Study design
The study was conducted in accordance with Helsinki Declaration II, and approved by local Ethical Committees (EudraCT number: 2006-002685-19), with the trial registration number NCT00411125. Written informed consent was obtained for all participants. The study was approved by the local Danish ethical committee, Copenhagen County.
The study was a randomized, double-blind, double-dummy, placebo-controlled study to assess the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of two different variants of 0.8 mg of oral sCT (SMC021) and the effect of timing of drug intake in healthy postmenopausal women. The two different variants consisted of two production batches of the same product: Batch 002 was a pilot scale equipment batch and batch 010 a full scale production. The inclusion criteria requested that generally healthy ambulatory female volunteers were aged between 40–70 years and having passed a natural or surgical menopause at least 5 years before entering the study and being without diseases or medications known to affect bone metabolism.
The study was divided into 3 separate parts with timing of dosing and intake of meal at pre-defined time-points as outlined in Table
1. After consenting the women were allocated to participate in one of the parts.
Table 1
described the study design in the 3 parts with the time of meal and dosing of oral calcitonin.
Part 1 Morning Dosing | Dose | Meal | Meal | - | Meal | - | End | x | x | x | x | x |
Part 2 Pre-dinner Dosing | x | x | Meal | Dose | Meal | - | - | Meal | Meal | End | x | x |
Part 3 Evening Dosing | x | x | x | x | Meal | Dose | - | Meal | Meal | - | Meal | End |
Part 1 included a total of 42 subjects and was aimed at investigation of PK and PD of two variants of 0.8 mg of sCT and placebo given in the morning at 8:00. At the start of the study, subjects were randomized to receive active treatment (the two production variants) and placebo in a specific order. Part 1 had 3 treatment periods of 3 days, with study drug intake in the morning of each day. The wash-out period was at least 3 days between treatment periods. After an overnight fast, subjects received the dose of drug with 200 ml of water and was sampled immediately before drug intake, and at 5, 10, 15, 30, 45 minutes, 1, 1 1/2, 2, 2 1/2, 3 hours, and every hour until the second dosing 24 hours after first dose. Meals were served at scheduled time points as outlined in Table
1. No other food consumption at any other time point was allowed, but intake of water was allowed from 1 hour after dosing.
Part 2 included a total of 20 subjects and was aimed at investigation of PK and PD of an oral dose of 0.8 mg of sCT (batch 010) and placebo given pre-dinner at 17:00. At the start of the study, subjects were randomized to receive active treatment and placebo in a specific order. The study included 2 treatment periods with a single dosing each and with a wash-out period of at least 3 days between treatment periods. On the first day of treatment, the subjects arrived at the clinic during the morning, had a meal at 13:00 and received the first dose at 17:00. Additional meals were served at the scheduled time points, and the 24-hour blood sampling schedule was similar to that described in Part 1.
Part 3 included a total of 19 subjects and was aimed at investigation of PK and PD of an oral dose of 0.8 mg of sCT (batch 010) and placebo given in the evening at 22:00. The randomization, treatment periods, and wash-out period were similar to Part 2. On the first day of treatment, the subjects arrived at the clinic at dinner time, had a meal at 18:00 and received the first dose at 22:00. Additional meals were served at the scheduled time points, and the 24-hour blood sampling schedule was similar to that described in Part 1.
The plasma and serum samples were stored at -20°C until analysis. For PK assessment, plasma sCT was measured in the blood samples collected in the period of 0–4 hours after drug intake.
The concentration of plasma sCT was measured by a chemiluminescence-based immunoassay as previously described [
6]. Values measured below the lower limit of quantification of 2.5 pg/ml was assigned the value of 2.5 pg/ml. The assay was a two-site immunometric type employing two antibodies, one biotinylated and the other acridium labeled. Specificity has been tested against synthetic fragments of sCT and against human as well as eel calcitonin and negligible interaction has been found over the range of standard curve. The lower limit of quantification (LLOQ) was 2.5 pg/ml. The quality control samples, ranging from 2.5 pg/ml to 700 pg/ml, were prepared daily and measured in 3 to 5 replicates. The overall accuracy and precision (CV) of the control samples measured on 11 different days was 101.3% and 10.1% for 2.5 pg/ml concentration and 94.3% and 6.0% for 700 pg/ml concentration, respectively.
The Serum CTX-I test is a sandwich enzyme enzyme-linked immunosorbent assay (ELISA) employing two monoclonal antibodies both recognizing the C-telopeptide of the α1-chain in type I collagen [
33]. The monoclonal antibodies, i.e. MAb F1103 and MAb F12, recognize the eight amino acid sequence EKAHD-β-GGR, where D- β -G denotes an isomerised bond between aspartate and glycine, and both antibodies require the presence of a free C-terminal arginine for binding. Cathepsin K, secreted by the osteoclast, is responsible for the proteolytic cleavage exposing the free C-terminal arginine [
30]. The sandwich construction assures that only cross-linked di-peptides, i.e. EKAHD-β-GGR × EKAHD-β-GGR, are measured by the Serum CTX-I ELISA. The measuring range is 0.020–3.380 ng/ml, and in this range the intra- and interassay coefficient of variation is < 3.0 and < 10.9%, respectively, and the dilution recovery 103%. The reference range (mean (95% confidence interval) for postmenopausal and premenopausal women as well as men is 0.439 ng/ml (0.142 – 1.351 ng/ml), 0.287 ng/ml (0.112 – 0.738 ng/ml), and 0.294 ng/ml (0.115 – 0.748 ng/ml), respectively, according to the manufacturer (Immunodiagnostic Systems Nordic, Herlev, Denmark)[
33].
Statistical analysis
The sample size was calculated separately for the three parts of the study. With the primary objective of investigating the PK profiles of the two variants of sCT in Part 1, a sample size of 42 subjects was necessary to have at least 85% power to reject the null hypothesis that the absolute difference in the loge-transformed Cmax of the two variants was above 0.693 using a one-sided t-tests at the 5% significance level, based on a two-arm cross-over design. Transformed back to the original scale, the null hypothesis corresponds to ratio less than 0.5 or greater than 2.0. The alternative hypothesis was that the difference in mean loge-transformed Cmax between the two variants was 0.05 (that is, the Cmax of the batch 010 variant was 95% of the batch 002 variant and that the between-subjects standard deviation on the loge scale was 1.0. Similar sample size calculations applied for AUC0–4 hrs.
For Parts 2 and 3, a sample size of 20 subjects was necessary to have at least 90% power to reject the null hypothesis that the difference in the serum CTX AUC0–24 hrs between sCT and placebo was less than 0.150 using a one-sided t-test at the 5% significance level using a two-arm cross-over design. The between-subjects common standard deviation was assumed to be 0.11.
No randomization of subjects was performed to Parts 1, 2 or 3 of the study, therefore comparisons between the three parts should be made with care as they may be affected by bias.
The trapezoidal method was applied for the calculation of AUC0–4 hrs of plasma sCT and relative change in serum CTX after dosing. The relative value of serum CTX was calculated as percentage of the individual pre-dose value within each treatment period. As a post-hoc unplanned analysis, the relative change of serum CTX was determined as 100% minus the relative value of serum CTX. The AUC of plasma sCT, and the time course data of plasma sCT, serum CTX, and relative value of serum CTX were logarithmically transformed to obtain normality and symmetry of variances.
Comparison of the two variants of sCT on the pharmacokinetic parameters of sCT Cmax and sCT AUC0–4 hrs investigated in part 1 was performed in a linear mixed effect model with parameter as response variable and variant (var002, var010) and treatment sequence (1,2,3) as fixed effects and subject as random effect. In each dose time group the treatment response on sCTX AUC0–24 hrs was assessed in a linear effect model with treatment group (var002, var010, placebo) and treatment sequence (1,2,3) as fixed effects and subject as random effect. Comparison of the parameters of sCT Cmax and sCT AUC0–4 hrs in the active treatment groups among the dose time groups was performed in a linear effect model with parameter as response variable and dosing time (part 1,2,3) and sequence (1,2,3) as fixed effects. In the comparison of the active treatment response of var010 on sCTX AUC0–24 hrs among the dose time groups, first the placebo-corrected values were calculated by subtracting the placebo AUC from the treatment AUC for each individual subject. The placebo-corrected AUC was then compared among the dose time groups in a linear effect model having dosing time (part 1,2,3) as fixed effect. The significance levels were Tukey-Kramer adjusted in the multiple comparisons.
A difference was considered significant if p-value was less than 5%. All statistical calculations were performed using the SAS software package (release 9.1, SAS Institute Inc., Cary, NC, USA).