Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

The isolation and purification of tris-2,2'-bipyridine complexes of ruthenium(II) containing unsymmetrical ligands

Monomeric ruthenium(II) complexes [Ru(L)3]2+ containing unsymmetric bipyridine ligands [Where L = 5-methyl-2,2'-bipyridine (L1), 5-ethyl-2,2'-bipyridine (L2), 5-propyl-2,2'-bipyridine (L3), 5-(2-methylpropyl)-2,2'-bipyridine (L4), 5-(2,2-dimethylpropyl)-2,2'-bipyridine (L5) and 5-(carbomethoxy)-2,2'-bipyridine (L6)] have been studied and the meridional and facial isomers isolated by the use of cation-exchange column chromatography (SP Sephadex C-25) eluting with either sodium toluene-4-sulfonate or sodium hexanoate. The relative yield of the facial isomer was found to decrease with increasing steric bulk, preventing the isolation of fac-[Ru(L5)3]2+. The two isomeric forms were characterized by 1H NMR, with the complexes [Ru(L1-3)3]2+ demonstrating an unusually large coupling between the H6 and H4 protons. Crystals suitable for X-ray structural analysis of [Ru(L1)3]2+ were obtained as a mixture of the meridional and facial isomers, indicating that separation of this isomeric mixture could not be achieved by fractional crystallisation. The optical isomers of the complex [Ru(L3)3]2+ were chromatographically separated on SP Sephadex C-25 relying upon the inherent chirality of the support. It is apparent that chiral interactions can inhibit geometric isomer separation using this technique.

Differential effects of the CCKA receptor ligands PD-140, 548 and A-71623 on latent inhibition in the rat.

Latent inhibition (LI) is a behavioural paradigm in which repeated exposure to a stimulus without consequence inhibits the formation of any new associations with that stimulus. To the extent that LI reflects a process of learning to ignore irrelevant stimuli, disrupted LI has been suggested as an animal model for the attentional deficits observed in schizophrenia. The antipsychotic potential of cholecystokinin (CCK) stems from its colocalization with dopamine (DA) in the mesolimbic pathway, where it demonstrates both excitatory and inhibitory effects on dopaminergic activity. This may be explained by mediation through different receptor subtypes. A variety of hypotheses has emerged regarding the potential clinical application of subtype-selective CCK-based drugs. The present experiments examined the effects on LI of two selective CCKA ligands: PD-140,548 (a CCKA antagonist, Experiment 1: 0.001, 0.01, and 0.1 mg/kg) and A-71623 (a CCKA agonist, Experiment 2: 0.02, 0.05, and 0.1 mg/kg). In both experiments, the effects of haloperidol (0.1 mg/kg) were also investigated. Animals receiving 0.1 mg/kg of haloperidol or 0.001 or 0.1 mg/kg (but not 0.01 mg/kg) of PD-140,548 treated the preexposed stimulus as irrelevant after a low number of preexposures. In contrast, no facilitatory effect on LI was detectable at any of the A-71623 doses. The finding that A-71623 failed to enhance LI indicates that it is unlikely that this compound would have any antipsychotic effect within the clinical setting. Considering the facilitatory effect exerted by PD-140,548 on LI, it is probable that the inhibition of CCK activity might prove a more promising strategy for the pharmacological treatment of schizophrenia.

Amyloid B-peptide 1-40 increases neuronal membrane fluidity: role of cholesterol and brain region

There is increasing evidence of an interaction between cholesterol dynamics and Alzheimer's disease (AD), and amyloid ß-peptide may play an important role in this interaction. Aß destabilizes brain membranes and this action of Aß may be dependent on the amount of membrane cholesterol. We tested this hypothesis by examining effects of Aß1-40 on the annular fluidity (i.e., lipid environment adjacent to proteins) and bulk fluidity of rat synaptic plasma membranes (SPM) of the cerebral cortex, cerebellum, and hippocampus using the fluorescent probe pyrene and energy transfer. Amounts of cholesterol and phospholipid of SPM from each brain region were determined. SPM of the cerebellum were significantly more fluid as compared with SPM of the cerebral cortex and hippocampus. Aß significantly increased (P 0.01) annular and bulk fluidity in SPM of cerebral cortex and hippocampus. In contrast, Aß had no effect on annular fluidity and bulk fluidity of SPM of cerebellum. The amounts of cholesterol in SPM of cerebral cortex and hippocampus were significantly higher (P 0.05) than amount of cholesterol in SPM of cerebellum. There was significantly less (P 0.05) total phospholipid in cerebellar SPM as compared with SPM of cerebral cortex. Neuronal membranes enriched in cholesterol may promote accumulation of Aß by hydrophobic interaction, and such an interpretation is consistent with recent studies showing that soluble Aß can act as a seed for fibrillogenesis in the presence of cholesterol.

Altered pericyte-endothelial relations in the rat retina during aging: Implications for vessel stability

Mural cells (smooth muscle cells and pericytes) regulate blood flow and contribute to vessel stability. We examined whether mural cell changes accompany age-related alterations in the microvasculature of the central nervous system. The retinas of young adult and aged Wistar rats were subjected to immunohistofluorescence analysis of a-smooth muscle actin (SMA), caldesmon, calponin, desmin, and NG2 to identify mural cells. The vasculature was visualized by lectin histochemistry or perfusion of horse-radish peroxidase, and vessel walls were examined by electron microscopy. The early stage of aging was characterized by changes in peripheral retinal capillaries, including vessel broadening, thickening of the basement membrane, an altered length and orientation of desmin filaments in pericytes, a more widespread SMA distribution and changes in a subset of pre-arteriolar sphincters. In the later stages of aging, loss of capillary patency, aneurysms, distorted vessels, and foci of angiogenesis were apparent, especially in the peripheral deep vascular plexus. The capillary changes are consistent with impaired vascular autoregulation and may result in reduced pericyte-endothelial cell contact, destabilizing the capillaries and rendering them susceptible to angiogenic stimuli and endothelial cell loss as well as impairing the exchange of metabolites required for optimal neuronal function. This metabolic uncoupling leads to reactivation of

Synthetic peptides interacting with the 67-kd laminin receptor can reduce retinal ischemia and inhibit hypoxia-induced retinal neovascularization.

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF33–42) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF33–42 significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P <0.0001). The agonist peptide (Lamß1925–933) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P <0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.