NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Whole transcriptome analysis and validation of metabolic pathways in subcutaneous adipose tissues during FGF21-induced weight loss in non-human primates.

Fibroblast growth factor 21 (FGF21) induces weight loss in mouse, monkey, and human studies. In mice, FGF21 is thought to cause weight loss by stimulating thermogenesis, but whether FGF21 increases energy expenditure (EE) in primates is unclear. Here, we explore the transcriptional response and gene networks active in adipose tissue of rhesus macaques following FGF21-induced weight loss. Genes related to thermogenesis responded inconsistently to FGF21 treatment and weight loss. However, expression of gene modules involved in triglyceride (TG) synthesis and adipogenesis decreased, and this was associated with greater weight loss. Conversely, expression of innate immune cell markers was increased post-treatment and was associated with greater weight loss. A lipogenesis gene module associated with weight loss was evaluated by testing the function of member genes in mice. Overexpression of NRG4 reduced weight gain in diet-induced obese mice, while overexpression of ANGPTL8 resulted in elevated TG levels in lean mice. These observations provide evidence for a shifting balance of lipid storage and metabolism due to FGF21-induced weight loss in the non-human primate model, and do not fully recapitulate increased EE seen in rodent and in vitro studies. These discrepancies may reflect inter-species differences or complex interplay of FGF21 activity and counter-regulatory mechanisms.

Colonic absorption of salmon calcitonin using tetradecyl maltoside (TDM) as a permeation enhancer.

Calcitonin is used as a second line treatment of postmenopausal osteoporosis, but widespread acceptance is somewhat limited by subcutaneous and intranasal routes of delivery. This study attempted to enable intestinal sCT absorption in rats using the mild surfactant, tetradecyl maltoside (TDM) as an intestinal permeation enhancer. Human Caco-2 and HT29-MTX-E12 mucus-covered intestinal epithelial monolayers were used for permeation studies. Rat in situ intestinal instillation studies were conducted to evaluate the absorption of sCT with and without 0.1 w/v% TDM in jejunum, ileum and colon. TDM significantly enhanced sCT permeation across intestinal epithelial monolayers, most likely due to combined paracellular and transcellular actions. In situ, TDM caused an increased absolute bioavailability of sCT in rat colon from 1.0% to 4.6%, whereas no enhancement increase was observed in ileal and jejunal instillations. Histological analysis suggested mild perturbation of colonic epithelia in segments instilled with sCT and TDM. These data suggest that the membrane composition of the colon is different to the small intestine and that it is more amenable to permeation enhancement. Thus, formulations designed to release payload in the colon could be advantageous for systemic delivery of poorly permeable molecules.

Evaluation of alkylmaltosides as intestinal permeation enhancers: comparison between rat intestinal mucosal sheets and Caco-2 monolayers.

Alkylmaltosides are a class of non-ionic surfactant currently in clinical trials to improve nasal permeation of peptide drugs, however few studies have detailed their potential effects on intestinal permeation enhancement. Tetradecyl maltoside (TDM, C(14)), was examined in Caco-2 monolayers and in isolated rat jejunal and colonic mucosae mounted in Ussing chambers. Dodecyl maltoside (DDM, C(12)) was examined in mucosae. Parameters measured included critical micelle concentration (CMC), transepithelial electrical resistance (TEER), and apparent permeability coefficients (P(app)) of paracellular and transcellular flux markers. TDM and DDM decreased TEER and increased the P(app) of [(14)C]-mannitol and FD-4 across Caco-2 monolayers and colonic mucosae in the concentration range of 0.01-0.1% w/v, concentrations much higher than the CMC. Remarkably, neither agent had any effect on the TEER or fluxes of jejunal mucosae. Histopathology, cell death assays (MTT and LDH) and sub-lethal high content cytotoxicity analyses (HCA) were carried out with TDM. Exposure of colonic mucosae to high concentrations of TDM had no major effects on gross histology and ion transport function was retained. In Caco-2, HCA data at sub-lethal concentrations of TDM was consistent with the action of a mild non-ionic surfactant. In conclusion, alkylmaltosides are effective non-toxic permeation enhancers in isolated colonic tissue and their inclusion in oral peptide formulations directed to that intestinal region warrants further study.

Human glucagon receptor antagonists with thiazole cores. A novel series with superior pharmacokinetic properties.

The aim of the work presented here was to design and synthesize potent human glucagon receptor antagonists with improved pharmacokinetic (PK) properties for development of pharmaceuticals for the treatment of type 2 diabetes. We describe the preparation of compounds with cyclic cores (5-aminothiazoles), their binding affinities for the human glucagon and GIP receptors, as well as affinities for rat, mouse, pig, dog, and monkey glucagon receptors. Generally, the compounds had slightly less glucagon receptor affinity compared to compounds of the previous series, but this was compensated for by much improved PK profiles in both rats and dogs with high oral bioavailabilities and sustained high plasma exposures. The compounds generally showed species selectivity for glucagon receptor binding with poor affinities for the rat, mouse, rabbit, and pig receptors. However, dog and monkey glucagon receptor affinities seem to reflect the human situation. One compound of this series, 18, was tested intravenously in an anesthetized glucagon-challenged monkey model of hyperglucagonaemia and hyperglycaemia and was shown dose-dependently to decrease glycaemia. Further, high plasma exposures and a long plasma half-life (5.2 h) were obtained.

Novel glucagon receptor antagonists with improved selectivity over the glucose-dependent insulinotropic polypeptide receptor.

Optimization of a new series of small molecule human glucagon receptor (hGluR) antagonists is described. In the process of optimizing glucagon receptor antagonists, we counter-screened against the closely related human gastric inhibitory polypeptide receptor (hGIPR), and through structure activity analysis, we obtained compounds with low nanomolar affinities toward the hGluR, which were selective against the hGIPR and the human glucagon-like peptide-1 receptor (hGLP-1R). In the best cases, we obtained a >50 fold selectivity for the hGluR over the hGIPR and a >1000 fold selectivity over the hGLP-1R. A potent and selective glucagon receptor antagonist was demonstrated to inhibit glucagon-induced glycogenolysis in primary rat hepatocytes as well as to lower glucagon-induced hyperglycemia in Sprague-Dawley rats. Furthermore, the compound was shown to lower blood glucose in the ob/ob mouse after oral dosing.

Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers.

In this study, we investigated bi-directional fluxes (i.e., in absorptive and secretive directions) of human serum proteins [albumin (HSA), transferrin (TF), and immunoglobulin G (IgG)] and peptides/proteins of potential therapeutic relevance [insulin (INS), glucagon-like peptide-1 (GLP-1), growth hormone (GH), and parathyroid hormone (PTH)] across tight monolayers of human alveolar epithelial cells (hAEpC) in primary culture. Apparent permeability coefficients (P(app); x10(-7)cm/s, mean+/-S.D.) for GLP-1 (6.13+/-0.87 (absorptive) versus 1.91+/-0.51 (secretive)), HSA (2.45+/-1.02 versus 0.21+/-0.31), TF (0.88+/-0.15 versus 0.30+/-0.03), and IgG (0.36+/-0.22 versus 0.15+/-0.16) were all strongly direction-dependent, i.e., net absorptive, while PTH (2.20+/-0.30 versus 1.80+/-0.77), GH (8.33+/-1.24 versus 9.02+/-3.43), and INS (0.77+/-0.15 versus 0.72+/-0.36) showed no directionality. Trichloroacetic acid precipitation analysis of tested molecules collected from donor and receiver fluids exhibited very little degradation. This is the first study on permeability data for a range of peptides and proteins across an in vitro model of the human alveolar epithelial barrier. These data indicate that there is no apparent size-dependent transport conforming to passive restricted diffusion for the tested substances across human alveolar barrier, in part confirming net absorptive transcytosis. The obtained data differ significantly from previously published reports utilising monolayers from different species. It can be concluded that the use of homologous tissue should be preferred to avoid species differences.

In silico prediction of membrane permeability from calculated molecular parameters.

A data set consisting of 712 compounds was used for classification into two classes with respect to membrane permeation in a cell-based assay: (0) apparent permeability (P(app)) below 4 x 10(-6) cm/s and (1) P(app) on 4 x 10(-6) cm/s or higher. Nine molecular descriptors were calculated for each compound and Nearest-Neighbor classification was applied using five neighbors as optimized by full cross-validation. A model based on five descriptors, number of flex bonds, number of hydrogen bond acceptors and donors, and molecular and polar surface area, was selected by variable selection. In an external test set of 112 compounds, 104 compounds were classified and 8 compounds were judged as "unknown". Among the 104 compounds, 16 were misclassified corresponding to a misclassification rate of 15% and no compounds were falsely predicted in the nonpermeable class.

Glutamate receptor ligands: synthesis, stereochemistry, and enantiopharmacology of methylated 2-aminoadipic acid analogs.

Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA) analogs, and the synthesis, stereochemistry, and enantiopharmacology of 3-methyl-AA (4a-d), 4-methyl-AA (5a-d), 5-methyl-AA (6a-d), and (E)-Delta(4)-5-methyl-AA (7a and 7b) are reported. The compounds were resolved using chiral HPLC and the configurational assignments of the enantiomers were based on X-ray crystallographic analyses, chemical correlation, and CD spectral analyses. The effects of the individual stereoisomers at ionotropic and metabotropic (S)-Glu receptors (iGluRs and mGluRs) were characterized. Compounds with S-configuration at the alpha-carbon generally showed mGluR2 agonist activity of similar or slightly lower potencies than (S)-AA [e.g., EC(50) = 76 microM for (2S,4S)-4-methyl-AA (5a) as compared to EC(50) = 35 microM for (S)-AA]. The position of the methyl substituent had a profound effect on the observed pharmacology, whereas the absolute stereochemistry at the methylated carbon atom had a very limited effect on pharmacology. Structure-activity relationships at iGluRs in the rat cortical wedge preparation showed a complex pattern, some compounds being NMDA receptor agonists [e.g., EC(50) =110 microM for (2S,5RS)-5-methyl-AA (6a,b)] and some compounds showing NMDA receptor antagonist effects [e.g., IC(50) = 300 microM for (2R,4S)-4-methyl-AA (5d)]. The two unsaturated analogs (S)- (7a) and (R)-(E)-Delta(4)-5-methyl-AA (7b) turned out to be a weak AMPA receptor agonist and a weak mixed NMDA/AMPA receptor antagonist, respectively.