The euglycemic glucose clamp technique has been regarded as the best available method for the assessment of PK/PD values of study insulin and its analogs. Sufficient suppression of endogenous insulin secretion in such assessment is of considerable importance as it affects the precision of PK/PD assessments of new insulin preparations in the healthy. C-peptide, cleaved from the proinsulin molecule in islet cells, is released into the circulation in amounts equimolar to insulin, and the hepatic extraction of C-peptide is negligible. Therefore, C-peptide is usually measured in parallel to insulin concentrations during euglycemic clamp studies to evaluate whether endogenous insulin secretion is inhibited. The half-life of C-peptide is longer than that of human insulin such that it cannot correct for rapid changes due to poor clamp technique. In the present study, human insulin was measured by a reliable ELISA method with a cross-reactivity of less than 0.006% with IAsp, allowing the interference of IAsp in the analysis of human insulin to be ruled out. Therefore, the endogenous insulin represented by human insulin might be more similar to the true situation. We considered human insulin to be roughly equivalent to endogenous insulin secretion. The study revealed that the method using C-peptide to predict endogenous insulin secretion had a sensitivity of 85.2% and a specificity of 79.2%. Increased C-peptide level can thus be regarded as a marker of insufficient inhibition of endogenous insulin.
In general, clamp studies aimed at evaluating short-acting insulin preparations require that the CVBG not exceed 10%, whereas those aimed at evaluating long-acting insulin preparations require that it not exceed 5%. The results of the present study showed that as the CVBG increased, the distance of C-peptide above baseline increased (Fig. 2C), and there was a positive correlation between the two measures. These findings indicate that large fluctuations in blood glucose may be one factor responsible for insufficient suppression of endogenous insulin secretion. However, since the overall CVBG values of group A and group B were comparable in this study, we speculate that there may be other factors associated with uninhibited endogenous insulin secretion. According to the European Medicines Agency (EMA) guidelines, the blood glucose is recommended to be clamped below the subject’s fasting glucose in healthy volunteers, and the blood glucose should be controlled to within ± 10% of the target value. Other researchers have reported that blood glucose could be clamped at the subject's own basal level[6–13]. Based on the AUCs of glucose excursion in our study, nearly 37% of excursions were above baseline when the ‘clamped’ glucose was maintained around baseline (group A), whereas fewer than 4% of glucose excursions were above baseline when the ‘clamped’ glucose was approximately 5% below baseline (group B). ‘Clamped’ glucose around baseline increased the possibility of over-baseline glucose excursion, which might stimulate endogenous insulin secretion.
Insufficient information is available to evaluate the accuracy of insulin PK/PD data obtained in the clamp in the context of an elevated postdosing C-peptide level. Some researchers have suggested if the C-peptide level is increased by > 200 pmol/L from baseline after dosing, the data are not suitable for analysis. One possible reason for the difference in AUCGIR, 0−8h after correction for ‘clamped’ glucose between the two groups in this study is the difference in C-peptide level between the two groups. Another possible explanation is the unequal extent of suppression of hepatic glucose production (HGP) between the groups due to the absence of a continuously high blood insulin level. Much stronger, almost complete, suppression of HGP has been observed at serum insulin levels higher than 40 uU/mL. We calculated the AUC of GIR for the period from 0.5 to 2 h, when insulin level was high, to minimize the interference due to HGP. The AUCGIR, 0.5−2h after ‘clamped’ glucose correction differed by approximately 14.6% between the two groups. This finding indicates that if a subject’s endogenous insulin secretion is not sufficiently inhibited, it will contribute to the observed GIR. Furthermore, we found that elevated postdosing C-peptide level was a marker of inappropriately excessive glucose infusion.
EMA guidelines state that the C-peptide correction method can be considered in the absence of endogenous insulin suppression or when commercially specific assays for insulin preparations are restricted. The calculation is based on the consistency of the ratio between human insulin and C-peptide after dosing and the initial basal period (F value). In a research where specific assays for the tested insulin are lacking, the primary PK parameter, total exogenous insulin exposure, can be calculated using the following equation: [AUC0 − t of exogenous insulin]=[AUC0 − t of observed insulin]-[AUC0 − t of endogenous insulin]. The results of the present study showed that in the absence of endogenous insulin inhibition (group A), the value of total endogenous insulin predicted using C-peptide was significantly different from that predicted by human insulin, whereas no such significant difference was detected when postdosing C-peptide was inhibited (group B). Therefore, estimates of the total exogenous insulin exposure might be inaccurate when C-peptide is used to correct endogenous insulin in a euglycemic clamp in the absence of suppressed endogenous insulin. This finding conflicts with the EMA guidelines mentioned above.
To our knowledge, this study is the first to quantitatively evaluate the effect of unsuppressed endogenous insulin on PD and to reveal the inaccurate results of the C-peptide correction method in the absence of endogenous insulin suppression. This study only included young healthy males, which limits the extrapolation of the results to subjects with diabetes with and without some preserved insulin secretion and to females. Further study may be needed to detect that to what extent CPpostdosing increasing significantly affect the accuracy of the C-peptide correction method.