2.1 Study Design
This randomised, open-label, five-period, single-centre (Profil, Germany), single-dose crossover trial was conducted in healthy subjects (ClinicalTrials.gov identifier: NCT01151072). The study protocol was reviewed and approved by the health authority (Bundesinstitut für Arzneimittel und Medizinprodukte) according to local regulations and by the ethics committee of Ärztekammer Nordrhein. The study was performed in accordance with the Declaration of Helsinki and its amendments, and in accordance with Good Clinical Practice as defined by the International Conference on Harmonisation. Subjects were informed of the risks and benefits of the trial and were informed that they could withdraw at any time for any reason. Consent was obtained in writing before any trial-related activities, and the investigator retained the consent forms.
2.2 Subjects
Study subjects were healthy males or females aged 18–55 years, with a body mass index (BMI) of 18.0–27.0 kg/m2 and fasting plasma glucose concentrations of ≤6.0 mmol/L (≤108 mg/dL). Key exclusion criteria for participation in the study included the use of prescription drugs within 3 weeks prior to screening, the use of non-prescription drugs (including over-the-counter medication, non-routine vitamins and herbal products) within 3 weeks prior to screening, and smoking.
2.3 Interventions and Pharmacokinetic Sampling
Following screening (Visit 1), subjects were randomised to predetermined dosing sequences consisting of five single doses of IDeg on five separate dosing visits (Visits 2–6). Dosing was conducted via SC injection of 0.4 U/kg body weight (BW) of IDeg in the thigh, abdomen or deltoid (upper arm); intramuscular (IM) injection of 0.4 U/kg BW of IDeg in the thigh area; or intravenous (IV) injection of 0.04 U/kg BW of IDeg. Only the methods and data from the SC dosing arms are reported here. IDeg was provided in 3 mL Penfill® cartridges (100 U/mL) (Novo Nordisk A/S, Bagsværd, Denmark) for dosing and administered (using a syringe and needle) into a lifted skinfold in either the anterior surface of the thigh, the lower abdominal wall (above the inguinal area) or the outer aspect of the deltoid area.
At each dosing visit, IDeg administration was followed by a 24-h euglycaemic clamp procedure (see below for description). Subjects attended dosing visits in a fasted state, and each subject remained in the clinic for 48 h after dosing, during which blood samples for pharmacokinetic analysis were taken frequently. Blood samples were also taken frequently for analysis of blood glucose concentrations. Subjects subsequently returned to the clinic at 24-h intervals. Blood samples were taken at these visits (at 72, 96 and 120 h post-dosing) for pharmacokinetic assessment. Dosing visits were separated by a washout period of 13–21 days. An interval of 7–21 days existed between the last of the five dosing visits and a subsequent follow-up visit (Visit 7).
2.4 Euglycaemic Clamp Procedure
Subjects remained fasted (with water ad libitum) and in a supine position for the euglycaemic clamp procedure (Biostator®, MTB Medizintechnik, Amstetten, Germany); target blood glucose: 4.5 mmol/L (81 mg/dL). One to 6 hours before dosing, subjects received a variable IV infusion of human insulin [15 (I)U Actrapid® (Novo Nordisk A/S, Bagsværd, Denmark), 100 (I)U/mL in 49 mL saline and 1 mL of subject’s blood] or glucose (20 %) to obtain the glucose clamp target concentration. The target glucose concentration was maintained for at least 1 h before dosing, without any glucose infusion. After dosing, the rate of insulin infusion, if any, was decreased gradually and terminated when glucose concentrations had declined by approximately 0.3 mmol/L (5 mg/dL). A variable IV glucose infusion was then initiated to maintain the clamp target concentration.
2.5 Data and Statistical Analyses
The primary objective of this study was to investigate the relative exposure among different SC administration regions in healthy subjects following single-dose administration. Secondary objectives were evaluation of the pharmacokinetic and pharmacodynamic profiles and the safety and tolerability of IDeg.
Serum concentrations of IDeg were measured using an IDeg-specific sandwich ELISA, with a lower limit of quantification of 20 pmol/L. The primary endpoint was the area under the IDeg serum concentration–time curve 0–120 h after a single dose (AUCIDeg,0–120h,SD) given by SC administration in the thigh, abdomen or deltoid area. AUCIDeg,0–120h,SD was derived by non-compartmental analysis using the linear trapezoidal technique based on observed values and actual measurement times between 0 and 120 h, with missing values interpolated. The log-transformed AUCIDeg,0–120h,SD was analysed using an ANOVA method with injection region and treatment period as fixed factors and subject as a random effect. In order to account for potential heteroscedasticity, the error-term was dependent on the injection region. The maximum IDeg serum concentration after a single dose (C
max,IDeg,SD) was also assessed. C
max,IDeg,SD was derived from individual concentration–time curves and analysed using the same approach as for AUCIDeg,0–120h,SD.
Pharmacodynamic endpoints included the area under the glucose infusion rate (GIR) curve 0–24 h after a single dose (AUCGIR,0–24h,SD) and maximum GIR after a single dose (GIRmax,SD). GIR data were smoothed using the Loess smoothing technique (fixed smoothing parameter of 0.25). Pharmacodynamic endpoints were summarised using descriptive statistics.
To predict the steady-state pharmacokinetic and pharmacodynamic profiles of IDeg following SC administration, a pharmacokinetic/pharmacodynamic model using single-dose IDeg data from the current study was applied, with area under the concentration–time curve (AUC) derived by non-compartmental analysis. The pharmacokinetic component of the model consisted of an absorption part with a depot compartment, a transit compartment, a bioavailability parameter, an absorption rate parameter and a transit rate parameter; and a disposition part with two compartments, two clearance parameters and two volume of distribution parameters. The pharmacodynamic component of the model linked the IDeg concentration to GIR by means of an effect compartment, a turnover parameter, an insulin sensitivity parameter and an underlying GIR baseline parameter. The parameters of the model were estimated in a population pharmacokinetic/pharmacodynamic setting, using a non-linear mixed-effects approach, which allowed individual sets of the ten parameters for each of the subjects included in the trial to be obtained. The bioavailability parameter and the absorption rate parameter were allowed to vary between injection regions for each subject. The values of the absorption rate parameter were subsequently calibrated based on information from the comprehensive clinical pharmacology programme of studies conducted with IDeg. The same calibration factor was applied for all subjects and all injection regions. Using the estimated individual parameters, a simulation of once-daily multiple dosing was conducted to obtain mean steady-state profiles. More specifically, once-daily multiple dosing for 6 days at a dose level of 0.4 U/kg was simulated by extrapolating the profile for each of the subjects, and for each injection region, and subsequently calculating the mean of the profiles on Day 6.
Safety endpoints, including adverse events (AEs), laboratory safety variables, physical examination, vital signs, ECG, hypoglycaemic episodes and local tolerability at injection site, were monitored and summarised using descriptive statistics.