Bioequivalence Study of Two Formulations Containing Irbesartan 300 Mg Tablets in Healthy Colombian Volunteers

Arterial Hypertension may be defined as the condition in which blood pressure in the arterial tree is higher than normal and may be considered as a disorder per se or as an expression of other diseases. In most cases, it is not possible to find a specific cause, thus, it is named as Essential or Primary Hypertension. In a lesser proportion (10 to 15% of cases) finding a specific causal factor is feasible, generally being a nephropathy, a vascular injury or an endocrine disorder. Studies performed worldwide noted that 15 to 20% of the population suffers from hypertension (when hypertension is considered as a blood pressure above 160/90 mm Hg). In Colombia, the available data through First National Morbidity Survey pointed out that 9.6% of the subjects older than 15 years had systolic blood pressure above 160 mm Hg and 9.2% of the subjects had diastolic pressure above 95 mm Hg. [1]


Introduction
Arterial Hypertension may be defined as the condition in which blood pressure in the arterial tree is higher than normal and may be considered as a disorder per se or as an expression of other diseases. In most cases, it is not possible to find a specific cause, thus, it is named as Essential or Primary Hypertension. In a lesser proportion (10 to 15% of cases) finding a specific causal factor is feasible, generally being a nephropathy, a vascular injury or an endocrine disorder. Studies performed worldwide noted that 15 to 20% of the population suffers from hypertension (when hypertension is considered as a blood pressure above 160/90 mm Hg). In Colombia, the available data through First National Morbidity Survey pointed out that 9.6% of the subjects older than 15 years had systolic blood pressure above 160 mm Hg and 9.2% of the subjects had diastolic pressure above 95 mm Hg. [1] The objective of this study was to establish the Bioequivalence of two formulations containing Irbesartan 300 mg tablets by comparing its bioavailability after a single dose between the Test product produced by Tecnoquímicas S.A. (Colombia) and the Reference product, Aprovel ® , produced by Sanofi Aventis.
Reference drug : Aprovel ® Irbesartan 300 mg tablets, manufactured and distributed by Sanofi Aventis. Lot 1A715 Physical and chemical properties like assay of active ingredient and dose uniformity were evaluated for both the Test and Reference products and results are summarized in (Table 1) in order to state the Pharmaceutical Equivalence of these medicinal products before the conduction of the in vivo study.
to be used to collect such samples, the staff in charge of sampling and monitoring, diet restrictions to comply with, and all the information they requested to freely decide on their participation in the study. Subsequently, each one of them signed an informed consent form.

Study design:
A randomized, open-label, two periods, two sequences, crossover design was used with an 8 days washout period between each period. Three days before each period initiation, volunteers must refrain from medications, alcohol and any food or beverage containing methylxanthines. These restrictions were maintained during the entire sampling period. All volunteers were randomized to be allocated to the treatment sequence.
Drug administration: Volunteers had 10 hours fasting prior administration of the drug, which was given with 200 mL of water at doses of Irbesartan 300 mg, [3] (i.e. 1 tablet of 300 mg) to each volunteer, and two hours later, each volunteer was given a standardized food. During hospital stay, they received three full meals (breakfast, lunch and dinner) and two snacks (one in the morning and one in the afternoon). These are shown in (Table 3).
The sampling team was comprised by a physician and one registered nurse. Using Vacutainer ® , a blood sample was obtained by venipuncture in the superior limb immediately prior to administering the medication. Such sample was called 'zero time point sample'. All volunteers received both the Test and Reference product based on randomization and 12 blood venous samples were collected according to the following time points: 0; 20 and 40 minutes; 1, 1.5, 2, 3, 6, 9, 12, 24 and 48 hours. Samples were labeled for identification and centrifuged at 3000 rpm for 30 minutes. Plasma was transferred to a previously labeled tube and frozen at -20°C for later analysis. After an 8-day washout period, administration was repeated completing the second study period.
Validation of analytical method: The bioanalytical method employed for Irbesartan quantification in plasma was high-performance liquid chromatography with UV detection (HPLC-UV). Irbesartan was quantified in plasma using a chromatographic bio-analytical method validated Acetonitrile was employed as proteins precipitating agent. Analyte separation was achieved with an L1 Phenomenex 4.6 x 15 mm, 5 μm Column, at a temperature of 25°C employing an Agilent Infinity 1256 chromatographer. Isocratic elution was performed with a mobile phase comprised by buffer solution: Acetonitrile 60:40, at a constant flow rate of 1.0 mL/min. Total run time was 9.4 min. [4,5] Pharmacokinetic analysis: The pharmacokinetic analysis was performed using WinNonlin 5.3 (Pharsight Corporation, Cary USA) software, by means of a non-compartmental analysis. Peak concentration (C max ) and time to peak concentration (t max ) were directly obtained from results of plasma concentrations, as currently recommended by the FDA [6] and the European Medicines Agency (EMA) for drug assessment [7]. AUC total was calculated by the sum of partial AUC: a) AUC 0-t , between zero time point and the last time point with detectable concentrations, calculated through the trapezoidal rule and guaranteeing the calculation of at least 80% of the AUC with the last sample, b) AUC t-∞ , calculated as the C/K ratio, where C is the last detectable concentration and K the slope obtained by linear regression from the points corresponding to the drug elimination phase through a linear regression of the natural logarithm of concentrations [8]. Bioavailability-adjusted elimination constant (Ke), half-life (t ½ ), clearance (Cl) and mean residence time (MRT) were calculated after performing the non-compartmental analysis. The results of pharmacokinetic variables are summarized in (Table 4) with C max , AUC 0-t , AUC 0-∞ , T max values and the elimination rate (Ke) of each one of the studied formulations.

Statistical analysis
An analysis of variance (ANOVA) was used to determine possible effects for each variation factor by sequence, period or subject. For this, F-test with a statistical significance level of 5% (α=0.05) was used. Statistical comparison of transformed pharmacokinetic parameters of both formulations was performed using the statistical software WinNonlin version 5.3. The following Bioequivalence criterion was established in the protocol: The 90% confidence interval of Test C max / Reference C max and last Test AUC/last Reference, ratios that should be within the range 80-125% acceptability. In addition, the last AUC parameter should not be less than 80% of total AUC parameter.
Adverse events report: Adverse events were recorded according

Related Compounds
There is no more than 0.2% of the related compound A of Irbesartan, there is no more than 0.2% of any individual impurity, and there is no more than 0.5% of total impurities.

Conforms Conforms
Not detected Not detected

Results
Analytical results of active ingredient content, dose uniformity and dissolution test met the required specifications for the Pharmaceutical Equivalence statement.
The study involved the participation of 24 healthy Colombian volunteers of both genders (50% women and 50% men) who completed both periods and were included in the pharmacokinetic and statistical analysis. Both treatments were well tolerated and no adverse events were observed (Table 4) shows the mean pharmacokinetic parameters obtained from all volunteers (mean ± SD) and 90% confidence intervals  of the log-transformed pharmacokinetic parameters, these analyses were performed to determine Bioequivalence between the Test product produced by Tecnoquímicas S.A. and Aprovel ® produced by Sanofi Aventis, as shown in (Table 5).

NORMAL, SEMI-BLAND, LOW-SALT DIET And/Or PATTERN
The analytical method showed to be specific, since no interferences arising from the matrix components were found in the identification and quantification of Irbesartan. It showed linearity in a concentration range from 500 ng/mL to 7000 ng/mL with a correlation coefficient of 0.9977. The precision was proven with variation coefficients lower than 2.0% for low levels (500 ng/mL), and lower than 1.5% for the medium (3000 ng/mL) and high levels (7000 ng/mL). The accuracy was determined by comparing the ratio between the areas of the samples against 5 calibration curves of the system obtaining deviations of less than 2.0% for the lowest concentration of the curves, and less than 1.5% for the rest of the concentrations, showing compliance with the specification. The system has a quantification limit of 60 ng/mL for Irbesartan and a detection limit of 30 ng/mL for Irbesartan. The lower limit of quantification (LLOQ) was 60 ng/mL, the lower quality control (LQC) 7000ng/mL, the minimum quantifiable concentration (MQC) was ≤ 2.0 % and the high quality control (HQC) was ≤ 2.0%.

Discussion
The reduction in costs of cardiovascular pathologies treatment using multisource products is a desired aim by governments [8] and, accordingly, Bioequivalence studies allow suggesting the interchangeability of generic products versus reference products without repeating clinical trials in patients [6,7].
WHO recommends in its guidelines for the Conduction of Comparative Bioavailability Studies to carry out in vivo testing in multisource products to assess one dose and a sudden increase of the medication in plasma concentration, which was evaluated in this study [9]. These findings are consistent with other studies, which assess the pharmacokinetics changes of Irbesartan when administered without food [4,5,10].
The Pharmaceutical Equivalence Statement allowed qualifying the in vitro quality attributes of both formulations. These two periods, two sequences, crossover, single-dose design with healthy volunteers minimizes the variability and allows to assess the formulation effects. The analytical method used was selective, precise, accurate and robust. All 24 volunteers completed the study and did not exhibit adverse events with any of the formulations. The washout period was higher than the recommended 7 elimination half-lives and guaranteed the absence of carryover effect between periods.
The objective of this study was to assess the Bioequivalence of two Irbesartan 300 mg formulations. (Figure 1) shows the curves of the graphic representation of mean plasma concentration vs. time where similarity can be observed. Furthermore, the mean AUC 0-t and C max were not significantly different and the 90% confidence intervals of ratios (Test/Reference) to the mean criteria of AUC 0-t and C max comply with the interval requested by the FDA and the EMA [7] (Table 4).
In response to this evidence, this study conducted between the Test product manufactured by Tecnoquímicas S.A. and Aprovel ® manufactured by Sanofi Aventis in Colombian population, demonstrates that it will be possible to exchange these two formulations.