Effect of Ambrisentan, Bosentan, Sitaxsentan, and Macitentan on Hepatic Uptake and Efflux Transporters.

a<p>Data presented as mean ± standard deviation for 3 independent studies performed in duplicate;</p>b<p>Data presented for a single experiment preformed in duplicate;</p>c<p>Data previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087548#pone.0087548-Ray1" target="_blank">[35]</a>. Ambrisentan, bosentan, and macitentan were tested in concentrations ranging from 0.14–100 µM.</p><p>ND = not determined.</p

Uptake of bosentan and macitentan into human hepatocytes.

<p>ERAs were evaluated either in the absence or presence of the transporter inhibitors rifampicin (40 µM) and cyclosporin A (5 µM). Data presented as mean (±SD) pmol/million cells; n = 4 donors; *P<0.05 for comparisons indicated.</p

Dose-dependent intracellular accumulation of test ERAs in sandwich-cultured human hepatocytes.

<p>Ambrisentan displayed the lowest intracellular accumulation followed by bosentan, sitaxsentan, and macitentan. Data are presented as mean (±SD) micromolar (µM) concentration; n = 3 donors; *P<0.05 vs. corresponding intracellular accumulation value for ambrisentan at the same test concentration.</p

d₈-Taurocholate (d₈-TCA) total (A) and cellular (B) accumulation in sandwich-cultured human hepatocytes exposed to ambrisentan, bosentan and macitentan.

<p>Bosentan and macitentan treatment resulted in a dose-dependent reduction in total accumulation of d₈-TCA. Ambrisentan, bosentan and macitentan treatment each resulted in a dose-dependent reduction in cellular accumulation of d₈-TCA. Data presented as mean (±SD) expressed as percent of control treated; n = 3 donors; *P<0.05 bosentan vs. control; # P<0.05 macitentan vs. control.</p

Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR‑2 (SLC19A3)

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH’s, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (<i>K</i>_m = 1.15 ± 0.2 mM) and other cationic compounds (MPP⁺ and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug–drug and drug–nutrient interactions

Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleosides

<div><p>Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (<em><u>mito</u></em>chondrial dysfunction caused by anti<em><u>vir</u></em>al ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.</p> </div

Predicting adverse effects of antiviral ribonucleosides during preclinical development: The mitovir score.

<p>Correlations between (<b>A</b>) cytotoxicity in Huh-7 cells and MT4 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a><b>, CC₅₀</b>), (<b>B</b>) cytotoxicity in MT4 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a><b>, CC₅₀</b>) and the efficiency of nucleotide incorporation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t002" target="_blank"><b>Table 2</b></a><b>, </b><b><i>k</i></b><b>_pol/</b><b><i>K</i></b><b>_d,app</b>), (<b>C</b>) cytotoxicity in MT4 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a><b>, CC₅₀</b>) and <i>mitovir score</i> for MT4 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t002" target="_blank"><b>Table 2</b></a>), (<b>D</b>) cytotoxicity in MT4 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a><b>, CC₅₀</b>) and the <i>mitovir score</i> for each analogue corrected to account for the presence of the nucleotide with which the analogue competes, ATP or CTP, and (<b>E</b>) cytotoxicity in Huh-7 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a><b>, CC₅₀</b>) and <i>mitovir score</i> for Huh-7 cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t002" target="_blank"><b>Table 2</b></a>). Error bars represent s.d. Nonparametric (Spearman) correlations with r values shown. In parentheses are one-tailed P-values calculated from Spearman coefficients to provide a measure of statistical significance of correlation.</p

Kinetic parameters for POLRMT-catalyzed nucleotide incorporation^a.

a<p>Values rounded to two significant figures. Standard errors are from non-linear regression fits of data to a hyperbolic model (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat.1003030.s001" target="_blank">Figures S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat.1003030.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat.1003030.s003" target="_blank">S3</a></b>).</p>b<p><i>mitovir score</i>: rate constant for incorporation calculated by using the experimentally determined kinetic parameters, <i>k</i>_pol and <i>K</i>_d,app and the intracellular concentration of nucleoside analog triphosphate [TP] (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat-1003030-t001" target="_blank"><b>Table 1</b></a>); <i>mitovir score</i> = <i>k</i>_eff (s⁻¹) = (<i>k_pol</i> * [TP])/(<i>K_d,app</i>+[TP]).</p>c<p><i>mitovir score</i> determined for Huh-7 cells.</p>d<p><i>mitovir score</i> determined for MT4 cells.</p>e<p>rate constant for incorporation calculated by using the experimentally determined kinetic parameters, <i>k</i>_pol and <i>K</i>_d,app and the intracellular concentration of nucleotide [TP] (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat.1003030.s009" target="_blank">Table S1</a></b>); <i>k</i>_eff (s⁻¹) = (<i>k_pol</i> * [TP])/(<i>K_d,app</i>+[TP]).</p>f<p>not determined.</p

Intracellular metabolism, cytotoxicity (CC₅₀), anti-HCV replicon activity (EC₅₀) and anti-NS5B activity (IC₅₀)^a.

a<p>Values rounded to two significant figures. All values are the mean ± s.d. of at least 3 independent experiments done in duplicate or triplicate except for CC₅₀ Huh-7 for 3-deazaadenosine and 6-methylpurine-riboside, which are the average of replicate wells from one experiment.</p>b<p>Intracellular metabolism [TP] is the amount of nucleoside triphosphate determined from LC/MS/MS analysis and converted from pmol per million cells to intracellular concentration (µM) using a cellular volume of 2 pL per cell. All data for 10 µM 24 h incubations except where noted otherwise.</p>c<p>Compounds that showed toxicity in MT4 cells at 10 µM. Incubations were done at 0.1 µM and the intracellular levels dose normalized assuming proportional increase in intracellular metabolites with extracellular concentrations.</p>d<p>not determined.</p>e<p>Tested in the form of monophosphate prodrug GS-7977.</p

TFIIS prevents accumulation of antiviral nucleotides in Pol II transcripts.

<p>(<b>A</b>) Schematic of synthetic nucleic scaffolds for transcription elongation complex (TEC) assembly with calf thymus Pol II. The first templating base is underlined. The TEC with 11-nt RNA (TEC-A11) was assembled using TDS50, NDS50 and RNA9 (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003030#ppat.1003030.s010" target="_blank">Table S2</a></b> for the complete oligonucleotide sequences) in the presence of 10 µM each GTP and ATP (TEC-A11) and purified from the unincorporated DNA, RNA and NTPs; TEC-C12 was obtained by addition of 10 µM CTP to TEC-A11. (<b>B</b>) Reaction products from Pol II-catalyzed nucleotide incorporation in the absence and presence of TFIIS. The concentration of unmodified substrate NTP and analogs were 500 µM; TFIIS was added at 10 µM. Reactions proceeded for 2 min. Reactions with ribavirin-TP proceeded for 10 min. (<b>C,D</b>) Reaction products from Pol II-catalyzed nucleotide incorporation in the presence of the next correct nucleotide substrate. The concentration of the unmodified substrate NTP and analogs were 500 µM; TFIIS was added at 10 µM. Reactions proceeded for 2 min. Reactions with ribavirin-TP, 4′-methyl-CTP and 4′-azido-CTP proceeded for 10 min. (<b>E</b>) Percent inhibition by TFIIS on Pol II nucleoside analog incorporation.</p