Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.—Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

Background:The enzyme myeloperoxidase produces chlorine bleach at sites of inflammation. Results: 2-Thioxanthines are potent mechanism-based inactivators of myeloperoxidase. Conclusion: 2-Thioxanthines block production of chlorine bleach during inflammation. Significance: Mechanism-based inactivators of myeloperoxidase should limit oxidative stress at sites of inflammation.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine’s antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Background:The 42 amino acid long amyloid beta peptide (A␤42) plays a pivotal role in Alzheimer disease. Results: A novel assay was developed and enabled us to describe the process of 〈␤42 production and modulation. Conclusion: A␤42 is generated through different pathways, which are differently affected by disease causing mutations and anti-amyloidogenic drugs. Significance: The data provide key information for therapeutic development in Alzheimer disease.

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Due to the small path length and low injection volume the concentration limit of detection is comparatively poor in capillary electrophoresis (CZE) with UV detection. This limitation can be overcome by means of preconcentration methods and/or improved detection techniques. This paper describes a strategy where isotachophoresis (ITP) is used to preconcentrate a new cholinesterase inhibitor (NXX-066) prior to a capillary zone electrophoresis analysis in the same single capillary. A hydrodynamic backpressure is used to prevent the analyte from migrating out of the capillary. Laser-induced fluorescence (LIF) is used to further increase the detectability. The total gain in detectability with ITP-CZE-LIF compared to CZE-UV was at least 5500-fold, and it is possible to determine NXX-066 at the 1 nM level. The ITP-CZE method was further evaluated for two beta-blockers; the mean coefficient of variation of the peak areas was 3.4% and the linearity of the calibration plots was satisfying.