The stability of insulin in solid formulations containing melezitose and starch. Effects of processing and excipients.

Solid insulin formulations obtained by different methods of preparation were compared with respect to chemical stability and morphology. Spray- and freeze-drying, solution enhanced dispersion by supercritical fluids (SEDS) and precipitation into starch microspheres were the methods used for preparation of solid powders. The excipients applied were melezitose, starch, and sodium taurocholate. The stability of the samples was evaluated after storage in open containers at 25 degrees C and 30% RH for 6 months. All samples were amorphous after processing and storage as detected by XRD, except for the starch microspheres which were semi-crystalline. The spray- and freeze-dried samples containing melezitose and sodium taurocholate experienced a significant water uptake during storage, resulting in changes in morphology and disappearance of Tg. However, the chemical stability of these samples did not seem to be affected by the water uptake. Changes in morphology were not observed for the SEDS powders and the starch microspheres. The chemical stability of the samples was assessed by HPLC. In general, conventional spray- and freeze drying resulted in samples with higher chemical stability compared to SEDS powders and starch microspheres. Nevertheless, the excipients applied were observed to be of major importance, and further optimization of the formulation as well as processing conditions may lead to slightly different conclusions.

Displacement of adsorbed insulin by Tween 80 monitored using total internal reflection fluorescence and ellipsometry.

PURPOSE: This study was conducted to investigate the mechanism of action in the displacement of adsorbed insulin from a hydrophobic surface by Tween 80 and of the competitive adsorption of the two species. METHODS: Total internal reflection fluorescence (TIRF) and ellipsometry were used as in situ methods to examine the processes taking place at hydrophobic model surfaces in the presence of insulin and Tween 80. RESULTS: TIRF studies showed that the displacement of insulin by Tween 80 could be fitted to a sigmoidal function, indicating a nucleation-dependent process. Furthermore, a linear dependence between the apparent rate constant and the logarithm of the Tween 80 concentration was found. Competitive adsorption from solution mixtures of insulin and Tween 80 indicated that insulin was adsorbed first, but subsequently displaced by the surfactant. This displacement proved also to be dependent on the concentration of Tween 80 in the mixture. CONCLUSIONS: The results indicate that Tween 80 at concentrations above critical micelle concentration can be used to protect insulin against surface adsorption. The presence of a lag phase in the displacement at low surfactant concentration indicates that the mechanism of action for Tween 80 to reduce adsorption of insulin may be by competing for sites at the surface.

Adsorption of human insulin and AspB28 insulin on a PTFE-like surface.

The interactions of human insulin, Zn-free human insulin, and AspB28 insulin with a hydrophobic surface were studied by ellipsometry. All three insulin types investigated adsorbed with high affinity onto the hydrophobic surface, as the plateau of the adsorption isotherm, represented by the irreversible bound fraction, was reached at concentrations >10(-3) mg/ml. The plateau values for human insulin and Zn-free human insulin could not be distinguished with statistical significance, whereas the plateau value for AspB28 insulin was lower than those for the two others, with an adsorbed amount corresponding to a monolayer of insulin monomers. The results observed may be explained by differences in self-association patterns of the insulin types or by enhanced charge repulsion between the AspB28 analog and the negatively charged surface.

A putative proton binding site of plasma membrane H(+)-ATPase identified through homology modelling.

We have used the 2.6 A structure of the rabbit sarcoplasmic reticulum Ca(2+)-ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. and Ogawa, H. (2000) Nature 405, 647-655], to build models by homology modelling of two plasma membrane (PM) H(+)-ATPases, Arabidopsis thaliana AHA2 and Saccharomyces cerevisiae PMA1. We propose that in both yeast and plant PM H(+)-ATPases a strictly conserved aspartate in transmembrane segment (M)6 (D684(AHA2)/D730(PMA1)), and three backbone carbonyls in M4 (I282(AHA2)/I331(PMA1), G283(AHA2)/I332(PMA1) and I285(AHA2)/V334(PMA1)) comprise a binding site for H3O(+), suggesting a previously unknown mechanism for transport of protons. Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region.

Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248.

Native carboxypeptidase A has been crystallized in a new crystal form, and the structure has been refined with X-ray data to 2.0 A resolution. In contrast to the previously published structure [Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983) J. Mol. Biol. 168, 367-387], no active-site amino acids are involved in the crystal packing. The important Tyr248 is stabilized inside the active site by a hydrogen bond and by interactions with Ile247. The proposed role of Tyr248 in the induced fit mechanism is therefore not supported by the findings in this structure of native carboxypeptidase A. The structure has a partly populated inhibitory Zn2+ site in close proximity to the catalytic Zn2+ as evident from X-ray anomalous dispersion data. A hydroxo bridge is found between the catalytic Zn2+ and the inhibitory Zn2+ with a Zn2+-Zn2+ distance of 3.48 A. In addition, the inhibitory Zn2+ has Glu270 as a monodentate ligand. No other protein ligands to the inhibitory Zn2+ are seen. The crystals were grown at 0.3 M LiCl and weak evidence for a binding site for partly competitive inhibitory anions is observed.