The protocol study was reviewed by the Committee of The Medical Research Ethics of the Faculty of Medicine, University of Indonesia (Jakarta, Indonesia) and was approved by the National Agency of Drug and Food Control (Jakarta, Indonesia).

This study was conducted in compliance with the ethical principles of the Declaration of Helsinki for biomedical research involving human volunteers and Good Clinical Practice (GCP). All participants signed a written informed consent after they had been informed of the nature and details of the study in accordance with Indonesian Guidelines for Bioequivalence Studies.

The study was based on single-dose, open-label, randomized two-way crossover design under fasting period with two weeks wash out period. Subjects were randomized to one of the two sequences to receive the formulations according to randomization scheme. The test preparation was 10 mg of Prohytens^® tablets, manufactured by PT. Novell Pharmaceutical Laboratories, Indonesia (Batch no. 10M035) and the reference formulation was 10 mg Triatec^® tablets, manufactured by PT. Aventis Pharma, Indonesia (Batch no. 065U014).

Based on previous study, the sample size n = 24 subjects was sufficient to ensure power of 80% for correctly concluding bioequivalence under the following assumption: α = 0.05, 0.95 < μT / μR < 1.05 and an intra-subject variability of 20% (Diletti et al., 1991; Mendes et al., 2006).

A total of 26 volunteers were selected among Indonesia residents and participated in this study. First twenty-four volunteers (19 males and 5 females) were available for pharmacokinetic evaluation. If volunteers could not complete the entire study (dropout/withdrawn), volunteers were replaced with two reversed volunteers based on the same randomization code. The demographic data of twenty-four volunteers are shown in Table 1. Volunteers were selected after passing a clinical screening procedure including a physical examination, ECG and clinical laboratory tests (hemoglobin, hematocrit, WBC, platelets, WBC differential, blood urea nitrogen, sGPT, sGOT, alkaline phosphatase, total bilirubin, total protein, fasting glucose, albumin, total cholesterol, creatinine, urine analysis, pregnancy test (for female subjects) and negative results of HBsAg, anti HBC and anti HIV. Volunteers were excluded if they had a history of peptic ulcer, any illness of the hepatic, renal and cardiovascular system, taken alcohol or other medications for a long period of time, had hypersensitivity to ramipril or related ACEi, had received any investigation drug within four weeks (or suitable longer period for slowly eliminated drugs) of enrollment and donation or loss more than 450 ml of blood within 3 months prior to the screening of the study.

All volunteers avoided using other drugs for at least two weeks prior to the study and until after its completion. They also refrain from ingesting alcohol, caffeine, chocolate, tea or cokecontaining beverages at least 48 hours before each dosing and until the collection of the last blood sample. The grapejuice was not prohibited (Bailey and Dresser, 2004).

Volunteers were confined to clinical unit of Clinisindo Laboratories one night before study to assure the fasting condition (10 hours before drug administration). On the study day, subjects were given one tablet of either product with 240 ml of water. No food was allowed until 4 hours after dose administration. Water intake was allowed 2 hours after the dose. Standard meals were served at 6 hours (±1008 calories) and 12 hours (±836 calories), snacks were served at 4 hours (±165 calories) and 8 hours (±160 calories) after drug administration. Total calories were calculated by nutritionist.

Subjects were remained upright (sitting or standing) for the first 4 hours. Subjects were confined at clinical unit of Clinisindo Laboratories for 24 hours after dosing and and did not permitted to take strenuous exercise during the sampling days. Blood pressure, heart rate, body temperature and adverse events were monitored during blood sampling.

5 ml of the venous blood were collected at pre dose, 10, 20, 40, 60, 80, 100, 120 minutes, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48 and 72 hours after drug administration in the heparinized tubes. After blood separation, plasma was frozen at -20°C until analysis (Mendes et al., 2006; van Griensven, 1993).

After two weeks wash out period, subjects returned to Clinisindo Laboratories and the blood sample analysis was repeated in the second period in the same manner to complete the crossover design.

The concentration of ramipril and ramiprilat in plasma was determined using LC-MS/MS method with TurboIonSpray mode. Enalapril (CAS# 75847-73-3, MW 376.4) was used as the internal standard (Bo Yuan et al., 2000). The method has already been validated in terms of selectivity, sensitivity, linearity, accuracy and precision, recovery and also has been verified just before being used in study. The limit of quantification for ramipril and ramiprilat was 0.1 ng/mL and 0.2 ng/mL, respectively. The standard calibration curves for ramipril and ramiprilat were ranged from 0.1-100 ng/mL and 0.2-100 ng/mL, respectively. The best linear fit and least-squares residual for the calibration curve were achieved with 1/x² weighing factor. The recoveries of ramipril and ramiprilat were 80.90%, 84.09%, 79.13% and 36.99%, 37.49%, 38.42% at low medium and high QC samples. The analytical separation was performed on a Synergi 4μ POLAR- RP-80A, 50 x 2.00 mm, 4 μm (Phenomenex^®, USA) and protected by guard column AQ C18, 4 x 2.0 mm (Phenomenex^®, USA). The mobile phase was used gradient of 0.1 % formic acid in acetonitrile and 0.1 % formic acid in water, pumped 0.6 mL/ min for 4.0 min run time. The column temperature was maintained at 40°C. Briefly, a 500 μL of human plasma in polypropylene tube was added with internal standard, 100 μL of 1 M phosphoric acid and 100 μL of methanol:water (50:50). After mixing, 3 mL of ethyl acetate was added and vortex mixed for 2 min. The mixture was centrifuged at 3000 rpm for 10 min. The organic phase was removed and evaporated to dryness under vacuum at 60°C for 15 min. The residue was reconstituted with methanol:water (1:1). A volume of 10 μL aliquot was injected into the LC MS/MS system. The retention time for ramipril and ramiprilat was 0.41 min and 0.34 min, respectively.

Analysis of safety-related data was considered using the more common adverse events which occurred after initiation of study treatment and supported by the following more detailed tabulations and analysis.

The bioequivalence was determined using the primary parameters, AUC_0-t, AUC_0-η, C_max. The maximum plasma concentration (C_max) and time to reach maximum plasma concentration (t_max) were obtained directly by inspection of the individual drug plasma concentration time data, and were used as measures of rate of absorption. The area under the plasma concentration time curve up to the last time (t) showing a measurable concentration (C_t) of the analyte (AUC_0-t) was calculated using the trapezoidal rule. The elimination rate constant (K_el) was calculated by the technique of least-squares regression from the data of the last 3-8 points of each plasma concentration data curve. The AUC_0-η values were determined by adding the quotient of C_t and the appropriate K_el to the corresponding AUC_0-t, that is:

where C is the estimated last plasma concentration. The apparent elimination half-life (t_1/2) of ramipril and ramiprilat in plasma was calculated by using the following equation:

For the parameters of AUC_0-t, AUC_0-η and C_max a multiplicative model was assumed, and analysis of variance (ANOVA) was applied using the respective ln-transformed data. For estimation of bioequivalence the 90% CI of the geometric mean ratio test/reference (T/R) for AUC_0-t, AUC_0-η and C_max were calculated assuming a multiplicative model. The accepted bioequivalence range for these parameters was 80-125%. All statistical analyses were performed using EquivTest version 2.0 software (Statistical Solution, Cork, Ireland).

Both ramipril formulations were well-tolerated at the administered dose and no significant adverse clinical events were observed. Twenty three out of 26 volunteers experienced 83 adverse events during the study. All adverse events were of mild intensity and recovered without concomitant medication. There were no serious adverse events. However, all events resolved completely. The disposition of adverse events is shown in Table 2.

A total of 26 volunteers were invited to participate in this study. First twenty-four volunteers were available for pharmacokinetic evaluation. The mean ramipril and ramiprilat concentration versus time profiles for both formulations are shown in Figure 2 and Figure 3. The pharmacokinetic parameters used to assess the bioequivalence of the test formulation versus the reference were AUC_0-t, AUC_0-η for the extent of the absorption and C_max and tmax for the rate of absorption. Descriptive statistics of the pharmacokinetic parameter for ramipril and ramiprilat test and reference are summarized in Table 3 where the geometric mean values and the range for the AUC_0-t, AUC_0-η, C_max and t_1/2 values obtained for each formulation are shown. The pharmacokinetic characteristic t_max was presented as mean values. For ramipril, the mean obtained values for test and reference products were 35.06 and 37.29 ng/mL for C_max; 21.62 and 23.19 ng.h/mL for AUC_0-t; 21.87 and 23.40 ng.h/mL for AUC_0-η. The median t_max for both formulations was 0.33 h. For ramiprilat, the mean obtained values for test and reference products were 11.17 and 12.21 ng/mL for C_max; 109.99 and 119.15 ng.h/mL for AUC_0-t; 134.48 and 141.63 ng.h/mL for AUC_0-η. The median t_max was 2.5 h (test) and 2.75 h (reference).

The results of the bioequivalence analysis for ramipril and ramiprilat are given in Table 4. The intra-subject variability of ramipril in the AUC_0-t, AUC_0-η, C_max, t_1/2 estimates from the coefficient of variables as determined by ANOVA were 17.00%, 17.03%, 32.36%, and 36.30%, respectively. The intra-subject variability of ramiprilat in the AUC_0-t, AUC_0-η , C_max, t_1/2 estimates from the coefficient of variables as determined by ANOVA were 10.05%, 10.63%, 15.23%, and 24.23%, respectively.