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. RESULTS: Niacinamide increased the rate of early insulin aspart absorption in pigs, and pharmacokinetic modelling revealed this effect to be most pronounced up to ~30-40 min after injection in humans. Niacinamide increased the relative monomer fraction of insulin aspart by ~35%, and the apparent permeability of insulin aspart across an endothelial cell barrier by ~27%. Niacinamide also induced a concentration-dependent vasorelaxation of porcine arteries, and increased skin perfusion in pigs. CONCLUSION: Niacinamide mediates the acceleration of initial insulin aspart absorption, and the mechanism of action appears to be multifaceted. Niacinamide increases the initial abundance of insulin aspart monomers and transport of insulin aspart after subcutaneous administration, and also mediates a transient, local vasodilatory effect.

Recombinant activated factor VII is reabsorbed in renal proximal tubules and is a ligand to megalin and cubilin.

BACKGROUND/AIMS: Recombinant activated factor VIIa (rFVIIa) is used for treatment of haemophilia patients with inhibitors. Tissue distribution studies in rats have shown that injected (125)I-rFVIIa accumulates in organs such as the liver and the kidneys. In this study, we explored which mechanism could be involved in renal clearance of rFVIIa. METHODS: Immunohistochemistry was used for examination of the renal distribution in detail after injection of rFVIIa to mice and rats. Surface plasmon resonance evaluated specific binding of rFVIIa to megalin and cubilin. The biological function of megalin and cubilin in rFVIIa endocytosis was explored in opossum kidney (OK) cells. RESULTS: Staining of rFVIIa was observed only in endosomes and lysosomes within proximal convoluted tubules from renal cortex of mice and rats. Specific binding of rFVIIa to megalin and cubilin was in the presence of receptor-associated protein (RAP) obliterated and reduced by approximately 50%, respectively. Immunofluorescence microscopy and a quantitative cellular endocytosis showed uptake in OK cells of either rFVIIa or (125)I-rFVIIa, and this uptake was significantly decreased in the presence of RAP. CONCLUSION: We suggest that the renal cortex plays a significant role in clearance of injected rFVIIa and that endocytosis and degradation of rFVIIa in proximal tubule cells is mediated via binding to megalin and cubilin.

Subcutaneous Administration of Insulin is Associated With Regional Differences in Injection Depot Variability and Kinetics in The Rat.

BACKGROUND: In humans, subcutaneous administration of insulin in the abdominal region or arm is associated with a faster absorption compared to the thigh or buttocks. We hypothesised that this is partly caused by differences in injection depot structure and kinetics and that the variability in insulin exposure differs between injection sites. MATERIAL AND METHODS: Regional effects on insulin pharmacokinetics were evaluated in a series of studies in Sprague Dawley rats dosed subcutaneously with insulin aspart in the neck or flank. Injection depots were visualised using µCT after subcutaneous dosing with insulin aspart mixed with the contrast agent iomeprol, and insulin exposure was determined between the scans by Luminescent Oxygen Channeling Immunoassay. RESULTS: Insulin absorption was significantly delayed by subcutaneous dosing in the flank compared to the neck region (p<0.01 or less). This delay was associated with smaller depots, as measured by reduced depot volume and surface area (p<0.001). Furthermore, the delayed absorption correlated with a slower depot disappearance (p<0.001). Regional differences in depot variability were not reflected by similar differences in pharmacokinetic variability. CONCLUSION: Structure and kinetics of subcutaneous injection depots-as detected by µCT scans-predict insulin exposure and may thus contribute to the regional differences in insulin pharmacokinetics. The present methodology is applicable for visualisation of insulin injection depots in vivo. Our results did however not support a link between the variability in depot size and insulin pharmacokinetics.

The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes.

Recombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ perfused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.