Immune-Mediated Hemolytic Anemia (IMHA) is a spontaneously occurring disease of man and dogs in which immunoglobulin-coated erythrocytes are destroyed in the spleen. Immunosuppressive therapies and splenectomy have been previously used to combat premature destruction of erythrocytes. Liposomal clodronate has been previously shown to be a potent anti-macrophage agent when administered intravenously in a murine model. Splenic macrophages were selectively depleted through the induction of apoptosis following phagocytosis of liposomes containing clodronate. We hypothesized that such an approach might be useful in blocking clearance of erythrocytes in dogs with spontaneous IMHA.

Clodronate (dichloromethyl bisphosphonate) was formulated in liposomes (phosphatidyl choline, cholesterol and mannose), according to a previously published technique. The liposomal clodronate was evaluated in vitro for activity using canine and murine histiocytic cell lines, as assessed by cell killing and blocking of erythrophagia. Cell killing was monitored using microscopic evaluation of cell change, MTT and by propidium iodide staining. Blocking of erythophagia was monitored using flow cytometry. In vivo in normal dogs, increasing doses of liposomal clodronate were administered intravenously and evaluated to determine efficacy in blocking erythrocyte clearance and toxicity. Twenty-four hours post infusion, dye-labeled erythrocytes were opsonized using rabbit anti-canine erythrocyte antibody. Erythrocytes clearance was monitored using flow cytometry. The safe and effective dose of liposomal clodronate was determined to be 0.5 ml/kg administered IV via CRI over a 90 minute time period. After administration of intravenous liposomal clodronate in normal dogs, clearance of opsonized erythrocytes was nearly completely blocked at the 0.5 ml/kg dose. In vitro assays indicated that liposomal clodronate induced death of canine macrophage and dendritic cell lines. Five dogs with spontaneous IMHA were treated once with liposomal clodronate at the 0.5 ml/kg dose. Though erythrocyte clearance was not completely blocked, the drug was well-tolerated and all 5 dogs survived to leave the hospital. These studies suggest that liposomal clodronate may be an effective agent for temporary suppression of erythrocyte destruction in IMHA. Additional studies, including evaluation of higher doses of liposomal clodronate, are warranted in dogs with spontaneous IMHA.

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