Laurent B. Nicolas

Development and validation of a fully automated online human dried blood spot analysis of bosentan and its metabolites using the Sample Card And Prep DBS System

This paper describes the development and validation of a liquid chromatography (LC)-electrospray ionization tandem mass spectrometry assay for the fully automated simultaneous determination of bosentan, a dual endothelin receptor antagonist used in the treatment of pulmonary arterial hypertension, and its three primary metabolites hydroxy bosentan (Ro 48-5033), desmethyl bosentan (Ro 47-8634), and hydroxy desmethyl bosentan (Ro 64-1056) in human dried blood spots (DBS) by use of the Sample Card And Prep (SCAP) DBS System. The system enabled the online extraction of compounds from filter paper cards without the need for punching and sample pretreatment. This was realized by automatic introduction of DBS sample cards into the LC flow via a pneumatically controlled clamp module. Using a three-column setup comprised of two pre columns for successive online DBS sample cleanup and a Synergi™ POLAR-RP C(18) analytical column for chromatographic separation under gradient conditions with a mobile phase A consisting of 1% acetic acid and a mobile phase B consisting of 1% acetic acid in methanol/2-propanol (80/20, v/v). MS/MS detection was performed in the positive multiple reaction monitoring mode using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray™ source. The total run time was 9.0min. The individual phases of online human DBS analysis were synchronized by automated valve switching. The analytical method was shown to be sensitive and selective with inter-day accuracy and precision of 91.6-108.0% and 3.4-14.6%, respectively, and it exhibited good linearity (r(2)≥0.9951 for all analytes) over the concentration range of 2ng/mL (5ng/mL for Ro 47-8634)-1500ng/mL. The analytes were stable in human DBS over 3.5 months at ambient temperature and accurate and precise results were obtained when using a blood spot volume between 20 and 30μL. Furthermore, no apparent (-8.9 to 12.6%) impact of hematocrit values ranging from 0.35 to 0.65 was observed on the quantification of the analytes. The system allowed very good recoveries of all analytes, between 83.0% and 92.3% for bosentan, between 94.4% and 100% for Ro 48-5033, between 98.0% and 100% for Ro 47-8634, and between 94.3% and 100% for Ro 64-1056. The validation demonstrated that the SCAP DBS System provides a robust automated platform for DBS analysis.

Examining face and construct validity of a noninvasive model of panic disorder in Lister-hooded rats.

RationaleIncreasing evidence suggests that defensive escape behavior in Lister-hooded (LH) rats induced by ultrasound application may be an animal model of panic disorder.ObjectiveThe objectives of this study were to further explore the face and construct validity of ultrasound-induced escape behavior by characterizing the autonomic and neuroendocrine response to ultrasound, and to examine the underlying neuronal structures by comparing the effects of the anxiolytic with panicolytic properties, diazepam, with a preclinical anxiolytic without panicolytic-like activity, the NOP agonist Ro 64-6198.Materials and methodsLH rats were implanted with telemetry transmitters to monitor heart rate and core body temperature before, during, and after ultrasound application. Blood samples were taken after ultrasound application for corticosterone analysis. Ultrasound-induced c-Fos expression was measured in different periaqueductal gray (PAG) and amygdala subregions after treatment with diazepam or Ro 64-6198.ResultsUltrasound application increased heart rate and body temperature, but did not alter plasma corticosterone levels. Ultrasound application increased c-Fos expression in the dorsal and dorsolateral PAG (dPAG, dlPAG) and amygdaloid subregions. Diazepam, but not Ro 64-6198, reduced c-Fos expression in the dPAG/dlPAG, while Ro 64-6198, but not diazepam, reduced c-Fos expression in the central amygdala.ConclusionsSimilar to human panic attacks, ultrasound application to LH rats activated the autonomic, but not the neuroendocrine, stress system. Also, like in humans, the current data confirm and extend that the dPAG/dlPAG plays a key role in ultrasound-induced escape behavior. These observations suggest that ultrasound-induced escape behaviors in LH rats have face and construct validity for panic disorders.

Comparative Pharmacokinetic, Pharmacodynamic, Safety, and Tolerability Profiles of 3 Different Formulations of Epoprostenol Sodium for Injection in Healthy Men

BACKGROUND Epoprostenol sodium for injection is approved for the treatment of severe cases of primary pulmonary arterial hypertension. Currently, there are 3 approved formulations of this drug containing the same active ingredient (epoprostenol sodium) but differing with regard to excipients. When compared with epoprostenol sodium formulated with glycine-mannitol (epoprostenol GM), 2 new formulations of epoprostenol sodium, one formulated with arginine-mannitol (epoprostenol AM) and one formulated with arginine-sucrose (epoprostenol AS), have improved stability after reconstitution and dilution. The biocomparability of epoprostenol AM and epoprostenol GM, with regard to pharmacokinetic (PK), pharmacodynamic (PD), safety, and tolerability profiles, has been shown previously. OBJECTIVE This study compared PK, PD, safety, and tolerability profiles of the 3 different formulations of epoprostenol sodium for injection. METHODS This was a prospective, single-center, open-label, 2-period, 2-treatment, randomized, crossover, ascending dose study in 2 parts. Twenty healthy men in part 1 and 20 different individuals in part 2 received epoprostenol AM and epoprostenol AS and epoprostenol GM and epoprostenol AS, respectively, in a crossover fashion, as sequential IV infusions of 2, 4, 6, and 8 ng/kg/min for 2 hours each. In each part, the PK profile of epoprostenol was characterized via analysis of the concentration-time profiles of its 2 primary metabolites: 6-keto-prostacyclin F1α and 6,15-diketo-13,14-dihydro-prostacyclin F1α. The effect of the formulations was assessed using the 90% CI of the geometric mean ratio calculated for the exposure PK parameters. The PD variables cardiac output, cardiac index, and heart rate were assessed using echocardiography. Adverse events were recorded through the study. RESULTS The plasma concentration versus time curves of epoprostenol AM and epoprostenol AS in part 1 and epoprostenol GM and epoprostenol AS in part 2 were similar in shape and almost superimposable. For each study part, the 90% CIs of ratios of geometric means for AUC0-∞ of the assessed epoprostenol formulations were within the range for bioequivalence (0.8-1.25). The increases in cardiac output, cardiac index, and heart rate resulting from infusion with epoprostenol sodium were comparable between all formulations, with maximum values attained after 8 hours. Almost all study participants reported at least one treatment-emergent adverse event, the most common being headache, which was reported in 80% to 85% of study participants. CONCLUSIONS Overall, the PK, PD, safety, and tolerability profiles of the 3 formulations of epoprostenol sodium for injection are comparable and meet the criteria of bioequivalence. Australian New Zealand Clinical Trials Registry identifier: ACTRN12612001086853.

Relative bioavailability of a newly developed pediatric formulation of bosentan vs. the adult formulation.

WHAT IS KNOWN Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension (PAH). Since bosentan is frequently used to treat pediatric PAH patients, a pediatric formulation was developed. AIM To evaluate the pharmacokinetic properties of bosentan and its active metabolite, Ro 48-5033, of the quadrisected, dispersible pediatric vs. the adult tablet after single-dose administration to healthy subjects. Secondary objectives of the study were to compare the pharmacokinetics of two inactive metabolites and the safety of both formulations. MATERIALS AND METHODS In this open-label, two-way crossover study, subjects (20 - 43 years) were randomized to receive single oral doses of 62.5 mg of bosentan as 1 adult tablet and 64 mg as 2 pediatric tablets of 32 mg. Blood samples were drawn over a 48-hour period to measure bosentan and its metabolites. RESULTS 16 subjects were enrolled and completed the study. Following treatment with the pediatric formulation, values for Cmax and AUC0-∞ of bosentan were lower than with the adult formulation with geometric mean ratios (90% confidence interval) of 0.82 (0.65, 1.04) and 0.87 (0.78, 0.97), respectively. Similar results were obtained for the primary active metabolite Ro 48-5033. Both treatments were well tolerated. WHAT IS NEW AND CONCLUSION Although the 90% confidence intervals of the geometric mean ratios of Cmax and AUC0-∞ were not entirely within the conventional bioequivalence range (0.80 - 1.25), no clinically relevant effect of formulation on the pharmacokinetics of bosentan and Ro 48-5033 was detected. Both formulations were well tolerated.

Determination of 6-keto prostaglandin F1α and its metabolites in human plasma by LC-MS/MS.

An HPLC-MS/MS method was developed and validated for the quantification of 6-keto prostaglandin F1α, the stable hydrolysis product of prostacyclin, and its metabolites 2,3-dinor-6-keto prostaglandin F1α and 6,15-diketo-13,14-dihydro prostaglandin F1α in human plasma. For sample preparation, a solid phase extraction step was combined with a column switching approach for analytes enrichment and further sample clean-up of the processed sample. The assay was validated in the concentration range 50.0-5000 pg/mL for 6-keto prostaglandin F1α and 6,15-diketo-13,14-dihydro prostaglandin F1α, and 100-10,000 pg/mL for 2,3-dinor-6-keto prostaglandin F1α. The inter-batch precision was better than 12.7%, 9.2%, and 9.4% for 6-keto prostaglandin F1α, 2,3-dinor-6-keto prostaglandin F1α, and 6,15-diketo-13,14-dihydro prostaglandin F1α, respectively. The inter-batch accuracy was between 97.3% and 100.8% for 6-keto prostaglandin F1α, between 97.5% and 103.0% for 2,3-dinor-6-keto prostaglandin F1α, and between 92.0% and 100.0% for 6,15-diketo-13,14-dihydro prostaglandin F1α. Further it has been demonstrated that the analytes were stable in plasma for 20 h at room temperature, during three freeze-and-thaw cycles, for 96 days at -25 °C storage temperature, and 50h in the autosampler tray at room temperature.

Integrated pharmacokinetics and pharmacodynamics of epoprostenol in healthy subjects

AIM The aim of the study was to report the first thorough characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of epoprostenol in an integrated manner. METHOD Twenty healthy male subjects received two formulations of i.v. epoprostenol, in a crossover design, in sequential infusions of 2, 4, 6 and 8 ng kg(-1)  min(-1) for 2 h each. A sensitive assay was developed which allowed accurate PK characterization of epoprostenol via analysis of the concentration-time profiles of its two primary metabolites, 6-keto-prostacyclin F(1α) and 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . PD parameters included cardiac output (CO), cardiac index (CIn) and heart rate (HR). RESULTS The pharmacokinetics of epoprostenol deviated slightly from dose-proportionality, probably due to a food effect. After infusion of the two formulations of epoprostenol, the t(1/2) values expressed as geometric mean (95% confidence interval) were 0.25 h (0.14, 0.46) and 0.22 h (0.13, 0.38) for 6-keto-prostacyclin F(1α) , and 0.32 h (0.22, 0.45) and 0.34 h (0.26, 0.46) for 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . A single compartment infusion model with first order elimination adequately described the PK of 6-keto-prostacyclin F(1α) . This model also characterized the food effect. Stepwise infusions with epoprostenol resulted in a progressive increase in CO, CIn and HR. CONCLUSION Of the two metabolites analyzed, the appearance of 6-keto-prostacyclin F(1α) in plasma was more closely associated with the haemodynamic effects of i.v. epoprostenol. PK and PD profiles showed that CIn relates proportionally and linearly to the plasma concentrations of 6-keto-prostacyclin F(1α) . These results suggest that 6-keto-prostacyclin F(1α) is a suitable surrogate marker of plasma concentrations of epoprostenol.

Pharmacokinetics, pharmacodynamics, and tolerability of ACT-077825, a new direct renin inhibitor after multiple-ascending doses in healthy subjects.

Abstract: This study was conducted to characterize the multiple-dose tolerability, pharmacokinetics, and pharmacodynamics of ACT-077825, a new direct renin inhibitor, in healthy male subjects. In this single-center, double-blind, placebo-controlled, active-controlled (20 mg of enalapril), randomized multiple-ascending dose study, ACT-077825 was administered once a day. for 7 days in the 50–1000 mg dose range to sodium- and potassium-restricted subjects. ACT-077825 pharmacokinetics on days 1 and 7 were characterized by dose-proportional increases in Cmax and AUC&tgr;. At steady state, accumulation was modest (1.5- to 1.7-fold). Enalapril caused an increase in plasma active renin concentration and plasma renin activity (PRA). ACT-077825 dose dependently increased active renin on days 1 and 7 and inhibited PRA dose dependently only on day 1. On day 7, the maximal PRA inhibition was attained after 250 mg of ACT-077825. In contrast to enalapril, ACT-077825 did not induce any consistent lowering effect on blood pressure when compared with placebo. Of the reported adverse events, diarrhea, headache, and postural dizziness were more frequent. The incidence of diarrhea was greater in the 1000-mg group and a dose of 500 mg of ACT-077825 was identified as the maximum tolerated dose. Overall, pharmacokinetic, pharmacodynamic, and tolerability profiles warrant the further investigation of ACT-077825 in patients with hypertension.

Clinical pharmacology of single- and multiple-ascending doses of ACT-178882, a new direct renin inhibitor, and its pharmacokinetic interaction with food and midazolam.

This study investigated the tolerability, safety, pharmacokinetics, and pharmacodynamics of ACT‐178882, a new direct renin inhibitor, as well as its interaction with food and midazolam. Healthy male subjects received either single (10–1000 mg) or multiple doses (30–600 mg) administered once daily for 14 days of ACT‐178882, placebo, or 20 mg enalapril in the fasted state. Following a 2‐week washout, the single dose of 30 mg ACT‐178882 was also administered in the fed state. In the multiple‐ascending‐dose part, subjects were dosed with midazolam on days −2, 2, and 12 to investigate interactions with CYP3A4. Dizziness and headache were the most frequently reported adverse events. No clinically relevant changes occurred for body weight, vital signs, clinical laboratory variables, and ECG although both enalapril and ACT‐178882 tended to decrease systolic blood pressure. Following single doses of ACT‐178882, t1/2 and tmax varied from 18.7 to 24.7 h and from 3 to 5 h, respectively, and food had no significant effect. Steady‐state conditions were achieved after 4–6 days of dosing and accumulation was minimal. ACT‐178882 pharmacokinetics were dose proportional. ACT‐178882 but not enalapril dose‐dependently increased Cmax and area under the concentration‐time curve of midazolam. Single and multiple doses of ACT‐178882 dose‐dependently increased active renin and decreased plasma renin activity, whereas enalapril increased both variables. No effects on urinary excretion of creatinine, potassium, and the 6β‐hydroxycortisol/cortisol ratio were observed, whereas sodium and aldosterone excretion was decreased by both ACT‐178882 and enalapril. The current results with ACT‐178882 warrant further clinical investigation of this renin inhibitor in hypertensive patients.

Age and sex effects on the single- and multiple-dose safety and pharmacokinetics of the new renin inhibitor ACT-178882.

AIM To investigate the effect of age and sex on the single- and multiple-dose safety and pharmacokinetics of ACT-178882, a new direct renin inhibitor. METHODS In the single-dose, open-label part of the study, healthy young and elderly male and female subjects received a dose of 300 mg ACT-178882. In the multiple-dose, double-blind, placebo-controlled part of the study, healthy elderly male and female subjects received ACT-178882 (300 or 600 mg) or placebo once-a-day for 14 days. RESULTS Mild headache and diarrhea were the most common adverse events in the single- and multiple-dose parts, respectively. The frequency of diarrhea increased dose dependently, and no placebo subjects reported this adverse event. Administration of ACT-178882 did not result in any clinically relevant changes in ECG, vial signs, clinical laboratory, body weight, or physical examination. In the single-dose part and independent of sex, values for Cmax and AUC0-∞ were about 85% higher in elderly compared with young subjects, and t1/2 was about 5 h longer (29.3 vs. 24.1 h). In both age groups, plasma ACT-178882 concentrations were slightly higher in female versus male subjects. Following multiple-dose administration to elderly subjects, accumulation of ACT-178882 was moderate, about 1.8-fold, t1/2 was unchanged as compared with single-dose administration, sex effects were minimal, and the increase in the exposure with increasing dose appeared less than dose proportional. CONCLUSION Overall, single- and multiple-dose administration of ACT-178882 was well tolerated. No dose adaptation based on sex is necessary, whereas a dose reduction based on age could be considered.

Effect of Multiple‐Dose Diltiazem on the Pharmacokinetics of the Renin Inhibitor ACT‐077825

This open‐label, randomized study evaluated the effects of steady‐state diltiazem on the pharmacokinetic, safety, and tolerability profile of a single dose of the novel renin inhibitor ACT‐077825. Twelve healthy Caucasian male subjects (20–50 years) received in treatment sequence A, a single dose of 100 mg ACT‐077825 (Days 1 and 17), and oral diltiazem 300 mg once daily (Days 14–26). In treatment sequence B, subjects received a single dose of 100 mg ACT‐077825 (Days 4 and 22) and oral diltiazem 300 mg once daily (Days 1–13). ACT‐077825 alone and combined with diltiazem was generally well tolerated. On average, the systemic exposure to ACT‐077825 was higher in the presence of diltiazem. For AUC0–∞ and t1/2, the upper limit of the 90% confidence interval (CI) of the geometric mean ratios was outside the study‐specific 0.5–2.0 equivalence boundaries, that is, 1.92 (90% CI: 1.30, 2.83) and 1.58 (90% CI: 1.22, 2.04), respectively. In conclusion, diltiazem markedly affected the pharmacokinetics of ACT‐077825, probably via inhibition of CYP3A4 activity, without changing its safety and tolerability profile in healthy male subjects. Whether such an interaction will require for therapeutic dose adjustment of ACT‐077825 co‐administered with diltiazem has to be assessed once the dose‐response relationship of ACT‐077825 in hypertensive patients is determined.