Yoshimi Hirasawa

Spontaneous nitric oxide release accounts for the potent pharmacological actions of FK409

(+-)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409), which was isolated from microbial products, has been reported to show a vasorelaxant effect through a mechanism similar to that of the organic nitrates such as isosorbide dinitrate. In solution at pH 7.4, FK409 decomposed and released nitric oxide (NO) spontaneously, while isosorbide dinitrate did not. In in vitro biological tests, FK409 inhibited norepinephrine-induced contraction in rat isolated aorta more potently than did isosorbide dinitrate (ED50 = 1.0 and 310 nM, respectively) and ADP-induced human platelet aggregation (IC50 = 0.75 and > 100 microM, respectively). Nitrite/nitrate was recovered in urine accumulated for 24 h after collection from rats given FK409 or isosorbide dinitrate (10 mg/kg p.o.). FK409 (10 mg/kg p.o.) increased the plasma cyclic GMP level and at the same time decreased the mean blood pressure in conscious rats, while isosorbide dinitrate (10 mg/kg p.o.) did not change these parameters significantly. These results suggest that FK409 produces these pharmacological actions via spontaneously released NO, unlike isosorbide dinitrate, and has a possibility of becoming a unique orally active drug for cardiovascular diseases as a new NO donor.

In vitro antiplatelet profile of FR171113, a novel non-peptide thrombin receptor antagonist.

Synthetic peptides (5 to 14 amino acids), identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, act as thrombin receptor agonist peptides. Whilst thrombin receptor antagonist peptides are known, non-peptide thrombin receptor antagonists have yet to be described. In the present study, we compared the antiplatelet effects of 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one (FR171113), a novel non-peptide thrombin receptor antagonist, with the known thrombin receptor antagonist 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-OH (C186-65), and argatroban, a specific protease inhibitor of thrombin. FR171113 and C186-65 inhibited thrombin-induced platelet aggregation (IC(50)=0.29 microM and 15 microM, respectively) and Ser-Phe-Leu-Leu-Arg-Asn-NH(2) [a synthetic thrombin receptor agonist peptide (TRAP-6)] induced platelet aggregation (0.15 microM and 20 microM, respectively) in human washed platelets. Argatroban potently inhibited thrombin-induced platelet aggregation (IC(50)=3.5 nM), but did not inhibit TRAP-6-induced aggregation even at 100 microM. In contrast, these compounds did not show inhibitory effects on ADP- and collagen-induced aggregation in human platelet-rich plasma even at 100 microM. FR171113 caused a parallel shift to the right of the concentration-response curve describing aggregation induced by TRAP-6. The Schild plot of the data had a slope of -0.840 (r=0.98) and the pA(2) was 7.29. In protease activity studies using a chromogenic substrate, argatroban inhibited thrombin protease activity in a dose-dependent manner, whereas FR171113 and C186-65 were inactive, even at 100 microM. Additionally, only argatroban displayed dose-dependent prolongation of thrombin time, activated partial thromboplastin time and prothrombin time. FR171113 and C186-65 showed no effects, even at a concentration of 100 microM. These results suggest that FR171113 has a similar mode of action to C186-65, but with more potent antiplatelet activity. In conclusion, FR171113 is suggested to be the first example of a non-peptide thrombin receptor antagonist.

New reagents for controlled release of nitric oxide. Structure-stability relationships

Abstract The synthesis and structure-stability relationships of a series of novel FK409 derivatives ( 1 , 2 , and 3 ) are described. The rates of decomposition in aqueous solution (pH 8.0, 30 °C) were parallel with those of spontaneous NO release measured by ESR spectroscopy using carboxy-PTIO. The compounds can cover a wide range of NO releasing rates by appropriate modification of the molecule.

A New Nitric Oxide (No) Releaser: Spontaneous No Release from Fk409

The remarkable vasorelaxant and anti-platelet effects of FK409 have been reported to be due to nitric oxide (NO) release. The purpose of the present study is to investigate the spontaneous NO-releasing pathway of FK409 in aqueous solutions. 1H-NMR spectra of FK409 suggested that the compound underwent a time-dependent elimination of the hydrogen atom at alpha-position of the nitro moiety (at the 5-position) in weakly alkaline solutions. In addition, the degradation of FK409 monitored by HPLC showed a pH-dependency accelerating with an increase of pH. These results revealed that the first step in the degradation of FK409 might be the hydroxyl ion-dependent subtraction of the hydrogen atom at the 5-position. On the other hand, NO release from FK409 also exhibited a pH-dependency, and the velocity of NO liberation was markedly enhanced above pH 6. Furthermore, a linear relationship between the rate of FK409 degradation and that of NO formation was observed, indicating that the rate-limiting step for NO formation is the same as that for degradation. Thus, the rate-limiting process of NO formation from FK409 is due to the deprotonation reaction of the hydrogen atom at the 5-position by hydroxyl ions. The deprotonation process appears to be an essential step for both FK409 degradation and NO release. On the basis of the results, a possible kinetic scheme for NO release from FK409 is proposed.

Antianginal effects of FK409, a new spontaneous NO releaser

1 The aim of this study was to compare antianginal effects of (±)‐(E)‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexeneamide (FK409), a new spontaneous nitric oxide releaser, with those of isosorbide dinitrate (ISDN). We used two types of rat angina model; methacholine‐ and arginine vasopressin (AVP)‐induced coronary vasospasm models. 2 In the in vitro study, FK409 showed 80 times more potent vasorelaxant effect in dog isolated coronary artery than ISDN (EC50= 16.7 ± 4.8 and 1340 ± 320 nm, respectively). 3 In the rat methacholine‐induced coronary vasospasm model, FK409 suppressed the elevation of ST segment dose‐dependently and significantly at 0.1 mg kg−1, i.d. On the other hand, ISDN suppressed it significantly at 3.2 mg kg−1, i.d. In addition, the efficacy of 3.2 mg kg−1 ISDN in the model was almost the same as that of 0.1 mg kg−1 FK409. 4 In the above experiments, FK409 and ISDN decreased mean blood pressure significantly at the maximum dose tested (1.0 mg kg−1, i.d. and 3.2 mg kg−1, i.d., respectively) but did not change heart rate at these doses. Therefore, the hypotensive effect of FK409 was 10 times weaker than the antianginal effect of the compound, while those of ISDN were almost the same. 5 In the rat AVP‐induced coronary vasospasm model, 32mg kg−1 FK409 significantly inhibited the depression of ST segment 60 min after oral administration. On the other hand, 32 mg kg−1 ISDN did not inhibit it at 60 and 120 min after oral administration. 6 In conclusion, FK409 inhibits coronary vasospasm more potently in two types of rat angina models than ISDN. In addition, FK409 shows an antianginal effect more selectively that a hypotensive effect, compared with ISDN.

FR144420, a novel, slow nitric oxide-releasing agent

We report that (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409) decomposes and releases nitric oxide (NO) spontaneously in solution. (+/-)-N-[(E)-4-Ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen-1- yl]-3- pyridinecarboxamide (FR144420) was synthesized with the aim of discovering a compound with longer duration of effects in vivo, compared with FK409. FR144420, like FK409, released NO spontaneously in solution, but the amount of NO released from FR144420 during a 5-min incubation was half the amount from FK409. In addition, FR144420 spontaneously decomposed and generated nitrite, which is an oxidative metabolite of NO, at half the rate of FK409. In a vasorelaxant study with isolated rat aorta, FR144420 had a weaker potency than FK409 (EC50 = 54 and 8.1 nM, respectively). In in vivo studies, FR144420 decreased mean blood pressure immediately after intravenous and oral administration to conscious rats. The maximum hypotensive effects of FR144420 were less than those of FK409. However, the durations of FR144420-induced (i.v. and p.o.) hypotensive effects were longer than those of FK409-induced effects. In conclusion, FR144420 is more stable and releases NO more slowly in solution than does FK409. In in vivo experiments, FR144420 showed a longer duration of effects than FK409. FR144420 may be very useful for investigating the in vivo actions of NO.

Close correlation of the cardioprotective effect of FK409, a spontaneous NO releaser, with an increase in plasma cyclic GMP level

FK409 ((±)‐(E)‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexeneamide), which has been reported by us to be a new spontaneous nitric oxide (NO) releaser, prevented myocardial infarction following occlusion and reperfusion in rat coronary artery and increased plasma cyclic GMP level in rats, dose‐dependently and significantly at 32 mg kg−1. Isosorbide dinitrate (ISDN), which is the most popular orally active NO donor used in the treatment of ischaemic cardiovascular diseases, did not show significant effects at 32 mg kg−1 in either experiment. Therefore, it is suggested that FK409 can attenuate myocardial injury during ischaemia and reperfusion in contrast to ISDN and a change in plasma cyclic GMP level may serve as an indicator of the cardioprotective effect of NO‐releasing drugs.

Antiplatelet activities of FK409, a new spontaneous NO releaser

1 We reported that (±)‐(E)‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexeneamide (FK409) released nitric oxide (NO) spontaneously with a chemiluminescence analyzer. The aim of this study was to compare antiplatelet activities of FK409, a new NO releaser, with those of isosorbide dinitrate (ISDN) in vivo and in vitro. In order to elucidate the differences in antiplatelet activities between FK409 and ISDN, we compared their modes of action. 2 In in vitro experiments, FK409 had a more potent inhibitory effect on rat platelet aggregation induced by adenosine 5′‐diphosphate (2.0 μm) than ISDN (IC50 = 4.32 ± 0.95μm and > 100 μm respectively). 3 In the rat extracorporeal shunt model (in vivo experiments), FK409 suppressed thrombus formation dose‐dependently from 0.32 mg kg‐1, p.o. and showed the maximum inhibition (52% inhibition vs. vehicle treatment) at 10 mg kg‐1, p.o., while ISDN showed no inhibition at 10 mg kg‐1 and only 17% inhibition at 32 mg kg‐1, p.o. 4 FK409 could generate nitrite, which is an oxidative product of NO, much faster than ISDN in phosphate buffer solution and rat plasma during 60‐min incubation at 37°C. 5 These data show that FK409 has more potent antiplatelet effects than ISDN, by acting through spontaneously released NO.

Comparison of antiplatelet effects of FK409, a spontaneous nitric oxide releaser, with those of TRK-100, a prostacyclin analogue

The anti-platelet effects of FK409 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide) , a new spontaneous nitric oxide releaser, and TRK-100 (sodium dl-4-[(1R,2R,3aS,8bS)-1,2,3a,8b-tetra-hydro-2-hydroxy-1-[(3S ,4RS)-3-hydroxy- 4-methyl-oct-6-yen-(E)-1-enyl]-5-cyclopenta[b]benzofuranyl]butyrate), a stable prostacyclin analogue, were studied both in vivo and in vitro. FK409 and TRK-100 inhibited ADP-induced platelet aggregation in rat platelet-rich plasma at 1.0 and 0.032 microM, respectively. In a rat extracorporeal shunt model, FK409 suppressed thrombus formation dose dependently and significantly at 1.0 mg/kg and showed the maximum inhibition (52% inhibition) at 10 mg/kg. TRK-100 showed 79% inhibition of thrombus formation at 1.0 mg/kg, but not at less than 1.0 mg/kg. At the doses required for antiplatelet effects, TRK-100 decreased mean blood pressure significantly but FK409 did not alter the blood pressure. These data suggest that FK409 shows more selective activities on platelets than TRK-100 in these experiments.

Acceleration of nitric oxide (NO) release from FK409, a spontaneous NO releaser, in the presence of sulfhydryl-bearing compounds.

AbstractPurpose. Recently, we have reported that FK409 spontaneously releases nitric oxide (NO) in solution. In the present study, the influence of L-cysteine (Cys) and glutathione (GSH), which are typical sulfhydryl group-bearing compounds, on NO release from FK409 and biological action of FK409 was examined. Methods. We evaluated the effects of Cys and GSH on NO release from FK409 by nitrite analysis or detection with a chemiiluminesence analyzer. In a biological study, the influence of Cys on inhibition of rat platelet aggregation of FK409 was investigated. In addition, the above mentioned characteristics of FK409 were compared with those of isosorbide dinitrate (ISDN). Results. FK409 decomposed spontaneously with generation of nitrite in solution. Both Cys and GSH accelerated decomposition of FK409 and nitrite generation from FK409 in a concentration-dependent manner. When the NO levels in the headspace of FK409 solutions (0.5 mM) reached equilibrium with and without 25 mM Cys, the constant rate for NO release from FK409 in the presence of Cys was 13 times larger than that in the absence of Cys. In biological study, FK409 (100 µM) showed 56 and 90% inhibition of rat platelet aggregation in the absence and presence of 10 mM Cys, respectively, whereas ISDN (100 µM) showed 10 and 23% inhibition, respectively. Conclusions. Decomposition of FK409 with generation of NO is spontaneous, and is accelerated in the presence of sulfhydryl group-bearing compounds, thereby potentiating the biological action of FK409.