Most cardiovascular events are attributed to high blood pressure. Therefore, antihypertensive therapy means reducing considerably the risk of developing cardiovascular complications that cause a high mortality rate in the patients with hypertension [1]. Many drugs belonging to different classes are commonly prescribed for the treatment of hypertension. Non-peptidergic angiotensin II receptor antagonists (AT1 blockers, ARBs, sartans) were the last additions to the antihypertensive treatments. They have an important position in the treatment of hypertensive disease, including diabetic nephropathy, because they can selectively block the angiotensin type 1 (AT1) receptor in the renin-angiotensin system [2-4]. Recenty, some of these agents, in particular candesartan and valsartan (CAS 137862-53-4), were introduced for the management of congestive heart failure, as an alternative to ACE inhibitors [4]. Valsartan, N-valeryl-N[[2-(1H-tetrazol-5-yl)biphenyl-4-yl] methyl] valine, is a antihypertensive drug belonging to the family of angiotensin II receptor antagonists acting at the ATI receptor, which mediates all known effects of angiotensin II on the cardiovascular system. Its empirical formula is C24H29N5O3 and its molecular weight is 435.5. Valsartan is widely used in the treatment of hypertension [2]. The drug in its unchanged form shows strong pharmacological activity with high affinity for the AT1 receptor [5, 6]. It was approved in adults for the treatment of hypertension, heart failure, and left ventricular failure or left ventricular dysfunction postmyocardial infarction. Its effects primarily result from selective blockade of the angiotensin type I receptor in vascular smooth muscle and adrenal gland. Valsartan effectively reduces systolic and diastolic blood pressure in adults, both as monotherapy and in combination with other antihypertensive agents, displaying similar antihypertensive efficacy to other antihypertensive drug classes. Given its effects on angiotensin blockade, valsartan may also reduce proteinuria and have other beneficial effects in patients with underlying kidney disease [7]. When given as a capsule, 19-23% of the dose of valsartan is absorbed [8, 9]. Oral bioavailability is about 19% (range 10- 35%) if given as a capsule, but 39-51% if given as a solution.[8-10] The Tmax of valsartan is 2-4 h after the administration of a capsule; the absorption is more rapid if the drug is given as a solution (Tmax = 1 h) [8]. Food decreases Cmax by 50% and AUC by 40%. The Cmax and AUC increase linearly with increasing dose. Protein binding of valsartan is high (94-97%, mainly to albumin)[8, 11], and its Vd is 17 L [8, 9, 11]. The total clearance of valsartan is 37 mL/min, and plasma elimination time is in the range of 6-10 h. The drug does not accumulate significantly after repeated dosing [8]. Valsartan is eliminated mainly by biliary excretion as unchanged drug in faeces ( 80 -85%). Urinary excretion (mainly in unchanged form) accounts for 7%, 13% and 29% of the dose given as a capsule, as a solution or intravenously, respectively [8, 9]. Valsartan is not metabolised by the cytochrome P450 enzymes, but is converted to inactive compounds (15-20% of the dose), with only 9% in the circulation [8, 11]. Many drugs are made and marketed by more than one pharmaceutic manufacturer. There is evidence that the method of preparation and the final formulation of the drug can markedly affect the bioavailability of the drug. Since there are many products containing the same amount of active drug, physicians and pharmacists must select products that produce equivalent therapeutic effect. For this purpose the bioavailability of drug products are determined and compared according the FDA guidelines. The aim of this study was to demonstrate that the rate and extent of absorption of a generic tablet formulation of valsartan and the reference tablet formulation are not statistically different to each other based on the obtained plasma concentration data following oral administration in healthy volunteers.
2. Methods:
2.1. Subjects:
Twenty four male healthy volunteers were enrolled in this study. They were all Iranians, aged between 20 and 27 years (23.6 ± 2.1 years) and weight from 62 to 100 kg (74.1 ± 10.7 kg). Exclusion criteria included: hypersensitivity to fexofenadine; alcohol dependency or drug addiction; smoking; treatment with other drugs during the last two weeks and undergoing a medical examination at another hospital during the study. Before the studies the subjects underwent physical examination where the status of the heart, lungs and blood circulation, as well as medical history was recorded. The volunteers were informed about possible risks and adverse effects of taking the drug, and written consent was obtained. The study was reviewed and approved by the local ethical review board of Tabriz University of Medical Sciences in Iran and conducted in accordance with the guidelines of the Declaration of Helsinki (World Medical Assembly 1964) as revised in Edinburgh (2000).
2.2. Study design:
An open label, randomized, single-blind two sequence, two period, crossover study with a two week washout period was conducted. The test (Exir Pharmaceutical Co, Boroujerd, Iran, batch no: 01) and reference (batch no: K0010) formulations were randomized and given to the volunteers. Both formulations were obtained from the respective manufacturers. One tablet (equivalent to 80 mg Valsartan) of the test and reference formulations was administered to each subject as a single dose. They were given a standard breakfast 2 h and lunch 6 h after the morning drug administration and were only allowed to drink mineral water during the day of experiment. Five milliliters of blood were drawn at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 hours after each administration. The blood samples were taken from subject's forearm veins. All samples were centrifuged in heparinated tube. The plasma samples were separated and kept frozen at -20 °C for subsequent analysis.
2.3. Analysis of plasma samples:
Valsartan plasma concentrations was determined using a modified high performance liquid chromatographic (HPLC) method which was validated for specificity, accuracy, precision and sensitivity [12]. A liquid chromatographic system (Knauer, Germany) comprising of Knauer K1000 solvent delivery module equipped with a Rheodyne (Cotati, CA) injector and a variable wavelength spectrofluorimetric detector (RF-551). EZ Chrom Elite version 2.1.7 was used for data acquisition, data reporting and analysis. Methanol (Merck Darmstadt, Germany) (1 ml) was added to 0.2 ml of plasma; the tube was vortex-mixed for 30 seconds and centrifuged at 5500 rpm for 5 min. One hundred microlitres of the supernatant was injected into the chromatographic system. The drug was eluted from a MZ ODS2 (125 - 4.6 mm) 5 µm column (MZ-Analysentechnik GmbH, Mainz, Germany) and a MZ ODS2 (10 - 4 mm) 5 µm precolumn (MZ-Analysentechnik GmbH, Mainz, Germany), using a mobile phase consisting of acetonitrile (Merck Darmstadt, Germany) - 15 mM potassium dihydrogenphosphate (Merck Darmstadt, Germany), pH 2.0 adjusted with o-phosphoric acid (Merck Darmstadt, Germany) (45:55, %v/v), at a flow rate of 1 ml/min. The spectrofluorimetric excitation and emission wavelength were set at 234 and 374 nm respectively. Under these conditions the retention time of valsartan was 4.1 min. There were no interfering peaks in the chromatogram of blank plasma at the retention time of valsartan. The external standard method was used to analyze the samples and each run had a separate daily calibration. The within-day and between-day accuracy and precision values of the method were determined using the quantitation of 4 quality control samples with different concentrations within the calibration range during 4 consecutive days. Recovery values for the extraction procedure were calculated by comparing chromatographic responses obtained from spiked extracted plasma and drug free plasma samples spiked with the same concentration immediately after extraction. The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated based on the equations mentioned in the literature [13-16]. The drug was stable in plasma for more than 4 weeks when stored at -20°C.
2.4. Pharmacokinetic study:
The pharmacokinetic parameters for valsartan were calculated using standard non-compartmental methods. The plasma concentration-time profile of each individual treatment was constructed. Pharmacokinetic analysis consisted of visual identification of the maximum plasma concentration (Cmax) and the time at which this occurred (Tmax) from the individual subject plasma concentration-time profiles. The area under the plasma concentration - time curve from time zero to t (AUC0-t) was calculated using the linear trapezoidal rule. The terminal first order constant (kel) was determined by a least squares fit of the terminal plasma concentrations (using Excel® for Windows®). The elemination half life (t1/2) was determined with the quotient of 0.693/ kel. The constant kel was used to extrapolate AUCt-∞. AUC0-∞ was obtained from AUC0-t plus AUCt-∞. Bioequivalence between the formulations was determined by calculating 90% confidence intervals (90% C.I.) for the ratio of Cmax, AUC0-t, and AUC0-∞ values for the test and reference products and were compared to the reference intervals (0.8-1.25) as suggested by the FDA [17-21]. Analysis of variance (ANOVA) was used to assess formulation, period, sequence and subject effects statistically.
3. Results and discussion:
The assay was linear over the concentration ranges of 62.5 to 4000 ng/ml, with a coefficient of correlation (r2) of 0.999. The limit of quantitation for Valsartan was 30 ng/ml. The analytical recovery of Valsartan was 96.14, 96.58, and 96.35 % at concentrations of 500, 1000 and 2000 ng/ml respectively. Moreover the intra-assay accuracy of the method ranged from 99.7 to 101.9%, while the inter-assay accuracy ranged from 96.9 to 101.6%. All 24 volunteers well tolerated both of the formulations and completed the study until the end. No adverse effects were reported which could have influenced the outcome of the study. The mean serum concentration-time profiles after single oral dose administration of reference and test formulations are illustrated in Fig.1. As it is seen, the mean serum concentration-time curves of both test and reference formulations are almost superimposable. Moreover, there was no significant difference between valsartan serum concentrations at each time point following oral administration of the two formulations. At the first sampling time (0.5 h), the drug was measurable in all subjects following the administration of both formulations. The resulting pharmacokinetic parameters are shown in Table 1. Mean maximum serum concentrations of 3067.7±1281.7 ngh/ml (90% CI: 2637.4-3498.0) and 3304.3± 1196.4 ng/ml (90% CI: 2902.6-3706.0) were obtained for the test and reference formulations, respectively. Tmax, the time required to reach the maximum serum concentration, was 2.33 ± 0.69 h (90% CI: 2.10-2.56) and 1.98± 0.43 h (90% CI: 1.84-2.12), respectively. In addition to Cmax and Tmax, the ratio of Cmax/ AUC0-∞ also can be used as a parameter for evaluating the absorption rates in bioequivalence studies [22, 23]. These calculated ratios were 16.3% and 17.2% for the test and reference formulations. The parameters used as measures of the extent of absorption are AUC0-t, AUC0-∞. The AUC0-t and AUC0-∞ for the test formulation were 17834.4± 7083.8 ngh/ml (90%CI: 15455.9-20212.8) and 18825.7± 7553.2 ngh/ml (90% CI: 16289.7-21361.7), respectively. The calculated values for the reference formulation were 18319.1± 7800.7 ngh/ml (90% CI: 15700.0-20938.2) and 19172.2± 8307.2 ngh/ml (90% CI: 16383.1-21961.4) in the order mentioned. The confidence limits shown in Table 2 reveal that these values are entirely within the bioequivalence acceptable range of 80-125% proposed by the FDA and EMEA [17-21]. The multivariate analysis accomplished through analysis of variance (ANOVA) indicated that there were no statistical differences between the two formulations with any of the pharmacokinetic parameters. Furthermore, periods and sequence effects did not influence the outcome of the statistical analysis.
4. Conclusion:
In the light of the obtained results of the studies reported here it can be concluded that the valsartan test and reference formulations are bioequivalent in terms of rate and extent of absorption.
Acknowledgements
The authors thank Exir Pharmaceutical Co, Boroujerd (Iran) for financial support. The authority of Drug applied research center, Tabriz University of Medical Sciences, is acknowledged for their support.
