Circulation Kinetics and Organ Distribution of Hb-Vesicles Developed as a Red Blood Cell Substitute

Abstract

Phospholipid vesicles encapsulating concentrated human hemoglobin (Hb-vesicles, HbV), also known as liposomes, have a membrane structure similar to that of red blood cells (RBCs). These vesicles circulate in the bloodstream as an oxygen carrier, and their circulatory half-life times (t 1/2 ) and biodistribution are fundamental characteristics required for representation of their efficacy and safety as a RBC substitute. Herein, we report the pharmacokinetics of HbV and empty vesicles (EV) that do not contain Hb, in rats and rabbits to evaluate the potential of HbV as a RBC substitute. The samples were labeled with technetium-99m and then intravenously infused into animals at 14 ml/kg to measure the kinetics of HbV elimination from blood and distribution to the organs. The t 1/2 values were 34.8 and 62.6 h for HbV and 29.3 and 57.3 h for EV in rats and rabbits, respectively. At 48 h after infusion, the liver, bone marrow, and spleen of both rats and rabbits had significant concentrations of HbV and EV, and the percentages of the infused dose in these three organs were closely correlated to the circulatory half-life times in elimination phase (t 1/2␤ ). Furthermore, the milligrams of HbV per gram of tissue correlated well between rats and rabbits, suggesting that the balance between organ weight and body weight is a fundamental factor determining the pharmacokinetics of HbV. This factor could be used to estimate the biodistribution and the circulation time of HbV in humans, which is estimated to be equal to that in rabbit.Hemoglobin (Hb) isolated and purified from red blood cells (RBCs) has been tested as a principal component of RBC substitutes for carrying oxygen. However, the plasma retention time of isolated Hb is particularly short (half-life of ϳ0.5-1.5 h) because of the dissociation of the Hb tetramer into the dimeric form, which is subsequently filtered by the kidney, and it is known that this dimeric form is nephrotoxic (Savitsky et al., 1978). The potential of phospholipid vesicles as effective carriers of proteins and other bioactive materials has previously been proposed, since the cellular structure of such vesicles can protect the entrapped material from degradation and improve the biodistribution of proteins and other bioactive materials (Gregoriadis and Neerunjun, 1974;Papahadjopoulos et al., 1991). Phospholipid vesicles encapsulating concentrated Hb (HbV) have been proposed as a promising candidate RBC substitute, because encapsulation of Hb within a lipid membrane decreases potential side effects and toxicity of Hb, thereby making vesicles more RBC-like (Djordjevich and Miller, 1980;Gaber and Farmer, 1984;Tsuchida, 1998). The study of the safety and efficacy of HbV formulations by our research group has led to the development of an HbV formulation as a promising candidate for introduction into clinical trials (Tsuchida, 1998;Sakai et al., 2000bSakai et al., , 2001Sakai et al., , 2004bTakeoka et al., 2002).Determination of the circulation time (half-life) of vesicles has been an important...

Repeated Injection of High Doses of Hemoglobin-Encapsulated Liposomes (Hemoglobin Vesicles) Induces Accelerated Blood Clearance in a Hemorrhagic Shock Rat Model

“…Because this membrane reduces interactions between Hb and NO, adverse effects, such as hypertension and histological damage in myocardial lesions, are not induced, as are found for acellular-type HBOCs (Sakai et al, 2000(Sakai et al, , 2004a. In addition, there are some distinct advantages associated with the membrane structure of HbV as follows; the oxygen affinity (P 50 ) of HbV can be easily regulated by manipulating the content of an allosteric effector such as pyridoxal 5Ј-phosphate , an enhanced lifetime in the blood circulation compared with other types of HBOCs (Sou et al, 2005;Taguchi et al, 2009c), guarantees long-term storage for periods of more than 2 years at room temperature . Moreover, HbV possesses oxygen transport characteristics that are comparable to those of RBCs.…”

Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

“…In fact, HbVs have functioned well as blood substitutes in animal models with no noteworthy adverse reactions either in vivo Yoshizu et al, 2004;Cabrales et al, 2005) or in vitro (Ito et al, 2001;Wakamoto et al, 2005;Abe et al, 2006). They also reportedly accumulate in components of the MPS soon after their administration (Sou et al, 2005).…”

Hemoglobin Vesicles, Polyethylene Glycol (PEG)ylated Liposomes Developed as a Red Blood Cell Substitute, Do Not Induce the Accelerated Blood Clearance Phenomenon in Mice

“…Furthermore, the diameter of HbV liposomes can be tailored to approximately 250 nm, and further modification by PEG leads to an enhanced lifetime in the blood circulation compared with other types of hemoglobin-based oxygen carriers (t 1/2 for cell-free Hb and PEGylated Hb in rats of 1.5 and 10.9 h, respectively) (Goins et al, 1995;Lee et al, 2006) because the encapsulation of Hb completely suppresses renal excretion, although HbVs in the circulation are eventually captured by phagocytes in the mononuclear phagocyte system (MPS) (Sakai et al, 2001). In fact, our group reported that HbV has a long circulation time in blood as an oxygen carrier in mouse, rat, rabbit, and a hemorrhagic shock model rat (Sou et al, 2005;Taguchi et al, 2009a,b). Because of these unique characteristics, such liposomes show an oxygen transport comparable with red blood cells (Sakai et al, 2008) and also show improved survival in hemorrhagic shock animal models (Sakai et al, 2004b(Sakai et al, , 2009.…”