Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia-reperfusion injury.

The complement system is a potent mediator of ischemia-reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Inhibition of complement C3 might rescue vascular hyporeactivity in a conscious hemorrhagic shock rat model.

BACKGROUND: Vascular hyporeactivity in severe hemorrhagic shock could induce refractory hypotension and is an important cause of death. The global acute inflammatory response induced in shock triggers the over-expression of reactive oxygen species, NO, ET1 and TNF-α, which play essential roles in the pathology of vascular hyporeactivity. This leads to a hypothesis that inhibition of the complement system, the mediator of the inflammatory cascade, might be a promising therapeutic exploration for vascular hyporeactivity. METHODS: We use cobra venom factor (CVF) and the soluble form of CR1 (sCR1) which deplete or inhibit complement C3 respectively to examine its role in vascular hyporeactivity in a conscious hemorrhagic shock rat model. RESULTS: We first confirmed the over-activation of C3 during shock and the down-regulation effects of CVF and sCR1 on C3. Then, both CVF and sCR1 could significantly mitigate the over-expression of serum NO, ET-1, TNF-α and reactive oxygen species. Finally, the vascular reactivity of superior mesenteric arteries (SMA) was examined in vitro, which confirmed the massive reduction of vascular reactivity in shock, which was significantly rescued by both CVF and sCR1. CONCLUSIONS: Inhibition of C3 might improve the reactivity of SMA to norepinephrine during hemorrhagic shock possibly through the downregulation of NO, ET1, TNF-α and reactive oxygen radicals.

Preclinical evaluation of the neuroprotective effect of soluble complement receptor type 1 in a nonhuman primate model of reperfused stroke.

OBJECT: Postischemic cerebral inflammatory injury has been extensively investigated in an effort to develop effective neuroprotective agents. The complement cascade has emerged as an important contributor to postischemic neuronal injury. Soluble complement receptor Type 1 (sCR1), a potent inhibitor of complement activation, has been shown to reduce infarct volume and improve functional outcome after murine stroke. Given numerous high-profile failures to translate promising antiinflammatory strategies from the laboratory to the clinic and given the known species-specificity of the complement cascade, the authors sought to evaluate the neuroprotective effect of sCR1 in a nonhuman primate model of stroke. METHODS: A total of 48 adult male baboons (Papio anubis) were randomly assigned to receive 15 mg/kg of sCR1 or vehicle. The animals were subjected to 75 minutes of middle cerebral artery occlusion/reperfusion. Perioperative blood samples were analyzed for total complement activity by using a CH50 assay. Infarct volume and neurological scores were assessed at the time the animals were killed, and immunohistochemistry was used to determine cerebral drug penetration and C1q deposition. An interim futility analysis led to termination of the trial after study of 12 animals. Total serum complement activity was significantly depressed in the sCR1-treated animals compared with the controls. Immunostaining also demonstrated sCR1 deposition in the ischemic hemispheres of treated animals. Despite these findings, there were no significant differences in infarct volume or neurological score between the sCR1--and vehicle-treated cohorts. CONCLUSIONS: A preischemic bolus infusion of sCR1, the most effective means of administration in mice, was not neuroprotective in a primate model. This study illustrates the utility of a translational primate model of stroke in the assessment of promising antiischemic agents prior to implementation of large-scale clinical trials.

Soluble complement receptor type 1 protects rats from lethal shock induced by anti-Crry antibody following lipopolysaccharide priming.

BACKGROUND: In rats primed with a trace amount of lipopolysaccharide (LPS), acute lethal shock is induced following the injection of monoclonal antibody against a membrane inhibitor of complement (anti-Crry). Administration of cobra venom factor to exhaust complement before the LPS priming can prevent the lethal reaction. Therefore, we evaluated whether soluble complement receptor type 1 (sCR1), which inhibits complement reaction, can interfere with lethal shock when administered after LPS priming or even after anti-Crry injection. METHODS: sCR1 was administered intravenously before or after the administration of anti-Crry, and the effects on blood pressure and acute lethality were determined. RESULTS: Administration of sCR1 could rescue rats from lethal shock even when it was administered after anti-Crry injection, which immediately causes a blood pressure decrease leading to lethal shock. CONCLUSION: sCR1 may be an effective treatment for acute shock involving complement activation.