U.S. patent application number 17/022526 was filed with the patent office on 2021-01-14 for peptides for treatment of diabetes.
The applicant listed for this patent is Follicum AB. Invention is credited to Jan Alenfall, Pontus Duner, Anna Hultgardh Nilsson, Bjorn Walse.
Application Number | 20210009648 17/022526 |
Document ID | / |
Family ID | 1000005109606 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009648 |
Kind Code |
A1 |
Alenfall; Jan ; et
al. |
January 14, 2021 |
PEPTIDES FOR TREATMENT OF DIABETES
Abstract
The present disclosure concerns agents and their use in the
treatment of endocrine, nutritional and/or metabolic diseases in a
mammal. The disclosure furthermore concerns novel peptides.
Inventors: |
Alenfall; Jan; (Lomma,
SE) ; Duner; Pontus; (Dalby, SE) ; Nilsson;
Anna Hultgardh; (Genarp, SE) ; Walse; Bjorn;
(Lund, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Follicum AB |
Lund |
|
SE |
|
|
Family ID: |
1000005109606 |
Appl. No.: |
17/022526 |
Filed: |
September 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16666960 |
Oct 29, 2019 |
10815283 |
|
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17022526 |
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PCT/EP2018/061547 |
May 4, 2018 |
|
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16666960 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/164 20130101;
C07K 2319/40 20130101; C12N 9/1088 20130101; A61K 38/45 20130101;
A61K 38/1709 20130101; A61P 3/10 20180101; C07K 14/4705 20130101;
C07K 2319/00 20130101; C12Y 205/01018 20130101; C07K 14/31
20130101; C07K 14/001 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61P 3/10 20060101 A61P003/10; A61K 38/16 20060101
A61K038/16; A61K 38/17 20060101 A61K038/17; A61K 38/45 20060101
A61K038/45; C07K 14/00 20060101 C07K014/00; C07K 14/31 20060101
C07K014/31; C12N 9/10 20060101 C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2017 |
EP |
17169500.0 |
Claims
1. An agent comprising: a) a peptide or peptide analog comprising
or consisting of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID
NO: 136), KCLAECDSIELSYGIK (SEQ ID NO: 141), KPLAEDISIELSYGIK (SEQ
ID NO: 145), KPLAEIGDIELSYGIK (SEQ ID NO: 146), KPLAEIDSIELTYGIK
(SEQ ID NO: 149), KPLAEIDGIELSYGIK (SEQ ID NO: 150),
KPLAEIDGIELTYGIK (SEQ ID NO: 151), KPLAEIGSIELSYGIK (SEQ ID NO:
152), KGLAEIDSIELSYGIK (SEQ ID NO: 153) and KPLAGIDSIGLSYGIK (SEQ
ID NO: 154); CLAEIDSC (SEQ ID NO: 142), CFKPLAEIDSIECSYGIK (SEQ ID
NO: 143), KPLAEGDIELSYGIK (SEQ ID NO: 147), KPLAEIELSYGIK (SEQ ID
NO: 148), KCLAEIDSCELSYGIK (SEQ ID NO: 155), or CFKPLAEIDSIEC (SEQ
ID NO: 156); b) a polynucleotide encoding upon expression, the
peptide of a); c) a vector comprising the polynucleotide of b); or
d) a cell comprising the polynucleotide of b), or the vector of
c).
2. The agent according to claim 1, wherein the peptide or peptide
analog comprises an amino acid sequence of the general formula:
TABLE-US-00056 (SEQ ID NO: 162)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LSYGIK
wherein: X.sub.2 is C, P or G; X.sub.5 is E or G; X.sub.5 is C, I
or absent; X.sub.7 is D, G or absent; X.sub.8 is S, G or absent;
X.sub.10 is E or G.
3. The agent according to claim 1, wherein the agent comprises no
more than 85, such as no more than 80, such as no more than 75,
such as no more than 70, such as no more than 65, such as no more
than 60, such as nor more than 55, such as no more than 50, such as
no more than 55, such as no more than 40 amino acids, such as no
more than 35, such as no more than 30, such as no more than 28,
such as no more than 26, such as no more than 24, such as no more
than 22, such as no more than 20, such as no more than 19, such as
no more than 18, such as no more than 17, such as no more than 16,
such as no more than 15, such as no more than 14, such as no more
than 13, such as no more than 12, such as no more than 11, such as
no more than 10 amino acids.
4. The agent according to claim 1, wherein the agent comprises at
least 2 additional amino acids, such as at least 3, such as at
least 4, such as at least 5, such as at least 6, such as at least
7, such as at least 8, such as at least 9, such as at least 10,
such as at least 15 or such as at least 20 amino acids conjugated
to the N- or C-terminus of the peptide.
5. The agent according to claim 1, wherein the agent is
non-naturally occurring.
6. The agent according to claim 1, wherein the agent is conjugated
to a moiety.
7. The agent according to claim 1, wherein the agent is further
modified such as being glycosylated or by PEGylation, amidation,
esterification, acylation, acetylation and/or alkylation.
8. The agent according to claim 1, wherein the agent comprises or
consists of tandem repeats.
9. The agent according to claim 1, wherein the agent is fused to
another polypeptide.
10. The agent according to claim 9, wherein the said polypeptide is
selected from the group consisting of glutathione-S-transferase
(GST) and protein A.
11. The agent according to claim 1, wherein the agent is fused to a
tag.
12. The agent according to claim 1, wherein the agent is
cyclic.
13. The agent according to claim 1, wherein the peptide or peptide
analog comprises or consists of the amino acid sequence
KPLAEIDSIELSYGIK (SEQ ID NO: 136), or a variant or fragment
thereof.
14. The agent according to claim 1, wherein one or more amino acids
are conservatively substituted.
15. The agent according to claim 1, wherein the peptide or peptide
analog comprises or consists of one or more additional amino acids,
inserted at the N- and/or C-terminus and/or internally within the
sequence.
16. The agent according to claim 1, wherein the agent further
comprises a detectable moiety.
17. An agent comprising: c) a peptide or peptide analog comprising
or consisting of the amino acid sequence GDPNDGRGDSVVYGLR (SEQ ID
NO: 137), VDTYDGGISVVYGLR (SEQ ID NO: 138), and VDTYDGDGSVVYGLR
(SEQ ID NO: 139). VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG
(SEQ ID NO: 158), GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR
(SEQ ID NO: 160) VDVPEGDISLAYRLR (SEQ ID NO: 161),
V(beta-D)TYDGDISVVYGLR (SEQ ID NO:167), VDTY(beta-D)GDISVVYGLR (SEQ
ID NO: 168), or VDTYDG(beta-D)ISVVYGLR (SEQ ID NO:169); b) a
polynucleotide encoding upon expression, the peptide of a); c) a
vector comprising the polynucleotide of b); or d) a cell comprising
the polynucleotide of b), or the vector of c).
18. The agent according to claim 17, wherein the peptide or peptide
analog comprises or consists of the amino acid sequence
VDTYDGGISVVYGLR (SEQ ID NO: 138), or a variant or fragment
thereof.
19. A method of treating an endocrine disease and/or a metabolic
disease, the method comprising administering to a subject in need
thereof a therapeutically effective amount of an agent comprising:
a) a peptide or a peptide analog selected from the group consisting
of: (i) a peptide comprising or consisting of an amino acid
sequence of the general formula: TABLE-US-00057 (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
wherein: X.sub.2 is C, P or G; X.sub.5 is E or G; X.sub.6 is C, D
or I; X.sub.7 is D, I, S or G; X.sub.8 is S, D or G; X.sub.10 is E
or G; X.sub.12 is S or T; with the proviso that if X.sub.12 is T,
the peptide comprises no more than 25 amino acid residues; (ii) a
peptide comprising or consisting of an amino acid sequence of the
general formula: TABLE-US-00058 (SEQ ID NO: 68)
VDZ.sub.3Z4Z.sub.5GZ.sub.7Z.sub.8SZ.sub.10Z.sub.11YGLR
wherein: Z.sub.3 is T or V; Z.sub.4 is Y or P; Z.sub.5 is D or N;
Z.sub.7 is D or G; Z.sub.8 is I or G; Z.sub.10 is V or L; Z.sub.11
is V or A; wherein the peptide is selected from the group
consisting of VDTYDGDISVVYGLR (SEQ ID NO: 1), VDTYDGDISVVYGL (SEQ
ID NO: 3), VDTYDGDISVVYG (SEQ ID NO: 6), GDISVVYGLR (SEQ ID NO:
26), VDTYDGDIS (SEQ ID NO: 28), VDTYDGRGDSVVYGLR (SEQ ID NO: 67),
VDVPNGDISLAYGLR (SEQ ID NO: 69), VDVPNGDISLAYGL (SEQ ID NO: 71)
DVPNGDISLAYGLR(SEQ ID NO: 72), VDVPNGDISLAYG (SEQ ID NO: 74),
PNGDISLAYGLR (SEQ ID NO: 81), VDVPNGDISLA (SEQ ID NO: 83),
GDISLAYGLR( (iii) a peptide comprising or consists of an amino acid
sequence selected from the group consisting of KCLAECDSIELSYGIK
(SEQ ID NO: 141), CLAEIDSC (SEQ ID NO: 142), CFKPLAEIDSIECSYGIK
(SEQ ID NO: 143), KPLAEGDIELSYGIK (SEQ ID NO: 147), KPLAEIELSYGIK
(SEQ ID NO: 148), KCLAEIDSCELSYGIK (SEQ ID NO: 155) or
CFKPLAEIDSIEC (SEQ ID NO: 156); b) a polynucleotide encoding upon
expression, the peptide of a); c) a vector comprising the
polynucleotide of b); or d) a cell comprising the polynucleotide of
b), or the vector of c).
20. The method according claim 19, wherein said agent comprises a
second or further active ingredient.
21. The method according to claim 20, wherein the second or further
active ingredient is selected from the group consisting of insulin,
glucagon-like peptide-1 (GLP-1), sulfonylurea, a dipeptidyl
peptidase-4 (DPP4) inhibitor, an alpha-glucosidase inhibitor, a
thiazolidinedione, a meglitidine and a sodium-glucose
cotransporter-2 (SGLT2) inhibitor.
22. The method according to claim 19, wherein the endocrine disease
and/or metabolic disease are selected from the group consisting of
diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes
mellitus, malnutrition-related diabetes mellitus, disorders of
glucose regulation and pancreatic internal secretion, insulin
resistance syndrome, impaired glucose tolerance, hyperglycemia,
hyperinsulinemia, and any combinations thereof.
23. The method according to claim 19, wherein the endocrine disease
and/or metabolic disease are selected from the group consisting of
diabetes mellitus, disorders of the thyroid gland, disorders of
glucose regulation and pancreatic internal secretion, disorders of
endocrine glands, malnutrition, nutritional deficiencies, obesity,
hyperalimentation, and metabolic disorders.
24. The method according to claim 19, wherein the endocrine disease
and/or a metabolic disease is diabetes mellitus selected from the
group consisting of type 1 diabetes mellitus, type 2 diabetes
mellitus, malnutrition-related diabetes mellitus, specified
diabetes mellitus, and unspecified diabetes mellitus.
25. The method according to claim 19, wherein the endocrine disease
and/or a metabolic disease is a disorder of glucose regulation and
pancreatic internal secretion selected from the group consisting of
nondiabetic hypoglycaemic coma and disorders of pancreatic internal
secretion.
26. The method according to claim 19, wherein the endocrine disease
and/or a metabolic disease is a disorder of obesity and
hyperalimentation selected from the group consisting of localized
adiposity, hyperalimentation, and sequelae of
hyperalimentation.
27. A method for delaying onset of diabetes and/or a diabetes
associated disorder or disease, the method comprising administering
a therapeutically effective amount of an agent according to claim
1, to an individual in need thereof.
28. The method according to claim 27, wherein insulin secretion is
increased.
29. The method according to claim 27, wherein cellular uptake of
glucose is increased.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/666,960, filed Oct. 29, 2019, which is a
continuation of PCT Application No: PCT/EP2018/061547, filed May 4,
2018, designating the United States of America, which claims
priority to European Patent Application No: 17169500.0, filed May
4, 2017. The entire content of each application is incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present disclosure relates to peptides useful for
treatment of diabetes and associated disorders.
BACKGROUND
[0003] The peptide hormone insulin, which is produced by
.beta.-cells in the islets of Langerhans in the pancreas, is
released in response to increasing blood glucose levels. Thus,
glucose is removed from the blood by insulin dependent stimulation
of glucose transporters located in the cell membranes of the target
tissue, e.g. adipose tissue, skeletal muscle and liver. Insulin
exerts its biological effects by binding to and activating the
membrane-bound insulin receptor (IR), thereby initiating a cascade
of intracellular signalling events, which regulate multiple
biological processes such as glucose and lipid metabolism.
[0004] Currently, the treatment of diabetes, both type 1 and type 2
diabetes, relies primarily on insulin treatment. A complement to
insulin treatment is long-acting glucagon-like peptide-1 (GLP-1)
receptor agonists, i.e. derivatives that act on the same receptor
as GLP-1. GLP-1 is a metabolic hormone that stimulates insulin
secretion. Besides increasing insulin secretion from the pancreas
in a glucose-dependent manner, GLP-1 is known to increase
insulin-sensitivity in both .alpha.- and .beta.-cells; to increase
.beta.-cell mass and insulin expression, post-translational
modification, and secretion; and to decrease glucagon secretion
from the pancreas. Other medications used complementary to insulin
treatment for the purpose of lowering plasma glucose levels include
DPP-IV inhibitors, Metformin, SGLT-2 inhibitors and
sulfonylurea.
[0005] Certain drawbacks are associated with long term use of
insulin, such as weight gain and increased risks of cancer and
hypoglycaemia. Thus, there is a growing demand in the field for
novel non-insulin compounds capable of, not only treating diabetes,
by addressing insulin resistance and hyperglycemia, but also
reducing associated and consequential complications.
[0006] Identification of novel compounds that can restore glucose
metabolism and treat diabetes and related disorders is thus highly
relevant. Multiple approaches can be contemplated, albeit none of
which are obvious to the person of skill in the art.
SUMMARY
[0007] The present inventors have found peptides which stimulate
.beta.-cell proliferation, have the ability to rescue .beta.-cell
from apoptosis induced by glucotoxic conditions, and stimulate
insulin secretion from rat INS-1 .beta.-cells as well as isolated
mouse pancreatic islets. Furthermore, the present inventors found
that in a glucose tolerance test, the peptides lowered plasma
glucose levels in vivo and delayed onset of diabetes disease in BB
lyp/lyp rats, a model for type 1 diabetes. Hence, the peptides of
the present disclosure are suitable for use in the treatment of
endocrine, nutritional and metabolic diseases and disorders.
[0008] In one aspect, the present disclosure relates to an agent
comprising or consisting of: [0009] a) a peptide or peptide analog,
wherein the peptide or peptide analog comprises an amino acid
sequence of the general formula:
TABLE-US-00001 [0009] (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0010] wherein: [0011] X.sub.2 is C, P or G; [0012] X.sub.5 is E or
G; [0013] X.sub.6 is C, D or I; [0014] X.sub.7 is D, I, S or G;
[0015] X.sub.8 is S, D or G; [0016] X.sub.12 is S or T; [0017] with
the proviso that if X.sub.12 is T then the peptide comprises no
more than 25 amino acids; and [0018] with the proviso that if
X.sub.2 is P, X.sub.5 is E, X.sub.5 is I, X.sub.7 is D, X.sub.8 is
S, X.sub.10 is E and X.sub.12 is S, the peptide comprises no more
than 85 amino acid residues. [0019] or a biologically active
fragment and/or variant thereof, wherein said biologically active
fragment and/or variant is selected from the group consisting of
CLAEIDSC (SEQ ID NO: 142), CFKPLAEIDSIECSYGIK (SEQ ID NO: 143),
KPLAEGDIELSYGIK (SEQ ID NO: 147), KPLAEIELSYGIK (SEQ ID NO: 148),
KCLAEIDSCELSYGIK (SEQ ID NO: 155), and CFKPLAEIDSIEC (SEQ ID NO:
156); [0020] b) a polynucleotide encoding upon expression, the
peptide of a); [0021] c) a vector comprising the polynucleotide of
b); and [0022] d) a cell comprising the polynucleotide of b), or
the vector of c).
[0023] In one aspect, the present disclosure relates to an agent
comprising: [0024] a) a peptide or peptide analog comprising or
consisting of the amino acid sequence GDPNDGRGDSVVYGLR (SEQ ID NO:
137), VDTYDGGISVVYGLR (SEQ ID NO: 138), and VDTYDGDGSVVYGLR (SEQ ID
NO: 139). VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG (SEQ ID
NO: 158), GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR (SEQ ID
NO: 160) VDVPEGDISLAYRLR (SEQ ID NO: 161), V(beta-D)TYDGDISVVYGLR
(SEQ ID NO:167), VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 168),
VDTYDG(beta-D)ISVVYGLR (SEQ ID NO:169); [0025] b) a polynucleotide
encoding upon expression, the peptide of a); [0026] c) a vector
comprising the polynucleotide of b); and [0027] d) a cell
comprising the polynucleotide of b), or the vector of c).
[0028] In one aspect, the present disclosure relates to a
composition comprising the agent described herein above.
[0029] In one aspect, the present disclosure relates to an agent or
a composition comprising said agent, for use as a medicament.
[0030] In one aspect, the present disclosure relates to an agent
comprising: [0031] a) (i) a peptide or a peptide analog, wherein
the peptide or the peptide analog comprises or consists of an amino
acid sequence of the general formula:
TABLE-US-00002 [0031] (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0032] wherein: [0033] X.sub.2 is C, P or G; [0034] X.sub.5 is E or
G; [0035] X.sub.6 is C, D or I; [0036] X.sub.7 is D, I, S or G;
[0037] X.sub.8 is S, D or G; [0038] X.sub.10 is E or G; [0039]
X.sub.12 is S or T; [0040] with the proviso that if X.sub.12 is T,
the peptide comprises no more than 25 amino acid residues; [0041]
(ii) a peptide, wherein the peptide comprises an amino acid
sequence of the general formula:
VDZ.sub.3Z.sub.4Z.sub.5GZ.sub.7Z.sub.8SZ.sub.10Z.sub.11YGLR (SEQ ID
NO: 68) [0042] wherein: [0043] Z.sub.3 is T or V; [0044] Z.sub.4 is
Y or P; [0045] Z.sub.5 is D or N; [0046] Z.sub.7 is D or G; [0047]
Z.sub.8 is I or G; [0048] Z.sub.10 is V or L; [0049] Z.sub.11 is V
or A; [0050] (iii) a peptide, wherein the peptide comprises or
consists of an amino acid sequence selected from the group
consisting of KCLAECDSIELSYGIK (SEQ ID NO: 141), CLAEIDSC (SEQ ID
NO: 142), CFKPLAEIDSIECSYGIK (SEQ ID NO: 143), KPLAEIELSYGIK (SEQ
ID NO: 148), KCLAEIDSCELSYGIK (SEQ ID NO: 155) and CFKPLAEIDSIEC
(SEQ ID NO: 156); [0051] b) a polynucleotide encoding upon
expression, the peptide of a); [0052] c) a vector comprising the
polynucleotide of b); and [0053] d) a cell comprising the
polynucleotide of b), or the vector of c). [0054] for use in the
treatment of an endocrine disease, a nutritional disease and/or a
metabolic disease in a mammal.
[0055] In one aspect, the present disclosure concerns a method for
treating an endocrine disease a nutritional disease and/or a
metabolic disease, the method comprising administering a
therapeutically effective amount of an agent described herein, to
an individual in need thereof.
[0056] In one aspect, the present disclosure concerns the use of an
agent as described herein for the manufacture of a medicament for
the treatment of an endocrine disease a nutritional disease and/or
a metabolic disease.
[0057] In one aspect, the present disclosure concerns a method for
delaying onset of diabetes, the method comprising administering a
therapeutically effective amount of an agent described herein, to
an individual in need thereof.
[0058] In one aspect, the present disclosure concerns a method for
decreasing blood glucose levels, the method comprising
administering a therapeutically effective amount of an agent
described herein, to an individual in need thereof.
[0059] In one aspect, the present disclosure concerns a method,
e.g. an in vitro method, for improving beta cell morphology, the
method comprising administering a therapeutically effective amount
of an agent described herein, to an individual in need thereof.
[0060] In one aspect, the present disclosure concerns a method for
improving beta cell viability, the method comprising administering
a therapeutically effective amount of an agent described herein, to
an individual in need thereof.
[0061] In one aspect, the present disclosure concerns the use of
agent described herein for the preparation of a diagnostic
composition for the diagnosis of a disease, disorder or damage of
the pancreas in an individual.
DESCRIPTION OF DRAWINGS
[0062] FIG. 1. FOL-005 and FOL-014 induced proliferation of
.beta.-cells
[0063] Addition of increasing concentrations of FOL-005 in solution
induced increasing proliferation of INS-1 cells after 48 hours
(FIG. 1A). Wells coated with FOL-005 and blocked with Bovine Serum
Albumin (BSA) induced more proliferation of .beta.-cells compared
to only BSA coated control (ctrl) wells (FIG. 1B). Wells pre-coated
with FOL-014 and blocked with BSA induced more proliferation
compared to only BSA coated wells (FIG. 10). Data is presented as
counts per minute (CPM) relative unstimulated control (ctrl) cells.
Mean.+-.SD are presented for 10-12 different observations in each
group.
[0064] FIG. 2. FOL-005 protected .beta.-cells against
glucotoxicity
[0065] INS-1 cells incubated during 48 h in 20 mM glucose displayed
more apoptotic cells (Annexin V positive) compared to cells
incubated at 5 mM glucose. Addition of FOL-005 to cells incubated
with 20 mM glucose reduced the level of apoptotic cells compared to
20 mM glucose alone (FIG. 2A). Apoptosis measured by caspase-3
activity was increased in INS-1 cells at 20 mM compared to 5 mM
glucose. Addition of FOL-005 diminished the rate of
glucotoxicity-induced caspse-3 activity (FIG. 2B). Mean.+-.SD are
presented for 4-8 different observations in each group.
[0066] FIG. 3. Insulin secretion was increased from islets and
.beta.-cells following FOL-005 stimulation
[0067] FOL-005 stimulated .beta.-cell and islet insulin secretion.
Insulin release from INS-1 cells was increased after FOL-005 (6
.mu.M) stimulation in-non glucose containing media compared to
non-stimulated control (ctrl) and to a scrambled control peptide
(FOL-015) (FIG. 3A). FOL-005 stimulated insulin release from INS-1
at both low (5 mM) and high (20 mM) glucose (FIG. 3B). Isolated
mouse pancreatic islets stimulated with FOL-005 (6 .mu.M) or GLP-1
(100 nM) secreted more insulin compared to unstimulated control
islets (FIG. 3C). Mean.+-.SD are presented for 5-6 different
observations in each group.
[0068] FIG. 4. Insulin secretion was increased from islets and
.beta.-cells following FOL-014 stimulation
[0069] FOL-014 stimulated insulin secretion from .beta.-cells and
pancreatic islets. INS-1 cells stimulated with FOL-014 (6 .mu.M)
secreted more insulin compared to unstimulated control cells (FIG.
4A). Isolated mouse pancreatic islets stimulated with FOL-014 (6
.mu.M) secreted more insulin compared to control islets (FIG. 4B).
Addition of GLP-1 (100 nM) or FOL-014 (0.6 .mu.M) had no effect on
insulin secretion. Mean.+-.SD are presented for 5-6 different
observations in each group.
[0070] FIG. 5. The effect of FOL-014 on insulin secretion was dose
dependent. Stimulation of INS-1 cells by increasing doses of
FOL-014 resulted in a significant increase in insulin secretion for
all concentrations tested. The insulin secretion increased in a
linear fashion in the presence of FOL-014 ranging from 0.6 nM to 60
nM. Higher concentrations appeared to result in a less pronounced
effect on insulin secretion. Furthermore, FOL-014 induced insulin
secretion was comparable to the effect of 100 nM GLP-1. Bars
represent mean values and standard error of the mean (SEM).
[0071] FIG. 6. The effect on insulin secretion of FOL-014 was
glucose concentration dependent. The insulin secretion from
untreated or FOL-014 exposed INS-1 cells was measured in the
presence of increasing glucose concentrations. At glucose levels
5.5 mM or higher, the insulin secretion was significantly higher in
the FOL-014 treated cells, as compared to untreated control cells.
Bars represent mean values and standard error of the mean
(SEM).
[0072] FIG. 7. FOL-005 and FOL-014 dosed together with native GLP-1
elicited an additive effect on insulin secretion. The insulin
release from INS-1 cells was measured following combination
treatment of GLP-1 together with FOL-005 and FOL-014 (all three
peptides in a concentration of 100 nM), respectively and compared
with the effect of each peptide alone. The combination of GLP-1 and
FOL-014 significantly increased the insulin secretion as compared
with each peptide alone. An increase was also observed for the
combination of FOL-005 and GLP-1. Bars represent mean values and
standard error of the mean (SEM).
[0073] FIG. 8. FOL-014 affected insulin and glucagon secretion in
pancreatic islets. Two different concentrations of FOL-014 were
tested and compared with the effect of 100 nM GLP-1 on isolated
mouse islets in low (2.8 mM) (A, C) and high (16.7 mM) (B, D)
concentrations of glucose. In the low glucose samples, the presence
of FOL-014 did not increase insulin secretion, but reduced glucagon
secretion as compared with control and GLP-1. In the high glucose
samples, 600 nM FOL-014 and GLP-1, but not 6 .mu.M FOL-014,
significantly increased insulin secretion (B), and GLP-1 as well as
both concentrations of FOL-014 efficiently reduced glucagon
secretion (D). Bars represent mean values and standard error of the
mean (SEM).
[0074] FIG. 9. FOL-014 lowered plasma glucose levels in vivo
following a glucose injection. An intraperitoneal glucose tolerance
test (IPGTT) was performed on wild type C57bl/6 mice. FOL-014 dosed
at 200 nmol/kg significantly lowered the plasma glucose levels as
compared to the control at 15 minutes, 30 minutes and 45 minutes
(P=0.0027). At the 30 nmol/kg dose, FOL-014 lowered the glucose
levels with a significant effect at 45 minutes after the glucose
injection. The dotted line corresponds to mean non-fasting glucose
levels. Data represents mean values and standard error of the mean
(SEM). Statistical analysis was performed using student's
t-test.
[0075] FIG. 10. FOL-014 delayed the onset of type-1 diabetes in BB
lyp/lyp rats. BB lyp/lyp rats treated with FOL-014 showed a
significant delay in the onset of diabetes defined as plasma
glucose<11.1 mmol/l. Age of onset of diabetes for each rat was
depicted in (A) with a significant difference between untreated and
treated groups. The percentage of animals developing type 1
diabetes each day was depicted in (B) with a significant difference
between groups. Error bars in (A) represent standard error of the
mean (SEM).
[0076] FIG. 11. The effect on insulin secretion of peptide
analogues derived from FOL-005 or FOL-014. Novel peptide analogues
were tested in two separate INS-1 cell lines (A and B) for their
ability to induce insulin secretion under high glucose (16.7 mM)
conditions. The effect was compared with that of native GLP-1,
FOL-005 and FOL-014 as well as the effect of high glucose alone.
Analogues inducing insulin release below the average of the high
glucose control were considered non-functional (not shown). The
level of insulin secretion is depicted in black, filled bars for
the novel analogues, and in contrasting patterns for the
comparators. Bars represent mean values and standard error of the
mean (SEM).
[0077] FIG. 12. FOL-005 and FOL-014 displayed specific distribution
patterns following injection in mouse. Following subcutaneous
administration of .sup.3H-FOL-005, the highest overall levels of
radioactivity were present in pancreas and at the injection site, 1
hour (A) and 2 hours (B) after injection. Accumulation of the
.sup.3H-FOL-005 is also visible in liver, kidney, salivary glands.
Using Pearl Trilogy Small Animal Imaging System in vivo
bio-distribution and tissue localization of Cy7.5 labelled FOL-005
(C) and FOL-014 (D) in NMRI nude mice via subcutaneous injection
was investigated. Following initial control imaging, a dose of 10
nmol per mouse was administered and live imaging was performed at 5
min, 20 min, 50 min, 60 min, 2 hrs, 4 hrs, 6 hrs, 24 hrs and 48
hrs. High accumulation of both peptides was evident in the
pancreatic region as well as at the injection site.
DETAILED DESCRIPTION
[0078] The disclosure is as defined in the claims.
[0079] In one aspect, the present disclosure concerns a peptide or
a peptide analog comprising an amino acid sequence of the general
formula:
TABLE-US-00003 a) (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0080] wherein: [0081] X.sub.2 is C, P or G; [0082] X.sub.5 is E or
G; [0083] X.sub.6 is C, D or I; [0084] X.sub.7 is D, I, S or G;
[0085] X.sub.8 is S, D or G; [0086] X.sub.10 is E or G; [0087]
X.sub.12 is S or T; with the proviso that if X.sub.12 is T, the
peptide comprises no more than 25 amino acids; and with the proviso
that if X.sub.2 is P, X.sub.5 is E, X.sub.6 is I, X.sub.7 is D,
X.sub.8 is S, X.sub.10 is E and X.sub.12 is S, the peptide
comprises no more than 85 amino acid residues;
[0088] b) a polynucleotide encoding upon expression, the peptide of
a);
[0089] c) a vector comprising the polynucleotide of b); and
[0090] d) a cell comprising the polynucleotide of b), or the vector
of c).
[0091] In one embodiment, the present disclosure concerns a peptide
or a peptide analog comprising an amino acid sequence of the
general formula:
TABLE-US-00004 (SEQ ID NO: 162)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LSYGIK
wherein: [0092] X.sub.2 is C, P or G; [0093] X.sub.5 is E or G;
[0094] X.sub.6 is C, I or absent; [0095] X.sub.7 is D, G or absent;
[0096] X.sub.8 is S, G or absent; [0097] X.sub.10 is E or G; [0098]
wherein absent means that the amino acid X.sub.5 is coupled to the
amino acid X.sub.10
[0099] In one embodiment, the present disclosure concerns a peptide
comprising an amino acid sequence of the general formula:
TABLE-US-00005 (SEQ ID NO: 163)
KX.sub.2LAX.sub.5IX.sub.10LSYGIK
wherein: [0100] X.sub.2 is C, P or G; [0101] X.sub.5 is E or G;
[0102] X.sub.10 is E or G.
[0103] In one embodiment, the present disclosure concerns an agent
comprising: [0104] a) a peptide, wherein the peptide is selected
from the group consisting of: [0105] i) a peptide comprising or
consisting of the amino acid sequence of SEQ ID NO: 136, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
and 156; [0106] ii) a biologically active sequence variant of any
one of the peptides of i), wherein any one amino acid has been
altered for another proteinogenic or non-proteinogenic amino acid,
with the proviso that no more than five amino acids are so altered;
[0107] iii) a biologically active fragment of the peptide of any
one of i) or ii), wherein the fragment comprises at least 10
consecutive amino acids of any one of i) or ii); [0108] b) a
polynucleotide encoding upon expression, the peptide of a); [0109]
c) a vector comprising the polynucleotide of b); and [0110] d) a
cell comprising the polynucleotide of b), or the vector of c).
[0111] In one embodiment, the present disclosure concerns an agent
comprising: [0112] a) a peptide, wherein the peptide comprises or
consists of an amino acid sequence selected from the group
consisting of GDPNDGRGDSVVYGLR (SEQ ID NO: 137), VDTYDGGISVVYGLR
(SEQ ID NO: 138), and VDTYDGDGSVVYGLR (SEQ ID NO: 139).
VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG (SEQ ID NO: 158),
GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR (SEQ ID NO: 160)
VDVPEGDISLAYRLR (SEQ ID NO: 161); [0113] b) a polynucleotide
encoding upon expression, the peptide of a); [0114] c) a vector
comprising the polynucleotide of b); and [0115] d) a cell
comprising the polynucleotide of b), or the vector of c).
[0116] In one embodiment, the present disclosure concerns a peptide
comprising an amino acid sequence of the general formula:
TABLE-US-00006 (SEQ ID NO: 164) VDVPZ.sub.5GDISLAYZ.sub.13LR
[0117] wherein: [0118] Z.sub.5 is E or N; [0119] Z.sub.13 is R or
G.
[0120] In one embodiment, the present disclosure concerns a peptide
comprising an amino acid sequence of the general formula:
TABLE-US-00007 (SEQ ID NO: 165) VDTYDGZ.sub.7Z.sub.8SVVYGLR
[0121] wherein: [0122] Z.sub.7 is D or G; [0123] Z.sub.8 is I or
G.
[0124] In one embodiment, the present disclosure concerns a peptide
comprising an amino acid sequence of the general formula:
TABLE-US-00008 (SEQ ID NO: 166)
GDPNZ.sub.5Z.sub.6Z.sub.7Z.sub.8Z.sub.9SVVYGLR
[0125] wherein: [0126] Z.sub.5 is D or G; [0127] Z.sub.6 is D or G
[0128] Z.sub.7 is I or R; [0129] Z.sub.8 is G or absent; [0130]
Z.sub.9 is D or absent.
[0131] The term `absent` as used herein, e.g. "X.sub.6 is C, I or
absent" is to be understood as that the amino acid residues
directly adjacent to the absent amino acid are directly linked to
each other by a conventional amide bond.
[0132] The term "peptide analog" described herein refers to a
peptide comprising or consisting of a non-naturally occurring
peptide.
[0133] The term `amino acid` as used herein includes the standard
twenty genetically-encoded amino acids and their corresponding
stereoisomers in the D' form (as compared to the natural 1' form),
omega-amino acids and other naturally-occurring amino acids,
unconventional amino acids (e.g., .alpha.,.alpha.-disubstituted
amino acids, N-alkyl amino acids, etc.) and chemically derivatized
amino acids (see below).
[0134] When an amino acid is being specifically enumerated, such as
`alanine` or `Ala` or `A`, the term refers to both L-alanine and
D-alanine unless explicitly stated otherwise. Other unconventional
amino acids may also be suitable components for peptides of the
present disclosure, as long as the desired functional property is
retained by the peptide. For the peptides shown, each encoded amino
acid residue, where appropriate, is represented by a single letter
designation, corresponding to the trivial name of the conventional
amino acid.
[0135] Chemical derivatives of one or more amino acids may be
achieved by reaction with a functional side group. Such derivatives
include, for example, those molecules in which free amino groups
have been derivatized to form amine hydrochlorides, p-toluene
sulphonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters and hydrazides. Free hydroxyl groups may be derivatized
to form O-acyl or O-alkyl derivatives. Also included as chemical
derivatives are those peptides which contain naturally occurring
amino acid derivatives of the twenty standard amino acids. For
example: 4-hydroxyproline may be substituted for proline;
5-hydroxylysine may be substituted for lysine; 3-methylhistidine
may be substituted for histidine; homoserine may be substituted for
serine and ornithine for lysine. Derivatives also include peptides
containing one or more additions or deletions as long as the
requisite activity is maintained. Other included modifications are
amidation, amino terminal acylation (e.g. acetylation or
thioglycolic acid amidation), terminal carboxylamidation (e.g. with
ammonia or methylamine), and the like terminal modifications.
[0136] Some of the peptides of the disclosure shares amino acid
sequence similarity with a sub-region of naturally occurring
osteopontin proteins. In some embodiments, said peptide may be
regarded as an active fragment of a naturally-occurring osteopontin
protein or a variant of such as a fragment.
[0137] Some of the peptides of the disclosure shares amino acid
sequence similarity with a sub-region of naturally occurring
tenascin proteins. In some embodiments, said peptide may be
regarded as an active fragment of a naturally-occurring tenascin
protein or a variant of such as a fragment.
[0138] By "fragment", at least 5 contiguous amino acids of the
amino acid sequence are included, for example at least 6, 7, 8, 9,
10, 11, 12, 13, 14 or 15 contiguous amino acids of the amino acid
sequence. Thus, the fragment may be 15 or fewer amino acids in
length, for example 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids
in length
[0139] In one embodiment, said peptide is of no more than no more
than 85, such as no more than 80, such as no more than 75, such as
no more than 70, such as no more than 65, such as no more than 60,
such as nor more than 55, such as no more than 50, such as no more
than 55, such as no more than 40 amino acids, such as no more than
35, such as no more than 30, such as no more than 28, such as no
more than 26, such as no more than 24, such as no more than 22,
such as no more than 20, such as no more than 19, such as no more
than 18, such as no more than 17, such as no more than 16, such as
no more than 15, such as no more than 14, such as no more than 13,
such as no more than 12, such as no more than 11, such as no more
than 10 amino acids in length.
[0140] In another embodiment, said peptide is between 5 and 30
amino acids in length, such as between 5 and 20, such as between 8
and 20, such as between 8 and 16, such as between 10 and 15 amino
acids in length.
[0141] In yet another embodiment, said fragment comprises 15 or
fewer amino acids in length, such as fewer than 14 amino acids,
such as fewer than 13 amino acids, such as fewer than 12 amino
acids, such as fewer than 11 amino acids, such as fewer than 10
amino acids, such as fewer than 9 amino acids, such as fewer than 8
amino acids, such as fewer than 7 amino acids, such as fewer than 6
amino acids, such as fewer than 5 amino acids in length.
[0142] The term "variant" refers to a peptide that does not share
100% amino acid sequence identity with the parent peptide, i.e. one
or more amino acids must be mutated.
[0143] "Mutated" refers to altering an amino acid at a specified
position in the parent peptide. For example, an amino acid at a
specified position may be deleted, altered, substituted or may be
the site of an insertion/addition of one or more amino acids. It
will be appreciated by persons skilled in the art that the
substitutions may be conservative or non-conservative.
[0144] In one embodiment, said peptide variant comprises or
consists of a sequence wherein no more than five amino acids are
altered for another proteinogenic or non-proteinogenic amino acid,
such as no more than 4 amino acids, such as no more than 3 amino
acids, such as no more than 2 amino acids, such as no more than 1
amino acid is altered. In one embodiment, one or more amino acids
are conservatively substituted. "Conservatively substituted" refers
to a substitution of one amino acid with another with similar
properties (size, hydrophobicity, etc), such that the function of
the peptide is not significantly altered. Thus, by "conservative
substitutions" is intended combinations such as Gly, Ala; Val, Ile,
Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
[0145] In another embodiment, said peptide comprises or consists of
one or more additional amino acids, inserted at the N- and/or
C-terminus and/or internally within the sequence. In one
embodiment, at least 2 additional amino acids, such as at least 3,
such as at least 4, such as at least 5, such as at least 6, such as
at least 7, such as at least 8, such as at least 9, such as at
least 10, such as at least 15 or such as at least 20 additional
amino acids are inserted. The additional amino acids may be the
amino acids from the corresponding positions of the wildtype human
osteopontin (SEQ ID NO: 66) or from the corresponding positions of
the wildtype murine osteopontin (SEQ ID NO: 134). The term
"corresponding positions" of the wildtype osteopontin we mean that
the additional amino acids are the same as those present in the
equivalent position in the above wildtype osteopontin (if one
imagines that the amino acid sequence of SEQ ID NO:1 replaces the
sequence underlined in italics in SEQ ID NO:66 In another
embodiment, the peptide is selected from the group consisting of
SEQ ID NO: 1, 136, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,
128, 129, 130, 131, 132, 133, 135, 137, 138, 139, 141, 142, 143,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 167, 168 and 169;
[0146] i. 15-Amino Acid Peptides:
TABLE-US-00009 SEQ ID NO: 1 VDTYDGDISVVYGLR SEQ ID NO: 2
VDTYDGDISVVYGLS
[0147] ii. 14-Amino Acid Peptides:
TABLE-US-00010 SEQ ID NO: 3 VDTYDGDISVVYGL SEQ ID NO: 4
DTYDGDISVVYGLR SEQ ID NO: 5 TYDGDISVVYGLRS
[0148] iii. 13-Amino Acid Peptides:
TABLE-US-00011 SEQ ID NO: 6 VDTYDGDISVVYG SEQ ID NO: 7
DTYDGDISVVYGL SEQ ID NO: 8 TYDGDISVVYGLR SEQ ID NO: 9
YDGDISVVYGLRS
[0149] iv. 12-Amino Acid Peptides:
TABLE-US-00012 SEQ ID NO: 10 VDTYDGDISVVY SEQ ID NO: 11
DTYDGDISVVYG SEQ ID NO: 12 TYDGDISVVYGL SEQ ID NO: 13 YDGDISVVYGLR
SEQ ID NO: 14 DGDISVVYGLRS
[0150] v. 11-Amino Acid Peptides:
TABLE-US-00013 SEQ ID NO: 15 VDTYDGDISVV SEQ ID NO: 16 DTYDGDISVVY
SEQ ID NO: 17 TYDGDISVVYG SEQ ID NO: 18 YDGDISVVYGL SEQ ID NO: 19
DGDISVVYGLR SEQ ID NO: 20 GDISVVYGLRS
[0151] vi. 10-Amino Acid Peptides:
TABLE-US-00014 SEQ ID NO: 21 VDTYDGDISV SEQ ID NO: 22 DTYDGDISVV
SEQ ID NO: 23 TYDGDISVVY SEQ ID NO: 24 YDGDISVVYG SEQ ID NO: 25
DGDISVVYGL SEQ ID NO: 26 GDISVVYGLR SEQ ID NO: 27 DISVVYGLRS
[0152] vii. 9-Amino Acid Peptides:
TABLE-US-00015 SEQ ID NO: 28 VDTYDGDIS SEQ ID NO: 29 DTYDGDISV SEQ
ID NO: 30 TYDGDISVV SEQ ID NO: 31 YDGDISVVY SEQ ID NO: 32 DGDISVVYG
SEQ ID NO: 33 GDISVVYGL SEQ ID NO: 34 DISDVVYGLR SEQ ID NO: 35
ISDVVYGLRS
[0153] viii. 8-Amino Acid Peptides:
TABLE-US-00016 SEQ ID NO: 36 VDTYDGDI SEQ ID NO: 37 DTYDGDIS SEQ ID
NO: 38 TYDGDISV SEQ ID NO: 39 YDGDISVV SEQ ID NO: 40 DGDISVVY SEQ
ID NO: 41 GDISVVYG SEQ ID NO: 42 DISVVYGL SEQ ID NO: 43
ISVVYGLR
[0154] ix. 7-Amino Acid Peptides:
TABLE-US-00017 SEQ ID NO: 44 VDTYDGD SEQ ID NO: 45 DTYDGDI SEQ ID
NO: 46 TYDGDIS SEQ ID NO: 47 YDGDISV SEQ ID NO: 48 DGDISVV SEQ ID
NO: 49 GDISVVY SEQ ID NO: 50 DISVVYG SEQ ID NO: 51 ISVVYGL
[0155] x. 6-Amino Acid Peptides:
TABLE-US-00018 SEQ ID NO: 52 DTYDGD SEQ ID NO: 53 TYDGDI SEQ ID NO:
54 YDGDIS SEQ ID NO: 55 DGDISV SEQ ID NO: 56 GDISVV SEQ ID NO: 57
DISVVY SEQ ID NO: 58 ISVVYG
[0156] xi. 5-Amino Acid Peptides:
TABLE-US-00019 SEQ ID NO: 59 TYDGD SEQ ID NO: 60 YDGDI SEQ ID NO:
61 DGDIS SEQ ID NO: 62 GDISV SEQ ID NO: 63 DISVV SEQ ID NO: 64
ISVVY SEQ ID NO: 65 SVVYG
[0157] xii. 16-Amino Acid Peptide:
TABLE-US-00020 SEQ ID NO: 67 VDTYDGRGDSVVYGLR
[0158] xiii. 15-Amino Acid Peptides:
TABLE-US-00021 SEQ ID NO: 69 VDVPNGDISLAYGLR SEQ ID NO: 70
DVPNGDISLAYGLRS
[0159] xiv. 14-Amino Acid Peptides:
TABLE-US-00022 SEQ ID NO: 71 VDVPNGDISLAYGL SEQ ID NO: 72
DVPNGDISLAYGLR SEQ ID NO: 73 VPNGDISLAYGLRS
[0160] xv. 13-Amino Acid Peptides:
TABLE-US-00023 SEQ ID NO: 74 VDVPNGDISLAYG SEQ ID NO: 75
DVPNGDISLAYGL SEQ ID NO: 76 VPNGDISLAYGLR SEQ ID NO: 77
PNGDISLAYGLRS
[0161] xvi. 12-Amino Acid Peptides:
TABLE-US-00024 SEQ ID NO: 78 VDVPNGDISLAY SEQ ID NO: 79
DVPNGDISLAYG SEQ ID NO: 80 VPNGDISLAYGL SEQ ID NO: 81 PNGDISLAYGLR
SEQ ID NO: 82 NGDISLAYGLRS
[0162] xvii. 11-Amino Acid Peptides:
TABLE-US-00025 SEQ ID NO: 83 VDVPNGDISLA SEQ ID NO: 84 DVPNGDISLAY
SEQ ID NO: 85 VPNGDISLAYG SEQ ID NO: 86 PNGDISLAYGL SEQ ID NO: 87
NGDISLAYGLR SEQ ID NO: 88 GDISLAYGLRS
[0163] xviii. 10-Amino Acid Peptides:
TABLE-US-00026 SEQ ID NO: 89 VDVPNGDISL SEQ ID NO: 90 DVPNGDISLA
SEQ ID NO: 91 VPNGDISLAY SEQ ID NO: 92 PNGDISLAYG SEQ ID NO: 93
NGDISLAYGL SEQ ID NO: 94 GDISLAYGLR SEQ ID NO: 95 DISLAYGLRS
[0164] xix. 9-Amino Acid Peptides:
TABLE-US-00027 SEQ ID NO: 96 VDVPNGDIS SEQ ID NO: 97 DVPNGDISL SEQ
ID NO: 98 VPNGDISLA SEQ ID NO: 99 PNGDISLAY SEQ ID NO: 100
NGDISLAYG SEQ ID NO: 101 GDISLAYGL SEQ ID NO: 102 DISLAYGLR SEQ ID
NO: 103 ISLAYGLRS
[0165] xx. 8-Amino Acid Peptides:
TABLE-US-00028 SEQ ID NO: 104 VDVPNGDI SEQ ID NO: 105 DVPNGDIS SEQ
ID NO: 106 VPNGDISL SEQ ID NO: 107 PNGDISLA SEQ ID NO: 108 NGDISLAY
SEQ ID NO: 109 GDISLAYG SEQ ID NO: 110 DISLAYGL SEQ ID NO: 111
ISLAYGLR
[0166] xxi. 7-Amino Acid Peptides:
TABLE-US-00029 SEQ ID NO: 112 VDVPNGD SEQ ID NO: 113 DVPNGDI SEQ ID
NO: 114 VPNGDIS SEQ ID NO: 115 PNGDISL SEQ ID NO: 116 NGDISLA SEQ
ID NO: 117 GDISLAY SEQ ID NO: 118 DISLAYG SEQ ID NO: 119
ISLAYGL
[0167] xxii. 6-Amino Acid Peptides:
TABLE-US-00030 SEQ ID NO: 120 DVPNGD SEQ ID NO: 121 VPNGDI SEQ ID
NO: 122 PNGDIS SEQ ID NO: 123 NGDISL SEQ ID NO: 124 GDISLA SEQ ID
NO: 125 DISLAY SEQ ID NO: 126 ISLAYG
[0168] xxiii. 5-amino acid peptides:
TABLE-US-00031 SEQ ID NO: 127 VPNGD SEQ ID NO: 128 PNGDI SEQ ID NO:
129 NGDIS SEQ ID NO: 130 GDISL SEQ ID NO: 131 DISLA SEQ ID NO: 132
ISLAY SEQ ID NO: 133 SLAYG
[0169] xxiv. 16-Amino Acid Peptides:
TABLE-US-00032 SEQ ID NO: 136 KPLAEIDSIELSYGIK SEQ ID NO: 137
GDPNDGRGDSVVYGLR
[0170] xxv. 15-Amino Acid Peptides:
TABLE-US-00033 SEQ ID NO: 138 VDTYDGGISVVYGLR SEQ ID NO: 139
VDTYDGDGSVVYGLR
[0171] xxvi. 16-Amino Acid Peptides:
TABLE-US-00034 SEQ ID NO: 141 KCLAECDSIELSYGIK
[0172] xxvii. 8-Amino Acid Peptides:
TABLE-US-00035 SEQ ID NO: 142 CLAEIDSC
[0173] xxviii. 18-Amino Acid Peptides:
TABLE-US-00036 SEQ ID NO: 143 CFKPLAEIDSIECSYGIK
[0174] xxix. 16-Amino Acid Peptides:
TABLE-US-00037 SEQ ID NO: 144 KPLAEDISIELSYGIK SEQ ID NO: 145
KPLAEISDIELSYGIK SEQ ID NO: 146 KPLAEIGDIELSYGIK
[0175] xxx. 15-amino acid peptides:
TABLE-US-00038 SEQ ID NO: 147 KPLAEGDIELSYGIK
[0176] xxxi. 13-Amino Acid Peptides:
TABLE-US-00039 SEQ ID NO: 148 KPLAEIELSYGIK
[0177] xxxii. 16-Amino Acid Peptides:
TABLE-US-00040 SEQ ID NO: 149 KPLAEIDSIELTYGIK SEQ ID NO: 150
KPLAEIDGIELSYGIK SEQ ID NO: 151 KPLAEIDGIELTYGIK SEQ ID NO: 152
KPLAEIGSIELSYGIK SEQ ID NO: 153 KGLAEIDSIELSYGIK SEQ ID NO: 154
KPLAGIDSIGLSYGIK SEQ ID NO: 155 KCLAEIDSCELSYGIK
[0178] xxxiii. 13-amino acid peptides:
TABLE-US-00041 SEQ ID NO: 156 CFKPLAEIDSIEC
[0179] xxxiv. 15-Amino Acid Peptides:
TABLE-US-00042 SEQ ID NO: 157 VDVPEGDISLAYGLR SEQ ID NO: 158
LDGLVRAYDNISPVG
[0180] xxxv. 14-Amino Acid Peptides:
TABLE-US-00043 SEQ ID NO: 159 GDPNGDISVVYGLR
[0181] xxxvi. 15-Amino Acid Peptides:
TABLE-US-00044 SEQ ID NO: 160 VDVPNGDISLAYRLR SEQ ID NO: 161
VDVPEGDISLAYRLR SEQ ID NO: 167 V(beta-D)TYDGDISVVYGLR SEQ ID NO:
168 VDTY(beta-D)GDISVVYGLR SEQ ID NO: 169
VDTYDG(beta-D)ISVVYGLR
[0182] In one embodiment said peptide is derived from osteopontin,
such as a mammalian osteopontin variant and/or fragment.
[0183] In one embodiment, said peptide is non-naturally occurring,
such as a peptide comprising non-proteinogenic amino acid
residues.
[0184] In some embodiments, said peptide is further conjugated to a
moiety, which may be selected from the group consisting of PEG,
monosaccharides, fluorophores, chromophores, radioactive compounds,
and cell-penetrating peptides. In one embodiment, the fluorophore
is selected from the group consisting of Lucifer yellow, biotin,
5,6-carboxyltetramethylrhodamine (TAMRA), indodicarbocyanine (C5)
Alexa Fluor.RTM. 488, Alexa Fluor.RTM. 532, Alexa Fluor.RTM. 647,
ATTO 488, ATTO 532, 6-carboxyfluorescein (6-FAM), Alexa Fluor.RTM.
350, DY-415, ATTO 425, ATTO 465, Bodipy.RTM. FL, fluorescein
isothiocyanate, Oregon Green.RTM. 488, Oregon Green.RTM. 514,
Rhodamine Green.TM., 5'-Tetrachloro-Fluorescein, ATTO 520,
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluoresceine, Yakima
Yellow.TM. dyes, Bodipy.RTM. 530/550, hexachloro-fluorescein, Alexa
Fluor.RTM. 555, DY-549, Bodipy.RTM. TMR-X, cyanine phosphoramidites
(cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7.5), ATTO
550, Rhodamine Red.TM., ATTO 565, Carboxy-X-Rhodamine, Texas Red
(Sulforhodamine 101 acid chloride), LightCycler.RTM. Red 610, ATTO
594, DY-480-XL, DY-610, ATTO 610, LightCycler.RTM. Red 640, Bodipy
630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY-649,
LightCycler.RTM. Red 670, ATTO 680, LightCycler.RTM. Red 705,
DY-682, ATTO 700, ATTO 740, DY-782, IRD 700, IRD 800, CAL
Fluor.RTM. Gold 540 nm, CAL Fluor.RTM. Gold 522 nm, CAL Fluor.RTM.
Gold 544 nm, CAL Fluor.RTM. Orange 560 nm, CAL Fluor.RTM. Orange
538 nm, CAL Fluor.RTM. Orange 559 nm, CAL Fluor.RTM. Red 590 nm,
CAL Fluor.RTM. Red 569 nm, CAL Fluor.RTM. Red 591 nm, CAL
Fluor.RTM. Red 610 nm, CAL Fluor.RTM. Red 590 nm, CAL Fluor.RTM.
Red 610 nm, CAL Fluor.RTM. Red 635 nm, Quasar.RTM. 570 nm,
Quasar.RTM. 548 nm, Quasar.RTM. 566 nm (Cy 3), Quasar.RTM. 670 nm,
Quasar.RTM. 647 nm, Quasar.RTM. 670 nm, Quasar.RTM. 705 nm,
Quasar.RTM. 690 nm, Quasar.RTM. 705 nm (Cy 5.5), Pulsar.RTM. 650
Dyes, SuperRox.RTM. Dyes.).
[0185] In another embodiment, said peptide is further modified such
as being glycosylated or by PEGylation, amidation, esterification,
acylation, acetylation and/or alkylation.
[0186] In one embodiment, said peptide comprises or consists of
tandem repeats, which may comprise or consist of the amino acid
sequence of any one or more of the sequences as described
herein.
[0187] In one embodiment, said peptide is cyclic. The cyclic
structure may be achieved by any suitable method of synthesis.
Thus, heterodetic linkages may include, but are not limited to
formation via disulphide, cysteine, alkylene or sulphide
bridges.
[0188] In a further embodiment, the peptide comprises or consists
of a fusion. For example, the peptide may comprise a fusion of the
amino acid sequence of SEQ ID NO: 1 or 136.
[0189] The term `fusion` of a peptide relates to an amino acid
sequence corresponding to, for example, SEQ ID NO: 1 or 136 (or a
fragment or variant thereof) fused to any other peptide. For
example, the said peptide may be fused to a polypeptide such as
glutathione-S-transferase (GST) or protein A in order to facilitate
purification of said peptide. Examples of such fusions are well
known to those skilled in the art. Similarly, the said peptide may
be fused to an oligo-histidine tag such as His6 or to an epitope
recognised by an antibody such as the well-known Myc tag epitope.
Fusions to any variant or derivative of said peptide are also
included in the scope of the disclosure.
[0190] Alternatively, the fused portion may be a lipophilic
molecule or peptide domain that is capable of promoting cellular
uptake of the polypeptide, as known to those skilled in the
art.
Novel Peptides
[0191] In one embodiment, the present disclosure relates to a
peptide comprising or consisting of an amino acid sequence selected
from the group consisting of KPLAEIDSIELSYGIK (SEQ ID NO: 136),
GDPNDGRGDSVVYGLR (SEQ ID NO: 137), VDTYDGGISVVYGLR (SEQ ID NO:
138), and VDTYDGDGSVVYGLR (SEQ ID NO: 139), VDVPEGDISLAYGLR (SEQ ID
NO: 157), LDGLVRAYDNISPVG (SEQ ID NO: 158), GDPNGDISVVYGLR (SEQ ID
NO: 159), VDVPNGDISLAYRLR (SEQ ID NO: 160) VDVPEGDISLAYRLR (SEQ ID
NO: 161), or a variant or fragment thereof.
[0192] In another embodiment, the present disclosure relates to a
peptide comprising or consisting of an amino acid sequence selected
from the group consisting of KCLAECDSIELSYGIK (SEQ ID NO: 141),
CLAEIDSC (SEQ ID NO: 142), CFKPLAEIDSIECSYGIK (SEQ ID NO: 143),
KPLAEDISIELSYGIK (SEQ ID NO: 145), KPLAEIGDIELSYGIK (SEQ ID NO:
146), KPLAEGDIELSYGIK (SEQ ID NO: 147), KPLAEIELSYGIK (SEQ ID NO:
148), KPLAEIDSIELTYGIK (SEQ ID NO: 149), KPLAEIDGIELSYGIK (SEQ ID
NO: 150), KPLAEIDGIELTYGIK (SEQ ID NO: 151), KPLAEIGSIELSYGIK (SEQ
ID NO: 152), KGLAEIDSIELSYGIK (SEQ ID NO: 153), KPLAGIDSIGLSYGIK
(SEQ ID NO: 154), KCLAEIDSCELSYGIK (SEQ ID NO: 155) and
CFKPLAEIDSIEC (SEQ ID NO: 156), or a variant or fragment
thereof.
[0193] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO:
136), or a variant or fragment thereof.
[0194] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KPLAGIDSIGLSYGIK (SEQ ID NO:
154), or a variant or fragment thereof.
[0195] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KGLAEIDSIELSYGIK (SEQ ID NO:
153), or a variant or fragment thereof.
[0196] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KCLAECDSIELSYGIK (SEQ ID NO:
141), or a variant or fragment thereof.
[0197] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KPLAEIDGIELTYGIK (SEQ ID NO:
151), or a variant or fragment thereof.
[0198] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence
[0199] KPLAEIGSIELSYGIK (SEQ ID NO: 152), or a variant or fragment
thereof.
[0200] In one embodiment, the present disclosure relates to the
agent comprising a peptide, wherein the peptide comprises or
consists of the amino acid sequence KPLAEIELSYGIK (SEQ ID NO: 148),
or a variant or fragment thereof.
[0201] In one embodiment, the present disclosure relates to an
agent comprising: [0202] b) a peptide or peptide analog comprising
or consisting of the amino acid sequence GDPNDGRGDSVVYGLR (SEQ ID
NO: 137), VDTYDGGISVVYGLR (SEQ ID NO: 138), and VDTYDGDGSVVYGLR
(SEQ ID NO: 139). VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG
(SEQ ID NO: 158), GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR
(SEQ ID NO: 160) VDVPEGDISLAYRLR (SEQ ID NO: 161),
V(beta-D)TYDGDISVVYGLR (SEQ ID NO:167), VDTY(beta-D)GDISVVYGLR (SEQ
ID NO: 168), VDTYDG(beta-D)ISVVYGLR (SEQ ID NO:169); [0203] b) a
polynucleotide encoding upon expression, the peptide of a); [0204]
c) a vector comprising the polynucleotide of b); and [0205] d) a
cell comprising the polynucleotide of b), or the vector of c).
[0206] In some embodiments, said variant comprises or consists of a
sequence wherein any one amino acid has been altered for another
proteinogenic or non-proteinogenic amino acid, with the proviso
that no more than five amino acids are so altered, such as no more
than 4 amino acids, such as no more than 3 amino acids, such as no
more than 2 amino acids, such as no more than 1 amino acid is
altered. In some embodiments, one or more amino acids are
conservatively substituted.
[0207] In some embodiments, said peptide comprises or consists of
one or more additional amino acids, inserted at the N- and/or
C-terminus and/or internally within the sequence. In one
embodiment, at least 2 additional amino acids, such as at least 3,
such as at least 4, such as at least 5, such as at least 6, such as
at least 7, such as at least 8, such as at least 9, such as at
least 10, such as at least 15 or such as at least 20 additional
amino acids are inserted.
[0208] In some embodiments, said peptide is no more than 85, such
as no more than 80, such as no more than 75, such as no more than
70, such as no more than 65, such as no more than 60, such as nor
more than 55, such as no more than 50, such as no more than 55,
such as no more than 40 amino acids, such as no more than 35, such
as no more than 30, such as no more than 28, such as no more than
26, such as no more than 24, such as no more than 22, such as no
more than 20, such as no more than 19, such as no more than 18,
such as no more than 17, such as no more than 16, such as no more
than 15, such as no more than 14, such as no more than 13, such as
no more than 12, such as no more than 11, such as no more than 10
amino acids in length.
[0209] In some embodiments, said peptide is further conjugated to a
moiety, which may be selected from the group consisting of PEG,
monosaccharides, fluorophores, chromophores, radioactive compounds,
and cell-penetrating peptides.
[0210] In one embodiment, said peptide is further modified such as
being glycosylated or by PEGylation, amidation, esterification,
acylation, acetylation and/or alkylation.
[0211] In some embodiments, said peptide comprises or consists of
tandem repeats, which may comprise or consist of the amino acid
sequence of any one or more of the sequences as described herein
above.
[0212] In one embodiment, said peptide is cyclic. The cyclic
structure may be achieved by any suitable method of synthesis.
Thus, heterodetic linkages may include, but are not limited to
formation via, cysteine, disulphide, alkylene or sulphide
bridges.
Indications
[0213] The agents of the present disclosure are suitable for use in
the treatment of endocrine, nutritional and metabolic diseases and
disorders.
[0214] In one embodiment, the mammal in need of treatment of an
endocrine disease, a nutritional disease and/or a metabolic disease
is a human.
[0215] In some embodiments, the endocrine disease, nutritional
disease and/or metabolic disease is selected from the group
consisting of diabetes mellitus, type 1 diabetes mellitus, type 2
diabetes mellitus, malnutrition-related diabetes mellitus,
disorders of glucose regulation and pancreatic internal secretion,
insulin resistance syndrome, impaired glucose tolerance,
hyperglycemia, hyperinsulinemia, and any combinations thereof.
[0216] In some embodiments, the endocrine disease, nutritional
disease and/or metabolic disease is selected from the group
consisting of diabetes mellitus, disorders of the thyroid gland,
disorders of glucose regulation and pancreatic internal secretion,
disorders of endocrine glands, malnutrition, nutritional
deficiencies, obesity, hyperalimentation, and metabolic
disorders.
[0217] In one embodiment, diabetes mellitus is selected from the
group consisting of type 1 diabetes mellitus, type 2 diabetes
mellitus, malnutrition-related diabetes mellitus, specified
diabetes mellitus, and unspecified diabetes mellitus.
[0218] In one embodiment, disorders of glucose regulation and
pancreatic internal secretion are selected from the group
consisting of nondiabetic hypoglycaemic coma and disorders of
pancreatic internal secretion.
[0219] In one embodiment, disorders of obesity and
hyperalimentation are selected from the group consisting of
localized adiposity, hyperalimentation, and sequelae of
hyperalimentation.
[0220] In one embodiment, disorders of nutritional deficiencies are
selected from the group consisting of disorders of aromatic
amino-acid metabolism, disorders of branched-chain amino-acid
metabolism and fatty-acid metabolism, disorders of amino-acid
metabolism, lactose intolerance, disorders of carbohydrate
metabolism, disorders of sphingolipid metabolism, disorders of
lipid storage disorders, disorders of glycosaminoglycan metabolism,
disorders of glycoprotein metabolism, disorders of lipoprotein
metabolism, lipidaemias, disorders of purine and pyrimidine
metabolism, disorders of porphyrin and bilirubin metabolism,
disorders of mineral metabolism, cystic fibrosis, amyloidosis,
volume depletion, disorders of fluid, electrolyte and acid-base
balance, and postprocedural endocrine and metabolic disorders.
Compositions
[0221] In one aspect, the present disclosure relates to a
composition comprising the agent described herein.
[0222] In one aspect, the present disclosure relates to an agent
selected from the group consisting of: [0223] a) a peptide or a
peptide analog selected from the group consisting of [0224] (i) a
peptide comprising or consisting of an amino acid sequence of the
general formula:
TABLE-US-00045 [0224] (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0225] wherein: [0226] X.sub.2 is C, P or G; [0227] X.sub.5 is E or
G; [0228] X.sub.6 is C, D or I; [0229] X.sub.7 is D, I, S or G;
[0230] X.sub.8 is S, D or G; [0231] X.sub.10 is E or G; [0232]
X.sub.12 is S or T [0233] with the proviso that if X.sub.12 is T,
the peptide comprises no more than 25 amino acid residues; and
[0234] (ii) a peptide comprising or consisting of an amino acid
sequence of the general formula:
TABLE-US-00046 [0234] (SEQ ID NO: 68)
VDZ.sub.3Z4Z.sub.5GZ.sub.7Z.sub.8SZ.sub.10Z.sub.11YGLR
[0235] wherein: [0236] Z.sub.3 is T or V; [0237] Z.sub.4 is Y or P;
[0238] Z.sub.5 is D or N; [0239] Z.sub.7 is D or G; [0240] Z.sub.8
is I or G; [0241] Z.sub.10 is V or L; [0242] Z.sub.11 is V or A;
and [0243] (iii) a peptide comprising or consists of an amino acid
sequence selected from the group consisting of KCLAECDSIELSYGIK
(SEQ ID NO: 141), CLAEIDSC (SEQ ID NO: 142), CFKPLAEIDSIECSYGIK
(SEQ ID NO: 143), KPLAEIELSYGIK (SEQ ID NO: 148), KCLAEIDSCELSYGIK
(SEQ ID NO: 155) and CFKPLAEIDSIEC (SEQ ID NO: 156); [0244] b) a
polynucleotide encoding upon expression, the peptide of a); [0245]
c) a vector comprising the polynucleotide of b); and [0246] d) a
cell comprising the polynucleotide of b), or the vector of c);
[0247] for use in the treatment of an endocrine disease, a
nutritional disease and/or a metabolic disease in a mammal.
[0248] In one aspect, the present disclosure relates to a
composition for use in treatment of an endocrine disease, a
nutritional disease and/or a metabolic disease, comprising an agent
described herein. In one embodiment, said composition is a
pharmaceutical composition.
[0249] In one embodiment, the agent further comprises a second
active ingredient. Said second active ingredient may be selected
from the group consisting of insulin, glucagon-like peptide-1
(GLP-1), biguanides, forskolin compounds, sulfonylurea, a
dipeptidyl peptidase-4 (DPP4) inhibitor, an alpha-glucosidase
inhibitor, a thiazolidinedione, a meglitidine and a sodium-glucose
cotransporter-2 (SGLT2) inhibitor.
Other Methods
[0250] In one aspect, the present disclosure concerns a method of
treating an endocrine disease, a nutritional disease and/or a
metabolic disease, the method comprising administering an agent
described herein to a subject in need thereof.
[0251] In one aspect, the present disclosure concerns the use of an
agent for the manufacture of a medicament for use in treatment of
an endocrine disease, a nutritional disease and/or a metabolic
disease in a mammal.
[0252] In one aspect, the present disclosure concerns a
polynucleotide encoding upon expression the peptide as described
herein. In one aspect, the present disclosure concerns a vector
comprising said polynucleotide encoding upon expression the peptide
as described herein. In one aspect, the present disclosure concerns
a cell comprising said polynucleotide or said vector encoding upon
expression the peptide as described herein
[0253] In one aspect, the present disclosure concerns a method for
increasing insulin secretion, the method comprising administering a
therapeutically effective amount of a peptide described herein, to
an individual in need thereof. In one embodiment, said method is an
in vitro method.
[0254] In one aspect, the present disclosure concerns a method for
decreasing blood glucose levels, the method comprising
administering a therapeutically effective amount of a peptide
described herein, to an individual in need thereof. In one
embodiment, said method is an in vitro method. In one embodiment,
insulin secretion is increased. In another embodiment, cellular
uptake of glucose is increased. In yet another embodiment, insulin
production is increased. In another embodiment glucagon production
is decreased.
[0255] In one aspect, the present disclosure concerns a method,
e.g. an in vitro method, for improving .beta.-cell morphology, the
method comprising administering a therapeutically effective amount
of a peptide described herein, to an individual in need
thereof.
[0256] In one aspect, the present disclosure concerns a method for
improving .beta.-cell viability, the method comprising
administering a therapeutically effective amount of a peptide
described herein, to an individual in need thereof.
[0257] In one aspect, the present disclosure concerns a method for
delaying onset of diabetes and diabetes associated disorders and
disease, the method comprising administering a therapeutically
effective amount of a peptide described herein, to an individual in
need thereof.
[0258] In one embodiment of the present disclosure, the agent may
further comprise a detectable moiety. For example, a detectable
moiety may comprise or consist of a radioisotope, such as a
radioisotope selected from the group consisting of .sup.99mTc,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, .sup.123I
and .sup.201Tl. The binding moieties may thus be coupled to
nanoparticles that have the capability of multi-imaging (for
example, SPECT, PET, MRI, Optical, or Ultrasound). Alternatively,
the detectable moiety may comprise or consist of a paramagnetic
isotope, such as a paramagnetic isotope is selected from the group
consisting of .sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Cr and
.sup.56Fe.
[0259] In the case that the agent comprises a detectable moiety,
then the detectable moiety may be detectable by an imaging
technique such as SPECT, PET, MRI, optical or ultrasound
imaging.
[0260] In one aspect, the present disclosure concerns the use of
agent described herein for the preparation of a diagnostic
composition for the diagnosis of a disease, disorder or damage of
the pancreas in an individual.
Items
[0261] 1. An agent comprising: [0262] a) a peptide, wherein the
peptide or peptide analog comprises an amino acid sequence of the
general formula:
TABLE-US-00047 [0262] (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0263] wherein: [0264] X.sub.2 is C, P or G; [0265] X.sub.5 is E or
G; [0266] X.sub.6 is C, D or I; [0267] X.sub.7 is D, I, S or G;
[0268] X.sub.8 is S, D or G; [0269] X.sub.10 is E or G; [0270]
X.sub.12 is S or T; [0271] with the proviso that if X.sub.12 is T,
the peptide comprises no more than 25 amino acid residues; and
[0272] with the proviso that if X.sub.2 is P, X.sub.5 is E, X.sub.5
is I, X.sub.7 is D, X.sub.5 is S, X.sub.10 is E and X.sub.12 is S,
the peptide comprises no more than 85 amino acid residues; [0273]
or a biologically active fragment and/or variant of SEQ ID NO: 140;
[0274] b) a polynucleotide encoding upon expression, the peptide of
a); [0275] c) a vector comprising the polynucleotide of b); and
[0276] d) a cell comprising the polynucleotide of b), or the vector
of c). [0277] 2. An agent comprising a peptide, wherein the peptide
comprises an amino acid sequence of the general formula:
TABLE-US-00048 [0277] (SEQ ID NO: 164)
VDVPZ.sub.5GDISLAYZ.sub.13LR
[0278] wherein: [0279] Z.sub.5 is E or N; [0280] Z.sub.13 is R or
G. [0281] 3. An agent comprising a peptide, wherein the peptide
comprises an amino acid sequence of the general formula:
TABLE-US-00049 [0281] (SEQ ID NO: 165)
VDTYDGZ.sub.7Z.sub.8SVVYGLR
[0282] wherein: [0283] Z.sub.7 is D or G; [0284] Z.sub.8 is I or G.
[0285] 4. An agent comprising a peptide, wherein the peptide
comprises an amino acid sequence of the general formula:
TABLE-US-00050 [0285] (SEQ ID NO: 166)
GDPNZ.sub.5Z.sub.6Z.sub.7Z.sub.8Z.sub.9SVVYGLR
[0286] wherein: [0287] Z.sub.5 is D or G; [0288] Z.sub.6 is D or G
[0289] Z.sub.7 is I or R; [0290] Z.sub.8 is G or absent; [0291]
Z.sub.9 is D or absent. [0292] 5. The agent according to item 2 to
4, wherein the agent comprising: [0293] a) a peptide, wherein the
peptide comprises or consists of an amino acid sequence selected
from the group consisting of GDPNDGRGDSVVYGLR (SEQ ID NO: 137),
VDTYDGGISVVYGLR (SEQ ID NO: 138), and VDTYDGDGSVVYGLR (SEQ ID NO:
139). VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG (SEQ ID NO:
158), GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR (SEQ ID NO:
160) VDVPEGDISLAYRLR (SEQ ID NO: 161); [0294] b) a polynucleotide
encoding upon expression, the peptide of a); [0295] c) a vector
comprising the polynucleotide of b); and [0296] d) a cell
comprising the polynucleotide of b), or the vector of c). [0297] 6.
The agent according to item 1, wherein the peptide comprises or
consists of an amino acid sequence of the general formula:
TABLE-US-00051 [0297] (SEQ ID NO: 162)
KX.sub.2LAX.sub.5X.sub.8X.sub.7X.sub.8IX.sub.10LSYGIK
[0298] wherein: [0299] X.sub.2 is C, P or G; [0300] X.sub.6 is C, I
or absent; [0301] X.sub.7 is D, G or absent; [0302] X.sub.8 is S, G
or absent; [0303] X.sub.10 is E or G. [0304] 7. The agent according
to item 6, wherein the peptide comprises an amino acid sequence of
the general formula:
TABLE-US-00052 [0304] (SEQ ID NO: 163)
KX.sub.2LAX.sub.5IX.sub.10LSYGIK
[0305] wherein: [0306] X.sub.2 is C, P or G; [0307] X.sub.5 is E or
G; [0308] X.sub.10 is E or G. [0309] 8. An agent comprising: [0310]
a) a peptide or peptide analog comprising or consisting of the
amino acid sequence GDPNDGRGDSVVYGLR (SEQ ID NO: 137),
VDTYDGGISVVYGLR (SEQ ID NO: 138), and VDTYDGDGSVVYGLR (SEQ ID NO:
139). VDVPEGDISLAYGLR (SEQ ID NO: 157), LDGLVRAYDNISPVG (SEQ ID NO:
158), GDPNGDISVVYGLR (SEQ ID NO: 159), VDVPNGDISLAYRLR (SEQ ID NO:
160) VDVPEGDISLAYRLR (SEQ ID NO: 161), V(beta-D)TYDGDISVVYGLR (SEQ
ID NO:167), VDTY(beta-D)GDISVVYGLR (SEQ ID NO: 168),
VDTYDG(beta-D)ISVVYGLR (SEQ ID NO:169); [0311] b) a polynucleotide
encoding upon expression, the peptide of a); [0312] c) a vector
comprising the polynucleotide of b); and [0313] d) a cell
comprising the polynucleotide of b), or the vector of c). [0314] 9.
The agent according to any one of the preceding items, wherein the
agent comprises non-naturally occurring, e.g. non-proteinogenic,
amino acid residues. [0315] 10. The agent according to any one of
the preceding items, wherein the agent is conjugated to a moiety.
[0316] 11. The agent according to any one of the preceding items,
wherein the moiety is selected from the group consisting of
polyethylene glycol (PEG), monosaccharides, fluorophores,
chromophores, radioactive compounds, and cell-penetrating peptides.
[0317] 12. The agent according to any one of the preceding items,
wherein the agent is further modified such as being glycosylated or
by PEGylation, amidation, esterification, acylation, acetylation
and/or alkylation. [0318] 13. The agent according to any one of the
preceding items, wherein the agent comprises or consists of tandem
repeats. [0319] 14. The agent according to any one of the preceding
items, wherein the tandem repeats comprise or consist of the amino
acid sequence of any one or more of the sequences as described in
the preceding items. [0320] 15. The agent according to any of the
preceding items, wherein the agent is fused to another polypeptide.
[0321] 16. The agent according to any one of the preceding items,
wherein the said polypeptide is selected from the group consisting
of glutathione-S-transferase (GST) and protein A. [0322] 17. The
agent according to any of the preceding items, wherein the agent is
fused to a tag. [0323] 18. The agent according to any one of the
preceding items, wherein the said tag is an oligo-histidine tag.
[0324] 19. The agent according to any of the preceding items,
wherein the agent is cyclic, such as wherein the peptide is cyclic.
[0325] 20. The agent according to any of the preceding items,
wherein the peptide or peptide analog is capable of forming at
least one intramolecular cysteine bridge, e.g. to form a cyclic or
partially cyclic peptide. [0326] 21. The agent according to any of
the preceding items, wherein the peptide or peptide analog
comprises or consists of an amino acid sequence selected from the
group consisting of KCLAECDSIELSYGIK (SEQ ID NO: 141), CLAEIDSC
(SEQ ID NO: 142), CFKPLAEIDSIECSYGIK (SEQ ID NO: 143),
KPLAEDISIELSYGIK (SEQ ID NO: 145), KPLAEIGDIELSYGIK (SEQ ID NO:
146), KPLAEGDIELSYGIK (SEQ ID NO: 147), KPLAEIELSYGIK (SEQ ID NO:
148), KPLAEIDSIELTYGIK (SEQ ID NO: 149), KPLAEIDGIELSYGIK (SEQ ID
NO: 150), KPLAEIDGIELTYGIK (SEQ ID NO: 151), KPLAEIGSIELSYGIK (SEQ
ID NO: 152), KGLAEIDSIELSYGIK (SEQ ID NO: 153), KPLAGIDSIGLSYGIK
(SEQ ID NO: 154), KCLAEIDSCELSYGIK (SEQ ID NO: 155) and
CFKPLAEIDSIEC (SEQ ID NO: 156), or a variant or fragment thereof.
[0327] 22. The agent according to any of the preceding items,
wherein the peptide or peptide analog comprises or consists of the
amino acid sequence KPLAEIDSIELSYGIK (SEQ ID NO: 136), or a variant
or fragment thereof. [0328] 23. The agent according to any of the
preceding items, wherein the peptide or peptide analog comprises or
consists of the amino acid sequence KPLAGIDSIGLSYGIK (SEQ ID NO:
154), or a variant or fragment thereof. [0329] 24. The agent
according to any of the preceding items, wherein the peptide or
peptide analog comprises or consists of the amino acid sequence
KGLAEIDSIELSYGIK (SEQ ID NO: 153), or a variant or fragment
thereof. [0330] 25. The agent according to any of the preceding
items, wherein the peptide or peptide analog comprises or consists
of the amino acid sequence KCLAECDSIELSYGIK (SEQ ID NO: 141), or a
variant or fragment thereof. [0331] 26. The agent according to any
of the preceding items, wherein the peptide or peptide analog
comprises or consists of the amino acid sequence KPLAEIDGIELTYGIK
(SEQ ID NO: 151), or a variant or fragment thereof. [0332] 27. The
agent according to any of the preceding items, wherein the peptide
or peptide analog comprises or consists of the amino acid sequence
KPLAEIGSIELSYGIK (SEQ ID NO: 152), or a variant or fragment
thereof. [0333] 28. The agent according to any of the preceding
items, wherein the peptide or peptide analog comprises or consists
of the amino acid sequence KPLAEIELSYGIK (SEQ ID NO: 148), or a
variant or fragment thereof. [0334] 29. The agent according to any
one of the preceding items, wherein the variant comprises or
consists of a sequence wherein any one amino acid has been altered
for another proteinogenic or non-proteinogenic amino acid, with the
proviso that no more than five amino acids are so altered. [0335]
30. The agent according to any one of the preceding items, wherein
the variant comprises or consists of a sequence wherein no more
than five amino acids are altered for another proteinogenic or
non-proteinogenic amino acid, such as no more than 4 amino acids,
such as no more than 3 amino acids, such as no more than 2 amino
acids, such as no more than 1 amino acid is altered. [0336] 31. The
agent according to any one of the preceding items, wherein one or
more amino acids are conservatively substituted. [0337] 32. The
agent according to any one of the preceding items, wherein the
peptide or peptide analog comprises or consists of one or more
additional amino acids, inserted at the N- and/or C-terminus and/or
internally within the sequence. [0338] 33. The agent according to
any one of the preceding items, wherein the peptide or peptide
analog comprises 1 additional amino acid conjugated to either N- or
C-terminal. [0339] 34. The agent according to any of the preceding
items, wherein the agent comprises no more than 85, such as no more
than 80, such as no more than 75, such as no more than 70, such as
no more than 65, such as no more than 60, such as nor more than 55,
such as no more than 50, such as no more than 55, such as no more
than 40 amino acids, such as no more than 35, such as no more than
30, such as no more than 28, such as no more than 26, such as no
more than 24, such as no more than 22, such as no more than 20,
such as no more than 19, such as no more than 18, such as no more
than 17, such as no more than 16, such as no more than 15, such as
no more than 14, such as no more than 13, such as no more than 12,
such as no more than 11, such as no more than 10 amino acids.
[0340] 35. The agent according to any one of the preceding items,
wherein the agent comprises at least 2 additional amino acids, such
as at least 3, such as at least 4, such as at least 5, such as at
least 6, such as at least 7, such as at least 8, such as at least
9, such as at least 10, such as at least 15 or such as at least 20
amino acids conjugated to the N- or C-terminus of the peptide.
[0341] 36. The agent according to any of the preceding items,
wherein the agent further comprises a detectable moiety. [0342] 37.
The agent according to any of the preceding items, wherein the
detectable moiety comprises or consists of a radioisotope. [0343]
38. The agent according to any of the preceding items, wherein the
radioisotope is selected from the group consisting of .sup.99mTc,
.sup.111In, .sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, .sup.123I
and .sup.201Tl. [0344] 39. The agent according to any of the
preceding items, wherein the detectable moiety is detectable by an
imaging technique such as SPECT, PET, MRI, optical or ultrasound
imaging. [0345] 40. Use of the agent of any of the preceding items,
for the preparation of a diagnostic composition for the diagnosis
of a disease, disorder or damage of the pancreas in an individual.
[0346] 41. A composition comprising the agent according to any of
the preceding items. [0347] 42. The composition according to any
one of the preceding items, wherein the composition is a
pharmaceutical composition. [0348] 43. The agent or the composition
according to any one of the preceding items, for use as a
medicament. [0349] 44. An agent selected from the group consisting
of: [0350] a) a peptide selected from the group consisting of
[0351] (i) a peptide comprising or consisting of an amino acid
sequence of the general formula:
TABLE-US-00053 [0351] (SEQ ID NO: 140)
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10LX.sub.12YGIK
[0352] wherein: X.sub.2 is C, P or G; X.sub.5 is E or G; X.sub.6 is
C, D or I; X.sub.7 is D, I, S or G; X.sub.8 is S, D or G; X.sub.10
is E or G; X.sub.12 is S or T; [0353] with the proviso that if
X.sub.12 is T, the peptide comprises no more than 25 amino acid
residues; [0354] or a biologically active fragment and/or variant
of SEQ ID NO: 140; [0355] (ii) a peptide comprising or consisting
of an amino acid sequence of the general formula:
TABLE-US-00054 [0355] (SEQ ID NO: 68)
VDZ.sub.3Z4Z.sub.5GZ.sub.7SZ.sub.10Z.sub.11YGLR
[0356] wherein: [0357] Z.sub.3 is T or V; [0358] Z.sub.4 is Y or P;
[0359] Z.sub.5 is D or N; [0360] Z.sub.7 is D or G; [0361] Z.sub.8
is I or G; [0362] Z.sub.10 is V or L; [0363] Z.sub.11 is V or A;
and [0364] b) a polynucleotide encoding upon expression, the
peptide of a); [0365] c) a vector comprising the polynucleotide of
b); and [0366] d) a cell comprising the polynucleotide of b), or
the vector of c); [0367] for use in the treatment of an endocrine
disease, a nutritional disease and/or a metabolic disease in a
mammal. [0368] 45. The agent or the composition for use according
to item 44, wherein the peptide comprises or consists of an amino
acid sequence selected from the group consisting of
KCLAECDSIELSYGIK (SEQ ID NO: 141), CLAEIDSC (SEQ ID NO: 142),
CFKPLAEIDSIECSYGIK (SEQ ID NO: 143), KPLAEIELSYGIK (SEQ ID NO:
148), KCLAEIDSCELSYGIK (SEQ ID NO: 155) and CFKPLAEIDSIEC (SEQ ID
NO: 156); [0369] 46. The agent or the composition for use according
to any one of the preceding items, wherein the peptide is selected
from the group consisting of SEQ ID NO: 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153, 154, 155 and 156. [0370]
47. The agent or the composition for use according to any one of
the preceding items, wherein the peptide is selected from the group
consisting of SEQ ID NO: 1, 136, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 67, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 137, 138, 139,
157, 158, 159, 160, 161, 167, 168 and 169. [0371] 48. The agent or
the composition for use according to any one of the preceding
items, wherein said agent comprises a second or further active
ingredient. [0372] 49. The agent or the composition for use
according to item 48, wherein the second or further active
ingredient is selected from the group consisting of insulin,
glucagon-like peptide-1 (GLP-1), sulfonylurea, a dipeptidyl
peptidase-4 (DPP4) inhibitor, an alpha-glucosidase inhibitor, a
thiazolidinedione, a meglitidine and a sodium-glucose
cotransporter-2 (SGLT2) inhibitor. [0373] 50. The agent or the
composition according to any of the preceding items for use in the
treatment of an endocrine disease, a nutritional disease and/or a
metabolic disease in a mammal. [0374] 51. The agent or the
composition for use according to item 50, wherein the mammal is a
human. [0375] 52. The agent or the composition for use according to
any one of the preceding items, wherein the endocrine disease,
nutritional disease and/or metabolic disease are selected from the
group consisting of diabetes mellitus, type 1 diabetes mellitus,
type 2 diabetes mellitus, malnutrition-related diabetes mellitus,
disorders of glucose regulation and pancreatic internal secretion,
insulin resistance syndrome, impaired glucose tolerance,
hyperglycemia, hyperinsulinemia, and any combinations thereof.
[0376] 53. The agent or the composition for use according to any
one of the preceding items, wherein the endocrine disease,
nutritional disease and/or metabolic disease are selected from the
group consisting of diabetes mellitus, disorders of the thyroid
gland, disorders of glucose regulation and pancreatic internal
secretion, disorders of endocrine glands, malnutrition, nutritional
deficiencies, obesity, hyperalimentation, and metabolic disorders.
[0377] 54. The agent or the composition for use according to any
one of the preceding items, wherein the diabetes mellitus is
selected from the group consisting of type 1 diabetes mellitus,
type 2 diabetes mellitus, malnutrition-related diabetes mellitus,
specified diabetes mellitus, and unspecified diabetes mellitus.
[0378] 55. The agent or the composition for use according to any
one of the preceding items, wherein the disorder of glucose
regulation and pancreatic internal secretion is selected from the
group consisting of nondiabetic hypoglycaemic coma and disorders of
pancreatic internal secretion. [0379] 56. The agent or the
composition for use according to any one of the preceding items,
wherein the disorder of obesity and hyperalimentation is selected
from the group consisting of localized adiposity,
hyperalimentation, and sequelae of hyperalimentation. [0380] 57.
The agent or the composition for use according to any one of the
preceding items, wherein the disorder of nutritional deficiencies
is selected from the group consisting of disorders of aromatic
amino-acid metabolism, disorders of branched-chain amino-acid
metabolism and fatty-acid metabolism, disorders of amino-acid
metabolism, lactose intolerance, disorders of carbohydrate
metabolism, disorders of sphingolipid metabolism, disorders of
lipid storage disorders, disorders of glycosaminoglycan metabolism,
disorders of glycoprotein metabolism, disorders of lipoprotein
metabolism, lipiemias, disorders of purine and pyrimidine
metabolism, disorders of porphyrin and bilirubin metabolism,
disorders of mineral metabolism, cystic fibrosis, amyloidosis,
volume depletion, disorders of fluid, electrolyte and acid-base
balance, and postprocedural endocrine and metabolic disorders.
[0381] 58. A method of treating an endocrine disease, a nutritional
disease and/or a metabolic disease, the method comprising
administering an agent according to any one of the preceding items
to a subject in need thereof. [0382] 59. Use of an agent according
to any one of the preceding items for the manufacture of a
medicament for use in treatment of an endocrine disease, a
nutritional disease and/or a metabolic disease in a mammal. [0383]
60. A method for delaying onset of diabetes and diabetes associated
disorders and diseases, the method comprising administering a
therapeutically effective amount of the agent as defined in any one
of the preceding items, to an individual in need thereof. [0384]
61. A method for decreasing blood glucose levels, the method
comprising administering a therapeutically effective amount of an
agent of any one of the preceding items, to an individual in need
thereof. [0385] 62. The method according to item 61, wherein
insulin secretion is increased. [0386] 63. The method according to
item 61, wherein cellular uptake of glucose is increased. [0387]
64. The method according to item 61, wherein the insulin production
is increased. [0388] 65. The method according to item 61, wherein
the glucagon production is decreased. [0389] 66. A method for
improving beta cell viability, the method comprising administering
a therapeutically effective amount of an agent of any one of the
preceding items, to an individual in need thereof. [0390] 67. A
method for improving beta cell morphology, the method comprising
administering a therapeutically effective amount of an agent of any
one of the preceding items, to an individual in need thereof.
[0391] 68. A method for stabilising or improving viability and/or
morphology of pancreatic islets, the method comprising
administering a therapeutically effective amount of an agent of any
one of the preceding items, to an individual in need thereof.
EXAMPLES
[0392] The disclosure is further illustrated by the following
examples, which however should not be construed as being limiting
for the disclosure. These examples demonstrate that exemplary
peptides of the present disclosure stimulate .beta.-cell
proliferation, and have the ability to protect and rescue
.beta.-cells from apoptosis induced by glucotoxic conditions. It is
also demonstrated that the exemplary peptides have the ability to
stimulate insulin secretion from rat .beta.-cells as well as
isolated mouse pancreatic islets, where the peptides also are
demonstrated to reduce glucagon levels. Furthermore, the examples
demonstrate that the peptides reduce plasma glucose levels in vivo
in a glucose tolerance test and that the peptides delay onset of
type 1 diabetes in BB lyp/lyp rats
Example 1: Peptide Design
[0393] The novel peptides were designed following rational
structure activity investigations. For FOL-005 (SEQ ID NO: 1) the
peptides were designed around the RGD site but mutated in order to
generate different structures that potentially could interact with
different integrins. A sequence similar to FOL-005 was identified
in the third fibronectin type III repeat domain (TNfn3) in
tenascin-C and found to be reasonably similar to the mutated RGD
site of FOL-005. A peptide was designed from this sequence denoted
FOL-014. The X-ray crystal structure of the tenascin-3 TNfn3 domain
(PDB code 1TEN, Leahy et al. (1992) Science 258(5084):987-91) was
analyzed. The FOL-014 (SEQ ID NO: 136) sequence span the beta-turn
before and the entire 3rd beta sheet. FOL-014 variants were
designed to allow for structural modification and stabilization of
the 3-dimensional molecular structure. Specifically, the peptides
variants covered the beta-turn region with exposed side chains and
some cyclized variants to maintain geometry.
[0394] All peptides were synthesized by solid phase peptide
synthesis using several peptide manufacturers. Mainly, the peptide
variants have been provided by Biopeptide Inc., California.
Example 2: FOL-005 and FOL-014 Induced Proliferation of INS-1
Cells
[0395] To investigate if FOL-005 and FOL-014 could induce
proliferation of .beta.-cells we used INS-1 cells. Rat INS-1 cells
were seeded in 96-well plates in RPMI medium with supplement and
after 2 hours the medium was changed to RPMI without supplement.
During the proliferation experiment the cells were incubated at
different test conditions (FOL-005, FOL-014, coated or in solution,
48 h incubation) and during the last 20 hours of culture period the
cells were pulsed with 1.mu. Ci/well of [methyl-3H] thymidine. The
cells were then harvested onto glass fiber filters using a
FilterMate harvester. The filters were air dried, and the bound
radioactivity was measured using a liquid scintillation counter. To
study whether FOL-005 influenced .beta.-cell proliferation, INS-1
cells were treated with increasing amounts of soluble FOL-005
(0.06-6 .mu.M) during 48 hours and proliferation was measured with
radiolabeled thymidine incorporation into newly synthesized DNA.
FOL-005 stimulated INS-1 cell proliferation (FIG. 1A). Wells coated
with either FOL-005 or FOL-014 and later blocked with bovine serum
albumin (BSA) before addition of INS-1 cells also stimulated
proliferation compared to control (ctrl) coated wells (FIG.
1B-C).
[0396] This demonstrated that FOL-005 and FOL-014 interacted with
.beta.-cells and induced proliferation.
Example 3: FOL-005 Protected n-Cells from Glucotoxicity
[0397] Since glucotoxicity in pancreatic .beta.-cells is a
well-established process in type 2 diabetes we next investigated
the protective effects of FOL-005 on .beta.-cells during glucotoxic
conditions. First we confirmed that 20 mM glucose induced cell
apoptosis in INS cells after 48 h of exposure. High glucose (20 mM)
containing RPMI medium induced more Annexin V positive cells and
more caspase-3 activity in INS cells compared to cells incubated
with medium containing 5 mM glucose (FIG. 2A-B). Exposure of INS-1
cells to 20 mM of glucose at the same time as FOL-005 decreased
cell apoptosis as detected both by Annexin V staining and by
caspase-3 activity (FIG. 2 A-B). The rate of apoptosis in INS-1
cells was measured with either Caspase-3 Assay Kit or stained with
Annexin V Apoptosis Detection Kit with 7-AAD. Caspase-3 activity
was measured with fluorescence at an excitation wavelength of 380
nm and an emission wavelength of 440 nm. Caspase-3 activity was
then normalized to protein concentration in each well. Measurements
of Annexin V stained cells were performed using a CyAn ADP flow
cytometer and analyzed with Summit V4.3 software.
[0398] In conclusion, it is well known that glucotoxicity induces
.beta.-cell apoptosis, however in the presence of FOL-005
glucotoxicity-induced apoptosis was diminished.
Example 4: FOL-005 Induced Insulin Secretion from INS-1 Cells
[0399] To investigate the stimulatory effect of FOL-005 on insulin
secretion, INS-1 .beta.-cells were used in the following
experiments. Cells were seeded overnight in cRPMI and then washed
with PBS before pre-incubation for 60 min at 37.degree. C. in
Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, supplemented with 10
mM HEPES, 0.1% bovine serum albumin. After pre-incubation, the
buffer was changed and the INS-1 cells were incubated at different
test conditions (0 mM, 5 mM or 20 mM glucose) and stimulated with
peptide FOL-005 or FOL-015 (SEQ ID NO: 158) or left untreated
during 60 min at 37.degree. C. Immediately after incubation, an
aliquot of the buffer was removed and frozen for subsequent assay
of insulin with an insulin radioimmunoassay kit.
[0400] The results demonstrated that .beta.-cells stimulated with
FOL-005 peptide secreted more insulin compared to unstimulated
control cells or to cells stimulated with the FOL-015 control
peptide (FIG. 3A) under conditions without glucose. INS-1
.beta.-cells subjected to glucose (5 mM or 20 mM) responded with
insulin secretion after FOL-005 peptide (6 .mu.M) stimulation (FIG.
3B). INS-1 cells stimulated with 6 .mu.M FOL-005 peptide in the
presence of 20 mM glucose responded with more insulin secretion
compared to FOL-005 stimulated cells incubated with 5 mM glucose
(FIG. 3B).
Example 5: FOL-005 Induced Insulin Secretion from Mouse Pancreatic
Islets
[0401] Mouse pancreatic islets were isolated from 8-week old
C57BL/6J male mice (Taconic). Mice were sacrificed by an overdose
of isoflurane and cervical dislocation. 3 ml of 0.9 U/ml
collagenase P was injected into the pancreatic duct to inflate the
pancreas. The pancreas was then removed and collagen digested for
19 min at 37.degree. C. The samples were vigorously shaken to
disrupt the tissue. The digest was transferred into ice cold Hank's
Balanced Salt Solution (HBSS) with Ca.sup.2+ and Mg.sup.2+. The
suspension was allowed to sit for 10 min to allow the islet to
sink, and the islets were washed in fresh HBSS four times. The
islets were then hand-picked and sorted according to size. Islets
(n=3 per well in a 96 well plate) were pre-incubated in KRB buffer
during 10 min 37.degree. C., pH 7.4, supplemented with 10 mM HEPES,
0.1%) bovine serum albumin. After pre-incubation, the buffer was
changed and islets were incubated at different test conditions in
new KRB buffer with 0.1%) bovine serum albumin (non-treated ctrl,
FOL-005 peptide, or GLP-1) for 60 min at 37.degree. C. Immediately
after incubation, an aliquot of the buffer was removed and frozen
for subsequent assay of insulin.
[0402] The results demonstrated that isolated mouse pancreatic
islets stimulated with GLP-1 (100 nM) or FOL-005 (6 .mu.M) secreted
more insulin compared to unstimulated control islets (FIG. 3C).
Example 6: FOL-014 Induced Insulin Secretion from INS-1 Cells
[0403] INS-1 .beta.-cells were used to investigate the stimulatory
effect of FOL-014 on insulin secretion. Cells were seeded overnight
and then washed with PBS before pre-incubation for 60 min at
37.degree. C. in Krebs-Ringer bicarbonate buffer (KRB), pH 7.4,
supplemented with 10 mM HEPES, 0.1% bovine serum albumin. After
pre-incubation, the buffer was changed and the INS-1 cells were
incubated in new KRB buffer supplemented with 10 mM HEPES, 0.1%
bovine serum albumin and stimulated with peptide FOL-014 or left
untreated during 60 min at 37.degree. C. Immediately after
incubation, an aliquot of the buffer was removed and frozen for
subsequent assay of insulin.
[0404] The results demonstrated that .beta.-cells stimulated with
FOL-014 peptide secreted more insulin compared to unstimulated
control cells (FIG. 4A).
Example 7: FOL-014 Induced Insulin Secretion from Mouse Pancreatic
Islets
[0405] Mouse pancreatic islets were isolated from 8-week old
C57BL/6J male mice as described under example 5. The islets were
then hand-picked and sorted according to size. Islets (n=5 per well
in a 96 well plate) were pre-incubated in 200 .mu.l KRB buffer
during 10 min 37.degree. C., pH 7.4, supplemented with 10 mM HEPES,
0.1% bovine serum albumin. Following pre-incubation, the buffer was
changed and islets were incubated in different test conditions in
new KRB buffer with 0.1% bovine serum albumin (non-treated ctrl,
FOL-014 peptide, and GLP-1) for 60 min at 37.degree. C. Immediately
after incubation, an aliquot of the buffer was removed and frozen
for subsequent assay of insulin. The result show that mouse
pancreatic islets stimulated with FOL-014 (6 .mu.M) secreted more
insulin compared to unstimulated control islets (FIG. 4B). GLP-1
(100 nM) or FOL-014 (0.6 .mu.M) did not affect insulin secretion
(FIG. 4B).
Example 8-11: Stimulation of Insulin Secretion from INS-1 Cell
Lines by FOL-014, FOL-005 and Related Peptides
[0406] Materials and methods: Rat INS-1 .beta.-cells (passages
60-70) were cultured at 37.degree. C. and 5% CO.sub.2 in cRPMI
media (RPMI 1640 supplemented with 10% fetal bovine serum, 50 IU/mL
penicillin, 50 mg/L streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1
mM sodium pyruvate, and 50 .mu.M beta-mercaptoethanol) unless
otherwise stated. INS-1 cells were seeded in 96-well plates
(2.times.10.sup.3 cells/well) in cRPMI medium and following
overnight incubation, the cells were washed in PBS before
pre-incubation for 120 min at 37.degree. C. in Krebs-Ringer
bicarbonate buffer, pH 7.4, supplemented with 10 mM HEPES, 0.1%
bovine serum albumin and 2.8 mM glucose. Following pre-incubation,
the buffer was exchanged with fresh Krebs-Ringer buffer as
described above and supplemented with specific glucose
concentrations and peptides for the individual experiments as
described below. Immediately after 60 minutes incubation at
37.degree. C., an aliquot of the buffer was removed and frozen for
subsequent insulin ELIZA assay.
Example 8. FOL-014 Induced Insulin Secretion is Dose-Dependent in a
Non-Linear Manner
[0407] Insulin release from INS-1 cells were measured following
exposure to increasing concentrations of FOL-014 and compared with
the stimulatory effect of GLP-1 and untreated control during high
glucose concentration (16.7 mM). All concentrations of FOL-014
tested elicited significantly higher insulin release as compared
with the untreated control. At 6 nM or higher, FOL-014 triggered
insulin release within the same range as 100 mM GLP-1. At
concentrations ranging from 0.6-60 nM, insulin secretion increased
in a linear fashion in relation to increasing FOL-014
concentrations. Exposure to FOL-014 concentrations 600 nM did not
increase the insulin secretion (FIG. 5).
[0408] The results demonstrated that FOL-014 significantly
increased insulin secretion from INS-1 .beta.-cells in vitro in a
non-linear dose dependent fashion.
Example 9. The Capacity of FOL-014 to Induce Insulin Secretion is
Glucose Dependent
[0409] Insulin release from INS-1 cells was measured following
exposure to 60 nM FOL-014 at increasing concentrations of glucose.
In untreated control samples, elevated glucose concentrations
increased the insulin secretion at 11.1 mM glucose or higher. In
the presence of FOL-014, insulin secretion increased significantly
in a glucose dependent fashion already from 5.5 mM glucose. (FIG.
6).
[0410] The results demonstrated that the presence of FOL-014
significantly increased insulin secretion from INS-1 .beta.-cells
in vitro in a glucose concentration dependent fashion and that
FOL-014 was effective also at marginally elevated glucose
levels.
Example 10. FOL-014 or FOL-005 in Combination with GLP-1 Increased
Insulin Secretion as Compared with Either Peptide Alone
[0411] Insulin secretion from INS-1 cells was measured following
exposure to FOL-005, FOL-014, GLP-1 or combinations of those,
expressed as percentage of untreated control. The combined effect
of GLP-1 and FOL-014 resulted in a significantly higher insulin
release than GLP-1 or FOL-014 alone. The additive effect of the
combination of FOL-005 and GLP-1 was less pronounced, but did
however increase the insulin secretion as compared with GLP-1
alone. The experiments were performed in the presence of 16.7 mM
glucose (FIG. 7).
[0412] The results demonstrated that the combination of GLP-1 and
FOL-014 could further potentiate the insulin secretion from INS-1
cells in vitro as compared with each peptide alone. Furthermore,
the combination of FOL-005 and GLP-1 tendentially increased insulin
secretion.
Example 11. The Ability of Novel Peptide Analogues to Induce
Insulin Secretion in Pancreatic .beta.-Cell-Lines was
Investigated
[0413] Novel peptide analogues, derived from either FOL-005 or
FOL-014 were tested concerning their ability to induce insulin
secretion in two separate INS-1 cell lines in the presence of 16.7
mM glucose. FOL-005, FOL-014 and GLP-1 as well as a high glucose
(16.7 mM) and a low glucose (2.8 mM) control (not shown) was
included in each experiment and the peptide concentration was 100
nM. In order to correct for the variance between experiments, all
values were normalized to, and expressed as percentage of the
average value of the high glucose control in the individual
experiments. The analogues were subsequently ranked according to
performance (FIGS. 11A and 11B). Peptide analogues eliciting an
insulin response below the high glucose control average value were
considered non-functional and were hence excluded (not shown).
[0414] The results demonstrated the capacity of several novel
peptide analogues to enhance insulin secretion from INS-1
.beta.-cells in vitro.
Example 12. FOL-014 Increase Insulin Secretion from Mouse-Derived
Pancreatic Islets
[0415] Twelve-week-old male C57/bl6 mice were euthanized with
isoflurane and cervical dislocation. After clamping the hepatic
ducts, 3 ml of 0.9 U/ml collagenase P was injected into the bile
duct to inflate the pancreas. The pancreas was then removed and
digested for 19 min at 37.degree. C. The samples were vigorously
shaken to disrupt the tissue. The digest was quickly transferred
into ice cold Hank's Balanced Salts Solution with Ca.sup.2+ and
Mg.sup.2+. The suspension was allowed to sit for 8 min to allow the
islet to sink, and the islets were washed in the same manner our
times. The islets were then handpicked and sorted according to
size.
[0416] Freshly isolated islets were seeded in groups of 5 in a
96-well plate and preincubated for 1 h at 37.degree. C. in a
Krebs-Ringer bicarbonate buffer (pH 7.4). The islets were incubated
for 1 h at 37.degree. C. in Krebs-Ringer buffered solution
supplemented with 0.6 or 6 .mu.M FOL-014 or 100 nM GLP-1 or left
unsupplemented for control. Immediately after incubation, the
medium was removed for assays of insulin and glucagon using
Mercodia's ELISA kits. The effect of FOL-014 on insulin (FIGS. 8A
and B) and glucagon (FIGS. 8C and D) secretion from isolated mouse
islets was measured in the presence of low glucose (2.8 mM; FIGS.
8A and C) or high glucose (16.7 mM; FIGS. 8B and D) concentrations.
A significant effect of FOL-014 was observed in the presence of
high glucose for insulin and in the presence of both high and low
glucose for glucagon. The effect of FOL-014 differed from that of
GLP-1, which enhanced insulin secretion also in low glucose samples
but failed to inhibit glucagon secretion in low glucose
conditions.
[0417] The results demonstrated that FOL-014 enhanced insulin
secretion and inhibited glucagon secretion in pancreatic
islets.
Example 13. FOL-014 Reduced Plasma Glucose Levels in an
Intraperitoneal Glucose Tolerance Test (IPGTT) in Mice
[0418] Whole blood was collected for glucose and insulin
measurements from 10-week-old wild type male C57bl/6 mice. After a
4 hour fast, the mice were divided into three groups and given an
intraperitoneal injection (ip) of either saline, 30 nmol/kg peptide
(FIG. 9A) or 200 nmol/kg peptide (FIG. 9B). 15 min after the
FOL-014 or saline (control) injections, the mice were administered
2 g of glucose/kg ip. Blood glucose concentrations were measured at
5, 15, 30, 45 and 60 minutes after the glucose injection.
Statistical calculations were performed using student's t-test.
FOL-014 dosed at 200 nmol/kg significantly lowered the plasma
glucose levels as compared to the control when measured as area
under the curve. In addition, the difference was significant at 15,
30 and 45 minutes. At the 30 nmol/kg dose, FOL-014 lowered the
plasma glucose levels with a significant effect at 45 minutes after
the glucose injection.
[0419] The results demonstrated that FOL-014 could lower plasma
glucose levels in a glucose tolerance test performed on healthy
wild type mice.
Example 14. FOL-014 Delayed Onset of Type 1 Diabetes in BB Lyp/Lyp
Rats
[0420] BB lyp/lyp rats were randomized for placebo (sodium
chloride, 9 mg/ml) or FOL-014 treatment 3 times/week from day 40
until onset of type 1 diabetes, defined as plasma glucose levels
11.1 mM. The dose of 100 nmol/kg FOL-014 peptide in saline or
placebo (saline) was administered subcutaneously and the animals
were terminated immediately upon exceeding critical plasma glucose
levels. The difference between FOL-014 treated animals and animals
receiving placebo treatment was significant both when expressed as
average age for onset of type 1 diabetes (FIG. 10A) and when
described as percentage of animals developing type 1 diabetes per
day (FIG. 10B).
[0421] The results demonstrated that FOL-014 treatment
significantly delayed the onset of type-1 diabetes in BB lyp/lyp
rats.
Example 15. FOL-005 and FOL-014 Displayed Organ Specific
Distribution Patterns in Mice
[0422] 057BI/6 mice were injected subcutaneously with H.sup.3
labelled FOL-005 and euthanized at 1 h (FIG. 12A) or 2 h (FIG. 12B)
after injection. Following whole body sectioning the distribution
of the labelled peptide was visualised. Strong binding was evident
in pancreas and at the site of injection. Using Pearl Trilogy Small
Animal Imaging System, in vivo bio-distribution and tissue
localization of two Cy7.5 labelled peptides, FOL-005 (FIG. 12C) and
FOL-014 (FIG. 12D) in NMRI nude mice via subcutaneous injection was
investigated. High accumulation of the peptide was evident in the
pancreatic tissue area. The same distribution pattern was found
after i.v. administrations (not shown). The dose of each peptide
was 10 nmol per mouse. The mice were imaged before injection, at 5
min, 20 min, 50 min, 60 min, 2 hrs, 4 hrs, 6 hrs, 24 hrs and 48 hrs
post administration of labelled peptide.
Example 16. Tissue Specific Imaging for Diagnostic Use
[0423] Agents prepared as defined herein above are labelled by
conjugation to suitable imaging probe or moiety, using methods
known by those of skill in the art. The conjugated peptide-probe
agents are subsequently administered to a subject and
biodistribution is subsequently monitored e.g. up to 48 h after
administration. The conjugated agent is thus used as a diagnostic
or prognostic tool for investigation of pancreatic status. As such,
the conjugated agents are suitable for detecting, diagnosing, or
monitoring disease, disease processes and progression,
susceptibility, as well as to determine efficacy of a treatment.
The agents are particularly suited for monitoring the diabetic
status of a subject. The conjugated agents are also used for
monitoring and/or predicting risk of developing a disease,
specifically diabetes. The test is used alone or in combination
with other tests known by those of skill in the art, such as blood
tests, genetic testing, urine test, and biopsies.
TABLE-US-00055 Example 17 Sequence overview SEQ ID NO Sequence
Notes 1 VDTYDGDISVVYGLR FOL-005 2 VDTYDGDISVVYGLS 3 VDTYDGDISVVYGL
FOL-025 4 DTYDGDISVVYGLR 5 TYDGDISVVYGLRS 6 VDTYDGDISVVYG FOL-024 7
DTYDGDISVVYGL 8 TYDGDISVVYGLR 9 YDGDISVVYGLRS 10 VDTYDGDISVVY 11
DTYDGDISVVYG 12 TYDGDISVVYGL 13 YDGDISVVYGLR 14 DGDISVVYGLRS 15
VDTYDGDISVV 16 DTYDGDISVVY 17 TYDGDISVVYG 18 YDGDISVVYGL 19
DGDISVVYGLR 20 GDISVVYGLRS 21 VDTYDGDISV 22 DTYDGDISVV 23
TYDGDISVVY 24 YDGDISVVYG 25 DGDISVVYGL 26 GDISVVYGLR FOL-009h 27
DISVVYGLRS 28 VDTYDGDIS FOL-019h 29 DTYDGDISV 30 TYDGDISVV 31
YDGDISVVY 32 DGDISVVYG 33 GDISVVYGL 34 DISVVYGLR 35 ISVVYGLRS 36
VDTYDGDI 37 DTYDGDIS 38 TYDGDISV 39 YDGDISVV 40 DGDISVVY 41
GDISVVYG 42 DISVVYGL 43 ISVVYGLR 44 VDTYDGD 45 DTYDGDI 46 TYDGDIS
47 YDGDISV 48 DGDISVV 49 GDISVVY 50 DISVVYG 51 ISVVYGL 52 DTYDGD 53
TYDGDI 54 YDGDIS 55 DGDISV 56 GDISVV 57 DISVVY 58 ISVVYG 59 TYDGD
60 YDGDI 61 DGDIS 62 GDISV 63 DISVV 64 ISVVY 65 SVVYG 66
MRIAVICFCLLGITCA Wildtype human IPVKQADSGSSEEKQL osteopontin, i.e.
YNKYPDAVATWLNPDP Gen Bank: SQKQNLLAPQTLPSKS AAA59974.1
NESHDHMDDMDDEDDD DHVDSQDSIDSNDSDD VDDTDDSHQSDESHHS DESDELVTDFPTDLPA
TEVFTPVVPTVDTYDG RGDSVVYGLRSKSKKF RRPDIQYPDATDEDIT SHMESEELNGAYKAIP
VAQDLNAPSDWDSRGK DSYETSQLDDQSAETH SHKQSRLYKRKANDES NEHSDVIDSQELSKVS
REFHSHEFHSHEDMLV VDPKSKEEDKHLKFRI SHELDSASSEVN 67 VDTYDGRGDSVVYGLR
FOL-002 68 VDZ.sub.3Z4Z.sub.5GZ.sub.7Z.sub.8S Z.sub.3 is T or V;
Z.sub.10Z.sub.11YGLR Z4 is Y or P; Z.sub.5 is D or N; Z.sub.7 is D
or G; Z.sub.8 is I or G; Z.sub.10 is V or L; Z.sub.11 is V or A 69
VDVPNGDISLAYGLR FOL-004 70 DVPNGDISLAYGLRS 71 VDVPNGDISLAYGL
FOL-016 72 DVPNGDISLAYGLR FOL-007 73 VPNGDISLAYGLRS 74
VDVPNGDISLAYG FOL-017 75 DVPNGDISLAYGL 76 VPNGDISLAYGLR 77
PNGDISLAYGLRS 78 VDVPNGDISLAY 79 DVPNGDISLAYG 80 VPNGDISLAYGL 81
PNGDISLAYGLR FOL-008 82 NGDISLAYGLRS 83 VDVPNGDISLA FOL-018 84
DVPNGDISLAY 85 VPNGDISLAYG 86 PNGDISLAYGL 87 NGDISLAYGLR 88
GDISLAYGLRS 89 VDVPNGDISL 90 DVPNGDISLA 91 VPNGDISLAY 92 PNGDISLAYG
93 NGDISLAYGL 94 GDISLAYGLR FOL-009 95 DISLAYGLRS 96 VDVPNGDIS
FOL-019 97 DVPNGDISL 98 VPNGDISLA 99 PNGDISLAY 100 NGDISLAYG 101
GDISLAYGL 102 DISLAYGLR 103 ISLAYGLRS 104 VDVPNGDI 105 DVPNGDIS 106
VPNGDISL 107 PNGDISLA 108 NGDISLAY 109 GDISLAYG 110 DISLAYGL
111 ISLAYGLR 112 VDVPNGD 113 DVPNGDI 114 VPNGDIS 115 PNGDISL 116
NGDISLA 117 GDISLAY 118 DISLAYG 119 ISLAYGL 120 DVPNGD 121 VPNGDI
122 PNGDIS 123 NGDISL 124 GDISLA 125 DISLAY 126 ISLAYG 127 VPNGD
128 PNGDI 129 NGDIS 130 GDISL 131 DISLA 132 ISLAY 133 SLAYG 134
MRLAVICFCLFGIASS Wildtype murine LPVKVTDSGSSEEKLY osteopontin, i.e.
SLHPDPIATWLVPDPS NCBI Reference QKQNLLAPQNAVSSEE Sequence:
KDDFKQETLPSNSNES NP_001191162.1 HDHMDDDDDDDDDDGD HAESEDSVDSDESDES
HHSDESDETVTASTQA DTFTPIVPTVDVPNGR GDSLAYGLRSKSRSFQ VSDEQYPDATDEDLTS
HMKSGESKESLDVIPV AQLLSMPSDQDNNGKG SHESSQLDEPSLETHR LEHSKESQESADQSDV
IDSQASSKASLEHQSH KFHSHKDKLVLDPKSK EDDRYLKFRISHELES SSSEVN 135
VDVPNGRGDSLAYGLR FOL-001 136 KPLAEIDSIELSYGIK FOL-014 137
GDPNDGRGDSVVYGLR FOL-003 138 VDTYDGGISVVYGLR FOL-026 139
VDTYDGDGSVVYGLR FOL-027 140
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10 X.sub.2 is C, P or
G; LX.sub.12YGIK X.sub.5 is E or G; X.sub.6 is C, D or I; X.sub.7
is D, I, S or G; X.sub.8 is S, D or G; X.sub.10 is E or G; X.sub.12
i S S or T; 141 KCLAECDSIELSYGIK FOL-032 (Cyclic) 142 CLAEIDSC
FOL-033 (Cyclic) 143 CFKPLAEIDSIECSYG FOL-036 IK (Cyclic) 144
KPLAEDISIELSYGIK FOL-037 145 KPLAEISDIELSYGIK FOL-038 146
KPLAEIGDIELSYGIK FOL-039 147 KPLAEGDIELSYGIK FOL-040 148
KPLAEIELSYGIK FOL-041 149 KPLAEIDSIELTYGIK FOL-042 150
KPLAEIDGIELSYGIK FOL-043 151 KPLAEIDGIELTYGIK FOL-044 152
KPLAEIGSIELSYGIK FOL-045 153 KGLAEIDSIELSYGIK FOL-046 154
KPLAGIDSIGLSYGIK FOL-047 155 Cyclic FOL-034 KCLAEIDSCELSYGIK 156
Cyclic FOL-035 CFKPLAEIDSIEC 157 VDVPEGDISLAYGLR FOL-010 158
LDGLVRAYDNISPVG FOL-015 159 GDPNGDISVVYGLR FOL-006 160
VDVPNGDISLAYRLR FOL-011 161 VDVPEGDISLAYRLR FOL-012 162
KX.sub.2LAX.sub.5X.sub.6X.sub.7X.sub.8IX.sub.10 X.sub.2 is C, P or
G; LSYGIK X.sub.5 is E or G; X.sub.6 is C, I or absent; X.sub.7 is
D, G or absent; X.sub.8 is S, G or absent; X.sub.10 is E or G; 163
KX.sub.2LAX.sub.5IX.sub.10LSYGIK X.sub.2 iS C, P or G; X.sub.5 is E
or G; X.sub.10 is E or G. 164 VDVPZ.sub.5GDISLAYZ.sub.13 Z.sub.5 is
E or N; LR Z.sub.13 is R or G. 165 VDTYDGZ.sub.7Z.sub.8SVVYGLR
Z.sub.7 is D or G; Z.sub.8 is I or G. 166
GDPNZ.sub.5Z6Z.sub.7Z.sub.8Z.sub.9SV Z.sub.5 is D or G; VYGLR Z6 is
D or G Z.sub.7 is I or R; Z.sub.8 is G or absent; Z.sub.9 is D or
absent. 167 VZ.sub.2TYDGDISVVYGLR Z.sub.2 is beta D FOL-005
(2betaAsp) 168 VDTY Z.sub.5 is beta D Z.sub.5GDISVVYGLR FOL-005
(5betaAsp) 169 VDTYDG FOL-005 (7betaAsp) Z.sub.7ISVVYGLR Z.sub.7 is
beta D
Sequence CWU 1
1
169115PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide, FOL-005 1Val Asp Thr Tyr Asp
Gly Asp Ile Ser Val Val Tyr Gly Leu Arg1 5 10 15215PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(15)peptide 2Val Asp Thr
Tyr Asp Gly Asp Ile Ser Val Val Tyr Gly Leu Ser1 5 10
15314PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(14)peptide, FOL-025 3Val Asp Thr Tyr Asp
Gly Asp Ile Ser Val Val Tyr Gly Leu1 5 10414PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(14)peptide 4Asp Thr Tyr
Asp Gly Asp Ile Ser Val Val Tyr Gly Leu Arg1 5 10514PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(14)peptide 5Thr Tyr Asp
Gly Asp Ile Ser Val Val Tyr Gly Leu Arg Ser1 5 10613PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(13)peptide, FOL-024
6Val Asp Thr Tyr Asp Gly Asp Ile Ser Val Val Tyr Gly1 5
10713PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 7Asp Thr Tyr Asp Gly Asp Ile
Ser Val Val Tyr Gly Leu1 5 10813PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 8Thr Tyr Asp Gly Asp Ile Ser
Val Val Tyr Gly Leu Arg1 5 10913PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 9Tyr Asp Gly Asp Ile Ser Val
Val Tyr Gly Leu Arg Ser1 5 101012PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 10Val Asp Thr Tyr Asp Gly Asp
Ile Ser Val Val Tyr1 5 101112PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 11Asp Thr Tyr Asp Gly Asp Ile
Ser Val Val Tyr Gly1 5 101212PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 12Thr Tyr Asp Gly Asp Ile Ser
Val Val Tyr Gly Leu1 5 101312PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 13Tyr Asp Gly Asp Ile Ser Val
Val Tyr Gly Leu Arg1 5 101412PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 14Asp Gly Asp Ile Ser Val Val
Tyr Gly Leu Arg Ser1 5 101511PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 15Val Asp Thr Tyr Asp Gly Asp
Ile Ser Val Val1 5 101611PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 16Asp Thr Tyr Asp Gly Asp Ile
Ser Val Val Tyr1 5 101711PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 17Thr Tyr Asp Gly Asp Ile Ser
Val Val Tyr Gly1 5 101811PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 18Tyr Asp Gly Asp Ile Ser Val
Val Tyr Gly Leu1 5 101911PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 19Asp Gly Asp Ile Ser Val Val
Tyr Gly Leu Arg1 5 102011PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 20Gly Asp Ile Ser Val Val Tyr
Gly Leu Arg Ser1 5 102110PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 21Val Asp Thr Tyr Asp Gly Asp
Ile Ser Val1 5 102210PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 22Asp Thr Tyr Asp Gly Asp Ile
Ser Val Val1 5 102310PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 23Thr Tyr Asp Gly Asp Ile Ser
Val Val Tyr1 5 102410PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 24Tyr Asp Gly Asp Ile Ser Val
Val Tyr Gly1 5 102510PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 25Asp Gly Asp Ile Ser Val Val
Tyr Gly Leu1 5 102610PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide, FOL-009h 26Gly Asp Ile Ser
Val Val Tyr Gly Leu Arg1 5 102710PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 27Asp Ile Ser Val Val Tyr Gly
Leu Arg Ser1 5 10289PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide, FOL-019h 28Val Asp Thr Tyr Asp
Gly Asp Ile Ser1 5299PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 29Asp Thr Tyr Asp Gly Asp Ile
Ser Val1 5309PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 30Thr Tyr Asp Gly Asp Ile Ser
Val Val1 5319PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 31Tyr Asp Gly Asp Ile Ser Val
Val Tyr1 5329PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 32Asp Gly Asp Ile Ser Val Val
Tyr Gly1 5339PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 33Gly Asp Ile Ser Val Val Tyr
Gly Leu1 5349PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 34Asp Ile Ser Val Val Tyr Gly
Leu Arg1 5359PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 35Ile Ser Val Val Tyr Gly Leu
Arg Ser1 5368PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 36Val Asp Thr Tyr Asp Gly Asp
Ile1 5378PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 37Asp Thr Tyr Asp Gly Asp Ile
Ser1 5388PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 38Thr Tyr Asp Gly Asp Ile Ser
Val1 5398PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 39Tyr Asp Gly Asp Ile Ser Val
Val1 5408PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 40Asp Gly Asp Ile Ser Val Val
Tyr1 5418PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 41Gly Asp Ile Ser Val Val Tyr
Gly1 5428PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 42Asp Ile Ser Val Val Tyr Gly
Leu1 5438PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 43Ile Ser Val Val Tyr Gly Leu
Arg1 5447PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 44Val Asp Thr Tyr Asp Gly Asp1
5457PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 45Asp Thr Tyr Asp Gly Asp Ile1
5467PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 46Thr Tyr Asp Gly Asp Ile Ser1
5477PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 47Tyr Asp Gly Asp Ile Ser Val1
5487PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 48Asp Gly Asp Ile Ser Val Val1
5497PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 49Gly Asp Ile Ser Val Val Tyr1
5507PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 50Asp Ile Ser Val Val Tyr Gly1
5517PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 51Ile Ser Val Val Tyr Gly Leu1
5526PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 52Asp Thr Tyr Asp Gly Asp1
5536PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 53Thr Tyr Asp Gly Asp Ile1
5546PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 54Tyr Asp Gly Asp Ile Ser1
5556PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 55Asp Gly Asp Ile Ser Val1
5566PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 56Gly Asp Ile Ser Val Val1
5576PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 57Asp Ile Ser Val Val Tyr1
5586PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 58Ile Ser Val Val Tyr Gly1
5595PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 59Thr Tyr Asp Gly Asp1
5605PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 60Tyr Asp Gly Asp Ile1
5615PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 61Asp Gly Asp Ile Ser1
5625PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 62Gly Asp Ile Ser Val1
5635PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 63Asp Ile Ser Val Val1
5645PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 64Ile Ser Val Val Tyr1
5655PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 65Ser Val Val Tyr Gly1
566300PRTHomo sapiensMISC_FEATURE(1)..(300)Wildtype human
osteopontin 66Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile
Thr Cys Ala1 5 10 15Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu
Glu Lys Gln Leu 20 25 30Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp
Leu Asn Pro Asp Pro 35 40 45Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln
Thr Leu Pro Ser Lys Ser 50 55 60Asn Glu Ser His Asp His Met Asp Asp
Met Asp Asp Glu Asp Asp Asp65 70 75 80Asp His Val Asp Ser Gln Asp
Ser Ile Asp Ser Asn Asp Ser Asp Asp 85 90 95Val Asp Asp Thr Asp Asp
Ser His Gln Ser Asp Glu Ser His His Ser 100 105 110Asp Glu Ser Asp
Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala 115 120 125Thr Glu
Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr Asp Gly 130 135
140Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys
Phe145 150 155 160Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp
Glu Asp Ile Thr 165 170 175Ser His Met Glu Ser Glu Glu Leu Asn Gly
Ala Tyr Lys Ala Ile Pro 180 185 190Val Ala Gln Asp Leu Asn Ala Pro
Ser Asp Trp Asp Ser Arg Gly Lys 195 200 205Asp Ser Tyr Glu Thr Ser
Gln Leu Asp Asp Gln Ser Ala Glu Thr His 210 215 220Ser His Lys Gln
Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser225 230 235 240Asn
Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser 245 250
255Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu Val
260 265 270Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe
Arg Ile 275 280 285Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
290 295 3006716PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide, FOL-002 67Val Asp Thr Tyr Asp
Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg1 5 10
156815PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptideMISC_FEATURE(3)..(3)Z is T or
VMISC_FEATURE(4)..(4)Z is Y or PMISC_FEATURE(5)..(5)Z is D or
NMISC_FEATURE(7)..(7)Z is D or GMISC_FEATURE(8)..(8)Z is I or
GMISC_FEATURE(10)..(10)Z is V or LMISC_FEATURE(11)..(11)Z is V or A
68Val Asp Glx Glx Glx Gly Glx Glx Ser Glx Glx Tyr Gly Leu Arg1 5 10
156915PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide, FOL-004 69Val Asp Val Pro Asn
Gly Asp Ile Ser Leu Ala Tyr Gly Leu Arg1 5 10 157015PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(15)peptide 70Asp Val
Pro Asn Gly Asp Ile Ser Leu Ala Tyr Gly Leu Arg Ser1 5 10
157114PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(14)peptide 71Val Asp Val Pro Asn Gly Asp
Ile Ser Leu Ala Tyr Gly Leu1 5 107214PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(14)peptide 72Asp Val
Pro Asn Gly Asp Ile Ser Leu Ala Tyr Gly Leu Arg1 5
107314PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(14)peptide 73Val Pro Asn Gly Asp Ile Ser
Leu Ala Tyr Gly Leu Arg Ser1 5 107413PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(13)peptide, FOL-017
74Val Asp Val Pro Asn Gly Asp Ile Ser Leu Ala Tyr Gly1 5
107513PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 75Asp Val Pro Asn Gly Asp Ile
Ser Leu Ala Tyr Gly Leu1 5 107613PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 76Val Pro Asn Gly Asp Ile Ser
Leu Ala Tyr Gly Leu Arg1 5 107713PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 77Pro Asn Gly Asp Ile Ser Leu
Ala Tyr Gly Leu Arg Ser1 5 107812PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 78Val Asp Val Pro Asn Gly Asp
Ile Ser Leu Ala Tyr1 5 107912PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 79Asp Val Pro Asn Gly Asp Ile
Ser Leu Ala Tyr Gly1 5 108012PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 80Val Pro Asn Gly Asp Ile Ser
Leu Ala Tyr Gly Leu1 5 108112PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 81Pro Asn Gly Asp Ile Ser Leu
Ala Tyr Gly Leu Arg1 5 108212PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(12)peptide 82Asn Gly Asp Ile Ser Leu Ala
Tyr Gly Leu Arg Ser1 5 108311PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 83Val Asp Val Pro Asn Gly Asp
Ile Ser Leu Ala1 5 108411PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 84Asp Val Pro Asn Gly Asp Ile
Ser Leu Ala Tyr1 5 108511PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 85Val Pro Asn Gly Asp Ile Ser
Leu Ala Tyr Gly1 5 108611PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 86Pro Asn Gly Asp Ile Ser Leu
Ala Tyr Gly Leu1 5 108711PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 87Asn Gly Asp Ile Ser Leu Ala
Tyr Gly Leu Arg1 5 108811PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(11)peptide 88Gly Asp Ile Ser Leu Ala Tyr
Gly Leu Arg Ser1 5 108910PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 89Val Asp Val Pro Asn Gly Asp
Ile Ser Leu1 5 109010PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 90Asp Val Pro Asn Gly Asp Ile
Ser Leu Ala1 5 109110PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 91Val Pro Asn Gly Asp Ile Ser
Leu Ala Tyr1 5 109210PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 92Pro Asn Gly Asp Ile Ser Leu
Ala Tyr Gly1 5 109310PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 93Asn Gly Asp Ile Ser Leu Ala
Tyr Gly Leu1 5 109410PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide, FOL-009 94Gly Asp Ile Ser Leu
Ala Tyr Gly Leu Arg1 5 109510PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(10)peptide 95Asp Ile Ser Leu
Ala Tyr Gly Leu Arg Ser1 5 10969PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptideMISC_FEATURE(1)..(9)peptide,
FOL-019 96Val Asp Val Pro Asn Gly Asp Ile Ser1 5979PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(9)peptide 97Asp Val Pro
Asn Gly Asp Ile Ser Leu1 5989PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 98Val Pro Asn Gly Asp Ile Ser
Leu Ala1 5999PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 99Pro Asn Gly Asp Ile Ser Leu
Ala Tyr1 51009PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 100Asn Gly Asp Ile Ser Leu Ala
Tyr Gly1 51019PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 101Gly Asp Ile Ser Leu Ala Tyr
Gly Leu1 51029PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 102Asp Ile Ser Leu Ala Tyr Gly
Leu Arg1 51039PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(9)peptide 103Ile Ser Leu Ala Tyr Gly Leu
Arg Ser1 51048PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 104Val Asp Val Pro Asn Gly Asp
Ile1 51058PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 105Asp Val Pro Asn Gly Asp Ile
Ser1 51068PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 106Val Pro Asn Gly Asp Ile Ser
Leu1 51078PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 107Pro Asn Gly Asp Ile Ser Leu
Ala1 51088PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 108Asn Gly Asp Ile Ser Leu Ala
Tyr1 51098PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 109Gly Asp Ile Ser Leu Ala Tyr
Gly1 51108PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 110Asp Ile Ser Leu Ala Tyr Gly
Leu1 51118PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(8)peptide 111Ile Ser Leu Ala Tyr Gly Leu
Arg1 51127PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 112Val Asp Val Pro Asn Gly Asp1
51137PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 113Asp Val Pro Asn Gly Asp Ile1
51147PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 114Val Pro Asn Gly Asp Ile Ser1
51157PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 115Pro Asn Gly Asp Ile Ser Leu1
51167PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 116Asn Gly Asp Ile Ser Leu Ala1
51177PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 117Gly Asp Ile Ser Leu Ala Tyr1
51187PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 118Asp Ile Ser Leu Ala Tyr Gly1
51197PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(7)peptide 119Ile Ser Leu Ala Tyr Gly Leu1
51206PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 120Asp Val Pro Asn Gly Asp1
51216PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 121Val Pro Asn Gly Asp Ile1
51226PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 122Pro Asn Gly Asp Ile Ser1
51236PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 123Asn Gly Asp Ile Ser Leu1
51246PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 124Gly Asp Ile Ser Leu Ala1
51256PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 125Asp Ile Ser Leu Ala Tyr1
51266PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(6)peptide 126Ile Ser Leu Ala Tyr Gly1
51275PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 127Val Pro Asn Gly Asp1
51285PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 128Pro Asn Gly Asp Ile1
51295PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 129Asn Gly Asp Ile Ser1
51305PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 130Gly Asp Ile Ser Leu1
51315PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 131Asp Ile Ser Leu Ala1
51325PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 132Ile Ser Leu Ala Tyr1
51335PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(5)peptide 133Ser Leu Ala Tyr Gly1
5134294PRTMus musculusMISC_FEATURE(1)..(294)Wildtype murine
osteopontin 134Met Arg Leu Ala Val Ile Cys Phe Cys Leu Phe Gly Ile
Ala Ser Ser1 5 10 15Leu Pro Val Lys Val Thr Asp Ser Gly Ser Ser Glu
Glu Lys Leu Tyr 20 25 30Ser Leu His Pro Asp Pro Ile Ala Thr Trp Leu
Val Pro Asp Pro Ser 35 40 45Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn
Ala Val Ser Ser Glu Glu 50 55 60Lys Asp Asp Phe Lys Gln Glu Thr Leu
Pro Ser Asn Ser Asn Glu Ser65 70 75 80His Asp His Met Asp Asp Asp
Asp Asp Asp Asp Asp Asp Asp Gly Asp 85 90 95His Ala Glu Ser Glu Asp
Ser Val Asp Ser Asp Glu Ser Asp Glu Ser 100 105 110His His Ser Asp
Glu Ser Asp Glu Thr Val Thr Ala Ser Thr Gln Ala 115 120 125Asp Thr
Phe Thr Pro Ile Val Pro Thr Val Asp Val Pro Asn Gly Arg 130 135
140Gly Asp Ser Leu Ala Tyr Gly Leu Arg Ser Lys Ser Arg Ser Phe
Gln145 150 155 160Val Ser Asp Glu Gln Tyr Pro Asp Ala Thr Asp Glu
Asp Leu Thr Ser 165 170 175His Met Lys Ser Gly Glu Ser Lys Glu Ser
Leu Asp Val Ile Pro Val 180 185 190Ala Gln Leu Leu Ser Met Pro Ser
Asp Gln Asp Asn Asn Gly Lys Gly 195 200 205Ser His Glu Ser Ser Gln
Leu Asp Glu Pro Ser Leu Glu Thr His Arg 210 215 220Leu Glu His Ser
Lys Glu Ser Gln Glu Ser Ala Asp Gln Ser Asp Val225 230 235 240Ile
Asp Ser Gln Ala Ser Ser Lys Ala Ser Leu Glu His Gln Ser His 245 250
255Lys Phe His Ser His Lys Asp Lys Leu Val Leu Asp Pro Lys Ser Lys
260 265 270Glu Asp Asp Arg Tyr Leu Lys Phe Arg Ile Ser His Glu Leu
Glu Ser 275 280 285Ser Ser Ser Glu Val Asn 29013516PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(16)peptide, FOL-001
135Val Asp Val Pro Asn Gly Arg Gly Asp Ser Leu Ala Tyr Gly Leu Arg1
5 10 1513616PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide, FOL-014 136Lys Pro Leu Ala
Glu Ile Asp Ser Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10
1513716PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide, FOL-003 137Gly Asp Pro Asn
Asp Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg1 5 10
1513815PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide, FOL-026 138Val Asp Thr Tyr
Asp Gly Gly Ile Ser Val Val Tyr Gly Leu Arg1 5 10
1513915PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide, FOL-027 139Val Asp Thr Tyr
Asp Gly Asp Gly Ser Val Val Tyr Gly Leu Arg1 5 10
1514016PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptideMISC_FEATURE(2)..(2)X is C, P
or GMISC_FEATURE(5)..(5)X is E or GMISC_FEATURE(6)..(6)X is C, D or
IMISC_FEATURE(7)..(7)X is D, I, S or GMISC_FEATURE(8)..(8)X is S, D
or GMISC_FEATURE(10)..(10)X is E or GMISC_FEATURE(12)..(12)X is S
or T 140Lys Xaa Leu Ala Xaa Xaa Xaa Xaa Ile Xaa Leu Xaa Tyr Gly Ile
Lys1 5 10 1514116PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 141Lys Cys Leu Ala Glu Cys Asp
Ser Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10 151428PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(8)peptide 142Cys Leu
Ala Glu Ile Asp Ser Cys1 514318PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(18)peptide 143Cys Phe Lys Pro Leu Ala Glu
Ile Asp Ser Ile Glu Cys Ser Tyr Gly1 5 10 15Ile
Lys14416PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 144Lys Pro Leu Ala Glu Asp Ile
Ser Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10 1514516PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(16)peptide 145Lys Pro
Leu Ala Glu Ile Ser Asp Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10
1514616PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 146Lys Pro Leu Ala Glu Ile Gly
Asp Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10 1514715PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(15)peptide 147Lys Pro
Leu Ala Glu Gly Asp Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10
1514813PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(13)peptide 148Lys Pro Leu Ala Glu Ile Glu
Leu Ser Tyr Gly Ile Lys1 5 1014916PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 149Lys Pro Leu Ala Glu Ile Asp
Ser Ile Glu Leu Thr Tyr Gly Ile Lys1 5 10 1515016PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(16)peptide 150Lys Pro
Leu Ala Glu Ile Asp Gly Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10
1515116PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 151Lys Pro Leu Ala Glu Ile Asp
Gly Ile Glu Leu Thr Tyr Gly Ile Lys1 5 10 1515216PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(16)peptide 152Lys Pro
Leu Ala Glu Ile Gly Ser Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10
1515316PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 153Lys Gly Leu Ala Glu Ile Asp
Ser Ile Glu Leu Ser Tyr Gly Ile Lys1 5 10 1515416PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(16)peptide 154Lys Pro
Leu Ala Gly Ile Asp Ser Ile Gly Leu Ser Tyr Gly Ile Lys1 5 10
1515516PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(16)peptide 155Lys Cys Leu Ala Glu Ile Asp
Ser Cys Glu Leu Ser Tyr Gly Ile Lys1 5 10 1515613PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(13)peptide 156Cys Phe
Lys Pro Leu Ala Glu Ile Asp Ser Ile Glu Cys1 5 1015715PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(15)peptide 157Val Asp
Val Pro Glu Gly Asp Ile Ser Leu Ala Tyr Gly Leu Arg1 5 10
1515815PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide 158Leu Asp Gly Leu Val Arg Ala
Tyr Asp Asn Ile Ser Pro Val Gly1 5 10 1515914PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(14)peptide 159Gly Asp
Pro Asn Gly Asp Ile Ser Val Val Tyr Gly Leu Arg1 5
1016015PRTArtificial SequenceSynthetic
sequenceMISC_FEATURE(1)..(15)peptide 160Val Asp Val Pro Asn Gly Asp
Ile Ser Leu Ala Tyr Arg Leu Arg1 5 10 1516115PRTArtificial
SequenceSynthetic sequenceMISC_FEATURE(1)..(15)peptide 161Val Asp
Val Pro Glu Gly Asp Ile Ser Leu Ala Tyr Arg Leu Arg1 5 10
1516216PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(16)peptideMISC_FEATURE(2)..(2)X is C, P or
GMISC_FEATURE(5)..(5)X is E or GMISC_FEATURE(6)..(6)X is C, I or
absentMISC_FEATURE(7)..(7)X is D, G or absentMISC_FEATURE(8)..(8)X
is S, G or absentMISC_FEATURE(10)..(10)X is E or G 162Lys Xaa Leu
Ala Xaa Xaa Xaa Xaa Ile Xaa Leu Ser Tyr Gly Ile Lys1 5 10
1516313PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(13)peptideMISC_FEATURE(2)..(2)X is C, P or
GMISC_FEATURE(5)..(5)X is E or GMISC_FEATURE(10)..(10)X is E or G
163Lys Xaa Leu Ala Xaa Ile Xaa Leu Ser Tyr Gly Ile Lys1 5
1016415PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(15)peptideMISC_FEATURE(1)..(5)Z is E or
NMISC_FEATURE(5)..(5)Z is E or NMISC_FEATURE(13)..(13)Z is R or G
164Val Asp Val Pro Glx Gly Asp Ile Ser Leu Ala Tyr Glx Leu Arg1 5
10 1516515PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(15)peptideMISC_FEATURE(7)..(7)Z is D or
GMISC_FEATURE(8)..(8)Z is I or G 165Val Asp Thr Tyr Asp Gly Glx Glx
Ser Val Val Tyr Gly Leu Arg1 5 10 1516616PRTArtificial
Sequencesynthetic
peptideMISC_FEATURE(1)..(16)peptideMISC_FEATURE(5)..(5)Z is D or
GMISC_FEATURE(6)..(6)Z is D or GMISC_FEATURE(7)..(7)Z is I or
RMISC_FEATURE(8)..(8)Z is G or absentMISC_FEATURE(9)..(9)Z is D or
absent 166Gly Asp Pro Asn Glx Glx Glx Glx Glx Ser Val Val Tyr Gly
Leu Arg1 5 10 1516715PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(15)peptideMISC_FEATURE(2)..(2)Z is beta D
167Val Glx Thr Tyr Asp Gly Asp Ile Ser Val Val Tyr Gly Leu Arg1 5
10 1516815PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(15)peptideMISC_FEATURE(5)..(5)Z is beta D
168Val Asp Thr Tyr Glx Gly Asp Ile Ser Val Val Tyr Gly Leu Arg1 5
10 1516915PRTArtificial Sequencesynthetic
peptideMISC_FEATURE(1)..(15)peptideMISC_FEATURE(7)..(7)Z is beta D
169Val Asp Thr Tyr Asp Gly Glx Ile Ser Val Val Tyr Gly Leu Arg1 5
10 15
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