Instability, stabilization, and formulation of liquid protein pharmaceuticals.

In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH versus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic ...

Effect of Environmental Factors on the Kinetics of Insulin Fibril Formation: Elucidation of the Molecular Mechanism

Conformational constraints for amyloid fibrillation: the importance of being unfolded.

The increasing use of recombinantly expressed therapeutic proteins in the pharmaceutical industry has highlighted issues such as their stability during long-term storage and means of efficacious delivery that avoid adverse immunogenic side effects. Controlled chemical modifications, such as substitutions, acylation and PEGylation, have fulfilled some but not all of their promises, while hydrogels and lipid-based formulations could well be developed into generic delivery systems. Strategies to curb the aggregation and misfolding of proteins during storage are likely to benefit from the recent surge of interest in protein fibrillation. This might in turn lead to generally accepted guidelines and tests to avoid unforeseen adverse effects in drug delivery.

Protein drug stability: a formulation challenge

Protein aggregation and its inhibition in biopharmaceutics

Toward understanding insulin fibrillation

A new insulin immunoassay specific for the rapid-acting insulin analog, insulin aspart, suitable for bioavailability, bioequivalence, and pharmacokinetic studies.

The objective of this study was to compare the pharmacokinetics and pharmacodynamics of insulin aspart (IA), a rapidly acting insulin analog, with those of human soluble (regular) insulin (HI) in animal models after s.c. and i.v. dosing. Single doses of IA and HI were administered i.v. and s.c. to rats and dogs at three dose levels, and at one dose level to pigs; rats and dogs also underwent repeated s.c. dosing for 1 week. Plasma insulin levels were assessed at predetermined time points after dosing; plasma glucose levels were measured in pigs only. There were no significant pharmacokinetic differences between IA and HI after a single s.c. or i.v. dose in rats or dogs, and no differences were observed after repeated s.c. dosing, implying there was no accumulation. In pigs, there was a strong trend toward more rapid absorption of IA compared with HI after s.c. dosing, whereas there were no differences after i.v. administration. After s.c. dosing in pigs, IA produced significantly lower plasma glucose levels compared with HI during the period 30 to 75 min after dosing (P <.05). In conclusion, IA was more rapidly absorbed than HI after s.c. administration only in the pig; this difference was reflected in earlier and more pronounced effects on plasma glucose levels.

Pharmacokinetics of the rapid-acting insulin analog, insulin aspart, in rats, dogs, and pigs, and pharmacodynamics of insulin aspart in pigs.

A radioimmunoassay (RIA) for insulin was validated for reliable measurement of the human insulin analogue, insulin aspart, by correction of non-linear measurements. Specificity was equivalent for several species of insulin, except insulin aspart. A non-linear hyperbolic model fitted insulin aspart with a correction formula for non-linearity of: z = 1503y/(1398 - y), where y denotes measured concentration and z denotes true concentration. Matrix-effects were insignificant for human, porcine, and canine heparin-plasma and for human and porcine serum. The coefficient of variation was below 15% for 80–800 pmol/L human and porcine insulin and for 80–600 pmol/L insulin aspart. The limit of detection for insulin aspart was 11.5 pmol/L with a lower limit of quantification of 17.5 pmol/L. Dilution of serum with Pharmacia dilution media introduced no significant error. In conclusion, this paper demonstrates that a non-parallel radioimmunoassay can be used to estimate accurate concentrations of insulin aspart.

Lennart Andersen

Papers

Toward understanding insulin fibrillation

A new insulin immunoassay specific for the rapid-acting insulin analog, insulin aspart, suitable for bioavailability, bioequivalence, and pharmacokinetic studies.

Pharmacokinetics of the rapid-acting insulin analog, insulin aspart, in rats, dogs, and pigs, and pharmacodynamics of insulin aspart in pigs.

Validity and use of a non-parallel insulin assay for pharmacokinetic studies of the rapid-acting insulin analogue, insulin aspart