Gαi-biased apelin analog protects against isoproterenol-induced myocardial dysfunction in rats.

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and ß-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the ß-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts.

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01-100 µM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 µM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 µM), the DP1 receptor antagonist BW A868C (1 µM) also inhibited relaxation reaching significance above 10 µM. In contrast, the EP3 receptor antagonist L798106 (1 µM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 µM) blocked transforming growth factor ß1 (TGF-ß1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 µM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-ß1 in the presence of TRE.

NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension.

BACKGROUND: NTP42 is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2 is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy of NTP42 in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs. METHODS: PAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar-Kyoto rats. Animals were assigned into groups: 1. 'No MCT'; 2. 'MCT Only'; 3. MCT + NTP42 (0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT. RESULTS: From haemodynamic assessments, NTP42 reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals. CONCLUSIONS: These findings suggest that NTP42 and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

Inhaled hexadecyl-treprostinil provides pulmonary vasodilator activity at significantly lower plasma concentrations than infused treprostinil.

INS1009 is a long acting pulmonary vasodilator prodrug of treprostinil (TRE) that is formulated in a lipid nanoparticle for inhaled delivery by nebulization. This study examined the ability of INS1009 to inhibit vasoconstriction in the pulmonary vasculature of rats and dogs and the extent to which local activity within the lung contributes to its activity. Rats received a single dose of INS1009 by nose-only inhalation or were given a continuous intravenous (i.v.) infusion of TRE, followed by an i.v. challenge of the thromboxane mimetic pulmonary vasoconstrictor U46619 and the increase in pulmonary arterial pressure (PAP) was measured. In beagle dogs, INS1009 was given by inhalation via face mask and TRE was given by continuous i.v. infusion; vasoconstriction was then induced by inhaled hypoxia with reduction of FIO2 to 0.10. Changes in the dog's right ventricular pulse pressure (RVPP) were measured using implanted telemetry probes. Blood samples were collected in rats and dogs immediately after the challenge to measure the plasma TRE concentration. Exposure of rats to inhaled INS1009 (0.5, 3.0 and 20.9 µg/kg) inhibited the U46619-induced increase in PAP at all doses up to 6 h with statistically significant inhibition up to 24 h with the pooled dose-response data. The concentration of TRE in the plasma at which PAP was reduced by 50% was approximately 60-fold lower for INS1009 (EC50 = 0.08 ng/mL) as compared to i.v. TRE (EC50 = 4.9 ng/mL). In dogs, INS1009 (2.7-80.9 µg/kg) inhibited the hypoxia-induced increase in RVPP at all doses up to 6 h with activity once again observed with the pooled dose-response of 10 µg/kg and higher at 24 h. The concentration of TRE in the plasma at which RVPP was reduced by 50% was approximately 550-fold lower for INS1009 (EC50 = 0.0075 ng/mL) as compared to i.v. TRE (EC50 = 4.1 ng/mL). These studies, in two species and by two different pulmonary vasoconstrictor challenges, demonstrate that inhaled INS1009 not only has long-acting vasodilatory effects but also that the local activity within the lung contributes to this response. Therefore, INS1009 may offer the opportunity to effect pulmonary vasodilation for long periods but with substantially lower systemic exposure than infused TRE.

Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug.

This article describes the preclinical pharmacology and pharmacokinetics (PK) of hexadecyl-treprostinil (C16TR), a prodrug of treprostinil (TRE), formulated in a lipid nanoparticle (LNP) for inhalation as a pulmonary vasodilator. C16TR showed no activity (>10 µM) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E2 receptor 2, prostaglandin D2 receptor 1, prostaglandin I2 receptor, and prostaglandin E2 receptor 4; TRE potently bound to each of these prostanoid receptors. C16TR had no effect (up to 200 nM) on platelet aggregation induced by ADP in rat blood. In hypoxia-challenged rats, inhaled C16TR-LNP produced dose-dependent (0.06-6 µg/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6 µg/kg) was active at earlier times but lost its effect by 3 hours. Single- and multiple-dose PK studies of inhaled C16TR-LNP in rats showed proportionate dose-dependent increases in TRE Cmax and area under the curve (AUC) for both plasma and lung; similar results were observed for dog plasma levels in single-dose PK studies. In both species, inhaled C16TR-LNP yielded prolonged plasma TRE levels and a lower plasma TRE Cmax compared with inhaled TRE. Inhaled C16TR-LNP was well tolerated in rats and dogs; TRE-related side effects included cough, respiratory tract irritation, and emesis and were seen only after high inhaled doses of C16TR-LNP in dogs. In guinea pigs, inhaled TRE (30 µg/ml) consistently produced cough, but C16TR-LNP (30 µg/ml) elicited no effect. These results demonstrate that C16TR-LNP provides long-acting pulmonary vasodilation, is well tolerated in animal studies, and may necessitate less frequent dosing than inhaled TRE with possibly fewer side effects.

Apelin Compared With Dobutamine Exerts Cardioprotection and Extends Survival in a Rat Model of Endotoxin-Induced Myocardial Dysfunction.

OBJECTIVE: Dobutamine is the currently recommended ß-adrenergic inotropic drug for supporting sepsis-induced myocardial dysfunction when cardiac output index remains low after preload correction. Better and safer therapies are nonetheless mandatory because responsiveness to dobutamine is limited with numerous side effects. Apelin-13 is a powerful inotropic candidate that could be considered as an alternative noncatecholaminergic support in the setting of inflammatory cardiovascular dysfunction. DESIGN: Interventional controlled experimental animal study. SETTING: Tertiary care university-based research institute. SUBJECTS: One hundred ninety-eight adult male rats. INTERVENTIONS: Using a rat model of "systemic inflammation-induced cardiac dysfunction" induced by intraperitoneal lipopolysaccharide injection (10 mg/kg), hemodynamic efficacy, cardioprotection, and biomechanics were assessed under IV osmotic pump infusions of apelin-13 (0.25 µg/kg/min) or dobutamine (7.5 µg/kg/min). MEASUREMENTS AND MAIN RESULTS: In this model and in both in vivo and ex vivo studies, apelin-13 compared with dobutamine provoked distinctive effects on cardiac function: 1) optimized cardiac energy-dependent workload with improved cardiac index and lower vascular resistance, 2) upgraded hearts' apelinergic responsiveness, and 3) consecutive downstream advantages, including increased urine output, enhanced plasma volume, reduced weight loss, and substantially improved overall outcomes. In vitro studies confirmed that these apelin-13-driven processes encompassed a significant and rapid reduction in systemic cytokine release with dampening of myocardial inflammation, injury, and apoptosis and resolution of associated molecular pathways. CONCLUSIONS: In this inflammatory cardiovascular dysfunction, apelin-13 infusion delivers distinct and optimized hemodynamic support (including positive fluid balance), along with cardioprotective effects, modulation of circulatory inflammation and extended survival.

ELABELA Improves Cardio-Renal Outcome in Fatal Experimental Septic Shock.

OBJECTIVES: Apelin-13 was recently proposed as an alternative to the recommended ß-adrenergic drugs for supporting endotoxin-induced myocardial dysfunction. Since Apelin-13 signals through its receptor (Apelin peptide jejunum) to exert singular inotropic/vasotropic actions and to optimize body fluid balance, this candidate pathway might benefit septic shock management. Whether the newly discovered ELABELA (ELA), a second endogenous ligand of the Apelin peptide jejunum receptor highly expressed in the kidney, further improves cardio-renal impairment remains unknown. DESIGN, SETTING, AND SUBJECTS: Interventional study in a rat model of septic shock (128 adult males) to assess the effects of ELA and Apelin-13 on vascular and cardio-renal function. Experiments were performed in a tertiary care University-based research institute. INTERVENTIONS: Polymicrobial sepsis-induced cardiac dysfunction was produced by cecal ligation puncture to assess hemodynamic efficacy, cardioprotection, and biomechanics under acute or continuous infusions of the apelinergic agonists ELA or Apelin-13 (39 and 15 µg/kg/hr, respectively) versus normal saline. MEASUREMENTS AND MAIN RESULTS: Apelinergic agonists improved 72-hour survival after sepsis induction, with ELA providing the best clinical outcome after 24 hours. Apelinergic agonist infusion counteracted cecal ligation puncture-induced myocardial dysfunction by improving left ventricular pressure-volume relationship. ELA-treated cecal ligation puncture rats were the only group to 1) display a significant improvement in left ventricular filling as shown by increased E-wave velocity and left ventricular end-diastolic volume, 2) exhibit a higher plasma volume, and 3) limit kidney injury and free-water clearance. These beneficial renal effects were superior to Apelin-13, likely because full-length ELA enabled a distinctive regulation of pituitary vasopressin release. CONCLUSIONS: Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture-induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.

Apelin-13 administration allows for norepinephrine sparing in a rat model of cecal ligation and puncture-induced septic shock.

BACKGROUND: Infusion of exogenous catecholamines (i.e., norepinephrine [NE] and dobutamine) is a recommended treatment for septic shock with myocardial dysfunction. However, sustained catecholamine infusion is linked to cardiac toxicity and impaired responsiveness. Several pre-clinical and clinical studies have investigated the use of alternative vasopressors in the treatment of septic shock, with limited benefits and generally no effect on mortality. Apelin-13 (APL-13) is an endogenous positive inotrope and vasoactive peptide and has been demonstrated cardioprotective with vasomodulator and sparing life effects in animal models of septic shock. A primary objective of this study was to evaluate the NE-sparing effect of APL-13 infusion in an experimental sepsis-induced hypotension. METHODS: For this goal, sepsis was induced by cecal ligation and puncture (CLP) in male rats and the arterial blood pressure (BP) monitored continuously via a carotid catheter. Monitoring, fluid resuscitation and experimental treatments were performed on conscious animals. Based on pilot assays, normal saline fluid resuscitation (2.5 mL/Kg/h) was initiated 3 h post-CLP and maintained up to the endpoint. Thus, titrated doses of NE, with or without fixed-doses of APL-13 or the apelin receptor antagonist F13A co-infusion were started when 20% decrease of systolic BP (SBP) from baseline was achieved, to restore SBP values ≥ 115 ± 1.5 mmHg (baseline average ± SEM). RESULTS: A reduction in mean NE dose was observed with APL-13 but not F13A co-infusion at pre-determined treatment time of 4.5 ± 0.5 h (17.37 ± 1.74 µg/Kg/h [APL-13] vs. 25.64 ± 2.61 µg/Kg/h [Control NE] vs. 28.60 ± 4.79 µg/Kg/min [F13A], P = 0.0491). A 60% decrease in NE infusion rate over time was observed with APL-13 co-infusion, (p = 0.008 vs NE alone), while F13A co-infusion increased the NE infusion rate over time by 218% (p = 0.003 vs NE + APL-13). Associated improvements in cardiac function are likely mediated by (i) enhanced left ventricular end-diastolic volume (0.18 ± 0.02 mL [Control NE] vs. 0.30 ± 0.03 mL [APL-13], P = 0.0051), stroke volume (0.11 ± 0.01 mL [Control NE] vs. 0.21 ± 0.01 mL [APL-13], P < 0.001) and cardiac output (67.57 ± 8.63 mL/min [Control NE] vs. 112.20 ± 8.53 mL/min [APL-13], P = 0.0036), and (ii) a reduced effective arterial elastance (920.6 ± 81.4 mmHg/mL/min [Control NE] vs. 497.633.44 mmHg/mL/min. [APL-13], P = 0.0002). APL-13 administration was also associated with a decrease in lactate levels compared to animals only receiving NE (7.08 ± 0.40 [Control NE] vs. 4.78 ± 0.60 [APL-13], P < 0.01). CONCLUSION: APL-13 exhibits NE-sparing benefits in the treatment of sepsis-induced shock, potentially reducing deleterious effects of prolonged exogenous catecholamine administration.

Maximizing insights from nonclinical safety studies in the context of rising costs and changing regulations.

The traditional paradigm of non-rodent safety assessment studies, primarily reliant on non-human primates (NHPs) and dogs, is undergoing a transformation. During the 2023 Safety Pharmacology Society Annual Meeting, scientists from leading nonclinical contract organizations discussed how traditional IND-enabling studies can benefit from employing underutilized alternative non-rodent models, such as the swine. Swine offer a cost-effective approach to drug development and share many anatomical and physiological similarities with humans. The inclusion of non-traditional species in safety assessments, coupled with advanced measurement techniques, aids in de-risking compounds early on and adapting projects to the evolving cost landscape.

In vivo and in vitro characterization of a first-in-class novel azole analog that targets pregnane X receptor activation.

The pregnane X receptor (PXR) is a master regulator of xenobiotic clearance and is implicated in deleterious drug interactions (e.g., acetaminophen hepatotoxicity) and cancer drug resistance. However, small-molecule targeting of this receptor has been difficult; to date, directed synthesis of a relatively specific PXR inhibitor has remained elusive. Here we report the development and characterization of a first-in-class novel azole analog [1-(4-(4-(((2R,4S)-2-(2,4-difluorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)ethanone (FLB-12)] that antagonizes the activated state of PXR with limited effects on other related nuclear receptors (i.e., liver X receptor, farnesoid X receptor, estrogen receptor α, peroxisome proliferator-activated receptor γ, and mouse constitutive androstane receptor). We investigated the toxicity and PXR antagonist effect of FLB-12 in vivo. Compared with ketoconazole, a prototypical PXR antagonist, FLB-12 is significantly less toxic to hepatocytes. FLB-12 significantly inhibits the PXR-activated loss of righting reflex to 2,2,2-tribromoethanol (Avertin) in vivo, abrogates PXR-mediated resistance to 7-ethyl-10-hydroxycamptothecin (SN-38) in colon cancer cells in vitro, and attenuates PXR-mediated acetaminophen hepatotoxicity in vivo. Thus, relatively selective targeting of PXR by antagonists is feasible and warrants further investigation. This class of agents is suitable for development as chemical probes of PXR function as well as potential PXR-directed therapeutics.

Evaluation of the effects of RP5063, a novel, multimodal, serotonin receptor modulator, as single-agent therapy and co-administrated with sildenafil, bosentan, and treprostinil in a monocrotaline-induced pulmonary arterial hypertension rat model.

Pulmonary arterial hypertension (PAH), a condition that is defined by pulmonary vasculature constriction and remodeling, involves dysfunctional signaling of the serotonin (5-HT) receptors, 5-HT2A/2B/7. In a rat model of monocrotaline (MCT)-induced PAH, the effectiveness of RP5063 (RP), a dopamine and 5-HT receptor modulator, was evaluated as monotherapy and as an adjunct to standard PAH treatments. After a single 60 mg/kg dose of MCT, rats received vehicle (MCT+Veh; gavage twice-daily [b.i.d.]), RP (10 mg/kg; gavage b.i.d.), bosentan (B; 100 mg/kg; gavage BID), sildenafil (S; 50 mg/kg; gavage, BID), treprostinil (T; 100 ng/kg/min over 24 h intravenous), RP+B, RP+S, and RP+T for 28 days. Single-agent RP limited the functional and structural effects of PAH seen in the MCT+Veh group, with significant improvements in pulmonary hemodynamics, right ventricular (RV) hypertrophy, SO2, and pulmonary blood vessel structural changes. These effects appeared comparable with those associated with B, S, and T. Adjunctive RP treatment resulted in significantly lower mean pulmonary arterial pressures, RV systolic pressure. It also improved SO2 measurements, as compared with MCT+Veh (P < 0.05), and diastolic pulmonary artery pressure (P < 0.05), as compared with single-agent B and S therapy (Bonferroni method adjusting for multiplicity). RP+S appeared to show the most consistent and extensive effects on pulmonary hemodynamics, respiratory parameters, and histopathologic changes. These results corroborate earlier preclinical findings supporting the efficacy of single-agent RP in PAH. RP, as mono and adjunctive therapy compared with induced-control, mitigated the functional and structural effects of MCT-induced PAH.

A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects.

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue 15, Ki 0.15 nM and t1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues 13 and 15, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system.

Inhaled Treprostinil-Prodrug Lipid Nanoparticle Formulations Provide Long-Acting Pulmonary Vasodilation.

Treprostinil (TRE), a prostanoid analogue approved in the USA for the treatment of pulmonary arterial hypertension, requires continuous infusion or multiple dosing sessions per day for inhaled and oral routes of administration due to its short half-life. The inhaled drug is known to induce adverse systemic and local effects including headache, nausea, cough, and throat irritation which may be due at least in part to transiently high drug concentrations in the lungs and plasma immediately following administration [1]. To ameliorate these side effects and reduce dosing frequency we designed an inhaled slow-release TRE formulation. TRE was chemically modified to be an alkyl prodrug (TPD) which was then packaged into a lipid nanoparticle (LNP) carrier. Preclinical screening in a rat model of hypoxia-induced pulmonary vasoconstriction led to selection of a 16-carbon alkyl ester derivative of TRE. The TPD-LNP demonstrated approximately 10-fold lower TRE plasma Cmax compared to inhaled TRE solution while maintaining an extended vasodilatory effect. The favorable PK profile is attributed to gradual dissociation of TPD from the LNP and subsequent conversion to TRE. Together, this sustained presentation of TRE to the lungs and plasma is consistent with a once- or twice-daily dosing schedule in the absence of high Cmax-associated adverse events which could provide patients with an improved treprostinil therapy.

RP5063, a novel, multimodal, serotonin receptor modulator, prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH), a condition characterized by pulmonary vasculature constriction and remodeling, involves dysregulation of the serotonin (5-HT) receptors 5-HT2A and 5-HT2B. A rat model of monocrotaline (MCT)-induced PAH was used to examine the potential beneficial effects of RP5063, a 5-HT receptor modulator. After a single 60mg/kg dose of MCT, rats were gavaged twice-daily (b.i.d.) with vehicle, RP5063 (1, 3, or 10mg/kg), or sildenafil (50mg/kg) for 28 days. RP5063 at a dose as low as 1mg/kg, b.i.d. reduced pulmonary resistance and increased systemic blood oxygen saturation. The highest dose of RP5063 (10mg/kg, b.i.d.) reduced diastolic, systolic, and mean pulmonary pressure, right systolic ventricular pressure, ventilatory pressure, and Fulton's index (ratio of right to left ventricular weight). Doses as low as 3mg/kg RP5063, b.i.d. also increased weight gain and body temperature, suggesting an improvement in overall health of MCT-treated animals. Similar reductions in pulmonary, right ventricular, and ventilatory pressure, pulmonary resistance, and Fulton's index as well as increased systemic blood oxygen saturation were observed in animals treated with the reference agent sildenafil at a higher dose (50mg/kg, b.i.d.). Histological examination revealed that RP5063 produced dose-dependent reductions in pulmonary blood vessel wall thickness and proportion of muscular vessels, similar to sildenafil. RP5063 completely blocked MCT-induced increases in the plasma cytokines TNFα, IL-1ß, and IL-6 at all doses. In summary, RP5063 improved pulmonary vascular pathology and hemodynamics, right ventricular pressure and hypertrophy, systemic oxygen saturation, and overall health of rats treated with MCT.

RP5063, a novel, multimodal, serotonin receptor modulator, prevents Sugen 5416-hypoxia-induced pulmonary arterial hypertension in rats.

RP5063, a multimodal dopamine (DA) and serotonin (5-HT) modulator with high affinity for DA2/3/4 and 5-HT2A/2B/7 receptors and moderate affinity for SERT, is a novel therapeutic of special interest in the treatment of pulmonary arterial hypertension (PAH). Evidence indicates that therapeutics targeting the 5-HT2A/2B receptors can influence the pathogenesis of PAH. However, the therapeutic effect of RP5063 in humans has yet to be investigated. A Sugen 5416-hypoxia (SuHx)-induced PAH model was used to evaluate twice-daily (b.i.d.) RP5063 at 10mg/kg (RP-10) and 20mg/kg (RP-20), as compared with positive (sildenafil 50mg/kg b.i.d.; Sil-50) and negative controls (SuHx+vehicle; SuHx+veh), in 24 adult male Wistar-Kyoto rats. RP5063 showed significantly lower systolic pulmonary arterial (both doses) and systolic right ventricular (RP-10) pressures, and improvement in oxygen saturation (RP-20). It significantly reduced small-vessel wall thickness (RP-20), lowered the percentage of muscular vessels (both doses). Both doses limited arterial obliteration due to endothelial cell proliferation, prevented plexiform lesion formation, and stemmed the release of leukotriene B4. Sildenafil showed statistically greater effects on vessel structure than that seen in both RP5063 groups and improved oxygen saturation. Additionally, Sildenafil did not demonstrate any significant effect on arterial obliteration, plexiform lesion development, or pulmonary arterial or right ventricular pressure. As PAH gains in severity, the impact of RP5063 inhibition of 5HT2B increases, preventing arterial constriction and improving pulmonary hemodynamics. Due to its functional, structural, and chemokine effects, RP5063 represents a promising candidate for investigation in late-phase PAH.

Therapeutic Antibody-Induced Vascular Toxicity Due to Off-Target Activation of Nitric Oxide in Cynomolgus Monkeys.

PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension, and hemorrhage.

Discovery and Structure-Activity Relationship of a Bioactive Fragment of ELABELA that Modulates Vascular and Cardiac Functions.

ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELA's structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and ß-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases.

Liposomes ameliorate Crizotinib- and Nilotinib-induced inhibition of the cardiac IKr channel and QTc prolongation.

Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinase inhibitors approved for the treatment of non-small cell lung cancer and chronic myeloid leukemia, respectively. Both have been shown to result in electrocardiogram rate-corrected Q-wave T-wave interval (QTc) prolongation in humans and animals. Liposomes have been shown to ameliorate drug-induced effects on the cardiac-delayed rectifier K(+) current (IKr, KV11.1), coded by the human ether-a-go-go-related gene (hERG). This study was undertaken to determine if liposomes would also decrease the effect of crizotinib and nilotinib on the IKr channel. Crizotinib and nilotinib were tested in an in vitro IKr assay using human embryonic kidney (HEK) 293 cells stably transfected with the hERG. Dose-responses were determined and the 50% inhibitory concentrations (IC50s) were calculated. When the HEK 293 cells were treated with crizotinib or nilotinib that were mixed with liposomes, there was a significant decrease in the IKr channel inhibitory effects of these two drugs. When isolated, rabbit hearts were exposed to crizotinib or nilotinib, there were significant increases in QTc prolongation. Mixing either of the drugs with liposomes ameliorated the effects of the drugs. Rabbits dosed intravenously (IV) with crizotinib or nilotinib showed QTc prolongation. When liposomes were injected prior to crizotinib or nilotinib, the liposomes decreased the effects on the QTc interval. The use of liposomal encapsulated QT-prolongation agents, or giving liposomes in combination with drugs, may decrease their cardiac liability.

Functional reconstitution of an eicosanoid-modulated Cl- channel from bovine tracheal smooth muscle.

We describe the biochemical properties of an eicosanoid-modulated Cl- channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability (P(o)). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl- channel is a 55-kDa glycosylated monomer with voltage- and Ca(2+) concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 microM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P(o) of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 microM but failed to alter the P(o) of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET- anions with the channel pore, whereas the alteration of P(o) requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.