Currently, the euglycemic clamp test is generally regarded as the optimum method for evaluating insulin PK and PD.1,7 During the euglycemic clamp test, the PK characteristics of insulin are evaluated by measuring the concentration of exogenous insulin, and the PD characteristics of insulin are evaluated using GIR. During the test, exogenous insulin is injected first, followed by glucose infusion. The GIR is adjusted according to the BG value to ensure that the BG of the participants is kept within the target range to obtain a high-quality clamp test.8–10 Therefore, the quality of the glucose clamp test directly affects the evaluation of insulin. Therefore, the purpose of this study is to discuss the quality evaluation standard of the euglycemic clamp test for long-acting insulin.
CVBG is currently used to evaluate the quality of the euglycemic clamp test by reflecting the variation in BG during the euglycemic clamp.11 When the CVBG value is lower (≤ 5%), 9,11 the BG oscillation is narrower, and the euglycemic clamp test quality is superior. However, there is a delay in the BG detection time and GIR regulation time; therefore, in the clamp test, it is difficult to maintain a stable BG level. Additionally, BG should be controlled near the target level, and deviations from the target level imply that a wrong GIR was used. For the short/fast-acting insulin euglycemic clamp test, maintaining BG within 10% of the target BG is an adequate criterion for a glucose clamp of sufficient quality. However, the peaks are flatter for long-acting insulin analogs; the action lasts longer, and the BG is better adjusted in the euglycemic clamp test than for short-acting/fast-acting insulin.12–14 In this study, we defined the target range as BG within 5% of the target BG value and evaluated the quality of the long-acting insulin analog euglycemic clamp test using GEFTR, the duration of GEFTR, the AUC of GEFTR, MEFTG, and the AUC of MEFT.
This study showed that with the decrease in CVBG, the GEFTR, duration of GEFTR, AUC of GEFTR, MEFTG, and AUC of MEFT gradually decreased, suggesting that they were positively correlated with CVBG (moderate correlation). Therefore, CVBG could be used as a quality evaluation standard for the euglycemic clamp test of long-acting insulin. Our results also showed that when CVBG was ≤ 3.5%, the clamp quality was superior (p < 0.05), and > 70% of the clamp tests showed a CVBG ≤ 3.5%. Therefore, this indicates that CVBG < 3.5% may be used as an evaluation criterion for the quality of the euglycemic glucose clamp test for long-acting insulin.
Similar to many other clamp test studies,15,16 this study was conducted on healthy volunteers. In healthy volunteers, in addition to the subcutaneous absorption of injected insulin, endogenous insulin secretion contributes significantly to plasma insulin concentration and interferes with PK and PD. Therefore, adequate endogenous insulin inhibition is the key to obtaining high-quality clamp tests.17–19
C-peptide is a polypeptide secreted by pancreatic beta cells. It shares a common precursor with insulin, and each proinsulin can release the same amount of insulin and C-peptide into the circulation.20,21 C-peptide has a long half-life and can reflect the secretion of endogenous insulin; therefore, it is frequently used to evaluate the index of endogenous insulin. Previous studies22,23 have indicated that endogenous insulin inhibition is sufficient if the C-peptide inhibition rate is > 50%. Therefore, we categorized the participants into two groups according to the C-peptide reduction ratio. In this study, we observed that C-peptide values were lower than baseline levels after insulin administration, indicating endogenous insulin inhibition. However, the reduction in the ratio of C-peptide in Group D (C-peptide reduction ≥ 50%) was significantly higher than that in Group C (C-peptide reduction < 50%), which is consistent with previous studies.22–24
The process of the euglycemic clamp test is affected by various factors, such as the basal metabolic state of volunteers, basal glucose and insulin levels before injection of exogenous insulin, the dosage of insulin preparations, insulin sensitivity, and BG regulation during the euglycemic clamp test.20,25 In this study, we screened healthy volunteers strictly according to the predefined inclusion and exclusion criteria. Eligible volunteers were grouped according to the C-peptide reduction rate after insulin injection. The results showed that there were no statistical differences between Groups C and D in demography, clinical characteristics (basal BG, target BG, and basal C-peptide), and dosage of insulin degludec, indicating that demography and clinical characteristics did not influence the clamp test.
Usually, when the glucose level fluctuates, the CVBG and AUC of GEFTR are higher, the C-peptide level is less stable, and the C-peptide inhibition is lower.26 In this study, we observed that the CVBG in Groups C and D was 3.06% and 3.38%, respectively, both of which were lower than 3.5%, suggesting that the quality of the clamp test was good. However, the GEFTR of AUC in Group D was statistically higher than in Group C. This may be because when the glucose fluctuation is small (CVBG is maintained at a low level) during the clamp test, even if the clamp test quality is slightly different, it is not sufficient to interfere with endogenous insulin secretion.
After receiving an equal dose of degludec for each participant, AUCGIR0−24 of Groups C and D showed no difference; however, the concentration of insulin degludec AUC0 − 24h of Group D was higher. PK is the rule that reflects the change in blood drug concentration with time. In contrast, the PD reaction, under the action of drugs, involves changes in organ physiological functions and cell metabolic activities, including the relationship between drug concentration and expected and adverse reactions over time.27 During the euglycemic clamp test, PK refers to the relationship between the blood concentration of exogenous insulin and the time after subcutaneous injection of exogenous insulin. PD refers to the time process of the influence of exogenous insulin on BG concentration after exogenous insulin injection.14 PK and PD of insulin were evaluated using the euglycemic clamp test. PD may be overestimated as the reduction rate of C-peptide in Group C was < 50%, and endogenous insulin inhibition was insufficient. Additionally, owing to the limited sample size, individual BG changes should not be ignored.28 This may be because the AUC0–24h changes in insulin degludec concentration and the AUCGIR0–24 are not parallel after insulin degludec administration.
This study had some limitations; 53 participants were included in this study, yet only 12 were assigned to Group B when categorized according to CVBG. The sample size may affect the verification of the study results; therefore, a larger sample size will be needed to assess the quality of the euglycemic clamp test.
Trial Status
The protocol version number and date: V1.0 (November 21, 2019)
The date recruitment began: November 11, 2020
The date recruitment complete: July 13, 2021