Todd R. Wiernicki

ARL 17477, a selective nitric oxide synthase inhibitor, with neuroprotective effects in animal models of global and focal cerebral ischaemia

In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.

Dipyridamole directly inhibits vascular smooth muscle cell proliferation in vitro and in vivo : implications in the treatment of restenosis after angioplasty

OBJECTIVES The effect of dipyridamole on smooth muscle cell proliferation and prevention of intimal thickening after arterial injury was investigated. BACKGROUND In addition to antiplatelet activity, dipyridamole also inhibits cell proliferation. We examined whether the antiproliferative action of dipyridamole on smooth muscle cells, as demonstrated here, has a direct effect on intimal thickening after vascular injury. METHODS Cell proliferation was determined by measuring deoxyribonucleic acid (DNA) synthesis and by cell counting. The in vivo effect of locally delivered dipyridamole was determined in a rabbit model with carotid or femoral artery injury. RESULTS Dipyridamole produced a dose-dependent inhibition of smooth muscle cell proliferation, producing 50% inhibition at 7 micrograms/ml. Structural analogues SH-869 and mopamidol were 10 to 100 times less effective than dipyridamole, suggesting that cell growth inhibition may be unrelated to the antiplatelet activity of dipyridamole. Inhibition of cell proliferation by dipyridamole was attenuated by increasing the serum concentration in the culture medium. Bypassing serum by local delivery of dipyridamole at the periadventitial site produced 63% inhibition (p < 0.05) of cell replication in balloon-injured arteries. Locally delivered dipyridamole also inhibited intimal thickening (20%, p < 0.05) after balloon injury. CONCLUSIONS Dipyridamole inhibited smooth muscle cell proliferation in vitro. This activity was attenuated by serum proteins. Locally delivered dipyridamole inhibited cell replication in arteries and intimal thickening after balloon injury. These results suggest that although systemic treatment with dipyridamole may not be efficacious because of inadequate serum levels, its antiproliferative action on smooth muscle cells may reduce restenosis when the drug is delivered locally after coronary angioplasty.

An Allosteric Potentiator of the Dopamine D1 Receptor Increases Locomotor Activity in Human D1 Knock-In Mice without Causing Stereotypy or Tachyphylaxis

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3–20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30–240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.