Dual near-infrared II laser modulates the cellular redox state of T cells and augments the efficacy of cancer immunotherapy.

Immunotherapy, including immune checkpoint inhibitors, has revolutionized cancer treatment, but only a minor fraction of patients shows durable responses. A new approach to overcome this limitation is yet to be identified. Recently, we have shown that photobiomodulation (PBM) with near-infrared (NIR) light in the NIR-II window reduces oxidative stress and supports the proliferation of CD8+ T cells, suggesting that PBM with NIR-II light could augment anti-cancer immunity. Here, we report a novel approach to support tumor-infiltrating CD8+ T cells upon PBM with NIR-II laser with high tissue penetration depth. Brief treatments of a murine model of breast cancer with dual 1064 and 1270 nm lasers reduced the expression of the programmed cell death protein 1 (PD-1) in CD8+ T cells in a syngeneic mouse model of breast cancer. The direct effect of the NIR-II laser treatment on T cells was confirmed by the enhanced tumor growth delay by the adoptive transfer of laser-treated CD8+ T cells ex vivo against a model tumor antigen. We further demonstrated that specific NIR-II laser parameters augmented the effect of the immune checkpoint inhibitor on tumor growth. PBM with NIR-II light augments the efficacy of cancer immunotherapy by supporting CD8+ T cells. Unlike the current immunotherapy with risks of undesirable drug-drug interactions and severe adverse events, the laser is safe and low-cost. It can be broadly combined with other therapy without modification to achieve clinical significance. In addition, our study established a path to develop a novel laser-based therapy to treat cancer effectively.

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.

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.

Experimental study of a novel method of cardiopulmonary resuscitation using a combination of percutaneous cardiopulmonary support and liposome-encapsulated hemoglobin (TRM645).

Percutaneous cardiopulmonary support (PCPS) has been applied for cardiopulmonary arrest (CPA). We have developed a novel method of cardiopulmonary resuscitation using PCPS combined with liposome-encapsulated hemoglobin (TRM645) to improve oxygen delivery to vital organs. Ventricular fibrillation was electrically induced to an adult goat for 10 min. Next, PCPS (30 ml/kg/min, V/Q: 1) was performed for 20 min. Then, external defibrillation was attempted and observed for 120 min. The TRM group (n5) was filled with 300 mL of TRM645 for the PCPS circuit. The control group (n5) was filled with the same volume of saline. The delivery of oxygen (DO2) and oxygen consumption (VO2) decreased markedly by PCPS after CPA, compared to the preoperative values. DO2 was kept at a constant level during PCPS in both groups, but VO2 slowly decreased at 5, 10, and 15 min of PCPS in the control groups, demonstrating that systemic oxygen metabolism decreased with time. In contrast, the decreases in VO2 were small in the TRM group at 5, 10, and 15 min of PCPS, demonstrating that TRM645 continuously maintained systemic oxygen consumption even at a low flow rate. AST and LDH in the TRM group were lower than the control. There were significant differences at 120 min after the restoration of spontaneous circulation (p<0.05).

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.

Experimental study of pegylated liposomal hemoglobin on norepinephrine release and reperfusion arrhythmias in isolated guinea pig hearts.

PURPOSE: Under myocardial reperfusion conditions, hemoglobin (Hb)-based artificial blood showed effectiveness for post-ischemic dysfunction. However, there are no studies about the effects of this product on reperfusion arrhythmias (ventricular fibrillation, VF) associated with norepinephrine (NE) release. This study was to evaluate the effects of the timing of the administration of pegylated liposomal Hb (LHb, P(50)=40-45 mmHg, 1 mg/mL) on NE release and VF. MATERIALS AND METHODS: Isolated guinea pig hearts (n=6 in each group) were randomly divided into four groups in Krebs-Henseleit solution being supplemented or not with LHb as follows: pre-ischemia (PRE), reperfusion (REP), or PRE+REP groups. The hearts were perfused for 30 min (preischemic period) and then subjected to 30 min of global ischemia, followed by 30 min of reperfusion with a normothermic Langendorff apparatus at 30 mm Hg aortic pressure in a constant pressure model. RESULTS: No differences were documented among the four groups in heart rate, left ventricular-developed pressure, or coronary flow rate. However, the REP group significantly decreased the duration of VF and NE release, but it did not inhibit the incidence of VF. CONCLUSION: These results suggest that the administration of LHb, especially with the timing of reperfusion, might prevent reperfusion arrhythmias linked to the inhibition of NE release.

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 degrees and 37 degrees 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.