Robert Letienne

Protease-Activated Receptor-1 Antagonist F 16618 Reduces Arterial Restenosis by Down-Regulation of Tumor Necrosis Factor α and Matrix Metalloproteinase 7 Expression, Migration, and Proliferation of Vascular Smooth Muscle Cells

Wound healing after angioplasty or stenting is associated with increased production of thrombin and the activation of protease-activated receptor 1 (PAR1). The aim of the present study was to examine the effects of a new selective PAR1 antagonist, 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)-penta-1,3-dienyl]-benzonitrile (F 16618), in restenosis and vascular smooth muscle cell (SMC) proliferation and migration using both in vivo and in vitro approaches. Daily oral administration of F 16618 inhibited the restenosis induced by balloon angioplasty on rat carotid artery in a dose-dependent manner. Furthermore, single intravenous administration of F 16618 during the angioplasty procedure was sufficient to protect the carotid artery against restenosis. In vitro, F 16618 inhibited the growth of human aortic SMCs in a concentration-dependent manner with maximal effects at 10 μM. At that concentration, F 16618 also prevented thrombin-mediated SMC migration. In vivo, oral and intravenous F 16618 treatments reduced by 30 and 50% the expression of the inflammatory cytokine tumor necrosis factor α (TNFα) 24 h after angioplasty. However, only acute intravenous administration prevented the induction of matrix metalloproteinase 7 expression. In contrast, F 16618 treatments had no effect on early SMC de-differentiation and transcription of monocyte chemoattractant protein-1 and interleukin-6 and late re-endothelialization of injured arteries. Furthermore, F 16618 compensated for the carotid endothelium loss by inhibiting PAR1-mediated contraction. Altogether, these data demonstrate that PAR1 antagonists such as F 16618 are a highly effective treatment of restenosis after vascular injury, by inhibition of TNFα, matrix metalloproteinase 7, and SMC migration and proliferation in addition to an antithrombotic effect.

Sodium late current blockers in ischemia reperfusion: is the bullet magic?

We describe the discovery of the first selective, potent, and voltage-dependent inhibitor of the late current mediated by the cardiac sodium channel Na V1.5. The compound 3,4-dihydro- N-[(2 S)-3-[(2-methoxyphenyl)thio]-2-methylpropyl]-2 H-(3 R)-1,5-benzoxathiepin-3-amine, 2a (F 15845), was identified from a novel family of 3-amino-1,5-benzoxathiepine derivatives. The late sodium current inhibition and antiischemic effects of 2a were studied in various models in vitro and in vivo. In a rabbit model of ischemia-reperfusion, 2a exhibited more potent antiischemic effects than reference compounds KC 12291, ranolazine, and ivabradine. Thus, after a single administration, 2a almost abolished ST segment elevation in response to a transient coronary occlusion. Further, the antiischemic activity of 2a is maintained over a wide range of doses and is not associated with any hemodynamic changes, contrary to conventional antiischemic agents. The unique pharmacological profile of 2a opens new and promising opportunities for the treatment of ischemic heart diseases.

Short-term haemodynamic variability in the conscious areflexic rat

1 Similtaneous measurements of arterial pressure and cardiac output (n= 8), mesenteric blood flow (n= 7) or hindquarters (n= 8) blood flow were performed during 1 h periods in conscious rats, before and after acute pharmacological blockade of the autonomic, renin‐angiotensin and vasopressin systems. In the latter condition (areflexic state), arterial pressure was maintained with a continuous infusion of noradrenaline. 2 In the areflexic state, spontaneous fluctuations in arterial pressure were markedly exaggerated, especially depressor episodes. At the onset of these falls in arterial pressure, there was an abrupt and transient decrease in stroke volume and cardiac output. Systemic vasodilatation then developed while cardiac output returned to normal. Regional vasodilatations were also delayed from the onset of the falls in arterial pressure and were usually large enough to maintain blood flow. 3 Both time and frequency domain analyses confirmed that changes in systemic and regional vascular conductances lagged by about 1 s behind arterial pressure changes. 4 These results indicate that, in the absence of neurohumoral influences, autoregulatory‐like mechanisms become dominant in the control of systemic and regional circulations and contribute to exaggeration of the spontaneous short‐term variability of arterial pressure.

Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

Myocardial protection by F 15845, a persistent sodium current blocker, in an ischemia-reperfusion model in the pig.

The specific persistent sodium current blocker F 15845 was tested in two myocardial ischemia-reperfusion models in the pig in order to evaluate its cardioprotective effects. In the first protocol, the left circumflex coronary artery was ligated for 60-min and then reperfused for 48-h. F 15845 (2.5+2.5 and 5+5mg/kg) was administered by i.v. infusion, starting before ischemia to the beginning of reperfusion. The second protocol attempted to evaluate F 15845 (5+5mg/kg) response in a more pathological state of the heart. To this end, a non necrotic ligation of the left circumflex coronary artery was applied for 15 min one week before the actual 60 min occlusion. For both protocols, infarct size was determined at the end of the reperfusion period and was assessed by histochemistry (tetrazolium staining). Plasma levels of biochemical markers (myoglobin and troponin I) were also evaluated. In protocol 1, F 15845 significantly reduced the infarct size by 27+/-3 and 43+/-5% at 2.5+2.5 and 5+5mg/kg, respectively. At 5+5mg/kg, F 15845 decreased plasma levels of myoglobin and cardiac troponin I. In protocol 2, F 15845 (5+5mg/kg) significantly reduced myocardial infarct size by 54+/-15% and lowered the plasma myoglobin and troponin I levels relative to vehicle-treated animals. In conclusion, the highly effective persistent sodium current blocker F 15845 exerts remarkable cardioprotective activities. It reduces both myocardial infarct size and the release of biochemical markers in healthy pigs as well in pigs previously exposed to an ischemic episode.

Antithrombotic activity of F 16618, a new PAR1 antagonist evaluated in extracorporeal arterio-venous shunt in the rat

The purpose of the present work was the evaluation of the antithrombotic activity of a new PAR1 antagonist, F 16618 in arterio-venous shunt in the rat. Arterial thrombosis was induced by insertion of a silk thread (thrombogenic substrate) into an extracorporeal shunt. F 16618 was administered either by intravenous route (0.63-2.5mg/kg) or by oral route (20-80mg/kg). Oral activity of F 16618 was compared to that of aspirin (20-80mg/kg) and clopidogrel (0.63-10mg/kg). Finally, F 16618 was associated to aspirin and/or clopidogrel to test for possible antithrombotic activity and its effects on bleeding time. SFLLR-induced human platelet aggregation was evaluated in the presence of F 16618, demonstrating the anti-aggregant activity of this compound. F 16618 (1.25mg/kg) significantly delayed the time leading to occlusion by 52+/-17%, without affecting bleeding time and in absence of hemodynamic effects. F 16618 given orally dose-dependently increased the time to occlusion. The maximal effect was observed at 40mg/kg (984+/-95s versus 644+/-17s in vehicle group). Aspirin and clopidogrel also dose-dependently lengthened time to occlusion, but this effect was associated with an increase of bleeding time. F 16618 (20mg/kg) orally associated with either aspirin (40mg/kg) or with clopidogrel (1.25mg/kg) potentiated the antithrombotic effects of both compounds without further increasing of bleeding time. In conclusion, F 16618 exerted a potent antithrombotic activity by intravenous and oral routes, without affecting bleeding time. Furthermore, the antithrombotic activity was potentiated when combined with aspirin or clopidogrel.

Na+ Currents in Cardioprotection: Better to Be Late

We report the discovery of a selective, potent inhibitor of the late current mediated by the cardiac isoform of the sodium channel (Na(V)1.5). The compound, 3,4-dihydro-N-[(2S)-3-[(2-hydroxy-3-methylphenyl)thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2d) (F 15741), blocks the late component of the Na(+) currents and greatly reduces veratridine- or ischemia-induced contracture in isolated tissue and whole heart. The cardioprotective action of 2d was further established in a model of myocardial infarction in the pig in which 2d prevents ischemia-reperfusion damage after 60 min of coronary occlusion and 48 h reperfusion. Under these experimental conditions, only 2d and cariporide reduce infarct size. Remarkably, myocardial protection afforded by 2d occurs in the absence of hemodynamic effects. These data expand the therapeutic potential of late I(Na) blockers and suggest that 2d could be useful in pathologies for which pharmacological treatments are not yet available.

Protease-activated receptor 1 antagonists prevent platelet aggregation and adhesion without affecting thrombin time.

The aim of this study was to investigate the in vitro antithrombotic effects of two PAR1 antagonists, ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation and on the thrombin time in human and guinea-pig platelets. ER121958 inhibited SFLLR-induced guinea-pig and human platelet adhesion with the IC(50) values of 1.73nM and 2.91nM, respectively and SFLLR-induced guinea-pig and human platelet aggregation with the IC(50) values of 2.74nM and 11.9nM, respectively. Similarly, SCH203099 exhibited a non competitive profile of inhibition on both SFLLR-induced guinea-pig and human platelet adhesion with the IC(50) values of 93nM and 127nM, respectively or SFLLR-induced guinea-pig and human platelet aggregation with the IC(50) values of 1.74microM and 2.36microM, respectively. These two antagonists failed to prolong the thrombin time. Altogether, these results highlighted the potent anti-platelets properties of both ER121958 and SCH203099 in an in vitro model of aggregation as well as in a static model of adhesion without any effect on the last step of coagulation cascade. Moreover, this work emphasized that guinea-pig is a suitable animal model to study the role of PAR1 antagonists since the magnitude of the effects of ER121958 and SCH203099 on both SFLLR-induced platelet adhesion and aggregation were similar in both species.

3-(R)-[3-(2-Methoxyphenylthio-2-(S)-methylpropyl]amino-3,4-dihydro-2H-1,5-benzoxathiepine Bromhydrate (F 15845) Prevents Ischemia-Induced Heart Remodeling by Reduction of the Intracellular Na+ Overload

The present study investigates whether 3-(R)-[3-(2-methoxyphenylthio-2-(S)-methylpropyl]amino-3,4-dihydro-2H-1,5-benzoxathiepine bromhydrate (F 15845), a new, persistent sodium current blocker, can reduce the ischemic Na+ accumulation and exert short- and long-term cardioprotection after myocardial infarction. First, F 15845 concentration-dependently reduced veratrine-induced diastolic contracture (IC50 = 0.14 μM) in isolated atria. Second, F 15845 from 1 μM preserved viability in 54.2 ± 12.5% of isolated cardiomyocytes exposed to lysophosphatidylcholine. Third, the effect of F 15845 on intracellular Na+ of isolated hearts from control and diabetic db/db mice was monitored using 23Na-nuclear magnetic resonance spectroscopy. F 15845 (0.3 μM) significantly counteracted [Na+]i increase during no-flow ischemia in control mouse hearts. In diabetic db/db mouse hearts, the reduction in [Na+]i was delayed relative to control. However, it was more marked and maintained upon reperfusion. The cardioprotective properties after myocardial infarction associated with short- (24-h) and long-term (14-day) reperfusion were measured in anesthetized rats. After 24-h reperfusion, F 15845 (5 mg/kg) significantly reduced infarct size (32.4 ± 1.7% with vehicle and 24.2 ± 3.4% with F 15845; P < 0.05) and decrease of troponin I levels (524 ± 93 μg/l with vehicle versus 271 ± 63 μg/l with F 15845; P < 0.05). It is important that F 15845 limits the long-term expansion of infarct size (35.2 ± 2.6%, n = 19 versus 46.7 ± 1.6%, n = 27 in the vehicle group; P < 0.001). Overall, F 15845 attenuates [Na+]i and prevents (or reverses) contractile and biochemical dysfunction in ischemic and remodeling heart. F 15845 constitutes a new generation of cardioprotective agent.

Soman‐induced hypertension in conscious rats is mediated by prolonged central muscarinic stimulation

Abstract— The acetylcholinesterase inhibitor, soman, induces marked and sustained hypertension and tachycardia associated with a convulsive syndrome in rats. The aims of the present study were to distinguish between the cardiovascular and convulsant effects of soman and to determine whether the maintenance of the soman‐induced hypertension and tachycardia depends solely on a central muscarinic effect. To this end, using a computerised analysis of blood pressure (BP) in conscious freely moving rats, we examined the consequences on the increase in mean BP (MBP) and heart rate (HR) induced by soman (60 μg/kg, i.v.) of 1) a pre‐treatment with the anticonvulsant drug diazepam (3 mg/kg, i.v.) and 2) atropine sulphate (10 mg/kg, i.v.) administered 10 or 60 min after the intoxication. Pretreatment with diazepam prevented the convulsions, assessed by electroencephalogram (EEG) recording, but modified neither the magnitude nor the kinetics of the pressor and tachycardic effects of soman (Δ MBP = 74 ± 2 and 73 ± 5 mmHg, Δ HR = 69 ± 10 and 79 ± 7 bpm, maximum MBP = 186 ± 3 and 182 ± 6 mmHg, maximum HR = 545 ± 9 and 522 ± 16 bpm in solvent‐ (n = 8) and diazepam‐ (n = 8) pre‐treated rats, respectively). Whatever its time of administration, atropine sulphate fully and immediately reversed the rise in BP induced by soman. The soman‐induced tachycardia was also suppressed by atropine administered 10 min after soman whereas it persisted when atropine was injected 60 min after the intoxication. These results show that the cardiovascular effects of soman can occur independently of the convulsive syndrome and that the maintenance of the soman‐induced hypertension depends entirely on a permanent central muscarinic stimulation.