Takanobu Ishizuka

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.

Oxygen metabolism during cardiopulmonary bypass with hemodilution using liposome-encapsulated hemoglobin in kid goats

Cardiopulmonary bypass (CPB) with hemodilution has been proposed as a useful method for many types of cardiovascular surgery. Although the harmful effects of severe hemodilution need to be prevented, blood transfusion should be avoided whenever possible. Therefore, we have been developing a new CPB technique using liposome-encapsulated hemoglobin (LEH). The purpose of this study was to evaluate the combined therapy of diluted CPB and LEH focusing on the influence of LEH on oxygen metabolism. Male kid goats (n = 8) were divided into two groups: the LEH and control groups. CPB was maintained at between 36° and 37°C. There was no significant difference in hemoglobin concentrations (6.3 ± 1.5 g/dl in the LEH group and 6.2 ± 1.3 g/dl in the control group) after initiation of CPB between the two groups. Thus, there was no distinction in oxygen deliveries between the two groups (11.0 ± 2.0 ml/kg/min in the LEH group and 11.0 ± 2.3 ml/kg/min in the control group). Oxygen consumption in the LEH group (2.5–2.7 ml/kg/min), however, had a tendency to be higher than that in the control group (2.4–2.5 ml/kg/min). In addition, the lactate/pyruvate ratio decreased earlier in the LEH group. These results suggest that the application of LEH in the pump-priming solution improves decreased aerobic oxygen metabolism during CPB without any serious adverse effects.

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.