Fatty acid derivatised analogues of glucose-dependent insulinotropic polypeptide with improved antihyperglycaemic and insulinotropic properties

C-terminal acylation of Lys³⁷ with myristic (MYR; tetradecanoic acid), palmitic (PAL; hexadecanoic acid) and stearic (octadecanoic acid) fatty acids with or without N-terminal acetylation was employed to develop long-acting analogues of the glucoregulatory hormone, glucose-dependent insulinotropic polypeptide (GIP). All GIP analogues exhibited resistance to dipeptidylpeptidase-IV (DPP-IV) and significantly improved in vitro cAMP production and insulin secretion. Administration of GIP analogues to ob/ob mice significantly lowered plasma glucose—GIP(Lys³⁷MYR), N-AcGIP(Lys³⁷MYR) and GIP(Lys³⁷PAL) increased plasma insulin concentrations. GIP(Lys³⁷MYR) and N-AcGIP(Lys³⁷MYR) elicited protracted glucose-lowering effects when administered 24 h prior to an intraperitoneal glucose load. Daily administration of GIP(Lys³⁷MYR) and N-AcGIP(Lys³⁷MYR) to ob/ob mice for 24 days decreased glucose and significantly improved plasma insulin, glucose tolerance and beta-cell glucose responsiveness. Insulin sensitivity, pancreatic insulin content and triglyceride levels were not changed. These data demonstrate that C-terminal acylation particularly with myristic acid provides a class of stable, longer-acting forms of GIP for further evaluation in diabetes therapy.     

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(Lys(37)PAL) and N-AcGIP(Lys(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25nmolkg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(Lys(37)PAL) or N-AcGIP(Lys(37)PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP.     

Acetaminophen normalizes glucose homeostasis in mouse models for diabetes

Loss of pancreatic beta cell insulin secretion is the most important element in the progression of type 1 and type 2 diabetes. Since oxidative stress is involved in the progressive loss of beta cell function, we evaluated the potential for the over-the-counter analgesic drug and antioxidant, acetaminophen (APAP), to intervene in the diabetogenic process. We used mouse models for type 1 diabetes (streptozotocin) and type 2 diabetes (high-fat diet) to examine the ability of APAP to intervene in the progression of diabetes. In C57BL/6J mice, streptozotocin caused a dosage dependent increase in fasting blood glucose (FBG), from 100 to >600mg/dl. Daily APAP (20mg/kg BW, gastric gavage), significantly prevented and partially reversed the increase in FBG levels produced by streptozotocin. After 10 weeks on a high-fat diet, mice developed fasting hyperinsulemia and impaired glucose tolerance compared to animals fed a control diet. APAP largely prevented these changes in insulin and glucose tolerance. Furthermore, APAP prevented most of the increase in body fat in mice fed the high-fat diet. One protective mechanism for APAP is suggested by studies using isolated liver mitochondria, where low micromolar concentrations abolished the production of reactive oxygen that might otherwise contribute to the destruction of pancreatic beta-cells. These findings suggest that administration of APAP to mice, in a dosage used safely by humans, reduces the production of mitochondrial reactive oxygen and concomitantly prevents the development of type 1 and type 2 diabetes in established animal models.     

Disturbance of zinc and glucose homeostasis by methyl tert-butyl ether (MTBE); evidence for type 2 diabetes

1.?The prevalence of diabetes and the other metabolic disorders has noticeably increased worldwide. A causal link between increasing risk of type 2 diabetes and exposure to environmental pollutants has been reported.

2.?We hypothesized that exposure to methyl tert-butyl ether (MTBE), an oxygenate additive to gasoline would hinder zinc and glucose homeostasis in rats.

3.?Male Sprague–Dawley rats received MTBE in drinking water for 90 days. At the end of the treatment, pancreas and blood samples were collected for biochemical and molecular examinations. Expression of four candidate genes, including Insulin1, Insulin2, MT1A, SLC30A8 by Real-Time Quantitative PCR (Q-PCR) as well as biochemical parameters, including fasting blood glucose (FBS), triglycerides (TG), cholesterol (CHO), low-density lipoprotein (LDL), high-density lipoprotein (HDL), copper (Cu²⁺) and calcium (Ca²⁺) levels as well as High-sensitive C-reactive protein were assessed as endpoints.

4.?This study suggested that MTBE exposure can be associated with disruption in zinc homeostasis and glucose tolerance.     

Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: a significant role of specific binding by serum albumin in the improved potency and stability

AS-3201 [(3R)-2'-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4'(1'H)-pyrrolo[1,2-a]pyrazine]-1',2,3',5(2'H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 (R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode (S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA-R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation. (13)C NMR observations of the succinimide ring (5-(13)C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and (19)F NMR of the pendent benzyl ring (2-(19)F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.     

Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties

Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.     

Improving the ex vivo stability of drug ester compounds in rat and dog serum: Inhibition of the specific esterases and implications on their identity

In drug development, it has been noticed that some drug compounds, especially esters, are unstable in serum samples ex vivo. This can lead to a substantial underestimation of the actual drug concentration.