Application of the euglycaemic clamp technique to bioassay of insulin analogues

Abstract The euglycaemic clamp method may offer a precise and clinically valid approach to assess the in vivo potency of new insulin analogues or derivatives relative to a human insulin standard. The proposed protocol was designed to overcome problems due to differences in pharmacokinetics between the test and standard preparations. An analogue of human insulin, Gly A21 + Arg B27 + Thr B30 -NH 2 , which is absorbed very slowly after subcutaneous injection, and human insulin were compared in intravenous clamp experiments in pigs. Both insulins were infused for 4 h to achieve steady state glucose metabolism. The infusion rate ranged from 2·5–8 pmol min −1 kg −1 . Parallel dose response curves were obtained with the mean glucose infusion rate from 180–240 min as the response and the logarithm of the insulin infusion rate as the dose. Standard bioassay analysis showed that the molar potency of the analogue relative to human insulin was 95·2% with a 95% confidence interval of 82·3–111·2%. To assess the clinical validity of the method a similar euglycaemic clamp study was carried out in human volunteers. The insulin infusion rates were 3 and 6 pmol min −1 kg −1 , and the mean glucose infusion rate over the final 180–240 min period of the clamp was used as response. The statistical analysis showed, as in the pig clamp bioassay, no significant deviations from steady state or from the assumption of parallelism. The resulting molar potency of the analogue relative to human insulin was 85·5% with a 95% confidence interval of 49·5–128·4%. This was in agreement with the result of the pig clamp bioassay. It is concluded that the euglycaemic clamp bioassay is a clinically valid method for assessing the potency of insulin analogues.