Shin-ichi Satoh

Pharmacological profile of hydroxy fasudil as a selective rho kinase inhibitor on ischemic brain damage.

The neuroprotective property and the effects on hemodynamics of hydroxy fasudil, an active metabolite of an antispastic drug, fasudil, were examined. In rats, hydroxy fasudil was found following intravenous infusion or intraperitoneal administration of fasudil, and the maximum plasma concentration of hydroxy fasudil was approximately 25 or 40% of the parent drug, respectively. The i.v. administration of hydroxy fasudil produced significant increases in regional cerebral blood flow in dogs. Hydroxy fasudil relaxed the KCl, PGF2alpha or U-46619-induced contraction in canine basilar or middle cerebral arterial strips, concentration-dependently. The neuroprotective property of hydroxy fasudil was examined on delayed neuronal death in gerbils. Hydroxy fasudil (3 mg/kg) significantly protected against the ischemia-induced neuronal loss. To further clarify the effect on neurological impairments, hydroxy fasudil was tested in a rat model of microembolization stroke. Intravenous administration of hydroxy fasudil improved neurological functions, significantly reduced the size of the infarct area and prevented the accumulation of neutrophils. The present findings suggest that hydroxy fasudil has an efficacy to improve the hemodynamic function and to inhibit neutrophil-mediated damage, and contributes to the potency and long duration of the cytoprotective properties of fasudil on ischemic brain damage, and also suggest a critical role for rho kinase in the pathogenesis of cerebral ischemic injury, and the potential utility of rho kinase inhibitor as a therapeutic agent in stroke.

Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase

1 The in vitro and in vivo vasorelaxant effects of HA1077, 1‐(5‐isoquinolinesulphonyl)‐homopiperazine HCl, a novel vasodilator were examined. 2 The inhibitory effects of HA1077 on contractile responses to various agonists were examined on strips of rabbit aorta. The concentration‐response curves to 5‐hydroxytryptamine, prostaglandin F2α, histamine, angiotensin II, noradrenaline and dopamine were concentration‐dependently shifted to the right in the presence of HA1077 (0.3‐3.0 μm). 3 The in vivo vasodilator effects of HA1077 were examined in the constant‐pressure autoperfused coronary vascular bed of dogs. Intra‐coronary administration of HA1077 (3–30 μg per dog) dose‐dependently increased coronary blood flow (CBF), with no effect on mean blood pressure (MBP) or heart rate (HR). Intra‐coronary infusion of atropine, propranolol or diphenhydramine did not modify the in vivo coronary vasodilator response to HA1077. 4 To determine the flow profile for HA1077 in dogs, blood flow in four vascular beds was measured, by use of noncannulating electromagnetic flow probes. HA1077 (0.01‐0.3 mg kg−1, i.v.) dose‐dependently decreased MBP and increased vertebral blood flow (VBF), CBF, renal blood flow (RBF) and femoral blood flow (FBF). 5 A haemodynamic analysis showed that continuous i.v. infusion of HA1077 (0.01 and 0.033 mg kg−1 min−1) dose‐dependently decreased peripheral vascular resistance and increased cardiac output. There were no significant changes in right atrial pressure, dP/dt or ventricular minute work. 6 The effects of HA1077 on various enzymes considered to be related to the regulation of smooth muscle contraction were examined. HA1077 had little effect on cyclic nucleotide phosphodiesterases, yet it potently inhibited protein kinases such as cyclic nucleotide dependent protein kinases and Ca2+/calmodulin dependent myosin light chain kinase. 7 The present study demonstrates that HA1077 is a novel type of arterial vasodilator.

Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction.

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure.

Endothelin: a potential modulator of cerebral vasospasm

1-(5-Isoquinolinesulfonyl)-homopiperazine, HA1077, is a calcium antagonist with anti-vasospastic properties. This compound blocks intracellular actions of calcium in a variety of experiments. In the present study, we examined the effects of HA1077 on the vascular actions of endothelin, an endothelium-derived vasoactive peptide, in dogs in vitro and in vivo. Intracisternal injections of endothelin (0.01 nmol) produced a significant vasospasm, as measured by angiography, similar to that seen in the canine hemorrhage model. Infusion of HA1077 led to a significant dilatation of the spastic basilar artery in endothelin-treated dogs. The rank order of in vitro contractile activity in canine cerebral arteries was a stable thromboxane A2 analog greater than endothelin greater than 5-hydroxytryptamine greater than prostaglandin F2 alpha greater than histamine greater than noradrenaline. HA1077 effectively antagonized the endothelin-induced contraction of canine basilar arterial strips in both calcium-containing and calcium-free medium. The present results indicate that HA1077 is an effective antagonist for endothelin in vitro and in vivo.

Neuroprotective properties of a protein kinase inhibitor against ischaemia-induced neuronal damage in rats and gerbils.

1 . The neuroprotective properties of fasudil (HA1077), a novel protein kinase inhibitor, were evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in Mongolian gerbils and cerebral microembolization in rats. 2 . The cytoprotective effect of fasudil on delayed neuronal death in gerbils was compared with the effects of nimodipine, a calcium channel antagonist and ozagrel, a thromboxane A2 synthetase inhibitor. The average of the neuronal cell density in the ischaemic control group was 17.8 ± 2.1 cells mm−1, whereas fasudil (30 mg kg−1) significantly diminished the loss of CA1 neurones with the average of the neuronal cell density of 101.0 ± 22.0 cells mm−1; nimodipine (10 mg kg−1) and ozagrel (30 mg kg−1) did not significantly protect against the ischaemia‐induced neuronal loss. 3 . In the rat model, the effects of fasudil on the histological and neurological consequences of cerebral microembolization produced via the injection of microspheres were examined. Twenty‐four hours after the injection of microspheres into the internal carotid artery, all animals in the control group showed typical symptoms of stroke. Neurological function was significantly improved in the fasudil‐treated animals. In the controls, the infarcted area in a cortical slice selected to include the hippocampal area was 0.25 ± 0.01 cm2 (mean ± s.e.mean) (43.9 ± 2.4% of cortical section of the half hemisphere); the difference was significant compared to the mean area of 32.7 ± 2.8 and 21.5 ± 4.8% observed in rats treated with fasudil (3, 10 mg kg−1), respectively. Fasudil (10 mg kg−1) significantly suppressed the increased water content in ischaemic brain tissues (saline‐treated rats, 82.4 ± 0.2% vs fasudil‐treated rats, 81.0 ± 0.4%). 4 . These results suggest that: (i) various protein kinases are involved in the pathogenesis of ischaemic injury; and (ii) the inhibition of protein kinases may be efficacious in preventing neuronal death, thus improving neurological function in the brain damage associated with ischaemic stroke.

Blockade of intracellular actions of calcium may protect against ischaemic damage to the gerbil brain

1 The brain cytoprotective effects of a putative calcium‐associated protein kinase inhibitor, HA1077, as well as a calcium entry blocker nicardipine were evaluated in models of cerebral ischaemia in Mongolian gerbils. Morphological changes characterizing delayed neuronal death of selectively vulnerable CA1 pyramidal neurones in the hippocampus of the Mongolian gerbil brain occurred 7 days after transient bilateral occlusion of the common carotid arteries. 2 A single injection of HA1077 (1 and 3 mg kg−1, i.p.) 5 min after the occlusion led to a dose‐dependent protection of the CA1 neurones. Repeated administrations of HA1077 (1 and 3 mg kg−1, i.p., twice daily for 7 days post‐ischaemia) revealed an increase in the number of normal cells, compared to findings with a single administration. 3 In contrast to HA1077, nicardipine (0.3 and 1 mg kg−1, i.p.) did not reduce neuronal degeneration. 4 HA1077 did not interact with the ion channel within which MK‐801 binds, as determined by receptor binding. 5 The calcium ionophore, A23187, caused a tonic contraction in canine cerebral arterial strips. HA1077, but not nicardipine, relaxed the A23187‐induced contraction, concentration‐dependently. 6 These results suggest that blockade of the intracellular actions of calcium may provide protection against ischaemic damage in the brain.

Antianginal effects of hydroxyfasudil, a Rho‐kinase inhibitor, in a canine model of effort angina

The effects of Rho‐kinase inhibitor, fasudil, and of a more specific Rho‐kinase inhibitor, hydroxyfasudil, on pacing‐induced myocardial ischaemia were determined in anaesthetized open‐chest dogs. The dogs were subjected to left anterior descending coronary artery (LAD) stenosis producing a sufficient ischaemia as measured by ST‐segment depression on electrocardiograms only when the hearts were paced 60 beats min−1 above the baseline. After a recovery (nonpacing) period, drugs or saline were infused intravenously over 30 min. The animals were again subjected to 5 min of pacing 25 min after the initiation of the treatment. Hydroxyfasudil (0.1 and 0.3 mg kg−1) and fasudil (0.3 mg kg−1) suppressed the ST‐segment depression. Hydroxyfasudil and fasudil also increased the regional blood flow of the LAD perfused endomyocardium region in the canine model of effort angina. To determine the flow profile for hydroxyfasudil in dogs, blood flow in three vascular beds was measured. Hydroxyfasudil (0.3 mg kg−1 for 30 min) significantly increased coronary blood flow and vertebral blood flow, without significantly changing the femoral blood flow. Hydroxyfasudil had no inotropic or chronotropic effect on the isolated hearts of guinea‐pigs. Hydroxyfasudil (2 mg kg−1 for 20 min) did not affect the PR or QTc interval in anaesthetized dogs. Inhibition of Rho‐kinase appears to protect myocardium subjected to pacing‐induced ischaemia through the increase in the regional myocardial blood flow. Hydroxyfasudil may be categorized as a novel type of anti‐anginal drug, without any inotropic or chronotropic effects.

Involvement of Rho-kinase in vascular remodeling caused by long-term inhibition of nitric oxide synthesis in rats.

Long-term inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces coronary vascular remodeling in rats. To determine the pathogenic mechanism involved in vascular remodeling, we examined the effects of fasudil, a Rho-kinase inhibitor, on vascular lesion formation. In rats treated with L-NAME at 10 mg/kg/day, vascular remodeling was evident in both large and small coronary arteries at the fourth week. Fasudil (3 mg/kg, p.o., twice daily) markedly prevented the development of vascular remodeling in small coronary arteries. Coronary flow was measured in Langendorff perfused isolated heart preparations. Long-term treatment with L-NAME caused a significant decrease in coronary flow, which was significantly inhibited by fasudil. Fasudil suppressed the structural and functional changes in coronary arteries by chronic blockade of NO synthesis. Thus, the Rho-kinase pathway may be substantially involved in the pathogenesis of vascular remodeling in this rat model.

Effects of Rho-kinase inhibitor on vasopressin-induced chronic myocardial damage in rats.

The aim of this study was to develop a new model of vasopressin-induced chronic myocardial damage based on sustained ST-segment depression in electrocardiogram (ECG) with progression of myocardial fibrosis in rats. Furthermore, using this model, we examined the prophylactic potential of fasudil, a Rho-kinase inhibitor, against myocardial damage induced by vasopressin. In 10-week old male Donryu rats, intravenous administration of arginine vasopressin (0.5 iu/kg) induced significant ST-segment depression. Two days and one week after the administration of vasopressin, ST-segment depression was -0.19 +/- 0.02 and -0.14 +/- 0.02 mV, respectively. Fasudil (10 and 30 mg/kg, p.o.) significantly attenuated the ST-segment depression induced by vasopressin. One week after the administration of vasopressin, the percent area of myocardial fibrosis in control animals (0.42 +/- 0.11%, p < 0.01) was significantly greater than that in normal animals (0.05 +/- 0.01%). Fasudil (10 and 30 mg/kg) significantly prevented the development of the fibrosis. We present a new model of chronic myocardial damage based on sustained ST-segment depression with progression of myocardial fibrosis in rats, and suggest that this model may be useful to investigate the treatment of chronic angina. Inhibition of Rho-kinase is efficacious in preventing the ECG change and development of fibrosis induced by vasopressin in this model.