Transporter tandems: precise tools for normalizing active transporter in the plasma membrane.

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification - as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

Metabolism and Pharmacokinetic Drug-Drug Interaction Profile of Vericiguat, A Soluble Guanylate Cyclase Stimulator: Results From Preclinical and Phase I Healthy Volunteer Studies.

BACKGROUND: Vericiguat is a stimulator of soluble guanylate cyclase currently under investigation as a first-in-class therapy for worsening chronic heart failure (NCT02861534). Patients with heart failure often require polypharmacy because of comorbidities. Hence, understanding the clearance mechanisms, elimination, and potential for pharmacokinetic drug-drug interactions of vericiguat is important for dose recommendations in this patient population. METHODS: Biotransformation and perpetrator properties of vericiguat were characterized in vitro using human hepatocytes, liver microsomes, and recombinant enzymes. This was complemented by a human mass balance study and ten drug-drug interaction studies in healthy volunteers wherein vericiguat was co-administered orally with omeprazole, magnesium/aluminum hydroxide, ketoconazole, rifampicin, mefenamic acid, midazolam, warfarin, digoxin, sacubitril/valsartan, aspirin, or sildenafil. RESULTS: In the human mass balance study, mean total radioactivity recovered was 98.3% of the dose administered (53.1% and 45.2% excreted via urine and feces, respectively). The main metabolic pathway of vericiguat is glucuronidation via uridine diphosphate-glucuronosyltransferase 1A9 and 1A1. In vitro studies revealed a low risk of vericiguat acting as a perpetrator by inhibiting cytochrome P450s, uridine diphosphate-glucuronosyltransferase isoforms, or major transport proteins, or by inducing cytochrome P450s. These observations were supported by phase I drug-drug interaction studies. Phase I studies that assessed the propensity of vericiguat as a victim drug showed changes in the range that did not warrant recommendations for dose adjustment in phase III. CONCLUSIONS: A low pharmacokinetic interaction potential of vericiguat was estimated from in vitro data and confirmed in vivo. Thus, vericiguat is suitable for a patient population with multiple comorbidities requiring polypharmacy.

Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity.

Pregnane X receptor upregulates ABC-transporter Abcg2 and Abcb1 at the blood-brain barrier.

ATP-driven efflux transporters are important, selective elements of the blood-brain barrier. Abcg2 (also brain multidrug resistance protein) and Abcb1 (P-glycoprotein) belong to the best known ABC-transporters which limit the access of therapeutic drugs to the brain and impair pharmacotherapy of central nervous system (CNS) disorders. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated, we analyzed the influence of the nuclear receptor, pregnane X receptor (PXR) on transporter expression. PXR is a xenobiotic-activated transcription factor that is highly expressed in barrier tissue. Xenobiotics like rifampicin and hyperforin activate PXR and induce Abcb1 expression. ABC-transporter up-regulation could have potential effects on pharmacokinetics of different drugs. To study the influence of PXR on the two most prominent efflux transporters we used a primary culture of porcine brain capillary endothelial cells (PBCEC) due to higher homologies to the human form of PXR. For activation of the pregnane X receptor the ligands hyperforin and rifampicin were used. We investigated the effects on the transporters on RNA level (quantitative real time PCR), protein level (Western blotting) and transport level (uptake assay, active transport). The stimulation of the PBCEC with rifampicin or hyperforin showed a significant up-regulation of both ABC-transporters on RNA level after 6h, whereas an increased protein expression was strongest after 12h. Also the transport activity intensified after a period of 12h for Abcg2 and Abcb1. In conclusion our data prove PXR activation by rifampicin and hyperforin lead to an increased ABC-transporter expression and transport activity.

Neutrophils cross the BBB primarily on transcellular pathways: an in vitro study.

The cerebral microcapillary endothelium forms a highly important barrier between the blood and the interstitial fluid of the brain (blood-brain barrier) that controls the passage of molecules and cells in and out of the CNS. Several CNS diseases include leukocyte extravasation through the endothelium via two mechanistically distinct routes, the paracellular and the transcellular pathway. We established a new in vitro model of the inflamed blood-brain barrier consisting of primary cultured porcine brain capillary endothelial cells which express a tight endothelial barrier even under inflammatory conditions. By means of this specialized blood-brain barrier model we extensively studied the transmigration of neutrophils. Electron and scanning force microscopy as well as immunofluorescence imaging captured the penetrating neutrophil on the endothelial cellular body in between the junctions clearly suggesting a transcellular migration pathway. Electric cell-substrate impedance sensing and transendothelial electrical resistance measurements in combination with expression analysis of tight junction proteins demonstrate that the neutrophil-endothelial interaction does not disrupt the barrier. In conclusion, this study, based on an in vitro model of the blood-brain barrier under inflammatory conditions, evidently implicates that neutrophils preferentially migrate across the BBB via the transcellular route without impairing endothelial barrier function whereas paracellular transmigration plays only a minor role if the barrier is strongly expressed.