Marcelo M. Gutierrez

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.

Cadazolid, a novel antibiotic with potent activity against Clostridium difficile: safety, tolerability and pharmacokinetics in healthy subjects following single and multiple oral doses

OBJECTIVES Current treatment options for Clostridium difficile-associated diarrhoea (CDAD) leave a high unmet medical need for new therapies. Cadazolid is a new antibiotic in development for the treatment of CDAD. The objectives of this study were to evaluate its tolerability and pharmacokinetics following single ascending doses (AC-061-101) and multiple ascending doses (AC-061-102). METHODS Single and multiple (twice daily for 10 days) oral doses of cadazolid between 30 mg and 3000 mg, or placebo, were tested in a total of 64 healthy male subjects. Safety assessments were conducted at regular intervals. Blood, urine and faeces were sampled, and cadazolid concentrations were measured. RESULTS Cadazolid was well tolerated up to 3000 mg given twice daily for 10 days. The most common adverse event was headache, with no observed relationship between dose or treatment duration and adverse events. Plasma concentrations of cadazolid were low. No plasma concentrations >3.3 ng/mL were observed after single doses or >6.9 ng/mL after 10 days of multiple doses. Food increased the mean C(max) from 0.73 to 1.87 ng/mL and mean AUC(0-t) from 3.13 to 15.69 ng ·h/mL after a single 300 mg dose. The increase in systemic exposure to cadazolid across doses was less than dose-proportional. The mean cumulative faecal recovery was 81.0%-93.5%. Urinary recovery of unchanged compound was <0.015%. CONCLUSIONS Cadazolid was well tolerated and its systemic exposure was low. The majority of compound was recovered unchanged in the faeces, thus resulting in high concentrations at the site of action (colon).

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.

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.

Setipiprant, a selective oral antagonist of human CRTH2: relative bioavailability of a capsule and a tablet formulation in healthy female and male subjects.

BACKGROUND CRTH2 is a prostaglandin D2 receptor that plays an important role in allergic inflammation. Setipiprant is a potent CRTH2 antagonist under development for the treatment of allergic diseases. OBJECTIVE The aim of this study was to evaluate the tolerability and pharmacokinetics of a single oral dose of a setipiprant capsule (reference) and a tablet formulation. METHODS This was an open-label, 2-period, 2-way crossover, randomized study in which 20 healthy women and men (1:1 ratio) received either 2 250-mg capsules or a 500-mg tablet of setipiprant. Subjects were between 18 and 45 years old, with a body mass index of 18.0 to 28.0 kg/m(2). Differences in pharmacokinetics of setipiprant formulations were explored overall and by sex. RESULTS All subjects completed the study. Both formulations were well tolerated, with headache the most frequently reported adverse event (25% of subjects), followed by flatulence (15%) and somnolence and fatigue (10%). The adverse event profile in men and women and between formulations was similar. The ratios of geometric means for Cmax (0.94; 95% CI, 0.79-1.12) and AUC0-∞ (1.01; 95% CI, 0.92-1.12) were mostly within the limits of 0.80 to 1.25. When corrected for weight, the differences observed between sexes, within each treatment, for Cmax (capsules: 1.01; 95% CI, 0.71-1.44; tablet: 0.89; 95% CI, 0.62-1.26) and AUC0-∞ (capsules: 1.12; 95% CI, 0.86-1.47; tablet: 0.96; 95% CI, 0.73-1.25) were minor. CONCLUSION Both the setipiprant formulations were well tolerated. Setipiprant pharmacokinetics were similar between formulations, overall, and between sexes. The new tablet formulation may constitute a valid alternative to the capsule formulation for later clinical development phases. ClinicalTrials.gov identifier: NCT01877629.

Tolerability and pharmacokinetics of ACT-280778, a novel nondihydropyridine dual L/T-type calcium channel blocker: early clinical studies in healthy male subjects using adaptive designs.

Abstract: ACT-280778 is a novel nondihydropyridine dual L/T-type calcium channel blocker. Two clinical studies (AC-067-101 and AC-067-102) were conducted to characterize its safety, tolerability, and pharmacokinetics in healthy male subjects after oral administration of single and multiple doses. Both trials were single-center, randomized, double-blind, placebo-controlled, adaptive design, ascending-dose studies, in which ACT-280778 was administrated as single doses of 2, 5, 15, or 40 mg, or as once-daily doses of 5 or 15 mg for 7 days. Single and multiple doses up to and including 15 mg were well tolerated, and no serious or severe adverse event was reported in either study. A single dose of 40 mg was associated with abnormal electrocardiogram findings resulting in the discontinuation of further treatment at this dose or higher doses. ACT-280778 was rapidly absorbed, and larger than dose-proportional increases of the maximum plasma concentration and area under the plasma concentration–time curve were observed. Food intake delayed the time to maximum plasma concentration and doubled exposure. Urinary excretion of unchanged ACT-280778 was negligible, and accumulation at steady state was modest. Overall, pharmacokinetic and tolerability profiles of ACT-280778 observed in these 2 studies warranted further evaluation of ACT-280778 in a proof-of-concept study in patients with hypertension.