Additional file 1 of Application of a new MDCKII-MDR1 cell model to measure the extent of drug distribution in vitro at equilibrium for prediction of in vivo unbound brain-to-plasma drug distribution

Additional file 1. Supplemental figures and tables

Effect of SMB-1 on K_V7.2-W236L.

<p>(<b>A</b>) Representative current traces for K_V7.2-W236L in the absence and presence of 10 µM SMB-1 (<b>B</b>) Effect of SMB-1 on current-voltage relationship. (<b>C</b>) Effect of SMB-1 on voltage-dependence of activation. (<b>D</b>) Effect of SMB-1 on deactivation kinetics. Statistical significance was determined by paired, two-tailed Student's <i>t</i>-test. Representative tail current traces in the absence and presence of 10 µM SMB-1 are shown in the inset. Effect of SMB-1 on the fast (<b>E</b>) and slow (<b>F</b>) component of the activation kinetics. Statistical significance was determined by two-way repeated measurements ANOVA followed by Bonferroni post-test. Y-values were log-transformed before the statistical analysis to meet the assumption of normality. * <i>p<</i>0.05, ** <i>p<</i>0.01, *** <i>p<</i>0.001. Bars represent S.E.M and <i>n = </i>4–6.</p

Effect of SMB-1 on channels with mutations in the refined retigabine binding site.

<p>Effect of 10 µM SMB-1 on the current-voltage relationship of (A) K_V7.2-L275V, (B) K_V7.2-L299V and (C) K_V7.4-L305V. Bars represent S.E.M. and <i>n = </i>4–6.</p

Inhibition of K_V7.2 by SMB-1.

<p>Chemical structure of (S)-2 (<b>A</b>) and SMB-1 (<b>B</b>). (<b>C</b>) Representative current traces for K_V7.2 in the absence and presence of 10 µM SMB-1 (<b>D</b>) Effect of SMB-1 on current-voltage relationship. (<b>E</b>) Effect of SMB-1 on voltage-dependence of activation. (<b>F</b>) Effect of SMB-1 on deactivation kinetics. Statistical significance was determined by paired, two-tailed Student's <i>t</i>-test. Representative tail current traces in the absence and presence of 10 µM SMB-1 are shown in the inset. Effect of SMB-1 on the fast (<b>G</b>) and slow (<b>H</b>) component of the activation kinetics. Statistical significance was determined by two-way repeated measurements ANOVA followed by Bonferroni post-test. Y-values were log-transformed before the statistical analysis to meet the assumption of normality. (<b>I</b>) Dose-response relationship for the effect of SMB-1 on K_V7.2. *** <i>p<</i>0.001. Bars represent S.E.M and <i>n = </i>5–9. Note that the error bars in some instances are too small to be visible.</p

Activation of K_V7.4 by SMB-1.

<p>(<b>A</b>) Representative current traces for K_V7.4 in the absence and presence of 10 µM SMB-1. (<b>B</b>) Effect of SMB-1 on current-voltage relationship. (<b>C</b>) Effect of SMB-1 on voltage-dependence of activation. (<b>D</b>) Effect of SMB-1 on deactivation kinetics. Statistical significance was determined by paired, two-tailed Student's <i>t</i>-test. Representative tail current traces in the absence and presence of 10 µM SMB-1 are shown in the inset. (<b>E</b>) Effect of SMB-1 on activation kinetics. Statistical significance was determined by two-way repeated measurements ANOVA followed by Bonferroni post-test. (<b>F</b>) Dose-response relationship of SMB-1 on K_V7.4. *** <i>p<</i>0.001. Bars represent S.E.M and <i>n = </i>5–8.</p

Effect of SMB-1 on K_V7.4-W242L.

<p>(<b>A</b>) Representative current traces for K_V7.4-W242L in the absence and presence of 10 µM SMB-1. (<b>B</b>) Effect of SMB-1 on current-voltage relationship. (<b>C</b>) Effect of SMB-1 on voltage-dependence of activation. (<b>D</b>) Effect of SMB-1 on deactivation kinetics. Statistical significance was determined by paired, two-tailed Student's <i>t</i>-test. Representative tail current traces in the absence and presence of 10 µM SMB-1 are shown in the inset (note that the traces are completely overlapping). (<b>E</b>) Effect of SMB-1 on activation kinetics. Statistical significance was determined by two-way repeated measurements ANOVA followed by Bonferroni post-test. Bars represent S.E.M and <i>n = </i>5.</p

Plasma and brain exposure of SMB-1 in rats following subcutaneous administration of 20 mg/kg.

<p>Data shown as mean total concentrations ± S.E.M (n = 3).</p

5‑HT_2A/5-HT_2C Receptor Pharmacology and Intrinsic Clearance of <i>N</i>‑Benzylphenethylamines Modified at the Primary Site of Metabolism

The toxic hallucinogen 25B-NBOMe is very rapidly degraded by human liver microsomes and has low oral bioavailability. Herein we report on the synthesis, microsomal stability, and 5-HT_2A/5-HT_2C receptor profile of novel analogues of 25B-NBOMe modified at the primary site of metabolism. Although microsomal stability could be increased while maintaining potent 5-HT₂ receptor agonist properties, all analogues had an intrinsic clearance above 1.3 L/kg/h predictive of high first-pass metabolism

Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter

The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABA_A receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [³H]­ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [³H]­ATPCA displayed a striking selectivity for BGT1 among the four GATs (<i>K</i>_m and <i>V</i>_max values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na⁺-dependent [³H]­ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [³H]­ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies

A Quantitative LC-MS/MS Method for Distinguishing the Tau Protein Forms Phosphorylated and Nonphosphorylated at Serine-396

Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer’s Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches. Here, we present an indirect LC-MS/MS method for quantification of pS396-tau. We take advantage of the reproducible miscleavage caused by S396 being preceded by a lysine (K395) and the proteolytic enzyme trypsin not cleaving when the following amino acid is phosphorylated. Therefore, treatment with trypsin discriminates between the forms of tau with and without phosphorylation at S396 and pS396-tau can be quantified as the difference between total S396-tau and nonphosphorylated S396-tau. To qualify the method, it was successfully applied for quantification of pS396-tau in human CSF from healthy controls and patients with Mild Cognitive Impairment and AD. In addition, the method was applied for rTg4510 mice where a clear dose dependent decrease in pS396-tau was observed in CSF following intravenous administration of a monoclonal antibody (Lu AF87908, hC10.2) targeting the tau epitope containing pS396. Finally, a formal validation of the method was conducted. In conclusion, this sensitive LC-MS/MS-based method for measurement of pS396-tau in CSF allows for quantitative translational biomarker applications for tauopathies including investigations of potential drug induced effects