This trial was a single-dose, double-blind, randomised, parallel-group, replicate-design study investigating within-subject variability in systemic exposure and glucodynamics of insulin glargine compared with NPH (Huminsulin basal 100) and ultralente (Ultralong) in healthy volunteers. Thirty-six healthy volunteers were allotted to three groups of 12 subjects per group. Each group received two consecutive injections of one of the study medications. Prior to the second injection, there was a wash-out period of at least 7 days. The study was performed at the Hoechst Marion Roussel Research Centre, Bloemfontein, South Africa, now Farmovs-Parexel.
Healthy, non-smoking, male volunteers aged 18–33 years (mean 23.1 years), weighing between 66 and 100 kg (mean 79.6 kg), with BMI values of 20.0–26.0 kg/m2 (mean 23.5 kg/m2), a normal oral glucose tolerance test and no clinically important abnormalities in their clinical chemistry, ECGs, vital signs and medical history or on physical examination were included in the study. The demographic and baseline characteristics of the study population were similar between groups: 18.3–29.3 years (mean 23.3), 18.9–32.5 years (mean 23.6) and 19.1–27.5 years (mean 22.3) in the insulin glargine (n=12), NPH (n=12) and ultralente (n=12) groups, respectively. Body weight ranges for the insulin glargine, NPH and ultralente groups were 67.1–97.2 kg (mean 77.9), 69.0–91.2 kg (mean 80.5) and 65.6–99.8 kg (mean 80.4), respectively. The BMI ranges were 20.0–25.8 kg/m2 (mean 22.8), 21.1–26.0 kg/m2 (mean 23.9) and 22.1–26.0 kg/m2 (mean 23.7), respectively. All volunteers provided written, informed consent prior to initiation of the investigation; the study was carried out in accordance with the Declaration of Helsinki.
Each volunteer received two consecutive (≥7-day wash-out) s.c. injections of insulin glargine (12 subjects), NPH (12 subjects) or ultralente (12 subjects) at a dose of 0.4 IU/kg body weight administered by a physician or nurse otherwise not involved in the study. The peri-umbilical abdominal area was chosen for injection with a 0.13×12 mm single-use syringe with integrated needle (Braun Omnican) for comparison with previous single-dose studies.
Subjects remained fasting from 22.00 hours the night before to the end of the entire study procedure. The injection time defined the time zero of the insulin action period, which was monitored for 24 h. Glucose infusion rate (GIR), blood glucose concentration, serum immunoreactive insulin and serum C-peptide concentrations were recorded for pharmacokinetic and pharmacodynamic evaluations. Blood samples were taken from an i.v. cannula, which was inserted into the hand or wrist vein, for measurement of blood glucose, serum insulin and serum C-peptide levels.
Blood glucose concentrations were measured every 10 min after administration of the study medication with a Yellow Springs Instruments 2300S Glucose Analyzer (Yellow Springs Instruments, Yellow Springs, OH, USA) using the glucose oxidase method. Baseline blood glucose concentration was calculated as the mean value of blood glucose measurements taken at 60, 30, 15 and 5 min prior to study medication. A drop in blood glucose up to a maximum of 10% from baseline signified the initiation of the glucose infusion. A 20% glucose solution was infused at a manually stepwise-adjusted variable rate to restore and maintain the subject’s baseline blood glucose concentration.
Pharmacokinetic data collection
Serum total immunoreactive insulin was measured at 30, 15 and 5 min prior to and every 60 min up to 24 h after administration of the study medication using an RIA for human insulin with an in vitro cross-reactivity with insulin glargine of about 50%. The lower limit of quantification (LLOQ) was 2.5 μIU/ml. Simultaneous serum C-peptide concentrations were also determined with an RIA with an LLOQ of 0.15 ng/ml (Analytical Services Division, Farmovs Research Centre, Bloemfontein, South Africa). Immunoreactive insulin concentrations (INS) were corrected for endogenous insulin using serum C-peptide levels to yield exogenous insulin concentration profiles and to observe maximum concentrations (INS-C
max), times to INS-C
max) and to calculate the area under the exogenous insulin concentration-time curve up to 24 h (INS-AUC0–24 h).
Pharmacodynamic data collection
The time-action profile was characterised by the area under the GIR-time curve up to 24 h (GIR-AUC0–24 h), by the maximum GIR (GIRmax), time to 50% of GIR-AUC0–24 h (GIR-t
50%) and the time to GIRmax (GIR-T
Adverse events were reported by the subject or noted by the investigator. Routine laboratory tests included haematology, clinical chemistry and urinalysis. Determination of human insulin antibodies, a physical examination and a 12-lead ECG were also carried out.
For insulin, area under the concentration-time curve (AUC0–24 h) was calculated according to the linear trapezoidal rule up to 24 h after injection of the study medication. INS-C
max was read directly from the derived serum concentrations of exogenous insulin. ANOVA for treatment and subject effect was applied on natural logarithmic (ln)-transformed data. Antilog point estimates with 90% CIs were obtained for the mean ratio ‘Clamp 2-Clamp 1’ on the ln-scale with period and subject effect per insulin, and for the respective ratios of treatment means (secondary analysis). Non-parametric analysis was performed and 90% CIs calculated for INS-T
max according to Steinijans and Diletti.
For glucose, area under the GIR-time curve was calculated as the exact area under the stepwise constant function for the respective time intervals of the 24-h GIR-time profile; the GIR-t
50% was extrapolated from this. The determination of GIRmax was based on a ‘smoothed three-point running mean’ GIR curve for each subject. For each measured value of GIR, a mean GIR value was calculated from the previous, actual and following GIR values (this corresponded to mean values of 20-min intervals from injection of the study medication up to the end of the clamp period). The GIRmax was read directly from the smoothed GIR-time curves and the times of GIRmax were reported as the blood sampling times corresponding with the GIRmax. ANOVA of untransformed data was applied to the area under the smoothed (three-point running means smoother) GIR curve over 24 h (GIR-AUC0–24 h), GIR-t
50% and GIRmax, and CIs calculated based on Fieller’s theorem. Non-parametric analysis was performed and 90% CIs calculated for GIR-T
max. Between-treatment comparisons were conducted as described above.
Variability in pharmacokinetics and pharmacodynamics
Within-subject variability was assessed as intra-individual CV values, taken from the mean sum of the error terms (MSE) as calculated by the ANOVA on untransformed values for GIR-AUC0–24 h, GIRmax, GIR-t
50% and INS-T
max, and on ln-transformed values for INS-AUC and INS-C
max. (CV% of untransformed data=SQRT[MSE]/LSM×100; CV% of ln-transformed data=SQRT[(EXP(MSE)–1]×100.) For comparison of variances between treatments, the statistical F-distribution was used to compute 90% CIs on the ratio of the two variances.
Profile reproducibility was assessed in two steps: by the absolute individual cumulative (CUM) between-day differences in insulin concentration (Δabsolute–INS–CUM) and GIR profiles (Δabsolute–GIR–CUM) and by individual SD values of hourly between-day differences (untransformed, raw data) in insulin concentrations (SD–Δraw–INS) and GIR (SD–Δraw–GIR) over 24 h. For comparison between treatments, non-parametric analysis was performed for these metrics. A stem-and-leaf plot for outlier detection was also accomplished. An additional comparison of Δabsolute–INS–CUM was performed with insulin glargine concentrations corrected for the observed underestimation relative to NPH (i.e. equivalence in INS–AUC0–24 h was assumed).