U.S. patent application number 12/651839 was filed with the patent office on 2010-10-21 for analogs of gastric inhibitory polypeptide and their use for treatment of diabetes.
Invention is credited to Peter Raymond Flatt, Finbarr Paul Mary O'Harte.
Application Number | 20100267628 12/651839 |
Document ID | / |
Family ID | 26315353 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100267628 |
Kind Code |
A1 |
O'Harte; Finbarr Paul Mary ;
et al. |
October 21, 2010 |
Analogs of Gastric Inhibitory Polypeptide and Their Use for
Treatment of Diabetes
Abstract
The present invention provides peptides which stimulate the
release of insulin. The peptides, based on GIP 1-42 include
substitutions and/or modifications which enhance and influence
secretion and/or have enhanced resistance to degradation. The
invention also provides a process of N terminally modifying GIP and
the use of the peptide analogues for treatment of diabetes.
Inventors: |
O'Harte; Finbarr Paul Mary;
(Ballymoney, GB) ; Flatt; Peter Raymond;
(Portrush, GB) |
Correspondence
Address: |
K&L Gates LLP
STATE STREET FINANCIAL CENTER, One Lincoln Street
BOSTON
MA
02111-2950
US
|
Family ID: |
26315353 |
Appl. No.: |
12/651839 |
Filed: |
January 4, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12001449 |
Dec 11, 2007 |
7666838 |
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12651839 |
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11090825 |
Mar 25, 2005 |
7326688 |
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12001449 |
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09937687 |
Jan 8, 2002 |
6921748 |
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PCT/GB00/01089 |
Mar 29, 2000 |
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11090825 |
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Current U.S.
Class: |
514/6.7 ;
514/6.8; 514/6.9; 530/324; 530/325; 530/326 |
Current CPC
Class: |
A61P 5/48 20180101; A61P
3/08 20180101; A61K 38/00 20130101; A61P 3/10 20180101; C07K 14/575
20130101 |
Class at
Publication: |
514/6.7 ;
530/324; 530/325; 530/326; 514/6.8; 514/6.9 |
International
Class: |
A61K 38/22 20060101
A61K038/22; C07K 14/575 20060101 C07K014/575; C07K 7/08 20060101
C07K007/08; A61P 3/08 20060101 A61P003/08; A61P 3/10 20060101
A61P003/10; A61P 5/48 20060101 A61P005/48; A61K 38/10 20060101
A61K038/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 1999 |
GB |
9907216.7 |
Jul 27, 1999 |
GB |
9917565.5 |
Claims
1-11. (canceled)
12. A peptide analogue of GIP (1-42) comprising at least 15 amino
acid residues from the N-terminal end of GIP (1-42), wherein those
amino acids present at positions 15-30 of the peptide analogue are
unsubstituted with respect to GIP (1-42), with the proviso that the
peptide analogue is not tyrosine.sup.1 glucitol GIP (1-42), and
wherein (a) the peptide analogue comprises two amino acid
substitutions or modifications selected from the group consisting
of: (i) an L- or D-amino acid substitution at position 1 selected
from Alanine, Arginine, Asparagine, Aspartic acid, Cysteine,
Glutamic acid, Glutamine, Glycine, Isoleucine, Leucine, Lysine,
Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan,
Tyrosine, Valine or an amino acid modification at position 1; and
one of an L- or D-amino acid substitution at position 2 or 3
selected from Alanine, Arginine, Asparagine, Aspartic acid,
Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine,
Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine,
Threonine, Tryptophan, Tyrosine, Valine; or an amino acid
modification at position 2 or 3; and (ii) an amino acid
substitution at position 2 selected from Alanine, Arginine,
Asparagine, Aspartic acid, Cysteine, Glutamic acid, Glutamine,
Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine,
Phenylalanine, Proline, Threonine, Tryptophan, Tyrosine, Valine or
an amino acid modification at position 2; and one of an L- or
D-amino acid modification at position 1, an amino acid modification
at position 1, an L- or D-amino acid modification at position 3, or
an amino acid substitution at position 3; or wherein (b) the
peptide analogue comprises one amino acid substitution or
modification selected from the group consisting of: an L- or
D-amino acid substitution at position 1; an amino acid modification
at position 1; an amino acid modification at position 2; an L-amino
acid substitution at position 2; an D-amino acid substitution at
position 2 by a D-amino acid selected from D-arginine,
D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid,
D-glutamine, D-glycine, D-histidine, D-isoleucine, D-leucine,
D-lysine, D-methionine, D-phenylalanine, D-proline, D-serine,
D-threonine, D-tryptophan, D-tyrosine and D-valine; an L- or
D-amino acid substitution at position 3, and an amino acid
modification at position 3; and wherein the peptide analogue is
DPP-IV resistant and is capable of binding a receptor of GIP.
13. The peptide analogue of claim 12, wherein the peptide analogue
comprises a peptide analogue consisting of up to between 15 to 30
amino acids of GIP(1-42).
14. The peptide analogue of claim 12, wherein the peptide analogue
activates the receptor of GIP to stimulate insulin release.
15. The peptide analogue of claim 12, wherein the peptide analogue
is capable of binding a receptor of GIP and wherein the peptide
analogue comprises at least one amino acid substitution or
modification at one of position 1, 2 or 3, with the proviso that
the peptide analogue is not tyrosine.sup.1 glucitol GIP (1-42),
wherein the amino acid substitution or modification is selected
from the group consisting of: substitution at position 1 by an
amino acid; substitution at position 2 by an L-amino acid, amino
isobutyric acid or sarcosine; substitution at position 3 by an
amino acid, amino isobutyric acid or sarcosine; conversion of the
Ala.sup.2-Glu.sup.3 bond to a .psi.[CH.sub.2NH] bond; conversion of
the Ala.sup.2-Glu.sup.3 bond to a stable isostere bond; and
substitution by beta-alanine, an omega-amino acid, 3-amino
propionic acid, 4-amino butyric acid, ornithine, citrulline,
homoarginine, t-butylalanine, t-butylglycine, N-methylisoleucine,
phenylglycine, cyclohexylalanine, norleucine, cysteic acid and
methionine sulfoxide.
16. The peptide analogue of claim 12, with the proviso that, when
the analogue comprises one amino acid modification at position 1,
the one modification is not glycation of the tyrosine residue at
position 1.
17. A method of stimulating insulin release, the method comprising
administering to an individual an effective amount of the peptide
analogue of claim 12.
18. The method of claim 17, wherein the peptide analogue comprises
a peptide analogue consisting of up to between 15 to 30 amino acids
of GIP(1-42).
19. A method of moderating blood glucose excursions, the method
comprising administering to an individual an effective amount of
the peptide analogue of claim 12.
20. The method of claim 19, wherein the peptide analogue comprises
a peptide analogue consisting of up to between 15 to 30 amino acids
of GIP(1-42).
21. A method of treating diabetes comprising administering to an
individual an effective amount of the peptide analogue of claim
12.
22. The method of claim 21, wherein the peptide analogue comprises
a peptide analogue consisting of up to between 15 to 30 amino acids
of GIP(1-42).
23. The method of claim 21, wherein the diabetes is type 2
diabetes.
24. The method of claim 22, wherein the diabetes is type 2
diabetes.
Description
[0001] The present invention relates to the release of insulin and
the control of blood glucose concentration. More particularly the
invention relates to the use of peptides to stimulate release of
insulin, lowering of blood glucose and pharmaceutical preparations
for treatment of type 2 diabetes.
[0002] 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.
[0003] 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. DPP IV is completely inhibited in serum by the
addition of diprotin A(DPA, 0.1 mmol/l). This occurs through the
rapid removal of the N-terminal dipeptides Tyr.sup.1-Ala.sup.2 and
His.sup.7-Ala.sup.8 giving rise to the main metabolites GIP(3-42)
and GLP-1(9-36)amide, respectively. These truncated peptides are
reported to lack biological activity or to even serve as
antagonists at GIP or tGLP-1 receptors. The resulting biological
half-lives of these incretin hormones in vivo are therefore very
short, estimated to be no longer than 5 min.
[0004] 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 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
reached 20 min) and the resulting need for both high dosage and
precise timing with meals. Recent therapeutic strategies have
focused on precipitated preparations to delay peptide absorption
and inhibition of GLP-1 degradation using specific inhibitors of
DPP IV. A possible therapeutic role is also suggested by the
observation that a specific inhibitor of DPP IV, isoleucine
thiazolidide, lowered blood glucose and enhanced insulin secretion
in glucose-treated diabetic obese Zucker rats presumably by
protecting against catabolism of the incretin hormones tGLP-1 and
GIP.
[0005] Numerous 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 IGT or NIDDM. Longer term studies also show
significant benefits of tGLP-1 injections in NIDDM and possibly
IDDM therapy, providing a major incentive to develop an orally
effective or long-acting tGLP-1 analogue. Several attempts have
been made to produce structurally modified analogues of tGLP-1
which are resistant to DPP IV degradation. A significant extension
of serum half-life is observed with His.sup.7-glucitol tGLP-1 and
tGLP-1 analogues substituted at position 8 with Gly, Aib, Ser or
Thr. However, these structural modifications seem to impair
receptor binding and insulinotrophic activity thereby compromising
part of the benefits of protection from proteolytic degradation. In
recent studies using His.sup.7-glucitol tGLP-1, resistance to DPP
IV and serum degradation was accompanied by severe loss of
insulin-releasing activity.
[0006] 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
RIA.
[0007] The present invention aims to provide effective analogues of
GIP. It is one aim of the invention to provide a pharmaceutical for
treatment of Type 2 diabetes.
[0008] According to the present invention there is provided an
effective peptide analogue of the biologically active GIP(1-42)
which has improved characteristics for treatment of Type 2 diabetes
wherein the analogue comprises at least 15 amino acid residues from
the N terminus of GIP(1-42) and has at least one amino acid
substitution or modification at position 1-3 and not including
Tyr.sup.1 glucitol GIP(1-42).
[0009] 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.
[0010] The analogue may include modification by fatty acid addition
at an epsilon amino group of at least one lysine residue.
[0011] The invention includes Tyr.sup.1 glucitol GIP(1-42) having
fatty acid addition at an epsilon amino group of at least one
lysine residue.
TABLE-US-00001 FIG 1. Primary structure of human gastric inhibitory
polypeptide (GIP) 1 5 10
NH.sub.2-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser- 15 20
Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val- 25
Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp- 30 35
Trp-Lys-His-Asn-Ile-Thr-Gln-COOH 40 FIG. 2. Primary structure of
porcine gastric inhibitory polypeptide (GIP) 1 5 10
NH2-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser- 15 20
Ile-Ala-Met-Asp-Lys-Ile-Arg-Gln-Gln-Asp-Phe-Val- 25
Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Ser-Asp- 30 35
Trp-Lys-His-Asn-Ile-Thr-Gln-COOH 40
[0012] 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.
[0013] Any of these properties will enhance the potency of the
analogue as a therapeutic agent.
[0014] 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.
[0015] Various amino acid substitutions at second and third amino
terminal residues are included, such as GIP(1-42)Gly2,
GIP(1-42)Ser2, GIP(1-42)Abu2, GIP(1-42)Aib, GIP(1-42)D-Ala2,
GIP(1-42)Sar2, and GIP(1-42)Pro3.
[0016] Amino-terminally modified GIP analogues include N-glycated
GIP(1-42), N-alkylated GIP(1-42), N-actylated GIP(1-42),
N-acetyl-GIP(1-42) and N-isopropyl GIP(1-42).
[0017] Other stabilised 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
Tyr1-Ala2.
[0018] 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: [0019] (a) substitution of Ala.sup.2 by
Gly [0020] (b) substitution of Ala.sup.2 by Ser [0021] (c)
substitution of Ala.sup.2 by Abu [0022] (d) substitution of
Ala.sup.2 by Aib [0023] (e) substitution of Ala.sup.2 by D-Ala
[0024] (f) substitution of Ala.sup.2 by Sar [0025] (g) substitution
of Glu.sup.3 by Pro [0026] (h) modification of Tyr.sup.1 by
acetylation [0027] (i) modification of Tyr.sup.1 by acylation
[0028] (j) modification of Tyr.sup.1 by alkylation [0029] (k)
modification of Tyr.sup.1 by glycation [0030] (l) conversion of
Ala.sup.2-Glu.sup.3 bond to a psi [CH2NH] bond [0031] (m)
conversion of Ala2-Glu3 bond to a stable peptide isotere bond
[0032] (n) (n-isopropyl-H) 1GIP.
[0033] The invention also provides the use of Tyr.sup.1-glucitol
GIP in the preparation of a medicament for the treatment of
diabetes.
[0034] The invention further provides improved pharmaceutical
compositions including analogues of GIP with improved
pharmacological properties.
[0035] 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.
[0036] 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.
[0037] The invention also provides a method of N-terminally
modifying GIP or analogues thereof the method comprising the steps
of synthesizing the peptide from the C terminal to the penultimate
N terminal amino acid, adding tyrosine to a bubbler system as a
F-moc protected Tyr(tBu)-Wang resin, deprotecting the N-terminus of
the tyrosine and reacting with the modifying agent, allowing the
reaction to proceed to completion, cleaving the modified tyrosine
from the Wang resin and adding the modified tyrosine to the peptide
synthesis reaction.
[0038] Preferably the agent is glucose, acetic anhydride or
pyroglutamic acid.
[0039] The invention will now be demonstrated with reference to the
following non-limiting example and the accompanying figures
wherein:
[0040] FIG. 1a illustrates degradation of GIP by DPP IV.
[0041] FIG. 1b illustrates degradation of GIP and Tyr.sup.1
glucitol GIP by DPP IV.
[0042] FIG. 2a illustrates degradation of GIP human plasma.
[0043] FIG. 2b illustrates degradation of GIP and
Tyr.sup.1-glucitol GIP by human plasma.
[0044] FIG. 3 illustrates electrospray ionization mass spectrometry
of GIP, Tyr.sup.1-glucitol GIP and the major degradation fragment
GIP(3-42).
[0045] FIG. 4 shows the effects of GIP and glycated GIP on plasma
glucose homeostasis.
[0046] FIG. 5 shows effects of GIP on plasma insulin responses.
[0047] FIG. 6 illustrates DPP-IV degradation of GIP 1-42.
[0048] FIG. 7 illustrates DPP-IV degradation of GIP
(Abu.sup.2).
[0049] FIG. 8 illustrates DPP-IV degradation of GIP
(Sar.sup.2).
[0050] FIG. 9 illustrates DPP-IV degradation of GIP
(Ser.sup.2),
[0051] FIG. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.
[0052] FIG. 11 illustrates DPP-IV degradation of glycated GIP.
[0053] FIG. 12 illustrates human plasma degradation of GIP.
[0054] FIG. 13 illustrates human plasma degradation of GIP
(Abu.sup.2).
[0055] FIG. 14 illustrates human plasma degradation of GIP
(Sar.sup.2).
[0056] FIG. 15 illustrates human plasma degradation of GIP
(Ser.sup.2).
[0057] FIG. 16 illustrates human plasma degradation of glycated
GIP.
[0058] FIG. 17 shows the effects of various concentrations of GIP
1-42 and GIP (Abu.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0059] FIG. 18 shows the effects of various concentrations of GIP
1-42 and GIP (Abu.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0060] FIG. 19 shows the effects of various concentrations of GIP
1-42 and GIP (Sar.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0061] FIG. 20 shows the effects of various concentrations of GIP
1-42 and GIP (Sar.sup.2) on insulin release from GRIN-BD11 cells
incubated at 16.7 mM glucose.
[0062] FIG. 21 shows the effects of various concentrations of GIP
1-42 and GIP (Ser.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0063] FIG. 22 shows the effects of various concentrations of GIP
1-42 and GIP (Ser.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0064] 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 5.6 mM glucose.
[0065] 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 16.7 mM glucose.
[0066] FIG. 25 shows the effects of various concentrations of GIP
1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0067] FIG. 26 shows the effects of various concentrations of GIP
1-42 and glycated GIP 1-42 on insulin release from GRIN-BD11 cells
incubated at 16.7 mM glucose.
[0068] FIG. 27 shows the effects of various concentrations of GIP
1-42 and GIP (Gly.sup.2) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0069] FIG. 28 shows the effects of various concentrations of GIP
1-42 and GIP (Gly.sup.2) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
[0070] FIG. 29 shows the effects of various concentrations of GIP
1-42 and GIP (Pro.sup.3) on insulin release from BRIN-BD11 cells
incubated at 5.6 mM glucose.
[0071] FIG. 30 shows the effects of various concentrations of GIP
1-42 and GIP (Pro.sup.3) on insulin release from BRIN-BD11 cells
incubated at 16.7 mM glucose.
EXAMPLE 1
[0072] Preparation of N-Terminally modified GIP and analogues
thereof.
[0073] The N-terminal modification of GIP is essentially a three
step process. Firstly, GIP is synthesised from its C-terminal
(starting from a Fmoc-Gln (Trt)-Wang resin, Novabiochem) up to the
penultimate N-terminal amino-acid (Ala2) on an automated peptide
synthesizer (Applied Biosystems, CA, 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 h as necessary to allow the reaction to go to completion.
The completeness of reaction will be monitored using the ninhydrin
test which will determine the presence of available free
.alpha.-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 .alpha.-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
[0074] The following example investigates preparation of
Tyr.sup.1-glycitol GIP together with evaluation of its
antihyperglycemic and insulin-releasing properties in vivo. The
results clearly demonstrate that this novel GIP analogue exhibits a
substantial resistance to aminopeptidase degradation and increased
glucose lowering activity compared with the native GIP.
Research Design and Methods
[0075] Materials. Human GIP was purchased from the American Peptide
Company (Sunnyvale, Calif., USA). HPLC grade acetonitrile was
obtained from Rathburn (Walkersburn, Scotland). Sequencing grade
trifluoroacetic acid (TFA) was obtained from Aldrich (Poole,
Dorset, UK). All other chemicals purchased including dextran T-70,
activated charcoal, sodium cyanoborohydride and bovine serum
albumin fraction V were from Sigma (Poole, Dorset, UK). Diprotin A
(DPA) was purchased from Calbiochem-Novabiochem (UK) Ltd. (Beeston,
Nottingham, UK) and rat insulin standard for RIA was obtained form
Novo Industria (Copenhagen, Denmark). Reversed-phase Sep-Pak
cartridges (C-18) were purchased from Millipore-Waters (Milford,
Mass., USA). All water used in these experiments was purified using
a Milli-Q, Water Purification System (Millipore Corporation,
Milford, Mass., USA).
[0076] Preparation of Tyr.sup.1-glucitol GIP. Human GIP was
incubated with glucose under reducing conditions in 10 mmol/l
sodium phosphate buffer at pH 7.4 for 24 h. The reaction was
stopped by addition of 0.5 mol/l acetic acid (30 .mu.l) and the
mixture applied to a Vydac (C-18)(4.6.times.250 mm) analytical HPLC
column (The Separations Group, Hesperia, Calif., USA) and gradient
elution conditions were established using aqueous/TFA and
acetonitrile/TFA solvents. Fractions corresponding to the glycated
peaks were pooled, taken to dryness under vacuum using an AES 1000
Speed-Vac concentrator (Life Sciences International, Runcorn, UK)
and purified to homogeneity on a Supelcosil (C-8) (4.6.times.150
mm) column (Supelco Inc., Poole, Dorset, UK).
[0077] Degradation of GIP and Tyr.sup.1-glucitol GIP by DPP IV.
HPLC-purified GIP or Tyr.sup.1-glucitol GIP were incubated at
37.degree. C. with DPP-IV (5 mU) for various time periods in a
reaction mixture made up to 500 .mu.l with 50 mmol/l
triethanolamine-HCl, pH 7.8 (final peptide concentration 1 mmol/l).
Enzymatic reactions were terminated after 0, 2, 4 and 12 hours by
addition of 5 .mu.l of 10% (v/v) TFA/water. Samples were made up to
a final volume of 1.0 ml with 0.12% (v/v) TFA and stored at
-20.degree. C. prior to HPLC analysis.
[0078] Degradation of GIP and Tyr.sup.1-glucitol GIP by human
plasma. Pooled human plasma (20 .mu.l) taken from six healthy
fasted human subjects was incubated at 37.degree. C. with GIP or
Tyr.sup.1-glucitol GIP (10 .mu.g) for 0 and 4 hours in a reaction
mixture made up to 500 .mu.l, containing 50 mmol/l
triethanolamine/HCL buffer pH 7.8. Incubations for 4 hours were
also performed in the presence of diprotin A (5 mU). The reactions
were terminated by addition of 5 .mu.l of TFA and the final volume
adjusted to 1.0 ml using 0.1% v/v TFA/water. Samples were
centrifuged (13,000 g, 5 min) 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 1.0 ml with
0.12% (v/v) TFA/water prior to HPLC purification.
[0079] HPLC analysis of degraded GIP and Tyr.sup.1-glucitol GIP.
Samples were applied to a Vydac C-18 widepore column equilibriated
with 0.12% (v/v) TFA/H.sub.2O at a flow rate of 1.0 ml/min. Using
0.1% (v/v) TFA in 70% acetonitrile/H.sub.2O, the concentration of
acetonitrile in the eluting solvent was raised from 0% to 31.5%
over 15 min, to 38.5% over 30 min and from 38.5% to 70% over 5 min,
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.
[0080] Electrospray ionization mass spectrometry (ESI-MS). Samples
for ESI-MS analysis containing intact and degradation fragments of
GIP (from DPP IV and plasma incubations) as well as
Tyr.sup.1-glucitol GIP, were further purified by HPLC. Peptides
were dissolved (approximately 400 .mu.mol) in 100 .mu.l of water
and applied to the LCQ benchtop mass spectrometer (Finnigan MAT,
Hemel Hempstead, UK) equipped with a microbore C-18 HPLC column
(150.times.2.0 mm, Phenomenex, UK, Ltd, Macclesfield). Samples (30
.mu.l direct loop injection) were injected at a flow rate of 0.2
ml/min, under isocratic conditions 35% (v/v) acetonitile/water.
Mass spectra were obtained from the quadripole ion trap mass
analyzer and recorded. Spectra were collected using full ion scan
mode over the mass-to-charge (m/z) range 150-2000. The molecular
masses of GIP and related structures were determined from ESI-MS
profiles using prominent multiple charged ions and the following
equation M.sub.r=iM.sub.i-iM.sub.h (where M.sub.r=molecular mass;
M.sub.i=m/z ratio; i=number of charges; M.sub.h=mass of a
proton).
[0081] In vivo biological activity of GIP and Try.sup.1-glucitol
GIP. Effects of GIP and Tyr.sup.1-glucitol GIP on plasma glucose
and insulin concentrations were examined using 10-12 week old male
Wistar rats. The animals were housed individually in an air
conditioned room and 22.+-.2.degree. C. with a 12 hour light/12
hour dark cycle. Drinking water and a standard rodent maintenance
diet (Trouw Nutrition, Belfast) were supplied ad libitum. Food was
withdrawn for an 18 hour period prior to intraperitoneal injection
of glucose alone (18 mmol/kg body weight) or in combination with
either GIP or Tyr.sup.1-glucitol GIP (10 nmol/kg). Test solutions
were administered in a final volume of 8 ml/kg body weight. Blood
samples were collected at 0, 15, 30 and 60 minutes from the cut tip
of the tail of conscious rats into chilled fluoride/heparin
microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Samples were
centrifuged using a Beckman microcentrifuge for about 30 seconds at
13,000 g. Plasma samples were aliquoted and stored at -20.degree.
C. prior to glucose and insulin determinations. All animal studies
were done in accordance with the Animals (Scientific Procedures)
Act 1986.
[0082] 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 curves (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.
Results
[0083] Degradation of GIP and Tyr.sup.1-glucitol GIP by DPP IV.
FIG. 1 illustrates the typical peak profiles obtained from the HPLC
separation of the products obtained from the incubation of GIP
(FIG. 1a) or Tyr.sup.1-glucitol GIP (FIG. 1b) with DPP IV for 0, 2,
4 and 12 hours. The retention times of GIP and Tyr.sup.1-glucitol
GIP at t=0 were 21.93 minutes and 21.75 minutes respectively.
Degradation of GIP was evident after 4 hours incubation (54%
intact), and by 12 hours the majority (60% of intact GIP was
converted to the single product with a retention time of 21.61
minutes. Tyr.sup.1-glucitol GIP remained almost completely intact
throughout 2-12 hours incubation. Separation was on a Vydac C-18
column using linear gradients of 0% to 31.5% acetonitrile over 15
minutes, to 38.5% over 30 minutes and from 38.5 to 70% acetonitrile
over 5 minutes.
[0084] Degradation of GIP and Tyr.sup.1-glucitol GIP by human
plasma. FIG. 2 shows a set of typical HPLC profiles of the products
obtained from the incubation of GIP or Tyr.sup.1-glucitol GIP with
human plasma for 0 and 4 h. GIP (FIG. 2a) with a retention time of
22.06 min was readily metabolised by plasma within 4 hours
incubation giving rise to the appearance of a major degradation
peak with a retention time of 21.74 minutes. In contrast, the
incubation of Tyr.sup.1-glucitol GIP under similar conditions (FIG.
2b) did not result in the formation of any detectable degradation
fragments during this time with only a single peak being observed
with a retention time of 21.77 minutes. Addition of diprotin A, a
specific inhibitor of DPP IV, to GIP during the 4 hours incubation
completely inhibited degradation of the peptide by plasma. Peaks
corresponding with intact GIP, GIP (3-42) and Tyr.sup.1-glucitol
GIP are indicated. A major peak corresponding to the specific DPP
IV inhibitor tripeptide DPA appears in the bottom panels with
retention time of 16-29 min.
[0085] Identification of GIP degradation fragments by ESI-MS. FIG.
3 shows the monoisotopic molecular masses obtained for GIP, (panel
A), Tyr.sup.1-glucitol GIP (panel B) and the major plasma
degradation fragment of GIP (panel C) using ESI-MS. The peptides
analyzed were purified from plasma incubations as shown in FIG. 2.
Peptides were dissolved (approximately 400 .mu.mol) in 100 .mu.l of
water and applied to the LC/MS equipped with a microbore C-18 HPLC
column. Samples (30 .mu.l direct loop injection) were applied at a
flow rate of 0.2 ml/min, under isocratic conditions 35%
acetonitrile/water. Mass spectra were recorded using a quadripole
ion trap mass analyzer. Spectra were collected using full ion scan
mode over the mass-to-charge (m/z) range 150-2000. The molecular
masses (M.sub.r) of GIP and related structures were determined from
ESI-MS profiles using prominent multiple charged ions and the
following equation M.sub.r=iM.sub.i-iM.sub.h. The exact molecular
mass (M.sub.r) of the peptides were calculated using the equation
M.sub.r=1M.sub.i-iM.sub.h as defined in Research Design and
Methods. After spectral averaging was performed, prominent multiple
charges species (M+3H).sup.3+ and (M+4H).sup.4+ were detected from
GIP at m/z 1661.6 and 1246.8, corresponding to intact M.sub.r
4981.8 and 4983.2 Da, respectively (FIG. 3A). Similarly, for
Tyr.sup.1-glucitol GIP ((M+4H).sup.4+ and (M+5H).sup.5+) were
detected at m/z 1287.7 and 1030.3, corresponding to intact
molecular masses of M.sup.r 5146.8 and 5146.5 Da, respectively
(FIG. 3B). The difference between the observed molecular masses of
the quadruply charged GIP and the N-terminally modified GIP species
(163.6 Da) indicated that the latter peptide contained a single
glucitol adduct corresponding to Tyr.sup.1-glucitol GIP. FIG. 3C
shows the prominent multiply charged species (M+3H).sup.3+ and
(M+4H).sup.4+ detected from the major fragment of GIP at m/z 1583.8
and 1188.1, corresponding to intact M.sup.r 4748.4 and 4748 Da,
respectively (FIG. 3C). This corresponds with the theoretical mass
of the N-terminally truncated form of the peptide GIP(3-42). This
fragment was also the major degradation product of DPP IV
incubations (data not shown).
[0086] Effects of GIP and Tyr.sup.1-glucitol GIP on plasma glucose
homeostasis. FIGS. 4 and 5 show the effects of intraperitoneal (ip)
glucose alone (18 mmol.kg) (control group), and glucose in
combination with GIP or Tyr.sup.1-glucitol GIP (10 nmol/kg) on
plasma glucose and insulin concentrations.
[0087] (4A) Plasma glucose concentrations after i.p. glucose alone
(18 mmol/kg) (control group), or glucose in combination with either
GIP of Tyr.sup.1-glucitol GIP (10 nmol/kg). The time of injection
is indicated by the arrow (0 min). (4B) Plasma glucose AUC calues
for 0-60 min post injection. Values are mean.+-.SEM for six rats.
**P<0.01, ***P<0.001 compared with GIP and Tyr.sup.1-glucitol
GIP; tP<0.05, ttP<0.01 compared with non-glucated GIP.
[0088] (5A). Plasma insulin concentrates after i.p. glucose along
(18 mmol/kg) (control group), or glucose in combination with either
with GIP or glycated GIP (10 nmol/kg). The time of injection is
indicated by the arrow. (5B) 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 min).
Plasma insulin AUC values for 0-60 min post injection. Values are
mean.+-.SEM for six rats. *P<0.05, **P<0.001 compared with
GIP and Tyr.sup.1-glucitol GIP; tP<0.05, ttP<0.01 compared
with non-glycated GIP.
[0089] Compared with the control group, plasma glucose
concentrations and area under the curve (AUC) were significantly
lower following administration of either GIP or Tyr.sup.1-glucitol
GIP (FIG. 4A, B). Furthermore, individual values at 15 and 30
minutes together with AUC were significantly lower following
administration of Tyr.sup.1-glucitol GIP as compared to GIP.
Consistent with the established insulin-releasing properties of
GIP, plasma insulin concentrations of both peptide-treated groups
were significantly raised at 15 and 30 minutes compared with the
values after administration of glucose alone (FIG. 5A). The overall
insulin responses, estimated as AUC were also signigicantly greater
for the two peptide-treated groups (FIG. 5B). Despite lower
prevailing glucose concentrations than the GIP-treated group,
plasma insulin response, calculated as AUC, following
Tyr.sup.1-glucitol GIP was significantly greater than after GIP
(FIG. 5B). The significant elevation of plasma insulin at 30
minutes is of particular note, suggesting that the
insulin-releasing action of Tyr.sup.1-glucitol GIP is more
protracted than GIP even in the face of a diminished glycemic
stimulus (FIGS. 4A, 5A).
Discussion
[0090] The forty two 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 and 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 fulfils one of the most desirable features
of any current or potentially new antidiabetic drug.
[0091] The present results demonstrate for the first time that
Tyr.sup.1-glucitol GIP displays profound resistance to serum and
DPP IV degradation. Using ESI-MS the present study showed that
native GIP was rapidly cleaved in vitro to a major 4748.4 Da
degradation product, corresponding to GIP(3-42) which confirmed
previous findings using matrix-assisted laser desorption ionization
time-of-flight mass spectrometry. Serum degradation was completely
inhibited by diprotin A (Ile-Pro-Ile), a specific competitive
inhibitor of DPP IV, confirming this as the main enzyme for GIP
inactivation in vivo. In contrast, Tyr.sup.1-glucitol GIP remained
almost completely intact after incubation with serum or DPP IV for
up to 12 hours. This indicates that glycation of GIP at the
amino-terminal Tyr.sup.1 residue masks the potential cleavage site
from DPP IV and prevents removal of the Tyr.sup.1-Ala.sup.2
dipeptide from the N-terminus preventing the formation of
GIP(3-42).
[0092] Consistent with in vitro protection against DPP IV,
administration of Tyr.sup.1-glucitol GIP significantly enhanced the
antihyperglycemic activity and insulin-releasing action of the
peptide when administered with glucose to rats. Native GIP enhanced
insulin release and reduced the glycemic excursion as observed in
many previous studies. However, amino-terminal glycation of GIP
increased the insulin-releasing and antihyperglycemic actions of
the peptide by 62% and 38% respectively, as estimated from AUC
measurements. Detailed kinetic analysis is difficult due to
necessary limitation of sampling times, but the greater insulin
concentrations following Tyr.sup.1-glucitol GIP as opposed to GIP
at 30 minutes post-injection is indicative of a longer half-life.
The glycemic rise was modest in both peptide-treated groups and
glucose concentrations following injection of Tyr.sup.1-glucitol
GIP were consistently lower than after GIP. Since the
insulinotropic actions of GIP are glucose-dependent, it is likely
that the relative insulin-releasing potency of Tyr.sup.1-glucitol
GIP is greatly underestimated in the present in vivo
experiments.
[0093] In vitro studies in the laboratory of the present inventors
using glucose-responsive clonal B-cells showed that the
insulin-releasing potency of Tyr.sup.1-glucitol GIP was several
order of magnitude greater than GIP and that its effectiveness was
more sensitive to change of glucose concentrations within the
physiological range. Together with the present in vivo
observations, this suggests that N-terminal glycation of GIP
confers resistance to DPP IV degradation whilst enhancing receptor
binding and insulin secretory effects on the B-cell. These
attributes of Tyr.sup.1-glucitol GIP are fully expressed in vivo
where DPP IV resistance impedes degradation of the peptide to
GIP(3-42), thereby prolonging the half-life and enhancing effective
concentrations of the intact biologically active peptide. It is
thus possible that glycated GIP is enhancing insulin secretion in
vivo both by enhanced potency at the receptor as well as improving
DPP IV resistance. Thus numerous studies have shown that GIP (3-42)
and other N-terminally modified fragments, including GIP(4-42), and
GIP (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).
[0094] In addition to its insulinotopic actions, a number of other
potentially important extrapancreatic actions of GIP may contribute
to the enhanced antihyperglycemic activity and other beneficial
metabolic effects of Tyr.sup.1-glucitol GIP. These include the
stimulation of glucose uptake in adipocytes, increased synthesis of
fatty acids and activation of lipoprotein lipase in adipose tissue.
GIP also promotes plasma triglyceride clearance in response to oral
fat loading. In liver, GIP has been shown to enhance
insulin-dependent inhibition of glycogenolysis. GIP also reduces
both glucagon-stimulated lipolysis in adipose tissue as well as
hepatic glucose production. Finally, recent findings indicate that
GIP has a potent effect on glucose uptake and metabolism in mouse
isolated diaphragm muscle. This latter action may be shared with
tGLP-1 and both peptides have additional benefits of stimulating
somatostatin secretion and slowing down gastric emptying and
nutrient absorption.
[0095] In conclusion, this study has demonstrated for the first
time that the glycation of GIP at the amino-terminal Tyr.sup.1
residue limits GIP catabolism through impairment of the proteolytic
actions of serum petidases 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 should be explored alongside tGLP-1 as
potentially useful therapeutic agents for NIDDM. Tyr.sup.1-glucitol
GIP appears to be particularly interesting in this regard since
such amino-terminal modification of GIP enhances rather than
impairs glucose-dependent insulinotropic potency as was observed
recently for tGLP-1.
EXAMPLE 3
[0096] 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
Reagents
[0097] High-performance liquid chromatography (HPLC) grade
acetonitrile was obtained from Rathburn (Walkersburn, Scotland).
Sequencing grade trifluoroacetic acid (TFA) was obtained from
Aldrich (Poole, Dorset, UK). Dipeptidyl peptidase IV was purchased
from Sigma (Poole, Dorset, UK), and Diprotin A was purchased from
Calbiochem Novabiochem (Beeston, Nottingham, UK). RPMI 1640 tissue
culture medium, foetal calf serum, penicillin and streptomycin were
all purchased from Gibco (Paisley, Strathclyde, UK). All water used
in these experiments was purified using a Milli-Q, Water
Purification System (Millipore, Millford, Mass., USA).
[0098] All other chemicals used were of the highest purity
available.
Synthesis of GIP and N-Terminally Modified GIP Analogues
[0099] GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), GIP(Gly2) and
GIP(Pro3) were sequentially synthesised 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 procedure being carried out at least
five times. The pellets were then dried in vacuo and judged pure by
reversed-phase HPLC on a Waters Millennium 2010 chromatography
system (Software version 2.1.5.). N-terminal glycated and
acetylated GIP were prepared by minor modification of a published
method.
[0100] Electrospray ionization-mass spectrometry (ESI-MS) was
carried out as described in Example 2.
[0101] Degradation of GIP and novel GIP analogues by DPP IV and
human plasma was carried out as described in Example 2.
Culture of Insulin Secreting Cells
[0102] BRIN-BD11 cells [30] were cultured in sterile tissue culture
flasks (Corning, Glass Works, UK) using RPMI-1640 tissue culture
medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics
(100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose.
The cells were maintained at 37.degree. C. in an atmosphere of 5%
CO.sub.2 and 95% air using a LEEC incubator (Laboratory Technical
Engineering, Nottingham, UK).
Acute Tests for Insulin Secretion
[0103] Before experimentation, the cells were harvested from the
surface of the tissue culture flasks with the aid of trypsin/EDTA
(Gibco), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark)
at a density of 1.5.times.105 cells per well, and allowed to attach
overnight at 37.degree. C. Acute tests for insulin release were
preceded by 40 min pre-incubation at 37.degree. C. in 1.0 ml Krebs
Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM
CaCl.sub.2, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 10 mM
NaHCO.sub.3, 5 g/l bovine serum albumin, pH 7.4) supplemented with
1.1 mM glucose. Test incubations were performed (n=12) at two
glucose concentrations (5.6 mM and 16.7 mM) with a range of
concentrations (10.sup.-13 to 10.sup.-8 M) of GIP or GIP analogues.
After 20 min incubation, the buffer was removed from each well and
aliquots (200 .mu.l) were used for measurement of insulin by
radioimmunoassay [31].
Statistical Analysis
[0104] 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<0.05.
Results and Discussion
[0105] Structural identification of GIP and GIP analogues by
ESI-MS
[0106] 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
[0107] 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 h. The
results summarised 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.
Degradation of GIP and GIP Analogues by Human Plasma
[0108] 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 h. Observations were also made after
incubation for 24 h in the presence of DPA. These results are
summarised in Table 2 are broadly comparable with DPP IV
incubations, but conditions which more closely mirror in vivo
conditions are less enzymatically severe. GIP was rapidly degraded
by plasma. In comparison, all analogues tested exhibited resistance
to plasma degradation, including GIP(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.
Dose-Dependent Effects of GIP and Novel GIP Analogues on Insulin
Secretion
[0109] FIGS. 17-30 show the effects of a range of concentrations of
GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), acetylated GIP, glycated GIP,
GIP(Gly2) and GIP(Pro3) on insulin secretion from GRIN-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(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
hyperglycaemic 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.
TABLE-US-00002 TABLE 1 % Intact peptide remaining after incubation
with DPPIV % Intact peptide remaining after time (h) Peptide 0 2 4
8 24 GIP 1-42 100 52 .+-. 1 23 .+-. 1 0 0 Glycated GIP 100 100 100
100 100 GIP (Abu.sup.2) 100 38 .+-. 1 28 .+-. 2 0 0 GIP (Ser.sup.2)
100 77 .+-. 2 60 .+-. 1 32 .+-. 4 0 GIP (Sar.sup.2) 100 28 .+-. 2 8
0 0 N-Acetyl-GIP 100 100 100 100 0
TABLE-US-00003 TABLE 2 % Intact peptide remaining after incubation
with human plasma % Intact peptide remaining after incubations with
human plasma Peptide 0 2 4 8 24 DPA GIP 1-42 100 52 .+-. 1 23 .+-.
1 0 0 68 .+-. 2 Glycated GIP 100 100 100 100 100 100 GIP
(Abu.sup.2) 100 38 .+-. 1 28 .+-. 2 0 0 100 GIP (Ser.sup.2) 100 77
.+-. 2 60 .+-. 1 32 .+-. 4 0 63 .+-. 3 GIP (Sar.sup.2) 100 28 .+-.
2 8 0 0 100
[0110] Tables represent 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.
Sequence CWU 1
1
2142PRTHomo sapiens 1Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys1 5 10 15Ile His Gln Gln Asp Phe Val Asn Trp Leu
Leu Ala Gln Lys Gly Lys 20 25 30Lys Asn Asp Trp Lys His Asn Ile Thr
Gln 35 40242PRTPig 2Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile
Ala Met Asp Lys1 5 10 15Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu
Ala Gln Lys Gly Lys 20 25 30Lys Ser Asp Trp Lys His Asn Ile Thr Gln
35 40
* * * * *