Mass balance, pharmacokinetics and metabolism of the selective S1P1 receptor modulator ponesimod in humans.

1. Ponesimod [(R)-5-[3-chloro-4-(-2,3-dihydroxy-propoxy)-benzylidene]-2-propylimino-3-o-tolyl-thiazolidin-4-one] is an orally administered, selective S1P1 receptor modulator that blocks the egress of lymphocytes from lymphoid organs and reduces the availability of circulating effector T/B-cells. 2. The mass balance, pharmacokinetics and metabolism of 40 mg (14)C-ponesimod were investigated in six healthy male subjects. The total radioactivity in whole blood, plasma, urine, faeces and expired CO2 was determined by liquid scintillation counting. Metabolite profiling was performed by high-performance liquid chromatography and detection by mass spectrometry. 3. The majority of the radioactivity (% of administered dose) was recovered in faeces (57.3-79.6%), followed by urine (10.3-18.4%) and a small proportion in CO2 from expired air (0.6-1.9%). The average cumulative recovery (mass balance) of (14)C-associated radioactivity in faeces and urine was 77.9% of the administered dose. Unchanged ponesimod made up 25.9% of total radioactivity in faeces; none was detected in urine. Ponesimod was extensively metabolised and two pharmacologically inactive metabolites, M12 (ACT-204426) and M13 (ACT-338375), were detected in the circulation. M12 corresponded to 8.1% and M13 to 25.7% of the total drug-related radioactive exposure (AUC0-∞) in plasma. M12 was highly abundant in faeces (22.3% of total radioactivity) and to a smaller extent in urine (2.5% of total radioactivity).

Elucidation of the metabolic pathways and the resulting multiple metabolites of almorexant, a dual orexin receptor antagonist, in humans.

Almorexant [(2R)-2-{(1S)-6, 7-dimethoxy-1-[2-(4-trifluoromethyl-phenyl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide], a tetrahydroisoquinoline derivative, is a dual orexin receptor antagonist with sleep-promoting properties in both animals and humans. This study investigated the disposition, metabolism, and elimination of almorexant in humans. After oral administration of a 200-mg dose of ¹4C-almorexant, almorexant was rapidly absorbed (Tmax = 0.8 hour), and the apparent terminal half-life (t(1/2)) was 17.8 hours. The radioactive dose was almost completely recovered with 78.0% of the administered radioactive dose found in feces and 13.5% in urine. Unchanged almorexant was not found in urine and represented 10% of the administered dose in feces. In total, 47 metabolites were identified of which 21 were shown to be present in plasma. There are four primary metabolites, the isomeric phenols M3 and M8, formed by demethylation, the aromatic isoquinolinium ion M5, formed by dehydrogenation, and M6, formed by oxidative dealkylation with loss of the phenylglycine moiety. Most of the subsequent products are formed by permutations of these primary metabolic reactions followed by conjugation of the intermediate phenols with glucuronic or sulfonic acid. The percentage of dose excreted in urine or feces for any of the metabolites did not exceed 10% of the administered radioactive dose, nor did any of the metabolites represent more than 10% of the total drug-related exposure. In conclusion, after rapid absorption, almorexant is extensively metabolized, and excretion of metabolites in feces is the predominant route of elimination in humans.

Simultaneous determination of capecitabine and its metabolite 5-fluorouracil by column switching and liquid chromatographic/tandem mass spectrometry.

A liquid chromatographic/tandem mass spectrometric method was developed and validated for the quantitation of capecitabine and its metabolite 5-fluorouracil in human plasma. The simultaneous determination of both analytes was achieved by a column switching method using a trapping column and two analytical columns with different stationary phases. Isocratic elution was used for the separation of capecitabine on a C18 column whereas 5-fluorouracil was separated using gradient elution on an non-polar carbon phase. The calibration curves were linear for both compounds with a correlation factor (R2) > 0.9993 for 5-fluorouracil and >0.9942 for capecitabine. The assay was validated in the concentration range 5.00-1000 ng ml(-1) for both compounds. The intra-day precision was better than 10% for 5-fluorouracil and better than 11% for capecitabine whereas the inter-day precision was better than 8% for 5-fluorouracil and better than 14% for capecitabine.

Validation of an LC-MS/MS method for the quantitative determination of the orexin receptor antagonist almorexant and its four primary metabolites in human plasma.

A sensitive and selective LC-MS/MS method has been developed to quantify almorexant and its four primary metabolites M3, M5, M6, and M8 in human plasma samples. The method involved protein precipitation with acetonitrile in the high calibration range and liquid/liquid extraction with ethyl acetate in the low calibration range. Labeled internal standards were available for four analytes. Separation was performed with an Eclipse XDB-C18 (2.1mm×150mm, particle size 3.5µm) and a XBridge C18 column (2.1mm×50mm, particle size 3.5µm). The mobile phases were mixtures of acetonitrile, methanol, and water containing 1% formic acid; flow rate was 400µL/min. The triple stage quadrupole mass spectrometer was operated in ESI mode and the methods were linear over a range of 0.400-100ng/mL (almorexant, M5, M6), 1.00-100ng/mL (M3, M8), and 50.0-1000ng/mL (all analytes). The inter-day coefficients of variation were equal to or smaller than 10.5%. The inter-day accuracies were between 92.1% and 105.2%. The validated method was successfully applied to the pharmacokinetic assessment of almorexant and its metabolites in several phase I studies.

Bioanalysis for plasma protein binding studies in drug discovery and drug development: views and recommendations of the European Bioanalysis Forum.

Plasma protein binding (PPB) is an important parameter for a drug's efficacy and safety that needs to be investigated during each drug-development program. Even though regulatory guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed instructions on how to perform and validate such studies. To explore how PPB studies involving bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has conducted three surveys among their member companies: PPB studies in drug discovery (Part I); in vitro PPB studies in drug development (Part II); and in vivo PPB studies in drug development. This paper reflects the outcome of the three surveys, which, together with the team discussions, formed the basis of the EBF recommendation. The EBF recommends a tiered approach to the design of PPB studies and the bioanalysis of PPB samples: 'PPB screening' experiments in (early) drug discovery versus qualified/validated procedures in drug development.

Disposition and metabolism of setipiprant, a selective oral CRTH2 antagonist, in humans.

BACKGROUND: Setipiprant, a tetrahydropyridoindole derivative, is a CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper [Th]-2 cells) antagonist that has the potential to be effective in the treatment of patients with diseases with an allergic etiology, such as allergic rhinitis and asthma. OBJECTIVES: This study investigated the disposition, metabolism, and elimination of setipiprant. STUDY DESIGN: In this open-label study, a single oral dose of 1,000 mg (14)C-labeled setipiprant was administered. PARTICIPANTS: Six healthy male subjects were enrolled in this study. RESULTS: The radioactive dose was almost completely recovered in feces (88.2 %) and to a smaller extent in urine (11.7 %). The main recovery route for unchanged setipiprant was feces (50 % of the radioactive dose). The recovered amount of unchanged setipiprant in urine accounted for 3.7 %. The two main metabolites were M7 and M9 with the intact tetrahydropyridoindole core of setipiprant. M7 and M9 are supposedly two distinct dihydroxy-dihydronaphthalene isomers assumed to be formed by intermediate epoxidation of the naphthyl ring followed by a hydrolytic epoxide ring-opening. M7 and M9 accounted for 20.0 and 15.3 % of the administered radioactive dose. Both metabolites were mainly excreted via feces and to a lesser extent via urine. M7 was the only metabolite quantifiable in plasma, but at concentrations consistently below 10 % of those of the parent drug. CONCLUSION: Setipiprant is mainly excreted in feces in the form of the parent drug and in smaller amounts as its metabolites M7 and M9.

Validated LC-MS/MS method for the quantitative determination of the glucosylceramide synthase inhibitor miglustat in mouse plasma and human plasma and its application to a pharmacokinetic study.

A sensitive liquid chromatography-tandem mass spectrometry method has been developed to quantify miglustat in mouse plasma and in human plasma. The method involved simple protein precipitation with methanol. N-(n-nonyl)deoxynojirimycin was used as internal standard. Separation was performed on a Gemini C18 column (2.1 mm × 50 mm, particle size 5 µm) with a binary gradient at a flow rate of 600 µl/min. The mobile phases were methanol and water both containing 0.01% of a 25% ammonium hydroxide solution. The triple stage quadrupole mass spectrometer was operated in APCI mode using the transitions m/z 220.1 ≥ 158.0 for miglustat and m/z 290.1 ≥ 228.0 for the internal standard. The method was linear over a range of 10-10,000 ng/ml. The intra-day coefficients of variation for mouse plasma were equal to or smaller than 14.1%. The intra- and inter-day accuracies were 84.5-107.2% and 90.9-104.0%, respectively. For human plasma the intra-day coefficients of variation were equal to or smaller than 13.5%, while accuracies ranged between 93.6% and 100.0%. The validated method offered increased sensitivity (10 times higher) and decreased cycle times compared to other methods. It was successfully applied to the pharmacokinetic assessment of miglustat during treatment of patients with cystic fibrosis.

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.