WO2005082928A2 - Analogues peptidiques de gip destines au traitement de diabetes, de la resistance a l'insuline et de l'obesite - Google Patents

Analogues peptidiques de gip destines au traitement de diabetes, de la resistance a l'insuline et de l'obesite Download PDF

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Publication number
WO2005082928A2
WO2005082928A2 PCT/GB2005/000710 GB2005000710W WO2005082928A2 WO 2005082928 A2 WO2005082928 A2 WO 2005082928A2 GB 2005000710 W GB2005000710 W GB 2005000710W WO 2005082928 A2 WO2005082928 A2 WO 2005082928A2
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WIPO (PCT)
Prior art keywords
gip
insulin
glucose
peptide analogue
mice
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PCT/GB2005/000710
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English (en)
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WO2005082928A3 (fr
Inventor
Victor A. Gault
Finbarr Paul Mary O'harte
Peter Raymond Flatt
Nigel Irwin
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University Of Ulster
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Priority to EP05717793A priority Critical patent/EP1730188A2/fr
Priority to JP2007500292A priority patent/JP2008500280A/ja
Priority to AU2005217198A priority patent/AU2005217198A1/en
Priority to CA2557151A priority patent/CA2557151C/fr
Priority to US11/090,787 priority patent/US20050272652A1/en
Publication of WO2005082928A2 publication Critical patent/WO2005082928A2/fr
Publication of WO2005082928A3 publication Critical patent/WO2005082928A3/fr
Priority to US11/713,892 priority patent/US7875587B2/en
Priority to US11/825,293 priority patent/US20080009603A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • 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.
  • GIP gastric inhibitory peptide
  • GIP Gastric inhibitory polypeptide
  • tGLP-1 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, J.J. et al, 2002, Regul. Pept. 107:1-13). GIP was initially discovered through its ability to inhibit gastric acid secretion (Brown, J.C. et al. 1969, Can. J. Physiol. Pharmacol.
  • GLP-1 glucagon-like peptide- 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. etal, 2003, Curr. Med. Chem. 10:2471-2483).
  • 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.
  • 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. etal, 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 1 - Ala 2 and His 7 - Ala 8 giving rise to the main metabolites GIP(3-42) and GLP-1 (9-36)amide, respectively. These truncated peptides are reported to lack biological activity or to even serve as antagonists at GIP or tGLP-1 receptors. The resulting biological half-lives of these incretin hormones in vivo are therefore very short, estimated to be no longer than 5 minutes.
  • DPP IV is completely inhibited in serum by the addition of diprotin A (DPA, 0.1 mmol/1).
  • DPA diprotin A
  • this short duration of action is advantageous in facilitating momentary adjustments to homeostatic control.
  • NIDDM non-insulin dependent diabetes
  • 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.
  • 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.
  • ITT impaired glucose tolerance
  • NIDDM impaired glucose tolerance
  • 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.
  • GIP glycosylcholine
  • SEQ ID NO:l peptide analogue of GIP(l-42) (SEQ ID NO:l), 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(l-42) (SEQ ID NO:l), which includes at least 12 amino acid residues from the N-terminal end of GIP(l-42) and having an amino acid substitution at Glu 3 .
  • the amino acid substituted at Glu 3 can be selected from the group consisting of: proline, hydroxyproline, lysine, tyrosine, phenylalanine and tryptophan.
  • a proline can be substituted for Glu 3 .
  • the peptide analogue can further include modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
  • the lysine residue can be Lys 1 , or Lys 37 .
  • the peptide analogue of GIP(l-42) (SEQ ID NO:l) can include at least 12 amino acid residues from the N-terminal end of GIP( 1 -42), and an amino acid modification at amino acid residues 1, 2 or 3.
  • the N-terminal amino acid residue can be acetylated. It can further comprising modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
  • the modification can be the linking of, e.g., a C-8, a C- 10, a C- 12, a C- 14, a C- 16, a C- 18 or a C-20 palmitate group to the epsilon amino group of a lysine residue.
  • the lysine residue can be Lys 16 , or Lys 37 .
  • the invention also includes a peptide analogue of GIP(l-42) (SEQ ID NO:l), 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(l-20), GIP(1-21), GIP(l-22), GIP(l-23), GIP(l-24), GIP(l-25), GIP(l-26), GIP(l-27), GIP(l-28), GIP(l-29), GIP(l-30), GIPQ-31), GIP(l-32), GIP(l-33), GIP(l-34), GIP(l-35), GIP(l-36), GIP(l-37), GIP(l-38), GIP(l-39), GIP(l-40), GIP(1-41) and GIP(l-42), where the base peptide
  • Such a peptide analogue can have a proline substituted for Glu 3 . It can also have a modification in the form of a C-16 palmitate group linked to the epsilon amino group of a lysine residue.
  • the modification can be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue.
  • the lysine residue can be Lys 16 , or Lys 37 .
  • the invention further includes a peptide analogue of GIP(l-42) (SEQ ID NO:l), 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(l-42) (SEQ ID NO:l), comprising at least 12 amino acid residues from the N-terminal end of GIP(l-42) and having an amino acid substitution at Glu 3 , wherein the peptide analogue is resistant to degradation by enzyme DPP IV when compared to naturally-occurring GIP.
  • the invention includes a peptide analogue of GIP(l-42) (SEQ ID NO:l), 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(l-42) (SEQ ID NO:l), comprising at least 12 amino acid residues from the N-terminal end of GIP(l-42) and having an amino acid substitution at Glu 3 , 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 ti-eatment 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.
  • 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.
  • an effective peptide analogue of the biologically active GIP(l-42) which has improved characteristics for treatment of Type 2 diabetes
  • the analogue comprises at least 15 amino acid residues from the N terminus of GIP(l-42) and has at least one amino acid substitution or modification at position 1-3 and not including Tyr 1 glucitol GIP(l-42).
  • the structures of human and porcine GIP(l-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 Tyr 1 glucitol GIP(l-42) having fatty acid addition at an epsilon amino group of at least one lysine residue.
  • Analogues of GIP(l-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.
  • GIP(l-42)Gly 2 GIP(l-42)Ser 2 , GIP( 1-42) Abu 2 , GIP(l-42)Aib 2 , GIP(l-42)D-Ala 2 , GIP(l-42)Sar 2 , and GIP(l-42)Pro 3 .
  • Amino-terminally modified GIP analogues include N-glycated GIP(l-42), N- alkylated GIP(l-42), N-acetylated GIP(l-42), N-acetyl-GIP(l-42) and N-isopropyl GIP(l-42).
  • DPP IV dipeptidyl-peptidase IV
  • the invention provides a peptide which is more potent than human or porcine GIP in moderating blood glucose excursions, said peptide consisting of GIP(l-42) or N-terminal fragments of GIP(l-42) consisting of up to between 15 to 30 amino acid residues from the N-terminus (i.e., GIP(1-15) GIP(l-3)) with one or more modifications selected from the group consisting of: (a) substitution of Ala 2 by Gly; (b) substitution of Ala 2 by Ser; (c) substitution of Ala 2 by Abu; (d) substitution of Ala 2 by Aib; (e) substitution of Ala 2 by D-Ala; (f) substitution of Ala 2 by Sar (sarcosine); (g) substitution of Glu 3 by Pro; (h) modification of Tyr 1 by acetylation; (i) modification of Tyr 1 by acylation; (j ) modification of Tyr 1 by alkylation; (k) modification of Tyr 1 by glycation; (1) conversion
  • the invention also provides the use of Tyr 1 -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-B ⁇ G), N-methylisoleucine (N-Melle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO), substitution of
  • 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 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.
  • the agent is glucose, acetic anhydride or pyroglutamic acid.
  • Fig. 1 a illustrates degradation of GIP by DPP IV.
  • Fig. lb illustrates degradation of GIP and Tyr 1 glucitol GIP by DPP IV.
  • Fig. 2a illustrates degradation of GIP human plasma.
  • Fig.2b illustrates degradation of GIP and Tyr 1 glucitol GIP by human plasma.
  • Fig. 3 illustrates electrospray ionization mass specfrometry of GIP, Tyr 1 - 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. 1 a illustrates degradation of GIP by DPP IV.
  • Fig. lb illustrates degradation of GIP and Tyr 1 glucitol GIP by DPP IV.
  • Fig. 2a illustrates degradation of GIP human plasma.
  • Fig.2b illustrates
  • FIG. 6 illustrates DPP-IV degradation of GIP (1-42).
  • Fig. 7 illustrates DPP-IV degradation of GIP (Abu 2 ).
  • Fig. 8 illustrates DPP-IV degradation of GIP (Sar 2 ).
  • Fig. 9 illustrates DPP-IV degradation of GIP (Ser 2 ).
  • 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 (Abu 2 ).
  • Fig. 14 illustrates human plasma degradation of GIP (Sar 2 ).
  • Fig. 15 illustrates human plasma degradation of GIP (Ser 2 ).
  • FIG. 16 illustrates human plasma degradation of glycated GIP.
  • Fig. 17 shows the effects of various concentrations of GIP 1-42 and GIP (Abu 2 ) on insulin release from BRIN-BDl 1 cells incubated at 5.6mM glucose.
  • Fig. 18 shows the effects of various concentrations of GIP 1-42 and GIP (Abu 2 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 19 shows the effects of various concentrations of GIP 1-42 and GIP (Sar 2 ) on insulin release from BRIN-BDl 1 cells incubated at 5.6mM glucose.
  • Fig. 17 shows the effects of various concentrations of GIP 1-42 and GIP (Abu 2 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 19 shows the effects of various concentrations of GIP 1-42 and GIP (Sar 2 ) on insulin release from BRIN-BDl 1 cells incuba
  • FIG. 20 shows the effects of various concentrations of GIP 1-42 and GIP (Sar 2 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 21 shows the effects of various concentrations of GIP 1-42 and GIP (Ser 2 ) on insulin release from BRIN-BDl 1 cells incubated at 5.6mM glucose.
  • Fig. 22 shows the effects of various concentrations of GIP 1-42 and GIP (Ser 2 ) on insulin release from BRIN-BDl 1 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-BDl 1 cells incubated at 5.6mM glucose.
  • Fig. 21 shows the effects of various concentrations of GIP 1-42 and GIP (Ser 2 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 23 shows the effects
  • FIG. 24 shows the effects of various concentrations of GIP 1-42 and N-Acetyl- GIP 1-42 on insulin release from BRIN-BDl 1 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-BDl 1 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-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 27 shows the effects of various concentrations of GIP 1-42 and GIP (Gly 2 ) on insulin release from BRIN-BDl 1 cells incubated at 5.6mM glucose.
  • Fig. 28 shows the effects of various concentrations of GIP 1-42 and GIP (Gly 2 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 29 shows the effects of various concentrations of GIP 1-42 and GIP (Pro 3 ) on insulin release from BRIN-BDl 1 cells incubated at 5.6mM glucose.
  • FIG. 30 shows the effects of various concentrations of CIP 1-42 and GIP (Pro 3 ) on insulin release from BRIN-BDl 1 cells incubated at 16.7mM glucose.
  • Fig. 31a shows the primary sfructure of human gastric inhibitory polypeptide
  • Figs. 32A and 32B are a line graph and a bar graph, respectively, showing the effects of (Pro 3 )GIP on GIP-stimulated cyclic AMP generation and insulin secretion in vitro.
  • Figs. 33 A - 33F are a set of six bar graphs showing the effects of Glu 3 - substituted forms of GIP and GIP(3-42) on GIP-stimulated insulin secretion in vitro.
  • Figs. 34A through 34D are a set of two line graphs (Figs. 34A, 34C) and two bar graphs (Figs.
  • Figs. 34A through 35D are a set of two line graphs (Figs. 35A, 35C) and two bar graphs (Figs. 35B, 35D) showing that acute administration of (Pro 3 )GIP impairs physiological meal-stimulated insulin release and worsens glycemic excursion 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 (Pro 3 )GIP impairs physiological meal-stimulated insulin release and worsens glycemic excursion 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 (Pro 3 )GIP impairs physiological meal-stimulated insulin release and worsens g
  • 36A and 36B are a set of two bar graphs showing that chronic administration of (Pro 3 )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 (Pro 3 )GIP for 11 days decreases glycated HbA ⁇ c , 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.
  • FIG. 39 is a line graph showing that chronic administration of (Pro 3 )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 adminisfration of (Pro 3 )GIP for 11 days in obese diabetic ob/ob mice are reversed 9 days after cessation of treatment. Figs.
  • FIGS. 41 A and 41B are a set of two line graphs showing that chronic administration of (Pro 3 )GIP for 11 days causes glucose intolerance in normal mice with reversal by 9 days after cessation of treatment.
  • Figs. 42 A through 42D are a set of two line graphs (Figs. 42 A, 42C) and two bar graphs (Figs. 42B, 42D) showing the effects of (Pro 3 )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 (Pro 3 )GIP administration on food intake (Fig. 43 A), body weight (Fig. 43B), plasma glucose (Fig. 43C) and insulin (Fig.
  • Figs. 44A through 44D are a set of four line graphs with inset bar graphs showing the effects of daily (Pro 3 )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 (Pro 3 )GIP administration (A; black bars) or saline (D; white bars) on glucose (Figs. 45 A, 45B) and insulin (Figs. 45C, 45D) responses to feeding in ob/ob mice fasted for 18 hours.
  • Figs. 45A, 45C two line graphs
  • FIGS. 46A through 46D are a set of two line graphs (Figs. 46A, 46C) and two bar graphs (Figs. 46B, 46D) showing the effects of daily (Pro 3 )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 (Pro 3 )GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice.
  • Figs. 48A through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs.
  • FIG. 49 is an illustration of how the GIP receptor ("GIP-R") antagonist, (Pro 3 )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.
  • Figs. 50A by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL 16 ) ( ⁇ ) and N-AcGIP(LysPAL 37 ) (•), 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(LysPAL 16 ) (grey bars) and N-AcGIP(LysPAL 37 ) (black bars) in the clonal pancreatic beta cell line, BRIN-BD 11.
  • Figs. 51A through 51D are a set of two line graphs (Figs.
  • Figs. 51A, 51C and two bar graphs (Figs. 51B, 51D) showing glucose lowering effects (Figs. 51A, 51B) and insulin-releasing activity (Figs. 51C, 5 ID) 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
  • Figs. 53 A through 53E are a set of graphs showing the effects of daily JV- AcGIP(LysPAL 37 ) (•; black bars) administration on food intake (Fig. 53A), body weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated hemoglobin N-AcGIP(LysPAL 37 ) (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.
  • Figs. 54B, 54D showing the effects of daily N-AcGIP(LysPAL 37 ) 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 JV-AcGIP(LysPAL 37 ) 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.
  • Figs. 56A, 56B and insulin releasing (Figs. 56C, 56D) activity of N-AcGIP(LysPAL 37 ) 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.
  • N-AcGIP(LysPAL 37 ) effects of N-AcGIP(LysPAL 37 ) after 14 daily injections of either N-AcGIP(LysPAL 37 ) (12.5 nmoles/kg/day; •; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; ⁇ ; white bars).
  • 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(l-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, gly cation, conversion of a bond between two amino acids, such as to a psi-[CH 2 NH] 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.
  • 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 Tyr 1 and linkage of a C-16 palmitate group to the epsilon amino group of a lysine residue (especially Lys 16 or Lys 37 ), or substitution of Glu 3 , especially by proline.
  • the modification can also be the linking of, e.g., a C-8, a C- 10, a C- 12, a C- 14, a C- 18 or a C-20 palmitate group to the epsilon amino group of a lysine residue.
  • GIP receptor antagonism using Glu 3 -substituted forms of GIP, such as (Pro 3 )GIP, improve obesity- related insulin resistance and associated glucose intolerance. This has uncovered an unexpected approach to the therapy of obesity, insulin resistance and type 2 diabetes.
  • 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.
  • 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.
  • 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.
  • AUC insulin area under the curve
  • Detailed kinetic analysis is difficult due to necessary limitation of sampling times, but the greater insulin concentrations following Tyr 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 Tyrl-glucitol GIP were consistently lower than after GIP.
  • 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.
  • 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 aminoterminal Tyr 1 residue limits GIP catabolism through impairment of the proteolytic actions of serum peptidases and thus prolongs its half-life in vivo.
  • N-terminal acetylated GIP exhibited a similar pattern and the GIP(Ser 2 ) analogue also evoked a strong response. From these tests, GIP(Gly 2 ) and GIP(Pro 3 ) appeared to the least potent analogues in terms of insulin release. Other stable analogues tested, namely GIP(Abu 2 ) and GIP(Sar 2 ), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed. Thus, very low concenfrations 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.
  • DPP-IV dipeptidylpeptidase-IV
  • Diabetologia 45:1111-1119 may reflect a generalized secretory dysfunction rather than a specific cellular defect (Meier, J.J. et al, 2003, Metabolism 52:1579-1585). Indeed, the insulin secretory response to all secretagogues, including GLP-1 is compromised in type 2 diabetes (Kjems, L.L. et al, 2003, Diabetes 52:380-386; Flatt, P.R. et al, "Defective insulin secretion in diabetes and insulinoma," in Nutrient regulation of insulin secretion, Flatt P.R., ed. London, Portland Press, 1992, p. 341- 386).
  • GLP-1 and GIP for diabetes therapy is reliant on peptide engineering to provide analogs of incretin hormones with improved potency due to DPP IV resistance, decreased renal clearance and/or enhanced GIP receptor and post-receptor activity (Gault, V.A. et al. , 2003, Biochem Biophys Res Commun 308:207-213).
  • GIP GIP receptor and post-receptor activity
  • GIP-R GIP receptor
  • Example 5 daily injections of the stable and specific GIP- R antagonist, (Pro 3 )GIP can be used to chemically ablate the GIP-R and evaluate the role of endogenous circulating GIP in obesity-diabetes as manifested in ob/ob mice.
  • the analogue (Pro )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).
  • (Pro 3 )GIP acutely, the results disclosd herein highlight the involvement of GIP in the plasma insulin response to feeding and the enteroinsular axis of ob/ob mice (Gault, V.A. et al, 2003, Diabetologia 46:222-230).
  • ob/ob mice treated with daily (Pro 3 )GIP for 11 days exhibited a marked improvement in diabetic status. This included decreased fasting and basal hyperglycemia, lowered glycated hemoglobin, improved glucose tolerance and a significantly diminished glycemic excursion following feeding. Notably, basal and glucose-stimulated plasma insulin concenfrations were decreased, suggesting that insulin sensitivity must have improved significantly following (Pro 3 )GIP in order to restrain the hyperglycemia. Indeed, insulin sensitivity tests conducted after 11 days of (Pro 3 )GIP administration revealed a 57% increase in the glucose-lowering action of exogenous insulin.
  • Substantial enteroendocrine stimulation results in K-cell hype ⁇ lasia and markedly elevated concentrations of intestinal and circulating GIP (Flatt, P.R. et al, 1983, Diabetes 32:433-435; Flatt, P.R. et al, 1984, J. Endocrinol. 101:249-256; Bailey, C.J. et al, 1986, Acta Endocrinol. (Copenh) 112:224-229).
  • This promotes islet hypertrophy and beta cell hyperplasia (Bailey, C.J., et al, 1982, Int. J. Obes.
  • Example 6 fatty acid derivatisation was used to develop two novel long- acting, N-terminally modified GIP analogues (N-AcGIP(LysPAL l ⁇ ) and N- AcGIP(LysPAL 37 )).
  • DPP IV dipeptidylpeptidase IV
  • Cyclic AMP production was assessed using GIP receptor transfected CHL fibroblasts.
  • In vitro insulin release was assessed in BRIN-BDl 1 cells. Insulinotropic and glycaemic responses to acute and long-term peptide administration were evaluated in obese diabetic (ob/ob) mice. 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.
  • Adminisfration of N- AcGIP(LysPAL 16 ) or -V-AcGIP(LysPAL 37 ) together with glucose in ob/ob mice significantly reduced the glycaemic excursion and improved the insulinotropic response compared to GIP.
  • Dose-response studies with N-AcGIP(LysPAL 37 ) revealed highly significant decreases in the overall glycaemic excursion and increases in circulating insulin even with 6.25 nmoles/kg.
  • these analogues display enhanced and protracted antihyperglycaemic and insulin-releasing activity when administered acutely to animals with obesity-diabetes (Hinke, S.A. et al, 2002, Diabetes 51 : 656-661; Gault, V.A. et al, 2002, Biochem. J. 367: 913-920; Gault, V.A. et al, 2003, J. Endocrinol. 176: 133-141 ; O'Harte, F.P.M. et al, 1999, Diabetes 48: 758-765; O'Harte, F.P.M. et al, 2002, Diabetologia 45: 1281-1291).
  • N-AcGIP has emerged as the most effective DPP IV-resistant analogue, substantially augmenting the plasma insulin response and curtailing the glycaemic excursion following conjoint administration with glucose to ob/ob mice (O'Harte, F.P.M. et al, 2002, Diabetologia 45: 1281- 1291).
  • Example 6 was designed to evaluate the metabolic stability, biological activity and antidiabetic potential of novel second generation fatty acid derivatised, N- terminally modified N-AcGIP analogues, N-AcGIP(LysPAL 16 ) andN- AcGIP(LysPAL 37 ).
  • Both GIP analogues contain a C-16 palmitate group linked to the epsilon-amino group of Lys at positions 16 or 37, in combination with an N-terminal (Tyr 1 ) acetyl group (O'Harte, F.P.M. et al, 2002, Diabetologia 45: 1281-1291).
  • Tyr 1 N-terminal acetyl group
  • N-AcGIP(LysPAL 37 ) The most effective analogue, N-AcGIP(LysPAL 37 ) was administered to ob/ob mice by once daily intraperitoneal injection for 14 days prior to evaluation of glucose homeostasis, pancreatic beta cell function and insulin sensitivity. Possible desensitization of GIP receptor action by prolonged exposure to elevated concentrations of N-AcGIP(LysPAL 37 ) was also examined. The results indicate the particular promise of the novel second generation N-terminally acetylated GIP analogue, N-AcGIP(LysPAL 37 ), as a potential therapeutic agent for the treatment of type 2 diabetes.
  • 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
  • the ob/ob syndrome is an extensively studied model of spontaneous obesity and diabetes, exhibiting hype ⁇ hagia, marked obesity, moderate hyperglycaemia and severe hyperinsulinemia (Bailey, C.J. et al, 1982, Int. J. Obesity 6: 11-21).
  • 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.
  • N-AcGIP(LysPAL 37 ) appeared to be the best fatty acid derivatised analogue displaying a more protracted, significantly enhanced insulin-releasing potency over N- AcGIP(LysPAL 1 ) in vivo.
  • Reasons for the increased potency of N- AcGIP(LysPAL 37 ) remain unclear, but one explanation is an extended half-life.
  • 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. Ada 1547: 143-55; Manhart, S. et al, 2003, Biochemistry 42: 3081-3088).
  • similarities between the in vitro biological activities of the two palmitate substituted analogues make this less likely.
  • N-AcGIP(LysPAL 37 ) was the more potent of the two analogues in vivo, it was further utilised in dose-response studies.
  • native GIP itself has only very modest effects in ob/ob mice, as sometimes observed with type 2 diabetic subjects ( ⁇ auck, M.A. et al, 1993, J. Clin. Invest. 91 : 301-307; Meier, J.J. et al, 2004, Diabetes 53: 220-224; Vilsb ⁇ ll, T.
  • N-AcGIP(LysPAL 37 ) even at the lowest dose of 6.25 nmoles/kg, exhibited significant glucose-lowering and insulinotropic activity when administered with glucose.
  • N-AcGIP(LysPAL 37 ) is subject to albumin binding, the fact that it is still highly biologically active even at lower concenfrations indicates striking potency.
  • N-AcGIP(LysPAL 37 ) Daily adminisfration of N-AcGIP(LysPAL 37 ) to young adult ob/ob mice by intraperitoneal injection (12.5 nmoles/kg) resulted in a progressive lowering of plasma glucose concentrations and a significant decrease of glycated haemoglobin by 14 days. This was associated with a substantial improvement of glucose tolerance. Importantly food intake and body weight were unchanged ruling out the possibility that improvement of glucose homeostasis was merely the consequence of body weight loss. These observations also indicate that N-AcGIP(LysPAL 37 ) did not exert any untoward toxic actions affecting feeding over the study period. This is in harmony with recent studies showing that GIP does not inhibit gasfric emptying (Meier, J.J.
  • 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 freatment 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.
  • a pharmaceutical composition may also contain (in addition to the analogue and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • 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.
  • Adminisfration 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.
  • 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.
  • 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.
  • the composition of the present invention 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.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
  • 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.
  • 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.
  • 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.
  • 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 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 gly colic acid), polylactide co-glycolide (co-polymers of lactic acid and gly colic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of gly colic acid), polylactide co-glycolide (co-polymers of
  • 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.
  • 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.
  • 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, frimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides
  • organic bases such as isopropylamine, frimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.
  • the salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable organic acid.
  • Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptonoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxymethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate
  • the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
  • long chain halides such as dec
  • 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.
  • compositions that contains active ingredients dissolved or dispersed therein are well understood in the art and need not be limited based on formulation.
  • 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.
  • 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.
  • 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.
  • 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.
  • peptide analogue may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. 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.
  • 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 (Ala 2 ) on an automated peptide synthesizer (Applied Biosystems, California, USA). The synthesis follows standard Fmoc peptide chemistry protocols.
  • N-terminal amino acid of native GIP 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.
  • the now structurally modified Tyr is cleaved from the Wang resin (95% TFA, and 5% of the appropriate scavengers.
  • N.B. Tyr is considered to be a problematic amino acid and may need special consideration) and the required amount of N-terminally modified-Tyr consequently added directly to the automated peptide synthesiser, which will carry on the synthesis, thereby stitching the N-terminally modified-Tyr to the a- amino of GIP (Ala 2 ), completing the synthesis of the GIP analogue.
  • This peptide is cleaved off the Wang resin (as above) and then worked up using the standard Buchner filtering, precipation, rotary evaporation and drying techniques.
  • Example 2 Preparation of Tyr ⁇ Glucitol GIP and Its Properties in vivo.
  • the following example investigates preparation of Ty ⁇ glucitol GIP together with evaluation of its antihyperglycemic and insulin-releasing properties in vivo. The results clearly demonstrate that this novel GIP analogue exhibits a substantial resistance to aminopeptidase degradation and increased glucose lowering activity compared with the native GIP.
  • 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- Q, Water Purification System (Millipore Corporation, Milford, Massachusetts, USA).
  • 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 ⁇ 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 x 2.0mm, Phenomenex, Ltd., Macclesfield, UK).
  • 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 -fc SEM and values were compared using the Student's unpaired t-test. Groups of data were considered to be significantly different if O.05.
  • Fig. 1 illustrates the typical peak profiles obtained from the HPLC separation of the products obtained from the incubation of GIP (Fig 1 a) or Tyr'-glucitol GIP (Fig 1 b) with DPP IV for 0, 2, 4 and 12 hours.
  • 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 ⁇ 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.
  • FIG. 2 shows a set of typical HPLC profiles of the products obtained from the incubation of GIP or Tyr 1 - glucitol GIP with human plasma for 0 and 4 hours.
  • GIP (Fig 2a) with a retention time of 22.06 minutes was readily metabolised by plasma within 4 hours incubation giving rise to the appearance of a major degradation peak with a retention time of 21.74 minutes.
  • the incubation of Tyr ⁇ glucitol GIP under similar conditions 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.
  • 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 1 OO ⁇ l of water and applied to the LC/MS equipped with a microbore C-18 HPLC column. Samples (30 ⁇ l direct loop injection) were applied at a flow rate of 0.2ml/min, under isocratic conditions 35% acetonifrile/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.
  • 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 1583.8 and 1188.1, corresponding to intact M 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).
  • FIGs. 4 and 5 show the effects of intraperitoneal (ip) glucose alone (18mmol/kg) (control group), and glucose in combination with GIP or Tyr' " glucitol GIP (lOnmol/kg) on plasma glucose and insulin concentrations.
  • Fig. 4A shows plasma glucose concenfrations after i.p. glucose alone
  • Fig. 4B shows plasma glucose AUC values for 0-60 minutes post injection. Values are mean -fc SEM for six rats. **P ⁇ 0.01, ***P ⁇ 0.001 compared with GIP and Tyr 1" glucitol GIP; t- ⁇ 0.05, f fPO.Ol 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 (lOnmol/kq).
  • 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 -fc SEM for six rats. *- ⁇ 0.05, **P ⁇ 0.001 compared with GIP and Tyr' glucitol GIP; f ⁇ 0.05, f f-? ⁇ 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 Tyr ⁇ glucitol GIP (Figs 4A, B).
  • 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.
  • High-performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK). Dipeptidyl peptidase IV was purchased from Sigma (Poole, Dorset, UK), and Diprotin A was purchased from Calbiochem Novabiochem (Beeston, Nottingham, UK). RPMI 1640 tissue culture medium, foetal calf serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). All water used in these experiments was purified using a Milli-Q, Water Purification System (Millipore, Milford, Massachusetts, USA). All other chemicals used were of the highest purity available.
  • GIP(Sar 2 ), GIP(Ser 2 ), GIP(Gly 2 ) and GIP(Pro 3 ) 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.
  • BRIN-BDl 1 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% C0 2 and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
  • 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 10 5 cells per well, and allowed to attach overnight at 37°C.
  • FIGs. 6-11 illustrate the typical peak profiles obtained from the HPLC separation of the reaction products obtained from the incubation of GIP, GIP(Abu 2 ), GIP(Sar 2 ), GIP(Ser 2 ), glycated GIP and acetylated GIP with DPP IV, for 0, 2, 4, 8 and 24 hours.
  • the results summarized in Table 1 indicate that glycated GIP, acetylated GIP, GIP(Ser 2 ) are GIP(Abu 2 ) more resistant than native GIP to in vitro degradation with DPP IV. From these data GIP(Sar 2 ) appears to be less resistant.
  • 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.
  • FIGs. 12-16 show a representative set of HPLC profiles obtained from the incubation of GIP and GIP analogues with human plasma for 0, 2, 4, 8 and 24 hours. Observations were also made after incubation for 24 hours in the presence of DPA. These results are summarized in Table 2 are broadly comparable with DPP IV incubations, but conditions which more closely mirror in vivo conditions are less enzymatically severe. GIP was rapidly degraded by plasma. In comparison, all analogues tested exhibited resistance to plasma degradation, including GIP(Sar 2 ) which from DPP IV data appeared least resistant of the peptides tested.
  • DPA substantially inhibited degradation of GIP and all analogues tested with complete abolition of degradation in the cases of GIP(Abu 2 ), GIP(Ser 2 ) and glycated GIP. This indicates that DPP IV is a key factor in the in vivo degradation of GIP.
  • 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.
  • Figs. 17-30 show the effects of a range of concenfrations of GIP, GIP(Abu 2 ), G ⁇ P(Sar), GIP(Ser 2 ), acetylated GIP, glycated GIP, GIP(Gly 2 ) and GIP(Pro 3 ) on insulin secretion from BRIN-BDl 1 cells at 5.6 and 16.7 mM glucose.
  • Native GIP provoked a prominent and dose-related stimulation of insulin secretion.
  • the glycated GIP analogue exhibited a considerably greater insulinotropic response compared with native peptide.
  • GIP(Gly 2 ) and GIP(Pro 3 ) appeared to be the least potent analogues in terms of insulin release.
  • Other stable analogues tested namely GIP(Abu 2 ) and GIP(Sar 2 ), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed.
  • very low concenfrations were extremely potent under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogues may prove therapeutically useful in the treatment of type 2 diabetes where insulinotropic capacity combined with in vivo degradation dictates peptide potency.
  • Example 4 Glu 3 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 Glu 3 -substituted form of GIP, namely, (Pro 3 )GIP.
  • the molecular masses of the purified peptide analogues were determined using Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectrometry. Samples were dissolved in 10 ⁇ l H 2 0 (approximately 40 pmol/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 -cyano-4-hydroxycinnamic acid) in acetonitrile/ethanol (1/1) and allowed to dry at room temperature. The molecular masses were then recorded as mass-to-charge (m/z) ratio versus relative peak intensity and compared using theoretical values on a Voyager-DE BioSpectrometry Workstation (PerSeptive Biosystems, Framingham, MA, USA).
  • MALDI-TOF Matrix Assisted Laser Desorption Ionisation-Time of Flight
  • CHL Chinese hamster lung
  • fibroblast cells stably transfected with the human GIP receptor were cultured in DMEM tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin).
  • BRIN-BDl 1 cells were cultured using RPMI- 1640 tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin).
  • Cells were maintained in sterile tissue culture flasks (Corning Glass Works, Sunderland, UK) at 37°C in an atmosphere of 5% C0 2 and 95%) air using an LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
  • Insulin release from BRIN-BDl 1 cells was determined using cell monolayers (McClenaghan, N.H. et al, 1996, Diabetes
  • 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 10 5 cells per well, and allowed to attach overnight at 37°C.
  • GIP receptor transfected CHL cells were seeded into 12- well plates (Nunc, Roskilde, Denmark) at a density of 1.0 x 10 5 cells per well. The cells were then allowed to grow for 48 hours before being loaded with tritiated adenine (2 ⁇ 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 HBS buffer (130 mM NaCl, 20 mM HEPES, 0.9 mM NaHP0 4 , 0.8 mM MgS0 4 , 5.4 mM KCl, 1.8 mM CaCl 2 , 25 mM glucose, 25 ⁇ M phenol red, pH 7.4).
  • HBS buffer 130 mM NaCl, 20 mM HEPES, 0.9 mM NaHP0 4 , 0.8 mM MgS0 4 , 5.4 mM KCl, 1.8 mM CaCl 2 , 25 mM glucose, 25 ⁇ 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 (Pro 3 )GIP in the absence (control) or presence of native GIP (10 "7 M).
  • TCA trichloroacetic acid
  • Plasma glucose and insulin responses were evaluated using 8- to 12-week old obese diabetic ob/ob mice following intraperitoneal (i.p.) injection of native GIP, (Pro 3 )GIP (25 nmol/kg body weight) or saline (0.9% (w/v) NaCl; control) immediately following the combined injection of GIP (25 nmol/kg body weight) with glucose (18 mmol/kg body weight). All test solutions were administered in a final volume of 8ml/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.
  • Plasma glucose and insulin responses were evaluated using 8- to 12-week old ob/ob mice where food was withdrawn for an 18-hour period prior to intraperitoneal injection of saline (0.9% (w/v) NaCl; control) or (Pro 3 )GIP (25 nmol/kg body weight). Following injection, the mice were allowed to re-feed for 15 minutes. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30, 60 and 120 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20° prior to glucose and insulin determinations.
  • pancreatic tissue was excised from non- fasted ob/ob mice after 11 days treatment with either saline (0.9% w/v NaCl) or (Pro 3 )GIP (25 nmol/kg body weight/day). Pancreatic samples were individually wrapped in aluminium foil and snap frozen in liquid nitrogen. Individual excised pancreatic samples were then either embedded, sectioned and immunohistochemically stained for insulin or permeabilised for determination of pancreatic insulin content.
  • HbAi c plasma glucose and insulin concentrations.
  • HbAj- 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
  • 32B is a bar graph showing insulin secretion (y-axis) with increasing peptide concentration (M) (x-axis) for 5.6 mM glucose (control) (white bar), GIP (gray bars), (Pro 3 )GIP (lined bars) and (Pro 3 )GIP+GIP( 10 "7 M) (black bars).
  • (Pro 3 )GIP inhibited GIP-induced cAMP formation with an IC 5 o value of 2.6 ⁇ M.
  • FIG. 33A shows 3 H-cAMP production as a percent of 10 "7 M GIP (y-axis) versus logio of GIP (10 "7 M) (white bar, control) and GIP (10 "7 M)+GIP(3-42) (black bars).
  • Figs. 33B through 33F show insulin secretion (in ng/10 6 cells/20 minutes) (y-axis) as a function of peptide concentration (M) (x-axis) for GIP (10 "7 M) (white bar, control) and a Glu 3 -substituted form of GIP (black bars), including (Hyp 3 )GIP (Fig. 33B), (Lys 3 )GIP (Fig. 33C), (Tyr 3 )GIP (Fig.
  • Figs. 34A through 34D are a set of two line graphs (Figs. 34A, 34C) and two bar graphs (Figs. 34B, 34D) showing that acute administration of (Pro 3 )GIP completely antagonises the actions of GIP on glucose tolerance (Figs. 34A, 34B) and plasma insulin (Figs. 34C, 34D) responses in obese diabetic ob/ob mice.
  • Figs. 34A and 34C are line graphs show plasma glucose levels (Fig.
  • Figs. 34A, y-axis) and plasma insulin levels (Fig. 34C, y-axis) over time (x-axis) for glucose (control; ⁇ ), glucose + GIP ( ⁇ ) and glucose + (GIP+Pro 3 GIP)) ( ⁇ ).
  • Figs. 34B and 34D are bar graphs showing plasmia glucose AUC for glucose alone (white bars), GIP (grey bars) and glucose + (GIP+Pro 3 GIP)) (black bars). Plasma glucose and insulin concentrations after i.p. adminisfration of glucose alone (18 mmol/kg body weight) or in combination with either native GIP or native GIP plus (Pro 3 )GIP (25 nmol/kg body weight). The time of injection is indicated by the arrow (0 minutes).
  • Plasma glucose and insulin AUC values are given for 0-60 minutes post-injection. Values are means ⁇ SEM for 8 mice. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 compared with glucose alone. ⁇ P ⁇ 0.01, ⁇ P ⁇ 0.001 compared with native GIP.
  • Acute administration of (Pro 3 )GIP completely antagonised the insulin- releasing action of GIP and the associated improvement of glucose tolerance in ob/ob mice. Indeed, the glycemic excursion following (Pro 3 )GIP ( ⁇ ) was worse than when glucose was administered alone ( ⁇ ).
  • Figs. 35A through 35D show the effects of (Pro 3 )GIP on physiological meal- stimulated insulin release and glycemic excursion in obese diabetic ob/ob mice.
  • Plasma glucose and insulin concenfrations were measured in mice allowed to re-feed for 15 minutes prior to i.p. administration of saline (0.9% (w/v) NaCl) as control or (Pro 3 )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 ( ⁇ ) and (Pro 3 )GIP (0), and plasma insulin AUC (Fig.
  • HbA ⁇ c pancreatic insulin content and average islet diameter were measured after 11 daily subcutaneous injections of either saline alone (white bars) or (Pro 3 )GIP (25 nmol/kg body weight; black bars) to obese diabetic ob/ob mice. Values are means ⁇ SEM for 6 mice and *P ⁇ 0.05, ***P ⁇ 0.001 compared with saline-treated group.
  • FIG. 38 shows the effects of chronic administration of (Pro 3 )GIP for 11 days on insulin sensitivity in obese diabetic ob/ob mice.
  • FIG. 41 A and 4 IB are a pair of line graphs showing the effects of chronic administration of (Pro 3 )GIP for 11 days on glucose tolerance in normal mice.
  • chronic daily freatment of normal mice with (Pro 3 )GIP ( ⁇ ) for 1 1 days resulted in a marked deterioration of glucose tolerance (Fig. 41 A) relative to controls ( ⁇ ), which was reversed 9 days after cessation of treatment (Fig. 4 IB).
  • Example 5 Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action By Daily (Pro 3 )GIP Administration Improves Glucose Tolerance and Ameliorates Insulin Resistance and Abnormalities of Islet Structure in Obesity-Diabetes.
  • Gastric inhibitory polypeptide GIP
  • GIP-R GIP receptor
  • Non-fasting plasma glucose levels and the overall glycemic excursion (AUC) to a glucose load were significantly reduced (1.6-fold; P ⁇ 0.05) in (Pro 3 )GIP-treated mice compared to controls.
  • GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P ⁇ 0.05) and insulin (1.5-fold; P ⁇ 0.05) responses to feeding.
  • These changes were associated with significantly enhanced (1.6-fold; P ⁇ 0.05) insulin sensitivity in the (Pro 3 )GIP-treated group.
  • Daily injection of (Pro 3 )GIP reduced pancreatic insulin content (1.3-fold; P ⁇ 0.05) and partially corrected the obesity-related islet hypertrophy and beta cell hype ⁇ lasia of ob/ob mice.
  • Obese diabetic mice derived from the colony maintained at Aston University, UK (Bailey, C.J., et al, 1982, Int. J. Obes. 6:11-21) were used at 12-16 weeks of age. Animals were age-matched, divided into groups and housed individually in an air-conditioned room at 22 ⁇ 2°C with a 12 hour light: 12 hour dark cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed following administration of (Pro 3 )GIP.
  • (Pro 3 )GIP was sequentially synthesized on an Applied Biosystems automated peptide synthesizer (Model 432 A).
  • (Pro 3 )GIP was purified by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterized using electrospray ionization mass spectrometry (ESI-MS).
  • mice received once daily i.p. injections (17:00 hours) of either saline vehicle (0.9% (w/v), NaCl) or (Pro 3 )GIP (25 nmol/kg body wt).
  • saline vehicle (0.9% (w/v), NaCl
  • Pro 3 )GIP 25 nmol/kg body wt.
  • Food intake and body weight were recorded daily whilst plasma glucose and insulin concentrations were monitored at intervals of 2-6 days.
  • Whole blood for the measurement of glycated hemoglobin was taken on days 11 and 20.
  • Intraperitoneal glucose tolerance (18 mmol/kg body wt), metabolic response to native GIP (25 nmol/kg body wt) and insulin sensitivity (50 U/kg body wt) tests were performed on days 11 and 20. Mice fasted for 18 hours were used to examine the metabolic response to 15 minutes feeding. In a separate series, pancreatic tissues were excised at the end of the 11-day treatment period or 9 days following discontinuation of (Pro 3 )GIP and processed for immunohistochemistry or measurement of insulin following extraction with 5 ml/g of ice-cold acid ethanol (750 ml ethanol, 235 ml water, 15 ml concentrated HCl).
  • Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Ada 32:199-201) using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma and pancreatic insulin were assayed by a modified dexfran-coated charcoal radioimmunoassay (Flatt, P.R. et al, 1981, Diabetologia 20:573-577).
  • Glycated hemoglobin was determined using cation-exchange columns (Sigma, Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash and eluting buffer using a VersaMax Microplate Spectrophotometer (Molecular Devices, Wokingham, Berkshire, UK).
  • Tissue fixed in 4% paraformaldehyde/PBS and embedded in paraffin was sectioned at 8 ⁇ m. After de-waxing, sections were incubated with blocking serum (Vector Laboratories, CA, USA) prior to exposure to insulin antibody. Tissue samples were then incubated consecutively with secondary biotinylated universal, pan-specific antibody (Vector Laboratories, CA, USA) and streptavidin/peroxidase preformed complex (Vector Laboratories, CA, USA).
  • pancreatic tissue was counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and then plunged into acid methanol (500 ml methanol, 500 ml H 2 0 and 2.5 ml concentrated HCl) prior to dehydration and mounting in Depex (BDH Chemicals, Dorset, UK).
  • acid methanol 500 ml methanol, 500 ml H 2 0 and 2.5 ml concentrated HCl
  • Depex BDH Chemicals, Dorset, UK
  • the stained slides were viewed under a microscope (Nikon Eclipse E2000, Diagnostic Instruments Incorporated, Michigan, USA) attached to a JVC camera Model KY-F55B (JVC, London, UK) and analyzed using Kromoscan imaging software (Kinetic Imaging Limited, Faversham, Kent, UK).
  • the average number and diameter of every islet in each section was estimated in a blinded manner using an eyepiece graticule calibrated with a stage micrometer (Graticules Limited, Tonbridge, Kent, UK). The longest and shortest diameters of each islet were determined with the graticule. Half of the sum of these two values was then considered to be the average islet diameter. Approximately 60- 70 random sections were examined from the pancreas of each mouse.
  • FIG. 43A through 43D are a set of two line graphs and two bar graphs showing the effects of daily (Pro 3 )GIP administration on food intake (Fig. 43 A), body weight (Fig. 43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in ob/ob mice.
  • Parameters were measured for 5 days prior to, 11 days during (indicated by black bar) and 9 days after treatment with saline or (Pro 3 )GIP (25 nmol/kg bw/day). Values are mean ⁇ SEM for eight mice. *P ⁇ 0.05 compared with saline group.
  • Administration of (Pro 3 )GIP had no effect on food intake and body weight (Fig. 43 A and 43B).
  • FIG. 44D are a set of four line graphs with inset bar graphs showing the effects of daily (Pro 3 )GIP administration on glucose tolerance and plasma insulin response to glucose in ob/ob mice.
  • Tests were conducted after daily treatment with (Pro 3 )GIP (25 nmoles/kg body weight/day; A ; black bars) or saline (control; o; white bars) for 11 days (Fig. 44A, 44C) or 9 days after cessation of freatment (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.
  • Plasma insulin concentrations were also significantly (P ⁇ 0.05) reduced 15, 30 and 60 minutes following intraperitoneal glucose injection in the (Pro 3 )GIP treated group (Fig. 44A). AUC, 0-60 minutes values were also significantly decreased (P ⁇ 0.001). Interestingly, an almost identical pattern was observed when 11 day treated ob/ob mice were administered glucose together with native GIP (25 nmoles/kg body weight) (data not shown). This supports the view that GIP action was effectively antagonized in the (Pro )GIP treated group. Discontinuation of (Pro 3 )GIP treatment for 9 days (day 20 of study) resulted in almost identical plasma glucose and insulin responses to intraperitoneal glucose (Fig.
  • Figs. 45A through 45D are a set of two line graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the effects of daily (Pro 3 )GIP adminisfration (A ; black bars) or saline ( ⁇ ; 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 (Pro 3 )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 (Pro 3 )GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice. Parameters were measured after daily treatment with (Pro 3 )GIP (25 nmol/kg body weight/day; black bars) or saline (white bars) for 11 days and 9 days after cessation of treatment (day 20). Values are mean ⁇ SEM for eight mice. *P ⁇ 0.05 and ***P ⁇ 0.001 compared with saline group. Figs.
  • FIG. 48A through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro 3 )GIP administration on islet size and mo ⁇ hology in ob/ob )mice.
  • (Pro 3 )GIP freatment had no effect on pancreatic weight (Fig. 47 A).
  • pancreatic insulin content was significantly (P ⁇ 0.05) decreased in ob/ob mice receiving (Pro 3 )GIP for 11 days compared to confrols (Fig. 47B). No significant differences were observed in islet number per pancreatic section (Fig.
  • Figure 48D presents similar analysis following cessation of freatment, 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 (Pro 3 )GIP treated ob/ob mice (Fig. 48B) and saline treated controls (Fig. 48C) illustrate the dramatic changes in pancreatic islet mo ⁇ hology induced by (Pro 3 )GIP treatment.
  • Pancreata immunohistologically stained for insulin on day 20 are also shown (Fig. 48E, 48F). Parameters were measured after daily freatment with (Pro 3 )GIP (25 nmol/kg body weight/day) or saline for 11 days (Fig.
  • FIG. 48A Proportion of islets classified as large (> 0.15 mm) diameter, medium (0.1 - 0.15 mm) diameter and small ( ⁇ 0.1 mm) diameter are shown. Values are mean ⁇ SEM for eight mice Figs. 48B, 48C, 48E and 48F are representative images (x 40 magnification) of pancreata stained for insulin following 11 days freatment with (Pro 3 )GIP (Fig. 48B) or saline (Fig. 48C). Corresponding images 9 days after cessation of treatment with (Pro 3 )GIP (Fig. 48E) or saline (Fig. 48F) are also shown. The arrows indicate islets.
  • Example 6 N-Terminally Acetylated and Ly l ⁇ and Lys 37 -substituted GIP This example examines the metabolic stability, biological activity and antidiabetic potential of fatty acid derivatized N-terminally modified GIP analogues. These are N-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ), which have an ⁇ -terminal Tyr 1 acetyl group, and a C-16 palmitate group linked to the epsilon-amino group of the lysine at either position 16 or position 37 of the GIP protein.
  • Obese diabetic mice derived from the colony maintained at Aston University, UK were used at 12-17 weeks of age. The genetic background and characteristics of the colony used have been outlined in detail elsewhere (Bailey, C.J. et al, 19S2, Int. J. Obesity 6:11-21 ; Gault, NA. 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.
  • High performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, UK).
  • Trifluoroacetic acid (TFA) and trichloroacetic acid (TCA) were obtained from Aldrich (Poole, Dorset, UK).
  • DPP IV, isobutylmethylxanthine (IBMX), alpha-cyano-4-hydroxycinnamic acid, cyclic AMP and ATP were all purchased from Sigma (Poole, Dorset, UK).
  • Fmoc-protected amino acids were from Calbiochem Novabiochem (Nottingham, UK).
  • RPMI- 1640 and DMEM tissue culture medium, foetal bovine serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK).
  • the chromatography columns used for cyclic AMP assay, Dowex AG50 WX and neutral alumina AG7 were obtained from Bio-Rad (Life Science Research, Alpha Analytical, Larne, UK). All water used in these experiments was purified using a Milli-Q Water Purification System (Millipore, Milford, MA, USA). All other chemicals used were of the highest purity available.
  • N-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ) were synthesised in the same way as native GIP but with the exception that the epsilon-amino groups of Lys at positions 16 or 37 were conjugated with a C-16 palmitate fatty acid.
  • GIP and fatty acid derivatised GIP analogues were incubated at 37°C with purified porcine dipeptidylpeptidase IV (5 mU in 50 mmol/1 friethanolamine-HCl; pH 7.8) for 0, 2, 4, 8 and 24 hours (final peptide concentration 2 mmol/1). The reactions were subsequently terminated by addition of 10% (v/v)
  • TF A/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) TF A/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.
  • CHL Chinese hamster lung
  • fibroblasts stably transfected with the human GIP receptor (Gremlich, S. et al, 1995, Diabetes 44: 1202-1208) were cultured in DMEM tissue culture medium containing 10% (v/v) FBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin).
  • DMEM tissue culture medium containing 10% (v/v) FBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin).
  • Clonal pancreatic BRIN- BDl 1 cells (McClenaghan, N.H.
  • Intracellular cyclic AMP production was measured using GIP-receptor transfected CHL fibroblasts (O'Harte, F.P.M. et al, 2002, Diabetologia 45: 1281-1291).
  • CHL cells were seeded into 12-well plates (Nunc, Roskilde, Denmark) at a density of 10 5 cells per well and allowed to grow for 48 hours before being loaded with tritiated adenine (2 ⁇ Ci; 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 mol/1) in HBS buffer in the presence of 1 mmol/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 mmol/1 unlabelled cyclic AMP and 0.1 mmol/1 unlabelled ATP. The intracellular cyclic AMP was then separated on Dowex and alumina exchange resins as described previously (O'Harte, F.P.M.
  • BRIN-BDl 1 cells were seeded into 24-well plates at a density of 10 5 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 mmol/1 glucose.
  • Test incubations were performed in the presence of 5.6 mmol/1 glucose with a range of concentrations (10 '13 to 10 "6 mol/1) of GIP and GIP analogues. After 20 minutes incubation, the buffer was removed from each well and aliquots (200 ⁇ l) used for measurement of insulin.
  • N-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ) Effects ofN-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ) in ob/ob mice.
  • Metabolic and dose-response effects of GIP and N-AcGIP(LysPAL) analogues (at 6.25 - 25 nmoles/kg bw) following glucose adminisfration (18 mmoles/kg bw) were examined in mice fasted for 18 hours.
  • mice received once daily intraperitoneal injections (17:00 h) for 14 days of either saline vehicle (0.9%, w/v, NaCl), native GIP or N-AcGIP(LysPAL 37 ) (both at 12.5 nmoles/kg body weight/day). Food intake and body weight were recorded daily.
  • Plasma glucose and insulin concenfrations were monitored at 2-6 day intervals. At 14 days, groups of animals were used to evaluate intraperitoneal glucose tolerance (18 mmoles/kg) and insulin sensitivity (50 U/kg). In a separate series, two experimental protocols were employed to examine the possibility of GIP receptor desensitization after 14 days treatment. Acute metabolic effects of the usual injection of either saline, GIP or N-AcGIP(LysPAL 37 ) were monitored when administered together with glucose (18 mmoles/kg). In the second, acute effects of N-AcGIP(LysPAL 37 ) given together with glucose were examined in all 3 groups of mice.
  • pancreatic tissues were excised for measurement of insulin following extraction with 5 ml/g ice-cold acid ethanol (75% ethanol, 2.35% H20, 1.5% HCl). Whole blood was taken for determination of glycated hemoglobin.
  • Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Ada 32:199-201) using a Beckman Glucose Analyser II (Beckman, Galway, Ireland). Plasma insulin was determined by dextran-charcoal RIA as described previously (Flatt, P.R. etal, 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).
  • Results are expressed as mean ⁇ SEM. Data were compared using the unpaired Student's t-test. Where appropriate, data were compared using repeated measures A ⁇ OVA or one-way A ⁇ OVA, followed by the Student- ⁇ ewman-Keuls post hoc test. Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer-generated program employing the trapezoidal rule (Burington, R.S., 1973, Handbook of Mathematical Tables and Formulae, McGraw- Hill, New York) with baseline subtraction. Groups of data were considered to be significantly different if ⁇ - ⁇ .05.
  • Peptide samples were mixed with mafrix (alpha-cyano-4-hydroxycinnamic acid) and m z ratio vs. relative peak intensity recorded using a Voyager-DE BioSpectrometry Workstation.
  • Fig. 50A shows intracellular cyclic AMP production by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL 16 ) (D) and N-AcGIP(LysPAL 37 ) (•), as determined by column chromatography, in CHL cells stably expressing the human GIP receptor.
  • GIP GIP
  • N-AcGIP(LysPAL 16 ) D
  • N-AcGIP(LysPAL 37 ) •
  • 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(LysPAL 16 ) (grey bars) and N-AcGIP(LysPAL 37 ) (black bars) in the clonal pancreatic beta cell line, BRIN-BDl 1.
  • N-AcGIP(LysPAL 16 ) grey bars
  • N-AcGIP(LysPAL 37 ) black bars
  • Figs. 51A through 5 ID are a set of two line graphs (Figs. 51 A, 51C) and two bar graphs (Figs. 5 IB, 5 ID) 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.
  • N-AcGIP(LysPAL 16 ) andN- AcGIP(LysPAL 37 ) produced a significant reduction in plasma glucose at each time point (p ⁇ 0.01 to pO.OOl) and significantly lowered glucose AUC (pO.OOl to pO.OOl) when compared to glucose alone. Additionally, N-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ) decreased the overall glucose excursion (p ⁇ 0.05 to pO.OOl) when compared to native GIP.
  • Figs. 51C and 51D The corresponding plasma insulin responses are illustrated in Figs. 51C and 51D.
  • glucose alone confrol
  • 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.
  • N- AcGIP(LysPAL 16 ) or N-AcGIP(LysPAL 37 ) where administered together with glucose, a maximum plasma insulin concentration was observed at 15 minutes. Profracted biological activity for both analogues was clearly evident from 30 to 60 minutes.
  • Glucose-mediated plasma insulin concenfrations were significantly higher compared in both confrol (p ⁇ 0.01 to pO.OOl) and GIP-treated animals (p ⁇ .05 to pO.OOl).
  • the corresponding AUC values forN-AcGIP(LysPAL 16 ) and N-AcGIP(LysPAL 37 ) revealed significant enhancements in overall glucose-mediated insulin release compared to native GIP (1.5-fold and 2.3-fold, respectively; pO.01 to pO.OOl).
  • N- AcGIP(LysPAL 37 ) was significantly more potent (1.5-fold: pO.OOl) than N- AcGIP(LysPAL 16 ) at stimulating insulin secretion.
  • Figs. 52A and 52B illustrate the dose-dependent antihyperglycaemic and insulinotropic effects of GIP and the more potent analogue ⁇ -AcGIP(LysPAL 37 ) when administered with glucose to ob/ob mice. They are are a pair of bar graphs showing dose-dependent effects of GIP andN-
  • Values represent means ⁇ SEM for 8 mice. **pO.01 and ***p .001 compared to glucose alone. ⁇ pO.01 and ⁇ pO.001 compared to native GIP at the same dose.
  • N-AcGIP(LysPAL 37 ) was substantially more potent than native GIP (pO.01 to pO.OOl) and exhibited prominent dose-dependent antihyperglycaemic and insulinotropic actions at all doses administered (Figs. 52A, 52B). Remarkably, even the lowest concentration of N-AcGIP(LysPAL 37 ) tested (6.25 nmoles/kg) had highly significant antihyperglycaemic properties compared to glucose alone (pO.OOl).
  • Figs. 53A through 53E are a set of graphs showing the effects of daily N- AcGIP(LysPAL 37 ) (•; black bars) adminisfration on food intake (Fig. 53A), body weight (Fig.
  • Fig. 53B plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated hemoglobin N-AcGIP(LysPAL 37 ) (12.5 nmoles/kg/day) (Fig. 53E).
  • Native GIP (12.5 nmoles/kg/day; A ; lined bars) or saline vehicle (control; D; 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, **pO.01 compared to control. ⁇ pO.01 compared to native GIP.
  • GIP or N-AcGIP(LysPAL 37 ) had no effect on body weight or food intake (Figs. 53A, 53B).
  • daily injection of N-AcGIP(LysPAL 37 ) resulted in a progressive lowering of plasma glucose, resulting in significantly (p ⁇ .05) lowered concentrations at 14 days (Fig. 53C).
  • Figs. 54A through 54D are a set of two line graphs (Figs. 54A, 54C) and two bar graphs (Figs. 54B, 54D) showing the effects of daily N-AcGIP(LysPAL 37 ) administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin response (Figs. 54C, 54D) to glucose.
  • Tests were conducted after 14 daily injections of either N-AcGIP(LysPAL 37 ) (12.5 nmoles/kg/day; •; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; ⁇ ; 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 .05, **pO.01, ***pO.001 compared to control.
  • ⁇ pO.05, ⁇ pO.01, ⁇ pO.001 compared to native GIP.
  • treatment of ob/ob mice for 14 days with ⁇ -AcGIP(LysPAL 37 ) resulted in a significant improvement in glucose tolerance (Figs. 54A, 54B).
  • Plasma glucose concenfrations throughout the test and the overall 0-60 minutes AUC values were decreased (pO.01 to pO.OOl). This was accompanied by increased insulin concentrations during the latter stages (p ⁇ .05) and a greater (pO.01) overall AUC insulin response (Figs. 54C, 54D).
  • 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).
  • Figs. 55A through 55D are a line graph and three bar graphs showing the effects of daily N-AcGIP(LysPAL 37 ) administration on insulin sensitivity (Figs. 55A, 55B) and pancreatic weight (Fig. 55C) and insulin content (Fig. 55D).
  • insulin 50 U/kg was administered by intraperitoneal injection at the time indicated by the arrow.
  • Plasma glucose AUC values for 0-60 minutes post injection are shown in the right panels. Values are means ⁇ SEM for 8 mice. *pO.05, **pO.01 compared to control. ⁇ pO.05, ⁇ A pO.01 compared to native GIP.
  • Insulin sensitivity of the 3 groups of mice after 14 days treatment is shown in Figs. 55A, 55B.
  • N-AcGIP(LysPAL 37 ) prompted a significant improvement of insulin sensitivity.
  • Both the individual glucose concentrations and 0-60 minutes AUC values were significantly different (pO.01) from the other two groups.
  • 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(LysPAL 37 ) did not affect pancreatic weight compared with saline-treated controls (Figs.
  • pancreatic insulin content was similar in the GIP and saline treated groups.
  • daily administration of N-AcGIP(LysPAL 37 ) significantly increased (p .01) insulin content compared with each of the other groups (Figs. 55C, 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(LysPAL 37 ) and native GIP after daily injection for 14 days.
  • Glucose (18 mmoles/kg) was administered by intraperitoneal injection alone (a; white bars) or in combination with either N-AcGIP(LysPAL 37 ) (•; 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.
  • 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.
  • N-AcGIP(LysPAL 37 ) effects of N-AcGIP(LysPAL 37 ) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; •; black bars), native GIP (12.5 mnoles/kg/day; A; lined bars) or saline vehicle (control; ⁇ ; white bars).
  • N-AcGIP(LysPAL 37 ) 25 nmoles/kg was administered by intraperitoneal injection with glucose (18 mmoles/kg) at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels. Values are means ⁇ SEM for 8 mice.

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Abstract

L'invention concerne des analogues peptidiques qui sont des antagonistes du peptide inhibiteur gastrique (GIP). Les peptides, sur la base du GIP 1-42, comportent des substitutions et/ou des modifications qui améliorent la résistance à la dégradation par la dipeptidylpeptidase IV (DPP IV). L'invention concerne également un procédé de modification de la terminaison N de GIP ainsi que l'utilisation d'analogues peptidiques destinés au traitement de diabètes.
PCT/GB2005/000710 1999-03-29 2005-02-25 Analogues peptidiques de gip destines au traitement de diabetes, de la resistance a l'insuline et de l'obesite WO2005082928A2 (fr)

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EP05717793A EP1730188A2 (fr) 2004-02-25 2005-02-25 Analogues peptidiques de gip destines au traitement de diabetes, de la resistance a l'insuline et de l'obesite
JP2007500292A JP2008500280A (ja) 2004-02-25 2005-02-25 糖尿病、インスリン抵抗性、及び肥満症の治療のためのｇｉｐペプチド・アナログ
AU2005217198A AU2005217198A1 (en) 2004-02-25 2005-02-25 Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
CA2557151A CA2557151C (fr) 2004-02-25 2005-02-25 Analogues peptidiques de gip destines au traitement de diabetes, de la resistance a l'insuline et de l'obesite
US11/090,787 US20050272652A1 (en) 1999-03-29 2005-03-25 Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
US11/713,892 US7875587B2 (en) 1999-03-29 2007-03-05 Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
US11/825,293 US20080009603A1 (en) 1999-03-29 2007-07-03 Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity

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WO2007028633A2 (fr) * 2005-09-08 2007-03-15 Uutech Limited Traitement de l'obesite associee aux diabetes
WO2007028632A2 (fr) * 2005-09-08 2007-03-15 Uutech Limited Analogues de polypeptide inhibiteur gastrique comme traitement de la fonction reduite des cellules beta pancreatiques du fait du vieillissement
WO2008021560A2 (fr) * 2006-08-17 2008-02-21 Amylin Pharmaceuticals, Inc. Analogue du gip et polypeptides hybrides possédant des propriétés sélectives
US7572451B2 (en) 2004-10-25 2009-08-11 Cytos Biotechnology Ag Gastric inhibitory polypeptide (GIP) antigen arrays and uses thereof
WO2010012495A1 (fr) * 2008-08-01 2010-02-04 University Of Ulster Immunisation active contre le gip
WO2010016940A2 (fr) 2008-08-07 2010-02-11 Ipsen Pharma S.A.S. Analogues de polypeptide insulinotrope glucose-dépendant
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WO2010016935A2 (fr) 2008-08-07 2010-02-11 Ipsen Pharma S.A.S. Analogues tronqués de polypeptide insulinotrope glucose-dépendant
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JP2010538030A (ja) * 2007-09-07 2010-12-09 イノベイション・ウルスター・リミテッド 機能障害性シナプス伝達に伴う障害の治療用ｇｉｐの使用
US20100317576A1 (en) * 2007-11-23 2010-12-16 Michael Rothkopf Methods of enhancing diabetes resolution
WO2012055770A1 (fr) * 2010-10-25 2012-05-03 F. Hoffmann-La Roche Ag Analogues de peptides insulinitropes glucodépendants
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US9790263B2 (en) 2009-06-16 2017-10-17 Indiana University Research And Technology Corporation GIP receptor-active glucagon compounds
WO2018181864A1 (fr) 2017-03-31 2018-10-04 Takeda Pharmaceutical Company Limited Peptide d'activation du récepteur gip
WO2019211451A1 (fr) * 2018-05-04 2019-11-07 Novo Nordisk A/S Dérivés de gip et leurs utilisations
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US11572399B2 (en) 2017-05-31 2023-02-07 University Of Copenhagen Long-acting GIP peptide analogues

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US7572451B2 (en) 2004-10-25 2009-08-11 Cytos Biotechnology Ag Gastric inhibitory polypeptide (GIP) antigen arrays and uses thereof
WO2007028632A2 (fr) * 2005-09-08 2007-03-15 Uutech Limited Analogues de polypeptide inhibiteur gastrique comme traitement de la fonction reduite des cellules beta pancreatiques du fait du vieillissement
WO2007028632A3 (fr) * 2005-09-08 2007-05-31 Uutech Ltd Analogues de polypeptide inhibiteur gastrique comme traitement de la fonction reduite des cellules beta pancreatiques du fait du vieillissement
WO2007028633A3 (fr) * 2005-09-08 2007-06-07 Uutech Ltd Traitement de l'obesite associee aux diabetes
WO2007028633A2 (fr) * 2005-09-08 2007-03-15 Uutech Limited Traitement de l'obesite associee aux diabetes
US8338368B2 (en) 2005-11-07 2012-12-25 Indiana University Research And Technology Corporation Glucagon analogs exhibiting physiological solubility and stability
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US9783592B2 (en) 2010-05-13 2017-10-10 Indiana University Research And Technology Corporation Glucagon superfamily peptides exhibiting nuclear hormone receptor activity
US9145451B2 (en) 2010-05-13 2015-09-29 Indiana University Research And Technology Corporation Glucagon superfamily peptides exhbiting G protein coupled receptor activity
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GB0404124D0 (en) 2004-03-31
AU2005217198A1 (en) 2005-09-09
WO2005082928A3 (fr) 2005-12-01
JP2008500280A (ja) 2008-01-10
EP1730188A2 (fr) 2006-12-13
CA2557151A1 (fr) 2005-09-09
CA2557151C (fr) 2015-02-17

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