) Biological, physiological,
pathophysiological, and pharmacological aspects of ghrelin. Endocr
Rev 25:426-457. [0154] 3. Gauna C, Delhanty P J, Hofland L J,
Janssen J A, Broglio F, Ross R J, Ghigo E, van der Lely A J; (2005)
Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose
output by primary hepatocytes. J Clin Endocrinol Metab
90:1055-1060. [0155] 4. Granata R, Settanni F, Biancone L, Trovato
L, Nano R, Bertuzzi F, Destefanis S, Annunziata M, Martinetti M,
Catapano F, Ghe C, Isgaard J, Papotti M, Ghigo E, Muccioli G;
(2007) Acylated and unacylated ghrelin promote proliferation and
inhibit apoptosis of pancreatic .beta. cells and human islets
involvement of CAMP/PKA, ERK1/2 and PI3K/AKT signaling.
Endocrinology 148:512-529. [0156] 5. Merglen A, Theander S, Rubi B,
Chaffard G, Wollheim C B, Maechler P.; (2004) Glucose sensitivity
and metabolism-secretion coupling studied during two-year
continuous culture in INS-1E insulinoma cells. Endocrinology
145:667-678. [0157] 6. Broglio F, Gottero C, Prodam F, Gauna C,
Muccioli G, Papotti M, Abribat T, Van Der Lely A J, Ghigo E; (2004)
Non-acylated ghrelin counteracts the metabolic but not the
neuroendocrine response to acylated ghrelin in humans. J Clin
Endocrinol Metab 89:3062-3065. [0158] 7. Asakawa A, Inui A,
Fujimiya M et al.; (2005) Stomach regulates energy balance via
acylated ghrelin and desacyl ghrelin. Gut 54:18-24. [0159] 8.
Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S,
Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico
N, Bussolino F, Isgaard J, Deghenghi R, Sinigaglia F, Prat M,
Muccioli G, Ghigo E, Graziani A; (2002) Ghrelin and des-acyl
ghrelin inhibit cell death in cardiomyocytes and endothelial cells
through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 159:1029-1037.
[0160] 9. Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal M S,
Hosoda H, Kojima M, Kangawa K, Arima T, Matsuo H, Yada T, Matsukura
S; (2002) Ghrelin is present in pancreatic alpha-cells of humans
and rats and stimulates insulin secretion. Diabetes 51:124-129.
[0161] 10. Delhanty P J, van Koetsveld P M, Gauna C, van de Zande
B, Vitale G, Hofland L J, van der Lely A J; (2007) Ghrelin and its
unacylated isoform stimulate the growth of adrenocortical tumor
cells via an anti-apoptotic pathway. Am J Physiol Endocrinol Metab.
293:E302-309. [0162] 11. Dezaki K, Kakei M, Yada T; (2007) Ghrelin
uses Galphai2 and activates voltage-dependent K+ channels to
attenuate glucose-induced Ca2+ signaling and insulin release in
islet beta-cells: novel signal transduction of ghrelin. Diabetes.
56:2319-2327. [0163] 12. Filigheddu N, Gnocchi V F, Coscia M,
Cappelli M, Porporato P E, Taulli R, Traini S, Baldanzi G, Chianale
F, Cutrupi S, Arnoletti E, Ghe C, Fubini A, Surico N, Sinigaglia F,
Ponzetto C, Muccioli G, Crepaldi T, Graziani A; (2007) Ghrelin and
des-acyl ghrelin promote differentiation and fusion of C2C12
skeletal muscle cells. Mol Biol Cell. 18:986-994. [0164] 13. Gauna
C, Kiewiet R M, Janssen J A, van de Zande B, Delhanty P J, Ghigo E,
Hofland L J, Themmen A P, van der Lely A J; (2007) Unacylated
ghrelin acts as a potent insulin secretagogue in glucose-stimulated
conditions. Am J Physiol Endocrinol Metab 293:E697-704. [0165] 14.
Granata R, Settanni F, Trovato L, Destefanis S, Gallo D, Martinetti
M, Ghigo E, Muccioli G; (2006) Unacylated as well as acylated
ghrelin promotes cell survival and inhibit apoptosis in HIT-T15
pancreatic beta-cells. J Endocrinol Invest 29:RC19-22. [0166] 15.
Granata R, Settanni F, Gallo D, Trovato L, Biancone L, Cantaluppi
V, Nano R, Annunziata M, Campiglia P, Arnoletti E, Ghe C, Volante
M, Papotti M, Muccioli G, Ghigo E; (2008) Obestatin promotes
survival of pancreatic .beta.-cells and human islets and induces
expression of genes involved in the regulation of -cell mass and
function. Diabetes 57:967-79. [0167] 16. Mandrup-Poulsen T; (2001)
beta-cell apoptosis: stimuli and signaling. Diabetes 50:S58-63.
[0168] 17. Muccioli G, Pons N, Ghe C, Catapano F, Granata R, Ghigo
E; (2004) Ghrelin and des-acyl ghrelin both inhibit
isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a
growth hormone secretagogue receptor. Eur J Pharmacol 498:27-35.
[0169] 18. Park S, Dong X, Fisher T L, Dunn S, Omer A K, Weir G,
White M F; (2006) Exendin-4 uses Irs2 signaling to mediate
pancreatic beta cell growth and function. J Biol Chem
281:1159-1168. [0170] 19. Prado C L, Pugh-Bernard A E, Elghazi L,
Sosa-Pineda B, Sussel L; (2004) Ghrelin cells replace
insulin-producing beta cells in two mouse models of pancreas
development. Proc Natl Acad Sci USA 101:2924-2929. [0171] 20.
Santerre R F, Cook R A, Crisel R M, Sharp J D, Schmidt R J,
Williams D C, Wilson C P; (1981) Insulin synthesis in a clonal cell
line of simian virus 40-transformed hamster pancreatic beta cells.
Proc Natl Acad Sci USA 78:4339-4343. [0172] 21. Wajchenberg B L;
(2007) beta-cell failure in diabetes and preservation by clinical
treatment. Endocr Rev. 28:187-218. [0173] 22. Wierup N, Svensson H,
Mulder H, Sundler F; (2002) The ghrelin cell: a novel
developmentally regulated islet cell in the human pancreas. Regul
Pept 107:63-69. [0174] 23. Zhang J V, Ren P G, Avsian-Kretchmer O,
Luo C W, Rauch R, Klein C, Hsuch A J; (2005) Obestatin, a peptide
encoded by the ghrelin gene, opposes ghrelin's effects on food
intake. Science 310:996-999. [0175] 24. Irako T, Akamizu T, Hosoda
H, Iwakura H, Ariyasu H, Tojo K, Tajima N, Kangawa K; (2006)
Ghrelin prevents development of diabetes at adult age in
streptozotocin-treated newborn rats. Diabetologia 49:1264-1273.
[0176] 25. Portha B, Levacher C, Picon L, Rosselin G.; (1974)
Diabetogenic effect of streptozotocin in the rat during the
perinatal period. Diabetes 23:889-895. [0177] 26. Tourrel C, Bailbe
D, Meile M J, Kergoat M, Portha B; (2001) Glucagon-like peptide-1
and exendin-4 stimulate beta-cell neogenesis in
streptozotocin-treated newborn rats resulting in persistently
improved glucose homeostasis at adult age. Diabetes 50:1562-1570.
[0178] 27. Menahan L A; (1983) Age-related changes in lipid and
carbohydrate metabolism of the genetically obese mouse. Metabolism
32:172-178. [0179] 28. Hayashi T, Boyko E J, McNeely M J, Leonetti
D L, Kahn S E, Fujimoto W Y; (2008) Visceral Adiposity, not
Abdominal Subcutaneous Fat Area, Is Associated with an Increase in
Future Insulin Resistance in Japanese Americans. Diabetes May;
57(5):1269-75. Epub 2008 Feb. 25. [0180] 29. Hamdy O, Porramatikul
S, Al-Ozairi E; (2006) Metabolic obesity: the paradox between
visceral and subcutaneous fat. Curr Diabetes Rev 2:367-373.
Sequence CWU 1
1
28128PRTHomo sapiens 1Gly Ser Ser Phe Leu Ser Pro Glu His Gln Arg
Val Gln Gln Arg Lys1 5 10 15Glu Ser Lys Lys Pro Pro Ala Lys Leu Gln
Pro Arg 20 25214PRTHomo sapiens 2Gly Ser Ser Phe Leu Ser Pro Glu
His Gln Arg Val Gln Gln1 5 10318PRTHomo sapiens 3Gly Ser Ser Phe
Leu Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys1 5 10 15Glu
Ser45PRTHomo sapiens 4Gly Ser Ser Phe Leu1 5512PRTHomo sapiens 5Glu
Ser Lys Lys Pro Pro Ala Lys Leu Gln Pro Arg1 5 1068PRTHomo sapiens
6Ser Pro Glu His Gln Arg Val Gln1 576PRTHomo sapiens 7Gly His Gln
Arg Val Gln1 585PRTHomo Sapiens 8Gly His Gln Arg Val1 5913PRTHomo
sapiens 9Ser Pro Glu His Gln Arg Val Gln Gln Arg Lys Glu Ser1 5
10104PRTHomo sapiens 10Glu His Gln Arg1114PRTHomo sapiens 11His Gln
Arg Val1128PRTHomo sapiens 12Ser Pro Asp His Gln Arg Val Gln1
5138PRTHomo sapiens 13Ser Pro Glu His Gln Lys Val Gln1 5148PRTHomo
sapiens 14Gly Pro Glu His Gln Arg Val Gln1 5158PRTHomo sapiens
15Ala Pro Glu His Gln Arg Val Gln1 5168PRTHomo sapiens 16Ser Ala
Glu His Gln Arg Val Gln1 5178PRTHomo sapiens 17Ser Pro Ala His Gln
Arg Val Gln1 5188PRTHomo sapiens 18Ser Pro Glu Ala Gln Arg Val Gln1
5198PRTHomo sapiens 19Ser Pro Glu His Ala Arg Val Gln1 5208PRTHomo
sapiens 20Ser Pro Glu His Gln Ala Val Gln1 5218PRTHomo sapiens
21Ser Pro Glu His Gln Arg Ala Gln1 5228PRTHomo sapiens 22Ser Pro
Glu His Gln Arg Val Ala1 5238PRTHomo
sapiensMOD_RES(1)..(1)ACETYLATION 23Ser Pro Glu His Gln Arg Val
Gln1 5248PRTHomo sapiensMOD_RES(1)..(1)ACETYLATION 24Ser Pro Glu
His Gln Arg Val Gln1 5258PRTHomo sapiensMISC_FEATURE(1)..(8)Cyclic
25Ser Pro Glu His Gln Arg Val Gln1 5268PRTHomo
sapiensMISC_FEATURE(3)..(6)Cyclic 26Ser Pro Glu His Gln Lys Val
Gln1 5278PRTHomo sapiensMOD_RES(1)..(1)ACETYLATION 27Ser Pro Glu
His Gln Lys Val Gln1 5288PRTHomo sapiensMOD_RES(1)..(1)ACETYLATION
28Ser Pro Glu His Gln Lys Val Gln1 5
* * * * *