Philippe Mennecier

Effect of vasodilators, including nitric oxide, on the release of cGMP and cAMP in the isolated perfused rat kidney

In isolated Tyrode-perfused rat kidneys, the release of the cyclic nucleotides cAMP and cGMP was measured in response to several vasodilators, including nitric oxide (NO). During vasoconstrictions induced by methoxamine, a basal release of both cyclic nucleotides was detected in the renal effluent (357 +/- 32 fmol/min for cGMP and 3097 +/- 219 fmol/min for cAMP). Injection of acetylcholine (ACh; 11 nmol), sodium nitroprusside (SNP; 0.8 nmol) and atrial natriuretic factor (ANF; 80 pmol) caused a marked release of cGMP. The cGMP release induced by ACh was not altered by indomethacin (3 microM) but was markedly reduced by the NO synthase inhibitor nitro-L-arginine (L-NNA; 200 microM). Authentic NO (0.16-80 nmol) caused dose-dependent vasodilatations that were accompanied by increases in the overflow of cGMP. The vasodilatations caused by forskolin (6 nmol) and prostacyclin (PGI2; 3-52 nmol) were not accompanied by an overflow of cGMP. The vasodilator responses to 5-hydroxytryptamine (5-HT; 0.25-2 mumol), obtained in presence of the 5-HT2 receptor blocker ritanserin (10 nM) and the 5-HT3 blocker ICS 205930 (10 nM), were markedly reduced by L-NNA; however, they were not accompanied by the renal release of cGMP. Both forskolin and PGI2 induced the release of cAMP from perfused rat kidneys; ACh, 5-HT and 5-carboxamidotryptamine (5-CT) also evoked a significant release of cAMP into the renal effluent. The release of cAMP induced by ACh and 5-HT was reduced by indomethacin and L-NNA. Higher doses of NO released cAMP from the perfused rat kidneys. Our data illustrate that both cAMP and cGMP can be released by vasodilator substances into the venous effluent of isolated perfused rat kidneys. The dilator responses to 5-HT were sensitive to the NO synthase inhibitor L-NNA and were accompanied by the release of cAMP and not by the release of cGMP. Our data suggest that the dilator responses may be due to NO released from endothelial cells, which then activates adenylyl cyclase either directly or indirectly.

5-hydroxytryptamine-induced vasodilatation in the isolated perfused rat kidney : are endothelial 5-HT1A receptors involved ?

Left kidneys obtained from male Wistar rats were perfused with Tyrode solution; the perfusion pressure was measured continuously and taken as an index of vascular resistance in the kidneys. 5-Hydroxytryptamine (5-HT; 3-50 nmol) caused dose-dependent dilator responses in kidneys preconstricted with noradrenaline (0.6 microM) and pretreated with ritanserin (10 nM) and ICS 205930 (10 nM). The 5-HT1 agonist 5-carboxamidotryptamine (5-CT; 16-64 nmol) also caused renal dilatations under similar conditions. The dilator responses to both 5-HT and 5-CT were antagonized by the non-selective 5-HT receptor antagonist metergoline (0.2 microM) and by the selective 5-HT1A receptor antagonist BMY 7378 (0.4 microM). The guanylate cyclase inhibitor methylene blue (30 microM) and the nitric oxide (NO) synthase inhibitor nitro-L-arginine (L-NNA; 100 microM) significantly attenuated the dilator responses to 5-HT and 5-CT. The 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.5-16 nmol) also caused dose-dependent dilator responses in preconstricted rat kidneys. These responses were antagonized by metergoline and BMY 7378 and significantly attenuated by the NO inhibitors hemoglobin (10 microM) and L-NNA. The renal dilator responses noted with the beta-adrenoceptor blocker tertatolol (1-32 nmol) were also antagonized by metergoline and BMY 7378 and significantly reduced by L-NNA and hemoglobin. Both 8-OH-DPAT and tertatolol (1-30 microM) significantly reduced the vasoconstrictor responses to angiotensin II (20 pmol). Our data indicate that 5-HT receptors located on the vascular endothelium of the renal circulation are involved in the dilator actions of 5-HT, 5-CT, 8-OH-DPAT and tertatolol, and suggest that these receptors resemble the 5-HT1A subtype.

S35225 is a direct inhibitor of Plasminogen Activator Inhibitor type-1 activity in the blood.

The increased risk of thrombotic events associated with disease states such as diabetes and hypertension has been correlated with elevated circulating levels of Plasminogen Activator Inhibitor type-1 (PAI-1). In the present study we evaluate the benzothiophene derivative S35225 in comparison with two recently described inhibitors of PAI-1 activity Tiplaxtinin and WAY140312 on a panel of PAI-1 activity assays in vitro and in vivo. In a direct chromogenic assay, S35225 has an IC50 value of 44+/-0.9 microM similar to that of Tiplaxtinin (34+/-7 microM) and of WAY140312 (39+/-1 microM). In a clot lysis assay however, S35225 has a significantly lower IC50 value than Tiplaxtinin and WAY140312 (0.6+/-0.3 versus 22+/-5 and 16+/-2 microM respectively). Using a tPA capture assay to quantify active PAI-1 in rat or human plasma, neither WAY140312, nor Tiplaxtinin attained 50% inhibition of PAI-1 activity at the highest concentration tested (1 mM); S35225 has an IC50 value of 194+/-30 microM against active rat PAI-1 and 260+/-41 microM against active human PAI-1. The ability of the compounds to inhibit endogenous active PAI-1 in the rat following intravenous administration was also tested using the tPA capture assay. Only S35225 reduced circulating active PAI-1 levels in vivo (maximum inhibition of 76+/-5% at 10 mg/kg and 53+/-5% at 3 mg/kg). In contrast to Tiplaxtinin and WAY140312, S35225 is a direct inhibitor of PAI-1 activity in vitro in rat and human plasmas where vitronectin is constitutively present as well as in vivo in the blood after an intravenous administration in the rat.

Design, synthesis, and biological evaluation of 1,5-benzothiazepine-4-one derivatives targeting factor VIIa/tissue factor

The 1,5-benzothiazepine-4-one scaffold was earlier shown to provide efficient protease inhibitors. In this contribution, we describe its use in the design of factor VIIa/tissue factor inhibitors. A series containing a scaffold non-substituted on its aryl part led to compound 20 with an IC(50) of 2.16 microM. Following molecular modelling studies of this compound, a second series was prepared, which necessitated the synthesis of protected 7- or 8-substituted 1,5-benzothiazepine-4-one derivatives.

Phosphoramidon inhibits the conversion of big ET-1 into ET-1 in the pithed rat and in isolated perfused rat kidneys

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that could be implicated in acute renal failure. The aim of this study was to test the effects of the endothelin-converting enzyme (ECE) inhibitor phosphoramidon on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In Tyrode-perfused rat kidneys, both big ET-1 (0.2-0.4 nmol) and ET-1 (0.01-0.03 nmol) evoked dose-dependent constrictions. Phosphoramidon (10 microM) selectively inhibited the pressor responses to big ET-1 without altering those to ET-1, norepinephrine, angiotensin I (AT-I), or angiotensin II (AT-II). The metalloprotease inhibitor thiorphan, but not the angiotensin-converting enzyme (ACE) inhibitor perindoprilate, also selectively inhibited the renal constrictions caused by big ET-1 but not those induced by ET-1. In vivo, both big ET-1 and ET-1 (0.5-2 nmol/kg) evoked pressor responses that were augmented by indomethacin (15 mg/kg) and L-NNA (1 mg/kg/min). Phosphoramidon selectively inhibited the pressor responses to big ET-1 (ID50: 78 micrograms/kg/min) without affecting those to ET-1, AT-I, or AT-II. These data illustrate that the pressor responses to big ET-1 in the rat, both in vivo and in vitro, are due to its conversion into ET-1 by a phosphoramidon-sensitive ECE. In the rat, phosphoramidon selectively inhibits ECE but not ACE both in vitro and in vivo.

S 17162 is a novel selective inhibitor of big ET-1 responses in the rat

Summary: Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that may be implicated in acute renal failure. The aim of the present study was to test the effects of the novel endothelin-converting enzyme inhibitor S 17162 (N-(2,3 dihydroxy propyl phosphonyl)-(S)-Leu-(S)-Trp-OH, disodium salt) on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In both models, phosphoramidon selectively inhibited the pressor responses to big ET-1 without influencing those to ET-1, angiotensins (AT-I and AT-II) or norepinephrine. S 17162 was active against big ET-1 in both test systems. It selectively inhibited the pressor responses to big ET-1 with ID50 values of 160 μg/kg/min (phosphoramidon: 120 μg/kg/min) in the spinal rat and 6 μM (phosphoramidon: 5μ,M) in the perfused rat kidney. In the nonanesthetized rat, S 17162 at 20 mg/kg p.o. inhibited the pressor responses to big ET-1, demonstrating its oral bioavailability. Therefore, S 17162 is a potential candidate for development as an orally active anti-endothelin drug.

A SCREENING PROCEDURE TO EVALUATE THE ANTICOAGULANT ACTIVITY AND THE KINETIC BEHAVIOUR OF DIRECT THROMBIN INHIBITORS.

The development of a fibrin clot microassay to define both the kinetic behaviour and the anticoagulant activity of direct thrombin inhibitors targeting various domains of thrombin (catalytic site, anion binding exosite or both) is described. Since classical kinetics studies are difficult to perform in a fibrin-clot assay, methodological conditions were selected in order to obtain a linear relationship between fibrin formation and the thrombin concentration i.e. 0.67 nM thrombin, 6 microM fibrinogen, 5 minutes reaction. Under those conditions, the concentration of the complex thrombin-inhibitor can easily be calculated from a standard curve performed with increasing concentrations of thrombin and fitted versus the total inhibitor concentration using adapted equations. To detect the slow establishment of the thrombin inhibition, results obtained with a protocol in which the inhibitor is pre-incubated with thrombin before the addition of fibrinogen is compared to a protocol in which the inhibitor is pre-incubated with fibrinogen before thrombin is added. Our assay which is validated using different types of thrombin inhibitors (classical competitive: NAPAP and hirudin 55-65; tight binding: r-hirudin; slow tight binding: DUP-714), provides a rapid screening protocol allowing to evaluate the biochemical and anticoagulant properties of any direct thrombin inhibitor.

S35972, a direct‐acting thrombin inhibitor with high oral bioavailability and antithrombotic efficacy

Summary.  Objectives: Dabigatran etexilate is the first oral thrombin inhibitor to demonstrate superior efficacy to warfarin for stroke prevention in patients with atrial fibrillation. This study describes the in vitro, ex vivo anticoagulant and in vivo antithrombotic effects of an oral thrombin inhibitor, S35972, in comparison with dabigatran etexilate. Methods: Enzyme assays with thrombin and related serine proteases were performed. Clotting times, including activated partial thromboplastin time (APTT) and thrombin time (TT), were measured in vitro in different species and ex vivo in dogs and rats to determine pharmacologic bioavailabilities. The formation of occlusive venous and arterial thrombi in the rat vena cava and aorta was induced with stasis plus thromboplastin or ferrous chloride, respectively. Results: S35972 inhibited human thrombin with an IC50 of 3.7 nm, and did not inhibit other serine proteases. The anticoagulant activities of S35972 in vitro were comparable in dog and human plasmas, and the sensitivity of the clotting times to S35972 was TT > APTT > prothrombin time. In the fasted dog, oral administration of 3 mg kg−1 S35972 increased TT rapidly and for at least 8 h, and its pharmacologic bioavailability was 75.4% ± 0.1%. In the rat venous thrombosis model, 3 mg kg−1 oral S35972 or dabigatran etexilate significantly decreased the thrombus weight. In the rat aortic thrombosis model, oral S35972 at 10 mg kg−1 significantly decreased thrombus weight, by approximately 50%, whereas, at this dose, no effect was obtained with dabigatran etexilate. Conclusions: S35972 is a non‐prodrug thrombin inhibitor with high selectivity, oral bioavailability, and antithrombotic efficacy.