Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu(9)-substituted analogues of glucagon-like peptide-1

Glucagonlike peptide-1(7 36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the Nterminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPPIV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4 10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP 1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IVmediated degradation.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala(8)-substituted GLP-1 analogues

The hormone glucagonlike peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucosedependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys(26) residue and to combine this modification with substitutions of the Ala(8) residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val(8),Lys(pal)(26)]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val(8),Lys(pal)(26)]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

Voltammetry of oxygen in the room-temperature ionic liquids 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide and hexyltriethylammonium bis((trifluoromethyl)sulfonyl)imide: One-electron reduction to form superoxide. Steady-state and transient behavior in the same cyclic voltammogram resulting from widely different diffusion coefficients of oxygen and superoxide

The electrochemical reduction of oxygen in two different room-temperature ionic liquids, 1-ethyl-3-methylimidazolium bis((trifluoromethyl)sulfonyl)imide ([EMIM][N(Tf)(2)]) and hexyltriethylammonium bis((trifluoromethyl)sulfonyl)imide ([N-6222][N(Tf)(2)]) was investigated by cyclic voltammetry at a gold microdisk electrode. Chronoamperometric measurements were made to determine the diffusion coefficient, D, and concentration, c, of the electroactive oxygen dissolved in the ionic liquid by fitting experimental transients to the Aoki model. [Aoki, K.; et al. J. Electroanal. Chem. 1981, 122, 19]. A theory and simulation designed for cyclic voltammetry at microdisk electrodes was then employed to determine the diffusion coefficient of the electrogenerated superoxide species, O-2(.-), as well as compute theoretical voltammograms to confirm the values of D and c for neutral oxygen obtained from the transients. As expected, the diffusion coefficient of the superoxide species was found to be smaller than that of the oxygen in both ionic liquids. The diffusion coefficients of O-2 and O-2(.-) in [N-6222][N(Tf)(2)], however, differ by more than a factor of 30 (D-O2 = 1.48 x 10(-10) m(2) s(-1), DO2.- = 4.66 x 10(-12) m(2) s(-1)), whereas they fall within the same order of magnitude in [EMIM][N(Tf)(2)] (D-O2 = 7.3 x 10(-10) m(2) s(-1), DO2.- = 2.7 x 10(-10) m(2) s(-1)). This difference in [N-6222][N(Tf)(2)] causes pronounced asymmetry in the concentration distributions of oxygen and superoxide, resulting in significant differences in the heights of the forward and back peaks in the cyclic voltammograms for the reduction of oxygen. This observation is most likely a result of the higher viscosity of [N-6222][N(Tf)(2)] in comparison to [EMIM][N(Tf)(2)], due to the structural differences in cationic component.

GLP-1 and related peptides cause concentration-dependent relaxation of rat aorta through a pathway involving KATP and cAMP

increasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentration-dependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients. (c) 2008 Elsevier Inc. All rights reserved.

The cyclic “silver-diphos” motif [Ag2(-diphosphine)2]2+ as a synthon for building up larger structure structures

The dinuclear, cyclic structural motif [Ag-2(diphosphine)(2)](2+), here termed the

Effects of antidiabetic drugs on dipeptidyl peptidase IV activity: Nateglinide is an inhibitor of DPP IV and augments the antidiabetic activity of glucagon-like peptide-1

Dipeptidyl peptidase IV (DPP IV) is the primary inactivator of glucoregulatory incretin hormones. This has lead to development of DPP IV inhibitors as a new class of agents for the treatment of type 2 diabetes. Recent reports indicate that other antidiabetic drugs, such as metformin, may also have inhibitory effects on DPP IV activity. In this investigation we show that high concentrations of several antidiabetic drug classes, namely thiazolidinediones, sulphonylureas, meglitinides and morphilinoguanides can inhibit DPP IV The strongest inhibitor nateglinide, the insulin-releasing meglitinide was effective at low therapeutically relevant concentrations as low as 25 mu mol/l. Nateglinide also prevented the degradation of glucagon-like peptide-1 (GLP-1) by DPP IV in a time and concentration-dependent manner. In vitro nateglinide and GLP-1 effects on insulin release were additive. In vivo nateglinide improved the glucose-lowering and insulin-releasing activity of GLP-1 in obese-diabetic ob/ob mice. This was accompanied by significantly enhanced circulating concentrations of active GLP-1(7-36)amide and lower levels of DPP IV activity. Nateglinide similarly benefited the glucose and insulin responses to feeding in ob/ob mice and such actions were abolished by coadministration of exendin(9-39) and (Pro(3))GIP to block incretin hormone action. These data indicate that the use of nateglinide as a prandial insulin-releasing agent may partly rely on inhibition of GLP-1 degradation as well as beta-cell K-ATP channel inhibition. (C) 2007 Elsevier B.V. All rights reserved.

1,2-Cyclic sulfite and sulfate furanoside diesters: improved syntheses and stability

The facile syntheses of 1,2- and 3,5-cyclic sulfite and sulfate furanoside diesters were conducted in molecular solvents and ionic liquids in the presence of immobilised morpholine. Molecular solvents and ionic liquids performed similarly with regards to overall yields. However, the use of ILs allowed for the reactions to be carried out under atmospheric conditions and showed good recyclability. Additionally, increases in product stability was achieved in ILs over organic solvents, in particular, in bis{(trifluoromethanesulfonyl)imide) and trispentafluoro-ethyltrifluorophosphate-based ionic liquids, which were also excellent media to control the hydrolysis of thionyl chloride and sulfuryl chloride. (C) 2009 Elsevier Ltd. All rights reserved.

Ligand differentiated complementary Rh-catalyst systems for the enantioselective desymmetrization of meso-cyclic anhydrides

Two distinct systems for the rhodium-catalyzed enantioselective desymmetrization of meso-cyclic anhydrides have been developed. Each system has been optimized and are compatible with the use of in situ prepared organozinc reagents. Rhodium/PHOX species efficiently catalyze the addition of alkyl nucleophiles to glutaric anhydrides, while a rhodium/phosphoramidite system is effective in the enantioselective arylation of succinic and glutaric anhydrides.