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.

Dry and moist heat sterilisation cannot inactivate pyrogenicity of Gram positive microorganisms.

In the monocytic cell line Mono Mac 6 pyrogens induce interleukin-6 secretion dose dependently. The aim of this study is to examine the interleukin-6 inducing capacity of Gram positive Staphylococcus aureus and Bacillus subtilis endospores after moist and dry heat sterilisation. Moist heat sterilisation of B. subtilis endospores for 15 min at 121 degrees C and 134 degrees C can only reduce the interleukin-6 inducing capacity to 57% and 63%, respectively, compared to untreated. Moist heat sterilisation of S. aureus for 60 min at 121 degrees C and 134 degrees C does not reduce the interleukin-6 inducing capacity of S. aureus. On the contrary moist heat sterilisation at 134 degrees C for 10, 20 and 40 min significantly increases the interleukin-6 inducing capacity compared to untreated S. aureus. This is confirmed in the rabbit pyrogen test. Dry heat sterilisation of B. subtilis endospores at 220 degrees C for 45 min reduces the interleukin-6 inducing capacity to 2% compared to untreated endospores. Dry heat treatment of S. aureus at 220 degrees C for 30 min only reduces the activity to 55%. However, after 250 degrees C for 30 min or 220 degrees C for 6h there is no detectable activity of S. aureus. In conclusion, neither the interleukin-6 inducing activity nor the pyrogenicity of S. aureus and endospores of B. subtilis can be inactivated by the heat sterilisation procedures described by the European Pharmacopoeia.

Identification and in vivo and in vitro characterization of long acting and melanocortin 4 receptor (MC4-R) selective α-melanocyte-stimulating hormone (α-MSH) analogues.

We report in vitro and in vivo data of new α-melanocyte-stimulating hormone (α-MSH) analogues which are N-terminal modified with a long chain fatty acid derivative. While keeping the pharmacophoric motif (d-Phe-Arg-Trp) fixed, we tried to improve selectivity and physicochemical parameters like solubility and stability of these analogues by replacing amino acids further away from the motif. Receptor specific changes in binding affinity to the melanocortin receptors were observed between the acetyl derivatives and the fatty acid analogues. Furthermore, amino acids at the N-terminal of α-MSH (Ser-Tyr-Ser) not considered to be part of the pharmacophore were found to have an influence on the MC4/MC1 receptor selectivity. While the acetyl analogues have an in vivo effect for around 7 h, the long chain fatty acid analogues have an effect up to 48 h in an acute feeding study in male Sprague-Dawley rats after a single subcutaneous administration.

Chemical and thermal stability of insulin: effects of zinc and ligand binding to the insulin zinc-hexamer.

PURPOSE: To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands. MATERIALS AND METHODS: The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry. RESULTS: An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn(B3) was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37 degrees C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated. CONCLUSIONS: Thermal stability can be used to predict the rate of Asn(B3) deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.

Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer.

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate >> p-aminobenzoate >> chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.

Thermal dissociation and unfolding of insulin.

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].