Cholesterol-Lowering Effect of Octaarginine-Appended ß-Cyclodextrin in Npc1-Trap-CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-ß-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of ß-CyD to NPC cells is expected. In this study, to efficiently deliver ß-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended ß-CyD with a spacer of γ-aminobutyric acid (R8-ß-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-ß-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-ß-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-ß-CyD significantly decreased intracellular cholesterol content compared with HP-ß-CyD. These results suggest that R8-ß-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

Evaluation of three-dimensional cultured HepG2 cells in a nano culture plate system: an in vitro human model of acetaminophen hepatotoxicity.

Overdoses of acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) cause severe liver injury, yet there is no common or high throughput in vitro human APAP model. This study examined the characteristics and usefulness of HepG2 cells grown in a nano culture plate (NCP) system, a three-dimensional culture method, as an in vitro human model for APAP-induced hepatotoxicity. The NCP-cultured HepG2 cells showed higher expression of mRNA and protein levels of cytochrome P450 2E1, which metabolizes APAP to a toxic metabolite, APAP-cysteine adduct formation, and higher sensitivity against APAP-induced cell injury compared with conventionally cultured cells. We demonstrated that treatment of APAP in NCP-cultured HepG2 cells shows key mechanistic features of APAP-induced hepatotoxicity, such as decreases in intracellular glutathione and mitochondrial membrane potential, activation of JNK, and cellular injury; and pharmacological agents, such as Cyclosporine A (a mitochondrial permeability transition inhibitor) and SP600125 (a JNK inhibitor), prevented cell injury induced by APAP exposure. In addition, the antidote of APAP-induced hepatotoxicity, N-acetylcysteine, could attenuate cellular injury induced by APAP in NCP-cultured HepG2 cells. We suggest that cellular injury induced by APAP treatment using an NCP-HepG2 system is a useful human model to study mechanisms and screen drug candidates of APAP-induced hepatotoxicity.

The ethical Kampo formulation Sho-seiryu-to (TJ-19) prevents bleomycin-induced pulmonary fibrosis in rats.

The effects of Sho-seiryu-to (TJ-19), an ethical Kampo formulation, on bleomycin (BLM)-induced pulmonary fibrosis in rats was examined. Pulmonary fibrosis was induced by intratracheal instillation of a single dose of BLM (5 mg/kg). The TJ-19 used consisted of at least 21 constituents, as determined by three-dimensional HPLC analysis, and was administered orally twice a day at a dose of 1.5 g/kg until the end of the study period. Changes in general appearance and body weight were monitored. Twenty-eight days after BLM instillation, the animals were sacrificed and the study parameters were measured. TJ-19 attenuated the loss in body weight, increase in lung/body weight ratio and concentration of hydroxyproline and malondialdehyde in the lung tissues induced by BLM administration. TJ-19 also prevented BLM-induced fibrotic changes in the lung histology. These protective effects of TJ-19 were observed when administration was started 1 week before and simultaneously with the instillation of BLM. These results suggest that TJ-19 has prophylactic potential against BLM-induced pulmonary fibrosis, and may therefore be a promising drug candidate and medicinal resource for preventing BLM-induced and idiopathic pulmonary fibrosis.

Protection afforded by a herbal medicine, Sho-seiryu-to (TJ-19), against oleic acid-induced acute lung injury in guinea-pigs.

OBJECTIVES: The effect of a herbal medicine, Sho-seiryu-to (TJ-19), on oleic acid-induced lung injury, an animal model of acute respiratory distress syndrome or acute lung injury (ARDS/ALI), was examined. METHODS: Acute lung injury was induced by an intravenous injection of 15 microl/kg oleic acid to guinea-pigs. TJ-19 was administered by a single oral dose (3 g/kg) or by multiple oral doses (0.75 g/kg). KEY FINDINGS: The decrease in partial oxygen pressure of arterial blood (Pao(2)) and the increase in airway vascular permeability induced by the oleic acid injection were attenuated by a single dose of TJ-19. When TJ-19 was administered orally twice a day for two weeks and then oleic acid was injected, a potent prophylactic effect of the drug was observed. TJ-19 also prevented airway vascular hyperpermeability, lung cell injury, oxidative stress and thromboxane A(2) generation, associated with the oleic acid injection. CONCLUSIONS: TJ-19 significantly attenuated the oleic acid-induced lung injury probably through the antioxidative effect and inhibitory effect of thromboxane A(2) generation, although the precise inhibitory mechanisms were not fully elucidated due to the diversity in constituents of the herbal medicine. We suggest that TJ-19 is a promising drug candidate and a medicinal resource for preventing ARDS/ALI.

Effects of bolus injection of soybean-based fat emulsion and fatty acids on pulmonary gas exchange function.

To determine whether or not a "bolus injection" of soybean-based fat emulsion (SFE), which contains oleic acid (OA), a potent lung-toxic unsaturated C-18 fatty acid, can induce pulmonary dysfunction, we examined the effect of SFE injection on the partial oxygen pressure of arterial blood (Pao2) and pulmonary vascular permeability. In addition, we compared the effect of an injection of SFE with that of OA, soybean oil (a source of SFE), emulsified OA and C-18 fatty acids. Bolus injection of SFE (0.3-4.8 ml/kg) had little effect on Pao2) and pulmonary vascular permeability. Injection of an equivalent amount of OA, on the other hand, significantly decreased Pao2 and increased pulmonary vascular hyper-permeability. This decrease in Pao2 was attenuated by emulsification. Unemulsified soybean oil also induced a decrease in Pao2, although the effect was weaker than that of OA. Other unsaturated C-18 fatty acids (linoleic and linolenic acid) induced a decrease in Pao2 as potent as OA while stearic acid, a C-18 saturated fatty acid, had little effect. Although we did not observe pulmonary toxicity as a result of "bolus injection" of SFE, the chemical form, for example, emulsification and the degree of saturability of the carbon chain, seems to influence the pulmonary toxicities of lipids and fatty acids. Furthermore, the potent pulmonary toxicity of OA seems to depend not only on pulmonary vascular embolization but also pharmacological and/or inflammation-inducing properties.

Involvement of calmodulin inhibition in analgesia induced with low doses of intrathecal trifluoperazine.

We examined which of the known properties of trifluoperazine, including calmodulin inhibition, are involved in its analgesic effect. Furthermore, we tried to find any possible interaction between opioidergic system and calmodulin inhibition-induced analgesia. Intrathecal trifluoperazine (1, 10, 100 microg) showed a biphasic effect in the formalin test; i.e., analgesia at relatively low doses (1, 10 microg) and hyperalgesia at a high dose (100 microg). No analgesic effects were observed after intrathecal injection of sulpiride (1, 10, 100 microg), atropine (0.1, 1, 10 microg), phentolamine (0.1, 1, 10 microg) and brompheniramine (0.1, 1, 10 microg). Meanwhile, intrathecal calmidazolium (10, 50, 250 microg) induced a dose-dependent analgesia. Histamine (1 microg), physostigmine (1 microg), bromocriptine (1 microg) and norepinephrine (1 microg) did not affect trifluoperazine-induced analgesia. Calcium (20 microg) attenuated the antinociceptive effect of trifluoperazine and inhibited the analgesic effect of calmidazolium. Finally, naloxone (2 mg/kg) decreased trifluoperazine-induced antinociception but did not have any effects on calmidazolium-induced analgesia. We concluded that calmodulin inhibition may be involved in the analgesia produced by trifluoperazine. With increasing doses of trifluoperazine, the algesic effect seems to overcome the analgesic effect. It is also suggested that the opioidergic system does not interact with calmodulin inhibition-induced analgesia even though this system has a possible role in trifluoperazine-induced analgesia.