A validated UHPLC-MS/MS method for rapid determination of senicapoc in plasma samples.

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-µL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.

Investigation of the effect of the dual orexin receptor antagonist almorexant on ophthalmological, spermatogenic, and hormonal variables in healthy male subjects.

BACKGROUND/AIMS: The aim of this single-center, double-blind study was to investigate the effect of a 4-week once daily administration of 200 mg almorexant on tear film break-up time, spermatogenesis, hormone levels, and pancreatic elastase in stool in healthy male subjects. METHODS: Almorexant 200 mg or matching placebo was administered in the evening for 4 weeks once daily to 56 healthy male subjects. Changes in ophthalmological variables, sperm composition, hormone levels, and pancreatic elastase levels in stool were evaluated periodically up to 8 weeks after discontinuation of drug administration. Blood samples for pharmacokinetic measurements were taken after 4 weeks to confirm compliance to study drug intake. RESULTS: The results of this study revealed no treatment effects of almorexant, neither on tear film break-up time nor on other ophthalmological variables investigated during this study. Furthermore, spermatogenesis, hormones of the hypothalamic-pituitary-adrenal and -gonadal axes, and endocrine pancreatic secretion were shown to be not affected by a 4-week once daily administration of almorexant. CONCLUSION: Almorexant was well tolerated and had no effect on the spectrum of pharmacodynamic variables assessed. Ophthalmology and testicular findings detected in preclinical studies were not observed in this clinical study. Therefore, these preclinical findings appear not to be relevant for humans and do not prevent from conducting larger clinical trials with either healthy subjects or patients.

68Ga-Labeled bismacrocyclic methylene phosphonate as potential bone seeking PET radiopharmaceutical.

Phosphonates-based agents are well-known bone-seeking radiopharmaceuticals with application in detection and therapy. With higher sensitivity and resolution offered by Positron Emission Tomography (PET), tracers based on this technique are gaining huge attention. 68Ga-based generator and radiotracers render independence from the on-site cyclotron. We report the development of 68Ga-labeled DOTA-based bismacrocyclic phosphonate derivative, for bone PET imaging. The synthesis and characterization of 68Ga- DO3P-AME-DO3P was carried out in > 95% purity. The radiotracer displayed high stability and low binding affinity (<3%) to blood serum. High in vitro binding affinity were observed for synthetic hydroxyapatite, SAOS-2, osteoclast and osteoblast cells. In vivo pharmacokinetics revealed fast washout with biphasic release pattern. The deposition of radiotracer in osseous tissues was high (Bone/Muscle ratio:18), as studied from the biodistribution studies. In vivo PET/CT and biodistribution analyses revealed the ability of 68Ga-DO3P-AME-DO3P to target and accumulate in bone, thus displaying its potential as a PET bone imaging agent.

Simultaneous quantification and pharmacokinetic investigation of selexipag and its main metabolite ACT-333679 in rat plasma by UPLC-MS/MS method.

In the present study, an accurate, simple and fast bioanalytical method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique for simultaneous quantification of plasma selexipag and its main metabolite ACT-333679 concentrations in rats was optimized and established. The purpose of chromatographic separation of selexipag, ACT-333679 and the internal standard (IS, diazepam) was accomplished using an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column. The mobile phase was consisted of acetonitrile (solution A) and 0.1 % formic acid in water (solution B) in a linear gradient elution procedure with a flow rate of 0.40 mL/min. The measurement of the analytes and IS was explored using a XEVO TQ-S triple quadrupole tandem mass spectrometer, which was comprised with electrospray ionization (ESI) source in positive ion mode. Selected multiple reaction monitoring (MRM) mode was employed to detect the parent-to-daughter ion transitions as follows: m/z 497.4 → 302.2 for selexipag, m/z 420.1 → 378.2 for ACT-333679, and m/z 285.0 → 154.0 for diazepam (IS), respectively. The new UPLC-MS/MS method showed good linearity respectively at the calibration curve range of 0.05-50 ng/mL for selexipag, and 0.05-250 ng/mL for ACT-333679. The intra- and inter-day of accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery and matrix effect were also met the requirements. The newly developed UPLC-MS/MS assay was forward successfully used to describe the pharmacokinetic profiles of selexipag and ACT-333679 in rats after oral treatment with 6.0 mg/kg selexipag.

Predicted values for human total clearance of a variety of typical compounds with differently humanized-liver mouse plasma data.

Prediction of human pharmacokinetics is important in the preclinical stage. Values for total clearance of compounds from plasma should be one of the most important pharmacokinetic parameters for predictions. Although several physiological and empirical methods including single-species allometry for prediction of values for human clearance of compounds using humanized-liver mice have been reported, further improvement of prediction accuracies would be still expected. To optimize these approaches, we proposed methods for unbound intrinsic clearance in virtually 100% humanized-liver mouse by incorporating unbound plasma fractions of compounds in differently humanized-liver mice. Comparisons of prediction accuracies of values for human clearance of 15 model compounds were performed among our current physiological and previously reported models and single-species allometry using humanized-liver mice. Incorporation of the actual unbound plasma fractions of compounds and correction of residual mice hepatocyte in humanized-liver mice showed comparable prediction accuracy to that by single-species allometry. After exclusion of 3 compounds with large species differences in values of clearance and unbound plasma fractions between mice and humans out of 15 compounds, prediction accuracies were improved in the methods investigated. The previously and present reported physiological methods could show the good prediction accuracy of values for clearance of drugs from plasma.

A novel variant of the 13C-methacetin liver function breath test that eliminates the confounding effect of individual differences in systemic CO2 kinetics.

The principle of dynamic liver function breath tests is founded on the administration of a 13C-labeled drug and subsequent monitoring of 13CO2 in the breath, quantified as time series delta over natural baseline 13CO2 (DOB) liberated from the drug during hepatic CYP-dependent detoxification. One confounding factor limiting the diagnostic value of such tests is that only a fraction of the liberated 13CO2 is immediately exhaled, while another fraction is taken up by body compartments from which it returns with delay to the plasma. The aims of this study were to establish a novel variant of the methacetin-based breath test LiMAx that allows to estimate and to eliminate the confounding effect of systemic 13CO2 distribution on the DOB curve and thus enables a more reliable assessment of the hepatic detoxification capacity compared with the conventional LiMAx test. We designed a new test variant (named "2DOB") consisting of two consecutive phases. Phase 1 is initiated by the intravenous administration of 13C-bicarbonate. Phase 2 starts about 30 min later with the intravenous administration of the 13C-labelled test drug. Using compartment modelling, the resulting 2-phasic DOB curve yields the rate constants for the irreversible elimination and the reversible exchange of plasma 13CO2 with body compartments (phase 1) and for the detoxification and exchange of the drug with body compartments (phase 2). We carried out the 2DOB test with the test drug 13C-methacetin in 16 subjects with chronic liver pathologies and 22 normal subjects, who also underwent the conventional LiMAx test. Individual differences in the systemic CO2 kinetics can lead to deviations up to a factor of 2 in the maximum of DOB curves (coefficient of variation CV ≈ 0.2) which, in particular, may hamper the discrimination between subjects with normal or mildly impaired detoxification capacities. The novel test revealed that a significant portion of the drug is not immediately metabolized, but transiently taken up into a storage compartment. Intriguingly, not only the hepatic detoxification rate but also the storage capacity of the drug, turned out to be indicative for a normal liver function. We thus used both parameters to define a scoring function which yielded an excellent disease classification (AUC = 0.95) and a high correlation with the MELD score (RSpearman = 0.92). The novel test variant 2DOB promises a significant improvement in the assessment of impaired hepatic detoxification capacity. The suitability of the test for the reliable characterization of the natural history of chronic liver diseases (fatty liver-fibrosis-cirrhosis) has to be assessed in further studies.

The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue.

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Species differences in metabolism of a new antiepileptic drug candidate, DSP-0565 [2-(2'-fluoro[1,1'-biphenyl]-2-yl)acetamide].

The metabolism and pharmacokinetics of DSP-0565 [2-(2'-fluoro[1,1'-biphenyl]-2-yl)acetamide], an antiepileptic drug candidate, was investigated in rats, dogs, and humans. In human hepatocytes, [14 C]DSP-0565 was primarily metabolized via amide bond hydrolysis to (2'-fluoro[1,1'-biphenyl]-2-yl)acetic acid (M8), while in rat and dog hepatocytes, it was primarily metabolized via both hydrolysis to M8 and hydroxylation at the benzene ring or the benzyl site to oxidized metabolites. After single oral administration of [14 C]DSP-0565 to rats and dogs, the major radioactivity fraction was recovered in the urine (71-72% of dose) with a much smaller fraction recovered in feces (23-25% of dose). As primary metabolites in their excreta, M8, oxidized metabolites, and glucuronide of DSP-0565 were detected. The contribution of metabolic pathways was estimated from metabolite profiles in their excreta: the major metabolic pathway was oxidation (57-62%) and the next highest was the hydrolysis pathway (23-33%). These results suggest that there are marked species differences in the metabolic pathways of DSP-0565 between humans and animals. Finally, DSP-0565 human oral clearance (CL/F) was predicted using in vitro-in vivo extrapolation (IVIVE) with/without animal scaling factors (SF, in vivo intrinsic clearance/in vitro intrinsic clearance). The SF improved the underestimation of IVIVE (fold error = 0.22), but the prediction was overestimated (fold error = 2.4-3.3). In contrast, the use of SF for hydrolysis pathway was the most accurate for the prediction (fold error = 1.0-1.4). Our findings suggest that understanding of species differences in metabolic pathways between humans and animals is important for predicting human metabolic clearance when using animal SF.

Polymorphisms in CYP1A2, CYP2C9 and ABCB1 affect agomelatine pharmacokinetics.

BACKGROUND: Agomelatine is an agonist of the melatoninergic receptors used for the treatment of depression. Our aim was to evaluate the effect of genetic polymorphisms in metabolising enzymes and the P-glycoprotein transporter on agomelatine pharmacokinetics and pharmacodynamics. METHODS: Twenty-eight healthy volunteers receiving a single 25 mg oral dose of agomelatine, were genotyped for nine polymorphisms in cytochrome P450 enzymes ( CYP1A2, CYP2C9 and CYP2C19) and adenosine triphosphate-binding cassette subfamily B member 1 ( ABCB1), by real-time polymerase chain reaction . Agomelatine concentrations were measured by high performance liquid chromatography coupled to a tandem mass spectrometry detector. RESULTS: We calculated a CYP1A2 activity score that was directly correlated with agomelatine pharmacokinetics. Individuals with a decreased enzyme activity (*1C carriers) had a lower clearance and accumulated higher concentrations of agomelatine. In contrast, individuals with a high CYP1A2 inducibility (*1F or *1B carriers) showed an extensive clearance and lower agomelatine concentrations. The apparently marked differences between races were due to the different CYP1A2 genotype distribution. CYP2C9 intermediate/poor metabolisers showed a higher area under the concentration-time curve and maximum concentration. ABCB1 G2677T/A polymorphism affected the time to reach maximum concentration, as subjects carrying A/A+A/T genotypes showed higher values. No association was found for CYP2C19 phenotype. Agomelatine did not produce any change in blood pressure, heart rate or QT interval. CONCLUSIONS: CYP1A2 polymorphisms affect agomelatine pharmacokinetics. CYP1A2 phenotype inferred from the genotyping of CYP1A2*1C, *1F and *1B alleles might be a potential predictor of agomelatine exposure. ABCB1 G2677T/A could affect agomelatine absorption, as subjects with A/A+A/T genotypes had lower agomelatine concentration and they take more time to reach the maximum concentration.

Optimized liquid chromatography-tandem mass spectrometry for Otaplimastat quantification in rat plasma and brain tissue.

An optimized liquid chromatography-tandem mass spectrometry method for simple and sensitive quantification of Otaplimastat in rat plasma and brain tissue was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation method based on recovery and matrix effect. The chromatographic separation of the sample was performed on a reverse-phase AQ column with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 4.0) and acetonitrile (50:50, v/v). The analyte was quantified by multiple reaction monitoring with a Waters Quattro micro™ API mass spectrometer. The lower limits of quantification were 20 ng/mL in plasma and 2 ng/g in brain, with the relative standard deviation % of 7.6 and 8.0% for plasma and brain samples, respectively. Acceptable intra-day and inter-day precisions and accuracies were obtained. Otaplimastat was sufficiently stable under all relevant analytical conditions, including a temperature of 4°C for 24 hr, room temperature 20°C for 24 hr, -80°C for 10 days and three freeze-thaw cycles (each at -80°C for 24 hr), for rat plasma and brain tissue. The validated method was successfully used to measure Otaplimastat concentrations in rat plasma and brain samples.

Reliable and Easy-To-Use Liquid Chromatography-Tandem Mass Spectrometry Assay for Quantification of Olorofim (F901318), a Novel Antifungal Drug, in Human Plasma and Serum.

A fast and easy-to-use liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of a novel antifungal drug, olorofim (F901318), a member of the novel class of orotomides, in human plasma and serum was developed and validated. Sample preparation was based on protein precipitation with acetonitrile and subsequent centrifugation. An isotope-labeled analogue of F901318 was employed as an internal standard. Chromatographic separation was achieved using a 50-mm by 2.1-mm, 1.9-µm, polar Hypersil Gold C18 column and isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (60%-40%, vol/vol) at a flow rate of 330 µl/min. The analyte was detected using a triple-stage quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode with positive heated electrospray ionization (HESI+) within a single runtime of 2.00 min. The present LC-MS/MS method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the U.S. Food and Drug Administration (FDA). Linearity of F901318 concentration ranges was verified by the Mandel test. The calibration curve was tested linear across the range and fitted using least-squares regression with a weighting factor of the reciprocal concentration. The limit of detection was 0.0011 mg/liter, and the lower limit of quantitation was 0.0033 mg/liter. Intraday and interday precisions ranged from 1.17% to 3.23% for F901318, and intraday and interday accuracies (percent bias) ranged from 0.75% to 5.01%. In conclusion, a method was established for the rapid quantitation of F901318 concentrations in serum and plasma samples in patient trials, and it optimizes therapeutic drug monitoring in applying an easy-to-use single method.

Determination of a PDE4 inhibitor Hemay005 in human plasma and urine by UPLC-MS/MS and its application to a PK study.

AIM: Hemay005 is a novel small-molecule inhibitor of phosphodiesterase-4 developed for the treatment of psoriasis. Measurement of Hemay005 in biological samples is critical for evaluation of its pharmacokinetics in clinical studies. Methodology & results: Plasma and urine samples were extracted and then chromatographed on an Acquity UPLC HSS T3 column with a gradient elution. Detection was performed on a Xevo TQ-S tandem mass spectrometer using negative ESI. CONCLUSION: For the first time, a sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the quantitative determination of Hemay005 in human plasma and urine, and it was successfully applied to evaluate the pharmacokinetics of Hemay005 in healthy subjects in a first-in-human study.

Development and full validation of an innovative HPLC-diode array detection technique to simultaneously quantify lacosamide, levetiracetam and zonisamide in human plasma.

AIM: To implement pharmacokinetic drug monitoring and individualize the posology of new antiepileptic drugs, the first HPLC-diode array detection method was developed and validated to simultaneously quantify lacosamide, levetiracetam and zonisamide in human plasma. MATERIALS & METHODS: Preceded by a reproducible liquid-liquid extraction, chromatographic separation was achieved by using a C18 column of 5 cm length and a mobile phase of water/acetonitrile. Full validation was performed according to international guidelines. RESULTS: The method was linear within 0.5-30, 2.5-40.0 and 0.5-50.0 µg ml-1 for lacosamide, levetiracetam and zonisamide, respectively (r2 ≥ 0.998), accurate (-12.411-8.303%), precise (≤8.875%). CONCLUSION: This innovative HPLC-diode array detection method was successfully employed in clinical practice and is expected to empower epileptic patients with a personalized pharmacotherapy service. [Formula: see text].

Stability-Indicating HPLC and HPTLC Methods for Determination of Agomelatine and its Degradation Products.

Two accurate, sensitive and highly selective stability-indicating methods are developed and validated for simultaneous determination of Agomelatine (AGM) and its forced degradation products (Deg I and II). The first method is High-Performance Liquid Chromatography for separation and quantitation of AGM, Deg I and II on a C18 column (250 mm × 4.6 mm, 5 µm p.s) in isocratic mode by using a binary mixture of Potassium dihydrogen phosphate (0.05 M, pH adjusted to 2.9 with orthophosphoric acid): acetonitrile (60:40, v/v) at a flow rate of 2 mL/min. The components were detected at 230 nm over a concentration range of 0.5-10 µg/mL for AGM and 0.5-5 µg/mL for both Deg I and II. The second method is High-Performance Thin-Layer Chromatography, where AGM, Deg I and II were separated on silica gel HPTLC F254 plates using chloroform:methanol:ammonia solution (9:1:0.1, by volume) as a developing system. The separated bands were scanned at 230 nm over the concentration range of 0.2-1.2 µg/band for AGM in pure form and human plasma and 0.1-1 µg/band for both Deg I and II. The proposed methods were successfully applied for analysis of AGM in pharmaceutical formulations. The results obtained by the proposed methods were statistically compared to the reported HPLC method revealing high accuracy and good precision.

The metabolism and drug-drug interaction potential of the selective prostacyclin receptor agonist selexipag.

1. The metabolism of selexipag has been studied in vivo in man and the main excreted metabolites were identified. Also, metabolites circulating in human plasma have been structurally identified and quantified. 2. The main metabolic pathway of selexipag in man is the formation of the active metabolite ACT-333679. Other metabolic pathways include oxidation and dealkylation reactions. All primary metabolites undergo subsequent hydrolysis of the sulphonamide moiety to their corresponding acids. ACT-333679 undergoes conjugation with glucuronic acid and aromatic hydroxylation to P10, the main metabolite detected in human faeces. 3. The formation of the active metabolite ACT-333679 is catalysed by carboxylesterases, while the oxidation and dealkylation reactions are metabolized by CYP2C8 and CYP3A4. CYP2C8 is the only P450 isoform catalysing the aromatic hydroxylation to P10. CYP2C8 together with CYP3A4 are also involved in the formation of several minor ACT-333679 metabolites. UGT1A3 and UGT2B7 catalyse the glucuronidation of ACT-333679. 4. The potential of selexipag to inhibit or induce cytochrome P450 enzymes or drug transport proteins was studied in vitro. Selexipag is an inhibitor of CYP2C8 and CYP2C9 and induces CYP3A4 and CYP2C9 in vitro. Also, selexipag inhibits the transporters OATP1B1, OATP1B3, OAT1, OAT3, and BCRP. However, due to its low dose and relatively low unbound exposure, selexipag has a low potential for causing drug-drug interactions.

Biocomparison Study of Adult and Paediatric Dose Strengths of the Prostacyclin Receptor Agonist Selexipag.

BACKGROUND AND OBJECTIVES: Selexipag is an oral, non-prostanoid, selective prostacyclin receptor agonist recently marketed for the treatment of pulmonary arterial hypertension (PAH) in adults. Selexipag may also be an effective treatment in children with PAH. The aim of this study was to compare the pharmacokinetics of selexipag and its active metabolite ACT-333679 following single oral administration of one tablet of 200 µg selexipag (Treatment A) vs. 4 paediatric tablets of 50 µg (Treatment B) in healthy adult male subjects. METHODS: This was an open-label, randomized, two-treatment, two-period, crossover biocomparison study. Bioequivalence criteria were explored and safety variables (vital signs, electrocardiogram, and laboratory parameters) were assessed. RESULTS: The exploratory analysis showed that the 90% confidence intervals of geometric mean ratio (Treatment B/Treatment A) for maximum plasma concentration (C max), area under the plasma concentration-time curve from zero to infinity (AUC0-∞) of ACT-333679, as well as AUC0-∞ of selexipag, were within the bioequivalence interval (0.80, 1.25). In addition, no relevant difference in C max for selexipag between the two treatments can be concluded. Single oral dose administration of 200 µg selexipag as one tablet of 200 µg or four tablets of 50 µg was well tolerated. CONCLUSIONS: Pharmacokinetic characteristics of selexipag and its metabolite ACT-333679 following administration of one adult tablet of 200 µg selexipag and four paediatric tablets of 50 µg selexipag were comparable. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02745860.

Evaluation of a Novel Immunoassay for Lacosamide Therapeutic Drug Monitoring: Comparison With a Liquid Chromatography-Mass Spectrometry Assay.

BACKGROUND: Monitoring serum levels of lacosamide, other than to establish individualized reference ranges may be helpful in several settings, including patients with liver and/or kidney failure or settings that may result in altered pharmacokinetic characteristics and to assess patients' compliance with therapy. In this study, the EurekaOne liquid chromatography-mass spectrometry (LC-MS/MS) method (in use method) and the ARK immunoassay method (new method) for lacosamide monitoring were compared. METHODS: Lacosamide concentrations were determined in 39 patient samples using (1) antiepileptic drug LC-MS/MS kit by EurekaOne on a Thermo Fisher Scientific TSQuantum Access Max system and (2) the lacosamide immunoassay by ARK Diagnostic Inc. (research use only kit), on a Abbott Architect System. RESULTS: Measured total imprecision of the new method is 6.29% at 6.59 µmol/L, 8.82% at 30.20 µmol/L, and 6.45% at 64.51 µmol/L. Passing-Bablok regression analysis showed a nonsignificant intercept of -0.03015 [95% confidence interval (CI), -1.2243 to 0.8593] and a slope of 1.05 (95% CI, 0.9973-1.1166), showing that the method does not deviate from linearity and absence of proportional systematic error. Bland-Altman analysis showed a systematic bias of -3.296% (95% CI, -5.81 to -0.78) with 95% of the LC-MS/MS-ARK mean % of differences ranging from -18.5 to 11.9. Despite this bias, data of the combined imprecision of the 2 methods show that the new method is still acceptable within the maximum allowable error of 15%. CONCLUSIONS: The performance of the new ARK method on the Architect system is acceptable and may be used routinely to measure serum lacosamide concentration in the clinic although the nature of the bias has to be carefully addressed.

Effect of gemfibrozil and rifampicin on the pharmacokinetics of selexipag and its active metabolite in healthy subjects.

AIMS: Based on in vitro data, there is evidence to suggest that cytochrome P450 (CYP) 2C8 is involved in the metabolism of selexipag and its active metabolite, ACT-333679. The present study evaluated the possible pharmacokinetic interactions of selexipag with gemfibrozil, a strong CYP2C8 inhibitor, and rifampicin, an inducer of CYP2C8. METHODS: The study consisted of two independent parts, each conducted according to an open-label, randomized, crossover design. The pharmacokinetics and safety of selexipag and ACT-333679 were studied following single-dose administration either alone or in the presence of multiple-dose gemfibrozil (part I) or rifampicin (part II) in healthy male subjects. RESULTS: Gemfibrozil had comparatively small effects on selexipag (less than 2-fold difference in any pharmacokinetic variable) but, with respect to ACT-333679, increased the maximum plasma concentration (Cmax ) 3.6-fold [90% confidence interval (CI) 3.1, 4.3] and the area under the plasma concentration-time curve from zero to infinity (AUC0-∞ ) 11.1-fold (90% CI 9.2, 13.4). The marked increased exposure to ACT-333679, which mediates the majority of the pharmacological activity of selexipag, was accompanied by significantly more adverse events such as headache, nausea and vomiting. Coadministration of rifampicin increased the Cmax of selexipag 1.8-fold (90% CI 1.4, 2.2) and its AUC0-∞ 1.3-fold (90% CI 1.1, 1.4); its effects on ACT-333679 were to increase its Cmax 1.3-fold (90% CI 1.1, 1.6), shorten its half-life by 63% and reduce its AUC0-∞ by half (90% CI 0.45, 0.59). CONCLUSION: Concomitant administration of selexipag and strong inhibitors of CYP2C8 must be avoided, whereas when coadministered with inducers of CYP2C8, dose adjustments of selexipag should be envisaged.

A pharmacokinetic drug-drug interaction study between selexipag and midazolam, a CYP3A4 substrate, in healthy male subjects.

PURPOSE: In vitro data showed that selexipag and its active metabolite (ACT-333679) have an inductive effect on CYP3A4, CYP2B6, and CYP2C9 at concentrations approximately 100-fold higher than the maximum plasma concentration (C max) measured under steady-state conditions. In order to confirm in vivo the lack of induction at the enterocyte level, we assessed the effect of selexipag on midazolam, a substrate of hepatic and intestinal CYP3A4. METHODS: This study was conducted according to an open-label, randomized, two-way crossover design. A total of 20 subjects received a single oral dose of 7.5 mg midazolam alone (treatment A) or on top of steady-state selexipag (treatment B). Selexipag was administered twice daily using an up-titration scheme consisting of three steps: 400, 600, 1000, and 1600 µg with increments every fourth day. A 24-h pharmacokinetic profile was performed following midazolam administration, and bioequivalence criteria were investigated on an exploratory basis. RESULTS: The C max of midazolam and 1-hydroxymidazolam was decreased by approximately 20 and 14%, respectively, following treatment B compared to A. The time to reach C max for midazolam and 1-hydroxymidazolam was similar between treatments. The terminal half-life was reduced in treatment B compared to A for both midazolam (16%) and 1-hydroxymidazolam (20%). Exposure (area under the curve) to midazolam and 1-hydroxymidazolam was similar between treatments, and the 90% confidence intervals of geometric mean ratios were within the bioequivalence interval. Treatment with midazolam, selexipag, and the combination was safe and well tolerated. CONCLUSION: Exposure to midazolam and 1-hydroxymidazolam was not affected by treatment with selexipag.

Intravenous lacosamide in status epilepticus: Correlation between loading dose, serum levels, and clinical response.

INTRODUCTION: Intravenous lacosamide (LCM) is increasingly used in the treatment of status epilepticus (SE), but optimal loading dose and target serum levels are unclear. We analysed the correlation between LCM serum levels after intravenous loading dose and clinical response. MATERIALS AND METHODS: Retrospective study in two centres from December 2014 to May 2016 including consecutive SE patients treated with LCM, in which trough serum levels after intravenous loading dose were available. Trough levels were correlated with the loading dose and the clinical response, defined as LCM introduction terminating SE without the need of further treatment. Correlations were adjusted for other SE characteristics. RESULTS: Among 40 patients, 16 (40%) responded to LCM. LCM serum concentrations within the reference interval (10-20mg/l) were associated with loading doses of >9mg/kg (p=0.003; χ2). However, we observed no difference between LCM serum levels in responders (median 10.4mg/l) versus non-responders (median 9.5mg/l; p=0.36; U test), even after adjusting for other predictors of clinical outcome (SE severity, aetiology, and number of previous treatment). DISCUSSION: High intravenous LCM loading doses (>9mg/kg) were associated with serum levels within the reference interval, there was however no correlation with the clinical response. Prospective studies are needed to evaluate the benefit of increasing the LCM loading dose in SE.