Yoichi Ishitsuka

Efficacy of 2-hydroxypropyl-β-cyclodextrin in Niemann-Pick disease type C model mice and its pharmacokinetic analysis in a patient with the disease

Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-β-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1(-/-)) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1(-/-) mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1(-/-) mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1(-/-) mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1(-/-) mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.

HPGCD Outperforms HPBCD as a Potential Treatment for Niemann‐Pick Disease Type C During Disease Modeling with iPS Cells

Niemann‐Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte‐like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC‐iPSC‐derived HLCs. These findings indicate that iPSC‐derived cells can phenocopy human NPC. We also newly found that 2‐hydroxypropyl‐γ‐cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient‐derived cells, and do so more effectively than 2‐hydroxypropyl‐β‐cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease. Stem Cells 2015;33:1075–1088

Involvement of thromboxane A2 (TXA2) in the early stages of oleic acid-induced lung injury and the preventive effect of ozagrel, a TXA2 synthase inhibitor, in guinea-pigs

An intravenous injection of oleic acid into animals can produce a lung injury with hypoxaemia and pulmonary vascular hyper‐permeability. Although oleic acid lung injury is used as a model of acute respiratory distress syndrome (ARDS), the precise mechanisms of the lung injury are still unclear. We have investigated whether thromboxane A2 (TXA2) participated in the lung injury and have evaluated the efficacy of ozagrel, a TXA2 synthase inhibitor, on the lung injury in guinea‐pigs. Oleic acid injection increased the plasma level of TXB2, a stable metabolite of TXA2, and the time‐course of plasma TXB2 was similar to that of the decreased partial oxygen pressure of arterial blood (Pao2) induced with oleic acid. Ozagrel administered intravenously 30 min before oleic acid injection prevented the decrease in Pao2 and pulmonary vascular hyper‐permeability. It also prevented increases in lactate dehydrogenase activity, a measure of lung cell injury, TXB2 and its weight ratio to 6‐keto prostaglandin F1 α in bronchoalveolar lavage fluid. Although ozagrel administered simultaneously with oleic acid ameliorated the decrease in Pao2, post treatment showed little effect. We suggest that TXA2 participated in the oleic acid lung injury, as an “early phase” mediator, and rapidly‐acting TXA2 synthase inhibitors were effective in the prevention of acute lung injury.

The exacerbating roles of CCAAT/enhancer-binding protein homologous protein (CHOP) in the development of bleomycin-induced pulmonary fibrosis and the preventive effects of tauroursodeoxycholic acid (TUDCA) against pulmonary fibrosis in mice.

The purpose of this study was to evaluate the role of CCAAT/enhancer-binding protein homologous protein (CHOP), an important transcription factor that regulates the inflammatory reaction during the endoplasmic reticulum (ER) stress response, in the development of pulmonary fibrosis induced by bleomycin (BLM) in mice. An intratracheal injection of BLM transiently increased the expression of CHOP mRNA and protein in an early phase (days 1 and 3) in mice lungs. BLM-induced pulmonary fibrosis was significantly attenuated in Chop gene deficient (Chop KO) mice, compared with wild-type (WT) mice. Furthermore, the inflammatory reactions evaluated by protein concentration, the total number of leucocytes and neutrophils in the bronchoalveolar lavage fluid (BALF), the mRNA expression of interleukin 1b and caspase 11, and the apoptotic cell death were suppressed in Chop KO mice compared with those in WT mice. In addition, administration of tauroursodeoxycholic acid (TUDCA), a pharmacological agent that can inhibit CHOP expression, inhibited the BLM-induced pulmonary fibrosis and inflammation, and the increase in Chop mRNA expression in WT mice in a dose-dependent manner. These results suggest that the ER stress-induced transcription factor, CHOP, at least in part, plays an important role in the development of BLM-induced pulmonary fibrosis in mice, and that the inhibition of CHOP expression by a pharmacological agent, such as TUDCA, may be a promising strategy for the prevention of pulmonary fibrosis.

Effects of intracerebroventricular administration of 2-hydroxypropyl-β-cyclodextrin in a patient with Niemann-Pick Type C disease

Niemann–Pick Type C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by progressive neurological deterioration. Previously, we reported that intravenous administration of 2-hydroxypropyl-β-cyclodextrin (HPB-CD) in two patients with NPC had only partial and transient beneficial effects on neurological function. The most likely reason for HPB-CD not significantly improving the neurological deficits of NPC is its inability to cross the blood–brain barrier. Herein, we describe the effects of intrathecal HPB-CD in an eight-year-old patient with a perinatal onset of NPC, administered initially at a dose of 10 mg/kg every other week and increased up to 10 mg/kg twice a week. Clinically, the patient maintained residual neurological functions for two years, at which time nuclear magnetic resonance spectroscopy showed a decreased choline to creatine ratio and increased N-acetylaspartate to creatine ratio, and positron emission tomography revealed increased standardized uptake values. Total-tau in the cerebrospinal fluid (CSF) was also decreased after two years. No adverse effects were observed over the course of treatment. The CSF concentrations of HPB-CD during the distribution phase after the injections were comparable with those at which HPB-CD could normalize cellular cholesterol abnormality in vitro. Further studies are necessary to elucidate the mechanisms of action of HPB-CD in NPC, and to determine the optimal dose and intervals of HPB-CD injection.

Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

BackgroundOverdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mice.MethodsHepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM) were evaluated by the WST-1 cell viability assay.ResultsOzagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16.ConclusionsWe demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury.

Protection afforded by pre- or post-treatment with 4-phenylbutyrate against liver injury induced by acetaminophen overdose in mice

Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) is a widely used analgesic/antipyretic drug with few adverse effects at therapeutic doses; suicidal or unintentional overdose of APAP frequently induces severe hepatotoxicity. To explore a new and effective antidote for APAP hepatotoxicity, this study examined the effects of sodium 4-phenylbutyrate (4-PBA) on liver injury induced by APAP overdose in mice. Liver injury was induced in C57BL/6 male mice by intraperitoneal injection of APAP (400mg/kg). The effects of 4-PBA (100-200mg/kg) treatment at 1h before the APAP injection were evaluated with serum alanine aminotransferase (ALT) and blood ammonia levels, hepatic pathological changes, including histopathology, DNA damage, nitrotyrosine formation, and mRNA or protein expression involved in the development of hepatotoxicity, such as X-box binding protein-1 (XBP1), c-Jun N-terminal kinase (JNK), C/EBP homologous protein (CHOP) and B-cell lymphoma 2 interacting mediator of cell death (Bim). In addition, glutathione depletion and CYP2E1 protein expression, which are measures of the metabolic conversion of APAP to a toxic metabolite, were examined. Furthermore, we examined the effects of post-treatment with 4-PBA against APAP-induced hepatotoxicity in mice. When administered at 1h before APAP injection, 4-PBA significantly prevented the increase in serum ALT and blood ammonia levels, centrilobular necrosis of hepatocytes, DNA fragmentation, and nitrotyrosine formation induced by APAP in mice. 4-PBA also inhibited hepatic Xbp1 mRNA splicing and JNK phosphorylation induced by APAP, but did not suppress CHOP and Bim mRNA and protein expression. In addition, 4-PBA had little effect on hepatic glutathione depletion and CYP2E1 expression, parameters of toxic APAP metabolite production. Post-treatment with 4-PBA administration at 1 or 2h after APAP injection also attenuated the increase in serum ALT and blood ammonia levels and hepatic pathological changes in APAP-induced hepatotoxicity in mice. Although post-treatment with 4-PBA did not show any effects on hepatic Xbp1 mRNA splicing and JNK phosphorylation, it drastically attenuated the DNA fragmentation induced by APAP. The precise molecular mechanisms of the protection afforded by 4-PBA against APAP hepatotoxicity in mice are unclear, but they seem to involve inhibition of hepatocellular DNA fragmentation. We suggest that 4-PBA is a promising candidate as an antidote against APAP-induced liver injury.

Influence of Npc1 genotype on the toxicity of hydroxypropyl-β- cyclodextrin, a potentially therapeutic agent, in Niemann-Pick Type C disease models

Hydroxypropyl-β-cyclodextrin (HPBCD) is an attractive drug candidate against Niemann–Pick Type C (NPC) disease. However, the safety of HPBCD treatment for NPC patients remains to be elucidated. In this study, we examined the acute toxicity of HPBCD in Npc1-deficient mice. When treated with HPBCD (20,000 mg/kg, subcutaneously), over half of the wild-type (Npc1+/+) or Npc1+/− mice died by 72 h after the injection. In contrast, all of the Npc1−/− mice survived. Marked pathophysiological changes, such as an elevation in serum transaminase and creatinine levels, hepatocellular necrosis, renal tubular damage, interstitial thickening, and hemorrhages in lungs, were induced by the HPBCD treatment in Npc1+/+ or Npc1+/− mice. However, these pathophysiological changes were significantly alleviated in Npc1−/− mice. In addition, in vitro analysis showed that the Npc1 gene deficiency and treatment with U18666A, an Npc1 inhibitor, remarkably attenuated the cytotoxicity of HPBCD in Chinese hamster ovary cells. These results suggest that the NPC1 genotype exacerbates the cytotoxicity of HPBCD and Npc1−/− mice have substantial resistance to the lethality and the organ injury induced by HPBCD injection compared with Npc1+/+ or Npc1+/− mice. We suggest that the Npc1 genotype should be considered in the safety evaluation of HPBCD using experimental animals and cells.

In vitro evaluation of 2-hydroxyalkylated β-cyclodextrins as potential therapeutic agents for Niemann-Pick Type C disease.

This study was conducted to evaluate the attenuating potential of 2-hydroxypropyl-β-cyclodextrin (HPBCD) against Niemann-Pick Type C (NPC) disease, as well as the physical and chemical properties, particularly the cholesterol-solubilizing ability, in an NPC disease model in vitro. As parameters of NPC abnormalities, intracellular free and esterified cholesterol levels and lysosome volume were measured in Npc1 null Chinese hamster ovary cells. HPBCD showed dose-dependent effects against dysfunctional intracellular cholesterol trafficking, such as the accumulation and shortage of free and esterified cholesterols, respectively, in Npc1 null cells. However, the effectiveness was gradually offset by exposure to ≥8mM HPBCD. The same effect was also observed for increasing lysosome volume in Npc1 null cells. The degree of substitution of the hydroxypropyl group had little influence on the attenuating effects of HPBCD against the NPC abnormalities, at least in the range between 2.8 and 7.4. Next, we compared the effects of other hydroxyalkylated β-cyclodextrin derivatives with different cholesterol-solubilizing abilities, such as 2-hydroxyethyl-β-cyclodextrin (HEBCD) and 2-hydroxybutyl-β-cyclodextrin (HBBCD). The cholesterol solubilizing potential, attenuating effects against NPC abnormalities and cytotoxicity induction were HBBCD≫HPBCD>HEBCD, HBBCD=HPBCD>HEBCD and HBBCD≫HPBCD=HEBCD, respectively. HPBCD may be superior in terms of safety and efficacy in Npc1 null cells compared with HEBCD and HBBCD. The results of this study will provide a rationale for the optimization of HPBCD therapy for NPC disease.

Anti-inflammatory Effects of Novel Polysaccharide Sacran Extracted from Cyanobacterium Aphanothece sacrum in Various Inflammatory Animal Models

The goal of this study was to investigate the topical anti-inflammatory effects of the megamolecular polysaccharide sacran extracted from cyanobacterium Aphanothece sacrum using various inflammatory animal models. Sacran showed potent anti-inflammatory effects with optimum effective concentrations at 0.01 and 0.05% (w/v). Sacran markedly inhibited paw swelling and neutrophil infiltration in carrageenan-induced rat paw edema. Additionally, 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionyl-carboxylic acid (DMEQ)-labeled sacran had the ability to penetrate carrageenan-induced rat paw skin rather than normal skin. Also, sacran significantly suppressed kaolin-induced and dextran-induced rat paw edema throughout the duration of the study. Furthermore, sacran significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and mRNA expression levels of cyclooxygenase (COX)-2 as well as pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Safety of sacran solution was verified by negligible cytotoxicity in HaCaT cells. These results suggest that sacran may be useful as a therapeutic agent against inflammatory skin diseases with no life-threatening adverse effects.