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Introduction The endothelin (ET) family consists of three 21-amino acid isopeptides, ET-1, ET-2, and ET-3. ET-1 is thought to serve an important physiological and pathological role in the regulation of [...], Endothelin is a potent vasoconstrictor often up-regulated in hypertension. Endothelin vasoconstriction is mediated via the G-protein coupled endothelin A ([ET.sub.A]) receptor present on vascular smooth muscle. Endothelin receptor antagonists (ERAs) have been shown to antagonize ET-induced vasoconstriction. We describe the primary pharmacology of darusentan, a propanoic acid based ERA currently in phase 3 clinical trials for resistant hypertension. Darusentan was tested in membrane-, cell-, and tissue-based assays to determine its biochemical and functional potency. Rat aortic vascular smooth muscle cells (RAVSMs) were characterized using flow cytometry. RAVSM membrane fractions tested in saturation experiments exhibited moderate endothelin receptor density. Receptor counting revealed that >95% of the endothelin receptors in these fractions were the [ET.sub.A] subtype. (5)-Darusentan competed for radiolabeled endothelin binding in RAVSM membranes with single-site kinetics, exhibiting a [K.sub.i] = 13 nmol/L. (R)-Darusentan exhibited no binding activity. In cultured RAVSMs, endothelin induced increases in inositol phosphate and [Ca.sup.2+] signaling, both of which were attenuated by (5)-darusentan in a concentration-dependent manner. In isolated endothelium- denuded rat aortic rings, (S)-darusentan inhibited endothelin-induced vascular contractility with a [pA.sub.2] = 8.1 ± 0.14 (n = 4 animals; mean ± SD). (R)-Darusentan had no effect. The vasorelaxant potency of (S)-darusentan did not change when determined in isolated denuded rat mesenteric arterioles, suggesting a similar mode of action in both conductance and resistance arteries. In vascular smooth muscle, (S)-darusentan is an ERA with high affinity for the ET receptor, which in this preparation is predominantly [ET.sub.A] receptors. (S)-Darusentan inhibits endothelin-induced signaling related to pro-contractile activity and is a potent inhibitor of vasoconstriction in large and small arteries. Key words: endothelin, darusentan, hypertension, ex vivo, potency. L'endotheline est un puissant vasoconstricteur souvent surexprime lors d'hypertension. La vasoconstriction induite par l'endotheline est vehicutee par le recepteur de l'endotheline A ([ET.sub.A]) couple a la proteine G present sur le muscle lisse vasculaire. Des travaux ont montre; que les antagonistes du recepteur de l'endotheline (ARE) repriment la vasoconstriction induite par l'ET-1. Nous decrivons la pharmacologie primaire du darusentan, un ARE base sur l'acide propanoique, actuellement evalue dans les essais cliniques de phase 3 pour le traitement de l'hypertension resistante. Nous avons examine; le darusentan dans des essais sur membranes, cellules et tissus afin de determiner sa puissance biochimique et fonctionnelle. Nous avons caracterise des cellules musculaires lisses vasculaire aortiques de rats (MLVAR) en utilisant la cytome;trie de flux. Les fractions membranaires des MLVAR examine;es dans les expe;riences de saturation ont montre; une densite; moyenne de re;cepteurs d'endothe;line. Le comptage des re;cepteurs a re;ve;le; que le sous-type [ET.sub.A] representait >95 % des recepteurs de l'endotheline dans ces fractions. Le (S)-darusentan a compeetitionne; pour la fixation de l'endotheline radiomarqueee dans les membranes des MLVAR avec une cinetique a un seul site, presentant une [K.sub.i] = 13 nmol/L. Le (R)-darusentan n'a montre aucune activite de fixation. Dans les MLVAR cultiveies, l'endotheline a induit des augmentations de signalisation de l'inositol phosphate et du [Ca.sup.2+], et ces augmentations ont eete attenuees d'une maniere concentration-deipendante par le (S)-darusentan. Dans les anneaux aortiques depourvus endothelium, le (S)-darusentan a inhibe la contractilite vasculaire induite par l'endotheline avec une valeur de [pA.sub.2] = 8,1 ± 0,14 (n = 4 animaux, moyenne ± E.-T.). Le (S)-darusentan n'a eu aucun effet. La puissance vasorelaxante du (S)- darusentan mesuree dans les arterioles meesenteeriques isotees depourvues d'endothelium est demeuree stable, ce qui laisse supposer un mode d'action similaire dans les arteres de conductance et de resistance. Dans le muscle lisse vasculaire, le (S)-darusentan est un ARE ayant une haute affinite pour les recepteurs de l'ET qui, dans cette preparation, sont principalement les recepteurs [ET.sub.A]. Le (S)-darusentan inhibe la signalisation par l'endotheline assocteea une activite pro-contractile et est un puissant inhibiteur de la vasoconstriction dans les petites et grosses arteres. Mots-cles: endotheeline, darusentan, hypertension, ex vivo, puissance. [Traduit par la Redaction]

Introduction The endothelin receptor antagonists (ERAs) ambrisentan (Letairis), bosentan (Tracleer), and sitaxsentan (Thelin) have been approved in several countries for the treatment of pulmonary arterial hypertension (PAH). PAH is a [...], To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [³H]estradiol-17β-D-glucuronide (for OATP), and [2-D-penicillamine, 5-D-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 µmol-[L.sup.-1] for inhibition and at 2 µmol-[L.sup.-1] as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan, Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%-100% of control), whereas bosentan was most affected (32%-58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting. Key words: endothelin, endothelin receptor antagonists, hepatic transport, pulmonary artery hypertension, transporter substrate, BSEP, OATP. Afin d'evaluer les mecanismes potentiels de l'hepalotoxicite clinique, on a examine quatre antagonistes des recepteurs de l'endotheline (ARE) relativement a l'activite de substrats et a l'inhibition de transporteurs de capture et d'efflux hepatiques (NTCP, OATP, BSEP, MRP2), en utilisant des hepatocytes humains cultives en configuration sandwich. On a examine l'inhibition des transporteurs par les ARE en utilisant les substrats taurocholate (NTCP et BSEP), [E.sub.2]-17βG (OATP) et DPDPE (MRP2). On a evalue l'activite de substrats des ARE en utilisant les inhibiteurs ritonavir (OATP et BSEP), bromosulfaleine (OATP), erythromycine (P-glycoproteine) et probenecide (MRP2 et OATP). On a examine la capacite d'inhibition a 2, 20 et 100 µmol/L, et l'activite de substrats a 2 µmol/L. L'ambrisentan et le darusentan n'ont pas diminue l'activite de transport d'OATP, de NTCP ou de BSEP. Le bosentan et le sitaxsentan ont attenue le transport de NTCP aux concentrations elevees. Seul le sitaxsentan a diminue le transport d'OATP (52%) ; seul le bosentan a diminue l'activite de transport de BSEP (78 %). L'activite de transport de MRP2 n'a pas ete modifiee. Les inhibiteurs d'OATP ont diminue l'influx de tous les ARE. L'influx de darusentan a ete le moins touche (84%-100% des valeurs temoins), alors que celui de bosentan a ete le plus affecte (32%-58% des valeurs temoins). Le NTCP n'a pas contribue a l'influx des ARE, Seuls le bosentan et le darusentan se sont demarques comme substrats des efflux de BSEP et de P-glycoproteine. Tous les ARE examines ont ete des substrats d'au moins un transporteur hepatique. Le bosentan et le sitaxsentan, mais pas l'ambrisentan ni le darusentan, ont inhibe les transporteurs hepatiques humains, ce qui fournit un mecanisme potentiel de l'augmentation d'hepatotoxicite de ces agents en milieu clinique. Mots-cles : endotheline, antagonistes des recepteurs de l'endotheline, transport hepatique, hypertension arterielle pulmonaire, substrat des transporteurs, BSEP, OATP, [Traduit par la Redaction]

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