Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with tandem liquid chromatography mass spectrometry

Fingolimod (Gilenya®; FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In vivo, FTY720 is phosphorylated by sphingosine kinase 2 to FTY720-phosphate (FTY720-P) which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. The quantitative analysis of FTY720 and that of FTY720-P in blood represents a great challenge. Two separate methods are commonly performed because of the different chemical properties of these two compounds and due the fact that low concentrations have to be measured. In the present study, we have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify FTY720 and FTY720-P in human blood. The sample preparation involves the sample dilution with a solution made of dimethylhexylamine (DMHA), ortho-phosphoric acid and methanol prior to the on-line solid phase extraction (SPE) on a C18 cartridge. The samples were then eluted on a C18 column with a gradient elution of DMHA solution and acetonitrile and analyzed by LC-MS/MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.5 minutes. Standard curves were linear over the ranges of 0.0800 ng/mL (LLOQ) to 16.0 ng/mL for FTY720 and 0.100 ng/mL (LLOQ) to 20.0 ng/mL for FTY720-P. The method was validated for selectivity, dilution and the accuracy and precision data were in accordance with those specified by the FDA guidance. In addition, stability data obtained during freeze-thaw (3 cycles), at room temperature (24 h), and in an auto-sampler were determined and reported. The method robustness was demonstrated by the consistent data obtained by reanalyzing human blood samples for several clinical studies. In addition comparative data for FTY720 and FTY720-P were obtained between our current method and those of two available separate LC-MS/MS assays. The results of the present work demonstrated that our bioanalytical LC-MS/MS method is rapid, sensitive, specific and reliable for the simultaneous quantitative analysis of FTY720 and FTY720-P in human blood

Absorption, distribution, metabolism, and excretion (ADME) of 14C-sonidegib (LDE225) in healthy volunteers

Purpose: The absorption, distribution, metabolism, and excretion of the hedgehog pathway inhibitor sonidegib (LDE225) were determined in healthy male subjects. Methods: Six subjects received a single oral dose of 800 mg 14C-sonidegib (74 kBq, 2.0 μCi) under fasting conditions. Blood, plasma, urine, and fecal samples were collected predose, postdose in-house (days 1-22), and during 24-h visits (weekly, days 29-43; biweekly, days 57-99). Radioactivity was determined in all samples using accelerator mass spectrometry (AMS). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine concentrations of sonidegib and its main circulating metabolite in plasma. Metabolite profiles and structures were determined in pooled plasma, urine, and fecal samples using high-performance LC-AMS and LC-MS/MS, respectively. Results: A single dose of 14C-sonidegib was well tolerated in healthy subjects. Unchanged sonidegib and total radioactivity reached peak concentration in plasma by 2 and 3 h, respectively, and demonstrated similarly long half-lives of 319 and 331 h, respectively. Absorbed sonidegib (estimated 6-7 %) was extensively distributed, and the approximate terminal volume of distribution was 2,500 L. Unchanged sonidegib and a metabolite resulting from amide hydrolysis were the major circulating components (36.4 and 15.4 % of radioactivity area under the curve, respectively). Absorbed sonidegib was eliminated predominantly through oxidative metabolism of the morpholine part and amide hydrolysis. Unabsorbed sonidegib was excreted through the feces. Metabolites in excreta accounted for 4.49 % of the dose (1.20 % in urine, 3.29 % in feces). The recovery of radioactivity in urine and feces was essentially complete (95.3 ± 1.93 % of the dose in five subjects; 56.9 % of the dose in one subject with incomplete feces collection suspected). Conclusions: Sonidegib exhibited low absorption, was extensively distributed, and was slowly metabolized. Elimination of absorbed sonidegib occurred largely by oxidative and hydrolytic metabolism. © 2014 Springer-Verlag

Population PK‐PD Model for Tolerance Evaluation to the p38 MAP Kinase Inhibitor BCT197

The p38 mitogen‐activated protein kinase (p38) is a key signaling pathway involved in regulation of inflammatory cytokines. Unexpectedly, several clinical studies using p38 inhibitors found no convincing clinical efficacy in the treatment of chronic inflammation. It was the objective of this study to characterize the population pharmacokinetics (PK) of BCT197 in healthy volunteers and to examine the relationship between BCT197 exposure and pharmacodynamics (PD) measured as inhibition of ex vivo lipopolysaccharide (LPS)‐induced tumor necrosis factor alpha (TNFα), a downstream marker of p38 activity. PK was characterized using a two‐compartment model with mixed‐order absorption and limited‐capacity tissue binding. The PK‐PD relationship revealed that suppression of TNFα was partly offset over time, despite continuous drug exposure. This may indicate a mechanism by which the inflammatory response acquires the ability to bypass p38. Simulations of posology dependence in drug effect suggest that an intermittent regimen may offer clinical benefit over continuous dosing and limit the impact of tolerance development

Singing abilities in children with Specific Language Impairment (SLI)

Specific Language impairment (SLI) is a heritable neurodevelopmental disorder diagnosed when a child has difficulties learning to produce and/or understand speech for no apparent reason (Bishop et al., 2012). The verbal difficulties of children with SLI have been largely documented, and a growing number of studies suggest that these children may also have difficulties in processing non-verbal complex auditory stimuli (Brandt et al., 2012; Corriveau et al., 2007). In a recent study, we reported that a large proportion of children with SLI present deficits in music perception (Planchou et al, submitted). Little is known, however, about the singing abilities of children with SLI. In order to investigate whether or not the impairments in expressive language extend to the musical domain, we assessed singing abilities in 8 children with SLI and 15 children with Typical Language Development (TLD) matched for age and non-verbal intelligence. To this aim, we designed a ludic activity consisting of two singing tasks: a pitch-matching and a melodic reproduction task. In the pitch-matching task, the children were requested to sing single notes. In the melodic reproduction task, children were asked to sing short melodies that were either familiar (FAM-SONG and FAM-TUNE conditions) or unfamiliar (UNFAM-TUNE condition). The analysis showed that children with SLI were impaired in the pitch-matching task, with a mean pitch error of 250 cents (mean pitch error for children with TLD: 154 cents). In the melodic reproduction task, we asked 30 healthy adults to rate the quality of the sung productions of the children on a continuous rating scale. The results revealed that singing of children with SLI received lower mean ratings than the children with TLD. Our findings thus indicate that children with SLI showed impairments in musical production and are discussed in light of a general auditory-motor dysfunction in children with SLI

Validation of on-line solid- phase extraction coupled with high-performance liquid chromatography–mass spectrometry assays for the determination of indacaterol, a potent β-adrenoceptor agonist in human serum

Adequate sample preparation is a key aspect of quantitative bioanalysis and can often be the cause of bottlenecks for the development of sensitive and high-throughput LC-MS/MS methods. Here, we have developed a highly sensitive method for the determination of indacaterol in human blood serum. Indacaterol is currently under development for the treatment of asthma and chronic obstructive pulmonary disease (COPD). The method involved was as follows: direct injection of the acidified serum into an on-line solid-phase extraction system with ion exchange MCX cartridge (Symbiosis™, Spark, Holland) followed by chromatography of indacaterol along with its stable isotope labeled internal standard on an analytical column, and analysis using LC–MS/MS (API 5000, Applied Biosystems, USA), in multiple reaction monitoring mode, with electron spray ionization. The results of assessments intended for validation viz., specificity, carry over, reproducibility of three days of validation, accuracy, precision, dilution effect, relative recovery and stability are presented. In addition, a second validation method with another cartridge (Hysphere C18 HD) was also studied. The methods were specific and selective for indacaterol in human serum and suitable for supporting clinical trials with indacaterol and allowed performing pharmacokinetic studies of indacaterol in humans

Single- and multiple-dose pharmacokinetics of inhaled indacaterol in healthy Chinese volunteers

Indacaterol is an inhaled, ultra-long-acting β2-agonist that provides 24-h bronchodilation with once-daily dosing in patients with chronic obstructive pulmonary disorder. This study evaluated the pharmacokinetics, safety, and tolerability of multiple daily inhaled doses of indacaterol 150 or 300 μg once daily in healthy Chinese volunteers. This was a single-center, randomized, double-blind, multiple-dose, parallel-group study, placebo-controlled trial including two doses of indacaterol: 150 and 300 μg. Serum indacaterol was quantified using high-performance liquid chromatography-mass spectrometry with a lower limit of quantification of 0.01 ng/mL. The pharmacokinetic parameters were analyzed using non-compartmental analysis and included Cmax, Tmax, and AUC0-24h on Day 1 and AUC0-24h,ss, Cmax,ss, Cmin,ss, Cav,ss, Tmax,ss, T1/2, T1/2,acc, CL/F, Vz/F, and Racc on Day 14 (after repeated once-daily doses). Safety analyses were recorded using physical examination, biochemical tests, and ECG. Indacaterol steady state was achieved after 12-14 days of daily dosing. The mean effective half-life of indacaterol (based on drug accumulation at steady state) was 33.9 and 35.8 h for 150 and 300 μg, respectively. Systemic exposure to indacaterol increased 1.27 and 1.34-fold between the 150- and 300-μg doses on Day 1 (first dose) and Day 14 (repeated dose), respectively. Indacaterol 150 and 300 μg were safe and well tolerated in these volunteers. The pharmacokinetics of multiple inhaled doses of indacaterol 150 and 300 μg (for 14 days) were consistent with moderate systemic accumulation at steady state after repeated once-daily inhalation in healthy Chinese volunteers

Pharmacokinetics of fingolimod and metabolites in subjects with severe renal impairment: An open-label, single-dose, parallel-group study

Objective: This study assessed the pharmacokinetics and tolerability of fingolimod and its metabolites in severe renal impairment and healthy subjects. Methods: In this single-dose, open-label study, 9 severe renal impairment subjects and 9 demographically matched healthy subjects were included. Each subject received a single oral dose of fingolimod 1.25 mg, and their blood and urine samples were assessed. The pharmacokinetics of fingolimod and its metabolites, fingolimod-phosphate (active metabolite, fingolimod- P), M2, and M3, were compared in both groups. Safety and tolerability were also assessed. Results: In severe renal impairment subjects, mean ± standard deviation values of Cmax (ng/mL) of fingolimod and fingolimod- P were 0.878 ± 0.256 and 1.13 ± 0.293 vs. 0.653 ± 0.138 and 0.904 ± 0.229 in healthy subjects, respectively. The overall drug exposures (AUCinf (ng×h/mL)) for fingolimod and fingolimod-P were 131 ± 90.7 and 75.5 ± 33.6 in severe renal impairment subjects vs. 82.3 ± 36.9 and 65.9 ± 30.6 in healthy subjects, respectively. t1/2 (hours) for fingolimod and fingolimod-P was comparable in severe renal impairment subjects (94 ± 53 and 95 ± 50) and healthy subjects (85 ± 25 and 101 ± 46). All adverse events were as expected for fingolimod 1.25 mg. Conclusions: The exposure to fingolimod and fingolimod-P was moderately increased (90% CI, 0.94 - 2.18) in severe renal impairment subjects, while half-lives and protein binding were similar to those in healthy subjects. Given that these changes are not clinically meaningful, fingolimod dose adjustment is considered unnecessary in patients with mild, moderate, or severe renal impairment. © 2015 Dustri-Verlag Dr. K. Feistle

Population PK-PD model for tolerance evaluation to the p38 MAP kinase inhibitor BCT197

The p38 mitogen-activated protein kinase (p38) is a key signaling pathway involved in regulation of inflammatory cytokines. Unexpectedly, several clinical studies using p38 inhibitors found no convincing clinical efficacy in treatment of chronic inflammation. It was the objective of this study to characterize the population pharmacokinetics (PK) of BCT197 in heathy volunteers and to examine the relationship between BCT197 exposure and pharmacodynamics (PD) measured as inhibition of ex-vivo LPS-induced TNF, a downstream surrogate marker of p38. PK was characterized using a two-compartment model with mixed-order absorption and limited-capacity tissue binding. The PK-PD relationship revealed that suppression of TNF was counteracted, at least partly, despite continuous drug exposure. This may indicate a mechanism by which the inflammatory response acquires the ability to bypass p38. Simulations of posology dependence in drug effect revealed that an intermittent regimen may offer clinical benefit over continuous dosing, by limiting the potential impact of tolerance development

Pharmacokinetics of fingolimod and metabolites in subjects with severe renal impairment: An open-label, single-dose, parallel-group study

Objective: This study assessed the pharmacokinetics and tolerability of fingolimod and its metabolites in severe renal impairment and healthy subjects. Methods: In this single-dose, open-label study, 9 severe renal impairment subjects and 9 demographically matched healthy subjects were included. Each subject received a single oral dose of fingolimod 1.25 mg, and their blood and urine samples were assessed. The pharmacokinetics of fingolimod and its metabolites, fingolimod-phosphate (active metabolite, fingolimod- P), M2, and M3, were compared in both groups. Safety and tolerability were also assessed. Results: In severe renal impairment subjects, mean ± standard deviation values of Cmax (ng/mL) of fingolimod and fingolimod- P were 0.878 ± 0.256 and 1.13 ± 0.293 vs. 0.653 ± 0.138 and 0.904 ± 0.229 in healthy subjects, respectively. The overall drug exposures (AUCinf (ng×h/mL)) for fingolimod and fingolimod-P were 131 ± 90.7 and 75.5 ± 33.6 in severe renal impairment subjects vs. 82.3 ± 36.9 and 65.9 ± 30.6 in healthy subjects, respectively. t1/2 (hours) for fingolimod and fingolimod-P was comparable in severe renal impairment subjects (94 ± 53 and 95 ± 50) and healthy subjects (85 ± 25 and 101 ± 46). All adverse events were as expected for fingolimod 1.25 mg. Conclusions: The exposure to fingolimod and fingolimod-P was moderately increased (90% CI, 0.94 - 2.18) in severe renal impairment subjects, while half-lives and protein binding were similar to those in healthy subjects. Given that these changes are not clinically meaningful, fingolimod dose adjustment is considered unnecessary in patients with mild, moderate, or severe renal impairment. © 2015 Dustri-Verlag Dr. K. Feistle