2015 Volume 38 Issue 10 Pages 1606-1616
The hemoglobin-vesicle (HbV), a vesicle in which a concentrated human hemoglobin solution is encapsulated, was developed as an artificial oxygen carrier. Although HbV has a favorable safety, metabolic, and excretion performance in healthy animals, the effect of a massive amount of HbV, which also contains a large amount of a lipid component including cholesterol, on physiological response and metabolic performance under hyperlipidemic conditions is unclear. The aim of this study was to evaluate whether administration of HbV causes toxicity in apolipoprotein E-deficient mice (hyperlipidemic model mice). Apolipoprotein E-deficient mice were given a single injection of HbV (2000 mg hemoglobin/kg), and physiological responses and metabolic profiles were monitored for 14 d thereafter. All the mice tolerated the massive amount of HbV and survived, and adequate biocompatibility was observed. Serum biochemical parameters indicate that liver and kidney function were not remarkably affected, and morphological changes in the liver and spleen were negligible. Lipid parameters in serum were significantly increased until 3 d after HbV administration, but recovered within 7 d after the administration. In a pharmacokinetic study, HbV was mainly found distributed in the liver and spleen, and disappeared from the body within 14 d. In conclusion, even under conditions of hyperlipidemia, a massive dose of HbV and its components resulted in favorable biological compatibility, metabolic, and excretion profiles. These findings provide further support for the safety of HbV for clinical use.
It is well known that liposomes (phospholipid vesicles) can be used as a carrier for some drugs, genes, proteins, and that they can enhance the blood circulation and specific targeting of encapsulated materials. Given these characteristics, many liposome type drugs have now been approved for clinical use in antifungal or anticancer therapies.1) In addition to their use in transporting drugs, phospholipid vesicles encapsulating concentrated hemoglobin (Hb), referred to as hemoglobin vesicles (HbV), have also been developed as artificial oxygen carriers. HbV has been shown to possess several superior characteristics to red blood cell (RBC) transfusions including the absence of viral contamination, a long-term storage period at room temperature of over 2 years and no need for cross-matching etc.2) Furthermore, it has been clearly shown that the transport of oxygen by HbV is comparable to RBC in hemorrhagic shock models.2,3) Based on these facts, HbV appears to have potential for use as an alternative to RBCs, and is expected to be of use in other clinical indications that cannot be solved with conventional blood transfusions in clinical settings.
It is very important to accumulate data related to the overall safety of HbV because high doses of HbV, in excess of hundreds of times higher than that of other commercially available liposomal formulations of doxorubicin and amphotericin B, would be required in some clinical settings. This means that a massive amount of HbV components, including Hb, lipids (cholesterol and phospholipids) and iron (heme) derived from Hb, would also enter the body. Free Hb molecules can trigger numerous side effects, such as renal failure, hypertension, and tissue damage induced by the Fenton reaction, which is mediated by heme (iron) derived from Hb, when the free Hb concentration exceeds the haptoglobin binding capacity.4,5) Encapsulation of Hb in phospholipid vesicles shields the above toxic effect of Hb. However, high levels of lipid components in the circulation, especially cholesterol, are risk factors for kidney disease, arterial sclerosis, and hyperlipidemia.6) Therefore, HbV and its components need to have favorable metabolic and excretion profiles in a wide variety of situations if they are to be used as a substitute for RBC in cases of emergencies.
In our previous studies, we reported that HbV has an appropriate metabolism and excretion profile after the administration of a putative clinical dose of HbV based on the histology, biochemical analysis and pharmacokinetic properties in healthy rodents and monkeys.7–10) In addition, the cholesterol contained in HbV behaves similar to endogenous cholesterol after the metabolism of HbV.9) Similar results were observed under conditions of hemorrhagic shock and chronic liver impairment.3,11,12) Therefore, these findings strongly indicate that HbV and its components appear to show favorable metabolic and excretion profiles not only in healthy but in certain types of diseases (hemorrhagic shock and chronic liver impairment).
As of one problems besetting modern society, the numbers of patients with hyperlipidemia or potential hyperlipidemia are continuously increasing world-wide. Some of these patients are not able to adequately metabolize lipid components including cholesterol, and the lipids accumulate in the blood stream and organs, which consequently increase the risk of developing serious disorders such as arterial sclerosis and cardiovascular disease. As mentioned above, a massive amount of lipids (cholesterol and phospholipids) are infused into body when HbV is used in a clinical situation, which means that the infusion of HbV at a putative clinical dose may show toxic properties under conditions of hyperlipidemia due to their poor metabolic and excretion profiles. Thus, before proceeding to a clinical evaluation, it becomes necessary to determine whether HbV could be safely used as a RBC alternative under conditions of hyperlipidemia. However, to date, this issue has not been addressed nor has it be studied extensively.
The aim of this study was to investigate the effects of a bolus administration of HbV on hyperlipidemic conditions, as well as to identify any potential side effects, for possible future clinical applications of HbV as a RBC alternative at emergency medical situations. For this purpose, we carried out a safety and toxicological assessment of HbV, including plasma biochemical parameters, histological staining and pharmacokinetics, after a bolus intravenous administration of HbV at a dose of 2000 mg Hb/kg using hyperlipidemic model mice (apolipoprotein E (ApoE) deficient mice: B6.KOR/StmSlc-Apoeshl mice), and subsequently compared the findings for corresponding data for healthy mice (C57BL/6N mice).
All animal experiments were reviewed and approved by the Animal Care and Use Committee of Sojo University (Permit number: 2013-P-021). The care and handling of the animals were in accordance with the NIH guidelines. Experiments involved the use of male C57BL/6N mice (15–18 g body weight; Kyudou Co., Kumamoto, Japan) and male B6.KOR/StmSlc-Apoeshl mice (18–21 g body weight; Japan SLC, Inc., Shizuoka, Japan). All animals were maintained under conventional housing conditions, with food and water ad libitum in a temperature-controlled room with a 12-h dark/light cycle.
Preparation of HbV ParticlesHbV particles were prepared under sterile conditions, as previously reported.13) Briefly, an Hb solution was purified from outdated RBCs donated by the Japanese Red Cross Society (Tokyo, Japan). Then, Hb was stabilized by carbonylation and concentrated using ultrafiltration to 38 g/dL. The carbonyl Hb solution was mixed with lipids and encapsulated in vesicles. The particle diameter was regulated using the extrusion method. The encapsulated carbonylHb was converted to oxyhemoglobin by exposing the liquid membranes of HbVs to visible light under an aerobic atmosphere. The encapsulated Hb (38 g/dL) contained 14.7 mM of Pyridoxal 5′-phosphate as an allosteric effector to regulate oxygen affinity. The lipid bilayer was a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, and 1,5-bis-O-hexadecyl-N-succinyl-L-glutamate (DHSG) at a molar ratio of 5/4/0.9, and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-N-polyethylene glycol (PEG)5000 (0.3 mol%). A concentrated Hb solution, nearly 35 wt%, is encapsulated within bilayler membrane. The HbV particles were suspended in a physiological salt solution at [Hb]=10 g/dL and [lipids]=8 g/dL. The particle diameter and zeta-potential were 250–280 nm and −18.7 mV, respectively. The resulting suspension was deoxygenated by exposure to nitrogen gas prior to storage.
Injection of a HbV Suspension and Sample (Blood and Organs) CollectionOn day 0, C57BL/6N mice (n=20) and B6.KOR/StmSlc-Apoeshl mice (n=20) received a single intravenous infusion of HbV, administered as a transfusion of 20 mL/kg (2000 mg Hb/kg) under ether anesthesia, or were injected with saline (20 mL/kg) as a control (C57BL/6N mice; n=20, B6.KOR/StmSlc-Apoeshl mice; n=20). The injected volume of HbV (2000 mg Hb/kg, 20 mL/kg) is the same as our previous safety and toxicology studies of HbV using rodent7,8,14) that corresponded to nearly 28% of mouse blood volume (72 mL/kg).15) At days 1, 3, 7 and 14 after the HbV injection, five mice were randomly selected from each group for collection of blood and organs. After collecting blood from the inferior vena cava, the mice were sacrificed by acute bleeding from the abdominal aorta and organs (kidneys, liver, spleen, lungs and heart) obtained. The organs were then weighed and resected en bloc for a histropathological study. The organs were fixed in 4% paraformaldehyde overnight.
Measurement of Hematology and Serum ChemistryHematology (white blood cell (WBC), RBC and platelet (PLT)) analyses of aliquots of blood samples were performed using Celltac α (MEK-6458, NIHON KOHDEN, Tokyo, Japan). The remaining venous blood was centrifuged (1710×g, 10 min) to obtain serum. The serum samples were then ultracentrifuged to remove HbV (50000×g, 30 min), because HbV interferes with some of the laboratory tests.16) All serum samples were stored at −80°C until used. The analyses performed were total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, triglyceride (TG), phospholipids, free fatty acid (FFA), total cholesterol (T-cholesterol), cholesterol ester, free cholesterol. BUN and creatinine were determined by using a urea nitrogen-B test kit and LabAssay creatinine test kit from Wako Pure Chemical Industries, Ltd. (Saitama, Japan), respectively. Others were determined by a commercial clinical testing laboratory (SRL, Tokyo, Japan).
Histopathological ExaminationThe organs, fixed in 4% paraformaldehyde overnight, were embedded in paraffin (kidneys, liver, spleen, lungs and heart) for hematoxylin/eosin (H&E) and Berlin blue staining, and a part of the liver and spleen were embedded in optical cutting temperature (O.C.T.) compound for oil red O staining. The organs were sectioned into 5-µm slices. Morphological changes in each organ were confirmed by H&E staining. The presence and location of hemosiderin, including free iron released by the metabolism of heme, were confirmed by Berlin blue staining. The neutral lipid deposition were examined by oil red O staining.
Pharmacokinetic Study3H-HbV, in which the lipid component (cholesterol) was radiolabeled, was prepared as previously reported.9) In a typical preparation, HbV (1 mL) was mixed with a [1,2-3H(N)]-cholesterol in ethanol (40 µL), (PerkinElmer, Inc., Yokohama, Japan) and the solution incubated for 12 h at 37°C. B6.KOR/StmSlc-Apoeshl mice (n=20) were anesthetized with ether and received a single injection of a 3H-HbV suspension (20 mL/kg, 2000 mg Hb/kg). Five mice were randomly selected at days 1, 3, 7 and 14 after the injection of 3H-HbV, and the blood samples were collected from the inferior vena cava. The blood was centrifuged (1710×g, 10 min), and the obtained plasma samples were then ultracentrifuged to separate HbV and other plasma phase (50000×g, 30 min). After collecting the blood samples, the mice were sacrificed and the organs (kidneys, liver, spleen, heart and lungs) were collected. The radioactivity was determined by liquid scintillation counting (LSC-5121, Aloka, Tokyo, Japan), as previously reported.9)
Data AnalysisData are shown as the mean±standard deviation (S.D.) for the indicated number of animals. Significant differences among each group were determined using the two-tail unpaired Student’s t-test. A probability value of p<0.05 was considered to indicate a statistical significance.
All of the mice survived up to 14 d after the HbV administration and none were on the verge of death. In addition, no abnormal behavior or appearance were observed, i.e., aggressive behavior or a pilomotor response after HbV administration. Body weights of the HbV group were significantly decreased at 1 d compared to that of the saline group in both C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice (Fig. 1A). However, the mice in the HbV group gained weight starting at 3 d after the HbV administration, as did the mice in the saline group. These data indicate that the effect of HbV on physiological functions and the suppression of growth was negligible under conditions of hyperlipidemia.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV (20 mL/kg, 2000 mg Hb/kg), and observed change in (A) body weight, (B) liver and (C) spleen weight at 1, 3, 7 and 14 d after saline or HbV administration. The values are the mean±S.D. (n=5). * p<0.05, ** p<0.01 vs. saline.
Figures 1B and C show the changes in liver and spleen weight, expressed as the percentage of organ weight relative to the body weight. The liver weight was significantly increased at 3 d after the HbV administration compared to saline administration in both the C57BL/6N and B6.KOR/StmSlc-Apoeshl mice (Fig. 1B). The spleen weight of the HbV group was increased by a maximum of 2-fold and 1.5-fold compared to that of saline group in C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice, respectively, but it was recovered within 14 d after the HbV administration (Fig. 1C). The other organ weights (kidney, lung and heart) relative to body weight remained essentially unchanged at each day after HbV administration (data not shown). Previous studies using healthy rats also showed transient splenomegaly and hepatomegaly after an HbV administration.7,8) It has been reported that HbV are mainly captured by liver Kupffer cells and splenic macrophages in mice and rats.8,9) Thus, the splenomegaly and hepatomegaly appear to be due to the effect of the entrapment of HbV by the mononuclear phagocyte system (MPS) in the liver and spleen.
HematologyFigure 2 shows the changes in hematology (WBC, RBC and PLT) after HbV administration in C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice. The number of WBC remained essentially unchanged after HbV administration in the case of the B6.KOR/StmSlc-Apoeshl mice, except for a slight decrease at 7 d after the HbV administration (Fig. 2A). On the other hand, the HbV group showed a reduction WBC count in C57BL/6N mice compared to the saline group. No abnormal changes in the number of red blood cells and PLT was observed in either the C57BL/6N mice or the B6.KOR/StmSlc-Apoeshl mice (Figs. 2B, C). Some anionic vesicles, including liposomes, are known to induce complement activation, which can lead to the development of thrombocytopenia.17,18) In a previous study, the transfusion of prototype HbV (containing no DHSG but 1,2-dipalmitoyl-sn-glycelo-phosphatidylglycerol (DPPG), no PEG modification) engendered transient thrombocytopenia due to complement activation.2) However, this problem could be overcome by changing the lipid component from DPPG to DHSG and by modifying the lipid membrane with PEG. In fact, the vesicle formulation for HbV used in this study did not induce thrombocytopenia in vivo in a rodent animal model19,20) or in vitro complement activation using human plasma.21) Furthermore, it was reported that 10 and 5% of the HbV was distributed in the bone marrow in rats and rabbits, respectively.22) Therefore, concern has arisen as to whether the HbV distributed in bone marrow might suppress hematopoiesis due to myelosuppression associated with the administration of HbV. However, it was demonstrated that HbVs, under conditions relevant to a clinical setting, have no adverse effects on human umbilical cord blood hematopoietic progenitor activity in vitro.23) Taking together with the present study and previous findings, HbV has adequate blood compatibility even under conditions of hyperlipidemia.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV (20 mL/kg, 2000 mg Hb/kg), and observed changes in (A) white blood cells (WBC), (B) red blood cells (RBC) and (C) platelets (PLT) at 1, 3, 7 and 14 d after saline or HbV administration. The values are the mean±S.D. (n=5). * p<0.05, ** p<0.01 vs. saline.
The parameters reflecting liver function (T-protein, albumin, AST and ALT) are shown in Fig. 3. No abnormal changes in T-proteins or albumin were found up to 14 d after HbV administration in C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice (Figs. 3A, B). While AST and ALT levels were maximally increased at 1 d after HbV administration in C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice, except for ALT in C57BL/6N mice, their values returned to levels comparable to this for the saline administration group within 3 d after HbV administration (Figs. 3C, D). A similar phenomenon was observed in previous studies using healthy rats and monkeys.7,10) Since the liver is one of the main organs for the metabolism of HbV,8,9) an extra load on the liver during the metabolism of the massive amounts of HbV might result in elevated AST and ALT levels. However, changes in the levels of aminotransferases were transient and no changes in any of the other parameters reflecting liver function were detected.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV (20 mL/kg, 2000 mg Hb/kg), and serum laboratory tests for liver function were performed at 1, 3, 7 and 14 d after saline or HbV administration. The values are the mean±S.D. (n=5). * p<0.05, ** p<0.01 vs. saline. T-protein; total protein, AST; aspartate aminotransferase, ALT; alanine aminotransferase.
It is well-known that the Hb derived from hemolysis causes renal toxicity by the dissociation of tetrameric Hb subunits into two dimers, extravasaion, and precipitation in tubules.5) Thus it becomes necessary to carefully monitor renal functions after a massive HbV administration. As shown in Fig. 4, BUN and creatinine, which reflect renal function, were in the normal range during the 14 d after the HbV administration, as compared to saline administration in both C57BL/6N mice and B6.KOR/StmSlc-Apoeshl mice. This nonrenal toxicity of HbV could be explained by the characteristics of HbV as follows: (i) the HbV structure is maintained intact in the circulation,9) (ii) Hb encapsulated into the HbV was completely degraded by MPS.7,9) These results indicate that HbV administration does not appear to induce either hepatic injury or renal injury under conditions of hyperlipidemia.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV administration (20 mL/kg, 2000 mg Hb/kg), and analyzed serum laboratory tests representing renal function at 1, 3, 7 and 14 d after saline or HbV administration. The values are the mean±S.D. (n=5). * p<0.05, ** p<0.01 vs. saline. BUN; blood urea nitrogen, CRE; creatinine.
Since HbV contains high amounts of lipids, including cholesterol and phospholipids, there is a concern that they could accumulate in the body due to a lack of lipid metabolism after HbV administration in the hyperlipidemic mice model (B6.KOR/StmSlc-Apoeshl mice). Therefore, we next examined the changes in lipid parameters that are related to metabolites of the lipid components of HbV.
The concentration of the TG and FFA remained essentially unchanged in C57BL/6N mice (Figs. 5A, C). The levels of phospholipids, T-cholesterol, cholesterol ester and free cholesterol were significantly increased at days 1 and 3 after HbV administration compared to the saline administration group, but returned to the same levels as the saline administration group within 7 d after the administration of HbV in C57BL/6N mice (Figs. 5B, D–F). These changes in lipid parameters are similar to results observed for healthy rats.7) On the other hand, the changing trend in the levels of lipid parameters in B6.KOR/StmSlc-Apoeshl mice were different than the values for the C57BL/6N mice after a massive administration of HbV. Before the HbV administration, a marked hypercholesterolemia was observed in the B6.KOR/StmSlc-Apoeshl mice, previously reported.24) After HbV administration to the B6.KOR/StmSlc-Apoeshl mice, phospholipids, FFA, T-cholesterol, cholesterol ester and free cholesterol were markedly increased from 1 d after the HbV administration and reached a maximum at day 3 (Figs. 5B–F). However, these values returned to levels comparable to those for the saline administration group at day 7 after the HbV injection. The concentration of TG was slightly increased at 3 d after the HbV administration, but decreased significantly compared to the saline administration group at day 7 (Fig. 5A). ApoE plays an important role in the transport of cholesterol to the liver via the circulation (metabolism of cholesterol), because ApoE is a ligand for receptors that clear remnants of lipoproteins. Thus, the level of cholesterol after HbV administration increased and its excretion from the circulation was delayed in ApoE deficient mice (B6.KOR/StmSlc-Apoeshl mice). However, these levels returned to levels comparable to the saline administration group within 7 d after the HbV injection. It is known that, not only ApoE, but also apolipoprotein B-100 (ApoB-100) are strongly related to plasma lipoprotein metabolism through their high affinity binding to lipoprotein receptors.25) Thus, the cholesterol derived from HbV would be transported and metabolized in B6.KOR/StmSlc-Apoeshl mice via ApoB-100 mediated pathways. Furthermore, several lines of evidence suggest that the affinity of binding of ApoE to lipoprotein receptors is higher than that of ApoB-100,26–28) accounting for the much longer residence time in the blood in the case of the B6.KOR/StmSlc-Apoeshl mice, compared to the C57BL/6N mice.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV (20 mL/kg, 2000 mg Hb/kg), and serum laboratory tests for lipid metabolism were performed at 1, 3, 7 and 14 d after saline or HbV administration. The values are the mean±S.D. (n=5). * p<0.05, ** p<0.01 vs. saline. TG; triglyceride, FFA; free fatty acid, T-cholesterol; total cholesterol.
The histological findings for the kidney, liver, spleen, lung and heart were observed by H&E staining. Morphological changes in the kidney, lung and heart were negligible after an injection of HbV at a dose of 2000 mg Hb/kg in B6.KOR/StmSlc-Apoeshl mice (data not shown). On the contrary, visible accumulation of HbV in the liver and spleen were observed days 1 and 3 after HbV administration, and they disappeared within 14 d after the HbV administration (Fig. 6). As shown in Figs. 1B, C, transient splenomegaly and hepatomegaly are observed after HbV administration, and this phenomena seems to be due to the accumulation of HbV in the liver and spleen. This speculation was supported by the histological examination shown in Fig. 6, because the increased hepatic and splenic weight was entirely consistent with the extent of hepatic and splenic accumulation of HbV observed in Fig. 6.
C57BL/6N mice (left) and B6.KOR/StmSlc-Apoeshl mice (right) received a single intravenous infusion of saline (20 mL/kg) or HbV (20 mL/kg, 2000 mg Hb/kg), and collected organs. Light micrographs of liver and spleen in B6.KOR/StmSlc-Apoeshl mice were observed at 1, 3, 7 and 14 d after HbV injection stained with H&E. HbV accumulation (arrow) was observed in the liver and spleen at 1 d and 3 d after injection of HbV. Scale bar: 100 µm.
In a previous study, using normal rats, lipid deposition was observed in the liver 3 d after an HbV injection,8) which might be generated during the metabolism of the phospholipid components of the bilayer membrane of HbV. Therefore, we also performed oil red O staining to evaluate the extent of lipid deposition in the liver in B6.KOR/StmSlc-Apoeshl mice. As a result, lipid deposition was not observed at 1, 3, 7 and 14 d after the HbV administration (data not shown). In addition, hemosiderin, excess iron (heme) stored as an inert form, was concentrated in the liver and spleen after a massive HbV injection in healthy rats.8) Thus, we also performed Berlin blue staining to evaluate the extent of decomposition of hemosiderin in the liver and spleen in B6.KOR/StmSlc-Apoeshl mice. No evidence of hemosiderin deposition was detected in the liver and spleen after the injection of HbV (data not shown). These results indicate that morphological changes and the accumulation of lipids and heme are negligible after the administration of HbV to B6.KOR/StmSlc-Apoeshl mice.
PharmacokineticsFinally, we carried out a pharmacokinetic analysis of the lipid component of HbV in B6.KOR/StmSlc-Apoeshl mice using 3H-HbV. Plasma samples were centrifuged in order to measure 3H radioactivity separately as intact HbV and its metabolites such as free cholesterol and lipoprotein (Fig. 7A). The findings show that most of the intact HbV was cleared from blood circulation within 3 d after a bolus injection of HbV (Fig. 7B). However, approximately 5% of the metabolites of the lipid components (cholesterol) of HbV remained in the blood stream at 14 d after the HbV administration in B6.KOR/StmSlc-Apoeshl mice (Fig. 7B). These results are in contrast to serum laboratory tests, as shown in Fig. 5, which revealed that the level of lipid parameters returned to levels comparable to those for the saline administration group within 7 d after HbV injection in B6.KOR/StmSlc-Apoeshl mice. The cholesterol of the vesicles should reappear in the blood mainly as lipoprotein cholesterol after entrapment in Kupffer cells and should then be excreted in the bile after entrapment of the lipoprotein cholesterol by hepatocytes,29) which is as same as endogenous cholesterol by physiological pathways. Therefore, this consistency between serum laboratory tests and a pharmacokinetic study can be explained by (i) the competitive excretion of endogenous cholesterol and exogenous cholesterol derived from HbV due to their overlapping excretion pathway and/or (ii) cholesterol derived from HbV is reused in materials such as cell membranes. Therefore, it would be desirable to have information concerning whether the lipid components (cholesterol) in HbV behaved the same as endogeneous cholesterol after the metabolism of HbV in MPS even under hyperlipidemic conditions.
Similar to other healthy animals such as mice, rats and rabbits,9,22) the major organs where HbV is distributed were the liver and spleen in B6.KOR/StmSlc-Apoeshl mice, and the maximum hepatic and splenic distributions of 3H-HbV in B6.KOR/StmSlc-Apoeshl mice were observed at 1 d after HbV injection (Figs. 7C, D). However, the 3H radioactivity had nearly disappeared from each organ by 14 d after the HbV injection. These biodistribution of labeled HbV are in good agreement with histological examination as shown in Fig. 6. These results indicate that HbV has a good metabolic and excretion performance even under hyperlipidemic conditions from the view point of pharmacokinetics.
(A) Scheme showing the centrifugation procedure used for the separation of plasma samples into intact HbV and its metabolites. The radioactivity (% of dose) in the (B) plasma, (C) organs (kidney, liver, spleen, lung, heart) at 1, 3, 7 and 14 d after 3H-HbV administration (20 mL/kg, 2000 mg Hb/kg) in B6.KOR/StmSlc-Apoeshl mice. (D) The radioactivity/gram of organ weight (% of dose /g tissue) at 1, 3, 7 and 14 d after 3H-HbV administration (20 mL/kg, 2000 mg Hb/kg) in B6.KOR/StmSlc-Apoeshl mice. The values are the mean±S.D. (n=5). The “Others” in (B) are corresponded to the “supernatant (lipoproteins, free cholesterol etc.)” in (A).
We demonstrate herein that HbV and its components perform favorably, in terms of biological compatibility, metabolism and excretion, under conditions of heperlipidemia. In addition to functioning as a RBC substitute, HbV would be expected to have a variety of other applications, based on its oxygen and carbon monoxide transport characteristics, such as its use in acute ischemic strokes,30) organ storage solutions,31) and as pulmonary fibrosis.32) Thus, the findings reported here not only clarify the safety of HbV based on physiological responses and pharmacokinetic profiles under conditions of hyperlipidemia but also provide the possibility that HbV has considerable promise for use as both a RBC substitute and other disorders in the clinic in the future.
We wish to thank the members of the RI Center in Kumamoto University for their important contributions to pharmacokinetic experiments. This work was supported in part by Health Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan and by a Grant-in-Aid for Young Scientist (B) from the Japan Society for the Promotion of Science (JSPS) (KAKENHI 26860121).
The authors declare no conflict of interest.
