Tetsuhiro Kimura

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.

Development of Neo Red Cells(NRC) with the enzymatic reduction system of methemoglobin

To assess the oxygen transport capacity and safety of Neo Red Cells (NRC) with the enzymatic reduction system of methemoglobin in vitro and in experimental animals. Stroma free hemoglobin (SFH) prepared without damage of enzymes from outdated human red blood cells, together with inositol hexaphosphate as an allosteric effector, NAD as a coenzyme and glucose, adenine and inosine as a substrate was encapsulated within liposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, myristic acid and alpha-tocopherol in the ratio of 7:7:2:0.28 respectively. NRC thus prepared with a mean diameter of 220 nm, encapsulation efficiency of 1.3 g-Hb:1 g-lipid and P50O2 of 50-60 mmHg were then coated with polyethylene glycol bound to hydrogenated soy phosphatidylethanolamine as a surface modifier to prevent aggregation of NRC in plasma. The methemoglobin formation of the NRC with enzymatic reduction system were evaluated by in-vitro examination and exchange transfusion with rats as in-vivo examination, then the methemoglobin formation was reduced from 1%/hr to 0.4%/hr by the addition of methemoglobin reduction system. The generation of the pyruvate and the lactate were observed within the NRC with enzymatic reduction system, then the activation of the Embden-Meyerhof pathway was confirmed. And we concerned about the availability of the NRC as a perfusate for the cardiopulmonary bypass during moderate or profound hypothermia, then we evaluated the oxygen transporting efficiency and capacity of the NRC under the using of the artificial lung system in vitro examination. The present investigation suggest that the effectiveness of the NRC with enzymatic reduction system, they restrained the formation of methemoglobin and they are efficient oxygen carriers as a perfusate of the artificial lung, and we suggest the new extracorporeal circulation system using of the NRC as a perfusate for the cardiopulmonary bypass.

Polyurethane membrane as an efficient immobilization carrier for high-density culture of rat hepatocytes in the fixed-bed reactor.

A fixed-bed bioreactor with a polyurethane membrane (PUM) as a cell-supporting material was developed for high-density culture of rat hepatocytes. The PUM has a heterogeneous porous structure of micropores (pore size <100 microm) and macropores (pore size >100 microm) with a porosity of 90%. One important feature of a PUM is that the macropores have finger-like structures and their diameters gradually decrease from the upper to the lower layer of the PUM. Most rat hepatocytes were readily immobilized in the micropores of PUM. Immobilized cell densities of 1-3 x 10(7) cells/cm(3) PUM were achieved within 5 min by natural downflow of cell suspension and their immobilization efficiencies were more than 99%. Using a syringe pump, a cell density of 5 x 10(7) cells/cm(3) PUM was achieved with more than 96% immobilization efficiency. Perfusion cultures using this reactor were performed for 7 days without cell leakage. The optimal cell density for albumin secretion was between 2 x 10(7) and 3 x 10(7) cells/cm(3) PUM. Albumin secretion in the perfusion culture was maintained for a relatively long period of time when compared to that in the monolayer culture. The rate of albumin secretion in the perfusion culture was about 50% of that in monolayer culture. Hepatocytes immobilized in PUM were slightly aggregated, but they maintained spherical form individually even after 7 days of cultivation. The above results show that PUM is a promising cell-supporting material for efficient immobilization of high cell density of hepatocytes.

Hemodilution with liposome-encapsulated low-oxygen-affinity hemoglobin facilitates rapid recovery from ischemic acidosis after cerebral ischemia in rats.

Liposome-encapsulated hemoglobin (LipoHb) with low oxygen affinity (P50 = 40–50 mmHg) has been developed. The purpose of this study was to evaluate the effects of the LipoHb on incomplete cerebral ischemia. Wistar rats were randomly assigned to one of the following three groups: (A) exchange transfusion with LipoHb solution (Hb = 6 g/dl) (LipoHb, n = 7), (B) exchange transfusion with rat red blood cell (RBC) solution (Hb = 6 g/dl) (RBC, n = 7), (C) no exchange transfusion (control, n = 7). Forebrain ischemia was induced for 9 min by bilateral carotid artery occlusion combined with a decrease in the mean arterial pressure (MAP) to 40 mmHg. 31P-magnetic resonance spectroscopy was performed during ischemia and 60 min of reperfusion. After exchange transfusion, the MAP increased in the LipoHb group and decreased in the RBC group (LipoHb versus RBC; P = 0.0028). During ischemia, intracellular pH (pHi) rapidly decreased in all groups; after reperfusion, the pHi recovery to preischemic levels was more rapid in the LipoHb group than in the RBC group (P < 0.05). Phosphocreatine and β-adenosine triphosphate decreased during ischemia and returned to the preischemic level in all groups following reperfusion. Inorganic phosphate (Pi) increased during ischemia and decreased to the normal value after reperfusion. The LipoHb group had a smaller production of Pi than the other two groups and demonstrated a rapid normalization, although the differences were not significant. Hemodilution with liposome-encapsulated low-oxygen-affinity hemoglobin facilitates rapid pHi recovery from incomplete forebrain ischemia in the rat.

Effect of liposome-encapsulated hemoglobin, neo red cells, on hemorrhagic shock.

We examined the effects of liposome-encapsulated hemoglobin, neo red cells (NRCs), on hemorrhagic shock in a canine model. The dogs were divided into the three groups according to treatment. In group 1, composed of six dogs, NRCs were substituted for blood without shock being induced; in group 2, composed of six dogs, NRCs were administered immediately after mild shock had been induced by exsanguination through the vein; and in group 3, composed of seven dogs, NRCs were administered after they had been left untreated for 30 min inducing severe shock. In group 2, administration of NRCs at a dose equivalent to the volume of exsanguinated blood improved the symptoms of shock; however, in group 3, a dose of NRCs 1.6-times the volume of exsanguinated blood was required. Peripheral vascular resistance (PVR) decreased after NRC administration in groups 1 and 2, but increased in group 3. On the other hand, the cardiac index (CI) increased in groups 1 and 2, and decreased in group 3. Concerning oxygen kinetics, there were no increases in the oxygen requirements or arteriovenous differences of the oxygen content per hemoglobin (AV/Hb) for NRCs in groups 1 and 2. Conversely, in group 3, the oxygen requirements increased and the NRCs compensated for the decrease in CI with an increase in AV/Hb by enhancing the oxygen transport efficiency to cope with the increased oxygen requirements.

Oxygen carrying capacity and oxygen supply rate of artificial oxygen carrier, Neo Red Cell (NRC).

Neo Red Cell (NRC), which is the liposome encapsulated hemolysate, has been developed as an artificial oxygen carrier. Oxygen carrying capacity and oxygen supply rate of NRC were estimated by continuous measurement of dissolved oxygen concentration in a spinner vessel. Oxygen carrying capacity of the medium was risen by adding NRC. The oxygen supply rate of the NRC medium containing hepatocytes was determined by the oxygen consumption rate of hepatocytes. The addition of NRC gave no effect on the oxygen transfer rate from gas phase to liquid phase (or kL a value) of the solution in the spinner vessel. The rate of oxygen absorption to NRC was limited by the oxygen transfer rate from gas phase to liquid phase in the spinner vessel. These results indicate that the oxygen supply from NRC may sustain the high-density culture of mammalian cells.

Hemodilution with liposome-encapsulated low-oxygen-affinity hemoglobin does not attenuate hypothermic cerebral ischemia in rats.

Hypothermia decreases cerebral metabolism and increases hemoglobin oxygen affinity. A hypothesis that the reversal of increased oxygen affinity would further attenuate hypothermic cerebral ischemia was tested by evaluating the effects of liposome-encapsulated hemoglobin (LipoHb) with low oxygen affinity (P50 = 40–50 mmHg) on hypothermic incomplete cerebral ischemia. Wistar rats were randomly assigned to one of the following two groups: (A) exchange transfusion with LipoHb solution (Hb = 6 g/dl) (LipoHb, n = 5), (B) no exchange transfusion (control, n = 5). After surface cooling to 22°C, forebrain ischemia was induced for 15 min by bilateral carotid artery occlusion combined with a decrease in the mean arterial pressure (MAP) to 40 mmHg. 31P-magnetic resonance spectroscopy was performed during ischemia and 45 min of reperfusion. After reperfusion, MAP was significantly higher in the control group than in the LipoHb group (P < 0.01), although there were no significant differences during ischemia. Intracellular pH and phosphocreatine (PCr) levels decreased during ischemia and returned to the preischemic level in both groups following reperfusion. The LipoHb group had a significantly larger decrease and smaller recovery in PCr than the control group (P < 0.0001). Althouth β-adenosine triphosphate decreased during ischemia in the LipoHb group, it increased in the control group (P < 0.0001). Inorganic phosphate (Pi) increased during ischemia and decreased to the normal value after reperfusion. The LipoHb group experienced a significantly larger production of Pi than the control group (P = 0.02). Hemodilution with high-P50 LipoHb does not reduce ischemic energy depletion induced by hypothermic incomplete forebrain ischemia in rats.

THE REDUCTION OF METHEMOGLOBIN IN NEO RED CELL

The hemoglobin oxidation factors that produce methemoglobin (metHb) were studied, for example, loss of enzymatic activity, lack of electron carrier and effect of lipids. The adverse effect of lipids is very pronounced and must be eliminated as much as possible in the producing Neo Red Cell (NRC). The process of swelling was studied. Liquid for swelling was varied from water to basic liquids. The pH of swollen lipids varied from acidic to neutral or basic, metHb was decreased and the enzymes in the Embden-Meyerhof pathway were activated. Phosphatidylcholine (PC) which is a component of mixed lipids is decomposed to lisophosphatidylcholine (liso-PC) in basic pH liquids, but liso-PC formation was not detected when the pH of swollen lipids was under 7.8. For both metHb formation and PC decomposition, swelling with the same quantity of 0.2 N aqueous sodium hydroxide proved optimal.

Assessment of the oxygen transport capacity of NRCs with a 70% blood exchange in rats

Neo red cells (NRCs) are a blood substitute representing stroma free hemolysate (SFHL) encapsulated in liposomes and containing NAD, glucose, adenine, and inosine, etc. as substrates or coenzymes of the Embden-Meyerhof pathway in RBCs and inositol hexaphosphate (IHP) as an allosteric effector. The oxygen transport efficiency (OTE) of NRCs was increased to > 45% when the oxygen partial pressure in arterial blood increased. We carried out exchange transfusion with NRCs in rats, monitored their functioning as a blood substitute in vivo, and demonstrated that NRCs worked sufficiently. First, we confirmed that the OTE of NRCs was augmented by increasing the oxygen supply to the rats by using oxygen masks. Second, we carried out 70% blood exchange with NRCs (with hemoglobin concentrations of 4, 5, 6, or 7 g/dl), and compared these experimental groups with control groups (RBC and saline). Each sample was contained bovine albumin (final volume of 5% (w/v)). As a result, the NRC groups with hemoglobin concentrations of 5, 6, and 7 g/dl showed stability after the exchange transfusion. On the other hand, the NRC group with hemoglobin concentration of 4 g/dl and the saline group showed respiratory acidosis and a drop of blood pressure and heart rate, and thus did not maintain an adequate oxygen supply. We concluded that a hemoglobin concentration of > 5 g/dl was sufficient in the NRC solution, and demonstrated in an experiment on animals that the OTE of NRCs was increased by using oxygen masks.

Optimized retrograde cerebral perfusion reduces ischemic energy depletion.

It has been reported that retrograde cerebral perfusion (RCP) provides minimal capillary flow; however, the extent to which RCP can provide aerobic metabolic support is unknown. We evaluated whether perfusate composition optimization for RCP would preserve brain energy metabolism during hypothermic circulatory arrest (HCA) at 20°C in rats. Three types of perfusates were prepared: hemoglobin-free saline, rat red blood cells, and artificial blood substitute (liposome-encapsulated hemoglobin); perfusates were made hypertonic, cooled to 20°C, and oxygenated and CO2 was administered (pH-stat management). Circulatory arrest was induced in 24 pH-stat-ventilated Wistar rats that had been surface cooled to 20°C; 18 were assigned to the RCP group in which one of the three (n = 6 each) perfusates was administered via the maxillary vein, and 6 received no perfusion. In two similarly surface-cooled rats (controls), brains were excised when the temperature reached 20°C. After 20 min of RCP or HCA, brains were excised and immediately frozen; brain high-energy phosphates, adenosine, and water content were measured. The liposome-encapsulated hemoglobin perfusate preserved levels of brain tissue adenosine triphosphates and energy charge, but not significantly better than rat red blood cells. Both maintained significantly higher levels than perfusion with oxygenated saline or hypothermic circulatory arrest alone (P = 0.0419–0.0001), under which regimes high-energy phosphates and energy charge declined to similar low values. RCP with hypertonic solution prevented brain edema. RCP with optimized composition perfusate (pH-stat, hypertonic rat red blood cells or liposome-encapsulated hemoglobin) reduced ischemic energy depletion during 20 min of HCA at 20°C in rats.