Amlodipine inhibits the proliferation and migration of esophageal carcinoma cells through the induction of endoplasmic reticulum stress

BACKGROUND L-type calcium channels are the only protein channels sensitive to calcium channel blockers,and are expressed in various cancer types.The Cancer Genome Atlas database shows that the mRNA levels of multiple L-type calcium channel subunits in esophageal squamous cell carcinoma tumor tissue are significantly higher than those in normal esophageal epithelial tissue.Therefore,we hypothesized that amlodipine,a long-acting dihydropyridine L-type calcium channel blocker,may inhibit the occurrence and development of esophageal cancer(EC).AIM To investigate the inhibitory effects of amlodipine on EC through endoplasmic reticulum(ER)stress.METHODS Cav1.3 protein expression levels in 50 pairs of EC tissues and corresponding paracancerous tissues were examined.Subsequently,the inhibitory effects of amlodipine on proliferation and migration of EC cells in vitro were detected using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and Transwell assays.In vivo experiments were performed using murine xenograft model.To elucidate the underlying mechanisms,in vitro cell studies were performed to confirm that ER stress plays a role in inhibition proliferation and migration of EC cells treated with amlodipine.RESULTS The expression level of Cav1.3 in esophageal carcinoma was 1.6 times higher than that in paracancerous tissues.Amlodipine treatment decreased the viability of esophageal carcinoma cells in a dose-and time-dependent manner.In vivo animal experiments also clearly indicated that amlodipine inhibited the growth of EC tumors in mice.Additionally,amlodipine reduces the migration of tumor cells by inhibiting epithelial-mesenchymal transition(EMT).Mechanistic studies have demonstrated that amlodipine induces ER stress-mediated apoptosis and suppresses EMT.Moreover,amlodipine-induced autophagy was characterized by an increase in autophagy lysosomes and the accumulation of light chain 3B protein.The combination of amlodipine with the ER stress inhibitor 4-phenylbutyric acid further confirmed the role of the ER stress response in amlodipine-induced apoptosis,EMT,and autophagy.Furthermore,blocking autophagy increases the ratio of apoptosis and migration.CONCLUSION Collectively,we demonstrate for the first time that amlodipine promotes apoptosis,induces autophagy,and inhibits migration through ER stress,thereby exerting anti-tumor effects in EC.

Simultaneous determination of telmisartan and amlodipine in human plasma by LC-MS/MS and its application in a human pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric （LC-MS/MS） assay method has been developed and fully validated for the simultaneous quantification of telmisartan and amlodipine in human plasma. Carbamazepine was used as an internal standard. Analytes and the internal standard were extracted from human plasma by solid-phase extraction technique using Waters Oasis HLB 1 cm3 （30 mg） extraction cartridge. The reconstituted samples were chromatographed on a Hypurity advance C18 column （50mm × 4.6mm, 5 gm） using a mixture of acetonitrile -5 mM ammonium acetate buffer （pH-4.0） （50：50, v/v） as the mobile phase at a flow rate of 0.8mL/min. The calibration curve obtained was linear （r_〉0.99） over the concentration range of 2.01-400.06 ng/mL for telmisartan and 0.05 -10.01 ng/mL for amlodipine. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.

Simultaneous determination of amlodipine,valsartan and hydrochlorothiazide by LC-ESIMS/MS and its application to pharmacokinetics in rats

Polypill is a fixed-dose combination that contains three or more active ingredients used as a single daily pill to achieve a large effect in preventing cardiovascular disease with minimal adverse effects.A novel and accurate liquid chromatography tandem mass spectrometry method using electrospray ionization mode has been developed and validated for the simultaneous determination of amlodipine（AMD）,valsartan（VAL） using losartan（LOS） as an internal standard（IS）,and hydrochlorothiazide（HCT） using furosemide（FSD） as an IS.The separation was carried on Aquasil C18（50 mm×2.1 mm,5μm） reversed phase column using acetonitrile and water containing 0.1%formic acid（50：50,v/v） as the mobile phase.The method was validated in terms of linearity,accuracy and precision over the concentration range of 1-1000 ng/mL.The intra and inter-day precision and accuracy,stability and extraction recoveries of all the analytes were in the acceptable range.This method can be successfully applied to the pharmacokinetic study of AMD,VAL and HCT when given as a polypill.

Application of an LC–MS/MS method for the analysis of amlodipine,valsartan and hydrochlorothiazide in polypill for a bioequivalence study

A sensitive and selective method has been proposed for the simultaneous determination of amlodipine(AML),valsartan(VAL) and hydrochlorothiazide(HCTZ) in human plasma by liquid chromatography–tandem mass spectrometry(LC–MS/MS). The analytes and their deuterated analogs were quantitatively extracted from100 μL human plasma by solid phase extraction on Oasis HLB cartridges. The chromatographic separation of the analytes was achieved on a Chromolith RP18 e(100 mm × 4.6 mm) analytical column within 2.5 min. The resolution factor between AML and VAL, AML and HCTZ, and VAL and HCTZ was 2.9, 1.5 and 1.4, respectively,under isocratic conditions. The method was validated over a dynamic concentration range of 0.02–20.0 ng/m L for AML, 5.00–10,000 ng/m L for VAL and 0.20–200 ng/m L for HCTZ. Ion-suppression/enhancement effects were investigated by post-column infusion technique. The mean IS-normalized matrix factors for AML, VAL and HCTZ were 0.992, 0.994 and 0.998, respectively. The intra-batch and inter-batch precision(% CV) across quality control levels was ≤ 5.56% and the recovery was in the range of 93.4%–99.6% for all the analytes. The method was successfully applied to a bioequivalence study of 5 mg AML + 160 mg VAL + 12.5 mg HCTZ tablet formulation(test and reference) in 18 healthy Indian males under fasting. The mean log-transformed ratios of C max, AUC0–120 h and AUC0-inf and their 90% CIs were within 90.2%–102.1%. The assay reproducibility was demonstrated by reanalysis of 90 incurred samples.

Clinical Utility of Amlodipine/Valsartan Fixed-Dose Combination in the Management of Hypertension in Chinese Patients

Amlodipine/valsartan(Aml/Val)single-pill combination(SPC)therapy has been widely used and studied in clinical practice in recent years.This article reviews the Chinese and English literature on the clinical use of Aml/Val SPC therapy in Chinese hypertensive patients.According to five studies concerning the efficacy and safety of this treatment,Aml/Val SPC therapy was more efficacious than monotherapy with valsartan,amlodipine,or the nifedipine gastrointestinal therapeutic system.This treatment showed greater blood pressure-lowering effects,a higher blood pressure control rate,and a higher response rate.Aml/Val SPC treatment was well tolerated,with adverse event rates similar to those of monotherapy with valsartan or amlodipine and significantly rarer adverse events compared with the nifedipine gastrointestinal therapeutic system.Aml/Val SPC is a highly efficacious and well-tolerated antihypertensive treatment in Chinese hypertensive patients.

Effect of Amlodipine on the Growth of Vascular SmoothMuscle Cells of Spontaneously Hypertensive Rats

The effect of anti-hypertensive drug amlodipine on regression of cardiovascular hypertrophy due to hypertension was studied by using cultured smooth muscle cells derived from arteries of spontaneously hypertensive rats (SHR) and measuring [3H]-TdR and [3H]-Leucine binding. 48 h after adding amlodipine.[3H] -TdR binding in arterial smooth muscle cells from SHR in vitro was reduced by 50.5% and [3H]-Leucine binding was reduced by 56. 2% as compared with neuropeptide Y (NPY)-treated group. However, there was no significant change in cell number. The results showed that amlodipine could effectively inhibit increase of DNA and protein synthesis of vascular smooth muscle cell (VSMC) due to NPY. It indicates that amlodipine is of great significance on regression of genesis and development of cardiovascular hypertrophy due to hypertension.

Spectrophotometric Methods for Simultaneous Determination of Amlodipine Besylate and Atenolol in Their Tablet Dosage Form

Three simple,specific,accurate and precise spectrophotometric methods are developed for simultaneous determination of amlodipine besylate(AM)and atenolol(AT)in tablets.The first method is dual wavelength spectrophotometry(DW).The second method is ratio subtraction(RS)which depends on subtraction of the plateau values from the ratio spectrum,coupled to first derivative of ratio spectra(1 DD).The third method applies bivariate calibration method using 210and 225nm as an optimum pair of wavelength for amlodipine and atenolol.The calibration curves are linear over the concentration range of 4~40μg·mL-1 for both drugs.The specificity of the developed methods is investigated by analyzing laboratory prepared mixtures of the two drugs and their combined dosage form.The two methods are validated as per ICH guidelines and can be applied for routine quality control testing.

Development and validation of an RP-HPLC method for simultaneous determination of Ramipril and Amlodipine in tablets

AnRP-HPLC method for the simultaneous determination ofRamipril （RP） andAmlodipine （AL） in tablets was developed and validated by Chinese Pharmacopoeia 2010. The linearity of the proposed method was investigated in the range of 0.01-0.25 mg/mL （r^2=0.9998） for RP and 0.014-0.36 mg/mL （r^2=0.9997） for AL. The limits of detection （LOD） were 0.06 lag/mL and 0.02 μg/mL for RP and AL, and the limits of quantitation （LOQ） were 0.2 Dg/mL and 0.07 μg/mL, respectively. Some major impurities and degradation products did not disturb the detection of RP and AL and the assay can thus be considered stability-indicating.

Development and Validation of HPLC Method for Simultaneous Determination of Amlodipine, Valsartan, Hydrochlorothiazide in Dosage Form and Spiked Human Plasma

A simple, sensitive, and specific method was developed for simultaneous determination of Amlodipine besylate (AML), Valsartan (Vals) and Hydrochlorothiazide (HCT) by high performance liquid chromatography without previous separation. Satisfactory resolution was achieved using a RP-C18 chromatographic column, Phenomenex Kinetex (150 mm × 4.6 mm i.d) and a mobile phase consisting of acetonitrile-phosphate buffer (0.05 M) with pH 2.8 in the proportion of (40/60, v/v) at a flow rate 0.8 mL/min and the wavelength detection was 227 nm. The retention time for HCT, AML and VAls was 2.26, 3.16 and 11.19 min;respectively. The described method was linear over a range of 4-28 μg /ml, 5-40 μg /ml and 1-12 μg /ml for AML, Vals and HCT;respectively. The mean percent recoveries were 99.94%, 99.96% and 99.78% for AML, Vals and HCT;respectively. F-test and t-test at 95%con?dence level were used to check the intermediate precision data obtained under different experimental setups. The method could be used for analysis of combined dose tablet formulation containing AML, Vals, HCT as well as spiked human plasma.

LC,MS^n and LC-MS/MS studies for the characterization of degradation products of amlodipine

In the present study,comprehensive stress testing of amlodipine（AM） was carried out according to International Conference on Harmonization（ICH） Q1A（R2） guideline.AM was subjected to acidic,neutral and alkaline hydrolysis,oxidation,photolysis and thermal stress conditions.The drug showed instability in acidic and alkaline conditions,while it remained stable to neutral,oxidative,light and thermal stress.A total of nine degradation products（DPs） were formed from AM.which could be separated by the developed gradient LC method on a C18 column.The products formed under various stress conditions were investigated by LC-MS/MS analysis.The previously developed LC method was suitably modified for LC-MS/MS studies by replacing phosphate buffer with ammonium acetate buffer of the same concentration（pH 5.0）.A complete fragmentation pathway of the drug was first established to characterize all the degradation products using LC-MS/MS and multi-stage mass（MS^n） fragmentation studies.The obtained mass values were used to study elemental compositions,and the total information helped with the identification of DPs.along with its degradation pathway.

New Stability Indicating Method for Quantification of Impurities in Amlodipine and Benazepril Capsules by Validated HPLC

A stability indicating LC method was developed for the simultaneous determination of Amlodipine and Benazepril capsules in pharmaceutical dosage form. Efficient chromatographic separation was achieved on Symmetry C18 stationary phase with simple combination of amobile phase containing 750 mL of DI Water, 250 mL of Acetonitrile and 2 mL of Octylamine into suitable container with adjusted pH to 2.50 ± 0.05 with the aid of Ortho phosphoric acid delivered in an isocratic mode and quantification was carried out using UV detection at 240 nm at a flow rate of 1.0 mL·min-1 with an injection volume of 20 μl and ambient column temperature. This method is capable to detect both the drug components of Amlodipine and Benazepril in presence of their degradation products (Amlodipine Imp-A and Benazepril Impurity-C) with a detection level of 0.05%. Amlodipine/Benazepril in their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the samples were analysed. Peak homogeneity data of Amlodipine and Benazeprilis were obtained using PDA detector, in the stressed sample chromatograms, demonstrating the specificity. The method shows excellent linearity over a range of 0.05%-2.0% for Amlodipine, Amlodipine Impurity-A and 0.05%-5.0% for Benazepril and Benazepril Impurity-C. The correlation coefficient for Amlodipine and Benazepril is 1. The relative standard deviation was always less than 2%. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of Amlodipine and Benazepril in pharmaceutical preparations. The developed HPLC method was validated with respect to linearity & range, accuracy, precision and robustness.

Rapid Simultaneous Determination of Olmesartan, —Amlodipine and Hydrochlorothiazide in Combined Pharmaceutical Dosage form by Stability-Indicating Ultra Performance Liquid Chromatography

A simple, precise and rapid stability-indicating ultra-performance liquid chromatography (UPLC) method was devel-oped for the simultaneous quantitative determination of Olmesartan, Amlodipine and Hydrochlorothiazide from their innovative Pharmaceutical combination drug product, with the presence of degradation products. It involved a 50 mm × 2.1 mm, 1.8 μm Phenyl column. The separation was achieved on simple gradient method. The mobile phase A contains a mixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile in the ratio 90:10, v/v, and mobile phase B con- tains a mixture of sodium perchlorate buffer pH 3.2(0.053M): acetonitrile in the ratio 10:90, v/v. The flow rate was 0.7 mL?min–1 and column temperature was maintained at 55?C.The gradient program (T/%B) was set as 0/10, 2/50, 4/80, and 6.0/10. The detector wavelength was 271 nm for Hydrochlorothiazide, 215 for Olmesartan and 237 nm for Amlodipine. The retention times of Olmesartan, Amlodipine, and Hydrochlorothiazide are 3.5, 3.3 and 0.9 minutes;respectively. The total runtime was 6.0 minutes within which three active compounds and their degradation products were separated. The described method was validated with respect to system suitability, specificity, linearity, precision and accuracy. The precision of the assay method was evaluated by carrying out six independent assays of Olmesartan, Amlodipine, and Hydrochlorothiazide (0.004 mg?mL–1, 0.001 mg?mL–1, 0.0025 mg?mL–1). The accuracy of the method was evaluated in triplicate at three concentration levels, i.e. 50%, 100%, and 150% of target test concentration. The described method was linear over the range, 2 to 6 μg?mL–1 for Olmesartan, 0.5 to 1.5 μg?mL–1 Amlodipine and 1.25 to 3.75 μg?mL–1 for Hydrochlorothiazide. The method is fast and is suitable for high-throughput analysis of the drug and one can analyze about 240 samples per working day, facilitating the processing of large-number batch samples.

The Effect of Food Thickeners on the Bitterness and Dissolution of Amlodipine Besilate Powder When Co-Administered with Thickeners to Patients with Dysphagia

Purpose: The present research was performed to evaluate the effect of food thickeners on the bitterness and dissolution of bitter drugs when co-administered to patients with dysphagia. Methods: Amlodipine besilate (AMPB) powder was used as a model drug. Starch- and xanthan gum-based food thickeners were examined, with swallowing-aid jelly as a reference. The line-spread test (LST), texture prolife analysis (TPA) were done firstly. In related to AMPB powder mixed with food thickeners solution, a conventional dissolution test simulating the oral cavity was performed, the amlodipine (AMP) concentration and taste sensor output for dissolved medium versus time profiles were developed. The dissolution test at pH 1.2 and 4.5, representing typical gastric conditions for younger or elderly people, was performed in two kinds of thickener solution and swallowing-aid jelly those were mixed with AMPB powder. Results: LST demonstrated that xanthan gum-based food thickeners fulfilled the requirements for patients with dysphagia but that starch-based food thickeners did not. In TPA, hardness and adhesiveness decreased proportionally as the concentration increased for both kinds of food thickener. Conventional dissolution test simulating oral cavity demonstrated the following bitterness ranking: xanthan gum-based food thickener Conclusion: Although xanthan gum-based food thickeners were successful in masking the bitterness of AMP, they may reduce its bioavailability in humans. The 7.1 and 4.7 (w/v) % starch-based thickener show bitterness inhibition under simulated oral cavity conditions and complete dissolution of AMP under simulated gastric conditions.

New HPLC Method with Experimental Design and Fluorescence Detection for Analytical Study of Antihypertensive Mixture,Amlodipine and Valsartan

New HPLC method was developed for determination of amlodipine and valsartan in their binary mixture as a part of routine control of combined formulations. The method was validated to meet official requirements including selectivity, stability, linearity, precision and accuracy. Chromatography was carried out using a LiChrospher RP-18 column, a mixture containing acetonitrile, phosphate buffer of pH 3.5 and methanol (45:45:10, v/v/v) and new fluorescence detection at 255 nm for excitation and 448 nm for emission. The effect of methanol content, pH of the buffer, flow rate, detection wavelengths and column temperature was estimated in robustness study, according to a plan defined by the Plackett-Burman design. For identification of significant effects, both graphical and statistical methods were used. Ro-bustness for dissolution test was checked estimating the effects of paddle speed, temperature and pH of dissolution medium. The method was proved to complying with all official guidelines. Therefore, it is suitable for determination of amlodipine and valsartan in their binary mixtures for different analytical and pharmaceutical purposes.

Amlodipine inhibits matrix metalloproteinases expression and secretion in mouse macrophage

To investigate whether the calcium channel blocker amlodipine could inhibit macrophage matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression and secretion. Methods Peritoneal macrophages were isolated from BALB/C mice and incubated with low (5μg/L), middle (15μg/L) and high (305μg/L) concentrations of amlodipine, or in the medium alone (controls) for 24 hours, and the expression and secretion of MMP-2 and MM-9 of the cells were analyzed by RT-PCR and gelatin zymography. Results Compared with controls, amlodipine at low concentration had no significant effects on the expression and secretion of either MMP-2 and MMP-9 (P＞0.05);at middle concentrationit it could inhibited MMP-2 and MMP-9 expressions completely and significantly reduced the secretion of MMP-9 (P＜0.05); but it had no effect on the secretion of MMP-2. At high concentration it also inhibited MMP-2 and MMP-9 expression completely. Conclusion Amlodipine at 15 ìg/L inhibited the expression of MMP-2 and MMP-9 and reduced the secretion of MMP-9, suggesting that amlodipine may stabilize atherosclerotic plaque.

Evaluation of stability and simultaneous determination of fimasartan and amlodipine by a HPLC method in combination tablets

A simple,rapid,accurate,precise and robust HPLC method was developed for the simultaneous determination of fimasartan and amlodipine in tablet dosage form.Furthermore,stability of active ingredients was evaluated under normal and stress conditions.The isocratic elution was accomplished by Nucleosil C18 column(250 mm×4.6 mm,5 mm)at 40℃.The mobile phase consisted of acetonitrile and 0.02 M monopotassium phosphate buffer(pH 2.2)in the ratio of 50:50(v/v)was eluted at 1.0 ml/min.The eluent was monitored by the UV detector for fimasartan and amlodipine at 237 nm for 8 min,detection time.The validation of HPLC method was carried out in accordance with the ICH guidelines.

MULTICENTER PARALLEL STUDY OF AMLODIPINE VERSUS NITRENDIPINE IN THE TREATMENT OF ESSENTIAL HYPERTENSION

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Simultaneous Determination of Amlodipine with H₁-Receptor Antagonists by Reversed Phase High Performance Liquid Chromatography and Application to Interaction Studies

A rapid, fast and precise method has been developed and validated for the simultaneous determination of amlodipine with H1-receptor antagonists (cetirizine, fexofenadine, and buclizine) from dosage forms. The chromatography was performed on a Purospher? Star, C18 (5 mm, 250 × 4.6 mm) column using acetonitrile: buffer (0.01 mM) (40:60, v/v, pH adjusted to 3.0), as a mobile phase. The mobile phase was pumped at a flow rate of 1.0 mL·min-1 and UV detection was performed at 240 nm. The method was validated for linearity, accuracy, precision and specificity. The method was applied to study the interaction between amlodipine and H1-receptor antagonists. These interactions were carried out in simulated gastric juice (pH 1), simulated full stomach (pH 4), blood pH (pH 7.4) and simulating GI (pH 9). The interacting drugs were heated at 37℃ with intermit-tent shaking and the samples were withdrawn every thirty minutes for three hours and drug contents were analyzed by RP-HPLC techniques. In most cases the in vitro availability of amlodipine was decreased. It was observed that the change in in vitro availability was pH dependent.

UPLCMS Method Development and Validation of Amlodipine, Hydrochlorthiazide and Losartan in Combined Tablet Dosage Form

A simple, rapid, sensitive and specific UPLCMS method was developed and validated following ICH guidelines for simultaneous estimation of tablet dosage form containing amlodipine (AMLO) hydrochlorothiazide (HCT) and losartan (LOSAT) using telmisartan (TELMI) as an internal standard (IS). The separation was achieved using Waters ACQUITY BEH C18 (1.7 μm, 2.1 × 50 mm) column with gradient mode, mobile phase containing acetonitrile (A) & 1% ammonium acetate (B) pH adjusted to 2.8 with trifluoro acetic acid with gradient mode. The flow rate was 0.4 mL·mL﹣1 and the injection volume 2 μl. The retention time for amlodipine, hydrochlorothiazide and losartan was found to be 3.7, 2.5 and 3.9 min, respectively. The developed method was found to be linear over the concentration range of 50 - 300 ng·mL﹣1, 125 - 750 ng·mL﹣1 and 500 - 3000 ng·mL﹣1 for AMLO, HCT and LOSAT respectively. The signal intensities obtained in ion mode for amlodipine, hydrochlorothiazide, losartan and telmisartan (IS) they were found to be much higher positive ion mode (M+)﹣ parent ion at m/z, 409.02, 297.97, 422.91 and 515.03, respectively, in QUATTROZQ full scan mass spectra.

Determination of Amlodipine in Human Plasma by LC-MS/MS and Its Bioequivalence Study in Healthy Chinese Subjects

A sensitive and reproducible liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of amlodipine in human plasma, with gliclazide as an internal standard (IS). The analyte was extracted with ethyl acetate and analyzed on a Diamond C18 (150 mm× 4.6 mm, 5 μm) column. The mobile phase was composed of methanol ?10 mM ammonium acetate with gradient flow rates and gradient conditions. Amlodipine and IS were ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring (MRM) mode using precursor→productions of m/z 409.2→238.1, 294.1 and m/z 324.2→127.3, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range of 0.05 - 12 ng/mL. The 90% confidence interval (CI) for AUC0-t, AUC0-∞ and Cmax ratios (test: reference) were all within 80% - 125% interval proposed by FDA. It is concluded that the validated method can successfully fulfill the requirement of clinical bioequivalence study of amlodipine in healthy Chinese subjects after administration of amlodipine/losartan combination tablets and amlodipine tablets, and the test and reference tablets were bioequivalent.