Background and Introduction
Although a variety of agents targeting a single mechanism have been studied as stroke therapies, the majority of these have failed. Many reasons can be attributed to the lack of successes, both pre-clinically and clinically, but one likely cause is the redundancy of pathways in the brain that lead to neuronal death. Novel therapies are clearly needed to overcome the multitude of cell death processes rapidly activated after cerebral ischemia. VELCADE® is a first-in-class, novel proteasome inhibitor which is approved for the treatment of relapsed, refractory multiple myeloma. Its specific target is the 26 s proteasome. Proteasomes are highly conserved multicatalytic enzyme complexes which are essential for the ubiquitin-dependent proteolysis of the majority of cellular proteins. By targeting the proteasome, a host of cellular effects are possible and the balance of these results in different phenotypes, depending on the cellular milieu. In rodent models of ischemia, there is an emerging body of evidence suggesting beneficial effects of proteasome inhibition 1 . Possible mechanisms of this protection include decreased NF-kB activation, upregulation of eNOS and HIF1alpha and decreases in inflammation.
Methods
In the setting of stroke, we evaluated the effect of proteasome inhibition with VELCADE® on a rat model of permanent middle cerebral artery occlusion (MCAO) using the intraluminal suture method. Male Wistar rats (n=10 per group) were treated with saline or VELCADE®, given as an i.v. bolus (0.2 mg/kg) one hour post-occlusion. Whole blood samples were drawn one hour after drug or vehicle injection for the determination of blood proteasome inhibition using an ex vivo fluorogenic pharmacodynamic assay 2 . At 24 h after ischemia, rats were assessed for neurologic dysfunction using a Neurological Rating Scale Score (NRSS). Rats were then sacrificed and brains were extracted for TTC staining and quantification of infarcts. Data are expressed as mean+SE and were analyzed using a paired t-test.
Results and Conclusions
A single dose of VELCADE® given 1 h post-MCAO, resulted in a 40% decrease in infarct volume (% infarction: 35.9+2.6 vs. 21.7+5.0; p<0.05) and a 38% decrease in neurologic deficits (NRSS: 2.6+0.163 vs. 1.625+0.183; p<0.01). There was no difference in body weight between the two groups. The functional and histopathologic protection was accompanied by a 67% inhibition of whole blood proteasome activity, a level of inhibition which is commonly achieved in cancer patients in the clinic 3 . Additional infarct volume and behavioral data will be presented assessing the dose-response of VELCADE® at 1 h post-stroke. Our study suggests that proteasome inhibition is beneficial in rat ischemic injury and that blocking ubiquitin-dependent proteolysis may be desirable in the acute setting following a stroke.