Comparative bioavailability of two formulations containing atenolol and chlortalidone associated in a 4:1 fixed combination.

Atenolol (CAS 29122-68-7) and chlortalidone (CAS 77-36-1) are marketed associated in a 4:1 strength ratio (100/25 and 50/12.5 mg) for the treatment of hypertension. According to EU guidelines, the bioequivalence of one dosage strength can also cover additional strengths when the pharmacokinetics of a given drug is linearly related with the dose. The kinetics of atenolol is linearly correlated with the dose and chlortalidone has linear kinetics with doses < or = 100 mg. Thus this trial carried out on the 100/25 mg strength also covers the 50/12.5 mg strength. The trial was carried out on 18 healthy volunteers (9 males and 9 females) according to a single dose, two-period, two-treatment, two-sequence study design with washout. Timed atenolol plasma concentrations and chlortalidone blood concentrations were used to assess primary pharmacokinetic parameters Cmax, tmax and AUC extrapolated to infinity by a non-compartmental model. The bioavailability of the two formulations was compared through the 90% confidence intervals (C.I.) of Cmax and AUC in accordance with operating guidelines. C.I. of chlortalidone were fully comprised in the 0.80-1.25 range. In the case of atenolol, which displayed a higher data dispersion, C.I. were comprised in the enlarged 0.70-1.43 range. Time to peak, tmax, did not show any statistically significant difference between the test and reference product with respect to both analytes. Pharmacodynamic measurements of the decrease in systolic blood pressure led to fully overlapping results with test and reference. The authors conclude that the test formulation should be considered bioequivalent with the reference with chlortalidone and in the borderline of bioequivalence with atenolol. As no safety problems were involved and pharmacodynamics led to overlapping results as between test and reference, the bioequivalence conclusion could be extended also to atenolol.

Amlodipine bioequivalence achieved with a very sensitive liquid chromatography tandem mass spectrometric bioassay.

Amlodipine (CAS 88150-42-9) is a 1,4-dihydropyridine derivative, one of the most widely used drugs for the management of essential hypertension. In developing manidipine (CAS 120092-68-4), a new antihypertensive drug, amlodipine was selected as the reference comparator drug in a Phase III double blind clinical trial. However, manidipine is formulated in hard gelatin capsules, whereas amlodipine is presented as a tablet. In order to respect the double blind design of the study, it was necessary to insert the amlodipine tablet into hard gelatin capsules matching those of the new test product. This called for an amlodipine bioequivalence study on two halves of one tablet inserted into a capsule (test formulation) and two halves of one tablet ingested as such (reference formulation). The bioequivalence trial was carried out on 18 healthy volunteers (9 males and 9 females). Subjects were administered a single 10 mg dose of test and reference products according to a two-treatment, two-period, two-sequence crossover design, with a wash-out period of three weeks. Plasma concentrations of the parent compound were monitored over a period of 6 days, considering the long half-life of amlodipine. The drug was quantified with a very sensitive, robust bioassay, which was set up and validated in our laboratory. Peak concentration and area under the curve of plasma concentrations were log-transformed and analyzed to obtain 90% confidence intervals which proved to be 0.94-1.06, and thus within the acceptable bioequivalence range of 0.80-1.25. Time to peak, analyzed according to a non-parametric test, did not show any statistically significant difference between the test and reference. Both the test and reference products showed a similar and very good safety profile. The conclusion is that one amlodipine tablet broken into two halves and administered as such (reference formulation) is bioequivalent with one amlodipine tablet broken into two halves and encapsulated (test formulation).

Bioequivalence of inhaled formoterol fumarate assessed from pharmacodynamic, safety and urinary pharmacokinetic data.

This paper deals with a crossover trial on healthy volunteers performed to obtain combined pharmacodynamic, safety and pharmacokinetic data in order to assess the bioequivalence of formoterol fumarate (CAS 43229-80-7) delivered by mono-dose dry powder inhalers, as test and reference. The trial was carried out on 24 Caucasian healthy male and female volunteers treated with 12 micrograms formoterol fumarate bihydrate capsules for inhalation route. Pharmacodynamics was evaluated through a challenge test with methacholine on the forced expiratory volume in 1 s (FEV1). Safety was achieved from glucose and potassium serum levels assayed on timed samples over a 12-h period cost-dosing and from blood pressure, heart rate and ECG recording. Pharmacokinetics was obtained from urinary excretion of formoterol, assessed by a highly sensitive analytical method (LC-MS-MS). Pharmacodynamic, safety and pharmacokinetic results evidenced the bioequivalence of the two formulations investigated. This investigation is an interesting approach how to assess bioequivalence when the classical approach based on the similarity of plasma concentrations can not be applied.

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry.

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC-MS-MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1-300 ng/ml for zofenopril and 2-600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.

Highly sensitive bioassay of lidocaine in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

A very sensitive HPLC-tandem mass spectrometric (LC-MS-MS) method for assaying lidocaine in human plasma was set up and fully validated. Lidocaine and an internal standard (bupivacaine) were extracted from 1 ml of alkalinized plasma with tert.-butylmethyl ether, back-extracted to a H3PO4 acidified solution and injected into a C18 column. Acetonitrile-26 mmol/l ammonium acetate, pH 4.5 (70:30, v/v) was the mobile phase at a flow-rate of 1 ml/min. The effluent was detected by PE Sciex API 365 LC-MS-MS system in positive ion mode. Ionisation was performed using an atmospheric pressure chemical ionization ion source operating at 400 degrees C. The multi reaction monitoring transition 235-->86 was monitored. Linearity was ascertained in the 0.2-30 ng/ml range with a limit of quantitation of 0.2 ng/ml. Intra- and inter-assay precision and accuracy were < or =3.8%. The high sensitivity and specificity achieved by the method allowed concentrations of lidocaine to be measured in plasma of healthy subjects topically treated with lidocaine (5% ointment) on normal skin over a 32-h period after dosing.

Pharmacokinetics and pharmacodynamics of zofenopril in healthy volunteers.

This study was carried out to investigate the pharmacokinetics of zofenopril (CAS 81938-43-4) and zofenoprilat, the behaviour of the angiotensin converting enzyme (ACE) (pharmacodynamics) following the administration of zofenopril calcium at the single oral dose of 60 mg in eighteen healthy volunteers. This open label, one-way study was carried out in a single centre on 18 healthy volunteers. The volunteers received an oral single 60 mg dose of zofenopril calcium following an overnight fast. The tablet was swallowed with 250 ml of water. Fasting continued for additional 4 h after dosing and no other liquid intake was allowed from 1 h before to 2 h after administration. Plasma concentrations of zofenopril and its active metabolite zofenoprilat as well as serum ACE activity were measured before drug intake (baseline) and on timed samples over a 36 h period after dosing by LC-MS-MS, a highly sensitive, validated method for active moiety concentrations. Peak plasma concentration was reached on average at 1.19 h with zofenopril and at 1.36 h with zofenoprilat. Concentrations then decreased reaching values under or close to the limit of quantitation (1 ng.ml-1 for zofenopril, 2 ng.ml-1 for zofenoprilat) from 8 to 16 h after dosing. Complete inhibition of ACE was seen at the first blood sampling time (1 h) and lasted on average up to 9.44 h. ACE activity then slowly reactivated, but enzyme inhibition continued and was estimated to be 74% and 56% at 24 and 36 h following drug administration, respectively. From these data a complete or almost complete enzyme inhibition is expected with zofenopril given in repeated dose regimen.