Quantitative determination of the antidepressant vortioxetine and its major human metabolite in plasma.

BACKGROUND: Vortioxetine is a novel antidepressant that has been developed in a joint partnership between H. Lundbeck A/S and the Takeda Pharmaceutical Company, Ltd. RESULTS: A number of bioanalytical methods have been developed in order to support the nonclinical and clinical development of the drug. Method performance, long-term stability, urine analysis, unspecific binding and metabolites analysis are presented and discussed. CONCLUSION: Two different method applications for the quantification of vortioxetine and its major human metabolite in human plasma, an isocratic cation exchange HPLC-MS/MS method utilizing C8-SPE sample extracts and a reversed-phase UPLC-MS/MS method with gradient elution of protein precipitated sample extracts, have been validated according to current regulatory standards and applied in support to a large number of nonclinical as well as clinical studies.

Tiered approach into practice: scientific validation for chromatography-based assays in early development - a recommendation from the European Bioanalysis Forum.

The principles of tiered approach have been part of the bioanalytical toolbox for some years. Nevertheless, an in spite of many valuable discussions in industry, they remain difficult to apply in a harmonized way for a broad array of studies in early drug development where these alternative approaches to regulated validation would make sense. The European Bioanalysis Forum has identified the need to proposes some practical workflows for five categories of studies for chromatography based assays where scientific validation will allow additional freedom while safeguarding scientific rigor and robust documentation: quantification of metabolites in plasma in relation to ICH M3(R2), urine analysis, tissue homogenate analysis, and preclinical and clinical studies in early stages of drug development. The recommendation would introduce a common language and harmonized best practice for these study categories and can help to refocus towards optimized scientific and resource investments for bioanalysis in early drug development.

Intestinal drug transport via the proton-coupled amino acid transporter PAT1 (SLC36A1) is inhibited by Gly-X(aa) dipeptides.

The oral absorption of some drug substances is mediated by nutrient transporters. As a consequence, nutrients and drugs may compete for available transporters, and interactions at the level of intestinal absorption are possible. Recently, we have identified δ-aminolevulinic acid, Gly-Gly, and Gly-Sar as substrates of the amino acid transporter PAT1. The aim of the present study is to investigate if other Gly-containing dipeptides interact with PAT1, and whether they can inhibit PAT1 mediated drug absorption, in vitro and in vivo. The in vitro methods included two-electrode voltage clamp measurements on hPAT1 expressing Xenopus laevis oocytes, which were used to investigate the PAT1-mediated transport of 17 different Gly-containing dipeptides (Gly-X(aa) or X(aa)-Gly). Also, the transepithelial transport of the PAT1 substrate gaboxadol was investigated across Caco-2 cell monolayers in the presence of different dipeptides. The in vivo part consisted of a pharmacokinetic study in rats following oral administration of gaboxadol and preadministration of 200 mg/kg dipeptide. The results showed that in hPAT1 expressing oocytes Gly-Tyr, Gly-Pro, and Gly-Phe inhibited currents induced by drug substances. In Caco-2 cell monolayers, Gly-Gly, Gly-Sar, and Gly-Pro significantly inhibited the PAT1 mediated absorptive transepithelial transport of gaboxadol; however, when orally administered to rats, Gly-Gly, Gly-Sar, Gly-Pro, or Gly-Tyr did not alter the pharmacokinetic profile of gaboxadol. In conclusion, the present study identifies selected dipeptides as inhibitors of PAT1 mediated drug absorption in various in vitro models.

Rectal absorption of vigabatrin, a substrate of the proton coupled amino acid transporter (PAT1, Slc36a1), in rats.

PURPOSE: To investigate the rectal absorption of vigabatrin in rats, based on the hypothesis that PAT1 (Slc36a1) is involved. METHODS: Male Sprague-Dawley rats were dosed rectally with five different gels, varying in buffer capacity, the amount of vigabatrin, and co-administration of proline or tryptophan. Western blotting was used to detect rPAT1 in rat rectal epithelium. X. Laevis oocytes were injected with SLC36A1 cRNA for the expression of hPAT1, prior to two-electrode voltage clamp measurements. RESULTS: rPAT1 protein was present in rat rectal epithelium. Approximately 7%-9% of a 1 mg/kg vigabatrin dose was absorbed after rectal administration, regardless of the formulation used. Increasing the dose of vigabatrin 10-fold decreased the absolute bioavailability to 4.2%. Co-administration of proline or tryptophan changed the pharmacokinetic profile, indicating a role of PAT1 in the rectal absorption of vigabatrin. Transport of vigabatrin via hPAT1 expressed in X. Laevis oocytes had a K(m) of 5.2 ± 0.6 mM and was almost completely inhibited by tryptophan. CONCLUSIONS: Although vigabatrin is a PAT1 substrate and the rPAT1 protein is expressed in the rectum epithelium, vigabatrin has low rectal absorption in rats.

Evaluation of an isochronic study design for long-term frozen stability investigation of drugs in biological matrices.

Long-term stability is a basic parameter in bioanalytical method validation; however, no criteria for conducting long-term stability studies are specified in current guidelines. We present an evaluation of a modified statistical approach applied to a study design utilizing an isochronic analysis (collection of samples to be analyzed at one time point) to determine the long-term stability and, further, a comparison with the most widely used continuous design. The presented approach has been used in regulated bioanalysis at Lundbeck for the past 7 years and has, in this period, been applied to 121 studies; all providing conclusive data. The isochronic approach eliminates day-to-day variation, reduces labor and adds to the flexibility in the laboratory. The statistical evaluation used is based on the relative difference between baseline samples and stability test samples as well as 90% confidence intervals for the mean concentration for each of the stability test points.

Development and validation of a selective and sensitive bioanalytical procedure for the quantitative determination of gaboxadol in human plasma employing mixed mode solid phase extraction and hydrophilic interaction liquid chromatography with tandem mass spectroscopic detection.

A selective and sensitive hydrophilic interaction liquid chromatography tandem mass spectrometric bioanalytical method for the quantitative determination of gaboxadol in human heparinized plasma was developed and validated. Gaboxadol and the stable isotope labeled internal standard were extracted from plasma by mixed mode solid phase extraction and analyzed on an Asahipak NH2P HPLC column with a mobile phase composed of 70% acetonitrile and 30% ammonium acetate (20 mM, pH 4). The analytes were detected by a SCIEX API 4000 triple quadropole instrument using turbo electrospray ionization and multiple reaction monitoring negative mode. The method was validated over the concentration range of 0.5-100 ng/mL. The intra-day precision of the assay, as measured by the coefficient of variation (CV%), was within 4%. The intra-day assay accuracy was found to be within 2.2% of the nominal concentration for all the standards. The average recovery of gaboxadol was about 87% and the ion suppression was approximately 8%. To eliminate late eluters including the glucuronides, a "front cut" column switching procedure was added to the chromatographic system. The effectiveness of the column switching in eliminating the absolute matrix effect caused by late eluters was demonstrated by the low variation (CV<3.5%) in the peak areas of the internal standard during the assessment of the inter-day precision and accuracy and no significant relative matrix effect was observed as illustrated by the excellent intra-day precision (CV<1.5%) from the assessment of standard samples prepared in five different lots of control plasma. The described bioanalytical method has been successfully utilized for the analysis of gaboxadol in post-dose samples (>8000) from various clinical studies. Inter-day precision and accuracy were assessed from the daily mean (n=2) of QC values from 52 runs, i.e. more than 3000 samples. The inter-day precision of the assay, based on the coefficient of variation of QC, ranged from 2.1 to 5.1%. The inter-day assay accuracy was found to be within 4% of the nominal concentration for all QC samples.

Electrophysiological safety of sertindole in dogs with normal and remodeled hearts.

Inhibition of the potassium current IKr and QT prolongation are associated with drug-induced torsades de pointes arrhythmias (TdP) and sudden cardiac death. We investigated the cardiac electrophysiological effects of sertindole, an antipsychotic drug reported to prolong the QT interval in schizophrenic patients. In cell cultures, sertindole seemed to be a selective blocker of IHERG over other ion currents. For IHERG, the IC50 value was 64 +/- 7 nM, whereas ISCN5A, ICa,L, ICa,T, IKCNQ1/KCNE1, and IKv4.3 were blocked in the micromolar range. In canine ventricular myocytes, the IC50 value for IKr inhibition by sertindole was 107 +/- 21 nM. Action potentials in these cells prolonged in a reverse rate- and concentration-dependent manner at 10 to 300 nM sertindole. In vivo, sertindole was administered to anesthetized dogs at clinically relevant (0.05-0.20 mg/kg) and high doses (1.0-2.0 mg/kg) i.v. At 0.05 to 0.20 mg/kg sertindole (plasma concentrations 30-157 nM), QTc was prolonged by 1 to 5% in normal dogs and by 9 to 20% in dogs with remodeled hearts due to chronic atrioventricular block (CAVB). TdP was not induced at these doses in normal dogs or in CAVB dogs with reproducible induction of TdP by dofetilide in previous experiments. At 1.0 to 2.0 mg/kg sertindole (plasma concentrations 0.5-3.1 microM), QTc prolonged by 6 to 11% in normal dogs and by 22% in dofetilide-sensitive CAVB dogs. TdP occurred in three of five animals in the latter group. Thus, at high i.v. doses sertindole can pose a serious proarrhythmic risk when electrical remodeling of the ventricles is present. At clinically relevant doses, however, sertindole does not cause TdP in anesthetized dogs with normal or remodeled hearts.

Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay.

Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.