2.1 Study design
We conducted an open-label, controlled, crossover, 2-treatment, 2-period, 2-sequence, monocentre study at the Sector for Bioequivalence Trials at MHAT Tokuda Hospital, (Sofia, Bulgaria) between Januar and Februar 2013 (EudraCT-No: 2011-002396-42. Each study period lasted 5 days, including two hospitalizations of approximately 24 h (days 0 to 1) and 4 out-patient visits.
The concentration of total (i.e., free, and protein-bound) DRSP was determined using liquid chromatography and double-sector mass spectrometry [LC/MS/MS] in accordance with the respective recommendation for determination of DRSP in PK studies .
DRSP was analyzed by the bioanalytical division of Anapharm Europe using the analytical method SOP ANE 5199.05 entitled “Determination of Drospirenone in Human EDTA Plasma over a Concentration Range of 0.25 to 100 ng/mL using a LC/MS/MS Method”. The method involved a solid-phase extraction procedure with reversed phase 60 mg cartridges and subsequent derivatization with Girard-P solution. DRSP and internal standard were measured by reversed phase high-performance liquid chromatography coupled to a tandem mass spectrometry detector (LC/MS/MS). The calibration range at on-line validation was 0.25-99.80 ng/mL. The lowest calibrator (and thus the limit of quantification) was 0.25 ng/mL. The on-line validation based on quality control samples at four concentration levels (0.75, 35.00, 75.00, 8.00 ng/mL) for DRSP measured twice per analytical run showed an inter-assay precision of 2.17-6.72% coefficient of variation (CV). All samples from the same subject were measured in a single analytical run to eliminate the influence of the inter-assay imprecision of the assessment [15, 16, 17].
2.2. Ethical conduct, study approval and timelines.
We performed the study in accordance with Good Clinical Practice (GCP), local requirements and the Declaration of Helsinki. The Bulgarian Drug Agency and the local ethics committee at MHAT Tokuda Hospital (Sofia, Bulgaria) approved the study, and all subjects gave written informed consent.
Date of first enrolment: 15-JAN-2013 date of first subject dosed: 20-JAN-2013
Date of last completion: 06-FEB-2013 (last regular final visit), on 22-FEB-2013, additional control visit in 1 subject.
Trial registration number: EudraCT-No: 2012-004309-28. https://www.clinicaltrialsregister.eu
The posted result-related information is made public through the EU Clinical Trials Register of Eudra Pharm in accordance with the Commission guidance documents set out under Section 1, i.e., only result-related information on non-paediatric Phase-I clinical trials is not made public.
Subjects and treatments
We conducted the study in pre-menopausal Caucasian women, aged between 18 and 40 years, with a body mass index of ≥18.5 to ≤30 kg/m2. The women were required to be physically and mentally healthy based on medical and standard laboratory examinations, non-smokers since at least 6 months (confirmed by urine cotinine test) and had to be using an effective non-hormonal method of contraception. According to the final version of the study protocol (dated 08-Oct-2012) 24 healthy pre-menopausal female volunteers were randomized and completed both study periods according to protocol.
The investigator administered the study medication to each volunteer in a random way a single oral dose of 4 mg drospirenone on two single occasions. Each dosing was performed in the morning of day 1 between 8:00 and 8:46 a.m. and thus either under fasting conditions (at least 10 hours overnight fasting) or after food intake (i.e., 30 minutes after the start of the standard high-fat breakfast) after check for exclusion criteria and diet, restrictions, and adverse event.
A second medical professional supervised the intake. On each day of administration and/or blood sampling, the identity of the subject was compared with their national identity card.
In 2 study periods, the subjects received an oral dose of 4 mg DRSP, with a wash-out period of 14 days between study periods.
The investigator administered the dose according to randomization (Figure 1) with the subjects standing, and on days 1 subjects had to remain in an upright position (walking, sitting, or standing) for 5 h after drug administration.
2.3. Sample size
We calculated the sample size based on residual variance data for the area under the concentration/time curve (AUC) (15%) and observed maximum concentration (Cmax) (20%) obtained in a preceding pilot PK study. Twenty-four (24) subjects were enrolled and dosed so that, and all 24 subjects completed the study, which was considered to provide at least 80% power at 5% alpha for an equivalence test with a geometric mean ratio of up to 1.05 and the corresponding confidence interval (CI) within the limits of 80-125%.
The following pharmacokinetic endpoints were defined for drospirenone:
AUC(0-72h) Area under the concentration/time curve, calculated by the trapezoidal rule from time 0 h to 72h:
Cmax Observed maximal concentration after administration
tmax Observed time point of maximal concentration
The highest concentration really measured and the time at which it was registered in any given volunteer was regarded as Cmax and tmax, respectively. In cases with two or more identical concentration maxima at different time points the first one was always regarded as tmax.
If differences between the planned and real blood sampling times (time deviations) were observed after the administration of the test product the real time intervals were used for the purpose of calculation of the pharmacokinetic endpoints.
In case of missing samples because of not coming to visit or in case of drop-out subject all available plasma samples of this subject had to be analysed in the bioanalytical center and the results were to be presented in the study report as concentrations and individual graphics. No dropouts and no missing samples were recorded in the present study. The PK/BA evaluations had to be done as far as possible for the concrete individual case.
All endpoints listed above were determined in a model-independent way according to the algorithm of the program NC_PKP.sas.
All pharmacokinetic endpoints had to be determined in a model-independent way. The highest concentration really measured and the time at which it has been registered after each dose in any given volunteer was regarded as Cmax and tmax respectively.
The primary endpoints in the present trial were AUC(0-72h) and Cmax of drospirenone. These endpoints had to undergo descriptive and comparative statistical evaluation.
Secondary endpoint was tmax of drospirenone and had to undergo descriptive statistical evaluation.
Descriptive statistical evaluation provided the arithmetic and geometric means, standard deviation, CV, minimum, maximum, and median of the following: safety and demographic data of all randomized subjects, blood concentrations per subject/treatment for all randomized subjects and PK endpoints for all randomized subjects.
We performed the analysis of variance of log-transformed data according to a general linear model (GLM-ANOVA). Fixed factors in the model were sequence, treatment, period and subject within sequence.
We carried out the biostatistical evaluation using SAS for Windows version 9.2 (Statistical Analysis System, SAS-Institute, Cary NC, USA).
For the pharmacokinetic endpoints a descriptive statistical evaluation for all PK endpoints after single dose administration with and without food intake was performed. A parametric method (ANOVA-log) for the primary endpoints AUC(0-72h) and Cmax of drospirenone was carried out.
A 90% confidence interval (CI) for the ratio (Test with food vs. Test without food) for the primary endpoints AUC(0-72h) and Cmax of drospirenone was used with following fixed factors in the model: sequence, treatment, period, volunteer within sequence. A non-parametric method (Hauschke et al. 1990) for tmax of drospirenone was used.
A descriptive statistical evaluation was used for the evaluation of the safety.
We selected the 90% CI in accordance with the Committee for Medicinal Products for Human Use (CHMP) Guideline on the Investigation of Bioequivalence (CPMP/EWP/QWP/1401/98 Rev. 1/Corr**), dated 20 January 2010, stating that in “studies to determine bioequivalence after a single dose, the parameters to be analyzed are AUC(0-t), or, when relevant, AUC(0-72h) and Cmax, and that for these parameters the 90% CI for the ratio of the test and reference products should be contained within the acceptance interval of 80.00-125.00%. For studies to determine bioequivalence of immediate-release formulations at steady-state, AUC(0-τ) and Cmax,ss should be analyzed using the same acceptance interval as stated above.”