Recombinant human C1-inhibitor protects against brain ischemia/reperfusion injury with a wide time-window of efficacy

We have previously shown that plasma derived C1-INH (pdC1-INH), the endogenous inhibitor of complement and contact/kinin system, has potent neuroprotective effects in ischemic mice, although with a narrow time-window of efficacy(1-3). This inhibitor, currently used to manage angioedema attacks in patients with hereditary or acquired C1-INH deficiency (angioedema, HAE or AAE), is purifed from human plasma. Using a plasma derived molecule can be disadvantageous, due to risks for human pathogenic viruses contamination and to difficulties in supply and purification. Due to these limitations, a recombinant approach was chosen to overcome these problems. Recombinant human C1-INH (rhC1-INH, Pharming Technologies B.V.) has been administrated intravenously to C57Bl/6 ischemic mice at different doses (5, 10 and 15U/mouse) and time-points (0, 3, 6, 18 and 24 hours) from transient ischemia. Forty-eight hours after ischemia, rhC1-INH, at the dose of 15U/mouse, was able to markedly improve the cerebral damage when administrated 0, 3 and 6h from ischemia (13.67±2.59 mm3, 11.71±0.63mm3 and 20.38±2.37mm3, respectively) compared to saline-treated ischemic mice (44.43±5.94mm3). Also when administrated 18 hours after ischemia, rhC1-INH was still significantly effective (27.13±2.58mm3). Fluoro jade staining showed that amelioration of cerebral damage was due to an effective sparing of neurons. Immunohistochemistry for macroglia/macrohages (anti-CD11b) and astrocytes (anti-GFAP) showed that rhC1-INH was able to counteract the microglial and astrocytic activation, as well as macrophage infiltration. Analysis of rhC1-INH localization in the brain by confocal microscopy revealed that rhC1-INH was confined to cerebral vessels and that it was able to bind endothelial cells as shown by colocalization with Claudin-5 and ICAM-1. These data demonstrate that rhC1-INH has an important neuroprotective action with a wider time-window of efficacy compared to pdC1-INH. This favorable profile of rhC1-INH is probably related to a different glycosylation of the native versus recombinant molecules.