Keizo Yoshida

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.

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.

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.

Platelet activating factor (PAF) involvement in endotoxin-induced thrombocytopenia in rabbits : Studies with FR-900452, a specific inhibitor of PAF

PAF (1 ug/kg) injected intravenously (i.v.) into anesthetized rabbits resulted in marked loss of circulating platelets and leukocytes. Administration of FR-900452 1-methyl-3-(1-(5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-y lidene)- 2-piperazinyl) ethyl)-2-indolinone, a specific PAF inhibitor, at a dose of 10 mg/kg i.v. with 10 min prior to the PAF injection significantly prevented both changes. On the other hand, PAF has been considered as a mediator of endotoxin shock. Therefore, in order to determine whether endogenous PAF contributes to the occurrence of thrombocytopenia or leukopenia in endotoxin shock, we assessed the effect of FR-900452 on the thrombocytopenia and the leukopenia following bolus i.v. injection of E.coli endotoxin (0.03 mg/kg) in rabbits. As a result, pretreatment with the compound (10 mg/kg, i.v.) significantly reduced the thrombocytopenia at 60 and 180 min after the endotoxin injection. In contrast, FR-900452 did not reduced the leukopenia at any time of after endotoxin. These results indicate that PAF might be involved in the occurrence of thrombocytopenia in rabbit endotoxemia and the contribution of PAF to the leukopenia is much less extent than that to the thrombocytopenia.

Discovery of FK453, a novel non-xanthine adenosine A1 receptor antagonist

Abstract Novel 2-phenylpyrazolo[1,5-a]pyridine-3-acryloylamides were synthesized and evaluated for diuretic activities. FK453 (1d), the most potent compound in this series, was found to be a potent and selective adenosine A1 receptor antagonist, whereas 1e, the (S)-enantiomer of FK453, was a weak and non-selective adenosine antagonist.

PAF inhibitory activity of diketopiperazines: Structure-activity relationships

FR900452, a natural product isolated from the culture broth ofStreptomyces phaeofaciens No. 7739, was found to inhibit PAF-induced rabbit platelet aggregation with an IC50 of 3.7×10−7M. FR900452, 1-methyl-3-[1-[5-methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-ylidene)-2-piperazinyl]ethyl]-2-indoline, has an oxocylopentylidene group incorporated as a vinylogous amide in a diketopiperazine skeleton. This unique structure led us to synthesize diketopiperazine derivatives, 3-arylalkyl-6-substituted-piperazine-2,5-diones. their observed PAF inhibitory activity suggest that the D-D configuration of diketopiperazine is an important factor for anti-PAF activity and that the hydrophobic aromatic portion may play a specific role in the binding of the diketopiperazine to the PAF receptor.

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.

The anti-platelet actions of FR122047, a novel cyclooxygenase inhibitor

The anti-platelet actions of 1-[(4,5-bis(4-methoxyphenyl)-2- thiazoyl)carbonyl]-4-methylpiperazine hydrochloride (FR122047) were investigated in vitro and in vivo. FR122047 was 100 times more potent than aspirin against arachidonic acid- and collagen-induced human and guinea-pig platelet aggregation in vitro. Its actions on platelets were a result of cyclooxygenase inhibition. The single oral dose of FR122047 inhibited arachidonic acid- and collagen-induced aggregation with an ED50 of 280 micrograms/kg and 530 micrograms/kg, respectively, in guinea-pigs. The anti-platelet action was augmented 5-10 times by repeated administration for 4 days. At 1 mg/kg the inhibitory actions were prolonged for 48 h and the drug concentration was < 0.1 ng/ml in platelet-poor plasma at 24 h and 0.282 ng/ml in platelet-rich plasma at 48 h. The safety margin in rats (minimum ulcerogenic dose/ED50 for anti-platelet aggregation) of FR122047 was more than 70, while that of aspirin was only 1.2. These results indicate that FR122047 is concentrated in platelets and may be a useful anti-platelet agent.

Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis.

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced contraction by a direct stimulatory effect on the smooth muscle of rat colon in a receptor-mediated manner.