Shinichi Kaneda

Liposome-Encapsulated Hemoglobin, TRM-645: Current Status of the Development and Important Issues for Clinical Application.

Clinical application of artificial oxygen carriers as a substitute for blood transfusion has long been expected to solve some of the problems associated with blood transfusion. Use for oxygen delivery treatment for ischemic disease by oxygen delivery has also been examined. These prospective applications of artificial oxygen carriers are, however, still in development. We have developed liposome-encapsulated hemoglobin (LEH), developmental code TRM-645, using technologies for encapsulation of concentrated hemoglobin (Hb) with high encapsulation efficiency as well as surface modification to achieve stability in circulating blood and a long shelf life. We have confirmed the basic efficacy and safety of TRM-645 as a red blood cell substitute in studies on the efficacy of oxygen delivery in vivo, and the safety of TRM-645 has been studied in some animal species. We are now examining various issues related to clinical studies, including further preclinical studies, management of manufacturing and the quality assurance for the Hb solution and liposome preparations manufactured by the GMP facility.

Efficacy of liposome-encapsulated hemoglobin in a rat model of cerebral ischemia.

Use of liposome-encapsulated hemoglobin (LEH) for oxygen delivery in the treatment of cerebral ischemia has been studied previously and its expected benefits confirmed. However, the relationship between the timing of administration and the efficacy of LEH in cerebral ischemia has not been studied in detail. We therefore investigated the therapeutic time window of LEH by using a rat model of cerebral ischemia, as well as evaluating the contribution of oxygen delivery to the efficacy of LEH. Dose-dependent effects and the therapeutic time window of LEH were studied using models of transient and permanent middle cerebral artery occlusion (MCAO), respectively, in SD rats. LEH was intravenously administered at 0.5 h after the onset of ischemia in the transient MCAO model and at 0.5, 2, 4, or 6 h in the permanent MCAO model. Efficacy of LEH treatment was evaluated using the infarct volume, which was examined with 2,3,5-triphenyltetrazolium chloride staining and estimated by integrating the unstained areas in serial sections of cerebral tissue. Effects of oxygen delivery by LEH were examined immunohistochemically with pimonidazole to stain for areas of low oxygen tension in the tissue. LEH treatment dose-dependently reduced the cerebral infarct volume, which was especially significant in the cortical region at doses of over 60 mg hemoglobin (Hb)/kg. In rats with permanent MCAO, LEH administration at a dose of 300 mg Hb/kg at 0.5 h and 2 h after the onset of cerebral ischemia significantly reduced cerebral infarct volume. Furthermore, immunohistochemical staining with pimonidazole showed that the areas of cerebral tissue that were hypoxic and had abnormal histological structure were reduced after LEH treatment. These results indicated that LEH is efficacious in the treatment of cerebral infarction secondary to MCAO and that oxygen delivery to ischemic cerebral tissues by LEH administered early after the onset of cerebral ischemia contributes to this effect.

Prednisolone phosphate-containing TRX-20 liposomes inhibit cytokine and chemokine production in human fibroblast-like synovial cells: a novel approach to rheumatoid arthritis therapy.

To evaluate the potential of using prednisolone phosphate (PSLP)‐containing 3,5‐dipentadecyloxybenzamidine hydrochloride (TRX‐20) liposomes to treat rheumatoid arthritis (RA), we examined their ability to bind human fibroblast‐like synovial (HFLS) cells and their effects in these cells. To test for binding, Lissamine rhodamine B‐1, 2‐dihexadecanoyl‐sn‐glycero‐3‐phosphoethanolamine (rhodamine)‐labelled PSLP‐containing TRX‐20 liposomes were added to HFLS cells, and the fluorescence intensity of the rhodamine bound to the cells was evaluated. Rhodamine‐labelled PSLP‐containing liposomes without TRX‐20 were used as a negative control. To evaluate the uptake of liposomes by the HFLS cells, we used TRX‐20 liposomes containing 8‐hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS) and p‐xylene‐bis‐pyridinium bromide (DPX), and observed the cells by fluorescence microscopy. The effects of the PSLP in TRX‐20 liposomes on HFLS cells were assessed by the inhibition of the production of two inflammatory cytokines (interleukin 6 and granulocyte macrophage colony‐stimulating factor) and one inflammatory chemokine (interleukin 8). The interaction of the PSLP‐containing TRX‐20 liposomes with HFLS cells was approximately 40 times greater than that of PSLP‐containing liposomes without TRX‐20. PSLP‐containing TRX‐20 liposomes bound to HFLS cells primarily via chondroitin sulfate. TRX‐20 liposomes taken up by the cell were localized to acidic compartments. Furthermore, the PSLP‐containing TRX‐20 liposomes inhibited the production of the inflammatory cytokines and the chemokine more effectively than did the PSLP‐containing liposomes without TRX‐20. These results indicate that PSLP‐containing TRX‐20 liposomes show promise as a novel drug delivery system that could enhance the clinical use of glucocorticoids for treating RA.

Efficacy of peritoneal oxygenation using a novel artificial oxygen carrier (TRM-645) in a rat respiratory insufficiency model.

PurposeSupplemental oxygenation is essentially important in critically ill patients with potentially reversible pulmonary insufficiency. An extracorporeal membrane oxygenator and percutaneous cardiopulmonary support have been used for these patients. However, these techniques are associated with so many complications that an additional new therapeutic modality is required. The purpose is to investigate if the peritoneal cavity can be used as “extrapulmonary respiration” that is analogous to peritoneal dialysis and utilizes the efficacy of liposome-encapsulated hemoglobin (artificial oxygen carrier; TRM-645).MethodsRats weighing an average of 300 g (n = 18) received an incision in the right chest to generate pneumothorax, which resulted in severe and lethal hypoxia. Oxygenated TRM-645 and human red blood cells (MAP group) were administered into the peritoneum in the experimental rats’ pneumothorax model. No treatment except the right pneumothorax was administered to the sham group.ResultsSurvival times from the pneumothorax were significantly longer in the TRM-645 and MAP groups than in the sham group (32.0 ± 6.9 and 22.0 ± 4.9 min vs 9.2 ± 1.9 min, P < 0.01). In addition, an arterial blood gas analysis showed that the oxygenation in levels significantly improved.ConclusionsThe abdomen (peritoneum) can potentially become an “artificial lung” that can be employed in critical care settings. TRM-645 provides an alternative to the use of washed human red blood cells.

Intraosseous Transfusion With Liposome-encapsulated Hemoglobin Improves Mouse Survival After Hypohemoglobinemic Shock Without Scavenging Nitric Oxide

Recently, we developed liposome-encapsulated hemoglobin (LEH), a novel cellular hemoglobin-based oxygen carrier. We hypothesized that the LEH effectively suppresses scavenging of nitrogen oxides by sequestering hemoglobin, thereby being useful for resuscitation from hemorrhagic shock, especially in prehospital settings where blood transfusion is not available. However, putting a catheter into the peripheral vessels is sometimes difficult in prehospital resuscitation, because these vessels collapse in patients with hemorrhagic shock. The intraosseous route does not collapse under such conditions. We here studied the resuscitation of severe hypohemoglobinemia following massive hemorrhage using intraosseous (intrafemur) transfusion with LEH in mice. First, we examined the effect of intravenous transfusion with LEH on the resuscitation of mice with fatal hypohemoglobinemia that was made with progressive hemodilution by blood exchanges. Despite a success in initial resuscitation without scavenging of NO2− or NO3−, LEH transfusion did not significantly improve mouse survival 72 h later as compared with red blood cell (RBC) transfusion. In other experiments, hypohemoglobinemic mice were also made with blood withdrawal and intraosseous infusion with 5% albumin. Thereafter, the mice were rescued with intraosseous transfusion of LEH or RBCs. Unlike intravenous transfusion, intraosseous transfusion with LEH (but not such transfusion with RBCs) significantly increased mouse survival without scavenging of NO2− or NO3−, presumably because LEH vesicles were much smaller than RBCs, thereby effectively flowing into the circulation from the femur. Thus, intraosseous transfusion with LEH may be a candidate strategy for efficient prehospital resuscitation from hemorrhagic shock.

Liposome-Encapsulated Hemoglobin as an Artificial Oxygen Carrier: Technological Features, Manufacturing and Issues for Practical Application

While the use of liposome-encapsulated hemoglobin (LEH) as artificial oxygen carriers (AOCs) has long been studied, its complicated manufacturing process and large production costs have hindered the progress in its development and, subsequently, its advancement to practical applications. However, we have been persistently developing LEH as an analogue of red blood cells (RBCs) for its use as AOCs, and to this end we have constructed a Good Manufacturing Practice (GMP) facility where LEH can be manufactured as biologics under quality assurance systems as well as establishing the manufacturing processes of LEH at this facility. We have also clarified the physicochemical properties and stability of LEH, and further confirmed the fundamental efficacy and safety of LEH as AOCs. Although the technology of LEH has already reached the stage of clinical testing, its high manufacturing costs remains an unsolved issue if LEH is to be used as a medicinal product.

Liposome-encapsulated hemoglobin attenuates cardiac dysfunction and sympathetic activity during hypohemoglobinemic shock.

ABSTRACT The use of liposome-encapsulated hemoglobin (LHb), which is a cellular Hb, has been demonstrated to be beneficial in the treatment of hypohemoglobinemic shock. As a molecule of appropriate size (220 nm) that can carry oxygen, LHb may ameliorate cardiac dysfunction during lethal hemodilation. The purpose of this study was to determine the efficacy of LHb transfusion in relieving cardiovascular dysfunction in a rat model of lethal progressive hemodilution. Over the course of 150 min, rats were subjected to blood withdrawal (0.2 mL/min) and simultaneously transfused with LHb, washed rat red blood cells, or 5% albumin. Temporal changes in cardiac function, heart-type fatty acid–binding protein levels, plasma levels of catecholamines, heart rate variability, and hypoxia-inducible factor 1&agr; expression were measured during lethal progressive hemodilution. More than 80% of the rats transfused with either LHb or washed rat red blood cells survived for 8 days. Liposome-encapsulated hemoglobin transfusion suppressed hypoxia-inducible factor 1&agr; expression in the heart, maintained low levels of heart-type fatty acid–binding protein, and attenuated sympathetic nerve activity as reflected by changes in heart rate variability and plasma levels of epinephrine and norepinephrine. The results indicate that LHb attenuates cardiac dysfunction and sympathetic overactivity during lethal hemorrhage.

Characteristic Changes in Heart Rate Variability Indices during Hemorrhagic Shock, and Effect of Liposome-Encapsulated Hemoglobin in Rats

Many compensatory mechanisms exit in hemorrhagic shock (HS). To characterize the efficacy of the new artificial oxygen carrier, liposome‐encapsulated hemoglobin (LHb), HS was induced by withdrawing 20% of the total blood volume from rats. Rats received one of five interventions: LHb resuscitation (LHb‐G, n = 7), normal saline (Saline‐G, n = 7), shed autologous blood (SAB‐G, n = 7), volume expander of 5% albumin (Albumin‐G, n = 7), or no treatment (Sham‐G, n = 7). Heart rate variability (HRV) indices were measured, including low frequency (LF, 0.10–0.60 Hz), high frequency (HF, 0.60–2.00 Hz), and the ratio of LF to HF (LF/HF). LF and LF/HF following HS were lower in the LHb‐G and SAB‐G groups when compared with the Saline‐G, Albumin‐G and Sham‐G groups. LF and LF/HF following HS in the LHb‐G group were comparable with that of the SAB‐G group. These data demonstrate that HS‐induced changes can be attenuated by resuscitation with LHb as well as SAB. LHb could be used as a substitute for blood transfusion for HS.