This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology
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Considering the endogenous insulin and glucose self-regulatory mechanisms, how to precisely evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) of insulin preparations has always been a challenge. According to the EMA, the endogenous insulin production of health volunteers can influence PK and PD measurements (
The hyperinsulinemic-euglycemic clamp technique has been widely used in previous studies, although it overestimates the effects of insulin preparations, especially long-acting preparations (
C-peptide is a polypeptide originating from proinsulin, which releases insulin and C-peptide in equimolar amounts into the circulation (
Blood glucose oscillations should be controlled within a specific range by infusion of glucose. According to the EMA, in healthy subjects, the target blood glucose value should be set below the subjects fasting glucose (e.g., 0.3 mmol/L or 10%); the closer the blood glucose concentration to the target, the more successful the clamp is in achieving its goal of maintaining the desired glycemic plateau (
In this study, healthy male volunteers were enrolled and underwent a 24-h euglycemic clamp study. Our objective was to investigate the different extent of inhibition of endogenous insulin secretion, which is reflected by the ratio of C-peptide reduction, and its effects on the PK and PD of long-acting insulin preparations, thus determining the best reduction range of C-peptide and exploring the way to improve the quality of clamp study, which could provide a theoretical basis for future empirical research.
The volunteers were screened and qualified for the entry standard. A 24-h euglycemic glucose clamp study was conducted in healthy male subjects after 0.4 iu/kg insulin glargine injection. During the trial, volunteers were requested to avoid strenuous exercises, smoking, drinking alcohol, or caffeinated drinks (e.g., tea and coffee). In order to investigate the best reduction extent of C-peptide, we divided the subjects into three groups according to the ratio of C-peptide reduction by using the empirical and quantile classification method, based on the values of 33.3% quantile, that is, group A: C-peptide reduction rate <30%; group B: C-peptide reduction rate between ≥30% and <50%; and group C: C-peptide reduction rate ≥50%. The trial was carried out in accordance with the principles of the Declaration of Helsinki. This study was approved by the Ethics Committee of the First Affiliated Hospital of ChongQing Medical University (No. 20190101).
Healthy Chinese male volunteers aged 18–45 years were enrolled. We selected individuals with a body mass index (BMI) of 19–24 kg/m2 without diabetes, insulin resistance, or a family history of diabetes, who did not have insulin resistance, and who did not have cardiovascular disease. The volunteers were non-smokers and non-alcohol users. Subjects had no abnormalities on routine OGTT (oral glucose tolerance test), insulin releasing test (IRT), blood and urine examinations, liver and kidney function tests, and electrocardiograms. All volunteers provided written informed consent prior to the start of the study.
All recruited volunteers underwent a single-dose euglycemic clamp test. Participants arrived at the ward on the day prior to the clamp test to ensure a 10-h fasting condition and to maintain fasting during euglycemic clamping. Insulin glargine was injected into a lifted abdominal skinfold (0.4 IU/kg). Intravenous access was obtained in one arm for a 20% glucose infusion and in the other for blood drawing. The arm for blood drawing was heated using a warming blanket to arterialize venous blood (55–65°C). Blood samples were collected before dosing and up to 24 h post-dosing to analyze glargine and C-peptide levels at the following time points: −30 min, −20 min, −10 min, and 0 min, and 0.5 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 21, and 24 h. PD variables were evaluated during euglycemic clamping lasting up to 24 h, which included blood samples drawn for biochemical analysis at 10-min intervals at 30 min before injection and up to 8 h after injection, at 20-min intervals from 8 to 16 h, and at 30-min intervals from 16 to 24 h.
Basal glucose (BG) was defined as the average blood glucose level before injection; the target glucose (TG) level was defined as the BG minus 0.28 mmol/L (
Blood glucose concentration was immediately analyzed using an automatic glucose oxidase analyzer Biosen C-line GP+(Germany) during clamping, whose qualification range was 0.5–50 mmol/L. The glargine and C-peptide samples were transferred to Covance Laboratories after processing and centrifugation. Insulin glargine is rapidly metabolized to its active metabolites M1 and M2; therefore, the glargine prototype drug and the concentrations of the metabolites M1 and M2 were used for pharmacokinetic analysis, which were evaluated by a validated liquid chromatography–tandem mass spectrometry (LC-MS/MS), whose qualification range was 0.07–2.5 ng/ml. The C-peptide concentration was analyzed by ELISA, the qualification range of which was 20–3,000 pmol/L.
SPSS 22.0 and WinNonlin 8.1 were used for statistical analysis. C-peptide concentration was quantified to monitor endogenous insulin secretion, and the C-peptide reduction rate was calculated as 1-mean CPt/CP0. The oscillation of glucose was calculated with formula 1-Gt (glucose at time t)/Gd (desired glucose). Parameter estimates were computed by non-compartmental analysis (NCA) of the total insulin glargine concentration versus time profiles and glucose infusion rate versus time profiles, and the pharmacokinetic parameters were area under the glargine concentration versus time curve (AUC0–24), peak glargine concentration (Cmax), and time to Cmax (Tmax). The pharmacodynamic parameters were area under the glucose infusion rate versus time curve (AUCGIR0–24 h), peak of glucose infusion rate (GIRmax), and time to GIRmax (TGIRmax).
For quality evaluation indicators, CVBG was calculated as the SD of the blood glucose/mean value of blood glucose. The GEFTR was calculated as the degree of glucose excursion from target range/total blood glucose at specific time points. The MEFTG was calculated as the mean excursion of target glucose.
Quantitative data were expressed as mean ± standard deviation (SD) or median values with interquartile ranges (25–75%). Normality was examined, and some data were natural log transformed prior to analysis. Statistical analysis was performed using the analysis of variance (ANOVA) or the Kruskal–Wallis test. The relationship between the ratio of C-peptide reduction and blood glucose was assessed using Pearson correlation coefficients.
Thirty-nine volunteers were enrolled after screening for eligibility. The demographics and clinical characteristics of the subjects in the three groups are summarized in
Demographics and clinical characteristics of the subjects.
Group A ( |
Group B ( |
Group C ( |
|
|
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Age (y) | 26.9 ± 5.2 | 29.3 ± 5.9 | 26.0 ± 4.0 | 0.238 |
BMI (kg/m2) | 22.5 ± 1.2 | 22.0 ± 1.0 | 21.9 ± 1.5 | 0.346 |
SBP (mmHg) | 120.2 ± 10.2 | 123.1 ± 9.4 | 117.7 ± 7.2 | 0.346 |
DBP (mmHg) | 72.0 ± 8.7 | 78.6 ± 7.5 | 73.7 ± 6.8 | 0.078 |
HR (times/min) | 73.2 ± 10.8 | 75.5 ± 11.7 | 72.7 ± 9.1 | 0.77 |
Fasting serum insulin (mmol/L) | 4.4 ± 2.3 | 4.3 ± 4.5 | 4.8 ± 3.5 | 0.887 |
Fasting glucose (mmol/L) | 5.3 ± 0.3 | 5.3 ± 0.3 | 5.0 ± 0.3 | 0.093 |
HOMA-IR | 0.9 ± 0.7 | 1.1 ± 1.3 | 1.0 ± 1.1 | 0.736 |
Dose (IU) | 25.7 ± 2.4 | 25.4 ± 1.6 | 25.1 ± 2.3 | 0.783 |
Ratio of C-peptide reduction (%) | 22.7 ± 5.4 | 39.8 ± 5.3 | 55.6 ± 3.9 | 0.000 |
Data are expressed as mean ± SD or medians (25–75%). Statistical analysis was performed using analysis of variance (ANOVA) or the Kruskal–Wallis test,
Volunteers were 27.6 ± 5.2 years old and had a BMI of 22.1 ± 1.2. There were no significant differences in the prevalence of risk factors for metabolic syndrome or cardiometabolic disease, including BMI, SBP (systolic blood pressure), DBP (diastolic blood pressure), HR (heart rate), fasting serum insulin, fasting glucose, and HOMA-IR. The doses of glargine in groups A, B, and C were 25.7 ± 2.4, 25.4 ± 1.6, and 25.1 ± 2.3 IU, respectively; there were no significant differences in administered doses (
Endogenous insulin secretion was restrained by euglycemic clamps, and the serum C-peptide levels were used to reflect the degree of restriction. The basal C-peptide levels were 484.6 ± 207.1 pmol/L in group A, 514.0 ± 184.4 pmol/L in group B, and 455.4 ± 154.6 pmol/L in group C. The profiles of C-peptide changes over time were shown in
Mean C-peptide concentration versus time profiles after 0.4 IU/kg doses of glargine insulin in healthy volunteers. The error bars represent the 95% confidence intervals. The extent of C-peptide reduction in group C was apparently higher than that in the others, and there were statistically significant differences among groups.
The average glucose target level achieved in groups A, B, and C were 4.99, 4.98, and 4.90 mmol/L, respectively. The GIR was adjusted in accordance with glucose values during the clamping procedure. The profiles of the oscillations of glucose and GIR over time were shown in
Blood glucose versus time after 0.4 IU/kg doses of glargine insulin in healthy volunteers. The error bars represent the 95% confidence intervals. The blood glucose in group C was obviously lower than that in the others, and significances were found in three groups.
Glucose infusion rate (GIR) versus time profiles after 0.4 IU/kg doses of glargine insulin in healthy volunteers. The error bars represent the 95% confidence intervals. GIR in group C was lower than that in the others, but there were no statistical differences among groups.
Relationship between the ratio of C-peptide reduction and blood glucose, in which revealed a negative correlation.
Oscillating range of blood glucose.
Group A | Group B | Group C |
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Lower limit (%) | −10.7 ± 7.9 | −11.4 ± 6.3 | −17.0 ± 6.6 | 0.070 | 0.035 |
Upper limit (%) | 10.2 ± 5.5 | 7.1 ± 5.1 | −1.1 ± 6.7* | 0.000 | 0.000 |
Data are expressed as means ± SD,
Pharmacokinetics (PK) and pharmacodynamics (PD) parameters in three groups.
Group A | Group B | Group C |
|
|
|
---|---|---|---|---|---|
PD | |||||
GIRmax (mg/kg/min) | 3.5 ± 1.3 | 3.4 ± 1.4 | 3.0 ± 1.5 | 0.586 | 0.326 |
TGIRmax (min) | 593.1 ± 198.1 | 584.0 ± 199.1 | 756.4 ± 228.0 | 0.088 | 0.062 |
AUCGIR0–24 h (mg/kg) | 3175.9 ± 1135.2 | 2862.9 ± 1176.0 | 2526.1 ± 1222.0 | 0.411 | 0.186 |
PK | |||||
Cmax (ng/ml) | 0.6 ± 0.2 | 0.6 ± 0.2 | 0.7 ± 0.2 | 0.293 | 0.120 |
Tmax (h) | 11.7 ± 4.0 | 10.4 ± 3.1 | 12.8 ± 2.2 | 0.182 | 0.403 |
AUC0–24 h (ng/ml × min) | 9.7 ± 2.2 | 11.0 ± 2.9 | 11.9 ± 2.1 | 0.112 | 0.041 |
Data are expressed as means ± SD,
The profile of plasma glargine insulin concentrations following the 0.4 IU/kg injection over time was shown in
Mean insulin glargine concentration versus time profiles after 0.4 IU/kg doses of glargine insulin in healthy volunteers. The error bars represent the 95% confidence intervals. Glargine concentration was higher than that in the others, but there were no statistical differences among groups.
We evaluated the quality of the euglycemic clamp by assessing mean glucose levels, SD, CVBG, MEFTG, and GEFTR (
Indexes of the quality assessment of euglycemic clamp in three groups.
Group A | Group B | Group C |
|
|
|
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Average glucose (mmol)/l | 5.2 | 5.1 | 4.8 | 0.000** | 0.000 |
standard deviation SD (mmol/l) | 0.2 | 0.2 | 0.2 | 0.148 | 0.053 |
CVBG (%) | 4.4 ± 1.5 | 3.7 ± 1.2 | 3.6 ± 0.6 | 0.182 | 0.097 |
MEFTG (mmol/l) | −0.01 ± 0.25 | −0.08 ± 0.26 | −0.45 ± 0.31 | 0.001* | 0.000 |
GEFTR (%) | 2.1 ± 3.2 | 1.8 ± 2.8 | 1.0 ± 1.3 | 0.592 | 0.316 |
Data are expressed as means ± SD; SD of average glucose was listed separately,
There were nine AEs observed in six subjects. Mild elevation of serum bilirubin (
Insulin analogs and their biosimilars are commonly used for the treatment of patients with diabetes; therefore, their safety and efficacy should be noticed (
Healthy volunteers were enrolled in this study, who presented homogenous characteristics and insulin sensitivity. Healthy volunteers exhibit lower intra-individual variability than patients with type 1 diabetes mellitus (T1DM). Furthermore, insulin secretion in women may vary during the menstrual cycle, although it is unclear whether this may influence study results (
Endogenous insulin secretion should be strictly controlled by the euglycemic clamp technique as endogenous insulin secretion interferes with PK and PD properties (
Factors that influence clamping include the metabolic activity of the subject, sensitivity to insulin, the dosage of the insulin preparation, and glucose regulation during clamping (
The results indicated that the ratio of C-peptide reduction in group C is the highest; thus, the contribution from the secretion of endogenous insulin was inhibited more thoroughly, and the PD could best reflect the real effect of exogenous administered insulin. According to the oscillating range of glucose, the ceiling and floor limits of glucose oscillation in group C were significantly lower than those in the other groups. In order to control the coefficient of variation, based on the results of this study, we suggest that the glucose regulating range should be maintained in −10% to 0. GIRmax and AUCGIR0–24 h in group C were lower than those in the other groups, and TGIRmax was more prolonged in group C than in the other groups. Although there were no significant differences in the PD parameters, values differed across the three groups. For PK parameters, Cmax, Tmax, and AUC0–24 h in group C were higher than those in the other groups. Thus, the extent of the reduction in the C-peptide influences the assessment of PD and PK properties, and adequate suppression of endogenous insulin secretion is crucial for successful clamping. Consequently, we suggest that the ratio of C-peptide reduction should be higher than 50%, with which the clamp study immunes to the disturbance of endogenous insulin, and this conclusion was consistent with previous studies (
There is currently no gold standard for evaluating euglycemic quality; the CVBG is commonly used as a quality indicator in euglycemic clamp studies, and a CVBG value ≤ 5% is considered superior (
Based on our findings, we propose that C-peptide levels should be below those of baseline values during the clamp, and the extent of the reduction in C-peptide levels will influence the PK/PD of insulin preparations and the quality of euglycemic clamps. Furthermore, the C-peptide reduction ratio should be greater than 50% to ensure better inhibition of endogenous insulin secretion. For glucose regulation, the oscillating glucose ranging from −10% to 0 is recommended. Finally, the ratio of C-peptide reduction should be considered a quality evaluation indicator of euglycemic clamp tests.
The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by the Ethics Committee of the first affiliated hospital of ChongQing Medical University (20190101). The patients/participants provided their written informed consent to participate in this study.
CT designed the study. YT, JP, PZ, and LW performed the experiment. MZ collected the data. YT drafted the manuscript. CT edited the manuscript. All authors approved the final version of the manuscript.
The data were collected from clinical trials funded by the Yichang HEC Changjiang pharmaceutical Co. Ltd. (No. CTR20191031). This research was funded by the Science and Technology Commission Foundation of Chongqing, China (Nos. cstc2019jscx-gksbX0005 and cstc2020jscx-msxmX0090), and the Science and Technology Commission and Health Commission Joint Research Project (No. 2020GDRC022).
This study received funding from the Yichang HEC Changjiang Pharmaceutical Co. Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. All authors declare no other competing interests.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
We thank all volunteers who participated in this study. We also thank QING F Cheng for his excellent technical assistance. We appreciate the successful implementation of this study by all researchers.