Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Bupivacaine-induced Vasodilation Is Mediated by Decreased Calcium Sensitization in Isolated Endothelium-denuded Rat Aortas Precontracted with Phenylephrine

BACKGROUND: A toxic dose of bupivacaine produces vasodilation in isolated aortas. The goal of this in vitro study was to investigate the cellular mechanism associated with bupivacaine-induced vasodilation in isolated endotheliumdenuded rat aortas precontracted with phenylephrine. METHODS: Isolated endothelium-denuded rat aortas were suspended for isometric tension recordings. The effects of nifedipine, verapamil, iberiotoxin, 4-aminopyridine, barium chloride, and glibenclamide on bupivacaine concentration-response curves were assessed in endothelium-denuded aortas precontracted with phenylephrine. The effect of phenylephrine and KCl used for precontraction on bupivacaine-induced concentration-response curves was assessed. The effects of verapamil on phenylephrine concentration-response curves were assessed. The effects of bupivacaine on the intracellular calcium concentration ([Ca2+]i) and tension in aortas precontracted with phenylephrine were measured simultaneously with the acetoxymethyl ester of a fura-2-loaded aortic strip. RESULTS: Pretreatment with potassium channel inhibitors had no effect on bupivacaine-induced relaxation in the endothelium-denuded aortas precontracted with phenylephrine, whereas verapamil or nifedipine attenuated bupivacaine-induced relaxation. The magnitude of the bupivacaine-induced relaxation was enhanced in the 100 mM KCl-induced precontracted aortas compared with the phenylephrine-induced precontracted aortas. Verapamil attenuated the phenylephrine-induced contraction. The magnitude of the bupivacaine-induced relaxation was higher than that of the bupivacaine-induced [Ca2+]i decrease in the aortas precontracted with phenylephrine. CONCLUSIONS: Taken together, these results suggest that toxic-dose bupivacaine-induced vasodilation appears to be mediated by decreased calcium sensitization in endothelium-denuded aortas precontracted with phenylephrine. In addition, potassium channel inhibitors had no effect on bupivacaine-induced relaxation. Toxic-dose bupivacaine- induced vasodilation may be partially associated with the inhibitory effect of voltage-operated calcium channels.

Mechanism of vasodilation by propofol in the rabbit renal artery

BACKGROUND: Propofol directly inhibits vascular reactivity. However, available information regarding the underlying mechanisms of propofol is poor. Therefore, mechanisms of the underlying relaxant action of propofol were investigated using rabbit renal arteries. METHODS: Propofol-induced relaxation of rabbit renal arteries was studied in contracted preparations with 50 mM KCl or 10microM histamine. Vessel tension was recorded with a pen recorder. We were interested in determining whether propofol-induced vasodilation is affected by endothelium-denudation, L-NG-nitroarginine methyl ester (L-NAME), tetraethylammonium (TEA), iberiotoxin, glibenclamide, 4-aminopyridine, 7-ethoxyresorufin, caffeic acid, baiclalein, ryanodine, and thapsigargin. RESULTS: Propofol-induced concentration-dependent vasodilation was not affected either by endothelium denudation or by L-NAME during histamine-induced contraction. The relaxing effect of propofol on histamine-induced contraction was inhibited by either TEA, a K+ channel inhibitor, or iberiotoxin (100 nM), a selective blocker of the large conductance Ca(2+)-activated K+ channel (BKCa channel). In contrast, the relaxing effect of propofol was unaffected by 10microM glibenclamide, an ATP-sensitive K+ channel blocker, by 5 mM 4-aminopyridine, a blocker of delayed rectifier, by 7-ethoxyresorufin, a cytochrome P450 inhibitor, by 10microM caffeic acid and 10microM baiclalein, lipooxygenase inhibitors, or by 10microM ryanodine and thapsigargin, Ca2+store inhibitors. CONCLUSIONS: These results suggest that the relaxant effect of propofol may result from activation of BKCa channels by inhibiting voltage-gated Ca2+ influx in a prolonged manner.

Lysophosphatidylcholine Attenuates Endothelium-dependent Relaxation Responses through Inhibition of ACh-induced Endothelial Ca2+i Increase

Lysophosphatidylcholine (LPC), which accumulates in atherosclerotic arteries, has been reported to inhibit endothelium-dependent relaxation (EDR) in many different species. However, the underlying mechanism of LPC-induced inhibition of EDR is still uncertain. In the present study, we measured simultaneously both isometric tension and cytosolic free Ca2+ ([Ca2+]i) in rabbit carotid strips, and examined the effect of LPC on tension and [Ca2+]i. In carotid strips with intact-endothelium, high K+ (70 mM) increased both tension and [Ca2+]i, and cumulative addition of acetylcholine (ACh) from 0.1 to 10microM induced dose dependent increase of [Ca2+]i with concomitant relaxation. In the presence of L-NAME (0.1 mM), ACh increased [Ca2+]i without affecting the amplitude of high K+-induced tension. These ACh-induced change of [Ca2+]i and tension was abolished by removal of endothelium or 10 nM 4-DAMP (muscarinic receptor antagonist) pretreatment. Pretreatment of LPC (10microM) inhibited ACh (10microM)-induced change of tension and [Ca2+]i in endothelium-intact carotid artery. On the other hand, LPC had no effect on ACh-induced change of tension and [Ca2+]i in endothelium denuded artery. In Ca2+-free external solution, ACh transiently increased [Ca2+]i, and pretreatment of LPC significantly inhibited ACh-induced transient [Ca2+]i change. Based on the above results, it may be concluded that LPC inhibits the ACh-induced [Ca2+]i change through inhibition of Ca2+ mobilization in vascular endothelial cells, resulting in decreased production of NO and concomitant inhibition of endothelium-dependent vascular relaxation.

Mechanism of Acetylcholine-induced Endothelium-dependent Relaxation in the Rabbit Carotid Artery by M3-receptor Activation

The present study were designed to characterize the action mechanisms of acetylcholine (ACh) -induced endothelium-dependent relaxation in arteries precontracted with high K (70 mM). For this, we simultaneously measured both muscle tension and cytosolic free Ca2 concentration ([Ca2 ]i), using fura-2, in endothelium-intact, rabbit carotid arterial strips. In the artery with endothelium, high K increased both [Ca2 ]i and muscle tension whereas ACh (10microM) significantly relaxed the muscle and increased [Ca2 ]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca2 ]i without relaxing the muscle. In the artery without endothelium, high K increased both [Ca2 ]i and muscle tension although ACh was ineffective. 4-DAMP (10 nM) or atropine (0.1microM) abolished ACh-induced increase in [Ca2 ]i and relaxation. The increase of [Ca2 ]i and vasorelaxation by ACh was siginificantly reduced by either 3microM gadolinium, 10microM lanthanum, or by 10microM SKF 96365. These results suggest that in rabbit carotid artery, ACh-evoked relaxation of 70 mM K -induced contractions appears to be mediated by the release of NO. ACh-evoked vasorelaxation is mediated via the M3 subtype, and activation of the M3 subtype is suggested to stimulate nonselective cation channels, leading to increase of [Ca2 ]i in endothelial cells.

Mechanism of L-NAME-resistant endothelium-dependent relaxation induced by acetylcholine in rabbit renal artery

In the rabbit renal artery, acetylcholine (ACh, 1 nM ~ 10 micrometer) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 micrometer) in a dose-dependent manner. NG-nitro-L-arginine (L-NAME, 0.1 mM), an inhibitor of NO synthase, or ODQ (1 micrometer), a soluble guanylate cyclase inhibitor, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was abolished in the presence of 25 mM KCl and L-NAME. The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10 micrometer), miconazole (10 micrometer), or 17-octadecynoic acid (17-ODYA, 10 micrometer), failed to inhibit the ACh-induced relaxation in the presence of L-NAME. 11,12-epoxyeicosatrienoic acid (11,12-EET, 10 micrometer) had no relaxant effect. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.3 micrometer) and apamin (1 micrometer), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 micrometer), in a dose-dependent manner. Furthermore, 2-methylthio-ATP (2MeSATP), a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue. In conclusion, in renal arteries isolated from rabbit, ACh produced non-NO relaxation that is mediated by an EDHF. The results also suggest that ACh may activate the release of ATP from endothelial cells, which in turn activates P2Y receptor on the endothelial cells. Activation of endothelial P2Y receptors induces a release of EDHF resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. The results further suggest that EDHF does not appear to be a cytochrome P450 metabolite.

Modulation of outward potassium currents by nitric oxide in longitudinal smooth muscle cells of guinea-pig ileum

To investigate the possible involvement of outward potassium (K+) currents in nitric oxide-induced relaxation in intestinal smooth muscle, we used whole-cell patch clamp technique in freshly dispersed guinea-pig ileum longitudinal smooth muscle cells. When cells were held at -60 mV and depolarized from - 40 mV to + 50 mV in 10 mV increments, sustained outward K+ currents were evoked. The outward K+ currents were markedly increased by the addition of 10 muM sodium nitroprusside (SNP). 10 muM S-nitroso-N-acetylpenicillamine (SNAP) and 1 mM 8-Bromo-cyclic GMP (8-Br-cGMP) also showed a similar effect to that of SNP. 1 mM tetraethylammonium (TEA) significantly reduced depolarization-activated outward K+ currents. SNP-enhanced outward K+ currents were blocked by the application of TEA. High EGTA containing pipette solution (10 mM) reduced the control currents and also inhibited the SNP-enhanced outward K+ currents. 5 mM 4-aminopyridine (4-AP) significantly reduced the control currents but showed no effect on SNP-enhanced outward K+ currents. 0.3 muM apamin and 10 muM glibenclamide showed no effect on SNP-enhanced outward K+ currents. 1 muM 1H-(1,2,4)oxadiazolo (4,3-a)quinoxaline-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, significantly blocked SNP-enhanced K+ currents. We conclude that NO donors activate the Ca2+-activated K+ channels in guinea-pig ileal smooth muscle via activation of guanylate cyclase.

The effect of sodium nitroprusside on muscle tension in guinea-pig ileum

Nitric oxide (NO) has been known as a mediator of nonadrenergic, noncholinergic inhibitory neurotransmitter in intestinal smooth muscles. It has been suggested that NO donor such as sodium nitroprusside (SNP) produces relaxation of smooth muscle via activation of guanylate cyclase and elevation of cGMP levels. We have therefore investigated the effects of NO, using SNP, on muscle tension in the longitudinal smooth muscle of guinea-pig ileum. The possible role of cGMP was also investigated as well as the involvement of K+ channel on SNP-induced inhibitory effect. The results are summarized as follows; high KCl-or CCh-activated contractions were inhibited by SNP in a concentration-dependent manner. 8-Br-cGMP also showed a similar effect in that of SNP. TEA (1 mM) significantly reduced the SNP-induced inhibitory effect. SNP-induced effect was further reduced by the presence of 10 mM TEA. On the other hand, 4-AP (0.1 mM), glibenclamide (10 muM) and apamin (0.1 muM) showed little effects on SNP-induced relaxation. Zaprinast significantly potentiated the SNP-induced inhibitory effect in all ranges. ODQ also significantly decreased the SNP-induced inhibitory effect. Pretreatment with CPA (10 muM) slightly reduced the SNP-induced inhibitory effect. From the above results, both effect mediated by NO and cGMP might be responsible for the activation of Ca2+/-activated K+ channel by SNP in guinea-pig ileum. And this K+ channel activation by SNP also contributes to the SNP-induced membrane hyperpolarization and relaxation.