AM-36

The plasma pharmacokinetics and brain uptake of the novel neuroprotective agent AM-36 (1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis-(1, 1dimethylethyl)-4-hydroxyphenyl) methylpiperazine) were assessed over 72 h following i.v. administration to male Sprague-Dawley rats. At nominal i.v. doses of 0.2, 1 and 3 mg kg−1, AM-36 exhibited an extremely large volume of distribution (18.2–24.6 L kg−1) and a long terminal elimination half-life, ranging from 25.2 to 37.7 h. Over this dose range, AM-36 exhibited linear pharmacokinetics, with no apparent change in clearance, volume of distribution or dose-normalised area under the plasma concentration-time curve. AM-36 was very highly bound to plasma proteins (> 99.6%); however, this did not appear to affect the ability of AM-36 to permeate the blood-brain barrier. Following a single i.v. dose of AM-36 at 3 mg kg−1 to rats, brain concentrations were detected for up to 72 h, and the brain-to-plasma ratios were high at all time points (ranging from 8.2 at 5 min post-dose to 0.9 at 72 h post-dose). The very high brain uptake of AM-36 supports previous in-vivo efficacy studies demonstrating the neuroprotective effects of this compound when administered to rats with middle cerebral artery occlusion.

Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM‐36, both a Na+ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin‐1 (ET‐1) induced middle cerebral artery occlusion (MCAo) in conscious rats.

AM‐36 was administered at 3 and 24 h after ET‐1‐induced MCAo. Functional recovery was determined using grid‐walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM‐36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double‐label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue.

AM‐36 reduced functional deficits in both tests such that no difference existed from pre‐ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM‐36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM‐36 reduced neutrophil infiltration by ∼50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM‐36 treatment.

These findings suggest that AM‐36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.British Journal of Pharmacology (2006) 149, 712–723. doi:10.1038/sj.bjp.0706918