JP2008500280A - GIP peptide analogs for the treatment of diabetes, insulin resistance, and obesity - Google Patents

Abstract

  The invention provides peptide analogs that are antagonists of gastric inhibitory peptide (GIP). Said peptides based on GIP1-42 contain substitutions and / or modifications that are highly resistant to degradation by the enzyme dipeptidyl peptidase IV (DPP IV). The present invention also provides a method for producing an N-terminal modified GIP and a method for using said peptide analog for the treatment of diabetes.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of UK Application No. GB0404124.0 filed February 25, 2004. The entire teachings of the previous application are incorporated herein.

FIELD OF THE INVENTION The present invention relates to insulin release and blood glucose concentration control. More particularly, the present invention relates to gastric inhibitory peptide (GIP) antagonists as pharmaceuticals for the treatment of type 2 diabetes.

Background Obesity and diabetes are predicted to be pandemic around the world within the next 20 years, and current treatments do not restore normal insulin sensitivity or glucose homeostasis, which is a debilitating diabetic complication, And bring about an early life.

  Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (7-36) amide (cleaved GLP-1; tGLP-1) are secreted by two important secreted endocrine cells in the intestine It is an insulin releasing hormone. Together with autonomic nerves, they play a pivotal role in supporting islets in the control of blood glucose homeostasis and nutrient metabolism.

  GIP is released into the bloodstream from intestinal endocrine K cells after ingestion of carbohydrates, proteins, and especially fat (Meier, J. J. et al., 2002, Regul. Pept. 107: 1-13). GIP was originally discovered through its ability to suppress gastric acid secretion (Brown, J. C et al., 1969, Can. J. Physio. Pharmacol. 47: 113-114), but its main physiological role is In general, it is now believed to be the role of incretin hormones that target the islets to increase insulin secretion and help lower postprandial hyperglycemia (Creutzfeldt, W., 2001, Exp. Clin. Endocrinol. Diabetes 109: S288-S303). GIP acts through binding to specific G proteins bound to GIP receptors on pancreatic beta cells (Wheeler, M. B. et al., 1995, Endocrinology 136: 4629-4639). In addition to this ability to stimulate insulin secretion, as well as its sister incretin hormone glucagon-like peptide-1 (GLP-1), there are other potentially beneficial effects on pancreatic beta cell proliferation and differentiation, It attracted great 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, GG et al., 2003, Curr. Med. Chem. 10: 2471-2483).

  Since GIP functions as a potent and natural stimulator of insulin secretion released from the intestine upon ingestion, antagonists that counter GIP action interfere with GIP's insulin-releasing action, and oral glucose tolerance and It was widely expected to reduce both the glycemic response to nutrient intake. In fact, all studies published to date show that GIP is a major physiological component of enteroinsular axis, and functional removal of GIP is metabolized towards a type 2 diabetes phenotype It has been shown to cause a glucose homeostasis defect that moves features (Gault, VA et al., 2002, Biochem. Biophys. Res. Comnun. 290: 1420-1426).

Dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) has been identified as an important enzyme involved in the inactivation of GIP and tGLP-1 in serum. This occurs by rapid removal of the N-terminal dipeptides Tyr ¹ -Ala ² and His ^7- Ala ⁸ which produce the main metabolites GIP (3-42) and GLP-1 (9-36) amide, respectively. These cleaved peptides have been reported to lack bioactivity or even serve as antagonists of the GIP or tGLP-1 receptor. The resulting biological half-life of these incretin hormones in vivo is estimated to be less than 5 minutes and is therefore very short. DPP IV is completely suppressed in serum by the addition of diprotin A (DPA, 0.1 mmol / l).

  In terms of normal glucose regulation and pancreatic B cell sensitivity, this short duration of action is advantageous to facilitate temporary adjustments to controls to maintain homeostasis. However, in addition to finding a convenient route of administration, the current goal of the potential therapeutic role of incretin hormones, particularly tGLP-1 in the treatment of non-insulin dependent diabetes mellitus (NIDDM) is hampered by a number of factors. ing. The most notable of these are the results for rapid peptide degradation and rapid absorption (to reach peak concentrations in 20 minutes), as well as for high dosages and for accurate timing with the diet As the need arises. Recent therapeutic strategies have focused on precipitated preparations to delay peptide absorption and to inhibit GLP-1 degradation using specific inhibitors of DPP IV. The promising therapeutic role is reduced in diabetic obese Zucker rats treated with glucose by protecting against specific catabolism of the specific inhibitor of DPP IV, isoleucine thiazolidide, possibly the incretin hormone tGLP-1 and GIP Observations of elevated blood glucose and elevated insulin secretion are suggested as well.

Studies have shown that tGLP-1 infusion restores pancreatic B cell sensitivity, fluctuations in insulin secretion, and improved glycemic control in various groups of patients with impaired glucose tolerance (IGT) or NIDDM. Longer-term studies also provide a significant motivation to develop oral effective or long-lasting tGLP-1 analogs, as well as the significant benefits of tGLP-1 injection in NIDDM treatment and possibly IDDM treatment The same is shown. Several attempts have been made to create structurally modified analogs of tGLP-1 that are resistant to DPP IV degradation. A significant increase in serum half-life is observed with His ⁷ -glucitol tGLP-1 and tGLP-1 analogs substituted at position 8 with Gly, Aib (aminoisobutyric acid), Ser, or Thr. However, these structural modifications appear to reduce receptor binding and insulin secretion activity, thereby hindering some of the benefits of protection from proteolysis. In a recent study using His ⁷ -glucitol tGLP-1, resistance to degradation by DPP IV and serum was accompanied by a significant loss of insulin releasing activity.

  GIP not only shares the same degradation pathway as tGLP-1, but also shares many similar physiological effects, including stimulation of insulin and somatostatin secretions, and increased glucose disposal. These effects are considered a major aspect in the anti-hyperglycemic properties of tGLP-1 and therefore there is good hope that GIP may have similar potential as NIDDM treatment. In practice, GIP compensation is used to reveal the slight glucose homeostasis observed in tGLP-1 knockout mice. Apart from early studies, the anti-diabetic potential of GIP has not been examined and is more effective from some people when injected at so-called physiological concentrations estimated by radioimmunoassay (RIA) TGLP-1 may seem more attractive because it is considered an insulin secretagogue.

  Thus, there is a need for diabetes treatment that includes GIP analogs that can cause the release of insulin and can also be resistant to rapid degradation by DPP IV.

Summary of the present invention
Disclosed herein are GIP antagonist peptides that are resistant to rapid degradation by DPP IV.

The present invention includes a peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (3-42). The present invention also applies to the peptide analog of GIP (1-42) (SEQ ID NO: 1) containing at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ^3. Included.

The amino acid substituted with Glu ³ may be selected from the group consisting of: proline, hydroxyproline, lysine, tyrosine, phenylalanine, and tryptophan. In particular, proline may replace the Glu ^3. The peptide analog may further comprise modification by fatty acid addition at the epsilon amino group of at least one lysine residue. The lysine residue may be Lys ¹⁶ or Lys ³⁷ .

The peptide analog of GIP (1-42) (SEQ ID NO: 1) has at least 12 amino acid residues from the N-terminus of GIP (1-42) and amino acid residues at position 1, 2, or 3. Of amino acid modifications. The N-terminal amino acid residue may be acetylated. It may further comprise modification by fatty acid addition at the epsilon amino group of at least one lysine residue. The modification is, for example, linking a C-8, C-10, C-12, C-14, C-16, C-18, or C-20 palmitate group to an epsilon amino group of a lysine residue. Also good. The lysine residue may be Lys ¹⁶ or Lys ³⁷ .

The present invention is a peptide analog of GIP (1-42) (SEQ ID NO: 1), which is: GIP (1-12), GIP (1-13), GIP (1-14), GIP (1 -15), GIP (1-16), GIP (1-17), GIP (1-18), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1- 22), GIP (1-23), GIP (1-24), GIP (1-25), GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29 ), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36) , GIP (1-37), GIP (1-38), GIP (1-39), GIP (1-40), GIP (1-41), and GIP (1-42) Also includes those that contain a base peptide {where the base peptide includes the following modifications: (1) amino acid substitution with Glu ³ , (2) fatty acid at the epsilon amino group of at least one lysine residue Modification by addition; and (3) having one or more of modifications by N-terminal acetylation. }. Such peptide analogs may have a proline replacing the 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 is a linkage of, for example, a C-8, C-10, C-12, C-14, C-16, C-18, or C-20 palmitate group to an epsilon amino group of a lysine residue. Also good. The lysine residue may be Lys ¹⁶ or Lys ³⁷ .

  The present invention is a peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (3-42), when compared to natural GIP, Further included are those that are resistant to degradation by the enzyme DPP IV.

A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ³ , This also includes those that are resistant to degradation by the enzyme DPP IV when compared to GIP.

  Furthermore, the present invention relates to a peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (3-42), which regulates insulin secretion including.

The present invention relates to a peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ^3. It also includes those that regulate insulin secretion.

  The invention also includes the use of any analog in the preparation of a medicament for the treatment of obesity, insulin resistance, insulin resistant metabolic syndrome (syndrome X), or type 2 diabetes.

  The present invention also includes a pharmaceutical composition comprising the peptide analog. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. The peptide analog can be in the form of a pharmaceutically acceptable salt or a pharmaceutically acceptable acid addition salt.

  In a further aspect, the invention includes a method of treating insulin resistance, obesity, or type 2 diabetes {wherein the method requires a therapeutically effective amount of the pharmaceutical composition in need of such treatment. Administration to a mammal. }.

According to the invention, an effective peptide analog of biologically active GIP (1-42) having improved characteristics for the treatment of type 2 diabetes, from the N-terminus of GIP (1-42) Provided are those containing at least 15 amino acid residues and having at least one amino acid substitution or modification at positions 1-3 and free of Tyr ¹ glucitol GIP (1-42).

  The structure of human and porcine GIP (1-42) is shown below. Porcine peptides differ by only two amino acid substitutions at positions 18 and 34.

  The analog may comprise a modification by fatty acid addition at the epsilon amino group of at least one lysine residue.

The present invention includes Tyr ¹ glucitol GIP (1-42) with fatty acid addition at the epsilon amino group of at least one lysine residue.

  GIP (1-42) analogues have increased ability to stimulate insulin secretion, increase glucose disposal, delay glucose absorption, or when compared to native GIP Stability may be exhibited. They may also have increased resistance to degradation as well.

  Any of these properties enhances the efficacy of the analog as a therapeutic agent.

  Analogs with D-amino acid substitutions at positions 1, 2, and 3 and / or N-glycated, N-alkylated, N-acetylated, or N-acylated amino acids at position 1 are resistant to degradation in vivo Have sex.

For example, GIP (1-42) Gly ² , GIP (1-42) Ser ² , GIP (1-42) Abu ² , GIP (1-42) Aib ² , GIP (1-42) D-Ala ² , GIP Various amino acid substitutions at the second and third amino terminal residues such as (1-42) Sar ² and GIP (1-42) Pro ³ are included.

  GIP analogs modified at the amino terminus include N-glycated GIP (1-42), N-alkylated GIP (1-42), N-acetylated GIP (1-42), N-acetyl-GIP (1-42) ), And N-isopropyl-GIP (1-42).

Other stabilized analogs include those with a peptide isostere bond between the amino terminal residues at the 2 and 3 positions. These analogs may be resistant to the plasma enzyme dipeptidyl peptidase IV (DPPIV), which is primarily responsible for inactivation of GIP by removal of the amino-terminal dipeptide Tyr ¹ -Ala ² .

In certain embodiments, the present invention is a peptide that is more potent than human or porcine GIP in suppressing glycemic excursion, comprising:
(A) replacement of Ala ² with Gly;
(B) Substitution of Ala ² by Ser;
(C) replacement of Ala ² with Abu;
(D) replacement of Ala ² with Aib;
(E) replacement of Ala ² with D-Ala;
(F) replacement of Ala ² with Sar (sarcosine);
(G) Replacement of Glu ³ with 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 saccharification;
(L) conversion of Ala ² -Glu ³ bond to psi [CH ₂ NH] bond;
(M) conversion of Ala ² -Glu ³ bond to stable peptide isostere bond; and (n) (n-isopropyl-H) 1GIP,
GIP (1-42) with one or more modifications selected from the group consisting of, or consisting of 15-30 amino acid residues from the N-terminus (ie, GIP (1-15) GIP (1-30) Provided consists of an N-terminal fragment of GIP (1-42).

The present invention also provides the use of Tyr ¹ -glucitol GIP in the preparation of a medicament for the treatment of diabetes.

  The present invention further provides an improved pharmaceutical composition comprising a GIP analog having improved pharmaceutical properties.

  Other possible analogs include certain commonly encountered amino acids that are not encoded by the general genetic code, such as β-alanine (β-ala), or other ω-amino acids, such as 3-aminopropionic acid , 4-aminobutyric acid, ornithine (Orn), citrulline (Cit), homoarginine (Har), t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine (N- MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO), substitution of neutral or acidic amino acids in D form, or substitution of tyrosine in D form , Including substitution by tyrosine.

  The invention also provides a pharmaceutical composition useful for the treatment of type 2 diabetes comprising an effective amount of a peptide described herein in admixture with a pharmaceutically acceptable excipient. To do.

  The present invention relates to a method for modifying the N-terminus of GIP or an analog thereof, comprising synthesizing a peptide from the C-terminus to the second N-terminal amino acid from the last to a bubbler as Fmoc-protected Tyr (tBu) -Wang resin. Add tyrosine to the system, deprotect the N-terminus of the tyrosine, and react with a denaturant, terminate the reaction, cleave the modified tyrosine from the Wang resin, and the modified tyrosine Said method comprising the step of adding to the peptide synthesis reaction is also provided.

  Preferably, the agent is glucose, acetic anhydride, or pyroglutamic acid.

  The invention will now be demonstrated by reference to the following non-limiting examples and the drawings attached therein.

DETAILED DESCRIPTION The peptide analogs disclosed herein exhibit resistance to degradation by the enzyme DPP IV. These analogs include those with changes in the first, second, and / or third residues of the native GIP (1-42) peptide, where the changes prevent DPP IV cleavage or Delay. The alteration may be made first by, for example, acylation, acetylation, alkylation, glycation, conversion of a bond between two amino acids to, for example, a psi- [CH ₂ NH] bond or a stable isostere bond, or addition of an isopropyl group. One or more chemical modifications of the three amino acids can be included. Said alteration can likewise include amino acid substitutions at positions 1, 2 and / or 3 to either different natural amino acids or amino acids not encoded by the genetic code. Other changes are possible as well, the purpose of which is still to allow insulin secretion, but to prevent peptide cleavage by DPP IV. This may be achieved by changes in other regions of the peptide, for example by changing the three-dimensional structure to prevent DPP IV resolution while still allowing insulin secretion.

  Preferred changes include chemical modifications consisting of amino acid substitutions of the first, second and third residues at the first, second and third residues, as well as chemical modifications of lysine residues throughout the protein.

Particularly preferred changes include acetylation of Tyr ¹ and linking of the C-16 palmitate group to an epsilon amino group of a lysine residue (especially Lys ¹⁶ or Lys ³⁷ ), or especially replacement of Glu ³ with proline. . The modification can also be, for example, linking a C-8, C-10, C-12, C-14, C-18, or C-20 palmitate group to an epsilon amino group of a lysine residue.

In contrast to acute, GIP receptor antagonism using long-term Glu ³ substituted forms of GIP, such as (Pro ³ ) GIP, is associated with insulin resistance and associated impaired glucose tolerance associated with obesity Was found to improve. This revealed an unexpected approach to the treatment of obesity, insulin resistance, and type 2 diabetes.

  As described in Example 1 below, the N-terminal modified version of the GIP protein is prepared similarly to the modified protein analog. The proteins and their analogs were then evaluated in Example 2 for their antihyperglycemic and insulin release properties in vivo, and had sufficient resistance to aminopeptidase degradation and higher glucose-lowering activity compared to native GIP. It was found to show.

  The 42 amino acid GIP is an important incretin hormone that is released into the circulation from endocrine K cells of the duodenum and jejunum upon food intake. The high degree of structural conservation of GIP among species supports the view that this peptide plays an important role in metabolism. GIP secretion is stimulated directly by nutrients actively carried into the intestinal lumen without any notable input from autonomic nerves. The most important stimulators of GIP release are monosaccharides and unsaturated long chain fatty acids with amino acids that exert a weaker effect. Similar to tGLP-1, the effect of GIP releasing insulin is strictly dependent on glucose. This provides protection against hypoglycemia, thereby realizing one of the most desirable properties of any current or potential new anti-diabetic drug.

The results demonstrate for the first time that Tyr ¹ -glucitol GIP is completely resistant to serum and DPP IV degradation. The study using ESI-MS showed that natural GIP is rapidly cleaved in vitro to a major degradation product of 4748.4 Da corresponding to GIP (3-42), and matrix-assisted laser desorption ionization time-of-flight The previous findings using mass spectrometry were verified. Serum degradation was completely suppressed by diprotin A (Ile-Pro-Ile), a specific antagonistic inhibitor of DPP IV that confirmed this as a major enzyme for GIP inactivation in vivo. In contrast, Tyr ¹ -glucitol GIP remained almost completely intact after incubation with serum or DPP IV for up to 12 hours. This hides the cleavage site where GIP glycation at the amino terminal Tyr ¹ residue may be possible from DPP IV and prevents the removal of the Tyr ¹ -Ala ² dipeptide from the N terminus. Indicates prevention of formation.

Consistent with protection against DPP IV in vitro, administration of Tyr ¹ -glucitol GIP significantly enhanced the antihyperglycemic activity and insulin release action of the peptide when administered to rats with glucose. Native GIP increased insulin release and decreased glycemic excursion, as observed in many previous studies. However, glycation at the amino terminus of GIP increased the insulin release and antihyperglycemic effect of the peptide by 62% and 38%, respectively, as estimated from the area under the curve (AUC) of insulin. Detailed kinetic analysis is difficult due to the inevitable limitation of sampling time, but the higher insulin concentration after Tyr ¹ -glucitol GIP is longer 30 minutes after injection, as opposed to GIP A half-life was suggested. The increase in blood glucose was small in both peptide treatment groups, and the glucose concentration after Tyr ¹ -glucitol GIP injection was consistently lower than after GIP. Since the insulin secretagogue action of GIP is dependent on glucose, the relative insulin release efficacy of Tyr ¹ -glucitol GIP appears to be greatly underestimated in the in vivo experiments.

In our in vitro studies using glucose-responsive cloned B cells, the insulin release efficacy of Tyr ¹ -glucitol GIP is several orders of magnitude greater than GIP, and its efficacy is within the physiological range. It was shown that it was more sensitive to changes in glucose concentration.

Together with the in vivo observations, this suggests that glycation at the N-terminus of GIP enhances receptor binding and insulin secretion effects on B cells while conferring resistance to DPP IV degradation. These qualities of Tyr ¹ -glucitol GIP make it possible for DPP IV resistance to prevent degradation of the peptide into GIP (3-42), thereby extending half-life and maintaining the intact biologically active peptide. It is well shown in vivo to increase the effective concentration. Thus, glycated GIP is capable of enhancing insulin secretion in vivo by both increased potency at the receptor as well as improved DPP IV resistance. Thus, many studies have shown that GIP (3-42) and other N-terminally modified fragments, including GIP (4-42) and GIP (17-42), have a weak effect on stimulating insulin release. It was only present or inactive. Furthermore, there is evidence that the N-terminal deletion of GIP produces receptor antagonist properties in Chinese hamster kidney cells [9] transfected with the GIP receptor, and inhibition of GIP catabolism has been shown to result in cleavage GIP (3-42 ) Suggests that the potential feedback antagonism at the receptor level is also reduced.

In addition to its insulin secretion-promoting action, many other possible important extrapancreatic actions of GIP contribute to increased antihyperglycemic activity and other beneficial metabolic effects of Tyr ¹ -glucitol GIP Might do. These include stimulation of glucose uptake into adipocytes, increased synthesis of fatty acids, and activation of lipoprotein lipase in adipose tissue. GIP also promotes plasma triglyceride clearance in response to oral fat load. In the liver, GIP has been shown to enhance insulin-dependent inhibition of glycogenolysis. GIP similarly reduces both glucagon-stimulated lipolysis in adipose tissue as well as hepatic glucose production.

  Finally, recent research results show that GIP has a potent effect on glucose uptake and metabolism in isolated diaphragm muscle of mice. This latter action can be shared with tGLP-1 and both peptides have the added benefit of stimulating somatostatin secretion and slowing gastric emptying and nutrient absorption.

This study demonstrates that glycation of GIP at the amino-terminal Tyr ¹ residue limits GIP catabolism through impaired proteolytic action of serum peptidases, thereby extending its half-life in vivo. This effect occurs simultaneously with increased antihyperglycemic activity and increased insulin concentration in vivo, suggesting that such DPP IV resistant analogs are potential useful therapeutics for NIDDM. Tyr ¹ -glucitol GIP seems particularly interesting in this regard because, as recently observed for tGLP-1, such GIP amino-terminal modification increases rather than impairs glucose-dependent insulinotropic effects Because.

As shown in Table 1 of Example 3, glycated GIP, acetylated GIP, and GIP (Ser ² ) are more resistant to in vitro degradation by DPP IV than natural GIP. From these data, GIP (Sar ² ) appears to be less resistant. As shown in Table 2, all analogs tested showed resistance to plasma degradation, including GIP (Sar ² ), which appears to be the least resistant of the peptides tested from DPP IV data. It was. DPA substantially inhibited degradation of GIP and of all analogs tested, including complete abolition of degradation in the case of GIP (Abu ² ), GIP (Ser ² ), and glycated GIP. This indicates that DPP IV is a major factor in the degradation of GIP in vivo.

As shown in FIGS. 17 to 30, the glycated GIP analog showed a considerably greater insulin secretion promoting response than the native GIP. N-terminally acetylated GIP showed a similar pattern and the GIP (Ser ² ) analog caused a strong response as well.

From these studies, GIP (Gly ² ) and GIP (Pro ³ ) appear to be analogs with minimal efficacy in terms of insulin release. The other stable analogs tested, GIP (Abu ² ) and GIP (Sar ² ), showed a complex pattern of reactivity depending on the glucose concentration and the dose used. Thus, very low concentrations were very strong under hyperglycemic conditions (16.7 mM glucose). This suggests that, even with these analogs, the ability to promote insulin secretion combined with in vivo degradation may prove useful as a treatment in the treatment of type 2 diabetes, which determines peptide efficacy.

A major limitation to the possible therapeutic use of GIP and GLP-1 as insulin-releasing agents for the treatment of diabetes is their rapid in vivo by dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5) It is a serious decomposition. This enzyme rapidly removes the amino-terminal dipeptide from the two peptides, creating GIP (3-42) and GLP-1 (9-36) lacking bioactivity (Gault, VA et al., 2002, Biochem. Bioplzys. Res. Commun. 290: 1420-1426). (Pro ³ ) GIP, a newly synthesized GIP analog with a single proline substitution at position 3 close to the cleavage site, is powerful in searching for stable, amino-terminally modified forms of GIP and GLP-1. It was found to function as a potent GIP receptor antagonist.

Below, as shown in Example 4, (Pro ³ ) GIP, other Glu ³ substituted forms of GIP, and GIP (3-42) are potent GIP receptor antagonists both in vivo and in vitro. is there. Experiments evaluating the effects of chronic GIP receptor antagonism in normal mice using (Pro ³ ) GIP have demonstrated extensive but reversible decreases in glucose tolerance. This is in complete agreement with the widely recognized physiological role of GIP as an important insulin-releasing intestinal hormone involved in the regulation of glucose disposal following feeding (Meier, JJ et al., 2002, Regul Pept. 107: 1-13.

Most strikingly and in stark contrast to normal mice, the experiments disclosed herein show that chronic (Pro ³ ) GIP administration to obese diabetic ob / ob mice for 11 days is impaired glucose tolerance and diabetes Indicates that the condition is not deteriorated at all. Surprisingly, GIP receptor antagonism in this obese insulin resistance model was associated with a very significant improvement in glycated HbA _1c , plasma glucose and insulin concentrations, glucose tolerance, and insulin sensitivity. Pancreatic insulin levels were similarly reduced, and the characteristic islet hypertrophy of obese mutants was partially recovered. These latter observations indicate a decreased secretion requirement of endogenous insulin after (Pro ³ ) GIP as a result of improved insulin resistance.

Indeed, an insulin sensitivity test with ob / ob mice treated with (Pro ³ ) GIP for 11 days not only compensates for the functional loss of the GIP element that releases insulin, consisting of enteroinsular axis. Revealed a significant improvement in tissue insulin insensitivity. The exact mechanism involved in this effect on insulin sensitivity is not known, but the loss of the direct effect of circulating GIP on adipose tissue metabolism is a promising candidate. Equally noteworthy was the fact that all of these beneficial effects of (Pro ³ ) GIP in obese diabetic ob / ob mice were reversed within 9 days after discontinuation of treatment.

These results show that (Pro ³ ) GIP, and other analogs based on Glu ³ substitution or N-terminal truncation of the gastrointestinal hormone GIP have been identified in human obesity, so-called insulin resistance (metabolic) syndrome, and type 2 diabetes. It shows that an important therapeutic means to reduce insulin resistance for treatment can be presented.

  Several studies have attempted to enhance incretin action using DPP IV inhibitors or stable analogs of GLP-1 and GIP for the treatment of type 2 diabetes (Green, BD et al., 2004, Curr Rharm. Des. 10: Printing; Drucker, DJ et al., Diabetes Care 10: 2929-2940). Such an approach relies on the possibility that incretin action is deficient in diabetes and that the underlying defects involved in metabolic disruption may be overcome by exogenous GLP-1 or GIP administration . There is some evidence regarding the beneficial and potential therapeutic role of GLP-1 and GIP analogs in diabetes (Meier, JJ et al., 2002, Regul. Pept. 107: 1-13; Gault, VA et al. , 2003, Biochem Biophys Res Commun 308: 207-213; Holst, JJ et al., 2004, Xm. J. Physiol. Endocrinol. Metab. 287: E199-E206; Green, BD et al., 2004, Curr. Pharm. Des. 10: Printing; Drucker, DJ et al., Diabetes Care 10: 2929-2940). Nonetheless, lack of understanding of the possible involvement of incretin hormones in the pathophysiology of diabetes, in part, the major truncated peptide form produced by DPP IV, especially circulating at high concentrations, GLP -1 (9-36) and GIP (3-42) resulting from a cross-reaction of classical GLP-1 and GIP radioimmunoassay (Meier, JJ et al., 2002, Regul. Pept. 107 : 1-13 pages). Several clinical studies appear to suggest the existence of a defective GLP-1 secretion and a defective GIP action in type 2 diabetes (Holst, JJ et al., 2004, Am. J. Playsiol. Endocrinol. Metab 287: pages E199-E206). However, the basis for such conclusions is that many previous contradictory human studies (Morgan, LM, "Insulin secretion and the enteroinsular axis,": Nutrient regulation of insulin secretion, Flatt, PR, London, Portland) Press, 1992, pages 1-22), and reported insensitivity of pancreatic beta cells to GIP (Vilsboll, T. et al., 2002, Diabetologia 45: 1111-1119) are general rather than specific cell defects Considering the possibility of reflecting sexual secretion dysfunction (Meier, JJ et al., 2003, Metabolism 52: 1579-1585), it is not impressive. In fact, the secretory response to all insulin secretagogues, including GLP-1, was impaired in type 2 diabetes (Kjems, LL et al., 2003, Diabetes 52: 380-386; Flatt, PR et al., "Defective insulin secretion in diabetes and insulinoma," Nutrient regulation of insulin secretion, Flatt PR, London, Portland Press, 1992, 341-386). Thus, the proposed use of GLP-1 and GIP for the treatment of diabetes has resulted in improved efficacy, reduced renal clearance, and / or increased GIP receptor activity and post-receptor activity due to DPP IV resistance. Rely on peptide engineering to provide analogs of incretin hormones (Gault, VA et al., 2003, Biochem Biophys Res Commun 308: 207-213).

  Although a single animal model cannot be matched to the complex etiology of human type 2 diabetes, mouse ob / ob syndrome studies have shown metabolism related to bulimia, hyperinsulinemia, and progressive obese diabetes Revealed a marked anomaly in GIP regarding interactions during decay (Flatt, PR et al., 1983, Diabetes 32: 433-435; Flatt, PR et al., 1984, J. Endocrinol. 101: 249- 256; Bailey, CJ et al., 1986, Acta Endocrinol. (Copenh) 112: 224-229). These animals are non-insulin dependent diabetes models with significant obesity and severe insulin resistance promoted by leptin deficiency (Bailey, CJ et al., “Animal syndromes resembling type 2 diabetes,” Textbook of Diabetes, 3rd edition, Pickup JC & Williams G., Oxford, Blackwell Science Ltd., 2003, pages 25.1-25.30). In addition, recent research studies have shown that the interaction between leptin and enteroinsular axis (Anini, Y. et al., 2003, Diabetes 52: 252-259) and the GIP receptor on adipocytes ("GIP- R ") overstimulation is suggested to be considered to be an important contributor to fat deposition in ob / ob mice (Miyawaki, K. et al., 2002, Nat. Med. 8: 738-742). ).

In the following, as shown in Example 5, daily injections of (Pro ³ ) GIP, a stable and specific GIP-R antagonist, demonstrated GIP- R can be chemically removed and used to assess the role of endogenous circulating GIP in obesity-diabetes. The results reveal a major role for GIP in insulin resistance and concomitant metabolic disorders, and a new and effective means of treating GIP-R antagonists in forms of type 2 diabetes promoted by obesity Provide the first experimental evidence that it might provide.

  GIP-R knockout in normal mice has been shown to result in significant impairment of glucose tolerance and diet-induced insulin secretion with no noticeable effect on food intake, body weight, or basal glucose or insulin concentrations (Miyawaki, K. et al., 1999, Proc. Nat. Acad. Soi. USA 96: 14843-14847). More recent studies using genetic GIP-R knockout mice supported these findings and showed that GIP is also heavily involved in enteroinsular axis (Pederson, RA et al., 1998, Diabetes 47 : 1046-1052; Pamir, N. et al., 2003, Am. J. Physiol. Endocrinol. Metab. 284: E931-939). However, double knockouts of GLP-1 and GIP receptors result in a surprisingly slight deterioration in glucose homeostasis (Hansotia, T. et al., 2004, Diabetes 53: 1326-1335; Preitner, F. et al., 2004, J. Clin. Invest. 113: 635-645), showing possible up-regulation of the compensatory mechanism of defects throughout the lifetime of GLP-1 and GIP receptors.

The analog (Pro ³ ) GIP is highly stable and can be used as a specific and potent GIP-R antagonist with resistance to degradation mediated by DPP IV ( Gault, VA et al., 2002, Biochenz. Biophys. Res. Commun. 290: 1420-1426). Using acutely (Pro ³ ) GIP, the results disclosed herein revealed a plasma insulin response to feeding in ob / ob mice and the involvement of GIP in enteroinsular axis (Gault, VA et al., 2003, Diabetologia 46: 222-230). Comparison with the effect of exendin (9-39), a GLP-1-R antagonist, shows that GIP contributes significantly to the insulin-releasing action of entero-insular axis and is the main physiological incretin (Gault, VA et al., 2003, Diabetologia 46: 222-230). Administration of (Pro ³ ) GIP to normal mice once daily for 11 days results in a reversible impairment of glucose tolerance associated with decreased insulin sensitivity (Irwin, N., 2004, Biol. Chem 385: 845-852). Basal and postprandial insulin secretion did not change with pancreatic insulin content and islet morphology. Thus, long-term GIP with daily (Pro ³ ) GIP, except to reveal an important additional effect of endogenous GIP on insulin action that appears unrelated to elevated insulin secretion Chemical loss of -R function can mimic the phenotype induced by genetic GIP-R knockout in mice.

Never reproduce this predicted scenario and the metabolic deterioration observed after genetic or chemical knockout of GIP-R in normal mice (Miyawaki, K. et al., 1999, Proc. Nat. Acad. Sci. USA 96: 14843-14847; Irwin, N., 2004, Biol. Chem. 385: 845-852), ob / ob mice treated with GIP for 11 days daily (Pro ³ ) It showed a marked improvement in the condition of diabetes. This includes reduced fasting and basal hyperglycemia, reduced glycated hemoglobin, improved glucose tolerance, and significantly reduced range of blood glucose after feeding. In particular, basal and glucose-stimulated plasma insulin concentrations decreased, suggesting that insulin sensitivity should have improved significantly after (Pro ³ ) GIP to suppress hyperglycemia. In fact, an insulin sensitivity test performed 11 days after (Pro3) GIP administration revealed a 57% increase in the glucose lowering effect of exogenous insulin. Considering that the severity of ob / ob syndrome represents a difficult trial for current anti-diabetic drugs including insulin, sulfonylurea, metformin, and thiazolidenedione (Flatt, PR et al., “Defective insulin secretion in diabetes and insulinoma, "Nutrient regulation of insulin secretion, Flatt PR, London, Portland Press, 1992, pages 341-386; Stevenson, RW et al., 1995, The Diabetes Annual 9: 175-191; Wiernsperger, NF , "Preclinical pharmacology of biguanides," Handbook of Experiniental Pharmacology 119: 305-358, 1996), (Pro ³ ) GIP-R blockade using GIP can induce such rapid and reversible changes There is no precedent.

It is important to note that the aforementioned effects were observed independently of changes in either food intake or body weight in (Pro ³ ) GIP treated ob / ob mice. This is consistent with the view that endogenous GIP lacks an effect on eating activity (Meier, JJ et al., 2002, Regul. Pept. 107: 1-13). However, the observations on body weight contrasted with the cross-bred findings of ob / ob mice to genetically knock out GIP-R function (Miyawaki, K. et al., 2002, Nat. Med. 8: 738- 742). Thus, in these transgenic mice, the depletion of GIP-R function from the time of birth resulted in decreased weight gain compared to control (Lep ^ob / Lep ^ob ) mice, as well as adiposity and insulin resistance. Related to significant improvement (Miyawaki, K. et al., 2002, Nat. Med. 8: 738-742). In this previous study, the improvement in insulin sensitivity may have been a simple consequence of reduced adipose tissue mass if this significantly increased peripheral glucose disposal (Bailey, CJ et al., “Animal syndromes resembling type 2 diabetes, "Textbook of Diabetes, 3rd edition, Pickup JC & Williams G., Oxford, Blackwell Science Ltd., 2003, pages 25.1-25.30). However, the results, which are observed immediately and have no effect on feeding or weight, clearly indicate the involvement of alternative mechanisms.

Ob / ob against the Aston background depicted in FIG. 49, which explains how GIP-R antagonist (Pro ³ ) GIP counters beta cell hyperplasia, hyperinsulinemia, and insulin resistance The most promising explanation for the data resulting from the correct recognition of the major milestones in the age-dependent progression of the syndrome results in improved impaired glucose tolerance and diabetes management. Potential long-term direct effects of GIP on adipocyte function and fat accumulation suggested by studies in GIP-R knockout ob / ob mice were omitted.

Because of the double recessive ob mutation and the resulting leptin deficiency, young ob / ob mice suffer from severe early binge eating (Bailey, CJ et al., 1982, Int. J. Obes. 6: 11-21 page). Extensive intestinal endocrine stimulation results in K cell hyperplasia and significantly increased concentrations of intestinal and circulating GIP (Flatt, PR et al., 1983 Diabetes 32: 433-435; Flatt, PR et al., 1984). J. Endocrinol 101: 249-256; Bailey, CJ et al., 1986, Acta Endocrinol (Copenh) 112: 224-229). This, along with marked hyperinsulinemia and worsening insulin resistance (Flatt, PR et al., 1981, Horm Metab Res 13: 556-560), in turn promotes islet hypertrophy and beta cell hyperplasia (Bailey, CJ et al., 1982, Int. J. Obes. 6: 11-21). This method becomes apparent with respect to elevated basal hyperglycemia and impaired glucose tolerance. Thus, a spiral vicious circle has been established in which beta cell compensation results in marked hyperinsulinemia that attempts to moderately increase insulin resistance (Bailey, CJ et al., 1982, Int. J. Obes. 6: 11-21; Flatt, PR et al., 1981, Horm Metab Res 13: 556-560). From this context, it is clear that daily chemical loss of GIP-R function by (Pro ³ ) GIP reduces beta cell stimulation and hyperinsulinemia. However, instead of causing further impairment of glucose homeostasis, a preferential and significant improvement in insulin sensitivity leads to a sufficient improvement of the metabolic syndrome. Further support for this scenario is the partial improvement of islet hypertrophy and beta cell hyperplasia in (Pro ³ ) GIP treated ob / ob mice (FIG. 48). In particular, the average islet diameter was reduced by the largest islet (> 15 mm) replaced by a higher proportion of small or medium diameter (0.1-15 mm). These effects were largely reversed by 9-day withdrawal of treatment, supporting the concept of active islets and beta cell proliferation in adult ob / ob mice (Bailey, CJ et al., 1982, Int. J. Obes 6: pages 11-21). Recent observations indicate that GIP plays a role as a mitotic stimulant and anti-apoptotic agent for beta cells (Pospisilik, JA et al., 2003, Diabetes 52: 741-750; Trumper, A. et al. 2001, Mol. Endocrinol. 15: 1559-1570; Ehses, JA et al. 2003, Endocrinology 144: 4433-4445; Trumper, A. et al., 2002, J. Endocrinol. 174: 233-246. ). Thus, the negative effect of (Pro ³ ) GIP on islet size appears to reflect a combination of decreased beta cell proliferation and increased apoptosis.

The results shown in Fig. 5 show that daily administration of the GIP-R antagonist (Pro ³ ) GIP improves glucose tolerance in ob / ob mice, as well as insulin resistance and abnormal islet structure and function. Proved to improve for the first time. In particular, these effects were overturned by a 9-day (Pro ³ ) GIP interruption. The absence of any obvious side effects has been genetically engineered from normal mice (Irwin, N., 2004, Biol. Chem. 385: 845-852) and GIP-R deficiency since birth Consistent with previous observations in mice (Miyawaki, K. et al., 2002, Nat. Med. 8: 738-742). The observation lists the major role of endogenous GIP in the development of obese insulin resistant diabetes. More importantly, the data show that GIP-R antagonists, such as (Pro ³ ) GIP, are novel, physiological and effective means for treating obese type 2 diabetes through reducing insulin resistance To provide.

In Example 6, fatty acid derivatization was used to develop two new long-acting N-terminally modified GIP analogs, N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ). was used by.

  Degradation studies were performed using dipeptidyl peptidase IV (DPP IV). Cyclic AMP production was assessed using GIP receptor transfected CHL fibroblasts. In vitro insulin release was assessed in BRIN-BD11 cells. Enhanced insulin secretion and glycemic response to acute and chronic peptide administration were evaluated in obese diabetic (ob / ob) mice.

In contrast to GIP, both analogs were resistant to DPP IV degradation. The analog stimulated cyclic AMP production as well, and showed significantly improved in vitro insulin secretion compared to the control. Administration of N-AcGIP (LysPAL ¹⁶ ) or N-AcGIP (LysPAL ³⁷ ) with glucose in ob / ob mice significantly reduced blood glucose excursion and improved insulin secretion response compared to GIP . Dose-response studies with N-AcGIP (LysPAL ³⁷ ) revealed a very significant decrease in overall glycemic range and an increase in circulating insulin, even at 6.25 nmoles / kg. Daily injection of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) for 14 days significantly reduced plasma glucose, glycated hemoglobin, compared to saline or natural GIP, and Improved glucose tolerance. Plasma and pancreatic insulin increased significantly, along with a marked increase in insulin response to glucose and a marked improvement in insulin sensitivity. No evidence for GIP receptor desensitization was found, and the metabolic effects of N-AcGIP (LysPAL ³⁷ ) were independent of any change in food intake or body weight.

These results indicate that novel fatty acid derivatized and N-terminally modified GIP analogs such as N-AcGIP (LysPAL ³⁷ ) may have important potential for the treatment of type 2 diabetes.

One approach to combat both renal clearance and enzymatic degradation of GIP involves the use of fatty acid derivatization along with N-terminal modification. Fatty acid derivatization has been reported in insulin (Kurtzhals, P. et al., 1995, Biochem. J. 312: 725-731) and sister incretin glucagon-like peptide-1 (GLP-1) (Knudsen, LB et al., 2000). J. Med. Clielit. 43: 1664-1669; Green, BD et al., 2004, Biol. Chem. 385: 169-177; Kim, JG et al., 2003, Diabetes 52: 751-759). Prolonging half-life has been shown previously. A number of N-terminally modified GIP analogs have been developed that exhibit significant resistance to DPP IV (Hinke, SA et al., 2002, Diabetes 51: 656-661; Gault, VA et al., 2002) Biochem. J. 367: 913-920; Gault, VA et al., 2003, J. Endocrinol. 176: 133-141; O'Harte, FPM et al., 1999, Diabetes 48: 758-765). Some of these, most particularly acetyl, glucitol, pyroglutamyl, or GIP Tyr ¹ modified with the addition of Fmoc adducts show enhanced activity at GIP receptors in vitro (Gault , VA et al., 2002, Biochem. J. 367: 913-920; O'Harte, FPM et al., 1999, Diabetes 48: 758-765; O'Harte, FPM et al., 2002, Diabetologia 45: 1281. ~ P. 1291). As a result of degradation resistance and increased cellular activity, these analogs are enhanced and prolonged antihyperglycemic activity and insulin release when administered acutely to obese-diabetic animals. Show activity (Hinke, SA et al., 2002 Diabetes 51: 656-661; Gault, VA et al., 2002, Biochem. J. 367: 913-920; Gault, VA et al., 2003, J. Endocrinol 176: 133-141; O'Harte, FPM et al., 1999, Diabetes 48: 758-765; O'Harte, FPM et al., 2002, Diabetologia 45: 1281-1129). Of these, N-AcGIP has emerged as the most effective DPP IV resistant analog that significantly increases plasma insulin response and reduces the glycemic excursion following the combined administration of glucose to ob / ob mice (O 'Harte, FPM et al., 2002, Diabetologia 45: 1281-1291).

Example 6 shows the metabolic stability of N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ), N-AcGIP analogs modified at the N-terminus, derivatized with novel second generation fatty acids. Designed to assess bioactive, anti-diabetic potential. Both GIP analogs contain a C-16 palmitate group linked to the epsilon amino group of Lys at position 16 or 37 in combination with an N-terminal (Tyr ¹ ) acetyl group (O'Harte, FPM et al., 2002, Diabetologia 45: pages 1281-1291). Relative stability to DPP IV degradation, insulin secretion, and cyclic AMP properties are investigated in vitro along with acute and dose response studies in obese diabetic ob / ob mice. The most effective analog, N-AcGIP (LysPAL ³⁷ ), was administered to ob / ob mice by intraperitoneal injection once a day for 14 days prior to evaluation for glucose homeostasis, pancreatic beta cell function, and insulin sensitivity. The Desensitization with potential GIP receptor action following prolonged exposure to elevated concentrations of N-AcGIP (LysPAL ³⁷ ) was also investigated. The results show detailed support for N-AcGIP (LysPAL ³⁷ ), a novel second generation N-terminal acetylated GIP analog as a potential therapeutic for the treatment of type 2 diabetes.

  Despite their many qualities, like their natural counterparts, DIP IV resistant analogs of GIP and GLP-1 are still exposed to renal filtration. To circumvent this problem, fatty acid derivatization was used to improve the duration of GLP-1 action (Knudsen, LB et al., 2000, J. Med. Chem. 43: 1664–1669; Green , BD et al., 2004, Biol. Chem. 385: 169-177; Kim, JG et al., 2003, Diabetes 52: 751-759). Despite the trend toward promoting nausea, which may be due to slowing gastric emptying, NN2211 (liraglutide), the most promising analog, is effective in improving glycemic control in subjects with type 2 diabetes (Agerso, H. et al., 2002, Diabetologia 45: 195-202).

Example 6 describes the results of introducing two specific modifications, N-terminal acetylation and C-terminal fatty acid derivatization, into the natural GIP hormone. N-terminal acetylation was utilized as previously described to significantly increase stability against DPP IV (O'Harte, FPM et al., 2002, Diabetologia 45: 1281-1291). In contrast, binding of C-16 palmitate residues at Lys ⁶ or Lys ³⁷ epsilon amino groups was introduced to extend biological half-life by binding to circulating proteins (Kurtzhals, P. et al. 1995, Biochem. J. 312: 725-731). Unlike the natural peptide, both GIP analogs appear to be completely resistant to oxygen degradation by DPP IV, supporting previous observations with N-AcGIP (O'Harte, FPM et al., 2002, Diabetologia 45: 1281-1291). Furthermore, both analogs showed similar or slightly better characteristics of insulin release and cyclic AMP production to natural GIP and N-AcGIP when tested in cell lines in vitro (O'Harte, FPM Et al., 2002, Diabetologia 45: 1281-1129).

Obese diabetic (ob / ob) mice were utilized to assess the potential of fatty acid derivatized GIP analogs in vivo for antihyperglycemia and insulin secretion promotion. ob / ob syndrome is a widely studied model of spontaneous obesity and diabetes that shows bulimia, marked obesity, moderate hyperglycemia, and severe hyperinsulinemia (Bailey, CJ et al., 1982, Int. J. Obesity 6: 11-21). As described in previous studies (Gault, VA et al., 2002, Biochem. J. 367: 913-920; Gault, VA et al., 2003, J. Endocrinol. 176: 133-141), natural Of GIP only slightly reduced glycemic excursion in ob / ob mice, reflecting the severe insulin resistance of this mutant animal model (Bailey, CJ et al., 1982, Int. J. Obesity 6: 11-21 pages). In marked contrast, both N-acetylated GIPs (N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ )), in which the analog is further substituted with a palmitate molecule at Lys ¹⁶ or Lys ³⁷ , are naturally occurring. Significantly reduced plasma glucose levels compared to the other peptides. This was achieved by significantly increased insulin release activity, especially in the case of N-AcGIP (LysPAL ³⁷ ). Despite the sufficiently low plasma glucose, a significantly sustained insulinotropic response to both fatty acid derivatized GIP analogs at 60 minutes indicates an extended plasma half-life. This may be because both palmitate derivatized GIP analogs bound serum albumin, thereby significantly impairing their clearance by the kidney (Meier, JJ et al., 2004, Diabetes 53: 654-662). ). However, further studies are needed to confirm such effects, including the establishment of sensitive and specific immunoassays for new GIP analogs.

N-AcGIP (LysPAL ³⁷ ) appeared to be the best fatty acid derivatized analog with more sustained and significantly enhanced insulin release potency over N-AcGIP (LysPAL ¹⁶ ) in vivo. The cause of the enhanced potency of N-AcGIP (LysPAL ³⁷ ) remains unclear, but one explanation is an extended half-life. Another possibility is that the fatty acid chain linked to Lys closer to the C-terminus of the peptide may have less detrimental effects on the bioactive site of the molecule known to be located at the N-terminus. (Gault, VA et al., 2002, Biosei. Rep. 22: 523-528; Hinke, SA et al., 2001, Biochim. Biophys. Acta 1547: 143-55; Manhart, S. Et al., 2003, Biochemistry 42: 3081-3088). However, the similarities between the in vitro bioactivity of the two palmitate substituted analogs make this less likely to occur.

Since N-AcGIP (LysPAL ³⁷ ) was more potent among the two analogs in vivo, it was further utilized in dose response studies. Considering that natural GIP itself has very little effect in ob / ob mice, as is sometimes observed in subjects with type 2 diabetes (Nauck, MA et al., 1993, J. Clin. Invest 91: 301-307; Meier, JJ et al., 2004, Diabetes 53: 220-224; Vilsbll, T. et al., 2002, Diabetologia 45: 1111-1119), N-AcGIP (LysPAL ³⁷ ) It should be noted that even the lowest dose of 6.25 nmoles / kg showed significant glucose lowering activity and insulin secretagogue activity when administered with glucose. Considering that N-AcGIP (LysPAL ³⁷ ) is exposed to albumin binding, the fact that it remains highly biologically active even at low concentrations suggests significant efficacy.

Daily administration of N-AcGIP (LysPAL ³⁷ ) to young adult ob / ob mice by intraperitoneal injection (12.5 nmoles / kg) resulted in a gradual drop in plasma glucose concentration and a significant decrease in glycated moglobin by 14 days. Brought. This was associated with a significant improvement in glucose tolerance. Importantly, food intake and body weight did not change, eliminating the possibility that improved glucose homeostasis was merely a result of weight loss. These observations also indicate that N-AcGIP (LysPAL ³⁷ ) did not have any inconvenient and toxic effects affecting feeding over the study period. This is consistent with a recent study showing that GIP does not inhibit gastric emptying (Meier, JJ et al., 2003, Am. J. Physiol. Endocrinol. Metab. 286: 621-625). Daily administration of natural GIP to ob / ob mice for 14 days had no effect on any index measurement, consistent with the very short half-life of natural GIP in vivo.

As expected, the major component of the beneficial action of N-AcGIP (LysPAL ³⁷ ) was related to its effect on beta cells. Thus, at the age studied, native GIP is a weak stimulator of insulin secretion in ob / ob mice, but plasma and pancreatic insulin concentrations increased in ob / ob mice given novel fatty acid derivatized analogs. . This is consistent with the action of GIP as a promoter of proinsulin gene expression (Wang, Y. et al., 1996, Mol. Cell. Endocrinol. 116: 81-87), and in this animal model of diabetes N-terminal Demonstrates the enhanced potency reported for GIP analogs modified with (Hinke, SA et al., 2002 Diabetes 51: 656-661; Gault, VA et al., 2002, Biochem. J. 367: 913- 920; Gault, VA et al., 2003, J. Endocrinol. 176: 133-141; O'Harte, FPM et al., 1999, Diabetes 48: 758-765; O'Harte, FPM et al., 2002, Diabetologia 45: pages 1281-1291). Furthermore, insulin response to glucose was significantly enhanced in ob / ob mice given N-AcGIP (LysPAL ³⁷ ). This ability to enhance or restore pancreatic beta cell glucose responsiveness was also observed with GLP-1 (Holz, GG et al., 1993, Nature 28: 362-365; Flamez, D. et al., 1998, Diabetes 47: 646-652). Similar to observations regarding glycemic control, none of these attributes were reproduced by daily injections of natural GIP.

The results of an insulin sensitivity test performed after 14 days of treatment indicate that the improvement in the diabetic state achieved in ob / ob mice by N-AcGIP (LysPAL ³⁷ ) was not only due to the potentiation of insulin secretion. Thus, these animals also showed a significant improvement in insulin sensitivity as compared to groups treated with GIP or saline. Considering that it is generally believed that hyperinsulinemia is a downregulation of insulin receptor function (Marshall, S. et al., 1981, Diabetes 30: 746-753), this is the case with N-AcGIP ( suggesting that LysPAL ³⁷⁾ is capable of exhibiting other compensatory effect. Further research is needed to assess this aspect, but possibilities include inhibition of reverse-regulated hormones and effects on sites other than the pancreas, such as muscle, adipose tissue, and liver (Morgan, LM et al. 1996, Biochem. Soc. Trans. 24: 585-591; O'Harte, FPM et al. 1998, J. Endocrinol. 156: 237-243; Yip, RG et al. 1998, Endocrinology 139: 4004 ~ 4007).

Regardless of the findings of any action that contributes to the antihyperglycemic effect of N-AcGIP (LysPAL ³⁷ ), the current envisaged problem with long-term treatment with stable analogues of GIP or GLP-1 is desensitization of hormone receptor action (Delmeire, D. et al., 2004, Biochem. PharmacoL 68: 33-39). This was observed during long-term exposure of pancreatic beta cells to GIP in rats (Tseng, CC et al., 1996, Am. J. Physiol. 270: E661-E666), but 14-day N-AcGIP ( treatment with LysPAL ³⁷⁾ There was no evidence that impair glucose lowering or insulin releasing action of N-AcGIP (LysPAL ^37). Thus, the anti-diabetic effect of N-AcGIP (LysPAL ³⁷ ) was unmistakably evident when the analog was administered acutely with glucose. In addition, the acute effect of N-AcGIP (LysPAL ³⁷ ) in such experiments is the ob / ob who received either 14 days of N-AcGIP (LysPAL ³⁷ ), natural GIP, or physiological saline injection. Identical in the group of mice.

Such data clearly shows that long-term exposure to N-AcGIP (LysPAL ³⁷ ) does not induce and possibly overcome congenital GIP receptor desensitization in ob / ob mice. Considering the high circulating concentrations of GIP in these obese diabetic rodents (Flatt, PR et al., 1983, Diabetes 32: 433-435; Flatt, PR et al., 1984, J. Endocrinol. 101: 249- (P. 256), we would like to link the apparent insensitivity of beta cells to GIP in ob / ob mice to simple receptor desensitization due to inappropriate secretion and metabolism of GIP.

  The data presented herein show that GIP acetylated at the N-terminus carrying a palmitate group linked to Lys at position 37 is against DPP IV in a commonly used obesity-diabetes animal model. It has been demonstrated that it exhibits remarkable properties of resistance, as well as bioactivity that is manifested by potent and long-acting glucose-lowering activity. This activity profile greatly encourages the development of long acting fatty acid derivatized N-terminally modified GIP analogs for once-daily treatment of type 2 diabetes.

  The peptide analogs of the invention treat diseases and conditions resulting from inappropriate regulation of insulin levels including diabetes, type 2 diabetes, insulin resistance, insulin resistant metabolic syndrome (syndrome X), and obesity Use to do.

  The peptide analog produced by the method of the invention can be used in the form of a pharmaceutical composition in which the analog is combined with a pharmaceutically acceptable carrier. Such compositions also include (in addition to the analog and carrier) diluents, extenders, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. But you can. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. The characteristics of the carrier will depend on the route of administration.

  Administration of the peptide analogues of the invention used in the form of pharmaceutical compositions or for carrying out the methods of the invention can be carried out in various conventional ways, for example by oral ingestion, inhalation, topical application, or cutaneous, subcutaneous. Can be performed by intraperitoneal, parenteral, or intravenous injection. Administration can be internal or external; and can be local (topical) or systemic.

  The composition comprising the peptide analog of the present invention can be administered intravenously, for example, as a unit dose injection. The term “unit dose” when used in reference to the therapeutic composition of the invention represents a physically discrete unit suitable as a unit dosage for a subject, wherein each unit requires a diluent, ie It includes a predetermined quantity of active agent calculated to cause the desired therapeutic effect in association with the carrier or vehicle.

  Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended beneficiary; and Includes aqueous or non-aqueous sterile suspensions that may include suspending and thickening agents. Formulations may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and lyophilized (frozen) requiring only the addition of a sterile liquid carrier, such as water for injection, just prior to use. It may be stored in a dry state.

  Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

  When a therapeutically effective amount of the composition of the invention is administered orally, the composition of the invention is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further comprise a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5-95% of the protein of the invention, and preferably about 25-90% of the protein of the invention. When administered in liquid form, a liquid carrier such as water, petroleum, animal or vegetable origin oils such as peanut oil, mineral oil, soybean oil or sesame oil or synthetic oil may be added. The liquid form of the pharmaceutical composition may further comprise a physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. When administered in liquid form, the pharmaceutical composition comprises about 0.5-90% by weight of the composition of the invention, and preferably about 1-50% of the composition of the invention.

  When a therapeutically effective amount of the composition of the invention is administered by intravenous, dermal, or subcutaneous injection, the composition of the invention is in the form of a parenterally acceptable aqueous solution that does not contain a pyrogen. It is. The preparation of such parenterally acceptable protein solutions with due consideration of pH, iso-osmotic pressure, stability, etc. is within the skill of the art. Preferred pharmaceutical compositions for intravenous, dermal or subcutaneous injection include isotonic media such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride in addition to the composition of the invention. Injectables, lactated Ringer's injections, or other media known in the art should be included. The pharmaceutical composition of the invention may also contain stabilizers, preservatives, buffer substances, antioxidants, or other additives known to those skilled in the art.

  Also included is the use of sustained release or sustained release delivery systems. As used herein, a sustained release matrix is a matrix made of a material, usually a polymer, that is degradable by oxygen or acid / base hydrolysis or dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained release matrix is preferably a biocompatible material such as liposome, polylactide (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide coglycolide (copolymer of lactic acid and glycolic acid), polyanhydride, 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, polyvinylpropylene, polyvinylpyrrolidone, and silicones Chosen from. A preferred biodegradable matrix is any one of polylactide, polyglycolide, or polylactide coglycolide (a copolymer of lactic acid and glycolic acid).

  The therapeutic composition can include therein pharmaceutically acceptable salts of said components that can be produced, for example, by inorganic or organic acids. “Pharmaceutically acceptable salts” are suitable for use in contact with human and lower animal tissues without excessive toxicities, hypersensitivity, allergic reactions, etc. within the scope of medical judgment. And those salts that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge., Et al. Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1 page et seq., Which is incorporated herein in its entirety. Pharmaceutically acceptable salts are acid addition salts formed with inorganic acids such as hydrogen chloride or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid (formed with the free amino group of the polypeptide). including. Salts formed with free carboxyl groups are inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Can be produced as well. Said salts may be prepared in situ during the final isolation and purification of the compounds of the invention or independently by reacting the free basic functional group 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, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- Hydroxymethanesulfonate (Isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectate, persulfate, 3 -Phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfone Salts, and undecanoate. Basic nitrogen-containing groups also include lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyls such as dimethyl, diethyl, dibutyl, and diamyl sulfates. Sulfates of long-chain halogen compounds such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and agents such as arylalkyl halides such as benzyl and phenethyl bromides. Can be classified. A product which is soluble or dispersible in water or oil is thereby obtained. Examples of acids that can be utilized to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and oxalic acid, maleic acid, succinic acid, and Contains organic acids such as citric acid.

  As used herein, the terms “pharmaceutically acceptable”, “physiologically acceptable”, and grammatical variations thereof are interchangeable when they represent compositions, carriers, diluents, and reagents. And represents a substance that can be administered to or on the surface of a mammal with minimal undesirable physiological effects such as nausea, dizziness, stomach upset and the like. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Usually such compositions are prepared as injections such as solutions or suspensions, but solid forms suitable for solutions or suspensions using liquids prior to use are prepared as well. Can. The preparation can be emulsified as well.

  The active ingredient can be mixed with excipients that 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, etc., or combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

  The amount of the peptide analog of the invention in the pharmaceutical composition of the invention depends on the nature and severity of the condition being treated, the nature of the prior treatment received by the patient, and the type of disorder, the age, gender of the individual, Depends on various other factors, including medical conditions. Ultimately, the attending physician will determine the amount of analog to treat each individual patient. Initially, the attending physician will administer a low dose of the peptide analog and observe the patient's response. Larger doses of peptide analog can be administered until the best therapeutic effect is obtained for the patient, at which point the dosage is not increased further.

  Additional guidance on how to determine dosages can be found in standard references such as Spilker, Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pages 7-13 and 54-60. Spilker, Guide to Clinical Trials, Raven Press, Ltd., New York, 1991, pp. 93-101; Craig et al., Modern Pharnaacology, 2nd edition, Little Brown and Co., Boston, 1986, pp. 127-133 Speight, Aoeiy's Dnug Treatnaent: Principles and Practices of Clinical Pharmacology and Therapeutics, 3rd edition, Williams and Wilkins, Baltimore, 1987, 50-56; Tallarida et al., Principles in General Phamacology, Springer-Verlag, New York, 1998 Years, pages 18-20; and Olson, Clinical Pharsnacology Made Ridiculously Simple, MedMaster, Inc., Miami, 1993, pages 1-5.

Example 1: Preparation of N-terminal modified GIP and its analogs
The N-terminal modification of GIP is in principle a three-step method. First of all, using an automated peptide synthesizer (Applied Biosystems, California, USA), GIP (starting with Fmoc-Gln (Trt) -Wang resin (Calbiochem Novabiochem, Beeston, Nottingham, UK)) To the last N-terminal amino acid (Ala ² ). The synthesis follows standard Fmoc peptide chemistry protocols. Second, the N-terminal amino acid of native GIP (Tyr) is added to the manual bubbler system as Fmoc protected Tyr (tBu) -Wang resin. This amino acid is deprotected at its N-terminus (piperidine in DMF (20% v / v)) and high concentrations of glucose (glycated, hydrogenated) up to 24 hours as needed to complete the reaction. (Under reducing conditions using sodium cyanoborohydride), acetic anhydride (acetylation), pyroglutamic acid (pyroglutamyl) and the like. The integrity of the reaction is monitored using a ninhydrin test that determines the presence of available free α-amino groups. Third (once the reaction is complete), the structurally modified Tyr is cleaved from the Wang resin (95% TFA and 5% suitable scavenger. Note: Tyr is a problematic amino acid. Are considered to be and may require special considerations), therefore, the required amount of N-terminal modified Tyr is added directly to the automated peptide synthesizer performing the synthesis, and thus the N-terminus The Tyr modified with is combined with the α-amino of GIP (Ala ² ) to complete the synthesis of the GIP analog. The peptide is cleaved from the Wang resin (as described above) and worked up using standard Buchner filtration, precipitation, rotary evaporation and drying techniques.

Example 2: Preparation of Tyr ¹ -glucitol GIP and its properties in vivo The following example investigates the preparation of Tyr ¹ glucitol GIP along with its evaluation for its antihyperglycemic and insulin release properties in vivo. The results clearly demonstrate that this novel GIP analog exhibits significant resistance to aminopeptidase degradation and enhanced glucose-lowering activity compared to native GIP.

Study Design and Methods Materials: Human GIP was purchased from the American Peptide Company (Sunnyvale, California, USA). Acetonitrile for HPLC was obtained from Rathburn (Walkersburn, Scotland). Sequencing trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK). All other chemicals including dextran T-70, activated carbon, sodium cyanoborohydride, and bovine serum albumin fraction V were purchased from Sigma (Poole, Dorset, UK). Diprotin A (DPA) was purchased from Calbiochem-Novabiochem (UK) Ltd. (Beeston, Nottingham, UK) and the rat insulin standard for RIA was Novo Industria (Copenhagen, Denmark). Reversed phase Sep-Pak cartridges (C-18) were purchased from Millipore-Waters (Milford, MA, USA). All water used in these experiments was purified using a Milli-Q water purification system (Millipore Corporation, Milford, Massachusetts, USA).

Preparation of Tyr ¹ -glucitol GIP: Human GIP was incubated with glucose under reducing conditions in 10 mmol / l sodium phosphate buffer at pH 7.4 for 24 hours. The reaction was terminated by the addition of 0.5 mol / l acetic acid (30 μl) and the mixture was applied to a Vydac (C18) (4.6 × 250 mm) analytical HPLC column (The Separations Group, Hesperia, California, USA). And gradient elution conditions were established using water / TFA and acetonitrile / TFA solvents. Fractions corresponding to saccharification peaks are pooled, dried under vacuum using an AES 1000 Speed-Vac concentrator (Life Sciences International, Runcorn, UK), and a Supelcosil (C-8) (4.6 × 150 mm) column (Supelco Inc., Poole, Dorset, UK).

Degradation of GIP and Tyr ¹ -glucitol GIP by DPP IV: HPLC purified GIP or Tyr ¹ glucitol GIP in a reaction mixture made up to 500 μl with 50 mmol / l triethanolamine-HCl, pH 7.8, at 37 ° C. And incubated with DPP-IV (5 mU) for various times at a final peptide concentration of 1 μmol / l. The oxygen reaction was terminated after 0, 2, 4, and 12 hours by addition of 5 μl of 10% (v / v) TFA / water. Samples were finished to a final volume of 1.0 ml using 0.12% (v / v) TFA and stored at −20 ° C. prior to HPLC analysis.

Degradation of GIP and Tyr ¹ -glucitol GIP by human plasma: pooled human plasma (20 μl) from 6 fasting healthy human subjects, 50 mmol / l triethanolamine / HCL buffer pH 7.8 Incubated at 37 ° C. with GIP or Tyr ¹ -glucitol GIP (10 μg) for 0 and 4 hours in 500 μl finished reaction mixture. A 4 hour incubation was similarly performed in the presence of diprotin A (5 mU). The reaction was terminated by the addition of 5 μl TFA and the final volume was adjusted to 1.0 ml using 0.1% v / v TFA / water. Samples were centrifuged (13,000 g, 5 min) and pre-prepared with 0.1% (v / v) TFA / water and washed onto a washed C-18 Sep-Pak cartridge (Millipore-Waters) Qing was applied. After washing with 20 ml 0.12% TFA / water, the bound material is released by elution with 2 ml 80% (v / v) acetonitrile / water and concentrated using a Speed-Vac concentrator (Runcorn, UK) did. Prior to HPLC purification, the volume was adjusted to 1.0 ml with 0.12% (v / v) TFA / water.

HPLC analysis of degraded GIP and Tyr ¹ -glucitol GIP: samples were applied to a Vydac C-18 widepore column equilibrated with 0.12% (v / v) TFA / H ₂ O at a flow rate of 1.0 ml / min . Using 0.1% (v / v) TFA / H ₂ O in 70% acetonitrile, the concentration of acetonitrile in the elution solvent from 0% to 31.5% in 15 minutes using a linear gradient for 30 minutes And increased from 38.5% to 70% in 5 minutes. Absorbance was observed at 206 nm and peak area was evaluated using a model 2221 LKB integrator. Samples collected manually were concentrated using a Speed-Vac concentrator.

Electrospray ionization mass spectrometry (ESI-MS): GIP (from DPP IV and plasma incubation), and samples for ESI-MS analysis containing intact and degraded fragments of Tyr ¹ -glucitol GIP, Further purification by HPLC. The LCQ tabletop mass spectrometer (Finnigan MAT, Hemel) dissolved in 100 μl water (approximately 400 pmol) and equipped with a microbore C-18 HPLC column (150 × 2.0 mm, Phenomenex, Ltd., Macclesfield, UK) Hempstead, UK). (30 μl direct loop injection) Samples were injected at a flow rate of 0.2 ml / min under isocratic conditions of 35% (v / v) acetonitrile / water. Mass spectra were obtained from a quadrupole ion trap mass analyzer and recorded. Spectra were collected using a full ion scan mode over a mass to charge (m / z) range of 150-2000. Multiple charged ions popped out and the following equation:
M _r = iM _i −iM _h
{Where M _r = molecular weight; M _i = m / z ratio; i = number of charges; M _h = proton mass. } Was used to determine the molecular weight of GIP and related structures from ESI-MS characteristics.

GIP and Tyr ¹ - physiology in vivo glucitol GIP activity: GIP on plasma glucose and insulin concentrations and Tyr ¹ - the effect of glucitol GIP, was investigated using a 10 to 12-week-old male Wistar rats. The animals were individually housed in a room air-conditioned at 22 ± 2 ° C. using a 12 hour light / 12 hour dark cycle. Drinking water and standard rodent maintenance diet (Trouw Nutrition, Belfast, Northern Ireland) were ad libitum. Food was withdrawn for 18 hours prior to intraperitoneal injection with glucose alone (18 mmol / kg body weight) or in combination with either GIP or Tyr ¹ -glucitol GIP (10 nmol / kg). The test solution was administered at a final volume of 8 ml / kg body weight. Blood samples were collected at 0, 15, 30, and 60 minutes in chilled fluoride / heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) from the tail tip of the wake rat. Samples were centrifuged at 13,000 g for about 30 seconds using a Beckman microcentrifuge. Plasma samples were aliquoted and stored at −20 ° C. prior to glucose and insulin measurements. All animal studies were conducted according to the animal (scientific treatment) method 1986.

  Analysis: Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman Glucose Analyzer II [33]. Plasma insulin was measured by dextran charcoal radioimmunoassay as previously described [34]. The area of increase in plasma glucose and insulin area under the curve (AUC) was calculated using a computer program (CAREA) utilizing the trapezoidal formula [35] with reference elimination. Results were expressed as mean ± SEM: values were compared using unpaired Student t-test. Data groups were considered significantly different when P <0.05.

Degradation of GIP and Tyr ¹ -glucitol GIP by DPP IV: FIG. 1 is obtained from 0, 2, 4, and 12 hours incubation of GIP (FIG. 1a) or Tyr ¹ -glucitol GIP (FIG. 1b) with DPP IV. The typical peak characteristics obtained from the HPLC separation of the resulting product are illustrated. The retention times for GIP and Tyr ¹ -glucitol GIP at t = 0 were 21.93 minutes and 21.75 minutes, respectively. GIP degradation was clearly evident after 4 hours of incubation (54% intact) and by 12 hours, the majority of retained GIP (60%) had a retention time of 21.61 minutes. Converted to one product. Tyr ¹ -glucitol GIP remained almost completely intact throughout the 2-12 hour incubation. The separation was on a Vydac C-18 column using a linear gradient of 0% to 31.5% acetonitrile in 15 minutes, 38.5% in 30 minutes, and 38.5 to 70% acetonitrile in 5 minutes.

Degradation of GIP and Tyr ¹ -glucitol GIP by human plasma: FIG. 2 shows a series of typical HPLC features of the products obtained from incubation of 0 and 4 hours of GIP or Tyr ¹ -glucitol GIP with human plasma . GIP with a retention time of 22.06 minutes (FIG. 2a) was readily metabolized by incubation with plasma within 4 hours resulting in the appearance of a major degradation peak with a retention time of 21.74 minutes. In contrast, incubation of Tyr ¹ -glucitol GIP under similar conditions (Fig. 2b) has any detectable degradation fragment peak with only a single peak observed with a retention time of 21.77 minutes during the period. The formation of did not occur. Addition of diprotin A, a specific inhibitor of DPP IV to GIP, during the 4 hour incubation completely suppressed peptide degradation by plasma. Peaks corresponding to the intact GIP, GIP (3-42), and Tyr ¹ -glucitol GIP are shown. The main peak corresponding to the specific DPP IV inhibitor tripeptide DPA is displayed in the bottom panel with a retention time of 16-29 minutes.

Identification of GIP degradation fragments by ESI-MS: Figure 3 is obtained for GIP using ESI-MS (Figure 3A), Tyr ¹ -Glucitol GIP (Figure 3B), and major plasma degradation fragments of GIP (Figure 3C) The obtained monoisotopic molecular weight is shown. The analyzed peptides were purified from the plasma incubation shown in FIG. The peptide was dissolved in 100 μl water (about 400 pmol) and applied to an LC / MS equipped with a microbore C-18 HPLC column. (30 μl direct loop injection) Samples were applied at a flow rate of 0.2 ml / min under 35% acetonitrile / water isocratic conditions. Mass spectra were recorded using a quadrupole ion trap mass spectrometer. Spectra were collected using a full ion scan mode over a mass to charge (m / z) range of 150-2000. The molecular weights (M _r ) of GIP and related structures were determined from ESI-MS characteristics using multiple ejected charged ions and the following equation M _r = iM _i −iM _h . The exact molecular weight (M _r ) of the peptide was calculated using the equation M _r = iM _i −iM _h as defined earlier in the study design and method. After averaging the spectrum, a plurality of charged species which jumped (M + 3H) ³⁺ and (M + 4H) ⁴⁺ and, M _r where intact each of 4981.8 and corresponds to 4983.2Da 1661.6 and 1,246.8 m Detected from GIP at / z (Figure 3A). Similarly, Tyr ¹ - For glucitol GIP, (M + 4H) ⁴⁺ and (M + 5H) ^5+, respectively, M _r is 1287.7 and 1030.3, corresponding to a molecular weight of intact of 5146.8 and 5146.5Da Detection was performed at m / z (FIG. 3B). The difference between the observed molecular weights of the quadruple charged GIP and the N-terminal modified GIP species (163.6 Da) is that the latter peptide contained a single glucitol adduct corresponding to Tyr ¹ -glucitol GIP showed that. Figure 3C is a plurality of charged species jumped (M + 3H) ³⁺ and (M + 4H) ⁴⁺ is, M _r respectively correspond to those intact of 4748.4 and 4748Da 1583.8 and 1188.1 m / z of From the main fragment of GIP (Fig. 3C). This is consistent with the theoretical mass of the form cleaved at the N-terminus of peptide GIP (3-42). This fragment was also the main degradation product of DPP IV incubation (data not shown).

Effect of GIP and Tyr ¹ -glucitol GIP on plasma glucose homeostasis: FIGS. 4 and 5 show intraperitoneal (ip) glucose alone (18 mmol / kg) (control group), and GIP or Tyr ^{1 on} plasma glucose and insulin concentrations. -Shows the effect of glucose in combination with glucitol GIP (10 nmol / kg).

FIG. 4A shows the plasma glucose concentration after ip of glucose alone (18 mmol / kg) (control group) or in combination with either GIP or Tyr ¹ -glucitol GIP (10 nmol / kg). The injection time is indicated by an arrow (0 minutes). FIG. 4B shows plasma glucose AUC values from 0-60 minutes after injection. Values are mean ± SEM for 6 rats. ^** P <0.01, ^*** P <0.001 compared to GIP and Tyr ¹ -glucitol GIP; † P <0.05, †† P <0.01 compared to non-glycated GIP. FIG. 5A shows the plasma insulin concentration after ip of glucose alone (18 mmol / kg) (control group) or glucose (10 nmol / kq) combined with either GIP or glycated GIP. The injection time is indicated by an arrow. FIG. 5B shows plasma insulin AUC values calculated for three groups each up to 90 minutes after injection. The injection time is indicated by an arrow (0 minutes). Plasma insulin AUC is assessed 0-60 minutes after injection. Values are mean ± SEM for 6 rats. ^* P <0.05, ^** P <0.001 compared to GIP and Tyr ¹ -glucitol GIP; † P <0.05, †† P <0.01 compared to non-glycated GIP.

Compared to the control group, plasma glucose concentration and area under the curve (AUC) were significantly lower after administration of GIP or Tyr ¹ -glucitol GIP (FIGS. 4A, B). Furthermore, individual values at 15 and 30 minutes with AUC were significantly lower after Tyr ¹ -glucitol GIP administration when compared to GIP. Consistent with the published insulin release profile of GIP, plasma insulin concentrations in both peptide treatment groups were significantly elevated at 15 and 30 minutes compared to values after administration of glucose alone (FIG. 5A). The overall insulin response, estimated as AUC, was also significantly higher for the two peptide treatment groups (FIG. 5B). Despite the normal glucose concentration lower than in the GIP treated group, the plasma insulin response calculated as AUC after Tyr ¹ -glucitol GIP was significantly higher than after GIP (FIG. 5B). A significant increase in plasma insulin at 30 minutes is particularly noteworthy, suggesting that Tyr ¹ -glucitol GIP's insulin-releasing action is even longer than GIP even in the face of reduced blood glucose stimulation (Figures 4A and 5A).

Example 3: Additional N-terminal structural modification of GIP This example demonstrates the ability of additional N-terminal structural modification of GIP to prevent inactivation by DPP and in plasma, as well as insulin release potency and potential We further consider their associated increase in potential therapeutic value. A number of GIP analogs with native human GIP, glycated GIP, acetylated GIP, and N-terminal amino acid substitution were tested.

  Materials and Methods: Acetonitrile for high performance liquid chromatography (HPLC) was obtained from Rathburn (Walkersburn, Scotland). Sequencing 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, fetal bovine 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 those with the highest purity available.

Synthesis of GIP and GIP analogs modified at the N-terminus: GIP, GIP (Abu ² ), GIP (Sar ² ), GIP (Ser ² ), GIP (Gly ² ), and GIP (Pro ³ ), Fmoc-Gln- Synthesized sequentially on an Applied Biosystems automated peptide synthesizer (model 432A) using a standard solid phase Fmoc procedure starting with Wang resin. Following cleavage from the resin with trifluoroacetic acid: water, thioanisole, ethanedithiol (90 / 2.5 / 5 / 2.5, total 20 ml / g resin), the resin is removed by filtration and the filtrate is filtered under reduced pressure. Reduced the amount. Anhydrous diethyl ether was added slowly until a precipitate was observed. The procedure is performed at least 5 times, collecting the precipitate by low speed centrifugation, resuspending in diethyl ether and centrifuging again. The pellet was then dried in vacuo and judged pure by reverse phase HPLC on a Waters Millennium 2010 chromatography system (software version 2.1.5). N-terminal glycated and acetylated GIPs were prepared with slight changes to published methods.

  Electrospray ionization mass spectrometry (ESI-MS) was performed as described in Example 2. Degradation of GIP and novel GIP analogs with DPP IV and human plasma was performed as described in Example 2.

Insulin-secreting cell culture: BRIN-BD11 cells [30], 10% (v / v) fetal calf serum, 1% (v / v) antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin And RPMI-1640 tissue culture medium containing 11.1 mM glucose in a sterile tissue culture flask (Corning, Glass Works, UK). The cells were maintained at 37 ° C. in an atmosphere consisting of 5% CO ₂ and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).

Acute test for insulin secretion: Prior to the experiment, the cells were collected from the surface of the tissue culture flask using trypsin / EDTA (Gibco) and 1.5 × 10 5 per well in a 24 multiwell plate (Nunc, Roskilde, Denmark). Seeds at a density of ⁵ cells and allowed to adhere overnight at 37 ° C. 1.0 ml Krebs-Ringer bicarbonate buffer supplemented with 1.1 mM glucose (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ before acute testing for insulin release. , 1.2 mM MgSO ₄ , 10 mM NaHCO ₃ , 5 g / l bovine serum albumin, pH 7.4), preceded by 40 min preincubation at 37 ° C. Test incubations were performed at two glucose concentrations (5.6 mM and 16.7 mM) with a concentration range of GIP or GIP analog (10 ⁻¹³ to 10 ⁻⁸ M) (n = 12). After a 20 minute incubation, the buffer was removed from each well and aliquots (200 μl) were used for insulin measurements by radioimmunoassay [31].

  Statistical analysis: Results were expressed as mean ± S.E.M. And values were compared using unpaired Student t-test. If P <05, the data groups were considered significantly different.

Structural identification of GIP and GIP analogs by ESI-MS: Monoisotopic molecular weight of the peptides was measured using ESI-MS. After averaging the spectra, multiple charged species (M + 3H) ³⁺ and (M + 4H) ⁴⁺ were detected for each peptide. The calculated molecular weight demonstrated the identity of the structure of each of the synthetic GIP and N-terminal analogs.

Decomposition of GIP and novel GIP analogs by DPP-IV: Figures 6-11 show DPP IV at 0, ² , 4, 8, and 24 hours, GIP, GIP (Abu ² ), GIP (Sar ² ), GIP ( The typical peak characteristics obtained from HPLC separation of reaction products obtained from incubation of Ser ² ), glycated GIP, and acetylated GIP are described. The results summarized in Table 1 show that glycated GIP, acetylated GIP, GIP (Ser ² ) are more resistant to degradation by DPP IV in vitro than natural GIP. From these data, GIP (Sar ² ) appears to be less resistant.

Degradation of GIP and GIP analogs by human plasma: FIGS. 12-16 show a representative series of HPLC characteristics resulting from incubation of GIP and GIP analogs with 0, 2, 4, 8, and 24 hours of human plasma . Similar observations were made after 24 hours of incubation in the presence of DPA. These results summarized in Table 2 were about the same as DPP IV incubation, but conditions that more closely reflect in vivo conditions are less rigorous enzymatically. GIP was rapidly degraded by plasma. In comparison, all analogs tested, including GIP (Sar ² ) that appeared to be the least resistant of the peptides tested from DPP IV data, were resistant to plasma degradation. DPA, with GIP (Abu ² ), GIP (Ser ² ), and glycated GIP, with complete abolition of degradation, significantly inhibited degradation of GIP and all analogs tested. This indicates that DPP IV is a major factor in the degradation of GIP in vivo.

Dose-dependent effects of GIP and novel GIP analogs on insulin secretion: Figures 17-30 show GIP, GIP (Abu ² ), GIP (Sar ² ) on insulin secretion from BRIN-BD11 cells at 5.6 and 16.7 mM glucose , GIP (Ser ² ), acetylated GIP, glycated GIP, GIP (Gly ² ), and GIP (Pro ³ ) concentration range effects. Natural GIP caused a significant and dose-dependent stimulation of insulin secretion. Consistent with previous studies [28], glycated GIP analogs showed a significantly greater insulin secretion-promoting response than the natural peptide. N-terminal acetylated GIP showed a similar pattern, and the GIP (Ser ² ) analog caused a strong response as well. From these studies, GIP (Gly ² ) and GIP (Pro ³ ) appeared to be the least potent analogs in terms of insulin release. The other stable analogs tested, GIP (Abu ² ) and GIP (Sar ² ), showed a complex pattern of reactivity depending on the glucose concentration and dose used. Thus, very low concentrations were very effective under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogs can prove to be useful as therapeutics in the treatment of type 2 diabetes, where the ability to promote insulin secretion combined with degradation in vivo affects peptide efficacy.

Example 4: GIP substituted with Glu ³ to improve insulin resistance and associated impaired glucose tolerance associated with obesity
This embodiment, GIP forms Glu ³ is substituted, i.e. (Pro ³⁾ GIP receptor antagonism using GIP, as well as to investigate the glucose intolerance that insulin resistance and merger related to obesity.

  Cell lines and animals: In vitro insulin secretion was assessed using the cloned pancreatic beta cell line BRIN-BD11 (McClenaghan, NH, et al., 1996, Diabetes 45: 1132-1140). Cyclic AMP production in vitro was measured using Chinese hamster lung (CHL) fibroblasts stably transfected with the human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44: 1202-1208). . In vivo studies were performed in obese diabetic ob / ob mice (Bailey C. J. et al., 1982, Int. J. Obesity 6: 11-21) and normal control mice, 8-12 weeks old.

Peptide synthesis and characterization: Standard Fmoc peptide chemistry (Fields, GB et al., 1990, Int. J. Pept. Protein) from pre-added Fmoc-Gln-Wang resin with Glu ³ substituted analogs Res. 35: 161-214) was continuously synthesized by an Applied Biosystems automated peptide synthesizer (model 432A). The synthetic peptide was judged pure by reverse phase HPLC on a Waters Millenium 2010 chromatography system (software version 2.1.5). The molecular weight of the purified peptide analog was measured using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Samples were dissolved in 10 μl H ₂ O (approximately 40 pmol / l), placed on a stainless sample plate and allowed to dry at room temperature. The sample was then mixed with a matrix solution (10 mg / ml α-cyano-4-hydroxycinnamic acid solution) in acetonitrile / ethanol (1/1) and dried at room temperature. The molecular weight was then recorded as a mass to charge (m / z) ratio to relative peak intensity and compared using theoretical values from a Voyager-DE BioSpectrometry Workstation (PerSeptive Biosystems, Framingham, MA, USA).

Tissue culture: Chinese hamster lung (CHL) fibroblasts stably transfected with human GIP receptor, 10% (v / v) fetal calf serum, 1% (v / v) antibiotics (100 U / ml Of penicillin, 0.1 mg / ml streptomycin). RPMI-1640 tissue culture medium containing 10% (v / v) fetal calf serum, 1% (v / v) antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin) in BRIN-BD11 cells Was used to culture. Use a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK) to sterilize the cells at 37 ° C in an atmosphere consisting of 5% CO ₂ and 95% air (Corning Glass Works, Sunderland, UK) Maintained within.

Acute study on insulin release: Insulin release from BRIN-BD11 cells was measured using a cell monolayer (McClenaghan, NH et al., 1996, Diabetes 45: 1132-1140). Cells are harvested using trypsin / EDTA (Gibco), seeded in 24 multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.0 × 10 ⁵ cells per well and allowed to attach overnight at 37 ° C. It was. Prior to acute testing, the cells were treated with 1.0 ml of Krebs-Ringer bicarbonate buffer supplemented with 11 mM glucose (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ , 10 mM NaHCO ₃ , 0.5% (w / v) bovine serum albumin, pH 7.4), and preincubated at 37 ° C. for 40 minutes. An acute test for insulin release was carried out using analogs with various concentrations of Glu ³ and GIP (3-42) in the presence of native GIP (10 ⁻⁷ M) and GIP (3-42) as shown in the figure. Performed at 37 ° C. for 20 minutes with mM glucose. After incubation, an aliquot of buffer was removed and stored at −20 ° C. for insulin radioimmunoassay (Flatt, PR et al., 1981, Diabetologia 20: 573-577).

Acute test for cyclic AMP production: GIP receptor transfected CHL cells were seeded in 12-well plates (Nunc, Roskilde, Denmark) at a density of 1.0 × 10 ⁵ cells per well. Cells were then grown for 48 hours before adding tritiated adenine (2 μCi; TRK311, Amersham, Buckinghamshire, UK) and 37 ml in 1 ml DMEM supplemented with 0.5% (w / v) fetal calf serum. Incubated for 6 hours at ° C. The cells were then washed with HBS buffer (130 mM NaCl, 20 mM HEPES, 0.9 mM NaHPO ₄ , 0.8 mM MgSO ₄ , 5.4 mM KCl, 1.8 mM CaCl ₂ , 25 mM glucose, 25 μM phenol red, Washed twice at pH 7.4). The cells were then exposed to forskolin (FSK, 10 μM) or various concentrations of (Pro ³ ) GIP for 10 minutes at 37 ° C. in the absence (control) or presence of native GIP (10 ⁻⁷ M). . After removing the medium, the cells were lysed with 1 ml 5% trichloroacetic acid (TCA) containing 0.1 mM unlabeled cAMP and 0.1 mM unlabeled ATP. The intracellular tritiated cAMP was then separated with Dowex and alumina exchange resin as previously described (Widmann, C. et al., 1993, Mol. Pharmacol. 45: 1029-1035).

Acute in vitro effect of (Pro ³ ) GIP administration in obese diabetic ob / ob mice: plasma Glucose and insulin response, natural GIP immediately after combined injection of GIP (25 nmol / kg body weight) with glucose (18 mmol / kg body weight) Obese diabetics 8-12 weeks of age after intraperitoneal (ip) injection of (Pro ³ ) GIP (25mnol / kg body weight) or saline (0.9% (w / v) NaCl; control) Evaluation was performed using ob mice. All test solutions were administered at a final volume of 8 ml / kg body weight. Blood samples were collected in chilled fluoride / heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately before injection, 15, 30, and 60 minutes after injection from the tip of the tail cut of awake mice. Blood samples were immediately centrifuged at 13000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK) and stored at −20 ° C. prior to glucose and insulin measurements.

Acute in vivo effect of (Pro ³ ) GIP on plasma glucose and insulin responses to feeding in obese diabetic ob / ob mice: plasma glucose and insulin responses were compared with saline (0.9% (w / v) NaCl; Evaluated using 8-12 week old ob / ob mice withdrawn food for 18 hours prior to intraperitoneal injection of (control) or (Pro ³ ) GIP (25 nmol / kg body weight). After the injection, the mice were fed again for 15 minutes. Blood samples were collected in chilled fluoride / heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately before injection, 15, 30, 60, and 120 minutes after injection from the tip of the tail cut of awake mice. . Blood samples were immediately centrifuged at 13000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK) and stored at −20 ° C. prior to glucose and insulin measurements.

Effect of chronic (Pro ³ ) GIP administration on plasma glucose, insulin, and glycated HbA _1c in obese diabetic ob / ob mice and normal mice: 8-12 weeks old obese diabetic ob / ob mice and normal control mice A daily subcutaneous injection of either saline (0.9% (w / v) NaCl) or (Pro ³ ) GIP (25 nmol / kg body weight in physiological saline) (17:00 h) Randomly divided into groups to receive. The treatment was stopped after 11 days. Food intake and body weight were recorded daily. Blood samples were collected from chilled mouse tail tips into chilled fluoride / heparin coated glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Blood samples were immediately centrifuged at 13000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK) before measuring glucose, insulin, and HbA _1c .

Effect of chronic treatment with (Pro ³ ) GIP on glucose tolerance in ob / ob mice and normal mice: plasma glucose and insulin concentrations were measured with saline (0.9% (w / v) NaCl) or (Pro ³ ) Measurements were made after intraperitoneal administration of glucose (18 mmol / kg body weight) in ob / ob mice and normal mice treated with either GIP (25 nmol / kg body weight / day) for 11 days. This study was repeated 9 days after withdrawal of chronic (Pro ³ ) GIP treatment. Blood samples were collected in chilled fluoride / heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30, and 60 minutes after injection from the tip of the cut of the tail of awake mice. Blood samples were immediately centrifuged at 13000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK) and stored at −20 ° C. prior to glucose and insulin measurements.

Effect of chronic treatment with (Pro ³ ) GIP on the glucose-lowering effect of exogenous insulin in ob / ob mice: the glucose-lowering effect of insulin, 11 days of physiology after acute intraperitoneal administration of insulin (50 U / kg body weight) Plasma glucose response was assessed in ob / ob mice treated with experimental saline (0.9% (w / v) NaCl) and (Pro ³ ) GIP (25 nmol / kg body weight / day). Blood samples were collected in fluorinated / heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately before injection and 30 and 60 minutes after injection from the tip of the tail cut of awake mice. Blood samples were immediately centrifuged at 13000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK) and stored at −20 ° C. before measuring glucose.

Effect of chronic treatment with (Pro ³ ) GIP on pancreatic insulin levels and combined islet hypertrophy in ob / ob mice: pancreatic tissue was either saline (0.9% (w / v) NaCl) or (Pro ³ ) GIP Enucleated from non-fasted ob / ob mice after 11 days of treatment at either (25 nmol / kg body weight / day). Pancreas samples were individually wrapped in aluminum foil and quickly frozen in liquid nitrogen. Individual excised pancreas samples were then embedded, cut into slices and immunohistochemically stained for insulin or permeabilized for measurement of pancreatic insulin levels.

Measurement of HbA _1c , plasma glucose and insulin concentrations: HbA _1c was measured in whole blood by ion exchange high performance liquid chromatography using the Menari HA-8140 kit (BIOMEN, Berkshire, UK). Plasma glucose was assayed by an automated glucose oxidase procedure using Beckman Glucose Analyzer II (Stevens, JF, 1971, Clinica Chemica Acta 32: 199-201) and plasma insulin was measured by RIA (Flatt, PR et al., 1981, Diabetologia 20: 573-577). Incremental area under the plasma glucose and insulin curves (AUC) is calculated using a trapezoidal formula with subtraction of the standard (Burington, RS, 1973, Handbook of Mathematical Tables and Formulae, New York, McGraw Hill) (CAREA) ) To calculate.

  Statistical analysis: Results were expressed as mean ± SEM. Values were compared using unpaired student t-tests and data groups were considered significantly different when P <0.05.

result
GIP-stimulated cyclic AMP production and insulin secretion were suppressed by (Pro ³ ) GIP in a dose-dependent manner, suggesting that (Pro ³ ) GIP is a potent functional GIP receptor antagonist.

Chinese hamster lung (CHL) fibroblasts transfected with the GIP receptor were incubated with 10 ⁻¹² to 10 ⁻⁶ M (Pro ³ ) GIP in the presence of native GIP (10 ⁻⁷ M). The results are shown in FIGS. 32A and 32B. FIG. 32A is a line graph showing ³ H-cAMP production as the percent maximum response (y axis) with increasing peptide concentration (M) (x axis). Figure 32B shows 5.6 mM glucose (control) (white bar), GIP (gray bar), (Pro ³ ) GIP (lined bar), and (Pro ³ ) GIP + GIP (10 ^-7 M) (black bar) ) Shows a bar graph of insulin secretion (y axis) with increasing peptide concentration (M) (x axis). ^{* P <0.05, ** P <} 0.01, *** P <0.001 is, compared to glucose ^{control, ΔΔ P <0.01, ΔΔΔ P} <0.001 was compared to the native GIP at the same concentration. Values are mean ± SEM for 3-8 observations.

(Pro ³ ) GIP inhibited GIP-induced cAMP formation with an IC ₅₀ value of 2.6 μM. Insulin releasing activity of BRIN-BD11 cells exposed to native GIP and (Pro ³ ) GIP (in the absence and presence of 10 ⁻⁷ M GIP).

GIP-stimulated insulin secretion, as shown in the bar graphs of FIGS. 33A-33F, GIP (3-42), (Hyp ³ ) GIP, (Lys ³ ) GIP, (Tyr ³ ) GIP, (Trp ³ ) GIP And (Phe ³ ) GIP suppressed in a dose-dependent manner. Figure 33A shows 10 ^-7 M GIP (y-axis) versus GIP (10 ^-7 M) log ₁₀ (white bar, control) and GIP (10 ^-7 M) + GIP (3-42) (black bar) ³ H-cAMP production is shown as a percentage (%). 33B-33F are GIP (10 ⁻⁷ M) (white bar, control) and (Hyp ³ ) GIP (FIG. 33B), (Lys ³ ) GIP (FIG. 33C), (Tyr ³ ) GIP (FIG. 33D), As a function of peptide concentration (M) (x axis) for Glu ³ substituted forms of GIP (black bars) including (Trp ³ ) GIP (FIG. 33E), and (Phe ³ ) GIP (FIG. 33F) ( Insulin secretion (y axis) is shown (ng / 10 ⁶ cells / 20 min). ^* P <0.05, ^** P <0.01 compared to GIP ( ^10-7 M) control. Values are mean ± SEM for 3-8 observations.

Figures 34A-34D show that acute administration of (Pro ³ ) GIP completely neutralizes the effects of GIP on glucose tolerance (Figure 34A, 34B) and plasma insulin (Figure 34C, 34D) responses in obese diabetic ob / ob mice It is a set of two line graphs (FIGS. 34A, 34C) and two bar graphs (FIGS. 34B, 34D) that show that FIGS. 34A and 34C show plasma glucose over time (x axis) for glucose (control; black inverted triangle), glucose + GIP (black diamond), and glucose + (GIP + (Pro ³ ) GIP) (white triangle). FIG. 34 is a line graph showing levels (FIG. 34A, y-axis) and plasma insulin levels (FIG. 34C, y-axis). FIGS. 34B and 34D are bar graphs showing plasma glucose AUC for glucose alone (white bars), GIP (grey bars), and glucose + (GIP + (Pro ³ ) GIP) (black bars).

Plasma glucose and insulin concentrations after ip administration either with glucose alone (18 mmol / kg body weight) or with either natural GIP or natural GIP plus (Pro ³ ) GIP (25 nmol / kg body weight). The injection time is indicated by the arrow (0 minutes). Plasma glucose and insulin AUC values are obtained for 0-60 minutes after injection. Values are mean ± SEM for 8 mice. ^* P <0.05, ^** P <0.01, ^*** P <0.001 compared to glucose alone. ^{ΔΔ P <0.01, ΔΔΔ P <} 0. 001 was compared to native GIP.

Acute administration of (Pro ³ ) GIP completely neutralized the insulin-releasing effect of GIP in ob / ob mice and related improvements in glucose tolerance. Indeed, the blood glucose excursion after (Pro ³ ) GIP (white triangle) was worse than when glucose was administered alone (black inverted triangle).

FIGS. 35A-35D show the effect of (Pro ³ ) GIP on physiological meal-stimulated insulin release and glycemic excursion in obese diabetic ob / ob mice. Plasma glucose and insulin concentrations were re-fed for 15 minutes prior to ip administration of saline (0.9% (w / v) NaCl) or (Pro ³ ) GIP (25 nmol / kg body weight) as a control. Measurements were made in mice. The injection time is indicated by an arrow (15 minutes).

The results are shown in FIGS. 35A-35D, which are a set of two line graphs (FIGS. 35A, 35C) and two bar graphs (FIGS. 35B, 35D). The figure shows plasma insulin (Figure 35A) and plasma glucose (Figure 35C) over time for saline control (black inverted triangle) and (Pro ³ ) GIP (◇), and saline control (white bar) And (Pro ³ ) GIP (black bars) show plasma insulin AUC (FIG. 35B) and plasma glucose AUC (FIG. 35D), respectively. Values are mean ± SEM for 8 mice. ^* P <0.05, ^** P <0.01, ^*** P <0.001 compared to physiological saline alone.

Acute administration of (Pro ³ ) GIP decreased the insulin response to feeding and aggravated the associated range of glycaemia in ob / ob mice. These effects, consisting of the functional loss of endogenous GIP by (Pro ³ ) GIP antagonists, are in complete agreement with the role of GIP recognized in the regulation of insulin secretion and glycemic excursion after feeding.

The effect of 11 days of chronic administration of (Pro ³ ) GIP on plasma glucose and insulin concentrations in obese diabetic ob / ob mice was also investigated. According to the classical idea and the experiments described above, and the results shown in FIGS. 32 to 35, the functional loss of endogenous GIP over 11 days of (Pro ³ ) GIP daily administration suppressed insulin secretion, And it is expected to cause significant deterioration of glucose tolerance.

However, the opposite occurred during chronic treatment with (Pro ³ ) GIP in ob / ob mice. After 11 days of daily subcutaneous injection of saline alone (0.9% (w / v) NaCl; as a control; white bar) or (Pro ³ ) GIP (25 nmol / kg body weight; black bar) A set of two bar graphs showing plasma glucose (FIG. 36A) and insulin (FIG. 36B) concentrations are shown in FIG. Values are mean ± SEM for 6 mice and ^* P <0.05 compared to saline alone. Chronic administration of (Pro ³ ) GIP (black bars) for 11 days reduces plasma glucose and plasma insulin concentrations in obese diabetic ob / ob mice relative to controls.

HbA _1c (FIG. 37A), pancreatic insulin levels (FIG. 37B), and combined islet hypertrophy (FIG. 37C) in obese diabetic ob / ob mice treated with saline (control, white bars) and (Pro ³ ) GIP The effects of chronic administration of (Pro ³ ) GIP for 11 days were investigated. HbA _1c , pancreatic insulin content, and mean islet diameter were either saline alone (white bars) or (Pro ³ ) GIP (25 nmol / kg body weight; black bars) in obese diabetic ob / ob mice Measurements were taken after 11 days of daily subcutaneous injections. Values are mean ± SEM for 6 mice and ^* P <0.05, ^*** P <0.001 compared to saline treatment group.

The beneficial effect of chronic (Pro ³ ) GIP administration in ob / ob mice was associated with a significant decrease in HbA _1c and pancreatic insulin stores with partial correction of significant islet hypertrophy of ob / ob mutants. There was about 7% weight loss in the (Pro ³ ) GIP treated ob / ob mice with no change in food intake. This effect did not achieve significance over the short study period, but this observation clearly suggests that GIP antagonism can have long-term anti-obesity effects as well.

The effects of chronic administration of (Pro ³ ) GIP for 11 days on glucose tolerance and plasma insulin in obese diabetic ob / ob mice were treated with saline (control, white) or (Pro ³ ) GIP (black). Set of line graphs (FIGS. 38A, 38C) and bar graphs (FIGS. 38B, 38D) showing plasma glucose levels (FIGS. 38A, 38B) and plasma insulin levels (FIGS. 38C, 38D) in obese diabetic ob / ob mice 38A-38D are shown. Plasma glucose and insulin concentrations were measured before and after the intraperitoneal administration of glucose (18 mmol / kg body weight). The arrow indicates the injection time (t = 0). Values are mean ± SEM for 6 mice and ^* P <0.05, ^** P <0.01, ^*** P <0.001 compared to the saline treatment group.

After 11 days of treatment with (Pro ³ ) GIP, glucose tolerance in ob / ob mice was significantly improved without changes in circulating insulin (FIG. 38).

FIG. 39 shows the effect of 11 days of chronic administration of (Pro ³ ) GIP on insulin sensitivity in obese diabetic ob / ob mice. Plasma glucose concentrations in ob / ob mice treated with saline and (Pro ³ ) GIP were measured before and after intraperitoneal administration of exogenous insulin (50 U / kg body weight; t = 0). Measured. Values are mean ± SEM for 6 mice and ^* P <0.05 compared to saline treatment group. As shown in FIG. 39, chronic administration of (Pro ³ ) GIP caused a significant improvement in insulin sensitivity.

Interestingly, the beneficial effects of chronic administration of (Pro ³ ) GIP for 11 days in obese diabetic ob / ob mice were reversed 9 days after withdrawal of treatment. This is consistent with the physiological effect and is shown in FIG. Plasma glucose concentrations were measured before and after intraperitoneal administration of glucose (18 mmol / kg body weight) for mice treated with saline (control, open squares) or (Pro ³ ) GIP (black triangles). . The arrow indicates the injection time (t = 0). Values are mean ± SEM for 6 animals.

41A and 41B are a pair of line graphs showing the effect of 11 days of chronic administration of (Pro ³ ) GIP on glucose tolerance in normal mice. Plasma glucose concentration was measured before and after intraperitoneal administration of glucose (18 mmol / kg body weight). The arrow indicates the injection time (t = 0). Values are mean ± SEM for 6 animals and ^* P <0.05, ^** P <0.01 compared to saline treatment group.

In contrast to the beneficial effects in ob / ob mice, chronic daily treatment of normal mice with (Pro ³ ) GIP (white triangles) for 11 days was reversed after 9 days of treatment discontinuation. (FIG. 41B) resulted in a marked deterioration in glucose tolerance compared to the control (black squares) (FIG. 41A).

Example 5: Chemical loss of gastric inhibitory polypeptide receptor action by daily (Pro ³ ) GIP administration improves glucose tolerance in obesity-diabetes and improves insulin resistance and islet structure abnormalities .

Gastric inhibitory polypeptide (GIP) is an important incretin hormone secreted by endocrine K cells in response to nutrient intake. This study uses (Pro ³ ) GIP, a stable and specific GIP-R antagonist, to demonstrate the effect of chemical loss of GIP receptor (GIP-R) action on the obesity-diabetes aspect. investigated. Young adult ob / ob mice received a daily ip injection of saline vehicle or (Pro ³ ) GIP for 11 days. Non-fasting plasma glucose levels and overall range of motion (AUC) relative to glucose load were significantly reduced (1.6 fold; P <0.05) in GIP-treated mice compared to controls (Pro ³ ). GIP-R loss also significantly decreased overall plasma glucose (1.4 fold; P <0.05) and insulin response to feeding (1.5 fold; P <0.05) as well. These changes are associated with significantly increased (1.6 fold; P <0.05) insulin sensitivity in the (Pro ³ ) GIP treated group. Daily injections of (Pro ³ ) GIP reduced the amount of pancreatic insulin in ob / ob mice (1.3 fold; P <0.05) and partially corrected islet hypertrophy and beta cell hyperplasia associated with obesity. These broad beneficial effects of (Pro ³ ) GIP were reversed after 9 days of treatment discontinuation and were independent of food intake and body weight. These studies highlight the role of GIP in impaired glucose tolerance related to obesity, and relieve specific GIP-R antagonists as a new class of drugs for alleviating insulin resistance and treating type 2 diabetes Emphasize the possibilities.

Study Design and Methods Animals: Obese diabetic (ob / ob) mice (Bailey, CJ et al., 1982, Int. J. Obes. 6: 11-21) from colonies maintained at the University of Aston, UK , Used at 12-16 weeks of age. Animals were age-matched, grouped, and individually housed in an air-conditioned room at 22 ± 2 ° C. using a 12 hour light: 12 hour dark cycle. Drinking water and standard rodent maintenance diet (Trouw Nutrition, Belfast, Northern Ireland) were ad libitum. All animal studies were conducted according to the British Animal (Scientific Treatment) Act 1986. No adverse events were observed after (Pro ³ ) GIP administration.

(Pro ³⁾ GIP synthesis, purification, and characterization: the (Pro ³⁾ GIP, were synthesized continuously by Applied Biosystems automated peptide synthesizer (Model 432 A). (Pro ³ ) GIP was purified by reverse phase HPLC on a Waters Millenium 2010 chromatography system (software version 2.1.5) followed by characterization using electrospray ionization mass spectrometry (ESI-MS).

Experimental protocol for ob / ob mouse studies: First, the prolonged bioactivity of (Pro ³ ) GIP was investigated in 18 hour fasted ob / ob mice 4 hours after dosing. Since then, for 11 days, mice were given a daily ip injection of either saline solution (0.9% (w / v) NaCl) or (Pro ³ ) GIP (25 nmol / kg body weight). 17:00). During the following 9 days, observations continued after (Pro ³ ) GIP administration was discontinued. Food intake and body weight were recorded daily, while plasma glucose and insulin concentrations were monitored at 2-6 day intervals. Whole blood for measurement of glycated hemoglobin was collected on days 11 and 20. Intraperitoneal glucose tolerance (18 mmol / kg body weight), metabolic response to native GIP (25 nmol / kg body weight), and insulin sensitivity (50 U / kg body weight) tests were performed on days 11 and 20. Mice fasted for 18 hours were used to investigate the metabolic response to 15 minutes of food intake. In another series, pancreatic tissue was removed at the end of the 11 day treatment period, or 9 days after (Pro ³ ) GIP withdrawal and immunohistochemistry, or 5 ml / g ice-cold acid-ethanol Processed for post-extraction insulin measurement (750 ml ethanol, 235 ml water, 15 ml concentrated HCl). A blood sample collected from the tip of the tail vein cut of awake mice at the time indicated in the drawing was immediately centrifuged at 13,000 g for 30 seconds using a Beckman microcentrifuge (Beckman Instruments, UK). The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at −20 ° C. prior to glucose and insulin measurements.

  Biochemical analysis: Plasma glucose was analyzed by automated glucose oxidase procedure (Stevens, JF, 1971, Clin. Chem. Acta 32: 199-201) using Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Assayed. Plasma and pancreatic insulin were assayed by a modified dextran-coated charcoal radioimmunoassay (Flatt, P. R. et al., 1981, Diabetologia 20: 573-577). Saccharification using a cation exchange column (Sigma, Poole, Dorset, UK) along with measurement of absorbance (415 nm) of washing and elution buffer using a VersaMax Microplate spectrophotometer (Molecular Devices, Wokingham, Berkshire, UK) Hemoglobin was measured.

Immunocytochemistry: Tissue fixed in 4% paraformaldehyde / PBS and embedded in paraffin was sliced to 8 μm. After removing the wax, the sections were exposed to insulin antibody after incubation with blocking serum (Vector Laboratories, CA, USA). Tissue samples are then serialized with a biotinylated universal, panspecific secondary antibody (Vector Laboratories, CA, USA), and a streptavidin / peroxidase preformed complex (Vector Laboratories, CA, USA) And incubated. Following washing, the stained pancreatic tissue is counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and then immersed in acid methanol (500 ml methanol, 500 ml H ₂ O, and 2.5 ml concentrated HCl). And then dehydrated and attached to Depex (BDH Chemicals, Dorset, UK). Stained slides are viewed under a microscope (Nikon Eclipse E2000, Diagnostic Instruments Incorporated, Michigan, USA) equipped with a JVC camera model KY-F55B (JVC, London, UK) and Kromoscan imaging software (Kinetic Imaging Limited) , Faversham, Kent, UK). The average number and diameter of each islet in each section was evaluated in a blinded fashion using an eyepiece scale calibrated with a stage micrometer (Graticules Limited, Tonbridge, Kent, UK). The longest and shortest diameters of each islet were measured on the scale. Thereafter, one half of the sum of these two values was considered the mean islet diameter. Approximately 60-70 random sections were examined from the pancreas of each mouse.

  Statistics: Results are expressed as mean ± SEM. Data were compared using ANOVA followed by Student-Newman-Kools post-test. The area under the curve (AUC) analysis was calculated using the trapezoidal formula with reference subtraction (Burington, R. S., Handbook of Formulas and Formulae, New York, McGraw-Hill, 1973). P <0.05 was considered statistically significant.

Results The effect of (Pro ³ ) GIP on plasma glucose and insulin concentrations 4 hours after administration was investigated. Results are shown in two line graphs (FIGS. 42A, 42C) and two bar graphs (FIGS. 42B, 42D) showing the effect of (Pro ³ ) GIP on plasma glucose and insulin responses to native GIP 4 hours after administration. The set is shown in FIGS. 42A-42D. The study was performed 4 hours after administration of (Pro ³ ) GIP (25 nmoles / kg body weight) or saline (0.9% NaCl) in 18 hour fasted ob / ob mice. Plasma glucose and insulin concentrations were measured before and after ip administration of glucose (18 mmoles / kg body weight) in combination with natural GIP (25 nmoles / kg body weight). The incremental area under the glucose or insulin curve (AUC) from 0-60 minutes is shown in the right panel. Values represent mean ± SEM for 8 mice. ^* P <0.05 and ^** P <0.01 compared to physiological saline alone group.

As shown in FIGS. 42A-42D, (Pro ³ ) GIP administration 4 hours ago impaired plasma glucose and insulin response to native GIP given with glucose. Compared to saline-treated controls, AUC glucose and insulin values were increased to 151% (P <0.05) and decreased to 25% (P <0.05), respectively. This supports an extended biological half-life and forms the basis for once-daily injections.

The effects of (Pro ³ ) GIP on food intake, body weight, and non-fasting plasma glucose and insulin concentrations were investigated. Results show the effect of daily (Pro ³ ) GIP administration on food intake (Figure 43A), body weight (Figure 43B), plasma glucose (Figure 43C), and insulin (Figure 43D) concentrations in ob / ob mice 2 A set of one line graph and two bar graphs is shown in FIGS. 43A-43D. Indicators were measured 5 days before treatment with saline or (Pro ³ ) GIP (25 nmol / kg body weight / day), 11 days in between (indicated by black bars), and 9 days thereafter. . Values are mean ± SEM for 8 mice. ^* P <0.05 compared to physiological saline group.

Administration of (Pro ³ ) GIP had no effect on food intake and body weight (FIGS. 43A and 43B). On day 11, plasma glucose decreased in ob / ob mice receiving (Pro ³ ) GIP to a significantly lower (P <0.05) concentration (FIG. 43C). Discontinuation of treatment returned plasma glucose concentration towards the control level. Consistent with this pattern, glycated hemoglobin was significantly lower (P <0.05) prior to daily injection or after 11 days of treatment with (Pro ³ ) GIP than 9 days after discontinuation (P <0.05), respectively. ± 0.3%, 6.9 ± 0.2%, 7.7 ± 0.4%). No significant change in plasma insulin levels was observed during or after the treatment period. However, there was an overall trend for plasma insulin concentrations to gradually decrease during (Pro ³ ) GIP treatment (FIG. 43D).

For glucose tolerance effects (Pro ³⁾ GIP, ob / glucose tolerant to glucose in ob mice and daily on plasma insulin response (Pro ³⁾ GIP sets of four line graphs with a bar graph narrowing points shows the effect of administration 44A-44D, which are The study was performed after daily treatment with (Pro ³ ) GIP (25 nmoles / kg body weight / day; black triangle; black bar) or saline (control; white square; white bar) for 11 days (FIGS. 44A, 44C). ), Or 9 days after discontinuation of treatment (FIGS. 44B and 44D). Glucose (18 mmoles / kg body weight) was administered at the time indicated by the arrow. (Pro ³ ) Plasma glucose (FIGS. 44A, 44B) and insulin (FIG. 44C) 0-60 minutes after injection, with the same criteria subtraction in each case to demonstrate the full effect of GIP treatment , 44D) is shown in the inset. Values are mean ± SEM for 8 mice. ^* P <0.05, ^** P <0.01, and ^*** P <0.001 were compared to the physiological saline group.

Daily administration of (Pro ³ ) GIP for 11 days resulted in a significant decrease (P <0.001) in plasma glucose concentration after 15, 30, and 60 minutes of intraperitoneal glucose (FIG. 44A). This was confirmed by a significantly reduced AUC value of 0-60 minutes (FIG. 44A), which was a 2.1-fold decrease compared to the control (P <0.01). Plasma insulin concentrations were also significantly (P <0.05) decreased at 15, 30, and 60 minutes after intraperitoneal glucose injection in the (Pro ³ ) GIP treated group (FIG. 44A). The AUC value from 0 to 60 minutes was also significantly decreased (P <0.001). Interestingly, almost the same pattern was observed when 11 day-treated ob / ob mice were given glucose with natural GIP (25 nmoles / kg body weight) (data not shown). This supports the view that GIP action was effectively neutralized in the (Pro ³ ) GIP treatment group. Discontinuation of (Pro ³ ) GIP treatment for 9 days (day 20 of study), intraperitoneal glucose, with low glucose-mediated plasma insulin concentrations (15 min; P <0.05) observed at one time point Resulted in almost the same plasma glucose and insulin responses to (FIG. 44).

The effects of (Pro ³ ) GIP on metabolic response to feeding and insulin sensitivity are shown in FIGS. FIG 45A~45D the glucose to feeding in ob / ob mice fasted for 18 hours (FIG. 45A, 45B) and insulin (FIG. 45C, 45D) daily (Pro ³⁾ on the reaction GIP administration (closed triangles; solid bars ) Or physiological saline (white squares; white bars) is a set of two line graphs (FIGS. 45A, 45C) and two bar graphs (FIGS. 45B, 45D). The study was performed after 11 days of daily treatment with (Pro ³ ) GIP (25 nmol / kg body weight / day) or saline. The arrow indicates the feeding time (15 minutes). AUC values from 0 to 105 minutes after feeding are also shown. Values are mean ± SEM for 8 mice. ^* P <0.05 compared to physiological saline group.

FIG 46A~46D is a set of daily (Pro ³⁾ GIP two line graphs showing the effect of administration (Fig. 46A, 46C) and two bars (FIG. 46B, 46D) on insulin sensitivity in ob / ob mice . Tests were performed for 11 days (FIGS. 46A, 46B) or 9 days after discontinuation of treatment (FIGS. 46C, 46D) (Pro ³ ) GIP (25 nmol / kg body weight / day; black inverted triangle; black bars) or physiological saline Performed after daily treatment with water (white squares; white bars). Insulin (50 U / kg body weight) was administered by intraperitoneal injection at the time indicated by the arrow. AUC values 0-60 minutes after injection are also shown. Values are mean ± SEM for 8 mice. ^* P <0.05 compared to physiological saline group.

Plasma glucose and insulin responses to 15 minutes of feeding were significantly reduced at 30 and 60 minutes in ob / ob mice treated with (Pro ³ ) GIP for 11 days (P <0.05) (FIG. 45). Similarly, AUC glucose and insulin were significantly (P <0.05) decreased in (Pro3) GIP-treated ob / ob mice despite similar food intake of 0.3-0.5 g / mouse / 15 min did. As shown in FIGS. 46A and 45B, the hypoglycemic effect of insulin was significantly increased (P <0.05) with respect to AUC measurement and post-injection evaluation in ob / ob mice treated with (Pro ³ ) GIP for 11 days did. The response following 9 days withdrawal of (Pro ³ ) GIP treatment was similar to the saline treated controls (FIGS. 45C, 45D).

The effects of (Pro ³ ) GIP on pancreatic insulin and islet morphology are shown in FIGS. 47A-47D and FIGS. 48A-48F. 47A-47D is the effect of daily (Pro ³ ) GIP administration on pancreas weight (Figure 47A), insulin content (Figure 47B), islet number (Figure 47C), and islet diameter (Figure 47D) in ob / ob mice Is a set of four bar graphs. Indicator measured after daily treatment with (Pro ³ ) GIP (25 nmol / kg body weight / day; black bar) or saline (white bar) for 11 days and 9 days after discontinuation of treatment (day 20) did. Values are mean ± SEM for 8 mice. ^* P <0.05 and ^*** P <0.001 compared to the physiological saline group. 48A-48F are two bar graphs (FIGS. 48A, 48D) and four micrographs (FIGS. 48B, 48C, 48E) showing the effect of daily (Pro ³ ) GIP administration on islet size and morphology in ob / ob mice. , 48F).

(Pro ³ ) GIP treatment had no effect on pancreas weight (FIG. 47A). However, pancreatic insulin levels were significantly (P <0.05) decreased in ob / ob mice given (Pro ³ ) GIP for 11 days compared to controls (FIG. 47B). No significant difference was observed in the number of islets per slice of pancreas (FIG. 47C), but the mean islet diameter was significantly and significantly (obtained in (Pro ³ ) GIP treated ob / ob mice) P <0.001) decreased (FIG. 47D). These effects were effectively reversed by the withdrawal of (Pro ³ ) GIP on day 20, but the mean islet diameter remained significantly reduced (P <0.05). As shown in FIG. 48A, a more detailed analysis shows that the decrease in islet diameter on day 11 shows an increase in the proportion of islets with small (<0.10 mm) and medium (0.1-0.15 mm) diameters. It was revealed that this was due to a significant reduction (P <0.001) in the percentage of larger diameter (> 0.15 mm) islets. FIG. 48D shows that a similar analysis after withdrawal of treatment still reveals a significant (P <0.05) increase in the percentage of small islets. Representative images of pancreas immunohistologically stained for insulin from 11 days (Pro ³ ) GIP treated ob / ob mice (FIG. 48B) and saline treated controls (FIG. 48C). (× 40 magnification) explains the dramatic changes in pancreatic islet morphology induced by (Pro ³ ) GIP treatment. The pancreas immunohistologically stained for day 20 insulin is also shown (FIGS. 48E, 48F).

Indicators were measured after 11 days of (Pro ³ ) GIP (25 nmol / kg body weight / day) or daily treatment with saline (FIG. 48A) and 9 days after discontinuation of treatment (FIG. 48D). . Percentage of islets classified as large (> 0.15 mm), medium (0.1-0.15 mm), and small (<0.1 mm) diameters. Values are mean ± SEM for 8 mice. 48B, 48C, 48E, and 48F are representative images of pancreas stained for insulin after 11 days of treatment with (Pro ³ ) GIP (FIG. 48B) or saline (FIG. 48C). (X40 magnification). The corresponding images 9 days after discontinuation of treatment with (Pro ³ ) GIP (FIG. 48E) or physiological saline (FIG. 48F) are also shown. Arrows indicate islets.

Example 6: GIP acetylated at the N-terminus and substituted with Lys ¹⁶ and Lys ³⁷
This example investigates the metabolic stability, bioactivity, and antidiabetic potential of fatty acid derivatized N-terminal modified GIP analogs. These include N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP with a C-16 palmitate group linked to the N-terminal Tyr ¹ acetyl group of the GIP protein and the epsilon amino group of either lysine at position 16 or 37 (LysPAL ³⁷ ).

Materials and Methods Animals: Obese diabetic (ob / ob) mice from colonies maintained at Aston University, UK, were used at 12-17 weeks of age. The genetic background and characteristics of the colonies used were reviewed in detail elsewhere (Bailey, CJ et al., 1982, Int. J. Obesity. 6: 11-21; Gault, VA et al., 2003, J Endocrinol. 176: 133-141). Animals were individually housed in air-conditioned rooms at 22 ± 2 ° C. using a 12 hour light: 12 hour dark cycle. Drinking water and standard rodent maintenance diet (Trouw Nutrition, Belfast, Northern Ireland) were ad libitum. All test solutions were administered by ip injection at a final volume of 5 ml / kg body weight. Blood was collected into a chilled fluoride / heparin microcentrifuge tube from the distal tip of the tail vein cut of conscious mice immediately before injection and at the time indicated in the figure. Plasma was separated at 13,000 g for about 30 seconds using a Beckman microcentrifuge and stored at −20 ° C. before measuring glucose and insulin. All animal studies were conducted according to the British Animal (Scientific Treatment) Act 1986. No adverse effects were observed after any acute or chronic administration of the peptide.

  Materials: Acetonitrile for high performance liquid chromatography (HPLC) was obtained from Rathburn (Walkersburn, UK). Trifluoroacetic acid (TFA) and trichloroacetic acid (TCA) were obtained from Aldrich (Poole, Dorset, UK). DPP IV, isobutylmethylxanthine (IBMX), α-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, fetal bovine serum, penicillin, and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). Chromatographic columns used for the cyclic AMP assay, Dowex AG50 WX, and neutralized 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 those with the highest purity available.

Synthesis, purification, and characterization of GIP and related analogs: Native GIP was synthesized sequentially using standard solid phase Fmoc peptide chemistry (ABI 432A peptide synthesizer) as previously described ( O'Harte, FPM et al., 2002, Diabetologia 45: 1281-1291). N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) were synthesized in the same manner as natural GIP except that the epsilon amino group of Lys at position 16 or 37 was linked to C-16 palmitate fatty acid. Furthermore, acetyl additives was incorporated at the N-terminal Tyr ^1. The synthetic peptide was determined to be pure by reverse phase HPLC on a Waters Millenium 2010 chromatography system (software version 2.1.5) and then matrix-assisted laser desorption ionization-time of flight as described above. Characterization was performed using mass spectrometry (MALDI-ToF MS) (Gault, VA et al., 2002, Cell. Biol. Int. 27: 41-46).

  Study of DPP IV degradation: GIP and fatty acid derivatized GIP analogs were purified at 37 ° C with purified porcine dipeptidyl peptidase IV (5 mU in 50 mmol / l triethanolamine-HCl; pH 7.8) at 37 ° C. , 4, 8, and 24 hours (final peptide concentration 2 mmol / l). Subsequently, the reaction was terminated by the addition of 10% (v / v) TFA / water and the reaction products were separated using HPLC. The reaction product was applied to a Vydac C-4 column (4.6 × 250 mm; The Separations Group, Hesparia, Calif.) And the main degradation product, GIP (3-42) was separated from the intact GIP. Using 0.1% (v / v) TFA in 70% acetonitrile / water with the acetonitrile concentration in the elution solvent raised from 0% to 40% in 10 minutes and then from 40% to 75% in 35 minutes And equilibrated with 0.12% (v / v) TFA / water at a flow rate of 1.0 ml / min. Absorbance was monitored at 206 nm using a SpectraSystem UV2000 detector (Thermoquest Limited, Manchester, UK) and peaks were collected manually prior to MALDI-ToF MS analysis. HPLC peak area data was used to calculate the percentage of the peptide that retained the original prototype that remained throughout the incubation.

Cells and cell culture: Chinese hamster lung (CHL) fibroblasts stably transfected with human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44: 1202-1208), 10% (v / v) Cultured in DMEM tissue culture medium containing 1% (v / v) antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin). Cloned pancreatic BRIN-BD11 cells (McClenaghan, NH et al., 1996, Diabetes 45: 1132-1140), 10% (v / v) FBS, 1% (v / v) antibiotics (100 U / ml Of penicillin, 0.1 mg / ml streptomycin), and RPMI-1640 medium containing 11.1 mmol / l glucose. Cells were maintained at 37 ° C. in an atmosphere consisting of 5% CO ₂ and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).

Bioactivity in vitro: Intracellular cyclic AMP production was measured using GIP receptor-transfected CHL fibroblasts (O'Harte, FPM et al., 2002, Diabetologia 45: 1281-11291). Briefly, CHL cells were seeded in 12-well plates (Nunc, Roskilde, Denmark) at a density of 10 ⁵ cells per well and allowed to grow for 48 hours before being tritiated adenine (2 μCi; TRK311; Amersham, Buckinghamshire, UK) was added. The cells were then incubated for 6 hours at 37 ° C. in 1 ml DMEM supplemented with 0.5% (w / v) BSA, followed by washing twice with HBS buffer (pH 7.4). The cells were then exposed to GIP / GIP analog (10 ⁻¹³ to 10 ⁻⁶ mol / l) in HBS buffer for 15 minutes at 37 ° C. in the presence of 1 mmol / l IBMX. Subsequently, the medium was removed and the cells were lysed with 1 ml of 5% TCA containing 0.1 mmol / l unlabeled cyclic AMP and 0.1 mmol / l unlabeled ATP. Subsequently, intracellular cyclic AMP was separated by Dowex and alumina exchange resin as previously described (O'Harte, FPM et al., 2002, Diabetologia 45: 1281-11291).

Insulin release studies were performed using cloned pancreatic BRIN-BD11 cells as previously described (O'Harte, FPM et al., 2002, Diabetologia 45: 1281-11291). Briefly, BRIN-BD11 cells were seeded in 24-well plates at a density of 10 ⁵ cells per well and allowed to adhere overnight at 37 ° C. Prior to acute testing for insulin release, incubation was carried out at 37 ° C. for 40 minutes in 1.0 ml Krebs-Ringer bicarbonate buffer supplemented with 1.1 mmol / l glucose. Test incubations were performed with GIP and GIP analogs in a concentration range of (10 ⁻¹³ to 10 ⁻⁶ mol / l) in the presence of 5.6 mmol / l glucose. After a 20 minute incubation, the buffer was removed from each well and an aliquot (200 μl) was used to measure insulin.

Effects of N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) in ob / ob mice: GIP and N-AcGIP (LysPAL) (from 6.25 to 25 nmoles / kg body weight) following glucose administration (18 mmoles / kg body weight) Analog metabolism and dose response effects were investigated in mice fasted for 18 hours. To assess long-term effects, ob / ob mice are treated with saline medium (0.9%, w / v, NaCl), natural GIP (both at 12.5 nmoles / kg body weight / day), or N-AcGIP (LysPAL ³⁷ ) was given an intraperitoneal injection (17:00 h) once a day for 14 days. Food intake and body weight were recorded daily. Plasma glucose and insulin concentrations were monitored at 2-6 day intervals. On day 14, groups of animals were used to assess intraperitoneal glucose tolerance (18 mmoles / kg) and insulin sensitivity (50 U / kg). In another series, two experimental protocols were utilized to investigate the possibility of GIP receptor desensitization after 14 days of treatment. The acute metabolic effects of normal injections consisting of either saline, GIP or N-AcGIP (LysPAL ³⁷ ) were monitored when administered with glucose (18 mmoles / kg). Second, the acute effect of N-AcGIP (LysPAL ³⁷ ) given with glucose was investigated in all three groups of mice. At the end of the 14 day treatment period, pancreatic tissue for measurement of insulin following extraction with 5 ml / g ice-cold acid-ethanol (75% ethanol, 2.35% H ₂ O, 1.5% HCl) Was extracted. Whole blood was collected for measurement of glycated hemoglobin.

  Biochemical analysis: Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, JF, 1971, Clin. Chem. Acta 32: 199-201) using a Beckman Glucose Analyzer II (Beckman, Galway, Ireland) . Plasma insulin was measured by dextran-charcoal RIA as previously described (Flatt, P. R. et al., 1981, Diabetologia 20: 573-577). Using cation exchange columns (Sigma, Poole, Dorset, UK) by measuring absorbance (415nm) of wash buffer and elution buffer using VersaMax microplate and spectrophotometer (Molecular Devices, Wokingham, Berkshire, UK) Then, glycated hemoglobin was measured.

  Statistics: Results are expressed as mean ± SEM. Data were compared using an unpaired student t-test. Data were compared by repeated measures ANOVA or one-way ANOVA, followed by Student-Newman-Kools post hoc test as needed. A computer program that uses the trapezoidal formula (Burington, RS, 1973, Handbook of Mathematical Tables and Formulae, McGraw-Hill, New York) to subtract the incremental area (AUC) under the plasma glucose and insulin curves. Calculated using. Data groups were considered significantly different when p <0.05.

result
Structural characterization by MALDI-ToF MS: Following synthesis and HPLC purification, MALDI-ToF MS is used to obtain molecular weights for GIP, N-AcGIP (LysPAL ¹⁶ ), and N-AcGIP (LysPAL ³⁷ ). (Table 3 below). The mass to charge (m / z) ratio for natural GIP was calculated to be 4983.7 Da, which corresponds very closely to the theoretical mass of 4982.4 Da. Similarly, the m / z ratios for N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) were 5268.9 Da and 5267.7 Da, respectively. These values were very closely correlated with the theoretical mass (5266.1 Da), thus confirming the correct structure for each of the synthetic peptides.

Degradation with DPP IV: Table 4 below describes the percentage of the peptide that retained the prototype remaining after incubation with DPP IV. It was clear that the degradation of native GIP was only 52 ± 3% of the peptide after 2 hours. After 8 hours of incubation, the native peptide was completely degraded to GIP (3-42). In contrast, both N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) remained completely intact even after 24 hours incubation with DPP IV (degraded fragments were evident). Not)

Changes in cyclic AMP production: FIG. 50A shows GIP (solid triangle) as well as N-AcGIP, a fatty acid derivatized GIP analog, measured by column chromatography in CHL cells stably expressing the human GIP receptor. Intracellular cyclic AMP production by (LysPAL ¹⁶ ) (white squares) and N-AcGIP (LysPAL ³⁷ ) (black circles) is shown. Each experiment was performed in triplicate (n = 3) and the results were expressed as a percentage of the maximum GIP response (mean ± SEM).

Concentration-dependent increase in cyclic AMP production (10 ^-6 to 10 ^-13 mol / l) using natural GIP (EC ₅₀ value 18.2 nmol / l) using CHL cells transfected with human GIP receptor (FIG. 50A). Similarly, both N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) have calculated EC ₅₀ values of 12.1 and 13.0 nmol / l, respectively, and stimulation similar to that of natural GIP Followed the pattern. The lower EC ₅₀ values for both analogs suggest increased cyclic AMP stimulation potency.

In vitro insulin release activity: FIG. 50B is glucose (5.6 mmo / l glucose; white bars), GIP (lined bars), and fatty acid derivatized GIP analogs in the cloned pancreatic beta cell line BRIN-BD11 N-AcGIP (LysPAL ¹⁶ ) (gray bar) and N-AcGIP (LysPAL ³⁷ ) (black bar) show insulin release activity. After preincubation (40 minutes), the effect of various concentrations of peptide was tested for insulin release during the 20 minute incubation. Values are mean ± SEM for 8 separate observations. ^* p <0.05, ^** p <0.01, ^*** p <0.001 compared to control (5.6 mmol / l glucose alone).

Consistent with its role as a potent insulinotropic hormone, native GIP stimulated insulin secretion in a dose-dependent manner (p <0.01-p <) compared to the control (5.6 mmol / l glucose alone). 0.001) (Figure 50B). Similarly, both N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) significantly stimulated glucose-induced insulin secretion (p <0.05 to p <0.001). Based on cyclic AMP and insulin secretion data, both GIP analogs appear to be at least as potent as the natural peptide.

Metabolic effects in ob / ob mice: FIGS. 51A-51D show glucose lowering effects of GIP and fatty acid derivatized GIP analogs in 18 / h fasted ob / ob mice (FIGS. 51A, 51B) and insulin release activity (FIGS. C51, 51D) Is a set of two line graphs (FIGS. 51A and 51C) and two bar graphs (FIGS. 51B and 51D). Plasma glucose and insulin concentrations were measured using glucose alone (18 mmoles / kg body weight; ○; white bar), or GIP (black triangle; lined bar), or GIP analog N-AcGIP (LysPAL ¹⁶ ) (white square). Gray bars) and N-AcGIP (LysPAL ³⁷ ) (black circles; black bars) before and after ip injection in combination with 25 nmoles / kg body weight. The incremental area (AUC) under the glucose or insulin curve from 0-60 minutes is shown in the right panel. Values represent mean ± SEM for 8 mice. ^{* P <0.05, ** p <} 0.01, *** p <0.001 is compared with glucose ^{alone, Δ p <0.05, ΔΔ p} <0.01 and ^ΔΔΔ p <0.001, is compared to native GIP, ^{Ganmaganmaganma} p <0.001 compared to N-AcGIP (LysPAL ¹⁶ ).

The basal blood glucose levels in the experimental group were not significantly different at the start of the study (p> 0.05). Following the injection of glucose alone, plasma glucose levels rose rapidly and reached a value of 40.3 ± 1.5 mmol / l in 60 minutes. Natural GIP lowered plasma glucose at each time point monitored, but this did not reach significance in terms of overall glucose excursion, as evidenced by AUC values ( Figure 52B). Administration of N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) resulted in a significant decrease in plasma glucose at each time point (p <0.01 to p <0.001) and significantly when compared to glucose alone Caused decreased glucose AUC (p <0.01-p <0.001). Moreover, N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) reduced the overall glucose excursion when compared to native GIP (p <0.05, p <0.001).

The corresponding plasma insulin response is illustrated in FIGS. 51C and 51D. After administration of glucose alone (control), a maximal increase in plasma insulin was observed at 15 minutes, after which they decreased towards basal levels over the remaining 45 minutes. Natural GIP administration significantly enhanced the overall insulin secretagogue response compared to glucose alone (p <0.05). N-AcGIP (LysPAL ¹⁶ ) or N-AcGIP (LysPAL ³⁷ ) administered with glucose observed a maximum plasma insulin concentration at 15 minutes. Prolonged bioactivity for both analogs was clearly evident for 30-60 minutes.

Glucose-mediated plasma insulin concentrations were significantly higher compared to both controls (p <0.01-p <0.001) and GIP-treated animals (p <0.05-p <0.001). The AUC values corresponding to N-AcGIP (LysPAL ¹⁶ ) and N-AcGIP (LysPAL ³⁷ ) are higher than that of natural GIP (1.5 times and 2.3 times, respectively; p <0.01 to p <0.001). A significant increase in mediated insulin release was revealed. N-AcGIP (LysPAL ³⁷ ) was significantly more potent than NAcGIP (LysPAL ¹⁶ ) in stimulating insulin secretion (1.5 fold: p <0.001).

Dose-dependent metabolic effects in ob / ob mice: FIGS. 52A and 52B show the dose of GIP and the more potent analog N-AcGIP (LysPAL ³⁷ ) when administered to ob / ob mice with glucose. The dependent antihyperglycemic effect and insulin secretion promoting effect will be described.

They are a set of bar graphs showing the dose-dependent effects of GIP and N-AcGIP (LysPAL ³⁷ ) in ob / ob mice fasted for 18 hours. Ip administration of glucose alone (18 mmoles / kg body weight; white bar), or GIP (lined bar), or N-AcGIP (LysPAL ³⁷ ) (6.25, 12.5, and 25 nmoles / kg body weight; black bar, respectively) Incremental area under the curve (AUC) for glucose (FIG. 52A) and insulin (FIG. 52B) 0-60 minutes after. Values are mean ± SEM for 8 mice. ^** p <0.01 and ^*** p <0.001 is compared with glucose alone, ^{.DELTA..delta} p <0.01 and ^ΔΔΔ p <0.001 were compared to native GIP at the same dose.

Data are expressed as an overall AUC response for convenience. Expressed in this manner, native GIP did not significantly affect AUC glucose and insulin at any dose tested. N-AcGIP (LysPAL ³⁷ ) is significantly more potent than native GIP (p <0.01-p <0.001) and has a significant dose-dependent antihyperglycemic effect and insulin secretion at all doses administered The promoting action was shown (FIGS. 52A and 52B). Indeed, even the lowest concentration of N-AcGIP (LysPAL ³⁷ ) (6.25 nmoles / kg) tested had significantly higher antihyperglycemic properties compared to glucose alone (p <0.001). Consistent with this observation, 6.25 nmoles / kg N-AcGIP (LysPAL ³⁷ ) elicited a significant insulin response (2.0 fold; p <0.01) compared to glucose alone.

Long-acting effects in ob / ob mice: Effect of 14-day daily injection of N-AcGIP (LysPAL ³⁷ ) on food intake, body weight, glycated hemoglobin, and nonfasting plasma glucose and insulin concentrations in ob / ob mice N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg /) for food intake (Figure 53A), body weight (Figure 53B), plasma glucose (Figure 53C), insulin (Figure 53D), and glycated hemoglobin (Figure 53E) FIGS. 53A to 53E, which are sets of graphs showing the effect of daily administration of (day) (black circle; black bar). Natural GIP (12.5 nmoles / kg / day; black triangle; lined bar) or saline medium (control; white square; white bar) was administered for 14 days as indicated by the horizontal black bar. Values are mean ± SEM for 8 mice. ^* p <0.05, ^** p <0.01 compared to control. ^ΔΔp <0.01 compared to natural GIP.

GIP or N-AcGIP (LysPAL ³⁷ ) had no effect on body weight or food intake (FIGS. 53A and 53B). Plasma glucose and insulin concentrations were similarly unchanged by 14 days of natural GIP treatment (FIGS. 53C, 53D). In contrast, daily injections of N-AcGIP (LysPAL ³⁷ ) resulted in a gradual drop in plasma glucose that resulted in a significantly (p <0.05) lowered concentration at day 14 (FIG. 53C). At this time, glycated hemoglobin was also significantly decreased (p <0.01) in ob / ob mice treated with N-AcGIP (LysPAL ³⁷ ) (FIG. 53E). These changes occur concomitant with a trend towards increased insulin concentrations, but they did not achieve statistical significance over the time frame study (FIG. 53D).

Effects of long-term treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) on glucose tolerance: FIGS. 54A-54D show glucose tolerance to glucose (FIGS. 54A, 54B) and plasma insulin response (FIGS. 54C, 54D) FIG. 5 is a set of two line graphs (FIGS. 54A and 54C) and two bar graphs (FIGS. 54B and 54D) showing the effects of daily N-AcGIP (LysPAL ³⁷ ) administration. The test was performed using N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg / day; black circle; black bar), natural GIP (12.5 nmoles / kg / day; black triangle; lined bar), or physiological saline medium ( Controls; white squares; white bars) were performed after 14 consecutive days of injection. Glucose (18 mmoles / kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose and insulin AUC 0-60 minutes after injection are shown in the right panel. Values are mean ± SEM for 8 mice. ^* p <0.05, ^** p <0.01, ^*** p <0.001 compared to control. ^{Δ p <0.05, ΔΔ p <} 0.01, ΔΔΔ p <0.001 were compared to native GIP.

Consistent with the effect on glycated hemoglobin, treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) for 14 days resulted in a significant improvement in glucose tolerance (FIGS. 54A, 54B). Plasma glucose concentrations throughout the study, and overall AUC of 60-60 minutes decreased (p <0.01-p <0.001). This occurred at the same time as the elevated insulin concentration (p <0.05) and higher (p <0.01) overall AUC insulin response during the late phase (FIGS. 54C, 54D). In contrast, daily administration of natural GIP had no effect on glucose tolerance or plasma insulin response to glucose compared to control ob / ob mice that received 14 days of saline injection (FIG. 54). .

Effects of long-term treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) on insulin sensitivity and long-term treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) on pancreatic insulin levels: FIGS. 55A-55D Is a single line graph and three bar graphs showing the effects of daily N-AcGIP (LysPAL ³⁷ ) administration on insulin sensitivity (FIGS. 55A, 55B) and pancreas weight (FIG. 55C) and insulin content (FIG. 55D) is there. Observations were made using N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg / day; black circles; black bars), natural GIP (12.5 nmoles / kg / day; black triangles; lined bars), or physiological saline medium ( Controls; white squares; white bars) were performed after 14 consecutive days of injection. In FIG. 55A, insulin (50 U / kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose AUC values from 0-60 minutes after injection are shown in the right panel. Values are mean ± SEM for 8 mice. ^* p <0.05, ^** p <0.01 compared to control. ^{Δ p <0.05, ΔΔ p <} 0.01 was compared to native GIP.

The insulin sensitivity of the three groups of mice after 14 days of treatment is shown in FIGS. 55A and 55B. Compared to ob / ob mice that received daily injections of saline or natural GIP, N-AcGIP (LysPAL ³⁷ ) caused a significant improvement in insulin sensitivity. Both individual glucose concentrations and 0-60 min AUC values were significantly different from the other two groups (p <0.01). In contrast, daily treatment with native GIP did not affect the characteristic insulin resistance of ob / ob mice (FIGS. 55A, 55B).

Treatment of ob / ob mice with native GIP or N-AcGIP (LysPAL ³⁷ ) for 14 days had no effect on pancreas weight compared to saline treated controls (FIGS. 55C, 55D). Similarly, pancreatic insulin levels were similar in groups treated with GIP and saline. However, daily administration with N-AcGIP (LysPAL ³⁷ ) significantly increased the amount of insulin compared to each of the other groups (p <0.01) (FIGS. 55C and 55D).

Evaluation of GIP receptor desensitization after long-term treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ): FIGS. 56A-56D show N-AcGIP (LysPAL ³⁷ ) and native after 14 days of daily injection FIG. 5 is a set of two line graphs (FIGS. 56A, 56C) and two bar graphs (FIGS. 56B, 56D) showing GIP glucose lowering (FIGS. 56A, 56B) and insulin release (FIGS. 56C, 56D) activity. Glucose (18 mmoles / kg) alone (white squares; white bars), or N-AcGIP (LysPAL ³⁷ ) (black circles; black bars) or natural GIP (black triangles; linear bars) (both 25 nmoles / kg) Were administered at the time indicated by the arrows by intraperitoneal injection in combination with any of the above. Plasma glucose and insulin AUC values 0-60 minutes after injection are shown in the right panel. Values are mean ± SEM for 8 mice. ^* P <0.05, ^** p <0.01 compared to glucose alone. ^{Δ p <0.05, ΔΔ p <} 0.01 was compared to native GIP. 57A-57D show N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg / day; black circle; black bar), natural GIP (12.5 mnoles / kg / day; black triangle; lined bar), or physiological salt Acute glucose lowering (FIGS. 57A, 57B) and insulin release effect (FIGS. 57C, 57D) of N-AcGIP (LysPAL ³⁷ ) after 14 days of daily injection in either aqueous medium (control; white square; white bar) ) Are two line graphs (FIGS. 57A and 57C) and two bar graphs (FIGS. 57B and 57D). N-AcGIP (LysPAL ³⁷ ) (25 nmoles / kg) was administered by intraperitoneal injection with glucose (18 mmoles / kg) at the time indicated by the arrows. Plasma glucose and insulin AUC values 0-60 minutes after injection are shown in the right panel. Values are mean ± SEM for 8 mice. ^* p <0.05, ^** p <0.01 compared to mice receiving control injection. ^{Δ p <0.05, ΔΔ p <} 0.01 was compared with the group that received injections of the native GIP.

As shown in FIGS. 56A-56D, treatment of ob / ob mice with N-AcGIP (LysPAL ³⁷ ) for 14 days significantly moderates the range of motion of blood glucose when administered acutely with intraperitoneal glucose. (P <0.01) and did not interfere with the peptide's ability to increase plasma insulin concentrations (p <0.01). In contrast, the response of ob / ob mice to acute administration of native GIP was almost the same in mice treated with GIP or saline for 14 days (FIGS. 56A-56D). N-AcGIP To further demonstrate a lack of GIP receptor desensitization after chronic treatment with (LysPAL ^37), the acute effects of the analog to be administered in conjunction with ^{glucose, N-AcGIP (LysPAL 37)} , Investigated in each of three groups after 14 days of treatment with natural GIP or saline (FIGS. 57A-57D). Aside from the lower basal values in the previously mentioned group, glucose and insulin responses were consistent with similar 0-60 minute AUC amounts for both plasma glucose and insulin concentrations.

  While the present invention has been shown and described in detail by reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail are encompassed by the appended claims. It may be added thereto without departing from the scope of the invention.

Describe the decomposition of GIP by DPP IV. The degradation of GIP and Tyr ¹ glucitol GIP by DPP IV is described. Describe the degradation of GIP by human plasma. The degradation of GIP and Tyr ¹ glucitol GIP by human plasma is described. Electrospray ionization mass spectrometry of the main degradation fragments of GIP, Tyr1-glucitol GIP, and GIP (3-42) will be described. The effects of GIP and glycated GIP on plasma glucose homeostasis are shown. The effect of GIP on plasma insulin response is shown. The DPP-IV decomposition of GIP (1-42) will be described. The DPP-IV decomposition of GIP (Abu ² ) will be described. The DPP-IV decomposition of GIP (Sar ² ) will be described. The DPP-IV decomposition of GIP (Ser ² ) will be described. The DPP-IV degradation of N-acetyl-GIP is described. The DPP-IV degradation of glycated GIP will be described. Describe human plasma degradation of GIP. Describe human plasma degradation of GIP (Abu ² ). Describe human plasma degradation of GIP (Sar ² ). The human plasma degradation of GIP (Ser ² ) will be described. The human plasma degradation of glycated GIP will be described. FIG. 6 shows the effect of various concentrations of GIP 1-42 and GIP (Abu ² ) on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. It shows the effects of various concentrations of GIP 1-42 and GIP (Abu ²⁾ on insulin release from BRIN-BD 11 cells incubated with 16.7mM glucose. Figure 3 shows the effect of various concentrations of GIP 1-42 and GIP (Sar ² ) on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. Figure 5 shows the effect of various concentrations of GIP 1-42 and GIP (Sar ² ) on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. Figure 3 shows the effect of various concentrations of GIP 1-42 and GIP (Ser ² ) on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. Figure 3 shows the effect of various concentrations of GIP 1-42 and GIP (Ser ² ) on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. Shows the effect of various concentrations of GIP 1-42 and N-acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. Shows the effect of various concentrations of GIP 1-42 and N-acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. Figure 5 shows the effect of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. Figure 6 shows the effect of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. FIG. 5 shows the effect of various concentrations of GIP 1-42 and GIP (Gly ² ) on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. FIG. 6 shows the effect of various concentrations of GIP 1-42 and GIP (Gly ² ) on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. FIG. 5 shows the effect of various concentrations of GIP 1-42 and GIP (Pro ³ ) on insulin release from BRIN-BD11 cells incubated with 5.6 mM glucose. FIG. 5 shows the effect of various concentrations of GIP 1-42 and GIP (Pro ³ ) on insulin release from BRIN-BD11 cells incubated with 16.7 mM glucose. 1 shows the primary structure of human gastric inhibitory polypeptide (GIP) (SEQ ID NO: 1). 1 shows the primary structure of porcine gastric inhibitory polypeptide (GIP) (SEQ ID NO: 2). They are a line graph and a bar graph showing the effects of (Pro ³ ) GIP on cyclic AMP production stimulated by GIP in vitro and insulin secretion, respectively. They are a line graph and a bar graph showing the effects of (Pro ³ ) GIP on cyclic AMP production stimulated by GIP in vitro and insulin secretion, respectively. A~F is, Glu ³ on insulin secretion stimulated by GIP in vitro is a set of six bar graph showing the effect of GIP and GIP form substituted (3-42). A-F is a set of 6 bar graphs showing the effect of GIP substituted for Glu ³ on GIP-stimulated insulin secretion in vitro and the effect of GIP (3-42). A-F is a set of 6 bar graphs showing the effect of GIP substituted for Glu ³ on GIP-stimulated insulin secretion in vitro and the effect of GIP (3-42). A-F is a set of 6 bar graphs showing the effect of GIP substituted for Glu ³ on GIP-stimulated insulin secretion in vitro and the effect of GIP (3-42). A-F is a set of 6 bar graphs showing the effect of GIP substituted for Glu ³ on GIP-stimulated insulin secretion in vitro and the effect of GIP (3-42). A-F is a set of 6 bar graphs showing the effect of GIP substituted for Glu ³ on GIP-stimulated insulin secretion in vitro and the effect of GIP (3-42). (Pro ³ ) showed that acute administration of GIP completely neutralizes the effects of GIP on glucose tolerance (Figure 34A, 34B) and plasma insulin (Figure 34C, 34D) responses in obese diabetic ob / ob mice , A set of two line graphs (FIGS. 34A, 34C) and two bar graphs (FIGS. 34B, 34D). (Pro ³ ) showed that acute administration of GIP completely neutralizes the effects of GIP on glucose tolerance (Figure 34A, 34B) and plasma insulin (Figure 34C, 34D) responses in obese diabetic ob / ob mice , A set of two line graphs (FIGS. 34A, 34C) and two bar graphs (FIGS. 34B, 34D). (Pro ³ ) showed that acute administration of GIP completely neutralizes the effects of GIP on glucose tolerance (Figure 34A, 34B) and plasma insulin (Figure 34C, 34D) responses in obese diabetic ob / ob mice , A set of two line graphs (FIGS. 34A, 34C) and two bar graphs (FIGS. 34B, 34D). (Pro ³ ) showed that acute administration of GIP completely neutralizes the effects of GIP on glucose tolerance (Figure 34A, 34B) and plasma insulin (Figure 34C, 34D) responses in obese diabetic ob / ob mice , A set of two line graphs (FIGS. 34A, 34C) and two bar graphs (FIGS. 34B, 34D). (Pro ³ ) Two line graphs showing that acute administration of GIP decreased physiological, diet-stimulated insulin release and worsened blood glucose excursions in obese diabetic ob / ob mice (Figure 35A, 35C) and two bar graphs (FIGS. 35B, 35D). (Pro ³ ) Two line graphs showing that acute administration of GIP decreased physiological, diet-stimulated insulin release and worsened blood glucose excursions in obese diabetic ob / ob mice (Figure 35A, 35C) and two bar graphs (FIGS. 35B, 35D). (Pro ³ ) Two line graphs showing that acute administration of GIP decreased physiological, diet-stimulated insulin release and worsened blood glucose excursions in obese diabetic ob / ob mice (Figure 35A, 35C) and two bar graphs (FIGS. 35B, 35D). (Pro ³ ) Two line graphs showing that acute administration of GIP decreased physiological, diet-stimulated insulin release and worsened blood glucose excursions in obese diabetic ob / ob mice (Figure 35A, 35C) and two bar graphs (FIGS. 35B, 35D). A and B are a set of two bar graphs showing that chronic administration of (Pro ³ ) GIP for 11 days reduces plasma glucose and insulin concentrations in obese diabetic ob / ob mice. A and B are a set of two bar graphs showing that chronic administration of (Pro ³ ) GIP for 11 days reduces plasma glucose and insulin concentrations in obese diabetic ob / ob mice. A to C of three bar graphs showing that chronic administration of (Pro ³ ) GIP for 11 days reduces glycated HbA _1c , pancreatic insulin levels, and concomitant islet hypertrophy in obese diabetic ob / ob mice Is a set. A to C of three bar graphs showing that chronic administration of (Pro ³ ) GIP for 11 days reduces glycated HbA _1c , pancreatic insulin levels, and concomitant islet hypertrophy in obese diabetic ob / ob mice Is a set. A to C of three bar graphs showing that chronic administration of (Pro ³ ) GIP for 11 days reduces glycated HbA _1c , pancreatic insulin levels, and concomitant islet hypertrophy in obese diabetic ob / ob mice Is a set. Two line graphs (Figures 38A and 38C) and two bar charts showing that chronic administration of (Pro ³ ) GIP for 11 days improves glucose tolerance in obese diabetic ob / ob mice without changes in circulating insulin (FIGS. 38B and 38D). Two line graphs (Figures 38A and 38C) and two bar charts showing that chronic administration of (Pro ³ ) GIP for 11 days improves glucose tolerance in obese diabetic ob / ob mice without changes in circulating insulin (FIGS. 38B and 38D). Two line graphs (Figures 38A and 38C) and two bar charts showing that chronic administration of (Pro ³ ) GIP for 11 days improves glucose tolerance in obese diabetic ob / ob mice without changes in circulating insulin (FIGS. 38B and 38D). Two line graphs (Figures 38A and 38C) and two bar charts showing that chronic administration of (Pro ³ ) GIP for 11 days improves glucose tolerance in obese diabetic ob / ob mice without changes in circulating insulin (FIGS. 38B and 38D). 11 is a line graph showing that chronic administration of (Pro ³ ) GIP for 11 days improves insulin sensitivity in obese diabetic ob / ob mice. 2 is a line graph showing that the beneficial effects of 11 days of chronic administration of (Pro ³ ) GIP in obese diabetic ob / ob mice are reversed 9 days after discontinuation of treatment. A and B are a set of two line graphs showing that chronic administration of (Pro ³ ) GIP for 11 days causes impaired glucose tolerance in normal mice that are subverted 9 days after discontinuation of treatment. A and B are a set of two line graphs showing that chronic administration of (Pro ³ ) GIP for 11 days causes impaired glucose tolerance in normal mice that are subverted 9 days after discontinuation of treatment. 4 is a set of two line graphs (FIGS. 42A, 42C) and two bar graphs (FIGS. 42B, 42D) showing the effect of (Pro ³ ) GIP on plasma glucose and insulin response to native GIP 4 hours after administration . Two lines showing the effects of daily (Pro ³ ) GIP administration on food intake (Figure 43A), body weight (Figure 43B), plasma glucose (Figure 43C), and insulin (Figure 43D) concentrations in ob / ob mice A set of graphs and two bar graphs. AD is a set of four line graphs with an inserted bar graph showing the effect of daily (Pro ³ ) GIP administration on glucose tolerance and plasma insulin response to glucose in ob / ob mice. AD are daily (Pro ³ ) GIP administration (black triangles; black bars) to glucose (Figures 45A, 45B) and insulin (Figures 45C, 45D) response to diet in ob / ob mice fasted for 18 hours or FIG. 4 is a set of two line graphs (FIGS. 45A and 45C) and two bar graphs (FIGS. 45B and 45D) showing the effect of physiological saline (white squares; white bars). AD is a set of two line graphs (FIGS. 46A, 46C) and two bar graphs (FIGS. 46B, 46D) showing the effect of daily (Pro ³ ) GIP administration on insulin sensitivity in ob / ob mice . AD is the effect of daily (Pro ³ ) GIP administration on pancreas weight (Figure 47A), insulin content (Figure 47B), islet number (Figure 47C), and islet diameter (Figure 47D) in ob / ob mice Is a set of four bar graphs. AF are two bar graphs (FIGS. 48A, 48D) and four micrographs (FIGS. 48B, 48C, 48E,) showing the effect of daily (Pro ³ ) GIP administration on islet size and morphology in ob / ob mice. 48F). How GIP Receptor (“GIP-R”) Antagonist, (Pro ³ ) GIP, Improves Glucose Tolerance and Diabetes Management Against Beta Cell Hyperplasia, Hyperinsulinemia, and Insulin Resistance It is an explanation. Intracellular cyclic AMP by GIP (black triangle) in cloned pancreatic beta cell line BRIN-BD11 and fatty acid derivatized GIP analogs N-AcGIP (LysPAL ¹⁶ ) (white square) and N-AcGIP (LysPAL ³⁷ ) (black circle) Production (FIG. 50A), and glucose (5.6 mmo / l glucose; white bars), GIP (lined bars), and fatty acid derivatized GIP analogs (FIG. 50B) N-AcGIP (LysPAL ¹⁶ ) (gray bars) 2 is a line graph and a bar graph showing the insulin releasing activity of N-AcGIP (LysPAL ³⁷ ) (black bar), respectively. Two line graphs (Fig. 51A, 51C) showing the glucose lowering effect of GIP and fatty acid derivatized GIP analogs (Figs. 51A, 51B) and insulin release activity (Figs. 51C, 51D) in ob / ob mice fasted for 18 hours. ) And two bar graphs (FIGS. 51B and 51D). 2 is a pair of bar graphs showing dose-dependent effects of GIP and N-AcGIP (LysPAL ³⁷ ) in ob / ob mice fasted for 18 hours. Daily N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg /) for food intake (Figure 53A), body weight (Figure 53B), plasma glucose (Figure 53C), insulin (Figure 53D), and glycated hemoglobin (Figure 53E) It is a set of graphs showing the effect of administration of (day) (black circle; black bar). Two line graphs (Figs. 54A, 54C) showing the effect of daily N-AcGIP (LysPAL ³⁷ ) administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin response to glucose (Figs. 54C, 54D) and two It is a set of bar graphs (FIGS. 54B and 54D). 1 line graph and 3 bar graphs showing the effect of daily N-AcGIP (LysPAL ³⁷ ) administration on insulin sensitivity (FIGS. 55A, 55B) and pancreas weight (FIG. 55C) and insulin content (FIG. 55D) . Two line graphs showing the activity of N-AcGIP (LysPAL ³⁷ ) and natural GIP glucose lowering (Figures 56A, 56B) and insulin release (Figures 56C, 56D) after 14 days of daily injection (Figures 56A, 56C) ) And two bar graphs (FIGS. 56B and 56D). N-AcGIP (LysPAL ³⁷ ) (12.5 nmoles / kg / day; black circle; black bar), natural GIP (12.5 nmoles / kg / day; black triangle; lined bar), or saline medium (control) Showed the effect of acute glucose reduction (Figures 57A, 57B) and insulin release (Figures 57C, 57D) of N-AcGIP (LysPAL ³⁷ ) after 14-day daily injection of any of the white squares; It is a set of two line graphs (FIGS. 57A and 57C) and two bar graphs (FIGS. 57B and 57D).

Claims (40)

  A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (3-42). A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ³ . The peptide analog according to claim 1 or 2, wherein the amino acid substituted with Glu ³ is selected from the group consisting of the following: proline, hydroxyproline, lysine, tyrosine, phenylalanine, and tryptophan. 4. The peptide analog of claim ³ , wherein the proline is substituted for Glu3.   The peptide analog according to claim 1 or 2, further comprising a modification by fatty acid addition in the epsilon amino group of at least one lysine residue.   6. The peptide analog according to claim 5, wherein the modification is a linkage of a C-16 palmitate group to an epsilon amino group of a lysine residue. The peptide analog according to claim 6, wherein the lysine residue is Lys ¹⁶ . 7. The peptide analog according to claim 6, wherein the lysine residue is Lys ³⁷ .   GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid modification at the first, second, or third amino acid residue ) Peptide analogs.   10. The peptide analog according to claim 9, wherein the N-terminal amino acid residue is acetylated.   11. The peptide analog of claim 10, further comprising a modification by fatty acid addition at the epsilon amino group of at least one lysine residue.   12. The peptide analog of claim 11, wherein the modification is a linkage of a C-16 palmitate group to an epsilon amino group of a lysine residue. 13. The peptide analog according to claim 12, wherein the lysine residue is Lys ¹⁶ . 13. The peptide analog according to claim 12, wherein the lysine residue is Lys ³⁷ .   15. Use of an analogue according to any one of claims 1-14 in the preparation of a medicament for the treatment of obesity, insulin resistance, insulin resistant metabolic syndrome (syndrome X), or type 2 diabetes.   A pharmaceutical composition comprising the peptide analog according to any one of claims 1 to 14.   The pharmaceutical composition according to claim 16, further comprising a pharmaceutically acceptable carrier.   17. The pharmaceutical composition according to claim 16, wherein the peptide analog is in the form of a pharmaceutically acceptable salt.   17. The pharmaceutical composition according to claim 16, wherein the peptide analog is in the form of a pharmaceutically acceptable acid addition salt.   17. A method of treating insulin resistance comprising administering a therapeutically effective amount of the composition of claim 16 to a mammal in need of insulin resistance treatment.   17. A method for treating obesity comprising administering a therapeutically effective amount of the composition of claim 16 to a mammal in need of treatment for obesity.   17. A method for treating type 2 diabetes comprising the step of administering a therapeutically effective amount of the composition of claim 16 to a mammal in need of treatment for type 2 diabetes. The following: GIP (1-12), GIP (1-13), GIP (1-14), GIP (1-15), GIP (1-16), GIP (1-17), GIP (1-18) ), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1-22), GIP (1-23), GIP (1-24), GIP (1-25) , GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36), GIP (1-37), GIP (1-38), GIP (1-39), GIP (1-40), GIP (1-41), and a base peptide consisting of one of GIP (1-42);
A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising:
The following modifications:
Amino acid substitution at one or more residues;
Amino acid substitution of lysine for one or more residues;
Amino acid substitution at Glu ³ ;
Modification by fatty acid addition at the epsilon amino group of at least one lysine residue; and
Modification by N-terminal acetylation,
The peptide analog having one or more of: The analog has a proline substituted for Glu ^3, peptide analogue according to claim 23.   24. The peptide analog of claim 23, further comprising a modification by fatty acid addition at the epsilon amino group of at least one lysine residue.   26. The peptide analog of claim 25, wherein the modification is a linkage of a C-16 palmitate group to an epsilon amino group of a lysine residue. 27. The peptide analog of claim 26, wherein the lysine residue is Lys ¹⁶ . 27. The peptide analog of claim 26, wherein the lysine residue is Lys ³⁷ .   29. Use of an analogue according to any one of claims 23 to 28 in the preparation of a medicament for the treatment of obesity, insulin resistance, insulin resistant metabolic syndrome (syndrome X) or type 2 diabetes.   A pharmaceutical composition comprising the peptide analog according to any one of claims 23 to 28.   32. The pharmaceutical composition according to claim 30, further comprising a pharmaceutically acceptable carrier.   32. The pharmaceutical composition according to claim 30, wherein the peptide analog is in the form of a pharmaceutically acceptable salt.   32. The pharmaceutical composition according to claim 30, wherein the peptide analog is in the form of a pharmaceutically acceptable acid addition salt.   32. A method of treating insulin resistance comprising administering a therapeutically effective amount of the composition of claim 30 to a mammal in need of insulin resistance treatment.   32. A method of treating obesity comprising administering a therapeutically effective amount of the composition of claim 30 to a mammal in need of treatment for obesity.   32. A method for treating type 2 diabetes comprising the step of administering a therapeutically effective amount of the composition of claim 30 to a mammal in need of treatment for type 2 diabetes.   A peptide analog of GIP (1-42) (SEQ ID NO: 1), comprising at least 12 amino acid residues from the N-terminus of GIP (3-42), when compared to native GIP, the enzyme DPP IV Said peptide analogues resistant to degradation by. A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ³ , The peptide analog, which is resistant to degradation by the enzyme DPP IV when compared to GIP.   A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (3-42), wherein said peptide analog regulates insulin secretion. A peptide analog of GIP (1-42) (SEQ ID NO: 1) comprising at least 12 amino acid residues from the N-terminus of GIP (1-42) and having an amino acid substitution at Glu ³ , comprising insulin Said peptide analogue that regulates secretion.

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