ABSTRACT
BACKGROUND AND PURPOSE: Rho-kinase (ROCK) has been implicated in the pathophysiology of altered vasoregulation leading to hypertension. Here we describe the pharmacological characterization of a potent, highly selective and orally active ROCK inhibitor, the derivative of a class of azaindoles, azaindole 1 (6-chloro-N4-{3,5-difluoro-4-[(3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]-phenyl}pyrimidine-2,4-diamine). EXPERIMENTAL APPROACH: Pharmacological characterization of azaindole 1 was performed with human recombinant ROCK in vitro. Vasodilator activity was determined using isolated vessels in vitro and different animal models in vivo. KEY RESULTS: This compound inhibited the ROCK-1 and ROCK-2 isoenzymes with IC50 s of 0.6 and 1.1 nM in an ATP-competitive manner. Although ATP-competitive, azaindole 1 was inactive against 89 kinases (IC50>10 microM) and showed only weak activity against an additional 21 different kinases (IC50=1-10 microM). Only the kinases TRK und FLT3 were inhibited by azaindole 1 in the sub-micromolar range, albeit with IC50 values of 252 and 303 nM, respectively. In vivo, azaindole 1 lowered blood pressure dose-dependently after i.v. administration in anaesthetized normotensive rats. In conscious normotensive and spontaneously hypertensive rats azaindole 1 induced a dose-dependent decrease in blood pressure after oral administration without inducing a significant reflex increase in heart rate. In anaesthetized dogs, azaindole 1 induced vasodilatation with a moderately elevated heart rate. CONCLUSIONS AND IMPLICATIONS: Azaindole 1 is representative of a new class of selective and potent ROCK inhibitors and is a valuable tool for the elucidation of the role of ROCK in the cardiovascular system.
Subject(s)
Cardiovascular System/drug effects , Diamines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Administration, Oral , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Binding Sites/drug effects , Blood Pressure/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Computer Simulation , Death-Associated Protein Kinases , Dogs , Dose-Response Relationship, Drug , Female , Humans , Injections, Intravenous , Male , Mice , Models, Animal , Models, Molecular , Organ Culture Techniques , Phosphorylation , Polymerase Chain Reaction/methods , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Rabbits , Rats , Rats, Inbred SHR , Rats, Wistar , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Time Factors , Vasodilator Agents/administration & dosage , Vasodilator Agents/chemistry , Vasodilator Agents/pharmacology , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
Enormous differences exist between rat strains with respect to the infarct volume induced by unilateral middle cerebral artery (MCA) occlusion. We performed three experiments to address the following questions. Firstly, whether the pattern of MCA-occlusion (MCA-O) induced sensorimotor impairments in rats are strain dependent; secondly, whether proximal (i.e., close to its origin) and distal occlusions (above the lenticulostriate branch) of the MCA affect infarct volume and the behavioral impairments to a different extent; and thirdly, whether there is a relationship between the infarct volume and behavioral deficits. We found that the pattern of sensorimotor malfunctions induced by proximal unilateral MCA-O were highly strain dependent. Of the eight strains tested, Winkelmann-Wistar rats, Spontaneously Hypertensive Stroke-Prone rats, and Wistar-Kyoto rats were most severely affected. By contrast, Brown-Norway rats showed only mild behavioral deficits after the MCA-O. The second experiment confirmed that proximal occlusions induced slightly more behavioral malfunctions than distal occlusions did. Histological evaluation of the brain damage caused by proximal and distal MCA-O, confirmed that distal MCA-O damaged nearly exclusively cortical areas, and spared the caudate/putamen. An exploratory analysis of the relationship between infarct volume and behavioral deficits did not indicate that the severity of sensorimotor malfunctions can be predicted from the size of the infarct.
Subject(s)
Arterial Occlusive Diseases/complications , Cerebral Arteries , Cerebral Infarction/etiology , Cerebral Infarction/physiopathology , Motor Activity/physiology , Sensation/physiology , Animals , Behavior, Animal/physiology , Cerebral Infarction/psychology , Male , Rats , Rats, Inbred Strains , Species SpecificityABSTRACT
Wistar Kyoto (WKY) rats with cerebral infarction induced by permanent unilateral occlusion of the middle cerebral artery (MCA) and sham-operated rats were tested in a series of simple behavioral test 2, 16 and 37 days after surgery. In addition, the motility of the animals was measured over a period of 62 h, after the third test series. A subset of the tests appeared to be suitable to assess the effects of cerebral infarction, namely, grasping reflex of contralateral hindpaw, circling behavior, forelimb flexion, hindlimb flexion, and latency to fall off a square bridge. Except for the impaired grasping reflex of the contralateral hindpaw, there was spontaneous complete recovery of function by the third test session, 37 days after surgery. Some of the other tests might not have been sensitive enough to detect the effects of the unilateral MCA-occlusion (MCA-O) on behavior. Moreover, the WKY rats were very inactive in some of the tests, so that reliable scoring of the effects was not always possible. A rat strain other than the WKY strain might be more suitable to study the behavioral consequences of MCA-O.
