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THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
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PEPTIDE ANALOGUES OF GIP FOR TREATMENT OF DIABETES,
INSULIN RESISTANCE AND OBESITY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.K. Application No. GB 0404124.0,
filed on 25 February 2004.
FIELD OF THE INVENTION
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
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.
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.
GIP is released from intestinal endocrine K-cells into the bloodstream
following ingestion of carbohydrate, protein and particularly fat (Meier, LI.
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
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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, Cum. Med. Chem. 10:2471-2483).
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).
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'-Ala 2 and His7-
A1a8
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 mmo1/1).
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
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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.
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 His7-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 His7-glucitol tGLP-1,
resistance to
DPP IV and serum degradation was accompanied by severe loss of insulin
releasing
activity.
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).
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.
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SUMMARY OF THE INVENTION
Disclosed herein are GIP antagonist peptides which are resistant to rapid
degradation by DPP IV.
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 G1u3.
The amino acid substituted at G1u3 can be selected from the group consisting
of: proline, hydroxyproline, lysine, tyrosine, phenylalanine and tryptophan.
Specifically, a proline can be substituted for G1u3. 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 Lys16, or Lys37.
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 Lys16,
or Lys37.
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 Glu3; (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 Glu3. 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
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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 Lys16, or Lys37.
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.
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 Glu3, wherein the peptide analogue is resistant to
degradation by enzyme DPP IV when compared to naturally-occurring GIP.
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.
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 G1u3, wherein the peptide analogue
modulates
insulin secretion.
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.
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.
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.
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
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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' glucitol
GIP(1-42).
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.
The analogue may include modification by fatty acid addition at an epsilon
amino group of at least one lysine residue.
The invention includes Tyri glucitol GIP(1-42) having fatty acid addition at
an
epsilon amino group of at least one lysine residue.
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.
Any of these properties will enhance the potency of the analogue as a
therapeutic agent.
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.
Various amino acid substitutions at second and third amino terminal residues
are included, such as GIP(1-42)G1y2, GIP(1-42)Ser2, GIP(1-42)Abu2, GIP(1-
42)Aib2,
GIP(1-42)D-A1a2, GIP(1-42)Sar2, and GIP(1-42)Pro3.
Amino-terminally modified GIP analogues include N-glycated GIP(1-42), N-
alkylated GIP(1-42), N-acetylated GIP(1-42), N-acetyl-GIP(1-42) and N-
isopropyl
GIP(1-42).
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 TyrI-A1a2.
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:
(a) substitution of Ala2 by Gly;
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(b) substitution of Ala2 by Ser;
(c) substitution of Ala2 by Abu;
(d) substitution of Ala2 by Aib;
(e) substitution of Ala2 by D-Ala;
(f) substitution of Ala2 by Sar (sarcosine);
(g) substitution of G1u3 by Pro;
(h) modification of Tyr1 by acetylation;
(i) modification of Tyr1 by acylation;
(j) modification of Tyr1 by alkylation;
(k) modification of Tyr1 by glycation;
(1) conversion of Ala2 - G1u3 bond to a psi [CH2NH] bond;
(m) conversion of Ala2-G1u3 bond to a stable peptide isotere bond; and
(n) (n-isopropyl-H) 1GIP.
The invention also provides the use of Tyr1-glucitol GIP in the preparation of
a medicament for the treatment of diabetes.
The invention further provides improved pharmaceutical compositions
including analogues of GIP with improved pharmacological properties.
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.
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.
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
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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.
Preferably the agent is glucose, acetic anhydride or pyroglutamic acid.
The invention will now be demonstrated with reference to the following non-
limiting examples and the accompanying figures wherein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la illustrates degradation of GIP by DPP IV.
Fig. lb illustrates degradation of GIP and Tyr' glucitol GIP by DPP IV.
Fig. 2a illustrates degradation of GIP human plasma.
Fig. 2b illustrates degradation of GIP and Tyr' glucitol GIP by human plasma.
Fig. 3 illustrates electrospray ionization mass spectrometry of GIP, Tyri-
glucitol GIP and the major degradation fragment GIP(3-42).
Fig. 4 shows the effects of GIP and glycated GIP on plasma glucose
homeostasis.
Fig. 5 shows effects of GIP on plasma insulin responses.
Fig. 6 illustrates DPP-IV degradation of GIP (1-42).
Fig. 7 illustrates DPP-IV degradation of GIP (Abu2).
Fig. 8 illustrates DPP-IV degradation of GIP (Sar2).
Fig. 9 illustrates DPP-IV degradation of GIP (Ser2).
Fig. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.
Fig. 11 illustrates DPP-IV degradation of glycated GIP.
Fig. 12 illustrates human plasma degradation of GIP.
Fig. 13 illustrates human plasma degradation of GIP (Abu2).
Fig. 14 illustrates human plasma degradation of GIP (Sar2).
Fig. 15 illustrates human plasma degradation of GIP (Ser2).
Fig. 16 illustrates human plasma degradation of glycated GIP.
Fig. 17 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 18 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD 11 cells incubated at 16.7mM glucose.
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Fig. 19 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2)
on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 20 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2)
on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 21 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2)
on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 22 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2)
on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 23 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 5.6mM glucose.
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 16.7mM glucose.
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 5.6mM glucose.
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 16.7mM glucose.
Fig. 27 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 28 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 29 shows the effects of various concentrations of GIP 1-42 and GIP (Pro3)
on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 30 shows the effects of various concentrations of CIP 1-42 and GIP (Pro3)
on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 31a shows the primary structure of human gastric inhibitory polypeptide
(GIP) (SEQ ID NO:1), and Fig. 31b shows the primary structure of porcine
gastric
inhibitory polypeptide (GIP) (SEQ ID NO:2).
Figs. 32A and 32B are a line graph and a bar graph, respectively, showing the
effects of (Pro3)GIP on GIP-stimulated cyclic AMP generation and insulin
secretion
in vitro.
Figs. 33A - 33F are a set of six bar graphs showing the effects of Glu3-
substituted forms of GIP and GIP(3-42) on GIP-stimulated insulin secretion in
vitro.
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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 (Pro3)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. 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 (Pro3)GIP
impairs
physiological meal-stimulated insulin release and worsens glycemic excursion
in
obese diabetic ob/ob mice.
Figs. 36A and 36B are a set of two bar graphs showing that chronic
administration of (Pro3)GIP for 11 days decreases plasma glucose and insulin
concentrations of obese diabetic ob/ob mice.
Figs. 37A through 37C are a set of three bar graphs showing that chronic
administration of (Pro3)GIP for 11 days decreases glycated HbAic, pancreatic
insulin
content and associated islet hypertrophy of obese diabetic ob/ob mice.
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 (Pro3)GIP
for 11
days improves glucose tolerance of obese diabetic ob/ob mice without change of
circulating insulin.
Fig. 39 is a line graph showing that chronic administration of (Pro3)GIP for
11
days improves insulin sensitivity in obese diabetic ob/ob mice.
Fig. 40 is a line graph showing that the beneficial effects of chronic
administration of (Pro3)GIP for 11 days in obese diabetic ob/ob mice are
reversed 9
days after cessation of treatment.
Figs. 41A and 41B are a set of two line graphs showing that chronic
administration of (Pro3)GIP for 11 days causes glucose intolerance in normal
mice
with reversal by 9 days after cessation of treatment.
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 (Pro3)GIP on plasma glucose
and
insulin response to native GIP 4 hours after administration.
Figs. 43A through 43D are a set of two line graphs and two bar graphs
showing the effects of daily (Pro3)GIP administration on food intake (Fig.
43A), body
weight (Fig. 43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D)
concentrations in
ob/ob mice.
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Figs. 44A through 44D are a set of four line graphs with inset bar graphs
showing the effects of daily (Pro3)GIP administration on glucose tolerance and
plasma insulin response to glucose in ob/ob mice.
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 (Pro3)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.
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 (Pro3)GIP
administration on
insulin sensitivity in ob/ob mice.
Figs. 47A through 47D are a set of four bar graphs showing the effects of
daily
(Pro3)GIP administration on pancreatic weight (Fig. 47A), insulin content
(Fig. 47B),
islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice.
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
(Pro3)GIP
administration on islet size and morphology in ob/ob)mice.
Fig. 49 is an illustration of how the GIP receptor ("GIP-R") antagonist,
(Pro3)GIP, counters beta cell hyperplasia, hyperinsulinemia and insulin
resistance lead
to improved glucose intolerance and diabetes control.
Figs. 50A and 50B are a line graph and a bar graph, respectively, showing
intracellular cyclic AMP production (Fig. 50A) by GIP (A) and fatty acid
derivatised
GIP analogues N-AcGIP(LysPAL16) (o) and N-AcGIP(LysPAL37) (9), and insulin-
releasing activity of glucose (5.6 mmo/1 glucose; white bars), GIP (lined
bars) and
fatty acid derivatised GIP analogues (Fig. 50B) N-AcGIP(LysPAL16) (grey bars)
and
N-AcGIP(LysPAL37) (black bars) in the clonal pancreatic beta cell line, BRIN-
BD11.
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.
Figs. 52A and 52B are a pair of bar graphs showing dose-dependent effects of
GIP and N-AcGIP(LysPAL37) in ob/ob mice fasted for 18 hours.
Figs. 53A through 53E are a set of graphs showing the effects of daily N-
AcGIP(LysP AL37) (9; black bars) administration on food intake (Fig. 53A),
body
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weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated
hemoglobin N-AcGIP(LysPAL37) (12.5 nmoles/kg/day) (Fig. 53E).
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(LysPAL37)
administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin
response
(Figs. 54C, 54D) to glucose.
Figs. 55A through 55D are a line graph and three bar graphs showing the
effects of daily N-AcGIP(LysPAL37) administration on insulin sensitivity
(Figs. 55A,
55B) and pancreatic weight (Fig. 55C) and insulin content (Fig. 55D).
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(Ly5PAL37) and
native GIP after daily injection for 14 days.
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(LysPAL37) after 14 daily
injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; fi; black bars),
native
GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; ID; white
bars).
DETAILED DESCRIPTION
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-
[CH2NFI]
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.
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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.
Particularly preferred alterations include acetylation of Tyr1 and linkage of
a
C-16 palmitate group to the epsilon amino group of a lysine residue
(especially Lys16
or Lys37), or substitution of G1u3, 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.
It has been found that longer-term, as opposed to acute, GIP receptor
antagonism using Glu3-substituted forms of GIP, such as (Pro3)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.
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.
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.
The present results demonstrate for the first time that Tyrl-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
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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'-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 Tyri residue masks the
potential
cleavage site from DPP IV and prevents removal of the Tyr'-Ala2 dipeptide from
the
N-terminus preventing the formation of GIP(3-42).
Consistent with in vitro protection against DPP IV, administration of Tyrl-
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'-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 Tyrl-
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 Tyrl-
glucitol GIP is greatly underestimated in the present in vivo experiments.
In vitro studies in the laboratory of the present inventors using glucose-
responsive clonal B-cells showed that the insulin-releasing potency of
Tyriglucitol
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 Tyri-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)
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and other N-terminally modified fragments, including GIP(4-42), and GIP(17-42)
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(3-42).
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 Tyrl-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.
This study demonstrates that the glycation of GIP at the aminotenninal Tyr'
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. Tyrl-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.
As shown in Table 1 in Example 3, glycated GIP, acetylated GIP, GIP(Ser2)
are GIP(Abu2) more resistant than native GIP to in vitro degradation with DPP
IV.
From these data GIP(Sar2) appears to be less resistant. As shown in Table 2,
all
analogues tested exhibited resistance to plasma degradation, including
GIP(Sar2)
which from DPP IV data appeared least resistant of the peptides tested. DPA
substantially inhibited degradation of GIP and all analogues tested with
complete
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abolition of degradation in the cases of GIP(Abu2), GIP(Ser2) and glycated
GIP. This
indicates that DPP IV is a key factor in the in vivo degradation of GIP.
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(Ser2) analogue also evoked a strong
response.
From these tests, GIP(Gly2) and GIP(Pro3) appeared to the least potent
analogues in
terms of insulin release. Other stable analogues tested, namely GIP(Abu2) and
GIP(Sar2), 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.
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,
(Pro3)GIP,
functioned as a potent GIP receptor antagonist.
As shown in Example 4, below, (Pro3)GIP, other G1u3-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 (Pro3)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 P ept. 107:1-13).
Most notably, and in complete contrast to normal mice, the experiments
disclosed herein show that chronic (Pro3)GIP administration to obese diabetic
ob/ob
mice for 11 days does not worsen glucose intolerance and diabetes status at
all.
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Surprisingly, GIP receptor antagonism in this obese insulin resistant model
was
associated with highly substantial improvements of glycated HbAic, 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 (Pro3)GIP as a result of
improved =
insulin resistance.
Indeed, insulin sensitivity tests conducted in ob/ob mice 11 days into
(Pro3)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 (Pro3)GIP in obese diabetic ob/ob mice were
reversed
within 9 days cessation of treatment.
These results clearly indicate that (Pro3)GIP and other analogues based on
G1u3-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.
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
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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. etal., 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 (Hoist, 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. etal.,
2002,
Diabetologia 45:1111-1119) may reflect a generalized secretory dysfunction
rather
than a specific cellular defect (Meier, J.J. etal., 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. etal., 2003, Diabetes 52:380-386;
Flatt,
P.R. etal., "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. etal., 2003, Biochem Biophys Res
Commun
308:207-213).
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. etal., 1983, Diabetes 32:433-435; Flatt, P.R. etal.,
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. etal., "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. etal., 2003, Diabetes 52:252-259)
and that
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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).
As shown in Example 5, below, daily injections of the stable and specific GIP-
S R antagonist, (Pro3)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.
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 9. 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.
The analogue (Pro3)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 (Pro3)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 (Pro3)GIP to normal mice for 11 days results in the
reversible
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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 (Pro3)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.
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. ScL USA 96:14843-14847; Irwin,
N.,
2004, Biol. Chem. 385:845-852), ob/ob mice treated with daily (Pro3)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 (Pro3)GIP in
order to
restrain the hyperglycemia. Indeed, insulin sensitivity tests conducted after
11 days of
(Pro3)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; Wiernsperger, 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 (Pro3)GIP is
unprecedented.
It is important to note that the above effects were observed independently of
any change in food intake or body weight in (Pro3)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
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weight gain and significant amelioration of both adiposity and insulin
resistance
compared with control (Lee/Lee) mice (Miyawaki, K. et at., 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 at., "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.
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, (Pro3)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.
Due to double recessive ob mutation and resulting leptin deficiency, young
ob/ob mice develop a profound early hyperphagia (Bailey, C.J., et at., 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 at., 1984, J EndocrinoL 101:249-
256;
Bailey, C.J. et at., 1986, Acta Endocrinol. (Copenh) 112:224-229). This in
turn
promotes islet hypertrophy and beta cell hyperplasia (Bailey, C.J., et at.,
1982, Int. J.
Obes. 6:11-21) together with marked hyperinsulinemia and mounting insulin
resistance (Flatt, P.R., et at., 1981, Horin Metab Res 13:556-560). This
process
manifests itself in ternis 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 at., 1982, Int. J. Obes. 6:11-21; Flatt, P.R., et at., 1981, Horni
Metab Res
13:556-560). Viewed in this context, it is clear that chemical ablation of GIP-
R
function with daily (Pro3)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
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substantial improvement of the metabolic syndrome. Further support for this
scenario, is the partial amelioration of islet hypertrophy and beta cell
hyperplasia in
(Pro3)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 (0.1-15mm). 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. 6:11-21). 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 (Pro3)GIP on islet size reflects a
combination of
decreased proliferation and increased apoptosis of beta cells.
The results shown in Example 5 have demonstrated for the first time that daily
administration of the GIP-R antagonist, (Pro3)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
(Pro3)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 (Pro3)GIP, provide a novel,
physiological and
effective means to treat obese type 2 diabetes through the alleviation of
insulin
resistance.
In Example 6, fatty acid derivatisation was used to develop two novel long-
acting, N-terminally modified GIP analogues (N-AcGIP(LysPAL16) and N-
AcG1P(LysP AL37)).
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.
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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(LysP AL16) or N-AcGIP(LysPAL37) 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(LysPAL37)
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(LysPAL37) 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(LysPAL37) were independent of any change in
feeding
or body weight.
These results show that novel fatty acid derivatised, N-terminally modified
analogues of GIP such as N-AcGIP(LysPAL37), may have significant potential for
the
treatment of type 2 diabetes.
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 at,
1999,
Diabetes 48: 758-765). Several of these, most notably those modified at Tyr1
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
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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. 1 367: 913-920; Gault, V.A. et at., 2003,1
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 at., 2002, Diabetologia 45:
1281-
1291).
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(LysPAL16) and N-
AcGIP(LysP AL37). 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
(Tyr1) acetyl group (O'Harte, F.P.M. et at., 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(LysPAL37) 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(LysPAL37) was also examined. The results
indicate the particular promise of the novel second generation N-terminally
acetylated
GIP analogue, N-AcGIP(LysPAL37), as a potential therapeutic agent for the
treatment
of type 2 diabetes.
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, 1 Med. Chem. 43: 1664-1669; Green, B.D. et
al.,
2004, BioL Chem. 385: 169-177; Kim, J.G. et at., 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
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promotion of nausea possibly due to slowing of gastric emptying (Agerso, H. et
al.,
2002, Diabetologia 45: 195-202).
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 Lys16 or Lys37 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 GIP 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).
To assess the antihyperglycaemic and insulinotropic potential of the fatty
acid
derivatised GIP 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 Lys16 or Lys37 (N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37))
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(LysPAL37). 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 GIP analogues to serum albumin,
therefore
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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.
N-AcGIP(LysPAL37) appeared to be the best fatty acid derivatised analogue
displaying a more protracted, significantly enhanced insulin-releasing potency
over N-
AcGIP(LysP AL16) in vivo. Reasons for the increased potency of N-
AcGIP(LysP AL37) 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, Biochemist/J.) 42: 3081-3088).
However,
similarities between the in vitro biological activities of the two palmitate
substituted
analogues make this less likely.
Given that N-AcGIP(LysPAL37) 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(LysPAL37), even at the lowest dose of
6.25
nmoles/kg, exhibited significant glucose-lowering and insulinotropic activity
when
administered with glucose. Considering N-AcGIP(LysPAL37) is subject to albumin
binding, the fact that it is still highly biologically active even at lower
concentrations
indicates striking potency.
Daily administration of N-AcGIP(LysPAL37) 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(LysPAL37) did not exert
any
untoward toxic actions affecting feeding over the study period. This is in
harmony
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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.
As expected, a key component of the beneficial action of N-AcGIP(LysPAL37)
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 at., 1996, Mot. 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 at., 2002, Diabetes 51: 656-661;
Gault, V.A.
et al., 2002, Biochein. 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 at.,
2002, Diabetologia 45: 1281-1291). Furthermore, the insulin response to
glucose was
significantly enhanced in ob/ob mice receiving N-AcGIP(LysPAL37). This ability
to
augment or restore pancreatic beta cell glucose responsiveness has been
similarly
observed with GLP-1 (Holz, G.G. et at., 1993, Nature 28: 362-365; Flamez, D.
et at.,
1998, Diabetes 47: 646-652). As with observations on glycaemic control, none
of
these attributes were reproduced by daily injections of native GIP.
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(LysP AL37) 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 at., 1981,
Diabetes 30:
746-753), this suggests that N-AcGIP(LysPAL37) 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 at., 1996, Biochem. Soc.
Trans.
24:585-591; O'Harte, F.P.M. et at., 1998,J. EndocrinoL 156: 237-243; Yip, R.G.
et
al., 1998, Endocrinology 139: 4004-4007).
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Irrespective of knowledge of the full range of actions contributing to the
antihyperglycaemic effect of N-AcGIP(Ly5PAL37), 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(LysPAL37) for 14 days compromised
the glucose lowering or insulin releasing actions of N-AcGIP(LysPAL37). Thus
the
antidiabetic actions of N-AcGIP(LysPAL37) were clearly evident when the
analogue
was administered acutely together with glucose. Furthermore, the acute effects
of N-
AcGIP(LysP AL37) in such experiments were identical in groups of ob/ob mice
receiving either N-AcGIP(LysPAL37), native GIP or saline injections for 14
days.
Such data clearly indicate that prolonged exposure to N-AcGIP(LysPAL37)
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 (Flat, 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.
The data shown herein demonstrate that N-terminally acetylated GIP 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 GIP for the once-
daily
treatment of type 2 diabetes.
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.
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
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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.
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.
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.
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.
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
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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.
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.
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,
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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).
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. 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, 'nydrobromide, hyciroiodide, 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 quatemized with such agents as lower alkyl halides such as
methyl,
ethyl, propyl, and butyl chlorides, bromides and iodides; diallcyl sulfates
like
dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as
decyl,
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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.
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.
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.
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
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administered until the optimal therapeutic effect is obtained for the patient,
and at that
point the dosage is not increased further.
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, Aveiy'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.
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 (Ala2) 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-
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amino of GIP (Ala2), 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'-Glucitol GIP and Its Properties in vivo.
The following example investigates preparation of Tyriglucitol GIP together
with evaluation of its antihyperglycemie 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.
Research Design and Methods
Materials. Human GIP was purchased from the American Peptide Company
(Sunnyvale, California, USA). HPLC grade acetonitrile was obtained from
Rathburn
(Walkersbum, 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, MA, USA). All water used in these experiments was purified using a
Milli-
WO, Water Purification System (Millipore Corporation, Milford, Massachusetts,
USA).
Preparation of Tyr' -glucitol GIP. Human GIP was incubated with glucose under
reducing conditions in 10 mmoi/1 sodium phosphate buffer at pH 7.4 for 24
hours.
The reaction was stopped by addition of 0.5 mo1/1 acetic acid (30 pl) and the
mixture
applied to a Vydac (C18)(4.6 x 250mm) analytical HPLC column (The Separations
Group, Hesperia, California, 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 ABS 1000
Speed-Vac concentrator (Life Sciences International, Runcorn, UK) and
purified to
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homogeneity on a Supelcosil (C-8) (4.6 x 150mm) column (Supelco Inc., Poole,
Dorset, UK).
Degradation of GIP and Tyri -glucitol GIP by DPP 1V. HPLC-purified GIP or Tyr1-
glucitol GIP were incubated at 37 C with DPP-IV (5mU) for various time periods
in a
reaction mixture made up to 5001.11 with 50 mmo1/1 triethanolamine-HC1, pH 7.8
(final peptide concentration 1 umo1/1). Enzymatic reactions were terminated
after 0,
2, 4 and 12 hours by addition of 5 1 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 C prior to
HPLC
analysis.
Degradation of GIP and Tyr) -glucitol GIP by human plasma. Pooled human plasma
(20 I) taken from six healthy fasted human subjects was incubated at 37 C
with GIP
or Tyrl-glucitol GIP (10 lig) for 0 and 4 hours in a reaction mixture made up
to 500
p1, containing 50 mmol/ltriethanolamine/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 pi of TFA and the final volume adjusted to 1.0 ml
using
0.1% v/v TFA/water. Samples were centrifuged (13,000g, 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 1.0m1 with 0.12% (v/v) TFA/water prior to HPLC
purification.
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HPLC analysis of degraded GIP and Tyr -glucitol GIP. Samples were applied to a
Vydac C-18 widepore column equilibriated with 0.12% (v/v) TFA/H20 at a flow
rate
of 1.0 ml/minute. Using 0.1% (v/v) TFA in 70% acetonitrile/H20, 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.
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'-glucitol GIP, were further purified by HPLC.
Peptides
were dissolved (approximately 400 pmol) in 100 ill of water and applied to the
LCQ
benchtop mass spectrometer (Finnigan MAT, Hemel Hempstead, UK) equipped with
a microhore C-18 HPLC column (150 x 2.0mm, Phenomenex , Ltd., Macclesfield,
UK). Samples (300 direct loop injection) were injected at a flow rate of
0.2m1/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 (rn/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
MT = iMi -
where Mr = molecular mass; Mi= m/z ratio; i = number of charges; Mh = mass of
a
proton.
In vivo biological activity of GIP and Tyr -glucitoi GIP. Effects of GIP and
Tyri-
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 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 (18mmol/kq body weight) or in
combination
with either GIP or Tyri-glucitol GIP (10 nmol/kg). Test solutions were
administered
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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 C prior to glucose and insulin
determinations. All animal studies were done in accordance with the Animals
(Scientific Procedures) Act 1986.
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.
Degradation of GIP and Tyr' -glueitol 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 la) or Tyrl-glucitol GIP (Fig lb) with DPP IV for 0, 2,
4 and
12 hours. The retention times of GIP and Tyr'-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. Tyrl-
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.
Degradation of GIP and Tyr' -glucitol GIP by human plasma. Fig. 2 shows a set
of
typical HPLC profiles of the products obtained from the incubation of GIP or
Tyri-
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
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rise to the appearance of a major degradation peak with a retention time of
21.74
minutes. In contrast, the incubation of Tyr'-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'-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.
Identification of GIP degradation fragments by ESI-MS. Fig. 3 shows the
monoisotopic molecular masses obtained for GIP (Fig. 3A), Tyr'-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 1 of water and applied to the
LC/MS
equipped with a microbore C-18 HPLC column. Samples (300 direct loop
injection)
were applied at a flow rate of 0.2m1/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 (Mr) of GIP and related structures
were
determined from ESI-MS profiles using prominent multiple charged ions and the
following equation Mr = iM, - iMh. The exact molecular mass (Mr) of the
peptides
were calculated using the equation Mr = iMi - iMh as defined above in Research
Design and Methods. After spectral averaging was performed, prominent multiple
charges species (M+3H )3+ and (M+4H )4+ were detected from GIP at m/z 1661.6
and
1246.8, corresponding to intact Mr 4981.8 and 4983.2 Da, respectively (Fig.
3A).
Similarly, for Tyrl-glucitol GIP ( (M+4H )4+ and (M+5H)5+) were detected at
m/z
1287.7 and 1030.3, corresponding to intact molecular masses of Mr 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 Tyri-glucitol GIP. Fig. 3C shows the prominent multiply
charged
species (M+3H )3+ and (M+4H)4+ detected from the major fragment of GIP at m/z
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1583.8 and 1188.1, corresponding to intact Mr 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).
Effects of GIP and Tyr' -glucitol GIP on plasma glucose homeostasis. Figs. 4
and 5
show the effects of intraperitoneal (ip) glucose alone (18mmol/kg) (control
group),
and glucose in combination With GIP or Tyri-glucitol GIP (10nmol/kg) on plasma
glucose and insulin concentrations.
Fig. 4A shows plasma glucose concentrations after i.p. glucose alone
(18mmol/kg) (control group), or glucose in combination with either GIP of Tyrl-
glucitol GIP (10nmol/kg). The time of injection is indicated by the arrow (0
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'"
glucitol GIP; TP<0.05, 11/3<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 (10nmol/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 (0 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 Tyri-glucitol GIP; fP<0.05, P<0.01 compared with non-
glycated GIP.
Compared with the control group, plasma glucose concentrations and area
under the curve (AUC) were significantly lower following administration of
either
GIP or Tyrl-glucitol GIP (Figs 4A, B). Furthermore, individual values at 15
and 30
minutes together with AUC were significantly lower following administration of
Tyrl-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
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insulin response, calculated as AUC, following Tyrl-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
Tyrl-
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.
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.
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). RPM'
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 Water Purification System (Millipore,
Milford, Massachusetts, USA). All other chemicals used were of the highest
purity
available.
Synthesis of GIP and N-terminally modified GIP analogues. GIP, GIP(Abu2),
GIP(Sku2), GIP(Ser2), GIP(G1y2) and GIP(Pro3) were sequentially synthesized on
an
Applied Biosystems automated peptide synthesizer (model 432A) 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
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=
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 Millenium 2010
chromatography system (Software version 2.1.5.). N-terminal glycated and
acetylated
GIP were prepared by minor modification of a published method.
Electrospray ionization-mass spectrometry (ESI-MS) was carried out as
described in Example 2. Degradation of GIP and novel Gil' analogues by DPP IV
and human plasma was carried out as described in Example 2.
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 C in an atmosphere of 5% CO2 and 95% air using a LEEC incubator
(Laboratory Technical Engineering, Nottingham, UK).
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
x 105
cells per well, and allowed to attach overnight at 37 C. Acute tests for
insulin release
were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer
bicarbonate buffer (115 mM NaC1, 4.7 mM KC I, 1.28 mM CaC12, 1.2 mM KH2PO4,
1.2 mM MgSO4, 10 mM NaHCO3, 5 g/1 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 (1(-13 to
10-8
M) of GIP or GIP analogues. After 20 minutes incubation, the buffer was
removed
from each well and aliquots (200 i) were used for measurement of insulin by
radioimmunoassay [31].
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.
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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)3+ and
(M+4H)4+ were detected for each peptide. Calculated molecular masses confirmed
the structural identity of synthetic GIP and each of the N-terminal analogues.
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(Abu2), GIP(Sar2), GIP(Ser2), 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(Ser2) are GIP(Abu2)
more
resistant than native GIP to in vitro degradation with DPP IV. From these data
GIP(Sar2) appears to be less resistant.
Table 1. Percent intact peptide remaining after incubation with DPPIV.
Peptide % Intact peptide remaining after time (h)
0 2 4 8 24
GIP 1-42 100 52 1 23+1 0 0
Glycated GIP 100 100 100 100 100
GIP(Abu2) 100 38 1 28 2 0 0
GIP(Ser2) 100 77+2 60+1 32+4 0
GIP(Sar2) 100 28 + 2 8 0 0
N-Acetyl-GIP 100 100 100 100
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.
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
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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(Sar2) 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(Abu2), GIP(Ser2) and glycated GIP. This indicates that DPP IV
is a
key factor in the in vivo degradation of GIP.
Table 2. Percent intact peptide remaining after incubation with human plasma.
Peptide % Intact peptide remaining after incubations
with human plasma
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 (Abu2) 100 38 1 28 2 0 0 100
GIP(Ser2) 100 77 2 60 1 32 .1 4 0 63 3
GIP(Sar2) 100 28 1 2 8 0 0 100
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.
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(Abu2),
GIP(Sar2),
GIP(Ser2), acetylated GIP, glycated GIP, GIP(G1y2) and GIP(Pro3) 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(Ser2) analogue also evoked a strong
response.
From these tests, GIP(G1y2) and GIP(Pro3) appeared to be the least potent
analogues
in terms of insulin release. Other stable analogues tested, namely GIP(Abu2)
and
GIP(Sar2), exhibited a complex pattern of responsiveness dependent on glucose
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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. Glu3 substituted GIP improves obesity-related insulin resistance
and
associated glucose intolerance.
This example examines GIP receptor antagonism and obesity-related insulin
resistance and associated glucose intolerance using a G1u3-substituted form of
GIP,
namely, (Pro3)GIP.
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.
Peptide synthesis and characterisation. Glu3-substituted analogues were
sequentially
synthesised on an Applied Biosystems automated peptide synthesiser (Model
432A)
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
lonisation-Time of Flight (MALDI-TOF) mass spectrometry. Samples were
dissolved in 10 [11 H20 (approximately 40 pmo1/1), 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 a-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
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and compared using theoretical values on a Voyager-DE BioSpectrometry
Workstation (PerSeptive Biosystems 0, Framingham, MA, USA).
Tissue culture. Chinese hamster lung (CHL) fibroblast cells stably fransfected
with
5 the human GIP receptor were cultured in DMEM tissue culture medium
containing
10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 Uhnl 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
10 flasks (Corning Glass Works, Sunderland, UK) at 37 C in an atmosphere of
5% CO2
and 95% air using an LEEC incubator (Laboratory Technical Engineering,
Nottingham, UK).
Acute studies of insulin release. Insulin release from BRIN-BD11 cells was
15 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 x
105cells per
well, and allowed to attach overnight at 37 C. Prior to acute test, cells were
preincubated for 40 minutes at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer
(115
20 mM NaCl, 4.7 mM KCI, 1.28 mM CaC12, 1.2 niM KH2PO4, 1.2 mM MgSO4, 10 mM
NaHCO3, 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 C
at 5.6
mM glucose using various concentrations of Glu3-substituted analogues and
GIP(3-
42) in the presence of native GIP (1 0-7 M) as indicated in the Figures. After
25 incubation, aliquots of buffer were removed and stored at -20 C for
insulin
radioimmunoassay (Flatt, P.R. et al., 1981, Diabetologia 20:573-577).
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 x 105
cells
30 per well. The cells were then allowed to grow for 48 hours before being
loaded with
tritiated adenine (2 p.Ci; TRK311, Amersham, Buckinghamshire, UK) and
incubated
at 37 C for 6 hours in 1 ml DMEM, supplemented with 0.5% (w/v) foetal bovine
serum. The cells were then washed twice with FIBS buffer (130 mM NaCl, 20 mM
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HEPES, 0.9 mM NaHPO4, 0.8 mM MgSO4, 5.4 mM KC1, 1.8 mM CaC12, 25 mM
glucose, 25 1.1.M phenol red, pH 7.4). The cells were then exposed for 10
minutes at
37 C to forskolin (FSK, 10 M) or varying concentrations of (Pro3)GIP in the
absence (control) or presence of native GIP (10-7 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).
Acute in vivo effects of (Pro3)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,
(Pro3)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
8m1/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 13000g and stored
at -
20 prior to glucose and insulin determinations.
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Acute in vivo effects of (Pro3)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
(Pro3)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 13000g and stored at -20 prior to glucose
and
insulin determinations.
Effects of chronic (Pro3 )GIP administration on plasma glucose, insulin and
glycated
HMI, 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 (17:00h) of either saline (0.9%
w/v NaC1)
or (Pro3)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 13000g prior to glucose, insulin and HbAie
determinations.
Effects of chronic treatment with (Pro3)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 NaC1) or (Pro3)GIP (25
nmol/kg
body weight/day) for 11 days. This test was repeated 9 days after cessation of
chronic
(Pro3)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
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microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored
at -
200 prior to glucose and insulin determinations.
Effects of chronic treatment with (Pro3)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
NaC1)
and (Pro3)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
13000g and stored at -20 prior to glucose determination.
Effects of chronic treatment with (Pro3)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% WIT NaC1)
or
(Pro3)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.
Determination of HbA lc, plasma glucose and insulin concentrations. HbAic 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.
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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.
Results
GIP-stimulated cyclic AMP production and insulin secretion were inhibited in
dose-dependent fashion by (Pro3)GIP, showing that (Pro3)GIP is a potent
functional
GIP receptor antagonist.
GIP receptor transfected Chinese hamster lung (CHL) fibroblasts were
incubated with 1012 to 10-6 M (Pro3)GIP in the presence of native GIP (10-7
M). The
results are shown in Figs. 32A and 32B. Fig. 32A is a line graph showing 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 inM glucose
(control)
(white bar), GIP (gray bars), (Pro3)GIP (lined bars) and (Pro3)GIP+GIP(10-7M)
(black
bars). *P<0.05, **P<0.01, ***P<0.001 compared to glucose control. A6P<0.01,
AAAP<0.001 compared with native GIP at the same concentration. Values are
means
SEM for 3-8 observations.
(Pro3)GIP inhibited GIP-induced cAMP formation with an 1050 value of 2.6
M. Insulin-releasing activity of BRIN-BD11 cells exposed to native GIP and
(Pro3)GIP (in the absence and presence of 10-7M GIP).
GIP-stimulated insulin secretion was inhibited in a dose-dependent fashion by
GIP(3-42), (Hyp3)GIP, (Lys3)GIP, (Tyr3)GIP, (Trp3)GIP, and (Phe3)GIP, as shown
in
Figs. 33A through 33F, which are bar charts. Fig. 33A shows 3H-cAMP production
as a percent of 10-7M GIP (y-axis) versus logio of GIP (10-7M) (white bar,
control)
and GIP (10-7M)+GIP(3-42) (black bars). Figs. 33B through 33F show insulin
secretion (in ng/106 cells/20 minutes) (y-axis) as a function of peptide
concentration
(M) (x-axis) for GIP (10-7M) (white bar, control) and a G1u3-substituted form
of GIP
(black bars), including (Hyp3)GIP (Fig. 33B), (Lys3)GIP (Fig. 33C), (Tyr3)GIP
(Fig.
33D), (Trp3)GIP (Fig. 33E), and (Phe3)GIP (Fig. 33F). *P<0.05, **P<0.01
compared
to GIP (10-7 M) control. Values are means SEM for 3-8 observations.
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 (Pro3)GIP
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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; =),
glucose +
GIP (4) and glucose + (GIP+Pro3GIP)) (A). Figs. 34B and 34D are bar graphs
showing plasmia glucose AUC for glucose alone (white bars), GIP (grey bars)
and
glucose + (GIP+Pro3GIP)) (black bars).
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 (Pro3)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. 6P<0.01, mP<0.001 compared with
native GIP.
Acute administration of (Pro3)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 (Pro3)GIP (A) was worse than
when
glucose was administered alone (V).
Figs. 35A through 35D show the effects of (Pro3)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) NaC1) as
control or
(Pro3)GIP (25 nmol/kg body weight). The time of injection is indicated by the
arrow
(15 minutes).
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
(V) and (Pro3)GIP (0), and plasma insulin AUC (Fig. 35B) and plasma glucose
AUC
(Fig. 35D) for saline control (white bars) and (Pro3)GIP (black bars),
respectively.
Values are means SEM for 8 mice. *P<0.05, "P<0.01, ***P<0.001 compared
with saline alone.
Acute administration of (Pro3)GIP decreased the insulin response to feeding
and worsened the associated glycemic excursion in ob/ob mice. These effects of
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functional ablation of endogenous GIP by the (Pro3)GIP antagonist are fully
consistent with the accepted role of GIP in the regulation of insulin
secretion and
glycemic excursion following feeding.
The effects of chronic administration of (Pro3)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 (Pro3)GIP over 11 days would be expected to inhibit insulin secretion and
cause a
marked deterioration in glucose tolerance.
However, the exact opposite occurred during chronic treatment with (Pro3)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 (Pro3)GIP (25 nmol/kg body weight; black bars) for 11 days. Values are
means
SEM for 6 mice and *P0.05 compared with saline alone. Chronic administration
of
(Pro3)GIP (black bars) for 11 days decreases plasma glucose and plasma insulin
concentrations of obese diabetic ob/ob mice, relative to controls.
The effects of chronic administration of (Pro3)GIP for 11 days on HbAlc, (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 (Pro3)GIP were examined. HbAic, pancreatic insulin content and
average
islet diameter were measured after 11 daily subcutaneous injections of either
saline
alone (white bars) or (Pro3)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.
Beneficial effects of chronic (Pro3)GIP administration in ob/ob mice were
associated with significant decreases in HbAlc 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 (Pro3)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.
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The effects of chronic administration of (Pro3)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
(Pro3)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.
After 11 days treatment with (Pro3)GIP, glucose tolerance of ob/ob mice was
substantially improved without change of circulating insulin (Fig. 38).
Fig. 39 shows the effects of chronic administration of (Pro3)GIP for 11 days
on insulin sensitivity in obese diabetic ob/ob mice. Plasma glucose
concentrations of
saline and (Pro3)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 (Pro3)GIP caused a significant
improvement of insulin sensitivity.
Interestingly, the beneficial effects of chronic administration of (Pro3)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,
ID) or (Pro3)GIP (A). Arrow indicates time of injection (t=0). Values are
means
SEM for 6.
Figs. 41A and 41B are a pair of line graphs showing the effects of chronic
administration of (Pro3)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 'I./)<0.05, **P<0.01 compared to saline-
treated
group.
In total contrast to beneficial actions in ob/ob mice, chronic daily treatment
of
normal mice with (Pro3)GIP (A) for 11 days resulted in a marked deterioration
of
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glucose tolerance (Fig. 41A) relative to controls (m), which was reversed 9
days after
cessation of treatment (Fig. 41B).
Example 5. Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action
By
Daily (Pro3)GIP Administration Improves Glucose Tolerance and Ameliorates
Insulin
Resistance and Abnormalities of Islet Structure in Obesity-Diabetes.
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, (Pro3)GIP. Young adult
oh/oh
mice received once daily i.p. injections of saline vehicle or (Pro3)GIP over
an 11-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 (Pro3)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 (Pro3)GIP-treated group. Daily injection of
(Pro3)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 (Pro3)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.
Research Design And Methods
Animals. Obese diabetic (oh/oh) mice derived from the colony maintained at
Aston
University, UK (Bailey, C.J., et al., 1982, Int. *I 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 222 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
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accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse
effects were observed following administration of (Pro3)GIP.
Synthesis, purification and characterization of (Pro3)GIP. (Pro3)GIP was
sequentially synthesized on an Applied Biosystems automated peptide
synthesizer
(Model 432 A). (Pro3)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).
Experimental protocols for ob/ob mouse studies. Initially, extended biological
activity of (Pro3)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
(Pro3)GIP (25
nmol/kg body wt). During a subsequent 9-day period, observations were
continued
following discontinuation of (Pro3)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 (Pro3)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 HC1). 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 C prior to glucose and insulin
determinations.
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
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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 VersaMaxo Microplate
Spectrophotometer (Molecular Devices, Wolcingham, Berkshire, UK).
Immunocytochemistry. Tissue fixed in 4% paraformaldehyde/PBS and embedded in
paraffin was sectioned at 8 um. 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 H20 and 2.5 ml concentrated HCI) 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 NC 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.
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.
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Results
Effects of (Pro3)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 (Pro3)GIP on plasma glucose and insulin response
to
native GIP 4 hours after administration. Tests were conducted 4 hours after
administration of (Pro3)GIP (25 nmoles/kg body weight) or saline (0.9% NaC1)
in 18
hour-fasted oh/oh 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.
As shown in Figs. 42A through 42D, administration of (Pro3)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.
The effects of (Pro3)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 (Pro3)GIP administration on food intake (Fig. 43A), body
weight (Fig.
43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in oh/oh
mice.
Parameters were measured for 5 days prior to, 11 days during (indicated by
black bar)
and 9 days after treatment with saline or (Pro3)GIP (25 nmol/kg bw/day).
Values are
mean SEM for eight mice. *P < 0.05 compared with saline group.
Administration of (Pro3)GIP had no effect on food intake and body weight
(Fig. 43A and 43B). On day 11, plasma glucose had declined to significantly
reduced
(P <0.05) concentrations in oh/oh mice receiving (Pro3)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 (Pro3)GIP than either before or 9 days following cessation
of
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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 (Pro3)GIP treatment (Fig. 43D).
The effects of (Pro3)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 (Pro3)GIP administration on glucose tolerance and plasma insulin
response to
glucose in ob/ob mice. Tests were conducted after daily treatment with
(Pro3)GIP (25
nmoles/kg body weight/day; = ; black bars) or saline (control; o; 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 (Pro3)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.
Daily administration of (Pro3)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
(Pro3)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 (Pro3)GIP treated group.
Discontinuation of (Pro3)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).
The effects of (Pro3)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
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graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the
effects of
daily (Pro3)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. Tests were conducted after daily treatment with (Pro3)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.
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 (Pro3)GIP
administration on
insulin sensitivity in ob/ob mice. Tests were conducted after daily treatment
with
(Pro3)GIP (25 nmol/kg body weight/day; = ; black bars) or saline (o; 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. *13 <0.05 compared with saline
group.
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 (Pro3)GIP
for 11
days (Fig. 45). Similarly, AUC glucose and insulin were significantly (P <
0.05)
decreased in (Pro3)GIP treated ob/ob mice, despite similar food intakes of 0.3
¨0.5
g/mouse/15 minutes. As shown in Fig. 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 (Pro3)GIP for 11 days. The
responses
following 9 days discontinuation of (Pro3)GIP treatment were similar to saline
treated
controls (Fig. 45C, 45D).
The effects of (Pro3)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 (Pro3)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 (Pro3)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 meanSEM 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
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(Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro3)GIP
administration on
islet size and morphology in ob/ob)mice.
(Pro3)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 (Pro3)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
(Pro3)GIP treated ob/ob mice (Fig. 47D). These effects were effectively
reversed by
discontinuation of (Pro3)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. Figure 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 (x40 magnification) of pancreata
immunohistologically stained for insulin from 11-day (Pro3)GIP treated ob/ob
mice
(Fig. 48B) and saline treated controls (Fig. 48C) illustrate the dramatic
changes in
pancreatic islet morphology induced by (Pro3)GIP treatment. Pancreata
immunohistologically stained for insulin on day 20 are also shown (Fig. 48E,
48F).
Parameters were measured after daily treatment with (Pro3)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 meanSEM for eight mice Figs. 48B, 48C, 48E and 48F are representative
images (x 40 magnification) of pancreata stained for insulin following 11 days
treatment with (Pro3)GIP (Fig. 48B) or saline (Fig. 48C). Corresponding images
9
days after cessation of treatment with (Pro3)GIP (Fig. 48E) or saline (Fig.
48F) are
also shown. The arrows indicate islets.
Example 6. N-Terminally Acetylated and Ly16 and Lys37-substituted GIP
This example examines the metabolic stability, biological activity and
antidiabetic potential of fatty acid derivatized N-terminally modified GIP
analogues.
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These are N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37), which have an N-terminal
Tyr' 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.
Materials and methods
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.
etal., 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 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 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.
Materials. High performance liquid chromatography (HPLC) grade acetonitrile
was
obtained from Rathburn (Walkersburn, UK). Trifluoroacefic 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, Larne, UK).
All
water used in these experiments was purified using a Water Purification
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System (Millipore, Milford, MA, USA). All other chemicals used were of the
highest
purity available.
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
at.,
2002, Diabetologia 45: 1281-1291). N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)
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'. 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 at., 2002,
Cell. Biol. Int. 27: 41-46).
DPP IV degradation studies. GIP and fatty acid derivatised GIP analogues were
incubated at 37 C with purified porcine dipeptidylpeptidase IV (5 mU in 50
mmo1/1
triethanolamine-HC1; pH 7.8) for 0, 2, 4, 8 and 24 hours (final peptide
concentration 2
mmo1/1). 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 x 250 mm; The Separations Group, Hesparia,
CA) and the major degradation product GIP(3-42) 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.
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)
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were cultured in DMEM tissue culture medium containing 10% (v/v) PBS, 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) PBS, 1% (v/v) antibiotics
(100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mmo1/1 glucose. Cells
were
maintained at 37 C in an atmosphere of 5% CO2 and 95% air using an LEEC
incubator (Laboratory Technical Engineering, Nottingham, UK).
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 (Niinc,
Roskilde,
Denmark) at a density of 105 cells per well and allowed to grow for 48 hours
before
being loaded with tritiated adenine (2 uCi; TRK311; Amersham, Buckinghamshire,
UK). The cells were then incubated at 37 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 (10-13 to 10-6 mo1/1)
in HBS
buffer in the presence of 1 mmo1/1 IBMX for 15 minutes at 37 C. The medium was
subsequently removed and the cells lysed with 1 ml of 5% TCA containing 0.1
mmo1/1 unlabelled cyclic AMP and 0.1 mmo1/1 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).
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 105
cells per well, and allowed to attach overnight at 37 C. Acute tests for
insulin release
were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer
bicarbonate buffer supplemented with 1.1 mmo1/1 glucose. Test incubations were
performed in the presence of 5.6 mmo1/1 glucose with a range of concentrations
(1043
to 10-6 mo1/1) of GIP and GIP analogues. After 20 minutes incubation, the
buffer was
removed from each well and aliquots (200 1.t1) used for measurement of
insulin.
Effects ofN-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) in ob/ob mice. Metabolic
and dose-response effects of GIP and N-AcGIP(LysPAL) analogues (at 6.25 ¨25
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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, NaC1), native GIP or N-AcGIP(LysPAL37) (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(LysPAL37) were monitored when administered together with
glucose (18 mmoles/kg). In the second, acute effects of N-AcGIP(LysPAL37)
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 mug ice-cold acid ethanol (75% ethanol, 2.35% H20,
1.5% HC1). Whole blood was taken for determination of glycated hemoglobin.
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 MA as described previously (Flaft, 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).
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-
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Hill, New York) with baseline subtraction. Groups of data were considered to
be
significantly different ifp<0.05.
Results
Structural characterisation by MALDI-ToF MS. Following synthesis and HPLC
purification, the molecular masses were obtained for GIP, N-AcGIP(LysPAL16)
and
N-AcGIP(LysPAL37) 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(LysPAL16) and N-AcGIP(LysPAL37) 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.
Table 3. Structural characterisation of GIP and GIP analogues by MALDI-ToF MS.
Peptide Experimental Theoretical Difference
Mr (Da) Mr (Da) (Da)
GIP 4983.7 4982.4 1.3
N-AcGIP(Ly5PAL16) 5268.9 5266.1 2.8
N-AcGIP(LysPAL37) 5267.7 5266.1 1.6
Peptide samples were mixed with matrix (alpha-cyano-4-hydroxycinnamic acid)
and
m/z ratio vs. relative peak intensity recorded using a Voyager-DE
BioSpectrometry
Workstation.
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 523% of the peptide remaining intact. After 8 hours incubation
the
native peptide was entirely degraded to GIP(3-42). In contrast, both N-
AcGIP(LysPAL16) and N-AcGIP(LysPAL37) remained completely intact (no
degradation fragment evident) even after 24 hours incubation with DPP IV.
Table 4. Percentage intact peptide remaining after incubation with DPP IV.
Peptide % Intact peptide remaining after time (hours)
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0 2 8 24
Native GIP 100 52 3 0 0
N-AcGIP(LysPAL116) 100 100 100 100
N-AcGIP(LysPAL37) 100 100 100 100
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.
Changes in Cyclic AMP production. Fig. 50A shows intracellular cyclic AMP
production by GIP (A) and fatty acid derivatised GIP analogues N-
AcGIP(LysPAL16)
(o) and N-AcGIP(LysPAL37) (*), 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.
A concentration-dependent (1043 to 10-6 mo1/1) increase in cyclic AMP
production was observed with native GIP (EC50 value 18.2 nmo1/1) using CHL
cells
transfected with the human GIP receptor (Fig. 50A). Likewise, both N-
AcGIP(Ly5PAL16) and N-AcGIP(LysPAL37) followed a similar pattern of
stimulation
to that of native GIP with calculated EC50 values of 12.1 and 13.0 nmo1/1,
respectively. The lower EC50 values for both analogues suggest an enhanced
cyclic
AMP-stimulating potency.
In vitro insulin-releasing activity. Fig. 50B shows insulin-releasing activity
of
glucose (5.6 mmo/1 glucose; white bars), GIP (lined bars) and fatty acid
derivatised
GIP analogues N-AcGIP(LysPAL16) (grey bars) and N-AcGIP(LysPAL37) (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 mmo1/1
glucose alone).
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
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mmo1/1 glucose alone) (Fig. 50B). Likewise, both N-AcGIP(LysPAL16) and N-
AcGIP(LysPAL37) 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.
Metaboliceffects 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/lcg bw; o; white bars) as a control, or in
combination with
GIP (A; lined bars) or GIP analogues N-AcGIP(LysPAL16) (a; grey bars) and N-
AcGIP(LysPAL37) (0; 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, Ap<0.05, AAp<0.01 and Amp<0.001 compared to native
GIP, rnp<0.001 compared with N-AcGIP(LysPAL16).
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 mmo1/1 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(LysPAL16) and N-
AcGIP(LysPAL37) 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(LysPAL16) and
N-AcGIP(Ly5PAL37) decreased the overall glucose excursion (p<0.05 to p<0.001)
when compared to native GIP.
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<0.05) compared with glucose alone. When N-
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AcGIP(LysPALI6) or N-AcGIP(LysPAL37) 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(LysPAL16) and N-AcGIP(LysPAL37)
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(LysP AL37) was significantly more potent (1.5-fold: p<0.001) than N-
AcGIP(LysPAL16) at stimulating insulin secretion.
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(LysPAL37) 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(LysP AL37) 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(LysPAL37) (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. Ap<0.01 and p<0.001 compared to
native
GIP at the same dose.
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(LysPAL37) 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(LysPAL37) 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(LysPAL37) elicited a
prominent insulin response (2.0-fold; p<0.01) compared to glucose alone.
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Long-acting effects in ob/ob mice. The effects of daily injection of N-
AcGIP(LysPAL37) 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(LysPAL37) (.;black bars) administration on food intake (Fig. 53A), body
weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated
hemoglobin N-AcGIP(LysPAL37) (12.5 nmoles/kg/day) (Fig. 53E). Native GIP (12.5
nmoles/kg/day; = ; lined bars) or saline vehicle (control; o; 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. mp<0.01
compared to native GIP.
GIP or N-AcGIP(LysPAL37) 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(LysPAL37) 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(LysPAL37) 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).
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Effects of long term treatment of ob/ob mice with N-AcGIP (LysPAL37) 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(LysPAL37)
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(Ly5PAL37) (12.5 nmoles/kg/day; 41; black bars), native GIP (12.5
nmoles/kg/day; A; lined bars) or saline vehicle (control; o; 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. 6p<0.05, mp<0.01, LiMp<0.001 compared to
native
GIP.
Consistent with effects on glycated hemoglobin, treatment of ob/ob mice for
14 days with N-AcGIP(LysPAL37) 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).
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(LysPAL37)
on pancreatic insulin content. Figs. 55A through 55D are a line graph and
three bar
graphs showing the effects of daily N-AcGIP(LysPAL37) 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-
AcG1P(LysP AL37) (12.5 nmoles/kg/day; *; black bars), native GIP (12.5
nmoles/kg/day; = ; lined bars) or saline vehicle (control; o; 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
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in the right panels. Values are means SEM for 8 mice. *p<0.05, **p<0.01
compared to control. A<0.05, mp<0.01 compared to native GIP.
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(LysPAL37) 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).
Treatment of ob/ob mice for 14 days with native GIP or N-AcGIP(LysPAL37)
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(LysPAL37) significantly
increased (p<0.01) insulin content compared with each of the other groups
(Figs. 55C,
55D).
Evaluation of GIP receptor desensitization after long term treatment of ob/ob
mice
with N-AcGIP (LysPAL37). 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(LysP AL37) and native GIP after daily injection for 14 days. Glucose (18
mmoles/kg) was administered by intraperitoneal injection alone (o; white bars)
or in
combination with either N-AcGIP(LysPAL37) (0; black bars) or native GIP (A;
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.
Ap<0.05, mp<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(LysPAL37) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5
nmoles/kg/day; 0; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars)
or
saline vehicle (control; o; white bars). N-AcGIP(LysPAL37) (25 nmoles/kg) was
administered by intraperitoneal injection with glucose (18 mmoles/kg) at the
time
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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. 6p<0.05,
'6"6'p<0.01
compared to group receiving injections of native GIP.
As shown in Figs. 56A through 56D, treatment of ob/ob mice with N-
AcGIP(LysPAL37) 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(LysPAL37), the acute effects of the analogue,
administered with glucose, were examined in each of the 3 groups after 14 days
treatment with N-AcGIP(LysPAL37), 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.
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.
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