Demonstration of a Common DPhe to DNal(2') Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression.

The effect of fatty diacid acylation of human PYY on Y receptor potency and half-life in minipigs.

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide.

Development of Cagrilintide, a Long-Acting Amylin Analogue.

A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide () and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication.

Molecular Engineering of Insulin Icodec, the First Acylated Insulin Analog for Once-Weekly Administration in Humans.

Here, we describe the molecular engineering of insulin icodec to achieve a plasma half-life of 196 h in humans, suitable for once-weekly subcutaneously administration. Insulin icodec is based on re-engineering of the ultra-long oral basal insulin OI338 with a plasma half-life of 70 h in humans. This systematic re-engineering was accomplished by (1) further increasing the albumin binding by changing the fatty diacid from a 1,18-octadecanedioic acid (C18) to a 1,20-icosanedioic acid (C20) and (2) further reducing the insulin receptor affinity by the B16Tyr → His substitution.

prediction of pseudo-allergenic response via MRGPRX2.

In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect.

Engineering of Orally Available, Ultralong-Acting Insulin Analogues: Discovery of OI338 and OI320.

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, , in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis.

Insulin binding to the analytical antibody sandwich pair OXI-005 and HUI-018: Epitope mapping and binding properties.

The insulin epitopes for two monoclonal antibodies (mAbs), OXI-005 and HUI-018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X-ray crystallography. The crystal structure of the HUI-018 Fab in complex with human insulin (HI) was determined and OXI-005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI-005 epitope comprises insulin residues 1,3,4,19-21 (A-chain) and 25-30 (B-chain) and for HUI-018 residues 7,8,10-14,17 (A-chain) and 5-7, 10, 14 (B-chain).

Elucidating the Mechanism of Absorption of Fast-Acting Insulin Aspart: The Role of Niacinamide.

Purpose: Fast-acting insulin aspart (faster aspart) is a novel formulation of insulin aspart containing two additional excipients: niacinamide, to increase early absorption, and L-arginine, to optimize stability. The aim of this study was to evaluate the impact of niacinamide on insulin aspart absorption and to investigate the mechanism of action underlying the accelerated absorption.

Methods: The impact of niacinamide was assessed in pharmacokinetic analyses in pigs and humans, small angle X-ray scattering experiments, trans-endothelial transport assays, vascular tension measurements, and subcutaneous blood flow imaging.

The tension of framed membranes from computer simulations.

We have analyzed the behavior of a randomly triangulated, self-avoiding surface model of a flexible, fluid membrane subject to a circular boundary by Wang-Landau Monte Carlo computer simulation techniques. The dependence of the canonical free energy and frame tension on the frame area is obtained for flexible membranes. It is shown that for low bending rigidities the framed membrane is only stable above a threshold tension, suggesting a discontinuous transition from the collapsed (branched polymer) state to a finite tension extended state.

Changes in glucose and fat metabolism in response to the administration of a hepato-preferential insulin analog.

Endogenous insulin secretion exposes the liver to three times higher insulin concentrations than the rest of the body. Because subcutaneous insulin delivery eliminates this gradient and is associated with metabolic abnormalities, functionally restoring the physiologic gradient may provide therapeutic benefits. The effects of recombinant human insulin (HI) delivered intraportally or peripherally were compared with an acylated insulin model compound (insulin-327) in dogs.

Efficient excitation of channel plasmons in tailored, UV-lithography-defined V-grooves.

We demonstrate the highly efficient (>50%) conversion of freely propagating light to channel plasmon-polaritons (CPPs) in gold V-groove waveguides using compact 1.6 μm long waveguide-termination coupling mirrors. Our straightforward fabrication process, involving UV-lithography and crystallographic silicon etching, forms the coupling mirrors innately and ensures exceptional-quality, wafer-scale device production.

The chromatographic separation of particles using optical electric fields.

We introduce a new field-flow fractionation (FFF) technique, whereby molecules are separated based on their differential interaction (dielectrophoresis (DEP)) with optical electric fields, i.e. electric fields with frequencies in the visible and near-infrared range.

Recombinant adiponectin does not lower plasma glucose in animal models of type 2 diabetes.

Aims/hypothesis: Several studies have shown that adiponectin can lower blood glucose in diabetic mice. The aim of this study was to establish an effective adiponectin production process and to evaluate the anti-diabetic potential of the different adiponectin forms in diabetic mice and sand rats.

Methods: Human high molecular weight, mouse low molecular weight and mouse plus human globular adiponectin forms were expressed and purified from mammalian cells or yeast.

Wafer scale imprint uniformity evaluated by LSPR spectroscopy: a high volume characterization method for nanometer scale structures.

We exploit the localized surface-plasmon resonance (LSPR) of terahertz gold gammadion structures for wafer scale critical dimension metrology of nanostructures. The proposed characterization method, LSPR spectroscopy, is based on optical transmission measurements and is benchmarked against numerical simulations of imprinted structures characterized by atomic force microscopy. There is a fair agreement between the two methods and the simulations enable the translation of optical spectra to critical dimensions of the physical structures, a concept known from scatterometry.

Electromagnetically induced transparency in metamaterials at near-infrared frequency.

We employ a planar metamaterial structure composed of a split-ring-resonator (SRR) and paired nano-rods to experimentally realize a spectral response at near-infrared frequencies resembling that of electromagnetically induced transparency. A narrow transparency window associated with low loss is produced, and the magnetic field enhancement at the center of the SRR is dramatically changed, due to the interference between the resonances with significantly different linewidths. The variation of the spectral response in terms of relative position of the bright and dark elements is evaluated with numerical simulations.

Comparison of a luminescent oxygen channeling immunoassay and an ELISA for detecting insulin aspart in human serum.

The study was a comparison between a Luminescent Oxygen Channeling Immunoassay (LOCI) and an enzyme-linked immunosorbent assay (ELISA) for quantification of Insulin Aspart (IAsp) in human serum. The advantage of LOCI compared to ELISA is reduced workload and higher throughput. The ELISA assay was performed as published (Andersen et al.

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.

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.

Design of a partial PPARdelta agonist.

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.

Novel selective PPARdelta agonists: optimization of activity by modification of alkynylallylic moiety.

Y-shaped molecules bearing alkynylallylic moieties were found to be potent and selective PPARdelta activators. The alkynylallylic moiety was synthesized from alkyn-1-ols by hydroalumination followed by a cross-coupling reaction. Series of active compounds 6 were obtained by stepwise changing the structure of the known PPARpan agonist 5 into Y-shaped compounds.

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors.

Design of potent PPARalpha agonists.

Computational analysis of the ligand binding pocket of the three PPAR receptor subtypes was utilized in the design of potent PPARalpha agonists. Optimum PPARalpha potency and selectivity were obtained with substituents having van der Waals volume around 260. Compound 6 had a PPARalpha potency of 0.

Identification and synthesis of a novel selective partial PPARdelta agonist with full efficacy on lipid metabolism in vitro and in vivo.

The aim was to identify a novel selective PPARdelta agonist with full efficacy on free fatty acid (FFA) oxidation in vitro and plasma lipid correction in vivo. Using the triple PPARalpha,gamma,delta agonist 1 as the structural starting point, we wanted to investigate the possibility of obtaining selective PPARdelta agonists by modifying only the acidic part of 1, while holding the lipophilic half of the molecule constant. The structure-activity relationship was guided by in vitro transactivation data using the human PPAR receptors, FFA oxidation efficacy performed in the rat muscle L6 cell line, and in vivo rat pharmacokinetic properties.

Antagonistic targeting of the histamine H3 receptor decreases caloric intake in higher mammalian species.

The main purpose of this study was to examine the effects of a selective histamine H(3) receptor antagonist, NNC 38-1202, on caloric intake in pigs and in rhesus monkeys. The compound was given intragastrically (5 or 15 mg/kg), to normal pigs (n=7) and subcutaneously (1 or 0.1mg/kg) to obese rhesus monkeys (n=9).

Small-molecule agonists for the glucagon-like peptide 1 receptor.

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs.