NIM811 prevents induction of mitochondrial permeability transition in cerebral ischemia

Introduction

In ischemic neurons, elevation of Ca2+ levels and formation of reactive oxygen species open an intermembranous pore in mitochondria, resulting in the activation of the mitochondrial permeability transition (MPT). Cyclosporin A (CsA) can block the MPT and is neuroprotective in animal models of cerebral ischemia, but its utility is hampered by limited penetration of the blood-brain barrier (BBB) and toxicity. NIM811 (Novartis Pharma, Basel, Switzerland) is a novel derivative of CsA that is a non-toxic and specific inhibitor of MPT induction. We report the first use of NIM811 to affect infarct volume and modulate cytochrome C release, a downstream effect of MPT induction that contributes to neuronal apoptosis.

Methods

To determine if NIM811 penetrates the BBB, rats were injected intraperitoneally with 50 mg/kg of drug. Blood and brain samples obtained 1 hr after drug injection were assayed for content of NIM811 by liquid chromatography/mass spectroscopy. To determine the effect of NIM811 on infarct volume, spontaneously hypertensive rats (SHRs) underwent reversible middle cerebral artery occlusion (MCAO) for 2 hrs. After 1 hr of reperfusion, animals were treated with NIM811 (50 mg/kg) or drug-free vehicle (100% DMSO). Brain was removed en bloc after 24 hrs for measurement of infarct volume corrected for ischemic edema. To determine the effect of NIM811 on release of cytochrome C from mitochondria in ischemic brain, SHRs underwent MCAO for 2 hrs, were treated with 50 mg/kg NIM811 or vehicle after 1 hr of reperfusion, and samples of ischemic core and penumbra were removed and snap-frozen 1 hr after treatment. Supernatants obtained after ultracentrifugation of brain mitochondrial fractions were assayed for cytochrome C content by ELISA.

Results

One hr after injection of 50 mg/kg NIM811 in non-ischemic rats (n=3), the brain level of NIM811 peaked at 169 mcg/mL, compared to 37 mcg/mL in whole blood. Treatment with NIM811 at 1 hr after post-ischemic reperfusion reduced infarct volume measured at 24 hrs by 40% (88 ± 3.8 mm³ [vehicle; n=4] vs. 53 ± 8.8 mm³ [NIM811; n=5]; *p<0.005, t-test). Cytochrome C levels were increased fivefold in ischemic core (74.4 ± 3.9 ng/mL) and penumbra (68.5 ± 3.6 ng/mL; n=4) vs. comparable areas in non-ischemic controls (sham-core 13.6 ± 0.56 ng/mL and sham-penumbra 14.2 ± 0.54; n=4; p<0.001 for both by ANOVA with Scheffe post hoc). Treatment with NIM811 after MCAO lowered the cytochrome C level to 49.2 ± 2.2 ng/mL in ischemic core and 42.4 ± 2.4 ng/mL in penumbra (p<0.001 compared to levels in vehicle-treated ischemic controls).

Conclusion

Our results show that NIM811 penetrates intact BBB, reduces infarct volume significantly after ischemia, and attenuates the release of cytochrome C from distressed mitochondria into the cytoplasm of ischemic neural cells. These data support the hypothesis that activation of MPT contributes to ischemia-reperfusion injury in brain and underscore the potential value of NIM811 as novel therapy for stroke.