C(1) inhibitor in canine intravascular hemolysis (C(1)INCH): study protocol for a randomized controlled trial

BACKGROUND: Immune-mediated hemolytic anemia (IMHA) is a common disease that affects all breeds of dogs and is associated with significant morbidity and mortality. Intravascular hemolysis of erythrocytes in IMHA is caused by complement activation and is often fatal. No current treatments target complement activation in canine IMHA. Human C(1) esterase (C(1)-INH) reduces canine complement-mediated hemolysis in vitro, and a recent pharmacokinetic analysis of an FDA licensed formulation of C(1)-INH in dogs confirmed that a 50 IU/kg dose of C(1)-INH is safe to administer to dogs, and effectively inhibits canine complement mediated hemolysis ex-vivo. The C(1)INCH randomized controlled trial will evaluate the efficacy of this drug in dogs with intravascular hemolysis. METHODS: We will conduct a multicenter, placebo-controlled double-blind randomized clinical trial of C(1)-INH in dogs with intravascular hemolysis due to IMHA. We will randomize 18 dogs to receive three doses of intravenous C(1)-INH or saline in 24 h. Immunosuppressive and antithrombotic therapies will be standardized. Primary outcome measures will be changes in plasma free hemoglobin, serum concentrations of LDH, bilirubin, and haptoglobin. Using patient samples, we will evaluate complement activation in canine IMHA using a novel C5b-9 ELISA assay, flow cytometric detection of C3b on RBC, and by measurement of residual plasma complement activity. Secondary outcome measures will be survival to hospital discharge, duration of hospitalization, number and volume of red blood cell transfusions, and rescue therapy requirements. We will monitor dogs for adverse drug reactions. Sample size was estimated from pilot data on LDH and hemolysis index (HI) in dogs with IMHA. To detect 2-way differences between the upper and lower 50% of the LDH and HI values of equivalent size with 80% power at P < 0.05 will require 9 dogs in each arm. DISCUSSION: We anticipate that IV administration of C1-INH will significantly inhibit complement mediated hemolysis in dogs with intravascular IMHA, as determined by blood biomarker measurements (decreased plasma hemoglobin, LDH and bilirubin, increased haptoglobin). We expect this will translate into significant reductions in transfusion requirements and duration of hospitalization. TRIAL REGISTRATION: This trial has been prospectively registered with the AVMA registry (AAHSD005025).