U.S. patent application number 11/090787 was filed with the patent office on 2005-12-08 for peptide analogues of gip for treatment of diabetes, insulin resistance and obesity.
Invention is credited to Flatt, Peter Raymond, Gault, Victor A., Harriott, Patrick, Irwin, Nigel, O'Harte, Finbarr Paul Mary.
Application Number | 20050272652 11/090787 |
Document ID | / |
Family ID | 35449761 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272652 |
Kind Code |
A1 |
Gault, Victor A. ; et
al. |
December 8, 2005 |
Peptide analogues of GIP for treatment of diabetes, insulin
resistance and obesity
Abstract
The present invention provides peptide analogues which are
antagonists of gastric inhibitory peptide (GIP). The peptides,
based on GIP 1-42 include substitutions and/or modifications which
have enhanced resistance to degradation by the enzyme dipeptidyl
peptidase IV (DPP IV). The invention also provides a process of N
terminally modifying GIP and the use of the peptide analogues for
treatment of diabetes.
Inventors: |
Gault, Victor A.; (Coleraine
Co Londonderry, IE) ; O'Harte, Finbarr Paul Mary;
(Coleraine Co Londonderry, IE) ; Irwin, Nigel;
(Coleraine Co Londonderry, IE) ; Harriott, Patrick;
(Coleraine Co Londonderry, IE) ; Flatt, Peter
Raymond; (Coleraine Co Londonderry, IE) |
Correspondence
Address: |
KIRKPATRICK & LOCKHART NICHOLSON GRAHAM LLP
(FORMERLY KIRKPATRICK & LOCKHART LLP)
75 STATE STREET
BOSTON
MA
02109-1808
US
|
Family ID: |
35449761 |
Appl. No.: |
11/090787 |
Filed: |
March 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11090787 |
Mar 25, 2005 |
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PCT/GB05/00710 |
Feb 25, 2005 |
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11090787 |
Mar 25, 2005 |
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09937687 |
Jan 8, 2002 |
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6921748 |
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09937687 |
Jan 8, 2002 |
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PCT/GB00/01089 |
Mar 29, 2000 |
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Current U.S.
Class: |
514/6.9 ;
514/11.7; 514/4.8; 514/5.3; 530/324 |
Current CPC
Class: |
Y10T 29/4935 20150115;
C07K 14/575 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/012 ;
530/324 |
International
Class: |
A61K 038/22 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 2004 |
GB |
GB0404124.0 |
Mar 29, 1999 |
GB |
GB9907216.7 |
Jul 27, 1999 |
GB |
GB9917565.5 |
Claims
1. A peptide analogue of GIP(1-42) (SEQ ID NO: 1), comprising at
least 12 amino acid residues from the N-terminal end of
GIP(3-42).
2. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(1-42)
and having an amino acid substitution at Glu.sup.3.
3. The peptide analogue of claim 2, wherein the amino acid
substituted at Glu.sup.3 is selected from the group consisting of:
proline, hydroxyproline, lysine, tyrosine, phenylalanine and
tryptophan.
4. The peptide analogue of claim 3, wherein proline is substituted
for Glu.sup.3.
5. The peptide analogue of claim 2, further comprising modification
by fatty acid addition at an epsilon amino group of at least one
lysine residue.
6. The peptide analogue of claim 5, wherein the modification is the
linking of a C-16 palmitate group to the epsilon amino group of a
lysine residue.
7. The peptide analogue of claim 6, wherein the lysine residue is
Lys.sup.16.
8. The peptide analogue of claim 6, wherein the lysine residue is
Lys.sup.37.
9. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(1-42),
and having an amino acid modification at amino acid residues 1, 2
or 3.
10. The peptide analogue of claim 9, wherein the N-terminal amino
acid residue is acetylated.
11. The peptide analogue of claim 10, further comprising
modification by fatty acid addition at an epsilon amino group of at
least one lysine residue.
12. The peptide analogue of claim 11, wherein the modification is
the linking of a C-16 palmitate group to the epsilon amino group of
a lysine residue.
13. The peptide analogue of claim 12, wherein the lysine residue is
Lys.sup.16.
14. The peptide analogue of claim 12, wherein the lysine residue is
Lys.sup.37.
15. A pharmaceutical composition comprising the peptide analogue of
claim 2.
16. The pharmaceutical composition of claim 15, further comprising
a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 15, wherein the peptide
analogue is in the form of a pharmaceutically acceptable salt.
18. The pharmaceutical composition of claim 15, wherein the peptide
analogue is in the form of a pharmaceutically acceptable acid
addition salt.
19. A method of treating insulin resistance, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
15.
20. A method of treating obesity, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
15.
21. A method of treating type 2 diabetes, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
15.
22. A peptide analogue of GIP(1-42) (SEQ ID NO: 1), wherein the
analogue comprises: a base peptide consisting of one of the
following: GIP(1-12), GIP(1-13), GIP(1-14), GIP(1-15), GIP(1-16),
GIP(1-17), GIP(1-18), GIP(1-19), GIP(1-20), GIP(1-21), GIP(1-22),
GIP(1-23), GIP(1-24), GIP(1-25), GIP(1-26), GIP(1-27), GIP(1-28),
GIP(1-29), GIP(1-30), GIP(1-31), GIP(1-32), GIP(1-33), GIP(1-34),
GIP(1-35), GIP(1-36), GIP(1-37), GIP(1-38), GIP(1-39), GIP(1-40),
GIP(1-41) and GIP(1-42); which possesses one or more of the
following modifications: an amino acid substitution at one or more
of the residues; an amino acid substitution of lysine for one or
more or the residues; an amino acid substitution at Glu.sup.3; a
modification by fatty acid addition at an epsilon amino group of at
least one lysine residue; and a modification by N-terminal
acetylation.
23. The peptide analogue of claim 22, wherein the analogue has a
proline substituted for Glu.sup.3.
24. The peptide analogue of claims 22, further comprising
modification by fatty acid addition at an epsilon amino group of at
least one lysine residue.
25. The peptide analogue of claim 24, wherein the modification is
the linking of a C-16 palmitate group to the epsilon amino group of
a lysine residue.
26. The peptide analogue of claim 25, wherein the lysine residue is
Lys.sup.16.
27. The peptide analogue of claim 25, wherein the lysine residue is
Lys.sup.37.
28. A pharmaceutical composition comprising the peptide analogue of
claim 22.
29. The pharmaceutical composition of claim 28, further comprising
a pharmaceutically acceptable carrier.
30. The pharmaceutical composition of claim 28, wherein the peptide
analogue is in the form of a pharmaceutically acceptable salt.
31. The pharmaceutical composition of claim 28, wherein the peptide
analogue is in the form of a pharmaceutically acceptable acid
addition salt.
32. A method of treating insulin resistance, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
28.
33. A method of treating obesity, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
28.
34. A method of treating type 2 diabetes, the method comprising
administering to a mammal in need of such treatment a
therapeutically effective amount of the composition of claim
28.
35. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(3-42),
wherein the peptide analogue is resistant to degradation by enzyme
DPP IV when compared to naturally-occurring GIP.
36. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(1-42)
and having an amino acid substitution at Glu.sup.3, wherein the
peptide analogue is resistant to degradation by enzyme DPP IV when
compared to naturally-occurring GIP.
37. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(3-42),
wherein the peptide analogue modulates insulin secretion.
38. A peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at
least 12 amino acid residues from the N-terminal end of GIP(1-42)
and having an amino acid substitution at Glu.sup.3, wherein the
peptide analogue modulates insulin secretion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
International Application No. PCT/GB2005/000710, which was filed on
Feb. 25, 2005, designated the United States and was published in
English, which claims benefit of U.K. Application No. GB 0404124.0,
filed on Feb. 25, 2004. The present application is also a
continuation-in-part of U.S. application Ser. No. 09/937,687, filed
Jan. 8, 2002, which is the U.S. National Phase Application of
International Application No. PCT/GB00/01089, which is designated
the United States and was filed on Mar. 29, 2000 and published in
English, which in turn claims the benefit of GB9907216.7, filed on
Mar. 29, 1999, and GB9917565.5, filed Jul. 27, 1999. The entire
teachings of the above applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the release of insulin and
the control of blood glucose concentration. More particularly the
invention relates to antagonists of gastric inhibitory peptide
(GIP) as pharmaceutical preparations for treatment of type 2
diabetes.
BACKGROUND
[0003] Obesity and diabetes are predicted to reach epidemic
proportions throughout the world in the next 20 years and current
treatments do not restore normal insulin sensitivity or glucose
homeostasis, therein resulting in debilitating diabetic
complications and premature death.
[0004] Gastric inhibitory polypeptide (GIP) and glucagon-like
peptide-1(7-36)amide (truncated GLP-1; tGLP-1) are two important
insulin-releasing hormones secreted from endocrine cells in the
intestinal tract in response to feeding. Together with autonomic
nerves they play a vital supporting role to the pancreatic islets
in the control of blood glucose homeostasis and nutrient
metabolism.
[0005] GIP is released from intestinal endocrine K-cells into the
bloodstream following ingestion of carbohydrate, protein and
particularly fat (Meier, J. J. et al., 2002, Regul. Pept.
107:1-13). GIP was initially discovered through its ability to
inhibit gastric acid secretion (Brown, J. C. et al. 1969, Can. J.
Physiol. Pharmacol. 47:113-114) but its major physiological role is
now generally believed to be that of an incretin hormone that
targets pancreatic islets to enhance insulin secretion and help
reduce postprandial hyperglycemia (Creutzfeldt, W., 2001, Exp.
Clin. Endocrinol. Diabetes 109:S288-S303). GIP acts through binding
to specific G-protein coupled GIP receptors located on pancreatic
beta-cells (Wheeler, M. B. et al., 1995, Endocrinology
136:4629-4639). Like its sister incretin hormone, glucagon-like
peptide-1 (GLP-1), this ability to stimulate insulin secretion plus
other potentially beneficial actions on pancreatic beta-cell growth
and differentiation have led to much interest in using GIP or GLP-1
receptor agonists in the treatment of type 2 diabetes (Creutzfeldt,
W., 2001, Exp. Clin. Endocrinol. Diabetes 109:S288-S303; Holz, G.
G. et al., 2003, Curr. Med. Chem. 10:2471-2483).
[0006] Since GIP functions as a potent and natural stimulator of
insulin secretion released from the intestine by feeding, it is
widely expected that antagonists opposing GIP action will block the
insulin-releasing actions of GIP and impair both oral glucose
tolerance and the glycemic response to nutrient ingestion. In fact,
all studies published to date indicate that GIP is a key
physiological component of the enteroinsular axis and that
functional ablation of GIP leads to impaired glucose homeostasis
moving the metabolic characteristic towards a type 2 diabetes
phenotype (Gault, V. A. et al., 2002, Biochem. Biophys. Res.
Commun. 290:1420-1426).
[0007] Dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) has been
identified as a key enzyme responsible for inactivation of GIP and
tGLP-1 in serum. This occurs through the rapid removal of the
N-terminal dipeptides Tyr.sup.1-Ala.sup.2 and His.sup.7-Ala.sup.8
giving rise to the main metabolites GIP(3-42) and GLP-1(9-36)amide,
respectively. These truncated peptides are reported to lack
biological activity or to even serve as antagonists at GIP or
tGLP-1 receptors. The resulting biological half-lives of these
incretin hormones in vivo are therefore very short, estimated to be
no longer than 5 minutes. DPP IV is completely inhibited in serum
by the addition of diprotin A (DPA, 0.1 mmol/l).
[0008] In situations of normal glucose regulation and pancreatic
B-cell sensitivity, this short duration of action is advantageous
in facilitating momentary adjustments to homeostatic control.
However, the current goal of a possible therapeutic role of
incretin hormones, particularly tGLP-1 in non-insulin dependent
diabetes (NIDDM) therapy is frustrated by a number of factors in
addition to finding a convenient route of administration. Most
notable of these are rapid peptide degradation and rapid absorption
(peak concentrations are reached in 20 minutes) and the resulting
need for both high dosage and precise timing with meals. Recent
therapeutic strategies have focused on precipitated preparations to
delay peptide absorption and inhibition of GLP-1 degradation using
specific inhibitors of DPP IV. A possible therapeutic role is also
suggested by the observation that a specific inhibitor of DPP IV,
isoleucine thiazolidide, lowered blood glucose and enhanced insulin
secretion in glucose-treated diabetic obese Zucker rats presumably
by protecting against catabolism of the incretin hormones tGLP-1
and GIP.
[0009] Studies have indicated that tGLP-1 infusion restores
pancreatic B-cell sensitivity, insulin secretory oscillations and
improved glycemic control in various groups of patients with
impaired glucose tolerance (IGT) or NIDDM. Longer term studies also
show significant benefits of tGLP-1 injections in NIDDM and
possibly IDDM therapy, providing a major incentive to develop an
orally effective or long-acting tGLP-1 analogue. Several attempts
have been made to produce structurally modified analogues of tGLP-1
which are resistant to DPP IV degradation. A significant extension
of serum half-life is observed with His.sup.7-glucitol tGLP-1 and
tGLP-1 analogues substituted at position 8 with Gly, Aib (amino
isobutyric acid), Ser or Thr. However, these structural
modifications seem to impair receptor binding and insulinotrophic
activity thereby compromising part of the benefits of protection
from proteolytic degradation. In recent studies using
His.sup.7-glucitol tGLP-1, resistance to DPP IV and serum
degradation was accompanied by severe loss of insulin releasing
activity.
[0010] GIP shares not only the same degradation pathway as tGLP-1
but many similar physiological actions, including stimulation of
insulin and somatostatin secretion, and the enhancement of glucose
disposal. These actions are viewed as key aspects in the
antihyperglycemic properties of tGLP-1, and there is therefore good
expectation that GIP may have similar potential as NIDDM therapy.
Indeed, compensation by GIP is held to explain the modest
disturbances of glucose homeostasis observed in tGLP-1 knockout
mice. Apart from early studies, the anti-diabetic potential of GIP
has not been explored and tGLP-1 may seem more attractive since it
is viewed by some as a more potent insulin secretagogue when
infused at so called physiological concentrations estimated by
radioimmunoassay (RIA).
[0011] There is therefore a need for a diabetes treatment that
includes an analogue of GIP which can cause release of insulin, yet
also be resistant to rapid degradation by DPP IV.
SUMMARY OF THE INVENTION
[0012] Disclosed herein are GIP antagonist peptides which are
resistant to rapid degradation by DPP IV.
[0013] The invention includes a peptide analogue of GIP(1-42) (SEQ
ID NO: 1), which includes at least 12 amino acid residues from the
N-terminal end of GIP(3-42). The invention also includes a peptide
analogue of GIP(1-42) (SEQ ID NO:1), which includes at least 12
amino acid residues from the N-terminal end of GIP(1-42) and having
an amino acid substitution at Glu.sup.3.
[0014] The amino acid substituted at Glu.sup.3 can be selected from
the group consisting of: proline, hydroxyproline, lysine, tyrosine,
phenylalanine and tryptophan. Specifically, a proline can be
substituted for Glu.sup.3. The peptide analogue can further include
modification by fatty acid addition at an epsilon amino group of at
least one lysine residue. The lysine residue can be Lys.sup.16 or
Lys.sup.37.
[0015] The peptide analogue of GIP(1-42) (SEQ ID NO: 1) can include
at least 12 amino acid residues from the N-terminal end of
GIP(1-42), and an amino acid modification at amino acid residues 1,
2 or 3. The N-terminal amino acid residue can be acetylated. It can
further comprising modification by fatty acid addition at an
epsilon amino group of at least one lysine residue. The
modification can be the linking of, e.g., a C-8, a C-10, a C-12, a
C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino
group of a lysine residue. The lysine residue can be Lys.sup.16, or
Lys.sup.37.
[0016] The invention also includes a peptide analogue of GIP(1-42)
(SEQ ID NO: 1), wherein the analogue comprises a base peptide
consisting of one of the following: GIP(1-12), GIP(1-13),
GIP(1-14), GIP(1-15), GIP(1-16), GIP(1-17), GIP(1-18), GIP(1-19),
GIP(1-20), GIP(1-21), GIP(1-22), GIP(1-23), GIP(1-24), GIP(1-25),
GIP(1-26), GIP(1-27), GIP(1-28), GIP(1-29), GIP(1-30), GIP(1-31),
GIP(1-32), GIP(1-33), GIP(1-34), GIP(1-35), GIP(1-36), GIP(1-37),
GIP(1-38), GIP(1-39), GIP(1-40), GIP(1-41) and GIP(1-42), where the
base peptide-possesses one or more of the following modifications:
(1) an amino acid substitution at Glu.sup.3; (2) a modification by
fatty acid addition at an epsilon amino group of at least one
lysine residue; and (3) a modification by N-terminal acetylation.
Such a peptide analogue can have a proline substituted for
Glu.sup.3. It can also have a modification in the form of a C-16
palmitate group linked to the epsilon amino group of a lysine
residue. The modification can be the linking of, e.g., a C-8, a
C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to
the epsilon amino group of a lysine residue. The lysine residue can
be Lys.sup.16, or Lys.sup.37.
[0017] The invention further includes a peptide analogue of
GIP(1-42) (SEQ ID NO: 1), comprising at least 12 amino acid
residues from the N-terminal end of GIP(3-42), wherein the peptide
analogue is resistant to degradation by enzyme DPP IV when compared
to naturally-occurring GIP.
[0018] Also included is a peptide analogue of GIP(1-42) (SEQ ID NO:
1), comprising at least 12 amino acid residues from the N-terminal
end of GIP(1-42) and having an amino acid substitution at
Glu.sup.3, wherein the peptide analogue is resistant to degradation
by enzyme DPP IV when compared to naturally-occurring GIP.
[0019] In addition, the invention includes a peptide analogue of
GIP(1-42) (SEQ ID NO:1), comprising at least 12 amino acid residues
from the N-terminal end of GIP(3-42), wherein the peptide analogue
modulates insulin secretion.
[0020] The invention also includes a peptide analogue of GIP(1-42)
(SEQ ID NO: 1), comprising at least 12 amino acid residues from the
N-terminal end of GIP(1-42) and having an amino acid substitution
at Glu.sup.3, wherein the peptide analogue modulates insulin
secretion.
[0021] The invention also includes use of any of the analogues in
the preparation of a medicament for the treatment of obesity,
insulin resistance, insulin resistant metabolic syndrome (Syndrome
X) or type 2 diabetes.
[0022] The invention also includes a pharmaceutical composition
including the peptide analogues. The pharmaceutical composition can
further comprise a pharmaceutically acceptable carrier. The peptide
analogue can be in the form of a pharmaceutically acceptable salt,
or a pharmaceutically acceptable acid addition salt.
[0023] In a further aspect, the invention includes a method of
treating insulin resistance, obesity, or type 2 diabetes, where the
method comprises administering to a mammal in need of such
treatment a therapeutically effective amount of the pharmaceutical
composition.
[0024] According to the present invention there is provided an
effective peptide analogue of the biologically active GIP(1-42)
which has improved characteristics for treatment of Type 2 diabetes
wherein the analogue comprises at least 15 amino acid residues from
the N terminus of GIP(1-42) and has at least one amino acid
substitution or modification at position 1-3 and not including
Tyr.sup.1 glucitol GIP(1-42).
[0025] The structures of human and porcine GIP(1-42) are shown
below. The porcine peptide differs by just two amino acid
substitutions at positions 18 and 34.
[0026] The analogue may include modification by fatty acid addition
at an epsilon amino group of at least one lysine residue.
[0027] The invention includes Tyr.sup.1 glucitol GIP(1-42) having
fatty acid addition at an epsilon amino group of at least one
lysine residue.
[0028] Analogues of GIP(1-42) may have an enhanced capacity to
stimulate insulin secretion, enhance glucose disposal, delay
glucose absorption or may exhibit enhanced stability in plasma as
compared to native GIP. They also may have enhanced resistance to
degradation.
[0029] Any of these properties will enhance the potency of the
analogue as a therapeutic agent.
[0030] Analogues having D-amino acid substitutions in the 1, 2 and
3 positions and/or N-glycated, N-alkylated, N-acetylated or
N-acylated amino acids in the 1 position are resistant to
degradation in vivo.
[0031] Various amino acid substitutions at second and third amino
terminal residues are included, such as GIP(1-42)Gly.sup.2,
GIP(142)Ser.sup.2, GIP(1-42)Abu.sup.2, GIP(1-42)Aib.sup.2,
GIP(1-42)D-Ala.sup.2, GIP(1-42)Sar.sup.2, and
GIP(1-42)Pro.sup.3.
[0032] Amino-terminally modified GIP analogues include N-glycated
GIP(1-42), N-alkylated GIP(142), N-acetylated GIP(142),
N-acetyl-GIP(1-42) and N-isopropyl GIP(1-42).
[0033] Other stabilized analogues include those with a peptide
isostere bond between amino terminal residues at position 2 and 3.
These analogues may be resistant to the plasma enzyme
dipeptidyl-peptidase IV (DPP IV) which is largely responsible for
inactivation of GIP by removal of the amino-terminal dipeptide
Tyr.sup.1-Ala.sup.2.
[0034] In particular embodiments, the invention provides a peptide
which is more potent than human or porcine GIP in moderating blood
glucose excursions, said peptide consisting of GIP(1-42) or
N-terminal fragments of GIP(1-42) consisting of up to between 15 to
30 amino acid residues from the N-terminus (i.e., GIP(1-15)
GIP(1-3)) with one or more modifications-selected from the group
consisting of:
[0035] (a) substitution of Ala.sup.2 by Gly;
[0036] (b) substitution of Ala.sup.2 by Ser;
[0037] (c) substitution of Ala.sup.2 by Abu;
[0038] (d) substitution of Ala.sup.2 by Aib;
[0039] (e) substitution of Ala.sup.2 by D-Ala;
[0040] (f) substitution of Ala.sup.2 by Sar (sarcosine);
[0041] (g) substitution of Glu.sup.3 by Pro;
[0042] (h) modification of Tyr.sup.1 by acetylation;
[0043] (i) modification of Tyr.sup.1 by acylation;
[0044] (j) modification of Tyr.sup.1 by alkylation;
[0045] (k) modification of Tyr.sup.1 by glycation;
[0046] (l) conversion of Ala.sup.2-Glu.sup.3 bond to a psi
[CH.sub.2NH] bond;
[0047] (m) conversion of Ala.sup.2-Glu.sup.3 bond to a stable
peptide isotere bond; and
[0048] (n) (n-isopropyl-H) 1GIP.
[0049] The invention also provides the use of Tyr.sup.1-glucitol
GIP in the preparation of a medicament for the treatment of
diabetes.
[0050] The invention further provides improved pharmaceutical
compositions including analogues of GIP with improved
pharmacological properties.
[0051] Other possible analogues include certain commonly
encountered amino acids, which are not encoded by the genetic code,
for example, beta-alanine (beta-ala), or other omega-amino acids,
such as 3-amino propionic, 4-amino butyric and so forth, ornithine
(Orn), citrulline (Cit), homoarginine (Har), t-butylalanine
(t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle),
phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle),
cysteic acid (Cya) and methionine sulfoxide (MSO), substitution of
the D form of a neutral or acidic amino acid or the D form of
tyrosine for tyrosine.
[0052] According to the present invention there is also provided a
pharmaceutical composition useful in the treatment of diabetes type
II which comprises an effective amount of the peptide as described
herein, in admixture with a pharmaceutically acceptable
excipient.
[0053] The invention also provides a method of N-terminally
modifying GIP or analogues thereof the method comprising the steps
of synthesizing the peptide from the C terminal to the penultimate
N terminal amino acid, adding tyrosine to a bubbler system as a
F-moc protected Tyr(tBu)-Wang resin, deprotecting the N-terminus of
the tyrosine and reacting with the modifying agent, allowing the
reaction to proceed to completion, cleaving the modified tyrosine
from the Wang resin and adding the modified tyrosine to the peptide
synthesis reaction.
[0054] Preferably the agent is glucose, acetic anhydride or
pyroglutamic acid.
[0055] The invention will now be demonstrated with reference to the
following non-limiting examples and the accompanying figures
wherein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1a illustrates degradation of GIP by DPP IV.
[0057] FIG. 1b illustrates degradation of GIP and Tyr.sup.1
glucitol GIP by DPP IV.
[0058] FIG. 2a illustrates degradation of GIP human plasma.
[0059] FIG. 2b illustrates degradation of GIP and Tyr.sup.1
glucitol GIP by human plasma.
[0060] FIG. 3 illustrates electrospray ionization mass spectrometry
of GIP, Tyr.sup.1-glucitol GIP and the major degradation fragment
GIP(3-42).
[0061] FIG. 4 shows the effects of GIP and glycated GIP on plasma
glucose homeostasis.
[0062] FIG. 5 shows effects of GIP on plasma insulin responses.
[0063] FIG. 6 illustrates DPP-IV degradation of GOP (1-42).
[0064] FIG. 7 illustrates DPP-IV degradation of GIP
(Abu.sup.2).
[0065] FIG. 8 illustrates DPP-IV degradation of GIP
(Sar.sup.2).
[0066] FIG. 9 illustrates DPP-IV degradation of GIP
(Ser.sup.2).
[0067] FIG. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.
[0068] FIG. 10 illustrates DPP-IV degradation of glycated GIP.
[0069] FIG. 12 illustrates human plasma degradation of GIP.
[0070] FIG. 13 illustrates human plasma degradation of GIP
(Abu.sup.2).
[0071] FIG. 14 illustrates human plasma degradation of GIP
(Sar.sup.2).
[0072] FIG. 15 illustrates human plasma degradation of GIP
(Ser.sup.2).
[0073] FIG. 16 illustrates human plasma degradation of glycated
GIP.
[0074] FIG. 17 shows the effects of various concentrations of GIP
1-42 and GIP (Abu.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0075] FIG. 18 shows the effects of various concentrations of GIP
1-42 and GP (Abu.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0076] FIG. 19 shows the effects of various concentrations of GIP
1-42 and GIP (Sar.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0077] FIG. 20 shows the effects of various concentrations of GIP
1-42 and GIP (Ser.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0078] FIG. 21 shows the effects of various concentrations of GIP
1-42 and GIP (Ser.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0079] FIG. 22 shows the effects of various concentrations of GIP
1-42 and GIP (Ser.sup.1) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0080] FIG. 23 shows the effects of various concentrations of GIP
142 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells
incubated at 16.7M glucose.
[0081] FIG. 24 shows the effects of various concentrations of GIP
1-42 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0082] FIG. 25 shows the effects of various concentrations of GIP
1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0083] FIG. 26 shows the effects of various concentrations of GIP
1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0084] FIG. 27 shows the effects of various concentrations of GIP
1-42 and GIP (Gly.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0085] FIG. 28 shows the effects of various concentrations of GIP
1-42 and GIP (Gly.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0086] FIG. 29 shows the effects of various concentrations of GIP
1-42 and GIP (Pro.sup.3) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0087] FIG. 30 shows the effects of various concentrations of GIP
1-42 and GIP (Pro.sup.3) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0088] FIG. 31a shows the primary structure of human gastric
inhibitory polypeptide (GIP) (SEQ ID NO: 1), and FIG. 3 lb shows
the primary structure of porcine gastric inhibitory polypeptide
(GIP) (SEQ ID NO:2).
[0089] FIGS. 32A and 32B are a line graph and a bar graph,
respectively, showing the effects of (Pro.sup.3)GIP on
GIP-stimulated cyclic AMP generation and insulin secretion in
vitro.
[0090] FIGS. 33A-33F are a set of six bar graphs showing the
effects of Glu.sup.3-substituted forms of GIP and GIP(3-42) on
GIP-stimulated insulin secretion in vitro.
[0091] FIGS. 34A through 34D are a set of two line graphs (FIGS.
34A, 34C) and two bar graphs (FIGS. 34B, 34D) showing that acute
administration of (Pro.sup.3)GIP completely antagonises the actions
of GIP on glucose tolerance (FIGS. 34A, 34B) and plasma insulin
(FIGS. 34C, 34D) responses in obese diabetic ob/ob mice.
[0092] FIGS. 35A through 35D are a set of two line graphs (FIGS.
35A, 35C) and two bar graphs (FIGS. 35B, 35D) showing that acute
administration of (Pro.sup.3)GIP impairs physiological
meal-stimulated insulin release and worsens glycemic excursion in
obese diabetic ob/ob mice.
[0093] FIGS. 36A and 36B are a set of two bar graphs showing that
chronic administration of (Pro.sup.3)GIP for 11 days decreases
plasma glucose and insulin concentrations of obese diabetic ob/ob
mice.
[0094] FIGS. 37A through 37C are a set of three bar graphs showing
that chronic administration of (Pro.sup.3)GIP for 11 days decreases
glycated HbA.sub.1c, pancreatic insulin content and associated
islet hypertrophy of obese diabetic ob/ob mice.
[0095] FIGS. 38A through 38D are a set of two line graphs (FIGS.
38A, 38C) and two bar graphs (FIGS. 38B, 38D) showing that chronic
administration of (Pro.sup.3)GIP for 11 days improves glucose
tolerance of obese diabetic ob/ob mice without change of
circulating insulin.
[0096] FIG. 39 is a line graph showing that chronic administration
of (Pro.sup.3)GIP for 11 days improves insulin sensitivity in obese
diabetic ob/ob mice.
[0097] FIG. 40 is a line graph showing that the beneficial effects
of chronic administration of (Pro.sup.3)GIP for 11 days in obese
diabetic ob/ob mice are reversed 9 days after cessation of
treatment.
[0098] FIGS. 41A and 41B are a set of two line graphs showing that
chronic administration of (Pro.sup.3)GIP for 11 days causes glucose
intolerance in normal mice with reversal by 9 days after cessation
of treatment.
[0099] FIGS. 42A through 42D are a set of two line graphs (FIGS.
42A, 42C) and two bar graphs (FIGS. 42B, 42D) showing the effects
of (Pro.sup.3)GIP on plasma glucose and insulin response to native
GIP 4 hours after administration.
[0100] FIGS. 43A through 43D are a set of two line graphs and two
bar graphs showing the effects of daily (Pro.sup.3)GIP
administration on food intake (FIG. 43A), body weight (FIG. 43B),
plasma glucose (FIG. 43C) and insulin (FIG. 43D) concentrations in
ob/ob mice.
[0101] FIGS. 44A through 44D are a set of four line graphs with
inset bar graphs showing the effects of daily (Pro.sup.3)GIP
administration on glucose tolerance and plasma insulin response to
glucose in ob/ob mice.
[0102] FIGS. 45A through 45D are a set of two line graphs (FIGS.
45A, 45C) and two bar graphs (FIGS. 45B, 45D) showing the effects
of daily (Pro.sup.3)GIP administration (A; black bars) or saline
(o; white bars) on glucose (FIGS. 45A, 45B) and insulin (FIGS. 45C,
45D) responses to feeding in ob/ob mice fasted for 18 hours.
[0103] FIGS. 46A through 46D are a set of two line-graphs (FIGS.
46A, 46C) and two bar graphs (FIGS. 46B, 46D) showing the effects
of daily (Pro.sup.3)GIP administration on insulin sensitivity in
ob/ob mice.
[0104] FIGS. 47A through 47D are a set of four bar graphs showing
the effects of daily (Pro.sup.3)GIP administration on pancreatic
weight (FIG. 47A), insulin content (FIG. 47B), islet number (FIG.
47C) and islet diameter (FIG. 47D) in ob/ob mice.
[0105] FIGS. 48A through 48F are a set of two bar graphs (FIGS.
48A, 48D) and four photomicrographs (FIGS. 48B, 48C, 48E, 48F),
showing the effects of daily (Pro.sup.3)GIP administration on islet
size and morphology in ob/ob) mice.
[0106] FIG. 49 is an illustration of how the GIP receptor ("GIP-R")
antagonist, (Pro.sup.3)GIP, counters beta cell hyperplasia,
hyperinsulinemia and insulin resistance lead to improved glucose
intolerance and diabetes control.
[0107] FIGS. 50A and 50B are a line graph and a bar graph,
respectively, showing intracellular cyclic AMP production (FIG.
50A) by GIP (.tangle-solidup.) and fatty acid derivatised GOP
analogues N-AcGIP(LysPAL.sup.16) (.quadrature.) and
N-AcGOP(LysPAL.sup.37) (.circle-solid.), and insulin-releasing
activity of glucose (5.6 mmo/l glucose; white bars), GIP (lined
bars) and fatty acid derivatised GIP analogues (FIG. 50B)
N-AcGIP(LysPAL.sup.16) (grey bars) and N-AcGIP(LysPAL.sup.37)
(black bars) in the clonal pancreatic beta cell line,
BRIN-BD11.
[0108] FIGS. 51A through 51D are a set of two line graphs (FIGS.
51A, 51C) and two bar graphs (FIGS. 51B, 51D) showing glucose
lowering effects (FIGS. 51A, 51B) and insulin-releasing activity
(FIGS. 51C, 51D) of GIP and fatty acid derivatised GIP analogues in
18 hour-fasted ob/ob mice.
[0109] FIGS. 52A and 52B are a pair of bar graphs showing
dose-dependent effects of GIP and N-AcGIP(LysPAL.sup.37) in ob/ob
mice fasted for 18 hours.
[0110] FIGS. 53A through 53E are a set of graphs showing the
effects of daily N-AcGIP(LysPAL.sup.37) (.circle-solid.; black
bars) administration on food intake (FIG. 53A), body weight (FIG.
53B), plasma glucose (FIG. 53C), insulin (FIG. 53D) and glycated
hemoglobin N-AcGIP(LysPAL.sup.37) (12.5 nmoles/kg/day) (FIG.
53E).
[0111] FIGS. 54A through 54D are a set of two line graphs (FIGS.
54A, 54C) and two bar graphs (FIGS. 54B, 54D) showing the effects
of daily N-AcGIP(LysPAL.sup.37) administration on glucose tolerance
(FIGS. 54A, 54B) and plasma insulin response (FIGS. 54C, 54D) to
glucose.
[0112] FIGS. 55A through 55D are a line graph and three bar graphs
showing the effects of daily N-AcGIP(LysPAL.sup.37) administration
on insulin sensitivity (FIGS. 55A, 55B) and pancreatic weight (FIG.
55C) and insulin content-(FIG. 55D).
[0113] FIGS. 56A through 56D are a set of two line graphs (FIGS.
56A, 56C) and two bar graphs (FIGS. 56B, 56D) showing the retention
of glucose lowering (FIGS. 56A, 56B) and insulin releasing (FIGS.
56C, 56D) activity of N-AcGIP(LysPAL.sup.37) and native GOP after
daily injection for 14 days.
[0114] FIGS. 57A through 57D are a set of two line graphs (FIGS.
57A, 57C) and two bar graphs (FIGS. 57B, 57D) showing the acute
glucose lowering (FIGS. 57A, 57B) and insulin releasing (FIGS. 57C,
57D) effects of N-AcGIP(LysPAL.sup.37) after 14 daily injections of
either N-AcGIP(LysPAL.sup.37) (12.5 nmoles/kg/day; .circle-solid.;
black bars), native GIP (12.5 nmoles/kg/day; .tangle-solidup.;
lined bars) or saline vehicle (control; .quadrature.; white
bars).
DETAILED DESCRIPTION
[0115] The peptide analogues disclosed herein display resistance to
degradation by the enzyme DPP IV. These analogues include those
with alterations at residues 1, 2 and/or 3 of the native GIP(1-42)
peptide, where the alterations interfere with or delay cleavage by
DPP IV. The alterations can include chemical modification of one or
more of the first three amino acids, such as by acylation,
acetylation, alkylation, glycation, conversion of a bond between
two amino acids, such as to a psi-[CH.sub.2NH] bond, or to a stable
isotere bond, or addition of an isopropyl group. The alterations
can also include amino acid substitutions at the 1, 2, and/or 3
position, to either a different naturally-occurring amino acid, or
an amino acid not encoded by the genetic code. Other alterations
are also possible, and the object is to prevent cleavage of the
peptide by DPP IV, yet still allow for insulin secretion. This may
be accomplished by alterations at other regions of the peptide,
such as by alterations that alter the three-dimensional structure
to prevent DPP IV cleavage, yet still allow insulin secretion.
[0116] Preferred alterations include chemical modifications of
residues 1, 2, and 3, amino acid substitutions at residues 1, 2,
and 3, and chemical modifications of lysine residues throughout the
protein.
[0117] Particularly preferred alterations include acetylation of
Tyr.sup.1 and linkage of a C-16 palmitate group to the epsilon
amino group of a lysine residue (especially Lys.sup.16 or
Lys.sup.37), or substitution of Glu.sup.3, especially by proline.
The modification can also be the linking of, e.g., a C-8, a C-10, a
C-12, a C-14, a C-18 or a C-20 palmitate group to the epsilon amino
group of a lysine residue.
[0118] It has been found that longer-term, as opposed to acute, GIP
receptor antagonism using Glu.sup.3-substituted forms of GIP, such
as (Pro.sup.3)GIP, improve obesity-related insulin resistance and
associated glucose intolerance. This has uncovered an unexpected
approach to the therapy of obesity, insulin resistance and type 2
diabetes.
[0119] As described in Example 1 below, an N-terminally modified
version of the GIP protein was prepared, as were analogues of the
modified protein. The protein and its analogues were then evaluated
in Example 2 for their antihyperglycemic and insulin-releasing
properties in vivo, and were found to exhibit a substantial
resistance to amino peptidase degradation and increased glucose
lowering activity relative to native GIP.
[0120] The 42 amino acid GIP is an important incretin hormone
released into the circulation from endocrine K-cells of the
duodenum and jejunum following ingestion of food. The high degree
of structural conservation of GIP among species supports the view
that this peptide plays an important role in metabolism. Secretion
of GIP is stimulated directly by actively transported nutrients in
the gut lumen without a notable input from autonomic nerves. The
most important stimulants of GIP release are simple sugars and
unsaturated long chain fatty acids, with amino acids exerting
weaker effects. As with tGLP-1, the insulin-releasing effect of GIP
is strictly glucose-dependent. This affords protection against
hypoglycemia and thereby fulfills one of the most desirable
features of any current or potentially new antidiabetic drug.
[0121] The present results demonstrate for the first time that
Tyr.sup.1-glucitol GIP displays profound resistance to serum and
DPP IV degradation. Using ESI-MS the present study showed that
native GIP was rapidly cleaved in vitro to a major 4748.4 Da
degradation product corresponding to GIP(3-42), which confirmed
previous findings using matrix-assisted laser desorption ionization
time-of-flight mass spectrometry. Serum degradation was completely
inhibited by diprotin A (Ile-Pro-Ile), a specific competitive
inhibitor of DPP IV, confirming this as the main enzyme for GIP
inactivation in vivo. In contrast, Tyr.sup.1-glucitol GIP remained
almost completely intact after incubation with serum or DPP IV for
up to 12 hours. This indicates that glycation of GIP at the
amino-terminal Tyr.sup.1 residue masks the potential cleavage site
from DPP IV and prevents removal of the Tyr.sup.1-Ala.sup.2
dipeptide from the N-terminus preventing the formation of
GIP(3-42).
[0122] Consistent with in vitro protection against DPP IV,
administration of Tyr.sup.1-glucitol GIP significantly enhanced the
antihyperglycemic activity and insulin-releasing action of the
peptide when administered with glucose to rats. Native GIP enhanced
insulin release and reduced the glycemic excursion as observed in
many previous studies. However, amino-terminal glycation of GIP
increased the insulin-releasing and antihyperglycemic actions of
the peptide by 62% and 38% respectively, as estimated from insulin
area under the curve (AUC) measurements. Detailed kinetic analysis
is difficult due to necessary limitation of sampling times, but the
greater insulin concentrations following Tyr.sup.1-glucitol GIP as
opposed to GIP at 30 minutes post-injection is indicative of a
longer half-life. The glycemic rise was modest in both
peptide-treated groups and glucose concentrations following
injection of Tyr.sup.1-glucitol GIP were consistently lower than
after GIP. Since the insulinotropic actions of GIP are
glucose-dependent, it is likely that the relative insulin-releasing
potency of Tyr.sup.1-glucitol GIP is greatly underestimated in the
present in vivo experiments.
[0123] In vitro studies in the laboratory of the present inventors
using glucose-responsive clonal B-cells showed that the
insulin-releasing potency of Tyr.sup.1 glucitol GIP was several
orders of magnitude greater than GIP and that its effectiveness was
more sensitive to change of glucose concentrations within the
physiological range. Together with the present in vivo
observations, this suggests that N-terminal glycation of GIP
confers resistance to DPP IV degradation whilst enhancing receptor
binding and insulin secretory effects on the B-cell. These
attributes of Tyr.sup.1-glucitol GIP are fully expressed in vivo
where DPP IV resistance impedes degradation of the peptide to
GIP(3-42), thereby prolonging the half-life and enhancing effective
concentrations of the intact biologically active peptide. It is
thus possible that glycated GIP is enhancing insulin secretion in
vivo both by enhanced potency at the receptor as well as improving
DPP IV resistance. Thus numerous studies have shown that GIP (3-42)
and other N-terminally modified fragments, including GIP(4-42), and
GIP(1742) are either weakly effective or inactive in stimulating
insulin release. Furthermore, evidence exists that N-terminal
deletions of GIP result in receptor antagonist properties in GIP
receptor transfected Chinese hamster kidney cells [9], suggesting
that inhibition of GIP catabolism would also reduce the possible
feedback antagonism at the receptor level by the truncated
GIP(342).
[0124] In addition to its insulinotopic actions, a number of other
potentially important extrapancreatic actions of GIP may contribute
to the enhanced antihyperglycemic activity and other beneficial
metabolic effects of Tyr.sup.1-glucitol GIP. These include the
stimulation of glucose uptake in adipocytes, increased synthesis of
fatty acids and activation of lipoprotein lipase in adipose tissue.
GIP also promotes plasma triglyceride clearance in response to oral
fat loading. In liver, GIP has been shown to enhance
insulin-dependent inhibition of glycogenolysis. GIP also reduces
both glucagon-stimulated lipolysis in adipose tissue as well as
hepatic glucose production. Finally, recent findings indicate that
GIP has a potent effect on glucose uptake and metabolism in mouse
isolated diaphragm muscle. This latter action may be shared with
tGLP-1 and both peptides have additional benefits of stimulating
somatostatin secretion and slowing down gastric emptying and
nutrient absorption.
[0125] This study demonstrates that the glycation of GIP at the
aminoterminal Tyr.sup.1 residue limits GIP catabolism through
impairment of the proteolytic actions of serum peptidases and thus
prolongs its half-life in vivo. This effect is accompanied by
enhanced antihyperglycemic activity and raised insulin
concentrations in vivo, suggesting that such DPP IV resistant
analogues are potentially useful therapeutic agents for NIDDM.
Tyr.sup.1-glucitol GIP appears to be particularly interesting in
this regard since such amino-terminal modification of GIP enhances
rather than impairs glucose-dependent insulinotropic potency as was
observed recently for tGLP-1.
[0126] As shown in Table 1 in Example 3, glycated GIP, acetylated
GIP, GIP(Ser.sup.2) are GIP(Abu.sup.2) more resistant than native
GIP to in vitro degradation with DPP IV. From these data
GIP(Sar.sup.2) appears to be less resistant. As shown in Table 2,
all analogues tested exhibited resistance to plasma degradation,
including GIP(Sar.sup.2) which from DPP IV data appeared least
resistant of the peptides tested. DPA substantially inhibited
degradation of GIP and all analogues tested with complete abolition
of degradation in the cases of GIP(Abu.sup.2), GIP(Ser.sup.2) and
glycated GIP. This indicates that DPP IV is a key factor in the in
vivo degradation of GIP.
[0127] As shown in FIGS. 17-30, the glycated GIP analogue exhibited
a considerably greater insulinotropic response relative to native
GIP. N-terminal acetylated GIP exhibited a similar pattern and the
GIP(Ser.sup.2) analogue also evoked a strong response. From these
tests, GIP(Gly.sup.2) and GIP(Pro.sup.3) appeared to the least
potent analogues in terms of insulin release. Other stable
analogues tested, namely GIP(Abu.sup.2) and GIP(Sar.sup.2),
exhibited a complex pattern of responsiveness dependent on glucose
concentration and dose employed. Thus, very low concentrations were
extremely potent under hyperglycemic conditions (16.7 mM glucose).
This suggests that even these analogues may prove therapeutically
useful in the treatment of type 2 diabetes where insulinotropic
capacity combined with in vivo degradation dictates peptide
potency.
[0128] A major limitation to the possible therapeutic use of both
GIP and GLP-1 as insulin-releasing agents for the treatment of
diabetes is their rapid degradation in vivo by
dipeptidylpeptidase-IV (DPP-IV; EC 3.4.14.5). This enzyme rapidly
removes the amino-terminal dipeptide from the two peptides
producing GIP(3-42) and GLP-1(9-36), which lack biological activity
(Gault, V. A. et al., 2002, Biochem. Biophys. Res. Commun.
290:1420-1426). In searching for stable amino-terminally modified
forms of GIP and GLP-1, it was discovered that a novel synthetic
GIP analogue with a single proline substitution at position 3 close
to the cleavage site, (Pro.sup.3)GIP, functioned as a potent GIP
receptor antagonist.
[0129] As shown in Example 4, below, (Pro.sup.3)GIP, other
Glu.sup.3-substituted forms of GIP and GIP(3-42) are potent GIP
receptor antagonists both in vitro and in vivo. Experiments
evaluating the effects of chronic GIP receptor antagonism in normal
mice using (Pro.sup.3)GIP demonstrated a substantial but reversible
deterioration of glucose tolerance. This is entirely consistent
with the widely recognised physiological role of GIP as an
important insulin-releasing intestinal hormone involved in the
regulation of glucose disposal following feeding (Meier, J. J. et
al., 2002, Regul. Pept. 107:1-13).
[0130] Most notably, and in complete contrast to normal mice, the
experiments disclosed herein show that chronic (Pro.sup.3)GIP
administration to obese diabetic ob/ob mice for 11 days does not
worsen glucose intolerance and diabetes status at all.
Surprisingly, GIP receptor antagonism in this obese insulin
resistant model was associated with highly substantial improvements
of glycated HbA.sub.1c, plasma glucose and insulin concentrations,
glucose tolerance and insulin sensitivity. Pancreatic insulin
content was also decreased and the characteristic islet hypertrophy
of the obese mutant was partially reversed. These latter
observations indicate a decreased secretory demand for endogenous
insulin following (Pro.sup.3)GIP as a result of improved insulin
resistance.
[0131] Indeed, insulin sensitivity tests conducted in ob/ob mice 11
days into (Pro.sup.3)GIP treatment revealed a substantial
improvement in tissue insulin insensitivity, which more than
compensated for the functional ablation of the insulin-releasing
GIP component of the enteroinsular axis. The exact mechanism
responsible for this effect on insulin sensitivity is unknown but
ablation of direct action of circulating GIP on adipose tissue
metabolism is a likely candidate. Also noteworthy was the fact that
all these beneficial actions of (Pro.sup.3)GIP in obese diabetic
ob/ob mice were reversed within 9 days cessation of treatment.
[0132] These results clearly indicate that (Pro.sup.3)GIP and other
analogues based on Glu.sup.3-substituted or N-terminally truncated
forms of the gastrointestinal hormone GIP can offer an important
therapeutic means of alleviating insulin resistance for the
treatment of obesity, the so-called insulin resistant (metabolic)
syndrome and type 2 diabetes in humans.
[0133] Some studies have attempted to enhance incretin action using
DPP IV inhibitors or stable analogs of GLP-1 and GIP for the
treatment of type 2 diabetes (Green, B. D. et al., 2004, Curr.
Pharm. Des. 10: In Press; Drucker, D. J. et al., Diabetes Care
10:2929-2940). Such an approach is reliant on the possibility that
incretin action is defective in diabetes and that the underlying
defects responsible for metabolic disarray might be over-ridden by
exogenous GLP-1 or GIP administration. There is some evidence for a
beneficial and possibly therapeutic role of both GLP-1 and GIP
analogs in diabetes (Meier, J. J. et al., 2002, Regul. Pept.
107:1-13; Gault, V. A. et al., 2003, Biochem Biophys Res Commun
308:207-213; Holst, J. J. et al., 2004, Am. J. Physiol. Endocrinol.
Metab. 287:E199-E206; Green, B. D. et al., 2004, Curr. Pharm. Des.
10: In Press; Drucker, D. J. et al., Diabetes Care 10:2929-2940).
Nevertheless, understanding of the possible involvement of incretin
hormones in the pathophysiology of diabetes is lacking, partly due
to cross-reaction of classical GLP-1 and GIP radioimmunoassays with
the predominant DPP IV-generated truncated peptide forms,
GLP-1(9-36) and GIP(3-42), which circulate at particularly high
concentrations (Meier, J. J. et al., 2002, Regul. Pept. 107:1-13).
Some clinical studies seems to suggest existence of a defect in the
secretion of GLP-1 and a defect in the action of GIP in type 2
diabetes (Holst, J. J. et al., 2004, Am. J. Physiol. Endocrinol.
Metab. 287:E199-E206). However, the basis for such a conclusion is
not impressive given the many previous contradictory human studies
(Morgan, L. M., "Insulin secretion and the enteroinsular axis," In:
Nutrient regulation of insulin secretion, Flatt, P. R., ed.,
London, Portland Press, 1992, p. 1-22), and the likelihood that the
reported insensitivity of pancreatic beta cells to GIP (Vilsboll,
T. et al., 2002, Diabetologia 45:1111-1119) may reflect a
generalized secretory dysfunction rather than a specific cellular
defect (Meier, J. J. et al., 2003, Metabolism 52:1579-1585).
Indeed, the insulin secretory response to all secretagogues,
including GLP-1 is compromised in type 2 diabetes (Kjems, L. L. et
al., 2003, Diabetes 52:380-386; Flatt, P. R. et al., "Defective
insulin secretion in diabetes and insulinoma," in Nutrient
regulation of insulin secretion, Flatt P. R., ed. London, Portland
Press, 1992, p. 341-386). Thus the proposed use of GLP-1 and GIP
for diabetes therapy is reliant on peptide engineering to provide
analogs of incretin hormones with improved potency due to DPP IV
resistance, decreased renal clearance and/or enhanced GIP receptor
and post-receptor activity (Gault, V. A. et al., 2003, Biochem
Biophys Res Commun 308:207-213).
[0134] Although no single animal model can match the complex
etiology of type 2 diabetes in man, studies of the ob/ob syndrome
in mice have highlighted notable abnormalities of GIP in relation
to the interplay between hyperphagia, hyperinsulinemia and the
metabolic demise associated with progressive obesity-diabetes
(Flatt, P. R. et al., 1983, Diabetes 32:433-435; Flatt, P. R. et
al., 1984, J. Endocrinol. 101:249-256; Bailey, C. J. et al., 1986,
Acta Endocrinol. (Copenh) 112:224-229). These animals constitute a
model of non-insulin dependent diabetes associated with gross
obesity and severe insulin resistance, driven by leptin deficiency
(Bailey, C. J. et al., "Animal syndromes resembling type 2
diabetes," in Textbook of Diabetes, 3rd ed. Pickup J. C. &
Williams G., eds. Oxford, Blackwell Science Ltd., 2003, p.
25.1-25.30). Furthermore, recent research suggests an interaction
between leptin and the enteroinsular axis (Anini, Y. et al., 2003,
Diabetes 52:252-259) and that over-stimulation of the GIP receptor
("GIP-R") on adipocytes appears to be an important contributor to
fat deposition in ob/ob mice (Miyawaki, K. et al., 2002, Nat. Med.
8:738-742).
[0135] As shown in Example 5, below, daily injections of the stable
and specific GIP-R antagonist, (Pro.sup.3)GIP can be used to
chemically ablate the GIP-R and evaluate the role of endogenous
circulating GIP in obesity-diabetes as manifested in ob/ob mice.
The results reveal a cardinal role for GIP in insulin resistance
and associated metabolic disturbances, and provide the first
experimental evidence that GIP-R antagonists might provide a novel
and effective means of treating obesity-driven forms of type 2
diabetes.
[0136] Knock-out of the GIP-R in normal mice has been shown to
result in significant impairment of glucose tolerance and
meal-induced insulin secretion without appreciable effects on food
intake, body weight or basal glucose or insulin concentrations
(Miyawaki, K. et al., 1999, Proc. Nat. Acad. Sci. USA
96:14843-14847). More recent studies with genetic GIP-R knockout
mice have corroborated these findings and additionally shown that
GIP has a significant involvement in the enteroinsular axis
(Pederson, R. A. et al., 1998, Diabetes 47:1046-1052; Pamir, N. et
al., 2003, Am. J. Physiol. Endocrinol. Metab. 284:E931-939).
However, double knockout of receptors for GLP-1 and GIP results in
a surprisingly modest deterioration of glucose homeostasis
(Hansotia, T., et al., 2004, Diabetes 53:1326-1335; Preitner, F.,
et al., 2004, J. Clin. Invest. 113:635-645), indicating possible
up-regulation of compensatory mechanisms during life-long deletion
of GLP-1 and GIP receptors.
[0137] The analogue (Pro.sup.3)GIP can be used as a specific and
potent antagonist of the GIP-R that is highly stable and resistant
to DPP IV-mediated degradation (Gault, V. A. et al., 2002, Biochem.
Biophys. Res. Commun. 290:1420-1426). Using (Pro.sup.3)GIP acutely,
the results disclosd herein highlight the involvement of GIP in the
plasma insulin response to feeding and the enteroinsular axis of
ob/ob mice (Gault, V. A. et al., 2003, Diabetologia 46:222-230).
Comparison with the effects of the GLP-1-R antagonist,
exendin(9-39), indicates that GIP contributes substantially to the
insulin releasing actions of the enteroinsular axis and represents
the major physiological incretin (Gault, V. A. et al., 2003,
Diabetologia 46:222-230). Once daily administration of
(Pro.sup.3)GIP to normal mice for 11 days results in the reversible
impairment of glucose tolerance associated with decreased insulin
sensitivity (Irwin, N., 2004, Biol. Chem. 385-845-852). Basal and
postprandial insulin secretion together with pancreatic insulin
content and islet morphology were unchanged. Thus longer-term
chemical ablation of GIP-R function with daily (Pro.sup.3)GIP can
mimic the phenotype induced by genetic GIP-R knockout in mice with
the exception of revealing a potentially important additional
effect of endogenous GIP on insulin action, which appears to be
independent of enhanced insulin secretion.
[0138] Far from reproducing this predicted scenario and the
metabolic deterioration observed following genetic or chemical
knockout of the GIP-R in normal mice (Miyawaki, K. et al., 1999,
Proc. Nat. Acad. Sci. USA 96:14843-14847; Irwin, N., 2004, Biol.
Chem. 385:845-852), ob/ob mice treated with daily (Pro.sup.3)GIP
for 11 days exhibited a marked improvement in diabetic status. This
included decreased fasting and basal hyperglycemia, lowered
glycated hemoglobin, improved glucose tolerance and a significantly
diminished glycemic excursion following feeding. Notably, basal and
glucose-stimulated plasma insulin concentrations were decreased,
suggesting that insulin sensitivity must have improved
significantly following (Pro.sup.3)GIP in order to restrain the
hyperglycemia. Indeed, insulin sensitivity tests conducted after 11
days of (Pro.sup.3)GIP administration revealed a 57% increase in
the glucose-lowering action of exogenous insulin. Bearing in mind
that the severity of the ob/ob syndrome represents a tough test for
current antidiabetic drugs, including insulin, sulfonylureas,
metformin and thiazolidenediones (Flatt, P. R. et al., "Defective
insulin secretion in diabetes and insulinoma," in Nutrient
regulation of insulin secretion, Flatt P. R., ed. London, Portland
Press, 1992, p. 341-386; Stevenson, R. W. et al., 1995, The
Diabetes Annual 9:175-191; Wiermsperger, N. F., "Preclinical
pharmacology of biguanides," Handbook of Experimental Pharmacology
119:305-358, 1996), induction of such rapid and reversible changes
by GIP-R blockade using (Pro.sup.3)GIP is unprecedented.
[0139] It is important to note that the above effects were observed
independently of any change in food intake or body weight in
(Pro.sup.3)GIP treated ob/ob mice. This accords with the view that
endogenous GIP lacks effects on feeding activity (Meier, J. J. et
al., 2002, Regul. Pept. 107:1-13). However, the observation on body
weight contrasts with findings in ob/ob mice cross-bred to
genetically knockout GIP-R function (Miyawaki, K. et al., 2002,
Nat. Med. 8:738-742). Thus in these transgenic mice, life-long
depletion of GIP-R function was associated with decreased body
weight gain and significant amelioration of both adiposity and
insulin resistance compared with control (Lep.sup.ob/Lep.sup.ob)
mice (Miyawaki, K. et al., 2002, Nat. Med. 8:738-742). In this
previous study, the improvement of insulin sensitivity may have
been a simple consequence of reduced adipose tissue mass as this
would significantly enhance peripheral glucose disposal (Bailey, C.
J. et al., "Animal syndromes resembling type 2 diabetes," in
Textbook of Diabetes, 3rd ed. Pickup J. C. & Williams G., eds.
Oxford, Blackwell Science Ltd., 2003, p. 25.1-25.30). However, the
present results observed in rapid time and without effects on
feeding or body weight clearly indicate the involvement of an
alternative mechanism.
[0140] The most plausible explanation for the present data stem
from appreciation of the key milestones in the age-dependent
progression of the ob/ob syndrome on the Aston background as
depicted in FIG. 49, which is an illustration of how the GIP-R
antagonist, (Pro.sup.3)GIP, counters beta cell hyperplasia,
hyperinsulinemia and insulin resistance lead to improved glucose
intolerance and diabetes control. Possible longer-term direct
actions of GIP on adipocyte function and fat stores, suggested by
studies in GIP-R knockout ob/ob mice have been omitted.
[0141] Due to double recessive ob mutation and resulting leptin
deficiency, young ob/ob mice develop a profound early hyperphagia
(Bailey, C. J., et al., 1982, Int. J. Obes. 6:11-21). Substantial
enteroendocrine stimulation results in K-cell hyperplasia and
markedly elevated concentrations of intestinal and circulating GIP
(Flatt, P. R. et al., 1983, Diabetes 32:433-435; Flatt, P. R. et
al., 1984, J. Endocrinol. 101:249-256; Bailey, C. J. et al., 1986,
Acta Endocrinol. (Copenh) 112:224-229). This in turn promotes islet
hypertrophy and beta cell hyperplasia (Bailey, C. J., et al., 1982,
Int. J. Obes. 6:11-21) together with marked hyperinsulinemia and
mounting insulin resistance (Flatt, P. R., et al., 1981, Horm Metab
Res 13:556-560). This process manifests itself in terms of rising
basal hyperglycemia and glucose intolerance. A vicious spiral is
thus established wherein beta cell compensation results in marked
hyperinsulinemia which attempts to moderate increasing insulin
resistance (Bailey, C. J., et al., 1982, Int. J. Obes. 6:11-21;
Flatt, P. R., et al., 1981, Horm Metab Res 13:556-560). Viewed in
this context, it is clear that chemical ablation of GIP-R function
with daily (Pro.sup.3)GIP will decrease beta cell stimulation and
hyperinsulinemia. However, instead of causing further impairment of
glucose homeostasis, a preferentially marked improvement of insulin
sensitivity results in a substantial improvement of the metabolic
syndrome. Further support for this scenario, is the partial
amelioration of islet hypertrophy and beta cell hyperplasia in
(Pro.sup.3)GIP treated ob/ob mice (FIG. 48). Notably, average islet
diameter was diminished with the largest islets (>15 mm) being
replaced by a greater proportion with small or medium diameters
(0.1-15 mm). These effects were largely reversed by 9 day cessation
of treatment, supporting the idea of active islet and beta cell
growth in adult ob/ob mice (Bailey, C. J., et al., 1982, Int. J.
Obes. 6:1 i-21). Recent observations indicate that GIP acts as a
mitotic stimulus and anti-apoptotic agent to the beta cell
(Pospisilik, J. A. et al., 2003, Diabetes 52:741-750; Trumper, A.
et al., 2001, Mol. Endocrinol. 15:1559-1570; Ehses, J. A. et al.,
2003, Endocrinology 144:4433-4445, Trumper, A. et al., 2002, J.
Endocrinol. 174:233-246). Thus, it is believed that negative
effects of (Pro.sup.3)GIP on islet size reflects a combination of
decreased proliferation and increased apoptosis of beta cells.
[0142] The results shown in Example 5 have demonstrated for the
first time that daily administration of the GIP-R antagonist,
(Pro.sup.3)GIP, improves glucose tolerance and ameliorates insulin
resistance and abnormalities of islet structure and function in
ob/ob mice. Notably, these effects were reversed by discontinuation
of (Pro.sup.3)GIP for 9 days. Freedom from any obvious side effects
also accords with earlier observations in normal mice (Irwin, N.,
2004, Biol. Chem. 385:845-852) and mice genetically engineered with
life-long GIP-R deficiency (Miyawaki, K. et al., 2002, Nat. Med.
8:738-742). The present observations point to a cardinal role of
endogenous GIP in the pathogenesis of obese-insulin
resistant-diabetes. More importantly, the data indicate that GIP-R
antagonists, such as (Pro 3)GIP, provide a novel, physiological and
effective means to treat obese type 2 diabetes through the
alleviation of insulin resistance.
[0143] In Example 6, fatty acid derivatisation was used to develop
two novel long-acting, N-terminally modified GIP analogues
(N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37)).
[0144] Degradation studies were carried out with
dipeptidylpeptidase IV (DPP IV). Cyclic AMP production was assessed
using GIP receptor transfected CHL fibroblasts. In vitro insulin
release was assessed in BRIN-BD11 cells. Insulinotropic and
glycaemic responses to acute and long-term peptide administration
were evaluated in obese diabetic (ob/ob) mice.
[0145] In contrast to GIP both analogues displayed resistance to
DPP IV degradation. The analogues also stimulated cyclic AMP
production and exhibited significantly improved in vitro insulin
secretion compared to control. Administration of
N-AcGIP(LysPAL.sup.16) or N-AcGIP(LysPAL.sup.37) together with
glucose in ob/ob mice significantly reduced the glycaemic excursion
and improved the insulinotropic response compared to GIP.
Dose-response studies with N-AcGIP(LysPAL.sup.37) revealed highly
significant decreases in the overall glycaemic excursion and
increases in circulating insulin even with 6.25 nmoles/kg. Once
daily injection of ob/ob mice with N-AcGIP(LysPAL.sup.37) over 14
days significantly decreased plasma glucose, glycated haemoglobin
and improved glucose tolerance compared with saline or native GIP.
Plasma and pancreatic insulin were significantly increased,
together with a significant enhancement in the insulin response to
glucose and a notable improvement of insulin sensitivity. No
evidence was found for GIP-receptor desensitization and the
metabolic effects of N-AcGIP(LysPAL.sup.37) were independent of any
change in feeding or body weight.
[0146] These results show that novel fatty acid derivatised,
N-terminally modified analogues of GIP such as
N-AcGIP(LysPAL.sup.37), may have significant potential for the
treatment of type 2 diabetes.
[0147] One approach to counter both renal clearance and enzyme
degradation of GIP concerns the utilisation of fatty acid
derivatisation together with N-terminal modification. Fatty acid
derivatisation has previously been shown to prolong the half-life
of insulin (Kurtzhals, P. et al., 1995, Biochem. J. 312: 725-731)
and the sister incretin glucagon-like peptide-1 (GLP-1) (Knudsen,
L. B. et al., 2000, J. Med. Chem. 43: 1664-1669; Green, B. D. et
al., 2004, Biol. Chem. 385: 169-177; Kim, J. G. et al., 2003,
Diabetes 52: 751-759). A number of N-terminally modified GIP
analogues have been developed which exhibit profound resistance to
DPP IV (Hinke, S. A. et al., 2002, Diabetes 51: 656-661; Gault, V.
A. et al., 2002, Biochem. J. 367: 913-920; Gault, V. A. et al.,
2003, J. Endocrinol. 176: 133-141; O'Harte, F. P. M. et al., 1999,
Diabetes 48: 758-765). Several of these, most notably those
modified at Tyr.sup.1 of GIP with an addition of an acetyl,
glucitol, pyroglutamyl or Fmoc adduct, exhibit enhanced activity at
the GIP receptor in vitro (Gault, V. A. et al., 2002, Biochem. J.
367: 913-920; O'Harte, F. P. M. et al., 1999, Diabetes 48: 758-765;
O'Harte, F. P. M. et al., 2002, Diabetologia 45: 1281-1291). As a
result of degradation resistance and enhanced cellular activity,
these analogues display enhanced and protracted antihyperglycaemic
and insulin-releasing activity when administered acutely to animals
with obesity-diabetes (Hinke, S. A. et al., 2002, Diabetes 51:
656-661; Gault, V. A. et al., 2002, Biochem. J. 367: 913-920;
Gault, V. A. et al., 2003, J. Endocrinol. 176: 133-141; O'Harte, F.
P. M. et al., 1999, Diabetes 48: 758-765; O'Harte, F. P. M. et al.,
2002, Diabetologia 45: 1281-1291). Of these, N-AcGIP has emerged as
the most effective DPP IV-resistant analogue, substantially
augmenting the plasma insulin response and curtailing the glycaemic
excursion following conjoint administration with glucose to ob/ob
mice (O'Harte, F. P. M. et al., 2002, Diabetologia 45:
1281-1291).
[0148] Example 6 was designed to evaluate the metabolic stability,
biological activity and antidiabetic potential of novel second
generation fatty acid derivatised, N-terminally modified N-AcGIP
analogues, N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37). Both
GIP analogues contain a C-16 palmitate group linked to the
epsilon-amino group of Lys at positions 16 or 37, in combination
with an N-terminal (Tyr.sup.1) acetyl group (O'Harte, F. P. M. et
al., 2002, Diabetologia 45: 1281-1291). The relative stability to
DPP IV degradation, insulin secretion and cyclic AMP properties
were examined in vitro together with acute and dose-response
studies in obese diabetic ob/ob mice. The most effective analogue,
N-AcGIP(LysPAL.sup.37) was administered to ob/ob mice by once daily
intraperitoneal injection for 14 days prior to evaluation of
glucose homeostasis, pancreatic beta cell function and insulin
sensitivity. Possible desensitization of GIP receptor action by
prolonged exposure to elevated concentrations of
N-AcGIP(LysPAL.sup.37) was also examined. The results indicate the
particular promise of the novel second generation N-terminally
acetylated GIP analogue, N-AcGIP(LysPAL.sup.37), as a potential
therapeutic agent for the treatment of type 2 diabetes.
[0149] Despite their many attributes, DPP IV-resistant analogues of
GIP and GLP-1, like their native counterparts, are still subject to
renal filtration. To circumvent this problem, fatty acid
derivatisation has been used to improve the duration of action of
GLP-1 (Knudsen, L. B. et al., 2000, J. Med. Chem. 43: 1664-1669;
Green, B. D. et al., 2004, Biol. Chem. 385: 169-177; Kim, J. G. et
al., 2003, Diabetes 52: 751-759). The most promising analogue,
NN2211 (Liraglutide), appears effective in improving blood glucose
control in type 2 diabetic subjects despite a tendency towards
promotion of nausea possibly due to slowing of gastric emptying
(Agers.o slashed., H. et al., 2002, Diabetologia 45: 195-202).
[0150] Example 6 describes the results of introducing two specific
modifications to the native GIP hormone, namely N-terminal
acetylation and C-terminal fatty acid derivatisation. N-terminal
acetylation was employed, as previously described (O'Harte, F. P.
M. et al., 2002, Diabetologia 45: 1281-1291), to significantly
enhance stability to DPP IV. In contrast, conjugation of a C-16
palmitate residue at the epsilon-amino group of Lys.sup.16 or
Lys.sup.37 was introduced to extend the biological half-life
through binding to circulating proteins (Kurtzhals, P. et al.,
1995, Biochem. J. 312: 725-731). Unlike the native peptide, both
GOP analogues appeared to be completely resistant to enzymatic
breakdown by DPP IV, which corroborates previous observations with
N-AcGIP (O'Harte, F. P. M. et al., 2002, Diabetologia 45:
1281-1291). Furthermore, both analogues displayed similar or
slightly better insulin-releasing and cyclic AMP generating
properties to native GIP and N-AcGIP when tested in the in vitro
cellular systems (O'Harte, F. P. M. et al., 2002, Diabetologia 45:
1281-1291).
[0151] To assess the antihyperglycaemic and insulinotropic
potential of the fatty acid derivatised GOP analogues in vivo,
obese diabetic (ob/ob) mice were employed. The ob/ob syndrome is an
extensively studied model of spontaneous obesity and diabetes,
exhibiting hyperphagia, marked obesity, moderate hyperglycaemia and
severe hyperinsulinemia (Bailey, C. J. et al., 1982, Int. J.
Obesity 6: 11-21). As described in previous studies (Gault, V. A.
et al., 2002, Biochem. J. 367: 913-920; Gault, V. A. et al., 2003,
J. Endocrinol. 176: 133-141), native GIP only modestly reduced the
glycaemic excursion in ob/ob mice reflecting the severe insulin
resistance of this mutant animal model (Bailey, C. J. et al., 1982,
Int. J. Obesity 6: 11-21). In sharp contrast, both N-acetylated GIP
analogues additionally substituted with a palmitate molecule at
Lys.sup.16 or Lys.sup.37 (N-AcGIP(LysPAL.sup.16) and
N-AcGIP(LysPAL.sup.37)) significantly lowered plasma glucose levels
compared to the native peptide. This was accompanied by
significantly enhanced insulin-releasing activity, especially in
the case of N-AcGIP(LysPAL.sup.37). The significantly protracted
insulinotropic response to both fatty acid derivatised GIP
analogues at 60 minutes despite substantially lower plasma glucose
is indicative of an extended plasma half-life. This may be due to
binding of both palmitate derivatised GOP analogues to serum
albumin, therefore significantly impairing their clearance via the
kidneys (Meier, J. J. et al., 2004, Diabetes 53: 654-662). However,
further studies including establishment of sensitive and specific
immunoassays for the novel GIP analogues would be needed to confirm
such actions.
[0152] N-AcGIP(LysPAL.sup.37) appeared to be the best fatty acid
derivatised analogue displaying a more protracted, significantly
enhanced insulin-releasing potency over N-AcGIP(LysPAL.sup.16) in
vivo. Reasons for the increased potency of N-AcGIP(LysPAL.sup.37)
remain unclear, but one explanation is an extended half-life.
Another possibility may be that a fatty acid chain linked to the
Lys closer to the C-terminus of the peptide may have less of a
detrimental effect upon the bioactive region of the molecule known
to be located within the N-terminus (Gault, V. A. et al., 2002,
Biosci. Rep. 22: 523-528; Hinke, S. A. et al., 2001, Biochim.
Biophys. Acta 1547: 143-55; Manhart, S. et al., 2003, Biochemistry
42: 3081-3088). However, similarities between the in vitro
biological activities of the two palmitate substituted analogues
make this less likely.
[0153] Given that N-AcGIP(LysPAL.sup.37) was the more potent of the
two analogues in vivo, it was further utilised in dose-response
studies. Considering that native GIP itself has only very modest
effects in ob/ob mice, as sometimes observed with type 2 diabetic
subjects (Nauck, M. A. et al., 1993, J. Clin. Invest. 91: 301-307;
Meier, J. J. et al., 2004, Diabetes 53: 220-224; Vilsboll, T. et
al., 2002, Diabetologia 45: 1111-1119), it is remarkable that
N-AcGIP(LysPAL.sup.37), even at the lowest dose of 6.25 nmoles/kg,
exhibited significant glucose-lowering and insulinotropic activity
when administered with glucose. Considering N-AcGIP(LysPAL.sup.37)
is subject to albumin binding, the fact that it is still highly
biologically active even at lower concentrations indicates striking
potency.
[0154] Daily administration of N-AcGIP(LysPAL.sup.37) to young
adult ob/ob mice by intraperitoneal injection (12.5 nmoles/kg)
resulted in a progressive lowering of plasma glucose concentrations
and a significant decrease of glycated haemoglobin by 14 days. This
was associated with a substantial improvement of glucose tolerance.
Importantly food intake and body weight were unchanged ruling out
the possibility that improvement of glucose homeostasis was merely
the consequence of body weight loss. These observations also
indicate that N-AcGIP(LysPAL.sup.37) did not exert any untoward
toxic actions affecting feeding over the study period. This is in
harmony with recent studies showing that GIP does not inhibit
gastric emptying (Meier, J. J. et al., 2003, Am. J. Physiol.
Endocrinol. Metab. 286: 621-625). Daily administration of native
GIP to ob/ob mice for 14 days had no effect on any of the
parameters measure, consistent with the very short half-life of the
native GIP in vivo.
[0155] As expected, a key component of the beneficial action of
N-AcGIP(LysPAL.sup.37) concerned effects on beta-cells. Thus
although native GIP is a weak stimulus to insulin secretion in
ob/ob mice at the age studied, plasma and pancreatic insulin
concentrations were raised in ob/ob mice receiving the novel fatty
acid derivatised analogue. This is consistent with the action of
GIP as a promoter of proinsulin gene expression (Wang, Y. et al.,
1996, Mol. Cell. Endocrinol. 116:81-87) and exemplifies the
increased potency reported for N-terminally modified GIP analogues
in this animal model of diabetes (Hinke, S. A. et al., 2002,
Diabetes 51: 656-661; Gault, V. A. et al., 2002, Biochem. J. 367:
913-920; Gault, V. A. et al., 2003, J. Endocrinol. 176: 133-141;
O'Harte, F. P. M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.
P. M. et al., 2002, Diabetologia 45: 1281-1291). Furthermore, the
insulin response to glucose was significantly enhanced in ob/ob
mice receiving N-AcGIP(LysPAL.sup.37). This ability to augment or
restore pancreatic beta cell glucose responsiveness has been
similarly observed with GLP-1 (Holz, G. G. et al., 1993, Nature 28:
362-365; Flamez, D. et al., 1998, Diabetes 47: 646-652). As with
observations on glycaemic control, none of these attributes were
reproduced by daily injections of native GIP.
[0156] Results of insulin sensitivity tests conducted after 14 days
treatment indicate that the improvement of diabetic status achieved
in ob/ob mice with N-AcGIP(LysPAL.sup.37) was not solely due to the
potentiation of insulin secretion. Thus, these animals also
exhibited a significant improvement of insulin sensitivity compared
to the GIP or saline treated groups. Given that hyperinsulinemia is
generally believed to down-regulate insulin receptor function
(Marshall, S. et al., 1981, Diabetes 30: 746-753), this suggests
that N-AcGIP(LysPAL.sup.37) may exert other compensatory effects.
Further study is necessary to evaluate this aspect but
possibilities include inhibition of counter-regulatory hormones and
effects on extrapancreatic sites such as muscle, adipose tissue and
liver (Morgan, L. M. et al., 1996, Biochem. Soc. Trans. 24:585-591;
O'Harte, F. P. M. et al., 1998, J. Endocrinol. 156: 237-243; Yip,
R. G. et al., 1998, Endocrinology 139: 4004-4007).
[0157] Irrespective of knowledge of the full range of actions
contributing to the antihyperglycaemic effect of
N-AcGIP(LysPAL.sup.37), a currently envisaged problem of long-term
treatment with stable analogues of GIP or GLP-1 concerns
desensitization of hormone receptor action (Delmeire, D. et al.,
2004, Biochem. Pharmacol. 68: 33-39). Although this has been
observed during prolonged exposure of pancreatic beta cells to GIP
in rats (Tseng, C. C. et al., 1996, Am. J. Physiol. 270:
E661-E666), there was no evidence that treatment with
N-AcGIP(LysPAL.sup.37) for 14 days compromised the glucose lowering
or insulin releasing actions of N-AcGIP(LysPAL.sup.37). Thus the
antidiabetic actions of N-AcGIP(LysPAL.sup.37) were clearly evident
when the analogue was administered acutely together with glucose.
Furthermore, the acute effects of N-AcGIP(LysPAL.sup.37) in such
experiments were identical in groups of ob/ob mice receiving either
N-AcGIP(LysPAL.sup.37), native GIP or saline injections for 14
days.
[0158] Such data clearly indicate that prolonged exposure to
N-AcGIP(LysPAL.sup.37) does not induce and possibly overcomes
inherent GIP receptor desensitization in ob/ob mice. Given the high
circulating concentrations of GIP in these obese-diabetic rodents
(Flatt, P. R. et al., 1983, Diabetes 32: 433-435; Flatt, P. R. et
al., 1984, J. Endocrinol. 101: 249-256), it is tempting to link
beta cell refractoriness to GIP evident in ob/ob mice to simple
receptor desensitization at the hands of inappropriate secretion
and metabolism of GIP.
[0159] The data shown herein demonstrate that N-terminally
acetylated GOP carrying a palmitate group linked to Lys at position
37 displays resistance to DPP IV and an impressive profile of
bioactivity manifested by potent and long-acting glucose-lowering
activity in a commonly employed animal model of obesity-diabetes.
This activity profile provides strong encouragement for the
development of long-acting fatty acid derivatised N-terminally
modified analogues of GOP for the once-daily treatment of type 2
diabetes.
[0160] The peptide analogues of the present invention have use in
treating diseases and conditions caused by improper modulation of
insulin levels, including diabetes, type 2 diabetes, insulin
resistance, insulin resistant metabolic syndrome (Syndrome X), and
obesity.
[0161] A peptide analogue produced by the methods of the present
invention can be used in a pharmaceutical composition, wherein the
analogue is combined with a pharmaceutically acceptable carrier.
Such a composition may also contain (in addition to the analogue
and a carrier) diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials well known in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredient(s). The characteristics of the carrier will
depend on the route of administration.
[0162] Administration of the peptide analogue of the present
invention used in the pharmaceutical composition or to practice the
method of the present invention can be carried out in a variety of
conventional ways, such as by oral ingestion, inhalation, topical
application or cutaneous, subcutaneous, intraperitoneal, parenteral
or intravenous injection. Administration can be internal or
external; or local, topical or systemic.
[0163] The compositions containing a peptide analogue of this
invention can be administered intravenously, as by injection of a
unit dose, for example. The term "unit dose" when used in reference
to a therapeutic composition of the present invention refers to
physically discrete units suitable as unitary dosage for the
subject, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect in
association with the required diluent, i.e., carrier or
vehicle.
[0164] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0165] When a therapeutically effective amount of the composition
of the present invention is administered orally, the composition of
the present invention will be in the form of a tablet, capsule,
powder, solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein of
the present invention, and preferably from about 25 to 90% protein
of the present invention. When administered in liquid form, a
liquid carrier such as water, petroleum, oils of animal or plant
origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition
contains from about 0.5 to 90% by weight of the composition of the
present invention, and preferably from about 1 to 50% of the
composition of the present invention.
[0166] When a therapeutically effective amount of the composition
of the present invention is administered by intravenous, cutaneous
or subcutaneous injection, the composition of the present invention
will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution. The preparation of such parenterally acceptable
protein solutions, having due regard to pH, isotonicity, stability,
and the like, is within the skill in the art. A preferred
pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to the
composition of the present invention, an isotonic vehicle such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art. The pharmaceutical
composition of the present invention may also contain stabilizers,
preservatives, buffers, antioxidants, or other additives known to
those of skill in the art.
[0167] Use of timed release or sustained release delivery systems
are also included. A sustained-release matrix, as used herein, is a
matrix made of materials, usually polymers, which are degradable by
enzymatic or acid/base hydrolysis or by dissolution. Once inserted
into the body, the matrix is acted upon by enzymes and body fluids.
The sustained-release matrix desirably is chosen from biocompatible
materials such as liposomes, polylactides (polylactic acid),
polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(co-polymers of lactic acid and glycolic acid) polyanhydrides,
poly(ortho)esters, polyproteins, hyaluronic acid, collagen,
chondroitin sulfate, carboxylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino acids, amino acids such
as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. A preferred
biodegradable matrix is a matrix of one of either polylactide,
polyglycolide, or polylactide co-glycolide (co-polymers of lactic
acid and glycolic acid).
[0168] The therapeutic compositions can include pharmaceutically
acceptable salts of the components therein, e.g., which may be
derived from inorganic or organic acids. By "pharmaceutically
acceptable salt" is meant those salts which are, within the scope
of sound medical judgement, suitable for use in contact with the
tissues of humans and lower animals without undue toxicity,
irritation, allergic response and the like and are commensurate
with a reasonable benefit/risk ratio. Pharmaceutically acceptable
salts are well-known in the art. For example, S. M. Berge, et al.,
describe pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences (1977) 66:1 et seq., which is incorporated
herein by reference in its entirety. Pharmaceutically acceptable
salts include the acid addition salts (formed with the free amino
groups of the polypeptide) that are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, tartaric, mandelic and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
procaine and the like. The salts may be prepared in situ during the
final isolation and purification of the compounds of the invention
or separately by reacting a free base function with a suitable
organic acid. Representative acid addition salts include, but are
not limited to acetate, adipate, alginate, citrate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,
camphorsufonate, digluconate, glycerophosphate, hemisulfate,
heptonoate, hexanoate, fumarate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxymethanesulfonate (isethionate), lactate,
maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate,
oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate,
picrate, pivalate, propionate, succinate, tartate, thiocyanate,
phosphate, glutamate, bicarbonate, p-toluenesulfonate and
undecanoate. Also, the basic nitrogen-containing groups can be
quaternized with such agents as lower alkyl halides such as methyl,
ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long
chain halides such as decyl, lauryl, myristyl and stearyl
chlorides, bromides and iodides; arylalkyl halides like benzyl and
phenethyl bromides and others. Water or oil-soluble or dispersible
products are thereby obtained. Examples of acids which may be
employed to form pharmaceutically acceptable acid addition salts
include such inorganic acids as hydrochloric acid, hydrobromic
acid, sulphuric acid and phosphoric acid and such organic acids as
oxalic acid, maleic acid, succinic acid and citric acid.
[0169] As used herein, the terms "pharmaceutically acceptable",
"physiologically tolerable" and grammatical variations thereof as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to or upon a mammal with a minimum of undesirable
physiological effects such as nausea, dizziness, gastric upset and
the like. The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
Typically such compositions are prepared as injectables either as
liquid solutions or suspensions, however, solid forms suitable for
solution, or suspensions, in liquid-prior to use can also be
prepared. The preparation can also be emulsified.
[0170] The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient and in amounts suitable for use in the
therapeutic-methods described herein. Suitable excipients include,
for example, water, saline, dextrose, glycerol, ethanol or the like
and combinations thereof. In addition, if desired, the composition
can contain minor amounts of auxiliary substances such as wetting
or emulsifying agents, pH buffering agents and the like which
enhance the effectiveness of the active ingredient.
[0171] The amount of peptide analogue of the present invention in
the pharmaceutical composition of the present invention will depend
upon the nature and severity of the condition being treated, on the
nature of prior treatments which the patient has undergone, and on
a variety of other factors, including the type of injury, the age,
weight, sex, medical condition of the individual. Ultimately, the
attending physician will decide the amount of the analogue with
which to treat each individual patient. Initially, the attending
physician will administer low doses of peptide analogue and observe
the patient's response. Larger doses of peptide analogue may be
administered until the optimal therapeutic effect is obtained for
the patient, and at that point the dosage is not increased
further.
[0172] Additional guidance on methods of determining dosages can be
found in standard references, for example, Spilker, Guide to
Clinical Studies and Developing Protocols, Raven Press Books, Ltd.,
New York, 1984, pp. 7-13 and 54-60; Spilker, Guide to Clinical
Trials, Raven Press, Ltd., New York, 1991, pp. 93-101; Craig et
al., Modern Pharmacology, 2d ed., Little Brown and Co., Boston,
1986, pp. 127-133; Speight, Avery's Drug Treatment: Principles and
Practices of Clinical Pharmacology and Therapeutics, 3d ed.,
Williams and Wilkins, Baltimore, 1987, pp. 50-56; Tallarida et al.,
Principles in General Pharmacology, Springer-Verlag, New York,
1998, pp. 18-20; and Olson, Clinical Pharmacology Made Ridiculously
Simple, MedMaster, Inc., Miami, 1993, pp. 1-5.
EXAMPLES
Example 1
Preparation of N-Terminally Modified GIP and Analogues Thereof
[0173] The N-terminal modification of GIP is essentially a three
step process. Firstly, GIP is synthesized from its C-terminal
(starting from a Fmoc-Gln (Trt)-Wang resin (Calbiochem Novabiochem,
Beeston, Nottingham, UK) up to the penultimate N-terminal
amino-acid (Ala.sup.2) on an automated peptide synthesizer (Applied
Biosystems, California, USA). The synthesis-follows standard Fmoc
peptide chemistry protocols. Secondly, the N-terminal amino acid of
native GIP (Tyr) is added to a manual bubbler system as a
Fmoc-protected Tyr(tBu)-Wang resin. This amino acid is deprotected
at its N-terminus (piperidine in DMF (20% v/v)) and allowed to
react with a high concentration of glucose (glycation, under
reducing conditions with sodium cyanoborohydride), acetic anhydride
(acetylation), pyroglutamic acid (pyroglutamyl) etc. for up to 24
hours as necessary to allow the reaction to go to completion. The
completeness of reaction is monitored using the ninhydrin test
which determines the presence of available free a-amino groups.
Thirdly (once the reaction is complete), the now structurally
modified Tyr is cleaved from the Wang resin (95% TFA, and 5% of the
appropriate scavengers. N. B. Tyr is considered to be a problematic
amino acid and may need special consideration) and the required
amount of N-terminally modified-Tyr consequently added directly to
the automated peptide synthesiser, which will carry on the
synthesis, thereby stitching the N-terminally modified-Tyr to the
a-amino of GIP (Ala.sup.2), completing the synthesis of the GIP
analogue. This peptide is cleaved off the Wang resin (as above) and
then worked up using the standard Buchner filtering, precipation,
rotary evaporation and drying techniques.
Example 2
Preparation of Tyr.sup.1-Glucitol GIP and its Properties In
Vivo
[0174] The following example investigates preparation of Tyr.sup.1
glucitol GIP together with evaluation of its antihyperglycemic and
insulin-releasing properties in vivo. The results clearly
demonstrate that this novel GIP analogue exhibits a substantial
resistance to aminopeptidase degradation and increased glucose
lowering activity compared with the native GIP.
[0175] Research Design and Methods
[0176] Materials. Human GIP was purchased from the American Peptide
Company (Sunnyvale, Calif., USA). HPLC grade acetonitrile was
obtained from Rathburn (Walkersburn, Scotland). Sequencing grade
trifluoroacetic acid (TFA) was obtained from Aldrich (Poole,
Dorset, UK). All other chemicals purchased including dextran T-70,
activated charcoal, sodium cyanoborohydride and bovine serum
albumin fraction V were from Sigma (Poole, Dorset, UK). Diprotin A
(DPA) was purchased from Calbiochem-Novabiochem (UK) Ltd. (Beeston,
Nottingham, UK) and rat insulin standard for RIA was obtained from
Novo Industria (Copenhagen, Denmark). Reversed-phase Sep-Pak
cartridges (C-18) were purchased from Millipore-Waters (Milford,
Mass., USA). All water used in these experiments was purified using
a Milli-Q, Water Purification System (Millipore Corporation,
Milford, Mass., USA).
[0177] Preparation of Tyr.sup.1-glucitol GIP. Human GIP was
incubated with glucose under reducing conditions in 10 mmol/l
sodium phosphate buffer at pH 7.4 for 24 hours. The reaction was
stopped by addition of 0.5 mol/l acetic acid (30 .mu.l) and the
mixture applied to a Vydac (C18)(4.6.times.250 mm) analytical HPLC
column (The Separations Group, Hesperia, Calif., USA) and gradient
elution conditions were established using aqueous/TFA and
acetonitrile/TFA solvents. Fractions corresponding to the glycated
peaks were pooled, taken to dryness under vacuum using an AES 1000
Speed-Vac concentrator (Life Sciences International, Runcorn, UK)
and purified to homogeneity on a Supelcosil (C-8) (4.6.times.150
mm) column (Supelco Inc., Poole, Dorset, UK).
[0178] Degradation of GIP and Tyr.sup.1-glucitol GIP by DPP IV.
HPLC-purified GIP or Tyr.sup.1-glucitol GIP were incubated at
37.degree. C. with DPP-IV (5 mU) for various time periods in a
reaction mixture made up to 500 .mu.l with 50 mmol/l
triethanolamine-HC 1, pH 7.8 (final peptide concentration 1
mmol/l). Enzymatic reactions were terminated after 0, 2, 4 and 12
hours by addition of 5 .mu.l of 10% (v/v) TFA/water. Samples were
made up to a final volume of 1.0 ml with 0.12% (v/v) TFA and stored
at -20.degree. C. prior to HPLC analysis.
[0179] Degradation of GIP and Tyr.sup.1-glucitol GIP by human
plasma. Pooled human plasma (20 .mu.l) taken from six healthy
fasted human subjects was incubated at 37.degree. C. with GIP or
Tyr.sup.1-glucitol GIP (10 .mu.g) for 0 and 4 hours in a reaction
mixture made up to 500 .mu.l, containing 50 mmol/l
triethanolamine/HCL buffer pH 7.8. Incubations for 4 hours were
also performed in the presence of diprotin A (5 mU). The reactions
were terminated by addition of 5 .mu.l of TFA and the final volume
adjusted to 1.0 ml using 0.1% v/v TFA/water. Samples were
centrifuged (13,000 g, 5 minutes) and the supernatant applied to a
C-18 Sep-Pak cartridge (Millipore-Waters) which was previously
primed and washed with 0.1% (v/v) TFA/water. After washing with 20
ml 0.12% TFA/water, bound material was released by elution with 2
ml of 80% (v/v) acetonitrile/water and concentrated using a
Speed-Vac concentrator (Runcorn, UK). The volume was adjusted to
11.0 ml with 0.12% (v/v) TFA/water prior to HPLC purification.
[0180] HPLC analysis of degraded GIP and Tyr.sup.1-glucitol GIP.
Samples were applied to a Vydac C-18 widepore column equilibriated
with 0.12% (v/v) TFA/H.sub.2O at a flow rate of 1.0 mil/minute.
Using 0.1% (v/v) TFA in 70% acetonitrile/H.sub.2O, the
concentration of acetonitrile in the eluting solvent was raised
from 0% to 31.5% over 15 min, to 38.5% over 30 minutes and from
38.5% to 70% over 5 minutes, using linear gradients. The absorbance
was monitored at 206 nm and peak areas evaluated using a model 2221
LKB integrator. Samples recovered manually were concentrated using
a Speed-Vac concentrator.
[0181] Electrospray ionization mass spectrometry (ESI-MS). Samples
for ESI-MS analysis containing intact and degradation fragments of
GIP (from DPP IV and plasma incubations) as well as
Tyr.sup.1-glucitol GIP, were further purified by HPLC. Peptides
were dissolved (approximately 400 pmol) in 100 .mu.l of water and
applied to the LCQ benchtop mass spectrometer (Finnigan MAT, Hemel
Hempstead, UK) equipped with a microbore C-18 HPLC column
(150.times.2.0 mm, Phenomenex, Ltd., Macclesfield, UK). Samples (30
.mu.l direct loop injection) were injected at a flow rate of 0.2
ml/min, under isocratic conditions 35% (v/v) acetonitile/water.
Mass spectra were obtained from the quadripole ion trap mass
analyzer and recorded. Spectra were collected using full ion scan
mode over the mass-to-charge (m/z) range 150-2000. The molecular
masses of GIP and related structures were determined from ESI-MS
profiles using prominent multiple charged ions and the following
equation
M.sub.r=iM.sub.i-iM.sub.h
[0182] where M.sub.r=molecular mass; M.sub.i=m/z ratio; i=number of
charges; M.sub.h=mass of a proton.
[0183] In vivo biological activity of GIP and Tyr.sup.1-glucitol
GIP. Effects of GIP and Tyr.sup.1-glucitol GIP on plasma glucose
and insulin concentrations were examined using 10-12 week old male
Wistar rats. The animals were housed individually in an air
conditioned room and 22.+-.2.degree. C. with a 12 hour light/12
hour dark cycle. Drinking water and a standard rodent maintenance
diet (Trouw Nutrition, Belfast, Northern Ireland) were supplied ad
libitum. Food was withdrawn for an 18 hour period prior to
intraperitoneal injection of glucose alone (18 mmol/kq body weight)
or in combination with either GIP or Tyr.sup.1-glucitol GIP (10
nmol/kg). Test solutions were administered in a final volume of 8
ml/kg body weight. Blood samples were collected at 0, 15, 30 and 60
minutes from the cut tip of the tail of conscious rats into chilled
fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht,
Germany). Samples were centrifuged using a Beckman microcentrifuge
for about 30 seconds at 13,000 g. Plasma samples were aliquoted and
stored at -20.degree. C. prior to glucose and insulin
determinations. All animal studies were done in accordance with the
Animals (Scientific Procedures) Act 1986.
[0184] Analyses. Plasma glucose was assayed by an automated glucose
oxidase procedure using a Beckman Glucose Analyzer II [33]. Plasma
insulin was determined by dextran charcoal radioimmunoassay as
described previously [34]. Incremental areas under plasma glucose
and insulin area under the curve (AUC) were calculated using a
computer program (CAREA) employing the trapezoidal rule [35] with
baseline subtraction. Results are expressed as mean.+-.SEM and
values were compared using the Student's unpaired t-test. Groups of
data were considered to be significantly different if
P<0.05.
[0185] Degradation of GIP and Tyr.sup.1-glucitol GIP by DPP IV.
FIG. 1 illustrates the typical peak profiles obtained from the HPLC
separation of the products obtained from the incubation of GIP
(FIG. 1a) or Tyr.sup.1-glucitol GIP (FIG. 1b) with DPP IV for 0, 2,
4 and 12 hours. The retention times of GIP and Tyr.sup.1-glucitol
GIP at t=0 were 21.93 minutes and 21.75 minutes respectively.
Degradation of GIP was evident after 4 hours incubation (54%
intact), and by 12 hours the majority (60%) of intact GIP was
converted to the single product with a retention time of 21.61
minutes. Tyr.sup.1-glucitol GIP remained almost completely intact
throughout 2-12 hours incubation. Separation was on a Vydac C-18
column using linear gradients of 0% to 31.5% acetonitrile over 15
minutes, to 38.5% over 30 minutes and from 38.5 to 70% acetonitrile
over 5 minutes.
[0186] Degradation of GIP and Tyr.sup.1-glucitol GIP by human
plasma. FIG. 2 shows a set of typical HPLC profiles of the products
obtained from the incubation of GIP or Tyr.sup.1-glucitol GIP with
human plasma for 0 and 4 hours. GIP (FIG. 2a) with a retention time
of 22.06 minutes was readily metabolised by plasma within 4 hours
incubation giving rise to the appearance of a major degradation
peak with a retention time of 21.74 minutes. In contrast, the
incubation of Tyr.sup.1-glucitol GIP under similar conditions (FIG.
2b) did not result in the formation of any detectable degradation
fragments during this time with only a single peak being observed
with a retention time of 21.77 minutes. Addition of diprotin A, a
specific inhibitor of DPP IV, to GIP during the 4 hours incubation
completely inhibited degradation of the peptide by plasma. Peaks
corresponding with intact GIP, GIP (3-42) and Tyr.sup.1-glucitol
GIP are indicated. A major peak corresponding to the specific DPP
IV inhibitor tripeptide DPA appears in the bottom peanels with
retention time of 16-29 minutes.
[0187] Identification of GIP degradation fragments by ESI-MS. FIG.
3 shows the monoisotopic molecular masses obtained for GIP (FIG.
3A), Tyr.sup.1-glucitol GIP (FIG. 3B) and the major plasma
degradation fragment of GIP (FIG. 3C) using ESI-MS. The peptides
analyzed were purified from plasma incubations as shown in FIG. 2.
Peptides were dissolved (approximately 400 pmol) in 100 .mu.l of
water and applied to the LC/MS equipped with a microbore C-18 HPLC
column. Samples (30%1 direct loop injection) were applied at a flow
rate of 0.2 ml/min, under isocratic conditions 35%
acetonitrile/water. Mass spectra were recorded using a quadripole
ion trap mass analyzer. Spectra were collected using full ion scan
mode over the mass-to-charge (m/z) range 150-2000. The molecular
masses (M.sub.r) of GIP and related structures were determined from
ESI-MS profiles using prominent multiple charged ions and the
following equation M.sub.r=iM.sub.i-iM.sub.h. The exact molecular
mass (M.sub.r) of the peptides were calculated using the equation
M.sub.r=iM.sub.i-iM.sub.h as defined above in Research Design and
Methods. After spectral averaging was performed, prominent multiple
charges species (M+3H).sup.3+ and (M+4H).sup.4+ were detected from
GIP at m/z 1661.6 and 1246.8, corresponding to intact M.sub.r4981.8
and 4983.2 Da, respectively (FIG. 3A). Similarly, for
Tyr.sup.1-glucitol GIP ((M+4H).sup.4+ and (M+5H).sup.5+) were
detected at m/z 1287.7 and 1030.3, corresponding to intact
molecular masses of M.sub.r 5146.8 and 5146.5 Da, respectively
(FIG. 3B). The difference between the observed molecular masses of
the quadruply charged GIP and the N-terminally modified GIP species
(163.6 Da) indicated that the latter peptide contained a single
glucitol adduct corresponding to Tyr.sup.1-glucitol GIP. FIG. 3C
shows the prominent multiply charged species (M+3H).sup.3+ and
(M+4H).sup.4+ detected from the major fragment of GIP at m/z 1583.8
and 1188.1, corresponding to intact M.sub.r 4748.4 and 4748 Da,
respectively (FIG. 3C). This corresponds with the theoretical mass
of the N-terminally truncated form of the peptide GIP(3-42). This
fragment was also the major degradation product of DPP IV
incubations (data not shown).
[0188] Effects of GIP and Tyr.sup.1-glucitol GIP on plasma glucose
homeostasis. FIGS. 4 and 5 show the effects of intraperitoneal (ip)
glucose alone (18 mmol/kg) (control group), and glucose in
combination with GIP or Tyr.sup.1-glucitol GIP (10 nmol/kg) on
plasma glucose and insulin concentrations.
[0189] FIG. 4A shows plasma glucose concentrations after i.p.
glucose alone (18 mmol/kg) (control group), or glucose in
combination with either GIP of Tyr.sup.1-glucitol GIP (10 nmol/kg).
The time of injection is indicated by the arrow (O minutes). FIG.
4B shows plasma glucose AUC values for 0-60 minutes post injection.
Values are mean.+-.SEM for six rats. **P<0.01, ***P<0.001
compared with GIP and Tyr.sup.1-glucitol GIP; .dagger.P<0.05,
.dagger..dagger.P<0.01 compared with non-glucated GIP. FIG. 5A
shows plasma insulin concentrates after i.p. glucose along (18
mmol/kg) (control group), or glucose in combination with either
with GIP or glycated GIP (10 nmol/kq). The time of injection is
indicated by the arrow. FIG. 5B shows plasma insulin AUC values
were calculated for each of the 3 groups up to 90 minutes post
injection. The time of injection is indicated by the arrow (O
minutes). Plasma insulin AUC values for 0-60 minutes post
injection. Values are mean.+-.SEM for six rats. *P<0.05,
**P<0.001 compared with GIP and Tyr.sup.1-glucitol GIP;
.dagger.P<0.05, .dagger..dagger.P<0.01 compared with
non-glycated GIP.
[0190] Compared with the control group, plasma glucose
concentrations and area under the curve (AUC) were significantly
lower following administration of either GIP or Tyr.sup.1-glucitol
GIP (FIGS. 4A, B). Furthermore, individual values at 15 and 30
minutes together with AUC were significantly lower following
administration of Tyr.sup.1-glucitol GIP as compared to GIP.
Consistent with the established insulin-releasing properties of
GIP, plasma insulin concentrations of both peptide-treated groups
were significantly raised at 15 and 30 minutes compared with the
values after administration of glucose alone (FIG. 5A). The overall
insulin responses, estimated as AUC were also significantly greater
for the two peptide-treated groups (FIG. 5B). Despite lower
prevailing glucose concentrations than the GIP-treated group,
plasma insulin response, calculated as AUC, following
Tyr.sup.1-glucitol GIP was significantly greater than after GIP
(FIG. 5B). The significant elevation of plasma insulin at 30
minutes is of particular note, suggesting that the
insulin-releasing action of Tyr.sup.1-glucitol GIP is more
protracted than GIP even in the face of a diminished glycemic
stimulus (FIGS. 4A, 5A).
Example 3
Additional N-Terminal Structural Modifications of GIP
[0191] This example further looked at the ability of additional
N-terminal structural modifications of GIP in preventing
inactivation by DPP and in plasma and their associated increase in
both the insulin-releasing potency and potential therapeutic value.
Native human GIP, glycated GIP, acetylated GIP and a number of GIP
analogues with N-terminal amino acid substitutions were tested.
[0192] Materials and Methods. High-performance liquid
chromatography (HPLC) grade acetonitrile was obtained from Rathburn
(Walkersburn, Scotland). Sequencing grade trifluoroacetic acid
(TFA) was obtained from Aldrich (Poole, Dorset, UK). Dipeptidyl
peptidase IV was purchased from Sigma (Poole, Dorset, UK), and
Diprotin A was purchased from Calbiochem Novabiochem (Beeston,
Nottingham, UK). RPMI 1640 tissue culture medium, foetal calf
serum, penicillin and streptomycin were all purchased from Gibco
(Paisley, Strathclyde, UK). All water used in these experiments was
purified using a Milli-Q, Water Purification System (Millipore,
Milford, Mass., USA). All other chemicals used were of the highest
purity available.
[0193] Synthesis of GIP and N-terminally modified GIP analogues.
GIP, GIP(Abu.sup.2), GIP(Sar.sup.2), GIP(Ser.sup.2), GIP(Gly.sup.2)
and GIP(Pro.sup.3) were sequentially synthesized on an Applied
Biosystems automated peptide synthesizer (model 432 A) using
standard solid-phase Fmoc procedure, starting with an Fmoc-Gln-Wang
resin. Following cleavage from the resin by trifluoroacetic
acid:water, thioanisole, ethanedithiol (90/2.5/5/2.5, a total
volume of 20 ml/g resin), the resin was removed by filtration and
the filtrate volume was decreased under reduced pressure. Dry
diethyl ether was slowly added until a precipitate was observed.
The precipitate was collected by low-speed centrifugation,
resuspended in diethyl ether and centrifuged again, this procedure
being carried out at least five times. The pellets were then dried
in vacuo and judged pure by reversed-phase HPLC on a Waters
Millennium 2010 chromatography system (Software version 2.1.5.).
N-terminal glycated and acetylated GIP were prepared by minor
modification of a published method.
[0194] Electrospray ionization-mass spectrometry (ESI-MS) was
carried out as described in Example 2. Degradation of GIP and novel
GIP analogues by DPP IV and human plasma was carried out as
described in Example 2.
[0195] Culture of insulin secreting cells. BRIN-BD11 cells [30]
were cultured in sterile tissue culture flasks (Corning, Glass
Works, UK) using RPMI-1640 tissue culture medium containing 10%
(v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin,
0.1 mg/ml streptomycin) and 11.1 mM glucose. The cells were
maintained at 37.degree. C. in an atmosphere of 5% CO.sub.2 and 95%
air using a LEEC incubator (Laboratory Technical Engineering,
Nottingham, UK).
[0196] Acute tests for insulin secretion. Before experimentation,
the cells were harvested from the surface of the tissue culture
flasks with the aid of trypsin/EDTA (Gibco), seeded into
24-multiwell plates (Nunc, Roskilde, Denmark) at a density of
1.5.times.10.sup.5 cells per well, and allowed to attach overnight
at 37.degree. C. Acute tests for insulin release were preceded by
40 minutes pre-incubation at 37.degree. C. in 1.0 ml Krebs Ringer
bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl.sub.2,
1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 10 mM NaHCO.sub.3, 5
g/l bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose.
Test incubations were performed (n=12) at two glucose
concentrations (5.6 mM and 16.7 mM) with a range of concentrations
(10.sup.-13 to 10.sup.-8 M) of GIP or GIP analogues. After 20
minutes incubation, the buffer was removed from each well and
aliquots (200 .mu.l) were used for measurement of insulin by
radioimmunoassay [31].
[0197] Statistical analysis. Results are expressed as
mean.+-.S.E.M. and values were compared using the Student's
unpaired t-test. Groups of data were considered to be significantly
different if P<05.
[0198] Structural identification of GIP and GIP analogues by
ESI-MS. The monoisotopic molecular masses of the peptides were
determined using ESI-MS. After spectral averaging was performed,
prominent multiple charged species (M+3H).sup.3+ and (M+4H).sup.4+
were detected for each peptide. Calculated molecular masses
confirmed the structural identity of synthetic GIP and each of the
N-terminal analogues.
[0199] Degradation of GIP and novel GIP analogues by DPP-IV. FIGS.
6-11 illustrate the typical peak profiles obtained from the HPLC
separation of the reaction products obtained from the incubation of
GIP, GIP(Abu.sup.2), GIP(Sar.sup.2), GIP(Ser.sup.2), glycated GIP
and acetylated GIP with DPP IV, for 0, 2, 4, 8 and 24 hours. The
results summarized in Table 1 indicate that glycated GIP,
acetylated GIP, GIP(Ser.sup.2) are GIP(Abu.sup.2) more resistant
than native GIP to in vitro degradation with DPP IV. From these
data GIP(Sar.sup.2) appears to be less resistant.
1TABLE 1 Percent intact peptide remaining after incubation with
DPPIV. % Intact peptide remaining after time (h) Peptide 0 2 4 8 24
GIP 1-42 100 52 .+-. 1 23 .+-. 1 0 0 Glycated GIP 100 100 100 100
100 GIP (Abu.sup.2) 100 38 .+-. 1 28 .+-. 2 0 0 GIP (Ser.sup.2) 100
77 .+-. 2 60 .+-. 1 32 .+-. 4 0 GIP (Sar.sup.2) 100 28 .+-. 2 8 0 0
N-Acetyl-GIP 100 100 100 100
[0200] Table represents the percentage of intact peptide (i.e., GIP
1-42) relative to the major degradation product GIP 3-42. Values
were taken from HPLC traces performed in triplicate and the mean
and S.E.M. values calculated. DPA is diprotin A, a specific
inhibitor of DPPIV.
[0201] Degradation of GIP and GIP analogues by human plasma. FIGS.
12-16 show a representative set of HPLC profiles obtained from the
incubation of GIP and GIP analogues with human plasma for 0, 2, 4,
8 and 24 hours. Observations were also made after incubation for 24
hours in the presence of DPA. These results are summarized in Table
2 are broadly comparable with DPP IV incubations, but conditions
which more closely mirror in vivo conditions are less enzymatically
severe. GIP was rapidly degraded by plasma. In comparison, all
analogues tested exhibited resistance to plasma degradation,
including GIP(Sar.sup.2) which from DPP IV data appeared least
resistant of the peptides tested. DPA substantially inhibited
degradation of GIP and all analogues tested with complete abolition
of degradation in the cases of GIP(Abu.sup.2), GIP(Ser.sup.2) and
glycated GIP. This indicates that DPP IV is a key factor in the in
vivo degradation of GIP.
2TABLE 2 Percent intact peptide remaining after incubation with
human plasma. % Intact peptide remaining after incubations with
human plasma Peptide 0 2 4 8 24 DPA GIP 1-42 100 52 .+-. 1 23 .+-.
1 0 0 68 .+-. 2 Glycated GIP 100 100 100 100 100 100 GIP
(Abu.sup.2) 100 38 .+-. 1 28 .+-. 2 0 0 100 GIP (Ser.sup.2) 100 77
.+-. 2 60 .+-. 1 32 .+-. 4 0 63 .+-. 3 GIP (Sar.sup.2) 100 28 .+-.
2 8 0 0 100
[0202] Table represents the percentage of intact peptide (i.e., GIP
1-42) relative to the major degradation product GIP 3-42. Values
were taken from HPLC traces performed in triplicate and the mean
and S.E.M. values calculated. DPA is diprotin A, a specific
inhibitor of DPPIV.
[0203] Dose-dependent effects of GIP and novel GIP analogues on
insulin secretion. FIGS. 17-30 show the effects of a range of
concentrations of GIP, GIP(Abu.sup.2), GIP(Sar.sup.2), GIP(Ser 2),
acetylated GIP, glycated GIP, GIP(Gly.sup.2) and GIP(Pro.sup.3) on
insulin secretion from BRIN-BD11 cells at 5.6 and 16.7 mM glucose.
Native GIP provoked a prominent and dose-related stimulation of
insulin secretion. Consistent with previous studies [28], the
glycated GIP analogue exhibited a considerably greater
insulinotropic response compared with native peptide. N-terminal
acetylated GIP exhibited a similar pattern and the GIP(Ser 2)
analogue also evoked a strong response. From these tests,
GIP(Gly.sup.2) and GIP(Pro.sup.3) appeared to be the least potent
analogues in terms of insulin release. Other stable analogues
tested, namely GIP(Abu.sup.2) and GIP(Sar.sup.2), exhibited a
complex pattern of responsiveness dependent on glucose
concentration and dose employed. Thus very low concentrations were
extremely potent under hyperglycemic conditions (16.7 mM glucose).
This suggests that even these analogues may prove therapeutically
useful in the treatment of type 2 diabetes where insulinotropic
capacity combined with in vivo degradation dictates peptide
potency.
Example 4
Glu.sup.3 Substituted GIP Improves Obesity-Related Insulin
Resistance and Associated Glucose Intolerance
[0204] This example examines GIP receptor antagonism and
obesity-related insulin resistance and associated glucose
intolerance using a Glu.sup.3-substituted form of GIP, namely,
(Pro.sup.3)GIP.
[0205] Cell lines and animals. In vitro insulin secretion was
evaluated using the clonal pancreatic beta-cell line, BRIN-BD11
(McClenaghan, N. H. et al., 1996, Diabetes 45:1132-1140). In vitro
cyclic AMP generation was measured using Chinese hamster lung (CHL)
fibroblast cells stably transfected with the human GIP receptor
(Gremlich, S. et al., 1995, Diabetes 44:1202-1208). In vivo studies
were conducted in 8-12 week-old obese diabetic ob/ob mice (Bailey
C. J. et al., 1982, Int. J. Obesity 6:11-21) and normal control
mice.
[0206] Peptide synthesis and characterisation.
Glu.sup.3-substituted analogues were sequentially synthesised on an
Applied Biosystems automated peptide synthesiser (Model 432 A)
using standard solid-phase Fmoc peptide chemistry (Fields, G. B. et
al., 1990, Int. J. Pept. Protein Res. 35:161-214), from a
pre-loaded Fmoc-Gln-Wang resin. The synthetic peptides were judged
pure by reversed-phase HPLC on a Waters Millenium
2010-chromatography system (Software version 2.1.5). The molecular
masses of the purified peptide analogues were determined using
Matrix Assisted Laser Desorption ionisation-Time of Flight
(MALDI-TOF) mass spectrometry. Samples were dissolved in 10 W
H.sub.2O (approximately 40 pmol/l), placed on a stainless steel
sample plate and allowed to dry at room temperature. Samples were
then mixed with a matrix solution (10 mg/ml solution of
.alpha.-cyano-4-hydroxycinnamic acid) in acetonitrile/ethanol (1/1)
and allowed to dry at room temperature. The molecular masses were
then recorded as mass-to-charge (m/z) ratio versus relative peak
intensity and compared using theoretical values on a Voyager-DE
BioSpectrometry Workstation (PerSeptive Biosystems, Framingham,
Mass., USA).
[0207] Tissue culture. Chinese hamster lung (CHL) fibroblast cells
stably transfected with the human GIP receptor were cultured in
DMEM tissue culture medium containing 10% (v/v) foetal bovine
serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml
streptomycin). BRIN-BD11 cells were cultured using RPMI-1640 tissue
culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v)
antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Cells
were maintained in sterile tissue culture flasks (Corning Glass
Works, Sunderland, UK) at 37.degree. C. in an atmosphere of 5%
CO.sub.2 and 95% air using an LEEC incubator (Laboratory Technical
Engineering, Nottingham, UK).
[0208] Acute studies of insulin release. Insulin release from
BRIN-BD11 cells was determined using cell monolayers (McClenaghan,
N. H. et al., 1996, Diabetes 45:1132-1140). Cells were harvested
with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell
plates (Nunc, Roskilde, Denmark) at a density of 1.0.times.10.sup.5
cells per well, and allowed to attach overnight at 37.degree. C.
Prior to acute test, cells were preincubated for 40 minutes at
37.degree. C. in 1.0 ml Krebs Ringer bicarbonate buffer (115 mM
NaCl, 4.7 mM KCl, 1.28 mM CaCl.sub.2, 1.2 mM KH.sub.2PO.sub.4, 1.2
mM MgSO.sub.4, 10 mM NaHCO.sub.3, 0.5% (w/v) bovine serum albumin,
pH 7.4) supplemented with 1.1 mM glucose. Acute tests for insulin
release were performed for 20 minutes at 37.degree. C. at 5.6 mM
glucose using various concentrations of Glu.sup.3-substituted
analogues and GIP(3-42) in the presence of native GIP (10.sup.-7 M)
as indicated in the Figures. After incubation, aliquots of buffer
were removed and stored at -20.degree. C. for insulin
radioimmunoassay (Flatt, P. R. et al., 1981, Diabetologia
20:573-577).
[0209] Acute studies of cyclic AMP generation. GIP receptor
transfected CHL cells were seeded into 12-well plates (Nunc,
Roskilde, Denmark) at a density of 1.0.times.10.sup.5 cells per
well. The cells were then allowed to grow for 48 hours before being
loaded with tritiated adenine (2 .eta.Ci; TRK311, Amersham,
Buckinghamshire, UK) and incubated at 37.degree. C. for 6 hours in
1 ml DMEM, supplemented with 0.5% (w/v) foetal bovine serum. The
cells were then washed twice with HBS buffer (130 mM NaCl, 20 mM
HEPES, 0.9 mM NaHPO.sub.4, 0.8 mM MgSO.sub.4, 5.4 mM KCl, 1.8 mM
CaCl.sub.2, 25 mM glucose, 25 .mu.M phenol red, pH 7.4). The cells
were then exposed for 10 minutes at 37.degree. C. to forskolin
(FSK, 10 .mu.M) or varying concentrations of (Pro.sup.3)GIP in the
absence (control) or presence of native GIP (10.sup.-7 M). After
removal of the medium, cells were lysed with 1 ml of 5%
trichloroacetic acid (TCA) containing 0.1 mM unlabelled cAMP and
0.1 mM unlabelled ATP. The intracellular tritiated cAMP was then
separated on Dowex and alumina exchange resins as previously
described (Widmann, C. et al., 1993, Mol. Pharmacol.
45:1029-1035).
[0210] Acute in vivo effects of (Pro.sup.3)GIP administration in
obese diabetic ob/ob mice. Plasma glucose and insulin responses
were evaluated using 8- to 12-week old obese diabetic ob/ob mice
following intraperitoneal (i.p.) injection of native GIP,
(Pro.sup.3)GIP (25 nmol/kg body weight) or saline (0.9% (w/v) NaCl;
control) immediately following the combined injection of GIP (25
nmol/kg body weight) with glucose (18 mmol/kg body weight). All
test solutions were administered in a final volume of 8 ml/kg body
weight. Blood samples were collected from the cut tip of the tail
of conscious mice into chilled fluoride/heparin microcentrifuge
tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection
and at 15, 30 and 60 minutes post injection. Blood samples were
immediately centrifuged using a Beckman microcentrifuge (Beckman
Instruments, UK) for 30 seconds at 13000 g and stored at
-20.degree. prior to glucose and insulin determinations.
[0211] Acute in vivo effects of (Pro.sup.3)GIP on plasma glucose
and insulin responses to feeding in obese diabetic ob/ob mice.
Plasma glucose and insulin responses were evaluated using 8- to
12-week old ob/ob mice where food was withdrawn for an 18-hour
period prior to intraperitoneal injection of saline (0.9% (w/v)
NaCl; control) or (Pro.sup.3)GIP (25 nmol/kg body weight).
Following injection, the mice were allowed to re-feed for 15
minutes. Blood samples were collected from the cut tip of the tail
of conscious mice into chilled fluoride/heparin microcentrifuge
tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection
and at 15, 30, 60 and 120 minutes post injection. Blood samples
were immediately centrifuged using a Beckman microcentrifuge
(Beckman Instruments, UK) for 30 seconds at 13000 g and stored at
-20.degree. prior to glucose and insulin determinations.
[0212] Effects of chronic (Pro.sup.3)GIP administration on plasma
glucose, insulin and glycated HbA.sub.1c in obese diabetic ob/ob
mice and normal mice. Obese diabetic ob/ob mice and normal control
mice aged 8-12 weeks were randomly divided into groups which
received once daily subcutaneous injections (17:00 h) of either
saline (0.9% w/v NaCl) or (Pro.sup.3)GIP (25 nmol/kg body weight in
saline). After 11 days, treatment was ceased. Food intake and body
weight were recorded daily. Blood samples were collected from the
cut tip of the tail of conscious mice into chilled fluoride/heparin
coated glucose microcentrifuge tubes (Sarstedt, Numbrecht,
Germany). Blood samples were immediately centrifuged using a
Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at
13000 g prior to glucose, insulin and HbA.sub.1c
determinations.
[0213] Effects of chronic treatment with (Pro.sup.3)GIP on glucose
tolerance in ob/ob mice and normal mice. Plasma glucose and insulin
concentrations were measured following intraperitoneal
administration of glucose (18 mmol/kg body weight) in ob/ob and
normal mice treated with either saline (0.9% w/v NaCl) or
(Pro.sup.3)GIP (25 nmol/kg body weight/day) for 11 days. This test
was repeated 9 days after cessation of chronic (Pro.sup.3)GIP
treatment. Blood samples were collected from the cut tip of the
tail of conscious mice into chilled fluoride/heparin
microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately
prior to injection and at 15, 30 and 60 minutes post injection.
Blood samples were immediately centrifuged using a Beckman
microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000 g
and stored at -20.degree. prior to glucose and insulin
determinations.
[0214] Effects of chronic treatment with (Pro.sup.3)GIP on the
glucose lowering effects of exogenous insulin in ob/ob mice. The
glucose lowering effects of insulin were evaluated by measuring
plasma glucose response in 11-day saline (0.9% w/v NaCl) and
(Pro.sup.3)GIP (25 nmol/kg body weight/day) treated ob/ob mice
following acute intraperitoneal administration of insulin (50 U/kg
bodyweight). Blood samples were collected from the cut tip of the
tail of conscious mice into chilled fluoride/heparin
microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately
prior to injection and at 30 and 60 minutes post injection. Blood
samples were immediately centrifuged using a Beckman
microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000 g
and stored at -20.degree. prior to glucose determination.
[0215] Effects of chronic treatment with (Pro.sup.3)GIP on
pancreatic insulin content and associated islet hypertrophy in
ob/ob mice. Pancreatic tissue was excised from non-fasted ob/ob
mice after 11 days treatment with either saline (0.9% w/v NaCl) or
(Pro.sup.3)GIP (25 nmol/kg body weight/day). Pancreatic samples
were individually wrapped in aluminium foil and snap frozen in
liquid nitrogen. Individual excised pancreatic samples were then
either embedded, sectioned and immunohistochemically stained for
insulin or permeabilised for determination of pancreatic insulin
content.
[0216] Determination of HbA.sub.1c, plasma glucose and insulin
concentrations. HbA.sub.1c was measured in whole blood by
ion-exchange high-performance liquid chromatography using the
Menari HA-8140 kit (BIOMEN, Berkshire, UK). Plasma glucose was
assayed by an automated glucose oxidase procedure using a Beckman
Glucose Analyzer II (Stevens, J. F., 1971, Clinica Chemica Acta
32:199-201) and plasma insulin was determined by RIA (Flatt, P. R.
et al., 1981, Diabetologia 20:573-577). Incremental areas under
plasma glucose and insulin curves (AUC) were calculated using a
computer generated program (CAREA) employing the trapezoidal rule
(Burington, R. S., 1973, Handbook of Mathematical Tables and
Formulae, New York, McGraw Hill) with baseline subtraction.
[0217] Statistical analysis. Results are expressed as means.+-.SEM.
Values were compared using Student's unpaired t-test and groups of
data were considered to be significantly different if
P<0.05.
[0218] Results
[0219] GIP-stimulated cyclic AMP production and insulin secretion
were inhibited in dose-dependent fashion by (Pro.sup.3)GIP, showing
that (Pro.sup.3)GIP is a potent functional GIP receptor
antagonist.
[0220] GIP receptor transfected Chinese hamster lung (CHL)
fibroblasts were incubated with 10.sup.-2 to 10.sup.-6
M(Pro.sup.3)GIP in the presence of native GIP (10.sup.-7 M). The
results are shown in FIGS. 32A and 32B. FIG. 32A is a line graph
showing .sup.3H-cAMP production as a percent of maximal response
(y-axis) with increasing peptide concentration (M) (x-axis). FIG.
32B is a bar graph showing insulin secretion (y-axis) with
increasing peptide concentration (M) (x-axis) for 5.6 mM glucose
(control) (white bar), GIP (gray bars), (Pro.sup.3)GIP (lined bars)
and (Pro.sup.3)GIP+GIP(10.sup.-7M) (black bars). *P<0.05,
**P<0.01, ***P<0.001 compared to glucose control.
.sup..DELTA..DELTA.P<0.01, .sup..DELTA..DELTA..DELTA.P<0.001
compared with native GIP at the same concentration. Values are
means.+-.SEM for 3-8 observations.
[0221] (Pro.sup.3)GIP inhibited GIP-induced cAMP formation with an
IC.sub.50 value of 2.6 .mu.M. Insulin-releasing activity of
BRIN-BD11 cells exposed to native GIP and (Pro.sup.3)GIP (in the
absence and presence of 10.sup.-7 M GIP).
[0222] GIP-stimulated insulin secretion was inhibited in a
dose-dependent fashion by GIP(3-42), (Hyp.sup.3)GIP,
(Lys.sup.3)GIP, (Tyr.sup.3)GIP, (Trp.sup.3)GIP, and (Phe.sup.3)GIP,
as shown in FIGS. 33A through 33F, which are bar charts. FIG. 33A
shows .sup.3H-cAMP production as a percent of 10.sup.-7M GIP
(y-axis) versus log.sub.10 of GIP (10.sup.-7M) (white bar, control)
and GIP (10.sup.-7M)+GIP(3-42) (black bars). FIGS. 33B through 33F
show insulin secretion (in ng/10.sup.6 cells/20 minutes) (y-axis)
as a function of peptide concentration (M) (x-axis) for GIP
(10.sup.-7M) (white bar, control) and a Glu.sup.3-substituted form
of GIP (black bars), including (Hyp.sup.3)GIP (FIG. 33B),
(Lys.sup.3)GIP (FIG. 33C), (Tyr.sup.3)GIP (FIG. 33D),
(Trp.sup.3)GIP (FIG. 33E), and (Phe.sup.3)GIP (FIG. 33F).
*P<0.05, **P<0.01 compared to GIP (10.sup.-7 M) control.
Values are means.+-.SEM for 3-8 observations.
[0223] FIGS. 34A through 34D are a set of two line graphs (FIGS.
34A, 34C) and two bar graphs (FIGS. 34B, 34D) showing that acute
administration of (Pro.sup.3)GIP completely antagonises the actions
of GIP on glucose tolerance (FIGS. 34A, 34B) and plasma insulin
(FIGS. 34C, 34D) responses in obese diabetic ob/ob mice. FIGS. 34A
and 34C are line graphs show plasma glucose levels (FIG. 34A,
y-axis) and plasma insulin levels (FIG. 34C, y-axis) over time
(x-axis) for glucose (control; .tangle-soliddn.), glucose+GIP
(.diamond-solid.) and glucose+(GIP+Pro.sup.3GIP)) (.DELTA.). FIGS.
34B and 34D are bar graphs showing plasma glucose AUC for glucose
alone (white bars), GIP (grey bars) and glucose+(GIP+Pro.sup.3GIP))
(black bars).
[0224] Plasma glucose and insulin concentrations after i.p.
administration of glucose alone (18 mmol/kg body weight) or in
combination with either native GIP or native GIP plus
(Pro.sup.3)GIP (25 nmol/kg body weight). The time of injection is
indicated by the arrow (0 minutes). Plasma glucose and insulin AUC
values are given for 0-60 minutes post-injection. Values are
means.+-.SEM for 8 mice. *P<0.05, **P<0.01, ***P<0.001
compared with glucose alone. .sup..DELTA..DELTA.P<0.01,
.sup..DELTA..DELTA..DELTA.P<0.001 compared with native GIP.
[0225] Acute administration of (Pro.sup.3)GIP completely
antagonised the insulin-releasing action of GIP and the associated
improvement of glucose tolerance in ob/ob mice. Indeed, the
glycemic excursion following (Pro.sup.3)GIP (.DELTA.) was worse
than when glucose was administered alone (.tangle-soliddn.).
[0226] FIGS. 35A through 35D show the effects of (Pro.sup.3)GIP on
physiological meal-stimulated insulin release and glycemic
excursion in obese diabetic ob/ob mice. Plasma glucose and insulin
concentrations were measured in mice allowed to re-feed for 15
minutes prior to i.p. administration of saline (0.9% (w/v) NaCl) as
control or (Pro.sup.3)GIP (25 nmol/kg body weight). The time of
injection is indicated by the arrow (15 minutes).
[0227] The results are shown in FIGS. 35A through 35D, which are a
set of two line graphs (FIGS. 35A, 35C) and two bar graphs (FIGS.
35B, 35D). The figures show plasma insulin (FIGS. 35A) and plasma
glucose (FIG. 35C) over time for saline control (.tangle-soliddn.)
and (Pro.sup.3)GIP (.diamond.), and plasma insulin AUC (FIG. 35B)
and plasma glucose AUC (FIG. 35D) for saline control (white bars)
and (Pro.sup.3)GIP (black bars), respectively. Values are
means.+-.SEM for 8 mice. *P<0.05, **P<0.01, ***P<0.001
compared with saline alone.
[0228] Acute administration of (Pro.sup.3)GIP decreased the insulin
response to feeding and worsened the associated glycemic excursion
in ob/ob mice. These effects of functional ablation of endogenous
GIP by the (Pro.sup.3)GIP antagonist are fully consistent with the
accepted role of GIP in the regulation of insulin secretion and
glycemic excursion following feeding.
[0229] The effects of chronic administration of (Pro.sup.3)GIP for
11 days on plasma glucose and insulin concentrations of obese
diabetic ob/ob mice were also studied. According to classical
thinking and the experiments described above and the results shown
in FIGS. 32-35, functional ablation of endogenous GIP by daily
administration of (Pro.sup.3)GIP over 11 days would be expected to
inhibit insulin secretion and cause a marked deterioration in
glucose tolerance.
[0230] However, the exact opposite occurred during chronic
treatment with (Pro.sup.3)GIP in ob/ob mice. This is shown in FIG.
36, which is a set of two bar graphs showing plasma glucose (FIG.
36A) and insulin (FIG. 36B) concentrations after daily subcutaneous
administration of saline alone (0.9% (w/v) NaCl; as control; white
bars) or (Pro.sup.3)GIP (25 nmol/kg body weight; black bars) for 11
days. Values are means.+-.SEM for 6 mice and *P<0.05 compared
with saline alone. Chronic administration of (Pro.sup.3)GIP (black
bars) for 11 days decreases plasma glucose and plasma insulin
concentrations of obese diabetic ob/ob mice, relative to
controls.
[0231] The effects of chronic administration of (Pro.sup.3)GIP for
11 days on HbA.sub.1C, (FIG. 37A), pancreatic insulin content (FIG.
37B) and associated islet hypertrophy (FIG. 37C) were examined in
obese diabetic ob/ob mice treated with saline (control, white bars)
and (Pro.sup.3)GIP were examined. HbA.sub.1c, pancreatic insulin
content and average islet diameter were measured after 11 daily
subcutaneous injections of either saline alone (white bars) or
(Pro.sup.3)GIP (25 nmol/kg body weight; black bars) to obese
diabetic ob/ob mice. Values are means.+-.SEM for 6 mice and
*P<0.05, ***P<0.001 compared with saline-treated group.
[0232] Beneficial effects of chronic (Pro.sup.3)GIP administration
in ob/ob mice were associated with significant decreases in
HbA.sub.1c and pancreatic insulin stores, with partial correction
of the marked islet hypertrophy of the ob/ob mutant. There was also
an approximate 7% decrease in body weight in (Pro 3)GIP-treated
ob/ob mice without any change in food intake. This effect did not
achieve significance over the short study period, but this
observation clearly suggests that GIP antagonism may also have a
longer-term anti-obesity action.
[0233] The effects of chronic administration of (Pro.sup.3)GIP for
11 days on glucose tolerance and plasma insulin in obese diabetic
ob/ob mice is shown in FIGS. 38A-38D, which are a set of line
graphs (FIGS. 38A, 38C) and bar graphs (FIGS. 38B, 38C) showing
plasma glucose levels (FIGS. 38A, 38B) and plasma insulin levels
(FIGS. 38C, 38D) in obese diabetic ob/ob mice treated with saline
(control, white) or (Pro.sup.3)GIP (black). Plasma glucose and
insulin concentrations were measured prior to and at intervals
after intraperitoneal administration of glucose (18 mmol/kg body
weight). Arrow indicates time of injection (t=0). Values are
means.+-.SEM for 6 mice and *P<0.05, **P<0.01, ***P<0.001
compared with saline-treated group.
[0234] After 11 days treatment with (Pro.sup.3)GIP, glucose
tolerance of ob/ob mice was substantially improved without change
of circulating insulin (FIG. 38).
[0235] FIG. 39 shows the effects of chronic administration of
(Pro.sup.3)GIP for 11 days on insulin sensitivity in obese diabetic
ob/ob mice. Plasma glucose concentrations of saline and
(Pro.sup.3)GIP treated ob/ob mice were measured prior to and at
intervals after intraperitoneal administration of exogenous insulin
(50 U/kg body weight; t=0). Values are means.+-.SEM for 6 mice and
*P<0.05 compared with saline-treated group. As shown in FIG. 39,
chronic administration of (Pro.sup.3)GIP caused a significant
improvement of insulin sensitivity.
[0236] Interestingly, the beneficial effects of chronic
administration of (Pro.sup.3)GIP for 11 days in obese diabetic
ob/ob mice was reversed 9 days after cessation of treatment. This
is consistent with a physiological effect, and is shown in FIG. 40.
Plasma glucose concentrations were measured prior to and after
intraperitoneal administration of glucose (18 mmol/kg body weight)
for mice that had been treated with saline (control, .quadrature.)
or (Pro.sup.3)GIP (.tangle-solidup.). Arrow indicates time of
injection (t=0). Values are means.+-.SEM for 6.
[0237] FIGS. 41A and 41B are a pair of line graphs showing the
effects of chronic administration of (Pro.sup.3)GIP for 11 days on
glucose tolerance in normal mice. Plasma glucose concentrations
were measured prior to and after intraperitoneal administration of
glucose (18 mmol/kg body weight). Arrow indicates time of injection
(t=0). Values are means.+-.SEM for 6 and *P<0.05, **P<0.01
compared to saline-treated group.
[0238] In total contrast to beneficial actions in ob/ob mice,
chronic daily treatment of normal mice with (Pro.sup.3)GIP
(.DELTA.) for 11 days resulted in a marked deterioration of glucose
tolerance (FIG. 41A) relative to controls (.box-solid.), which was
reversed 9 days after cessation of treatment (FIG. 41B).
Example 5
Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action
By Daily (Pro.sup.3)GIP Administration Improves Glucose Tolerance
and Ameliorates Insulin Resistance and Abnormalities of Islet
Structure in Obesity-Diabetes
[0239] Gastric inhibitory polypeptide (GIP) is an important
incretin hormone secreted by endocrine K-cells in response to
nutrient ingestion. This study investigated the effects of chemical
ablation of GIP receptor (GIP-R) action on aspects of
obesity-diabetes using a stable and specific GIP-R antagonist,
(Pro.sup.3)GIP. Young adult ob/ob mice received once daily i.p.
injections of saline vehicle or (Pro.sup.3)GIP over an 1'-day
period. Non-fasting plasma glucose levels and the overall glycemic
excursion (AUC) to a glucose load were significantly reduced
(1.6-fold; P<0.05) in (Pro.sup.3)GIP-treated mice compared to
controls. GIP-R ablation also significantly lowered overall plasma
glucose (1.4-fold; P<0.05) and insulin (1.5-fold; P<0.05)
responses to feeding. These changes were associated with
significantly enhanced (1.6-fold; P<0.05) insulin sensitivity in
the (Pro.sup.3)GIP-treated group. Daily injection of (Pro.sup.3)GIP
reduced pancreatic insulin content (1.3-fold; P<0.05) and
partially corrected the obesity-related islet hypertrophy and beta
cell hyperplasia of ob/ob mice. These comprehensive beneficial
effects of (Pro.sup.3)GIP were reversed following 9 days cessation
of treatment and were independent of food intake and body weight,
which were unchanged. These studies highlight a role for GIP in
obesity-related glucose intolerance and emphasize the potential of
specific GIP-R antagonists as a new class of drugs for the
alleviation of insulin resistance and treatment of type 2
diabetes.
[0240] Research Design And Methods
[0241] Animals. Obese diabetic (ob/ob) mice derived from the colony
maintained at Aston University, UK (Bailey, C. J., et al., 1982,
Int. J. Obes. 6:11-21) were used at 12-16 weeks of age. Animals
were age-matched, divided into groups and housed individually in an
air-conditioned room at 22.+-.2.degree. C. with a 12 hour light: 12
hour dark cycle. Drinking water and a standard rodent maintenance
diet (Trouw Nutrition, Cheshire, UK) were freely available. All
animal experiments were carried out in accordance with the UK
Animals (Scientific Procedures) Act 1986. No adverse effects were
observed following administration of (Pro.sup.3)GIP.
[0242] Synthesis, purification and characterization of
(Pro.sup.3)GIP. (Pro.sup.3)GIP was sequentially synthesized on an
Applied Biosystems automated peptide synthesizer (Model 432 A).
(Pro.sup.3)GIP was purified by reversed-phase HPLC on a Waters
Millenium 2010 chromatography system (Software version 2.1.5) and
subsequently characterized using electrospray ionization mass
spectrometry (ESI-MS).
[0243] Experimental protocols for ob/ob mouse studies. Initially,
extended biological activity of (Pro.sup.3)GIP was examined in
18-hour fasted ob/ob mice 4 hours after administration. Thereafter,
over an 11-day period, mice received once daily i.p. injections
(17:00 hours) of either saline vehicle (0.9% (w/v), NaCl) or
(Pro.sup.3)GIP (25 nmol/kg body wt). During a subsequent 9-day
period, observations were continued following discontinuation of
(Pro.sup.3)GIP administration. Food intake and body weight were
recorded daily whilst plasma glucose and insulin concentrations
were monitored at intervals of 2-6 days. Whole blood for the
measurement of glycated hemoglobin was taken on days 11 and 20.
Intraperitoneal glucose tolerance (18 mmol/kg body wt), metabolic
response to native GIP (25 nmol/kg body wt) and insulin sensitivity
(50 U/kg body wt) tests were performed on days 11 and 20. Mice
fasted for 18 hours were used to examine the metabolic response to
15 minutes feeding. In a separate series, pancreatic tissues were
excised at the end of the 11-day treatment period or 9 days
following discontinuation of (Pro.sup.3)GIP and processed for
immunohistochemistry or measurement of insulin following extraction
with 5 ml/g of ice-cold acid ethanol (750 ml ethanol, 235 ml water,
15 ml concentrated HCl). Blood samples taken from the cut tip of
the tail vein of conscious mice at the times indicated in the
Figures were immediately centrifuged using a Beckman
microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13,000
g. The resulting plasma was then aliquoted into fresh Eppendorf
tubes and stored at -20.degree. C. prior to glucose and insulin
determinations.
[0244] Biochemical analysis. Plasma glucose was assayed by an
automated glucose oxidase procedure (Stevens, J. F., 1971, Clin.
Chem. Acta 32:199-201) using a Beckman Glucose Analyzer II (Beckman
Instruments, Galway, Ireland). Plasma and pancreatic insulin were
assayed by a modified dextran-coated charcoal radioimmunoassay
(Flatt, P. R. et al., 1981, Diabetologia 20:573-577). Glycated
hemoglobin was determined using cation-exchange columns (Sigma,
Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash
and eluting buffer using a VersaMax Microplate Spectrophotometer
(Molecular Devices, Wokingham, Berkshire, UK).
[0245] Immunocytochemistry. Tissue fixed in 4% paraformaldehyde/PBS
and embedded in paraffin was sectioned at 8 .mu.m. After de-waxing,
sections were incubated with blocking serum (Vector Laboratories,
CA, USA) prior to exposure to insulin antibody. Tissue samples were
then incubated consecutively with secondary biotinylated universal,
pan-specific antibody (Vector Laboratories, CA, USA) and
streptavidin/peroxidase preformed complex (Vector Laboratories, CA,
USA). Following washing, the stained pancreatic tissue was
counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and
then plunged into acid methanol (500 ml methanol, 500 ml H.sub.2O
and 2.5 ml concentrated HCl) prior to dehydration and mounting in
Depex (BDH Chemicals, Dorset, UK). The stained slides were viewed
under a microscope (Nikon Eclipse E2000, Diagnostic Instruments
Incorporated, Michigan, USA) attached to a JVC camera Model KY-F55B
(JVC, London, UK) and analyzed using Kromoscan imaging software
(Kinetic Imaging Limited, Faversham, Kent, UK). The average number
and diameter of every islet in each section was estimated in a
blinded manner using an eyepiece graticule calibrated with a stage
micrometer (Graticules Limited, Tonbridge, Kent, UK). The longest
and shortest diameters of each islet were determined with the
graticule. Half of the sum of these two values was then considered
to be the average islet diameter. Approximately 60-70 random
sections were examined from the pancreas of each mouse.
[0246] Statistics. Results are expressed as mean.+-.SEM. Data were
compared using ANOVA, followed by a Student-Newman-Keuls post hoc
test. Area under the curve (AUC) analyzes were calculated using the
trapezoidal rule with baseline subtraction (Burington, R. S.,
Handbook of Mathematical Tables and Formulae, New York,
McGraw-Hill, 1973). P<0.05 was considered to be statistically
significant.
[0247] Results
[0248] Effects of (Pro.sup.3)GIP on plasma glucose and insulin
concentrations 4 hours after administration were examined. The
results are shown in FIGS. 42A through 42D, which are a set of two
line graphs (FIGS. 42A, 42C) and two bar graphs (FIGS. 42B, 42D)
showing the effects of (Pro.sup.3)GIP on plasma glucose and insulin
response to native GIP 4 hours after administration. Tests were
conducted 4 hours after administration of (Pro.sup.3)GIP (25
nmoles/kg body weight) or saline (0.9% NaCl) in 18 hour-fasted
ob/ob mice. Plasma glucose and insulin concentrations were measured
prior to and after i.p. administration of glucose (18 mmoles/kg
body weight) in combination with native GIP (25 nmoles/kg body
weight). The incremental area under the glucose or insulin curves
(AUC) between 0 and 60 min are shown in the right panels. Values
represent means.+-.SEM for 8 mice. *P<0.05 and **P<0.01
compared with saline alone group.
[0249] As shown in FIGS. 42A through 42D, administration of
(Pro.sup.3)GIP for 4 hours previously impaired the plasma glucose
and insulin responses to native GIP, given together with glucose.
AUC glucose and insulin values were increased by 151% (P<0.05)
and decreased by 25% (P<0.05); respectively, compared with
saline-treated controls. This supports a protracted biological
half-life and forms the basis of the once-daily injection.
[0250] The effects of (Pro.sup.3)GIP on food intake, body weight
and non-fasting plasma glucose and insulin concentrations were
studied. The results are shown in FIGS. 43A through 43D, which are
a set of two line graphs and two bar graphs showing the effects of
daily (Pro.sup.3)GIP administration on food intake (FIG. 43A), body
weight (FIG. 43B), plasma glucose (FIG. 43C) and insulin (FIG. 43D)
concentrations in ob/ob mice. Parameters were measured for 5 days
prior to, 11 days during (indicated by black bar) and 9 days after
treatment with saline or (Pro.sup.3)GIP (25 nmol/kg bw/day). Values
are mean.+-.SEM for eight mice. *P<0.05 compared with saline
group.
[0251] Administration of (Pro.sup.3)GIP had no effect on food
intake and body weight (FIGS. 43A and 43B). On day 11, plasma
glucose had declined to significantly reduced (P<0.05)
concentrations in ob/ob mice receiving (Pro.sup.3)GIP (FIG. 43C).
Cessation of treatment returned plasma glucose concentrations
towards control levels. Consistent with this pattern, glycated
hemoglobin was significantly lower (P<0.05) after 11 days
treatment with (Pro.sup.3)GIP than either before or 9 days
following cessation of daily injection (8.0.+-.0.3%, 6.9.+-.0.2%,
7.7.+-.0.4%, respectively). No significant changes in plasma
insulin levels were noted during or after the treatment period.
However, there was a general trend for plasma insulin
concentrations to decrease progressively during (Pro.sup.3)GIP
treatment (FIG. 43D).
[0252] The effects of (Pro.sup.3)GIP on glucose tolerance are shown
in FIGS. 44A through 44D, which are a set of four line graphs with
inset bar graphs showing the effects of daily (Pro.sup.3)GIP
administration on glucose tolerance and plasma insulin response to
glucose in ob/ob mice. Tests were conducted after daily treatment
with (Pro.sup.3)GIP (25 nmoles/kg body weight/day;
.tangle-solidup.; black bars) or saline (control; .quadrature.;
white bars) for 11 days (FIG. 44A, 44C) or 9 days after cessation
of treatment (FIG. 44B, 44B). Glucose (18 mmoles/kg body weight)
was administered at the time indicated by the arrow. Plasma glucose
(FIG. 44A, 44B) and insulin (FIG. 44C, 44D) AUC values for 0-60
minutes post injection, with identical baseline subtractions in
each case to demonstrate the complete effect of (Pro.sup.3)GIP
treatment, are shown in insets. Values are mean.+-.SEM for eight
mice. *P<0.05, **P<0.01 and ***P<0.001 compared with
saline group.
[0253] Daily administration of (Pro.sup.3)GIP for 11 days resulted
in significantly reduced (P<0.001) plasma glucose concentrations
at 15, 30 and 60 minutes following intraperitoneal glucose (FIG.
44A). This was corroborated by a significantly decreased 0-60
minutes AUC value (FIG. 44A) which was 2.1-fold reduced (P<0.01)
compared to controls. Plasma insulin concentrations were also
significantly (P<0.05) reduced 15, 30 and 60 minutes following
intraperitoneal glucose injection in the (Pro.sup.3)GIP treated
group (FIG. 44A). AUC, 0-60 minutes values were also significantly
decreased (P<0.001). Interestingly, an almost identical pattern
was observed when 11 day treated ob/ob mice were administered
glucose together with native GIP (25 nmoles/kg body weight) (data
not shown). This supports the view that GIP action was effectively
antagonized in the (Pro.sup.3)GIP treated group. Discontinuation of
(Pro.sup.3)GIP treatment for 9 days (day 20 of study) resulted in
almost identical plasma glucose and insulin responses to
intraperitoneal glucose (FIG. 44), with lower glucose-mediated
plasma insulin concentrations noted at one time point (15 minutes;
P<0.05).
[0254] The effects of (Pro.sup.3)GIP on metabolic response to
feeding and insulin sensitivity are shown in FIGS. 45 and 46. FIGS.
45A through 45D are a set of two line graphs (FIGS. 45A, 45C) and
two bar graphs (FIGS. 45B, 45D) showing the effects of daily
(Pro.sup.3)GIP administration (.tangle-solidup.; black bars) or
saline (.quadrature.; white bars) on glucose (FIGS. 45A, 45B) and
insulin (FIGS. 45C, 45D) responses to feeding in ob/ob mice fasted
for 18 hours. Tests were conducted after daily treatment with
(Pro.sup.3)GIP (25 nmol/kg body weight/day) or saline for 11 days.
The arrow indicates the time of feeding (15 minutes). AUC values
for 0-105 minutes post-feeding are also shown. Values are
mean.+-.SEM for eight mice. *P<0.05 compared with saline
group.
[0255] FIGS. 46A through 46D are a set of two line graphs (FIGS.
46A, 46C) and two bar graphs (FIGS. 46B, 46D) showing the effects
of daily (Pro.sup.3)GIP administration on insulin sensitivity in
ob/ob mice. Tests were conducted after daily treatment with
(Pro.sup.3)GIP (25 nmol/kg body weight/day; .tangle-soliddn.; black
bars) or saline (.quadrature.; white bars) for 11 days (FIG. 46A,
46B) or 9 days after cessation of treatment (FIG. 46C, 46D).
Insulin (50 U/kg body weight) was administered by intraperitoneal
injection at the time indicated by the arrow. AUC values for 0-60
minutes post-injection are also shown. Values are mean.+-.SEM for
eight mice.*P<0.05 compared with saline group.
[0256] Plasma glucose and insulin responses to 15 minutes feeding
were significantly lowered (P<0.05) at 30 and 60 minutes in
ob/ob mice treated with (Pro.sup.3)GIP for 11 days (FIG. 45).
Similarly, AUC glucose and insulin were significantly (P<0.05)
decreased in (Pro.sup.3)GIP treated ob/ob mice, despite similar
food intakes of 0.3-0.5 g/mouse/15 minutes. As shown in FIGS. 46A
and 45B, the hypoglycemic action of insulin was significantly
(P<0.05) augmented in terms of AUC measures and post injection
values in ob/ob mice treated with (Pro.sup.3)GIP for 11 days. The
responses following 9 days discontinuation of (Pro.sup.3)GIP
treatment were similar to saline treated controls (FIG. 45C,
45D).
[0257] The effects of (Pro.sup.3)GIP on pancreatic insulin and
islet morphology are shown in FIGS. 47A through 47D, and 48A
through 48F. FIGS. 47A through 47D are a set of four bar graphs
showing the effects of daily (Pro.sup.3)GIP administration on
pancreatic weight (FIG. 47A), insulin content (FIG. 47B), islet
number (FIG. 47C) and islet diameter (FIG. 47D) in ob/ob mice.
Parameters were measured after daily treatment with (Pro.sup.3)GIP
(25 nmol/kg body weight/day; black bars) or saline (white bars) for
11 days and 9 days after cessation of treatment (day 20). Values
are mean.+-.SEM for eight mice. *P<0.05 and ***P<0.001
compared with saline group. FIGS. 48A through 48F are a set of two
bar graphs (FIGS. 48A, 48D) and four photomicrographs (FIGS. 48B,
48C, 48E, 48F), showing the effects of daily (Pro.sup.3)GIP
administration on islet size and morphology in ob/ob) mice.
[0258] (Pro.sup.3)GIP treatment had no effect on pancreatic weight
(FIG. 47A). However, pancreatic insulin content was significantly
(P<0.05) decreased in ob/ob mice receiving (Pro.sup.3)GIP for 11
days compared to controls (FIG. 47B). No significant differences
were observed in islet number per pancreatic section (FIG. 47C),
but average islet diameter was markedly and significantly reduced
(P<0.001) in (Pro.sup.3)GIP treated ob/ob mice (FIG. 47D). These
effects were effectively reversed by discontinuation of
(Pro.sup.3)GIP on day 20, however average islet diameter was still
significantly reduced (P<0.05). As shown in FIG. 48A, more
detailed analysis revealed that the reduction is islet diameter on
day 11 was due to a significant decrease (P<0.001) in the
percentage of larger diameter (>0.15 mm) islets with increases
in the proportion of islets with small (<0.10 mm) and medium
(0.1-0.15 mm) diameters. FIG. 48D presents similar analysis
following cessation of treatment, with a significant (P<0.05)
increase in the percentage of small islets still apparent.
Representative images (.times.40 magnification) of pancreata
immunohistologically stained for insulin from 11-day (Pro.sup.3)GIP
treated ob/ob mice (FIG. 48B) and saline treated controls (FIG.
48C) illustrate the dramatic changes in pancreatic islet morphology
induced by (Pro.sup.3)GIP treatment. Pancreata immunohistologically
stained for insulin on day 20 are also shown (FIG. 48E, 48F).
[0259] Parameters were measured after daily treatment with
(Pro.sup.3)GIP (25 nmol/kg body weight/day) or saline for 11 days
(FIG. 48A) and 9 days after cessation of treatment (FIG. 48D).
Proportion of islets classified as large (>0.15 mm) diameter,
medium (0.1-0.15 mm) diameter and small (<0.1 mm) diameter are
shown. Values are mean.+-.SEM for eight mice FIGS. 48B, 48C, 48E
and 48F are representative images (.times.40 magnification) of
pancreata stained for insulin following 11 days treatment with
(Pro.sup.3)GIP (FIG. 48B) or saline (FIG. 48C). Corresponding
images 9 days after cessation of treatment with (Pro.sup.3)GIP
(FIG. 48E) or saline (FIG. 48F) are also shown. The arrows indicate
islets.
Example 6
N-Terminally Acetylated and Ly.sup.16 and Lys.sup.37-Substituted
GIP
[0260] This example examines the metabolic stability, biological
activity and antidiabetic potential of fatty acid derivatized
N-terminally modified GIP analogues. These are
N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37), which have an
N-terminal Tyr.sup.1 acetyl group, and a C-16 palmitate group
linked to the epsilon-amino group of the lysine at either position
16 or position 37 of the GIP protein.
[0261] Materials and Methods
[0262] Animals. Obese diabetic (ob/ob) mice derived from the colony
maintained at Aston University, UK were used at 12-17 weeks of age.
The genetic background and characteristics of the colony used have
been outlined in detail elsewhere (Bailey, C. J. et al., 1982, Int.
J. Obesity 6:11-21; Gault, V. A. et al., 2003, J. Endocrinol. 176:
133-141). Animals were housed in an air-conditioned room at
22.+-.2.degree. C. with a 12 hours light: 12 hours dark cycle.
Drinking water and standard rodent maintenance diet (Trouw
Nutrition, Cheshire, UK) were freely available. All test solutions
were administered by i.p. injection in a final volume of 5 ml/kg
bw. Blood was collected from the cut tip of the tail vein of
conscious mice into chilled fluoride/heparin microcentrifuge tubes
immediately prior to injection and at the times indicated in the
Figures. Plasma was separated using a Beckman microcentrifuge
(Beckman Instruments, UK) at 13,000 g for 30 second and stored at
-20.degree. C. prior to glucose and insulin determinations. All
animal experiments were carried out in accordance with the UK
Animals (Scientific Procedures) Act 1986. No adverse effects were
observed following acute or long-term administration of any of the
peptides.
[0263] Materials. High performance liquid chromatography (HPLC)
grade acetonitrile was obtained from Rathburn (Walkersburn, UK).
Trifluoroacetic acid (TFA) and trichloroacetic acid (TCA) were
obtained from Aldrich (Poole, Dorset, UK). DPP IV,
isobutylmethylxanthine (IBMX), alpha-cyano-4-hydroxycinnamic acid,
cyclic AMP and ATP were all purchased from Sigma (Poole, Dorset,
UK). Fmoc-protected amino acids were from Calbiochem Novabiochem
(Nottingham, UK). RPMI-1640 and DMEM tissue culture medium, foetal
bovine serum, penicillin and streptomycin were all purchased from
Gibco (Paisley, Strathclyde, UK). The chromatography columns used
for cyclic AMP assay, Dowex AG50-WX and neutral alumina AG7 were
obtained from Bio-Rad (Life Science Research, Alpha Analytical,
Lame, UK). All water used in these experiments was purified using a
Milli-Q Water Purification System (Millipore, Milford, Mass., USA).
All other chemicals used were of the highest purity available.
[0264] Synthesis, purification and characterisation of GIP and
related analogues. Native GIP was sequentially synthesised using
standard solid-phase Fmoc peptide chemistry (ABI 432A Peptide
Synthesiser) as described previously (O'Harte, F. P. M. et al.,
2002, Diabetologia 45: 1281-1291). N-AcGIP(LysPAL.sup.16) and
N-AcGIP(LysPAL.sup.37) were synthesised in the same way as native
GIP but with the exception that the epsilon-amino groups of Lys at
positions 16 or 37 were conjugated with a C-16 palmitate fatty
acid. In addition, an acetyl adduct was incorporated at the
N-terminal Tyr.sup.1. The synthetic peptides were judged pure by
reversed-phase HPLC on a Waters Millenium 2010 chromatography
system (Software version 2.1.5) and subsequently characterised
using matrix assisted laser desorption ionisation-time of flight
mass spectrometry (MALDI-ToF MS) as described previously (Gault, V.
A. et al., 2002, Cell. Biol. Int. 27: 41-46).
[0265] DPP IV degradation studies. GIP and fatty acid derivatised
GIP analogues were incubated at 37.degree. C. with purified porcine
dipeptidylpeptidase IV (5 mU in 50 mmol/l triethanolamine-HCl; pH
7.8) for 0, 2, 4, 8 and 24 hours (final peptide concentration 2
mmol/l). The reactions were subsequently terminated by addition of
10% (v/v) TFA/water and the reaction products separated using HPLC.
Reaction products were applied to a Vydac C-4 column (4.6.times.250
mm; The Separations Group, Hesparia, Calif.) and the major
degradation product GIP(342) separated from intact GIP. The column
was equilibrated with 0.12% (v/v) TFA/water at a flow rate of 1.0
ml/minute using 0.1% (v/v) TFA in 70% acetonitrile/water with the
concentration of acetonitrile in the eluting solvent being raised
from 0% to 40% over 10 minutes, and then from 40% to 75% over 35
minutes. The absorbance was monitored at 206 nm using a
SpectraSystem UV 2000 Detector (Thermoquest Limited, Manchester,
UK) and the peaks collected manually prior to MALDI-ToF MS
analysis. HPLC peak area data were used to calculate % intact
peptide remaining throughout the incubation.
[0266] Cells and cell culture. Chinese hamster lung (CHL)
fibroblasts stably transfected with the human GIP receptor
(Gremlich, S. et al., 1995, Diabetes 44: 1202-1208) were cultured
in DMEM tissue culture medium containing 10% (v/v) FBS, 1% (v/v)
antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Clonal
pancreatic BRIN-BD11 cells (McClenaghan, N. H. et al., 1996,
Diabetes 45: 1132-1140) were cultured using RPMI-1640 culture
medium containing 10% (v/v) FBS, 1% (v/v) antibiotics (100 U/ml
penicillin, 0.1 mg/ml streptomycin) and 11.1 mmol/l glucose. Cells
were maintained at 37.degree. C. in an atmosphere of 5% CO.sub.2
and 95% air using an LEEC incubator (Laboratory Technical
Engineering, Nottingham, UK).
[0267] In vitro biological activity. Intracellular cyclic AMP
production was measured using GIP-receptor transfected CHL
fibroblasts (O'Harte, F. P. M. et al., 2002, Diabetologia 45:
1281-1291). In brief, CHL cells were seeded into 12-well plates
(Nunc, Roskilde, Denmark) at a density of 10.sup.5 cells per well
and allowed to grow for 48 hours before being loaded with tritiated
adenine (2 .mu.Ci; TRK311; Amersham, Buckinghamshire, UK). The
cells were then incubated at 37.degree. C. for 6 hours in 1 ml DMEM
supplemented with 0.5% (w/v) BSA and subsequently washed twice with
HBS buffer (pH 7.4). Cells were then exposed to GIP/GIP analogues
(10.sup.-13 to 10.sup.-6 mol/l) in HBS buffer in the presence of 1
mmol/l IBMX for 15 minutes at 37.degree. C. The medium was
subsequently removed and the cells lysed with 1 ml of 5% TCA
containing 0.1 mmol/l unlabelled cyclic AMP and 0.1 mmol/l
unlabelled ATP. The intracellular cyclic AMP was then separated on
Dowex and alumina exchange resins as described previously (O'Harte,
F. P. M. et al., 2002, Diabetologia 45: 1281-1291).
[0268] Insulin-release studies were carried out using clonal
pancreatic BRIN-BD11 cells as described previously (O'Harte, F. P.
M. et al., 2002, Diabetologia 45: 1281-1291). Briefly, BRIN-BD11
cells were seeded into 24-well plates at a density of 10.sup.5
cells per well, and allowed to attach overnight at 37.degree. C.
Acute tests for insulin release were preceded by 40 minutes
pre-incubation at 37.degree. C. in 1.0 ml Krebs Ringer bicarbonate
buffer supplemented with 1.1 mmol/l glucose. Test incubations were
performed in the presence of 5.6 mmol/l glucose with a range of
concentrations (10.sup.-13 to 10.sup.-6 mol/l) of GIP and GIP
analogues. After 20 minutes incubation, the buffer was removed from
each well and aliquots (200 .mu.l) used for measurement of
insulin.
[0269] Effects of N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37)
in ob/ob mice. Metabolic and dose-response effects of GIP and
N-AcGIP(LysPAL) analogues (at 6.25-25 nmoles/kg bw) following
glucose administration (18 mmoles/kg bw) were examined in mice
fasted for 18 hours. To evaluate long-term effects, groups of ob/ob
mice received once daily intraperitoneal injections (17:00 h) for
14 days of either saline vehicle (0.9%, w/v, NaCl), native GIP or
N-AcGIP(LysPAL.sup.37) (both at 12.5 nmoles/kg body weight/day).
Food intake and body weight were recorded daily. Plasma glucose and
insulin concentrations were monitored at 2-6 day intervals. At 14
days, groups of animals were used to evaluate intraperitoneal
glucose tolerance (18 mmoles/kg) and insulin sensitivity (50 U/kg).
In a separate series, two experimental protocols were employed to
examine the possibility of GIP receptor desensitization after 14
days treatment. Acute metabolic effects of the usual injection of
either saline, GIP or N-AcGIP(LysPAL.sup.37) were monitored when
administered together with glucose (18 mmoles/kg). In the second,
acute effects of N-AcGIP(LysPAL.sup.37) given together with glucose
were examined in all 3 groups of mice. At the end of the 14-day
treatment period, pancreatic tissues were excised for measurement
of insulin following extraction with 5 ml/g ice-cold acid ethanol
(75% ethanol, 2.35% H.sub.2O, 1.5% HCl). Whole blood was taken for
determination of glycated hemoglobin.
[0270] Biochemical analyses. Plasma glucose was assayed by an
automated glucose oxidase procedure (Stevens, J. F., 1971, Clin.
Chem. Acta 32:199-201) using a Beckman Glucose Analyser II
(Beckman, Galway, Ireland). Plasma insulin was determined by
dextran-charcoal RIA as described previously (Flatt, P. R. et al.,
1981, Diabetologia 20: 573-577). Glycated hemoglobin was determined
using cation-exchange columns (Sigma, Poole, Dorset, UK) with
measurement of absorbance (415 nm) in wash and eluting buffers
using a VersaMax microplate spectrophotometer (Molecular Devices,
Wokingham, Berkshire, UK).
[0271] Statistics. Results are expressed as mean.+-.SEM. Data were
compared using the unpaired Student's t-test. Where appropriate,
data were compared using repeated measures ANOVA or one-way ANOVA,
followed by the Student-Newman-Keuls post hoc test. Incremental
areas under plasma glucose and insulin curves (AUC) were calculated
using a computer-generated program employing the trapezoidal rule
(Burington, R. S., 1973, Handbook of Mathematical Tables and
Formulae, McGraw-Hill, New York) with baseline subtraction. Groups
of data were considered to be significantly different if
p<0.05.
[0272] Results
[0273] Structural characterisation by MALDI-ToF MS. Following
synthesis and HPLC purification, the molecular masses were obtained
for GIP, N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37) using
MALDI-ToF MS (Table 3, below). The mass-to-charge (m/z) ratio for
native GIP was calculated to be 4983.7 Da, corresponding very
closely to the theoretical mass of 4982.4 Da. Similarly, the m/z
ratios for N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37) were
5268.9 Da and 5267.7 Da, respectively. These values correlate very
closely to the theoretical mass (5266.1 Da), therefore, confirming
the correct structures for each of the synthetic peptides.
3TABLE 3 Structural characterisation of GIP and GIP analogues by
MALDI-ToF MS. Experimental Theoretical Difference Peptide M.sub.r
(Da) M.sub.r (Da) (Da) GIP 4983.7 4982.4 1.3 N-AcGIP(LysPAL.sup.16)
5268.9 5266.1 2.8 N-AcGIP(LysPAL.sup.37) 5267.7 5266.1 1.6
[0274] Peptide samples were mixed with matrix
(alpha-cyano-4-hydroxycinnam- ic acid) and m/z ratio vs. relative
peak intensity recorded using a Voyager-DE BioSpectrometry
Workstation.
[0275] Degradation by DPP IV. Table 4, below, illustrates the %
intact peptide remaining after incubation with DPP IV. Degradation
of native GIP was evident after just 2 hours with only 52.+-.3% of
the peptide remaining intact. After 8 hours incubation the native
peptide was entirely degraded to GIP(3-42). In contrast, both
N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37) remained
completely intact (no degradation fragment evident) even after 24
hours incubation with DPP IV.
4TABLE 4 Percentage intact peptide remaining after incubation with
DPP IV. % Intact peptide remaining after time (hours) Peptide 0 2 8
24 Native GIP 100 52 .+-. 3 0 0 N-AcGIP(LysPAL1.sup.16) 100 100 100
100 N-AcGIP(LysPAL.sup.37) 100 100 100 100
[0276] Values represent the % intact peptide remaining relative to
the major degradation product GIP(3-42) following incubation with
DPP IV as determined from HPLC peak area data. The reactions were
performed in triplicate and the means.+-.SEM values calculated.
[0277] Changes in Cyclic AMP production. FIG. 50A shows
intracellular cyclic AMP production by GIP (.tangle-solidup.) and
fatty acid derivatised GIP analogues N-AcGIP(LysPAL.sup.16)
(.quadrature.) and N-AcGIP(LysPAL.sup.37) (.circle-solid.), as
determined by column chromatography, in CHL cells stably expressing
the human GIP receptor. Each experiment was performed in triplicate
(n=3) and the results expressed (means.+-.SEM) as a percentage of
the maximum GIP response.
[0278] A concentration-dependent (10.sup.-13 to 10.sup.-6 mol/l)
increase in cyclic AMP production was observed with native GIP
(EC.sub.50 value 18.2 nmol/l) using CHL cells transfected with the
human GIP receptor (FIG. 50A). Likewise, both
N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37) followed a
similar pattern of stimulation to that of native GIP with
calculated EC.sub.50 values of 12.1 and 13.0 nmol/l, respectively.
The lower EC.sub.50 values for both analogues suggest an enhanced
cyclic AMP-stimulating potency.
[0279] In vitro insulin-releasing activity. FIG. 50B shows
insulin-releasing activity of glucose (5.6 mmol/l glucose; white
bars), GIP (lined bars) and fatty acid derivatised GIP analogues
N-AcGIP(LysPAL.sup.16) (grey bars) and N-AcGIP(LysPAL.sup.37)
(black bars) in the clonal pancreatic beta cell line, BRIN-BD11.
After a pre-incubation (40 minutes), the effects of various
concentrations of peptide were tested on insulin-release during a
20 minutes incubation. Values are means.+-.SEM for 8 separate
observations. *p<0.05, **p<0.01, ***p<0.001 compared to
control (5.6 mmol/l glucose alone).
[0280] Consistent with its role as a potent insulinotropic hormone,
native GIP dose-dependently stimulated insulin secretion (p<0.01
to p<0.001) compared to control (5.6 mmol/l glucose alone) (FIG.
50B). Likewise, both N-AcGIP(LysPAL 6) and N-AcGIP(LysPAL.sup.37)
significantly stimulated glucose-induced insulin secretion
(p<0.05 to p<0.001). On the basis of cyclic AMP and insulin
secretory data, both GIP analogues appear to be at least equipotent
to the native peptide.
[0281] Metabolic effects in ob/ob mice. FIGS. 51A through 51D are a
set of two line graphs (FIGS. 51A, 51C) and two bar graphs (FIGS.
51B, 51D) showing glucose lowering effects (FIGS. 51A, 51B) and
insulin-releasing activity (FIGS. 51C, 51D) of GIP and fatty acid
derivatised GIP analogues in 18 hour-fasted ob/ob mice. Plasma
glucose and insulin concentrations were measured prior to and after
i.p. administration of glucose alone (18 mmoles/kg bw;
.smallcircle.; white bars) as a control, or in combination with GIP
(.tangle-solidup.; lined bars) or GIP analogues
N-AcGIP(LysPAL.sup.16) (.quadrature.; grey bars) and
N-AcGIP(LysPAL.sup.37) (.circle-solid.; black bars) (25 nmoles/kg
bw). The incremental area under the glucose or insulin curves (AUC)
between 0 and 60 minutes are shown in the right panels. Values
represent means.+-.SEM for 8 mice. *p<0.05, **p<0.01,
***p<0.001 compared to glucose alone, .sup..DELTA.p<0.05,
.sup..DELTA..DELTA.p<0.01 and
.sup..DELTA..DELTA..DELTA.p<0.001 compared to native GIP,
.sup..gamma..gamma..gamma.p<0.001 compared with
N-AcGIP(LysPAL16).
[0282] Basal blood glucose levels of the experimental groups were
not significantly different at the start of the study (p>0.05).
After injection of glucose alone, plasma glucose levels increased
rapidly, attaining values of 40.3.+-.1.5 mmol/1 at 60 min. Native
GIP reduced plasma glucose at each of the time points monitored,
however, this failed to reach significance in terms of overall
glucose excursion as revealed by the AUC values (FIG. 52B).
Administration of N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37)
produced a significant reduction in plasma glucose at each time
point (p<0.01 to p<0.001) and significantly lowered glucose
AUC (p<0.001 to p<0.001) when compared to glucose alone.
Additionally, N-AcGIP(LysPAL.sup.16) and N-AcGIP(LysPAL.sup.37)
decreased the overall glucose excursion (p<0.05 to p<0.001)
when compared to native GIP.
[0283] The corresponding plasma insulin responses are illustrated
in FIGS. 51C and 51D. After administration of glucose alone
(control) the maximal rise in plasma insulin was observed at 15
minutes, which then fell towards basal levels over the remaining 45
minutes. Administration of native GIP significantly elevated the
overall insulinotropic response (p<0.001) compared with glucose
alone. When N-AcGIP(LysPAL.sup.16) or N-AcGIP(LysPAL.sup.37) where
administered together with glucose, a maximum plasma insulin
concentration was observed at 15 minutes. Protracted biological
activity for both analogues was clearly evident from 30 to 60
minutes. Glucose-mediated plasma insulin concentrations were
significantly higher compared in both control (p<0.01 to
p<0.001) and GIP-treated animals (p<0.05 to p<0.001). The
corresponding AUC values for N-AcGIP(LysPAL.sup.16) and
N-AcGIP(LysPAL.sup.37) revealed significant enhancements in overall
glucose-mediated insulin release compared to native GIP (1.5-fold
and 2.3-fold, respectively; p<0.01 to p<0.001).
N-AcGIP(LysPAL.sup.37) was significantly more potent (1.5-fold:
p<0.001) than N-AcGIP(LysPAL.sup.16) at stimulating insulin
secretion.
[0284] Dose-dependent metabolic effects in ob/ob mice. FIGS. 52A
and 52B illustrate the dose-dependent antihyperglycaemic and
insulinotropic effects of GIP and the more potent analogue
N-AcGIP(LysPAL.sup.37) when administered with glucose to ob/ob
mice. They are are a pair of bar graphs showing dose-dependent
effects of GIP and N-AcGIP(LysPAL.sup.37) in ob/ob mice fasted for
18 hours. The incremental area under the curve (AUC) for glucose
(FIG. 52A) and insulin (FIG. 52B) between 0 and 60 minutes after
i.p. administration of glucose alone (18 mmoles/kg bw; white bars)
or in combination with GIP (lined bars) or N-AcGIP(LysPAL.sup.37)
(each at 6.25, 12.5 and 25 nmoles/kg bw; black bars). Values
represent means.+-.SEM for 8 mice. **p<0.01 and ***p<0.001
compared to glucose alone. .sup..DELTA..DELTA.p<0.01 and
.sup..DELTA..DELTA..DELTA.p<0.001 compared to native GIP at the
same dose.
[0285] Data are presented as overall AUC responses for convenience.
Expressed in this manner, native GIP did not significantly affect
AUC glucose and insulin at any of the doses tested.
N-AcGIP(LysPAL.sup.37) was substantially more potent than native
GIP (p<0.01 to p<0.001) and exhibited prominent
dose-dependent antihyperglycaemic and insulinotropic actions at all
doses administered (FIGS. 52A, 52B). Remarkably, even the lowest
concentration of N-AcGIP(LysPAL.sup.37) tested (6.25 nmoles/kg) had
highly significant antihyperglycaemic properties compared to
glucose alone (p<0.001). Consistent with this observation, 6.25
nmoles/kg N-AcGIP(LysPAL.sup.37) elicited a prominent insulin
response (2.0-fold; p<0.01) compared to glucose alone.
[0286] Long-acting effects in ob/ob mice. The effects of daily
injection of N-AcGIP(LysPAL.sup.37) for 14 days on food intake,
body weight, glycated hemoglobin and non-fasting plasma glucose and
insulin concentrations of ob/ob mice are shown in FIGS. 53A through
53E, which are a set of graphs showing the effects of daily
N-AcGIP(LysPAL.sup.37) (.circle-solid.; black bars) administration
on food intake (FIG. 53A), body weight (FIG. 53B), plasma glucose
(FIG. 53C), insulin (FIG. 53D) and glycated hemoglobin
N-AcGIP(LysPAL.sup.37) (12.5 nmoles/kg/day) (FIG. 53E). Native GIP
(12.5 nmoles/kg/day; .tangle-solidup.; lined bars) or saline
vehicle (control; .quadrature.; white bars) were administered for
the 14-day period indicated by the horizontal black bar. Values are
means.+-.SEM for 8 mice. *p<0.05, **p<0.01 compared to
control. .sup..DELTA..DELTA.p<0.01 compared to native GIP.
[0287] GIP or N-AcGIP(LysPAL.sup.37) had no effect on body weight
or food intake (FIGS. 53A, 53B). Plasma glucose and insulin
concentrations were also unchanged by treatment with native GIP for
14 days (FIGS. 53C, 53D). In contrast, daily injection of
N-AcGIP(LysPAL.sup.37) resulted in a progressive lowering of plasma
glucose, resulting in significantly (p<0.05) lowered
concentrations at 14 days (FIG. 53C). At this time, glycated
hemoglobin was also significantly (p<0.01) decreased in
N-AcGIP(LysPAL.sup.37) treated ob/ob mice (FIG. 53E). These changes
were accompanied by a tendency towards elevated insulin
concentrations, but these did not achieve statistical significance
over the time frame studies (FIG. 53D).
[0288] Effects of long term treatment of ob/ob mice with
N-AcGIP(LysPAL.sup.37) on glucose tolerance. FIGS. 54A through 54D
are a set of two line graphs (FIGS. 54A, 54C) and two bar graphs
(FIGS. 54B, 54D) showing the effects of daily
N-AcGIP(LysPAL.sup.37) administration on glucose tolerance (FIGS.
54A, 54B) and plasma insulin response (FIGS. 54C, 54D) to glucose.
Tests were conducted after 14 daily injections of either
N-AcGIP(LysPAL.sup.37) (12.5 nmoles/kg/day; .circle-solid.; black
bars), native GIP (12.5 nmoles/kg/day; .tangle-solidup.; lined
bars) or saline vehicle (control; .quadrature.; white bars).
Glucose (18 mmoles/kg) was administered by intraperitoneal
injection at the time indicated by the arrow. Plasma glucose and
insulin AUC values for 0-60 minutes post injection are shown in the
right panels. Values are means.+-.SEM for 8 mice. *p<0.05,
**p<0.01, ***p<0.001 compared to control.
.sup..DELTA.p<0.05, .sup..DELTA..DELTA.p<0.01,
.sup..DELTA..DELTA..DELTA.p<0.001 compared to native GIP.
[0289] Consistent with effects on glycated hemoglobin, treatment of
ob/ob mice for 14 days with N-AcGIP(LysPAL.sup.37) resulted in a
significant improvement in glucose tolerance (FIGS. 54A, 54B).
Plasma glucose concentrations throughout the test and the overall
0-60 minutes AUC values were decreased (p<0.01 to p<0.001).
This was accompanied by increased insulin concentrations during the
latter stages (p<0.05) and a greater (p<0.01) overall AUC
insulin response (FIGS. 54C, 54D). In contrast, daily
administration of native GIP had no effect on glucose tolerance or
the plasma insulin response to glucose compared with control ob/ob
mice receiving saline injections for 14 days (FIG. 54).
[0290] Effects long term treatment of ob/ob mice with
N-AcGIP(LysPAL37) on insulin sensitivity, and effects of long term
treatment of ob/ob mice with N-AcGIP(LysPAL.sup.37) on pancreatic
insulin content. FIGS. 55A through 55D are a line graph and three
bar graphs showing the effects of daily N-AcGIP(LysPAL.sup.37)
administration on insulin sensitivity (FIGS. 55A, 55B) and
pancreatic weight (FIG. 55C) and insulin content (FIG. 55D).
Observations were conducted after 14 daily injections of either
N-AcGIP(LysPAL.sup.37) (12.5 nmoles/kg/day; .circle-solid.; black
bars), native GIP (12.5 nmoles/kg/day; .tangle-solidup.; lined
bars) or saline vehicle (control; .quadrature.; white bars). In
FIG. 55A, insulin (50 U/kg) was administered by intraperitoneal
injection at the time indicated by the arrow. Plasma glucose AUC
values for 0-60 minutes post injection are shown in the right
panels. Values are means.+-.SEM for 8 mice. *p<0.05, **p<0.01
compared to control. .sup..DELTA.p<0.05,
.sup..DELTA..DELTA.p<0.01 compared to native GIP.
[0291] Insulin sensitivity of the 3 groups of mice after 14 days
treatment is shown in FIGS. 55A, 55B. Compared with ob/ob mice
receiving daily injections of saline or native GIP,
N-AcGIP(LysPAL.sup.37) prompted a significant improvement of
insulin sensitivity. Both the individual glucose concentrations and
0-60 minutes AUC values were significantly different (p<0.01)
from the other two groups. In contrast, daily treatment with native
GIP did not affect the characteristic insulin resistance of ob/ob
mice (FIG. 55A, 55B).
[0292] Treatment of ob/ob mice for 14 days with native GIP or
N-AcGIP(LysPAL.sup.37) did not affect pancreatic weight compared
with saline-treated controls (FIGS. 55C, 55D). Similarly,
pancreatic insulin content was similar in the GIP and saline
treated groups. However, daily administration of
N-AcGIP(LysPAL.sup.37) significantly increased (p<0.01) insulin
content compared with each of the other groups (FIGS. 55C,
55D).
[0293] Evaluation of GIP receptor desensitization after long term
treatment of ob/ob mice with N-AcGIP(LysPAL.sup.37). FIGS. 56A
through 56D are a set of two line graphs (FIGS. 56A, 56C) and two
bar graphs (FIGS. 56B, 56D) showing the retention of glucose
lowering (FIGS. 56A, 56B) and insulin releasing (FIGS. 56C, 56D)
activity of N-AcGIP(LysPAL.sup.37) and native GIP after daily
injection for 14 days. Glucose (18 mmoles/kg) was administered by
intraperitoneal injection alone (.quadrature.; white bars) or in
combination with either N-AcGIP(LysPAL.sup.37) (.circle-solid.;
black bars) or native GIP (.tangle-solidup.; lined bars) (both at
25 nmoles/kg) at the time indicated by the arrow. Plasma glucose
and insulin AUC values for 0-60 minutes post injection are shown in
the right panels. Values are means.+-.SEM for 8 mice. *p<0.05,
**p<0.01 compared to glucose alone. .sup..DELTA.p<0.05,
.sup..DELTA..DELTA.p<0.01 compared to native GIP. FIGS. 57A
through 57D are a set of two line graphs (FIGS. 57A, 57C) and two
bar graphs (FIGS. 57B, 57D) showing the acute glucose lowering
(FIGS. 57A, 57B) and insulin releasing (FIGS. 57C, 57D) effects of
N-AcGIP(LysPAL.sup.37) after 14 daily injections of either
N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; .circle-solid.; black bars),
native GIP (12.5 nmoles/kg/day; .tangle-solidup.; lined bars) or
saline vehicle (control; .quadrature.; white bars).
N-AcGIP(LysPAL.sup.37) (25 nmoles/kg) was administered by
intraperitoneal injection with glucose (18 mmoles/kg) at the time
indicated by the arrow. Plasma glucose and insulin AUC values for
0-60 minutes post injection are shown in the right panels. Values
are means.+-.SEM for 8 mice. *p<0.05, **p<0.01 compared to
mice receiving control injections. .sup..DELTA.p<0.05,
.sup..DELTA..DELTA.p<0.01 compared to group receiving injections
of native GIP.
[0294] As shown in FIGS. 56A through 56D, treatment of ob/ob mice
with N-AcGIP(LysPAL.sup.37) for 14 days did not prevent the ability
of the peptide to significantly moderate the glycaemic excursion
(p<0.01) and enhance plasma insulin concentrations (p<0.01)
when administered acutely with intraperitoneal glucose. In
contrast, the responses of ob/ob mice to acute administration of
native GIP were almost identical in mice receiving treatment with
GIP or saline for 14 days (FIGS. 56A-56D). To further substantiate
the lack of GIP receptor desensitization following chronic
treatment with N-AcGIP(LysPAL.sup.37), the acute effects of the
analogue, administered with glucose, were examined in each of the 3
groups after 14 days treatment with N-AcGIP(LysPAL.sup.37), native
GIP or saline (FIGS. 57A-57D). Apart from lower basal values in the
former group, the glucose and insulin responses were identical with
similar 0-60 minutes AUC measures for both plasma glucose and
insulin concentrations.
[0295] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
2 1 42 PRT Homo sapiens 1 Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Asp Lys 1 5 10 15 Ile His Gln Gln Asp Phe Val Asn
Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30 Lys Asn Asp Trp Lys His
Asn Ile Thr Gln 35 40 2 42 PRT Pig 2 Tyr Ala Glu Gly Thr Phe Ile
Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Ile Arg Gln Gln Asp
Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30 Lys Ser Asp
Trp Lys His Asn Ile Thr Gln 35 40
* * * * *