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The precise regulation of blood-brain barrier (BBB) permeability for immune cells and blood-borne substances is essential to maintain brain homeostasis. Sphingosine-1-phosphate (S1P), a lipid signaling molecule enriched in plasma, is known to affect BBB permeability. Previous studies focused on endothelial S1P receptors 1 and 2, reporting a barrier-protective effect of S1P1 and a barrier-disruptive effect of S1P2. Here, we present novel data characterizing the expression, localization, and function of the S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). Hitherto, the receptor was deemed to be exclusively immune cell associated. We detected a robust expression of S1P4 in homeostatic murine BMECs (MBMECs), bovine BMECs (BBMECs), and porcine BMECs (PBMECs) and pinpointed its localization to abluminal endothelial membranes via immunoblotting of fractionated brain endothelial membrane fragments. Apical S1P treatment of BMECs tightened the endothelial barrier in vitro, whereas basolateral S1P treatment led to an increased permeability that correlated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels (MBMVs) after stroke, a neurologic disease associated with BBB impairment. RNA sequencing and qPCR analysis of BMECs suggested the involvement of S1P4 in endothelial homeostasis and barrier function. Using S1P4 knock-out (KO) mice and S1P4 siRNA as well as pharmacological agonists and antagonists of S1P4 both in vitro and in vivo, we demonstrate an overall barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic potential in CNS diseases associated with BBB dysfunction. [ABSTRACT FROM AUTHOR]

3,4‐Methylenedioxymethamphetamine (MDMA, Ecstasy) tablets are widely used recreationally, and not only vary in appearance, but also in MDMA content. Recently, the prevalence of high‐content tablets is of concern to public health authorities. To compare UK data with other countries, we evaluated MDMA content of 412 tablets collected from the UK, 2001–2018, and investigated within‐batch content variability for a sub‐set of these samples. In addition, we investigated dissolution profiles of tablets using pharmaceutical industry‐standard dissolution experiments on 247 tablets. All analyses were carried out using liquid chromatography−tandem mass spectrometry (LC–MS/MS). Our data supported other studies, in that recent samples (2016–2018) tend to have higher MDMA content compared to earlier years. In 2018, the median MDMA content exceeded 100 mg free‐base for the first time. Dramatic within‐batch content variability (up to 136 mg difference) was also demonstrated. Statistical evaluation of dissolution profiles at 15‐minutes allowed tablets to be categorized as fast‐, intermediate‐, or slow‐releasing, but no tablet characteristics correlated with dissolution classification. Hence, there would be no way of users knowing a priori whether a tablet is more likely to be fast or slow‐releasing. Further, within‐batch variation in dissolution rate was observed. Rapid assessment of MDMA content alone provides important data for harm reduction, but does not account for variability in (a) the remainder of tablets in a batch, or (b) MDMA dissolution profiles. Clinical manifestations of MDMA toxicity, especially for high‐content, slow‐releasing tablets, may be delayed or prolonged, and there is a significant risk of users re‐dosing if absorption is delayed. [ABSTRACT FROM AUTHOR]

Sample preparation for the analysis of clinical samples with the mass spectrometer (MS) can be extensive and expensive. Simplifying and speeding up the process would be very beneficial. This paper reports sponge spray--a novel sampling and direct MS analysis approach--attempting exactly that. It enables direct analysis without any sample preparation from dried blood, plasma, and urine. The tip of a volumetric absorptive microsampling device is used to collect an exact amount of sample and from that same tip an electrospray can be directed into a mass spectrometer. We demonstrate here that, although with significant matrix effects, quantitation of penicillin G, a common antimicrobial, is possible in plasma and in urine, with essentially no sample preparation. [ABSTRACT FROM AUTHOR]

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10–10,000 ng/mL for plasma, and 1–50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP. [ABSTRACT FROM AUTHOR]