CIRCULATION PERSISTENCE AND BIODISTRIBUTION OF LYOPHILIZED LIPOSOME-ENCAPSULATED HEMOGLOBIN - AN OXYGEN-CARRYING RESUSCITATIVE FLUID

Objective: To evaluate the circulation persistence and organ biodistri bution of a freeze-dried, oxygen carrying resuscitative fluid: liposom e-encapsulated hemoglobin. Design: Randomized, animal studies. Setting : Accredited animal research facilities. Subjects: Normal female Balb/ c mice and male New Zealand rabbits. Interventions: Two groups of norm al female Balb/c mice were injected in the tail vein with either lyoph ilized liposome-encapsulated hemoglobin (n = 9) that was reconstituted just before administration, or with unlyophilozed liposome-encapsulat ed hemoglobin (n = 9) as a comparison. Two groups of male New Zealand rabbits were injected in the ear vein with either lyophilized Tc-99m-l iposome-encapsulated hemoglobin (n = 6) or unlyophilized Tc-99m-liposo me-encapsulated hemoglobin as a comparison (n = 6). After injection, m ice were anesthetized by brief inhalation of halothane followed by blo od sampling through the retro-orbital sinus. Rabbits were anesthetized 30 mins before liposome-encapsulated hemoglobin administration with a n intramuscular injection of a 5:1 mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Rabbits were then dynamically imaged for 90 mins, housed, and at 20 hrs, imaging again followed by autopsy and tissue s ampling to validate imaged organ biodistributions. Measurements: Circu lation persistence in the mouse was measured by removing a blood sampl e at various time points up to 24 hrs after injection. The blood sampl e was centrifuged in a hematocrit capillary tube and the disappearance of the sedimented liposome-encapsulated hemoglobin fraction was measu red. The change in the sedimented fraction of the liposomes with time was used to generate circulation persistence profiles in mice. The cir culation persistence and organ biodistribution of Tc-99m-liposome-enca puslated hemoglobin was measured by circling regions of interest on co mputer-generated gamma camera images. These image intensities were the n calculated as a function of total injected dose which was measured f rom a known volume and activity of Tc-99m-liposome-encapsulated hemogl obin. Actual tissue uptake was estimated from images by subtracting bl ood pool contribution which was measured by injecting Tc-99m-labeled r abbit red cells. Imaged organ biodistribution was validated at 20 hrs by measuring activity in weighed portions of tissue after autopsy. Mai n Results: The mean circulation half-life of liposome-encapsulated hem oglobin in mice injected at a dose of 1.0 g phospholipid/kg mouse and 1.95 g hemoglobin/kg was approximately 10.4 +/- 0.5 (SD) hrs. The circ ulation half-life of lyophilized liposome-encapsulated hemoglobin was 10.7 +/- 0.7 hrs. The circulation profiles demonstrate a rapid removal phase over the first 4 hrs after injection, followed by a secondary s low removal measured up to 24 hrs. The rapid removal phase of liposome -encapsulated hemoglobin and lyophilized liposome-encapsulated hemoglo bin in the rabbit (injected at the same dose) indicated that lyophiliz ed liposome-encapsulated hemoglobin persists longer than the unlyophil ized form in the first 4 hrs after injection. The organ biodistributio ns of unlyophilized Tc-99m-liposome-encapsulated hemoglobin and lyophi lized Tc-99m-liposome-encapsulated hemoglobin in the rabbit demonstrat e that the reticuloendothelial system is the primary site of removal, with significant uptake of lyophilized Tc-99m-liposome-encapsulated he moglobin by the liver (15.6 +/- 1.0%), bone marrow (12.6 +/- 1.6%), an d spleen (9.7 +/- 1.1%). The kidneys showed little accumulation of unl yophilized Tc-99m-liposome-encapsulated hemoglobin or lyophilized Tc-9 9m-liposome-encapsulated hemoglobin (1.6 +/- 0.2% and 1.8 +/- 0.1%, re spectively), an important result for the efficacy and safety of this h emoglobin-based blood substitute. Conclusion: The present results sugg est that liposome-encapsulated hemoglobin (and lyophilized Liposome-en capsulated hemoglobin) have pharmacokinetics that enable oxygen delive ry during early treatment for hemorrhagic shock. The organ biodistribu tion demonstrates that the monocyte phagocytic system is principally i nvolved with the slow removal of liposome-encapsulated hemoglobin (and lyophilized liposome-encapsulated hemoglobin) over the course of 24 h rs. The lyophilized liposome-encapsulated hemoglobin has similar pharm acokinetics to freshly prepared Liposome-encapsulated hemoglobin and c ould be an important storage strategy for the utilization of liposome- encapsulated hemoglobin in areas where stored blood is unavailable.