田中 健一郎

Neuronal cell death is a key mechanism involved in the development and exacerbation of Parkinson's disease (PD). The excessive production of reactive oxygen species (ROS) is a major cause leading to neuronal death; therefore, compounds that prevent oxidative stress-dependent neuronal death may be promising as a preventive method for PD. Ergothioneine is a natural amino acid with antioxidant properties, and its protective functions in the body are attracting attention. However, there has been no investigation into the protective functions of ergothioneine using in vivo and in vitro PD models. Thus, in this study, we analyzed the efficacy of ergothioneine against 6-hydroxydopamine (6-OHDA)-dependent neuronal cell death using immortalized hypothalamic neurons (GT1-7 cells). First, we found that ergothioneine prevents 6-OHDA-dependent neuronal cell death by suppressing ROS overproduction in GT1-7 cells. The cytoprotective effect of ergothioneine was partially abolished by verapamil, an inhibitor of OCTN1, which is involved in ergothioneine uptake. Furthermore, ergothioneine-rich Rice-koji (Ergo-koji) showed cytoprotective and antioxidant effects similar to those of ergothioneine. Taken together, these results suggest that ergothioneine or foods containing ergothioneine may be an effective method for preventing the development and progression of PD.

As overproduction of reactive oxygen species (ROS) causes various diseases, antioxidants that scavenge ROS, or inhibitors that suppress excessive ROS generation, can be used as therapeutic agents. From a library of approved drugs, we screened compounds that reduced superoxide anions produced by pyocyanin-stimulated leukemia cells and identified benzbromarone. Further investigation of several of its analogues showed that benziodarone possessed the highest activity in reducing superoxide anions without causing cytotoxicity. In contrast, in a cell-free assay, benziodarone induced only a minimal decrease in superoxide anion levels generated by xanthine oxidase. These results suggest that benziodarone is an inhibitor of NADPH oxidases in the plasma membrane but is not a superoxide anion scavenger. We investigated the preventive effect of benziodarone on lipopolysaccharide (LPS)-induced murine lung injury as a model of acute respiratory distress syndrome (ARDS). Intratracheal administration of benziodarone attenuated tissue damage and inflammation via its ROS-reducing activity. These results indicate the potential application of benziodarone as a therapeutic agent against diseases caused by ROS overproduction.

Parkinson's disease (PD) is a neurodegenerative disorder caused by oxidative stress-dependent loss of dopaminergic neurons in the substantia nigra and elevated microglial inflammatory responses. Recent studies show that cell loss also occurs in the hypothalamus in PD. However, effective treatments for the disorder are lacking. Thioredoxin is the major protein disulfide reductase in vivo. We previously synthesized an albumin-thioredoxin fusion protein (Alb-Trx), which has a longer plasma half-life than thioredoxin, and reported its effectiveness in the treatment of respiratory and renal diseases. Moreover, we reported that the fusion protein inhibits trace metal-dependent cell death in cerebrovascular dementia. Here, we investigated the effectiveness of Alb-Trx against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Alb-Trx significantly inhibited 6-OHDA-induced neuronal cell death and the integrated stress response. Alb-Trx also markedly inhibited 6-OHDA-induced reactive oxygen species (ROS) production, at a concentration similar to that inhibiting cell death. Exposure to 6-OHDA perturbed the mitogen-activated protein kinase pathway, with increased phosphorylated Jun N-terminal kinase and decreased phosphorylated extracellular signal-regulated kinase levels. Alb-Trx pretreatment ameliorated these changes. Furthermore, Alb-Trx suppressed 6-OHDA-induced neuroinflammatory responses by inhibiting NF-κB activation. These findings suggest that Alb-Trx reduces neuronal cell death and neuroinflammatory responses by ameliorating ROS-mediated disruptions in intracellular signaling pathways. Thus, Alb-Trx may have potential as a novel therapeutic agent for PD.

Trace elements such as iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) are absorbed from food via the gastrointestinal tract, transported into the brain, and play central roles in normal brain functions. An excess of these trace elements often produces reactive oxygen species and damages the brain. Moreover, increasing evidence suggests that the dyshomeostasis of these metals is involved in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, prion diseases, and Lewy body diseases. The disease-related amyloidogenic proteins can regulate metal homeostasis at the synapses, and thus loss of the protective functions of these amyloidogenic proteins causes neurodegeneration. Meanwhile, metal-induced conformational changes of the amyloidogenic proteins contribute to enhancing their neurotoxicity. Moreover, excess Zn and Cu play central roles in the pathogenesis of vascular-type senile dementia. Here, we present an overview of the intake, absorption, and transport of four essential elements (Fe, Zn, Cu, Mn) and one non-essential element (aluminum: Al) in food and their connections with the pathogenesis of neurodegenerative diseases based on metal-protein, and metal-metal cross-talk.

Reducing the health hazards caused by air pollution is a global challenge and is included in the Sustainable Development Goals. Air pollutants, such as PM2.5, induce respiratory and cardiovascular disorders by causing various inflammatory responses via oxidative stress. Catechins and polyphenols, which are components of green tea, have various protective effects, owing to their antioxidant ability. The main catechin in green tea, epigallocatechin gallate (EGCG), is potentially effective against respiratory diseases, such as idiopathic pulmonary fibrosis and asthma, but its effectiveness against air-pollution-dependent lung injury has not yet been investigated. In this study, we examined the effect of EGCG on urban aerosol-induced acute lung injury in mice. Urban aerosol treatment caused increases in inflammatory cell counts, protein levels, and inflammatory cytokine expression in the lungs of ICR mice, but pretreatment with EGCG markedly suppressed these responses. Analyses of oxidative stress revealed that urban aerosol exposure enhanced reactive oxygen species (ROS) production and the formation of ROS-activated neutrophil extracellular traps (NETs) in the lungs of mice. However, ROS production and NETs formation were markedly suppressed by pretreating the mice with EGCG. Gallocatechin gallate (GCG), a heat-epimerized form of EGCG, also markedly suppressed urban aerosol-dependent inflammatory responses and ROS production in vivo and in vitro. These findings suggest that EGCG and GCG prevent acute lung injury caused by urban aerosols through their inhibitory effects on ROS production. Thus, we believe that foods and medications containing EGCG or GCG may be candidates to prevent the onset and progression of acute lung injury caused by air pollutants.

Copper and zinc are essential for normal brain functions. Both are localized in presynaptic vesicles and are secreted into synaptic clefts during neuronal excitation. Despite their significance, excesses of copper and zinc are neurotoxic. In particular, excess zinc after transient global ischemia plays a central role in the ischemia-induced neurodegeneration and pathogenesis of vascular type senile dementia. We previously found that sub-lethal concentrations of copper remarkably exacerbated zinc-induced neurotoxicity, and we investigated the molecular pathways of copper-enhanced zinc-induced neurotoxicity. The endoplasmic reticulum stress pathway, the stress-activated protein kinases/c-‍Jun amino-terminal kinases pathway, and mitochondrial energy production failure were revealed to be involved in the neurodegenerative processes. Regarding the upstream factors of these pathways, we focused on copper-derived reactive oxygen species and the disruption of calcium homeostasis. Because excess copper and zinc may be present in the synaptic clefts during ischemia, it is possible that secreted copper and copper-induced reactive oxygen species may enhance zinc neurotoxicity and eventually contribute to the pathogenesis of vascular type senile dementia.

Although the pathogenesis of chronic obstructive pulmonary disease (COPD) is not yet fully understood, recent studies suggest that the disruption of the intracellular balance of oxidative (such as reactive oxygen species (ROS)) and antioxidant molecules plays an important role in COPD development and progression. Metallothionein is an endogenous metal-binding protein with reported ROS scavenging activity. Although there have been many publications on the protective effects of metallothionein in the kidney and liver, its role in COPD models such as elastase- or cigarette smoke (CS)-induced lung injury is unknown. Thus, in the present study, we analyzed the elastase-induced lung injury model using metallothionein-knockout (MT-KO; MT-1 and -2 gene deletion) mice. The expression of MT-1 and MT-2 in the lungs of MT-KO mice was markedly lower compared with that in the lungs of wildtype (WT) mice. Porcine pancreatic elastase (PPE)-induced lung injury (alveolar enlargement and respiratory impairment) was significantly exacerbated in MT-KO mice compared with WT mice. Additionally, PPE-induced increases in the number of inflammatory cells, inflammatory cytokines, and cell death in lung tissue were significantly more pronounced in MT-KO mice compared with WT mice. Finally, using an in vivo imaging system, we also found that PPE-induced ROS production in the lungs was enhanced in MT-KO mice compared with WT mice. These results suggest that metallothionein may act as an inhibitor against elastase-induced lung injury by suppressing ROS production. These results suggest that metallothionein protein, or compounds that can induce metallothionein, could be useful in the treatment of COPD.

Although the exact pathogenesis of idiopathic pulmonary fibrosis (IPF) is still unknown, the transdifferentiation of fibroblasts into myofibroblasts and the production of extracellular matrix components such as collagen, triggered by alveolar epithelial cell injury, are important mechanisms of IPF development. In the lungs of IPF patients, apoptosis is less likely to be induced in fibroblasts than in alveolar epithelial cells, and this process is involved in the pathogenesis of IPF. We used a library containing approved drugs to screen for drugs that preferentially reduce cell viability in LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human alveolar epithelial cell line). After screening, we selected eperisone, a central muscle relaxant used in clinical practice. Eperisone showed little toxicity in A549 cells and preferentially reduced the percentage of viable LL29 cells, while pirfenidone and nintedanib did not have this effect. Eperisone also significantly inhibited transforming growth factor-β1-dependent transdifferentiation of LL29 cells into myofibroblasts. In an in vivo study using ICR mice, eperisone inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory dysfunction, and fibroblast activation. In contrast, pirfenidone and nintedanib were less effective than eperisone in inhibiting BLM-induced pulmonary fibrosis under this experimental condition. Finally, we showed that eperisone did not induce adverse effects in the liver and gastrointestinal tract in the BLM-induced pulmonary fibrosis model. Considering these results, we propose that eperisone may be safer and more therapeutically beneficial for IPF patients than current therapies.

Renal ischemia/reperfusion (I/R) injury is a major clinical problem because it can cause acute kidney injury (AKI) or lead to the transition from AKI to chronic kidney disease (CKD). Oxidative stress, which involves the production of reactive oxygen species (ROS), plays an important role in the development and exacerbation of I/R-induced kidney injury. However, we have previously reported that lecithinized superoxide dismutase (PC-SOD), a SOD derivative with high tissue affinity and high stability in plasma, has beneficial effects in various disease models because of its inhibitory effect on ROS production. Therefore, we aimed to determine the effects of intravenous PC-SOD administration in a mouse model of renal injury induced by I/R. PC-SOD markedly ameliorated the I/R-induced increases in markers of renal damage (urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, and interleukin-6) and tubular necrosis 48 h after the intervention. We also found that PC-SOD significantly ameliorated the I/R-induced increase in ROS production, using an ex vivo imaging system. Furthermore, PC-SOD inhibited the increases in expression of markers of fibrosis (α-smooth muscle actin and collagen 1A1) 96 h after, and renal fibrosis 25 days after I/R was induced. Finally, we found that PC-SOD ameliorated the I/R-induced AKI in mice with high-fat diet-induced prediabetes. These results suggest that PC-SOD inhibits AKI and the transition from AKI to CKD through the inhibition of ROS production. Therefore, we believe that PC-SOD may represent an effective therapeutic agent for I/R-induced renal injury.

Copper is an essential trace element and possesses critical roles in various brain functions. A considerable amount of copper accumulates in the synapse and is secreted in neuronal firings in a manner similar to zinc. Synaptic copper and zinc modulate neuronal transmission and contribute to information processing. It has been established that excess zinc secreted during transient global ischemia plays central roles in ischemia-induced neuronal death and the pathogenesis of vascular dementia. We found that a low concentration of copper exacerbates zinc-induced neurotoxicity, and we have demonstrated the involvement of the endoplasmic reticulum (ER) stress pathway, the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) signaling pathway, and copper-induced reactive oxygen species (ROS) production. On the basis of our results and other studies, we discuss the collaborative roles of copper in zinc-induced neurotoxicity in the synapse and the contribution of copper to the pathogenesis of vascular dementia.

Prion diseases are progressive and transmissive neurodegenerative diseases. The conformational conversion of normal cellular prion protein (PrPC) into abnormal pathogenic prion protein (PrPSc) is critical for its infection and pathogenesis. PrPC possesses the ability to bind to various neurometals, including copper, zinc, iron, and manganese. Moreover, increasing evidence suggests that PrPC plays essential roles in the maintenance of homeostasis of these neurometals in the synapse. In addition, trace metals are critical determinants of the conformational change and toxicity of PrPC. Here, we review our studies and other new findings that inform the current understanding of the links between trace elements and physiological functions of PrPC and the neurotoxicity of PrPSc.

The number of deaths from air pollution worldwide is estimated at 8.8 million per year, more than the number of deaths from smoking. Air pollutants, such as PM2.5, are known to induce respiratory and cardiovascular diseases by inducing oxidative stress. Thioredoxin (Trx) is a 12-kDa endogenous protein that exerts antioxidant activity by promoting dithiol disulfide exchange reactions. We previously synthesized human serum albumin-fused thioredoxin (HSA-Trx), which has a longer half-life in plasma compared with Trx, and demonstrated its efficacy against various diseases including respiratory diseases. Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice. Urban aerosols induced lung injury and inflammatory responses in ICR mice, but intravenous administration of HSA-Trx markedly inhibited these responses. We next analyzed reactive oxygen species (ROS) production in murine lungs using an in vivo imaging system. The results show that intratracheal administration of urban aerosols induced ROS production that was inhibited by intravenously administered HSA-Trx. Finally, we found that HSA-Trx inhibited the urban aerosol-induced increase in levels of neutrophilic extracellular trap (NET) indicators (i.e., double-stranded DNA, citrullinated histone H3, and neutrophil elastase) in bronchoalveolar lavage fluid (BALF). Together, these findings suggest that HSA-Trx prevents urban aerosol-induced acute lung injury by suppressing ROS production and neutrophilic inflammation. Thus, HSA-Trx may be a potential candidate drug for preventing the onset or exacerbation of lung injury caused by air pollutants.

Excessive zinc ion (Zn2+) release is induced in pathological situations and causes neuronal cell death. Previously, we have reported that copper ions (Cu2+) markedly exacerbated Zn2+-induced neuronal cell death by potentiating oxidative stress, the endoplasmic reticulum (ER) stress response, and the activation of the c-Jun amino-terminal kinase (JNK) signaling pathway. In contrast, selenium (Se), an essential trace element, and amino acids containing selenium (such as seleno-l-methionine) have been reported to inhibit stress-induced neuronal cell death and oxidative stress. Thus, we investigated the effect of seleno-l-methionine on Cu2+/Zn2+-induced neuronal cell death in GT1-7 cells. Seleno-l-methionine treatment clearly restored the Cu2+/Zn2+-induced decrease in the viable cell number and attenuated the Cu2+/Zn2+-induced cytotoxicity. Accordingly, the levels of ER stress-related factors (especially, CHOP and GADD34) and of phosphorylated JNK increased upon CuCl2 and ZnCl2 co-treatment, whereas pre-treatment with seleno-l-methionine significantly suppressed these upregulations. Analysis of reactive oxygen species (ROS) as upstream factors of these pathways revealed that Cu2+/Zn2+-induced ROS production was clearly suppressed by seleno-l-methionine treatment. Finally, we found that seleno-l-methionine induced the antioxidative protein, glutathione peroxidase. Taken together, our findings suggest that seleno-l-methionine suppresses Cu2+/Zn2+-induced neuronal cell death and oxidative stress via induction of glutathione peroxidase. Thus, we think that seleno-l-methionine may help prevent refractory neurological diseases.

Increasing evidence suggests that the metal homeostasis is involved in the pathogenesis of various neurodegenerative diseases including senile type of dementia such as Alzheimer's disease, dementia with Lewy bodies, and vascular dementia. In particular, synaptic Zn2+ is known to play critical roles in the pathogenesis of vascular dementia. In this article, we review the molecular pathways of Zn2+-induced neurotoxicity based on our and numerous other findings, and demonstrated the implications of the energy production pathway, the disruption of calcium homeostasis, the production of reactive oxygen species (ROS), the endoplasmic reticulum (ER)-stress pathway, and the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) pathway. Furthermore, we have searched for substances that protect neurons from Zn2+-induced neurotoxicity among various agricultural products and determined carnosine (β-alanyl histidine) as a possible therapeutic agent for vascular dementia.

Conformational changes in amyloidogenic proteins, such as β-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with β-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca2+-permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca2+ homeostasis in amyloid-associated neurodegenerative diseases.

The adenosine A2B receptor is a critical protein in intestinal water secretion. In the present study, we screened compound libraries to identify inhibitors of the A2B receptor and evaluated their effect on adenosine-induced intestinal fluid secretion. The screening identified the dihydropyridine calcium antagonists nifedipine and nisoldipine. Their respective affinities for the A2B receptor (Ki value) were 886 and 1,399 nM. Nifedipine and nisoldipine, but not amlodipine or nitrendipine, inhibited both calcium mobilization and adenosine-induced cAMP accumulation in cell lines. Moreover, adenosine injection into the lumen significantly increased fluid volume in the colonic loop of wild-type mice but not A2B receptor-deficient mice. PSB-1115, a selective A2B receptor antagonist, and nifedipine prevented elevated adenosine-stimulated fluid secretion in mice. Our results may provide useful insights into the structure-activity relationship of dihydropyridines for A2B receptor. As colonic fluid secretion by adenosine seems to rely predominantly on the A2B receptor, nifedipine could be a therapeutic candidate for diarrhoea-related diseases.

Parkinson's disease is a progressive neurodegenerative disease for which prevention and effective treatments are lacking. The pathogenesis of Parkinson's disease is not clearly understood. It is thought to be caused by oxidative stress-dependent loss of dopamine neurons in the substantia nigra and the promotion of inflammatory responses by microglia at the lesion site. In addition, cell loss occurs in the hypothalamus of Parkinson's disease patients. Carnosine is an endogenous dipeptide that can exert many beneficial effects, including an antioxidant action, metal ion chelation, proton buffering capacity, and inhibition of protein carbonylation and glycolysis. Previously, we found that carnosine inhibits trace metal-induced death of immortalized hypothalamic neuronal GT1-7 cells. In this study, we analyzed the efficacy of carnosine on 6-hydroxydopamine (6-OHDA)-dependent GT1-7 cell death and inflammatory responses. We found that carnosine significantly prevented 6-OHDA-dependent GT1-7 cell death in a dose-dependent manner. Moreover, carnosine significantly suppressed the expression of 6-OHDA-induced integrated stress response (ISR)-related factors and pro-inflammatory cytokines. Carnosine also significantly inhibited 6-OHDA-dependent reactive oxygen species (ROS) production and c-Jun amino-terminal kinase (JNK) pathway activation in GT1-7 cells. These results indicate that carnosine inhibits hypothalamic neuronal cell death and inflammatory responses by inhibiting the ROS-JNK pathway. We therefore suggest that carnosine may be effective in preventing the onset or the exacerbation of Parkinson's disease.

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(-)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1 and 5, (R)-(-)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(-)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(-)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.

Zinc (Zn) plays an important role in many organisms in various physiological functions such as cell division, immune mechanisms and protein synthesis. However, excessive Zn release is induced in pathological situations and causes neuronal cell death. Previously, we reported that Cu ions (Cu2+) markedly exacerbates Zn2+-induced neuronal cell death by potentiating oxidative stress and the endoplasmic reticulum stress response. In contrast, the stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK) signaling pathway is important in neuronal cell death. Thus, in this study, we focused on the SAPK/JNK signaling pathway and examined its involvement in Cu2+/Zn2+-induced neurotoxicity. Initially, we examined expression of factors involved in the SAPK/JNK signaling pathway. Accordingly, we found that phosphorylated (ie, active) forms of SAPK/JNK (p46 and p54) are increased by CuCl2 and ZnCl2 co-treatment in hypothalamic neuronal mouse cells (GT1-7 cells). Downstream factors of SAPK/JNK, phospho-c-Jun, and phospho-activating transcription factor 2 are also induced by CuCl2 and ZnCl2 co-treatment. Moreover, an inhibitor of the SAPK/JNK signaling pathway, SP600125, significantly suppressed neuronal cell death and activation of the SAPK/JNK signaling pathway induced by CuCl2 and ZnCl2 cotreatment. Finally, we examined involvement of oxidative stress in activation of the SAPK/JNK signaling pathway, and found that human serum albumin-thioredoxin fusion protein, an antioxidative protein, suppresses activation of the SAPK/JNK signaling pathway. On the basis of these results, our findings suggest that activation of ZnCl2-dependent SAPK/JNK signaling pathway is important in neuronal cell death, and CuCl2-induced oxidative stress triggers the activation of this pathway.

Zinc (Zn) is abundantly present in the brain, and accumulates in the synaptic vesicles. Synaptic Zn is released with neuronal excitation, and plays essential roles in learning and memory. Increasing evidence suggests that the disruption of Zn homeostasis is involved in various neurodegenerative diseases including Alzheimer's disease, a vascular type of dementia, and prion diseases. Our and other numerous studies suggest that carnosine (β-alanyl histidine) is protective against these neurodegenerative diseases. Carnosine is an endogenous dipeptide abundantly present in the skeletal muscles and in the brain, and has numerous beneficial effects such as antioxidant, metal chelating, anti-crosslinking, and anti-glycation activities. The complex of carnosine and Zn, termed polaprezinc, is widely used for Zn supplementation therapy and for the treatment of ulcers. Here, we review the link between Zn and these neurodegenerative diseases, and focus on the neuroprotective effects of carnosine. We also discuss the carnosine level in various foodstuffs and beneficial effects of dietary supplementation of carnosine.

Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer's disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu2+/Zn2+-induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu2+/Zn2+-induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu2+/Zn2+-induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu2+ and Zn2+ after Cu2+/Zn2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu2+/Zn2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu2+/Zn2+-induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases.

The standard treatment for chronic obstructive pulmonary disease is a combination of anti-inflammatory drugs and bronchodilators. We recently found that mepenzolate bromide (MP), an antagonist for human muscarinic M3 receptor (hM3R), has both anti-inflammatory and short-acting bronchodilatory activities. To obtain MP derivatives with longer-lasting bronchodilatory activity, we synthesized hybrid compounds based on MP and two other muscarinic antagonists with long-acting bronchodilatory activity glycopyrronium bromide (GC) and aclidinium bromide (AD). Of these three synthesized hybrid compounds (MP-GC, GC-MP, MP-AD) and MP, MP-AD showed the highest affinity for hM3R and had the longest lasting bronchodilatory activity, which was equivalent to that of GC and AD. Both MP-GC and MP-AD exhibited an anti-inflammatory effect equivalent to that of MP, whereas, in line with GC and AD, GC-MP did not show this effect. We also confirmed that administration of MP-AD suppressed elastase-induced pulmonary emphysema in a mouse model. These findings provide important information about the structure-activity relationship of MP for both bronchodilatory and anti-inflammatory activities.

Zinc (Zn) is known as a co-factor for over 300 metalloproteins or enzymes, and has essential roles in many physiological functions. However, excessively high Zn concentrations are induced in pathological conditions such as interruption of blood flow in stroke or transient global ischemia-induced neuronal cell death. Furthermore, we recently found that copper (Cu2+) significantly exacerbates Zn2+ neurotoxicity in mouse hypothalamic neuronal cells, suggesting that Zn2+ interaction with Cu2+ is important for the development of neurological disease. Meanwhile, organic acids such as pyruvic acid and citric acid are reported to prevent neuronal cell death induced by various stresses. Thus, in this study, we focused on organic acids and searched for compounds that inhibit Cu2+/Zn2+-induced neurotoxicity. Initially, we examined the protective effect of various organic acids on Cu2+/Zn2+-induced neurotoxicity, and found that pyruvic acid clearly suppresses Cu2+/Zn2+-induced neurotoxicity in GT1-7 cells. Next, we examined the protective mechanisms of pyruvic acid against Cu2+/Zn2+-induced neurotoxicity. Specifically, we examined the possibilities that pyruvic acid chelates Cu2+ and Zn2+ or suppresses the ER stress response, but found that neither was suppressed by pyruvic acid treatment. In contrast, pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury, in a dose-dependent manner. These results suggest that pyruvic acid prevents Cu2+/Zn2+-induced neuronal cell death by suppressing mitochondrial injury. Based on our results, we assume that pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death such as vascular dementia.

BACKGROUND AND PURPOSE Visceral hypersensitivity is responsible for pathogenesis of irritable bowel syndrome (IBS). Therefore, its prevention can help avoid abdominal pain and discomfort in IBS. To find candidate drugs for visceral hypersensitivity, we screened existing medicines for their ability to prevent visceral sensitivity induced by colorectal distension (CRD) in rats and identified chlorpromazine, a typical antipsychotic drug, as a candidate compound. In this study, we investigated the effect of chlorpromazine on visceral hypersensitivity. EXPERIMENTAL APPROACH Visceral sensitivity (visceromotor response) was monitored by measuring the electrical activity of the abdominal external oblique muscle contraction in response to CRD using a barostat apparatus. Visceral hypersensitivity was induced by a colonic instillation of sodium butyrate or acetic acid in neonates. KEY RESULTS Oral administration of chlorpromazine suppressed butyrate-induced visceral hypersensitivity to CRD. Interestingly, atypical antipsychotic drugs, quetiapine and risperidone, ameliorated butyrate-induced visceral hypersensitivity, whereas the typical antipsychotic drugs, haloperidol and sulpiride, did not. Pharmacological analysis using specific inhibitors showed that a selective 5-HT2A receptor antagonist, ketanserin, suppressed butyrate-induced visceral hypersensitivity, whereas a selective dopamine D-2 receptor antagonist, L-741626, did not. Furthermore, the 5-HT2A receptor agonist AL-34662 stimulated visceral sensitivity to CRD in healthy control rats but not in butyrate-treated rats. These findings suggest that increased 5-HT levels in the colon contribute to the induction of visceral hypersensitivity. CONCLUSIONS AND IMPLICATIONS Our results indicate that chlorpromazine ameliorates visceral hypersensitivity and that the 5-HT2A receptor is a potential therapeutic target for treating abdominal pain and discomfort in IBS.

Increasing evidence suggests that disruption of metal homeostasis contributes to the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease, prion diseases, Lewy body diseases, and vascular dementia. Conformational changes of disease-related proteins (amyloidogenic proteins), such as beta-amyloid protein, prion proteins, and alpha-synuclein, are well-established contributors to neurotoxicity and to the pathogenesis of these diseases. Recent studies have demonstrated that these amyloidogenic proteins are metalloproteins that bind trace elements, including zinc, iron, copper, and manganese, and play significant roles in the maintenance of metal homeostasis. We present a current review of the role of trace elements in the functions and toxicity of amyloidogenic proteins, and propose a hypothesis integrating metal homeostasis and the pathogenesis of neurodegenerative diseases that is focused on the interactions among metals and between metals and amyloidogenic proteins at the synapse, considering that these amyloidogenic proteins and metals are co-localized at the synapse.

Idiopathic pulmonary fibrosis (IPF) involves alveolar epithelial injury and abnormal collagen production caused by activated fibroblasts; transforming growth factor (TGF)-beta 1 is implicated in this activation. In this study, we screened for chemicals capable of inhibiting TGF-beta 1-induced collagen production in cultured fibroblasts from medicines already in clinical use. We selected felodipine based on its extent of collagen production inhibition, clinical safety profile, and other pharmacological activity. Felodipine is a dihydropyridine Ca2+ channel blocker that has been used clinically to treat patients with high blood pressure. Felodipine suppressed collagen production within LL29 cells in the presence of TGF-beta 1, but not in its absence. Intratracheal administration of felodipine prevented bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction. Felodipine also improved pulmonary fibrosis, as well as lung and respiratory function when administered after fibrosis development. Furthermore, administration of felodipine suppressed a bleomycin-induced increase in activated fibroblasts in the lung. We also found other dihydropyridine Ca2+ channel blockers (nifedipine and benidipine) inhibited collagen production in vitro and partially prevented bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction in vivo. We propose that these Ca2+ channel blockers may be therapeutically beneficial for IPF patients.

Zinc (Zn), an essential trace element, is secreted by synaptic vesicles during neuronal excitation and plays several critical roles in neuronal information processing. However, excess Zn ion (Zn2+) is neurotoxic and has a causative role in the pathogenesis of vascular dementia. Here, we investigated the molecular mechanism of Zn2+-induced neurotoxicity by using immortalized hypothalamic neurons (GT1-7 cells), which are more vulnerable than other neuronal cells to Zn2+. We examined the effects of other metal ions on the Zn2+-induced neurotoxicity in these cells and found that sub-lethal concentrations of copper ion (Cu2+) markedly exacerbated Zn2+-induced neurotoxicity. The co-administration of Cu2+ and Zn2+ also significantly increased the expression of genes related to the endoplasmic reticulum's stress response, including CHOP, GADD34, and ATF4. Similar to Zn2+, Cu2+ is stored in presynaptic vesicles and secreted during neuronal excitation. Thus, based on our results, we hypothesize here that Cu2+ interacts with Zn2+ in the synapse to synergistically promote neuronal death and significantly influence the pathogenesis of vascular dementia.

For acute respiratory distress syndrome (ARDS), mechanical ventilation (MV) is a life-saving intervention without alternative; however, MV can cause ventilator-induced lung injury. Reactive oxygen species (ROS) play important roles in the pathogenesis of both ARDS and ventilator-induced lung injury. Lecithinized superoxide dismutase (PC-SOD) overcomes the limitations of superoxide dismutase such as low tissue affinity and low stability in plasma. In this study, we examined the effect of PC-SOD on tissue injury, edema, and inflammation in the lung and other organs of mice subjected to cecal ligation and puncture (CLP), LPS administration, or MV. The severity of the lung injury was assessed on the basis of vascular permeability, histopathologic evaluation, and lung mechanics. Intravenous PC-SOD administration (the first administered just before CLP) increased the survival rate and decreased vascular permeability in mice subjected to CLP. PC-SOD, but not dexamethasone or sivelestat sodium hydrate (sivelestat), suppressed CLP-induced kidney injury and systemic inflammation. PC-SOD also suppressed vascular permeability, tissue injury, and inflammation in the lung induced by LPS administration. Moreover, PC-SOD, but not dexamethasone or sivelestat, suppressed vascular permeability, edema, tissue injury, and mechanical alterations in the lung induced by MV. In vivo imaging analysis of ROS revealed that CLP, LPS administration, and MV increased the level of ROS and that this increase was suppressed by PC-SOD. The results of this study thus suggest that, on the basis of its ROS-reducing properties, intravenous administration of PC-SOD may be beneficial for patients at high risk of developing ARDS.

Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute lung injury, which involves neutrophilic inflammation and pulmonary cell death. Reactive oxygen species (ROS) play important roles in ARDS development. New compounds for inhibiting the onset and progression of ARDS are required. Carnosine (beta-alanyl-L-histidine) is a small di-peptide with numerous activities, including antioxidant effects, metal chelation, proton buffering capacity and the inhibition of protein carbonylation and glycoxidation. We have examined the preventive effects of carnosine on tissue injury, oedema and inflammation in a murine model for ARDS. Oral administration of carnosine suppressed lipopolysaccharide (LPS)-induced vascular permeability, tissue injury and inflammation in the lung. In vivo imaging analysis revealed that LPS administration increased the level of ROS and that this increase was inhibited by carnosine administration. Carnosine also suppressed LPS-induced neutrophilic inflammation (evaluated by activation of myeloperoxidase in the lung and increased extracellular DNA in bronchoalveolar lavage fluid). Furthermore, carnosine administration suppressed the LPS-induced endoplasmic reticulum stress response in vivo. These results suggest that the oral administration of carnosine suppresses LPS-induced lung injury via carnosine's ROS-reducing activity. Therefore, carnosine may be beneficial for suppressing the onset and progression of ARDS.

Pharmacological therapy for irritable bowel syndrome (IBS) has not been established. In order to find candidate drugs for IBS with diarrhea (IBS-D), we screened a compound library of drugs clinically used for their ability to prevent stress-induced defecation and visceral hypersensitivity in rats. We selected the bronchodilator aminophylline from this library. Using a specific inhibitor for each subtype of adenosine receptors (ARs) and phosphodiesterases (PDEs), we found that both A(2B)ARs and PDE4 are probably mediated the inhibitory effect of aminophylline on wrap restraint stress (WRS)-induced defecation. Aminophylline suppressed maternal separation-and acetic acid administration-induced visceral hypersensitivity to colorectal distension (CRD), which was mediated by both A(2A)ARs and A(2B)ARs. We propose that aminophylline is a candidate drug for IBS-D because of its efficacy in both of stress-induced defecation and visceral hypersensitivity, as we observed here, and because it is clinically safe.

Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized beta ENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-beta ENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease-and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-beta ENaC-Tg mice. Treatments of C57/BL6J-beta ENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-beta ENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-beta ENaC-Tg mice, consistent with the characteristics of human COPD/CF.

In the stress response, activation of the hypothalamic-pituitary-adrenal axis, and particularly the release of glucocorticoids, plays a critical role. However, dysregulation of this system and sustained high plasma levels of glucocorticoids can result in depression. Recent studies have suggested the involvement of reactive oxygen species (ROS), such as superoxide anion, in depression. However, direct evidence for a role of ROS in the pathogenesis of this disorder is lacking. In this study, using transgenic mice expressing human Cu/Zn-superoxide dismutase (SOD1), an enzyme that catalyzes the dismutation of superoxide anions, we examined the effect of SOD1 overexpression on depressive-like behavioral phenotypes in mice. Depressive-like behaviors were induced by daily subcutaneous administration of the glucocorticoid corticosterone for 4 weeks, and was monitored with the social interaction test, the sucrose preference test and the forced swim test. These tests revealed that transgenic mice overexpressing SOD1 are more resistant to glucocorticoid-induced depressive-like behavioral disorders than wild-type animals. Furthermore, compared with wild-type mice, transgenic mice showed a reduction in the number of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress)-positive cells in the hippocampal CA3 region following corticosterone administration. These results suggest that overexpression of SOD1 protects mice against glucocorticoid-induced depressive-like behaviors by decreasing cellular ROS levels. (C) 2015 Elsevier Inc. All rights reserved.

In the stress response, activation of the hypothalamic-pituitary-adrenal axis, and particularly the release of glucocorticoids, plays a critical role. However, dysregulation of this system and sustained high plasma levels of glucocorticoids can result in depression. Recent studies have suggested the involvement of reactive oxygen species (ROS), such as superoxide anion, in depression. However, direct evidence for a role of ROS in the pathogenesis of this disorder is lacking. In this study, using transgenic mice expressing human Cu/Zn-superoxide dismutase (SOD1), an enzyme that catalyzes the dismutation of superoxide anions, we examined the effect of SOD1 overexpression on depressive-like behavioral phenotypes in mice. Depressive-like behaviors were induced by daily subcutaneous administration of the glucocorticoid corticosterone for 4 weeks, and was monitored with the social interaction test, the sucrose preference test and the forced swim test. These tests revealed that transgenic mice overexpressing SOD1 are more resistant to glucocorticoid-induced depressive-like behavioral disorders than wild-type animals. Furthermore, compared with wild-type mice, transgenic mice showed a reduction in the number of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress)-positive cells in the hippocampal CA3 region following corticosterone administration. These results suggest that overexpression of SOD1 protects mice against glucocorticoid-induced depressive-like behaviors by decreasing cellular ROS levels. (C) 2015 Elsevier Inc. All rights reserved.

Delayed gastric emptying and impaired gastric accommodation (decreased gastric compliance) play important roles in functional dyspepsia (FD). Here we screen for a clinically used drug with an ability to improve delayed gastric emptying in rats. Oral administration of aldioxa (dihydroxyaluminum allantoinate) partially improved clonidine-or restraint stress-induced delayed gastric emptying. Administration of allantoin, but not aluminium hydroxide, restored the gastric emptying. Both aldioxa and allantoin inhibited clonidine binding to the alpha-2 adrenergic receptor, suggesting that antagonistic activity of the allantoin moiety of aldioxa on this receptor is involved in the restoration of gastric emptying activity. Aldioxa or aluminium hydroxide but not allantoin restored gastric compliance with restraint stress, suggesting that aluminium hydroxide moiety is involved in this restoration. We propose that aldioxa is a candidate drug for FD, because its safety in humans has already been confirmed and its ameliorating effect on both of delayed gastric emptying and impaired gastric compliance are confirmed here.

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. (C) 2015 Elsevier Ltd. All rights reserved.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and airflow limitations. We recently proposed that the muscarinic antagonist mepenzolate bromide (mepenzolate) would be therapeutically effective against COPD due to its muscarinic receptor-dependent bronchodilatory activity as well as anti-inflammatory properties. Mepenzolate has an asymmetric carbon atom, thus providing us with the opportunity to synthesize both of its enantiomers ((R)- and (S)-mepenzolate) and to examine their biochemical and pharmacological activities. (R)- or (S)-mepenzolate was synthesized by condensation of benzilic acid with (R)- or (S)-alcohol, respectively, followed by quaternization of the tertiary amine. As predicted by computational simulation, a filter-binding assay in vitro revealed that (R)-mepenzolate showed a higher affinity for the muscarinic M3 receptor than (S)-mepenzolate. In vivo, the bronchodilatory activity of (R)-mepenzolate was superior to that of (S)-mepenzolate, whereas anti-inflammatory activity was indistinguishable between the two enantiomers. We confirmed that each mepenzolate maintained its original stereochemistry in the lung when administered intratracheally. These results suggest that (R)-mepenzolate may have superior properties to (S)-mepenzolate as a drug to treat COPD patients given that the former has more potent bronchodilatory activity than the latter. (C) 2014 Elsevier Ltd. All rights reserved.

Idiopathic pulmonary fibrosis is thought to involve lung injury caused by reactive oxygen species (ROS), which in turn is followed by abnormal fibrosis. A transforming growth factor (TGF)-beta 1-induced increase in myofibroblast number plays an important role in this abnormal fibrosis. We recently found that mepenzolate bromide (mepenzolate), which has been used clinically to treat gastrointestinal disorders, has ROS-reducing properties. In the present study, we examined the effect of mepenzolate on bleomycin-induced pulmonary fibrosis and lung dysfunction in mice. The severity of pulmonary fibrosis was assessed by histopathologic evaluation and determination of hydroxyproline levels. Lung mechanics (elastance) and respiratory function [forced vital capacity (FVC)] were assessed using a computer-controlled ventilator. Respiratory function was also evaluated by monitoring percutaneous arterial oxygen saturation (SpO(2)). Intratracheal administration of mepenzolate prior to bleomycin treatment reduced the extent of pulmonary fibrosis and changes in lung mechanics and led to a significant recovery of both FVC and SpO(2) compared with control. Furthermore, mepenzolate produced a therapeutic effect even when it was administered after the development of fibrosis. Administration of mepenzolate also prevented bleomycin-induced pulmonary cell death and inflammatory responses and increased myofibroblast number. Mepenzolate also decreased NADPH oxidase activity and active TGF-b1 level or increased glutathione S-transferase (GST) activity in the presence of bleomycin treatment. These results show that the intratracheal administration of mepenzolate reduced bleomycin-induced pulmonary fibrosis and lung dysfunction in mice. These effects may be due to this drug's inhibitory effect on NADPH oxidase and TGF-b1 activities and its stimulatory effect on GST.

We recently proposed that mepenzolate bromide (mepenzolate) would be therapeutically effective against chronic obstructive pulmonary disease (COPD) due to its both anti-inflammatory and bronchodilatory activities. In this study, we examined the benefits and adverse effects associated with different routes of mepenzolate administration in mice. Oral administration of mepenzolate caused not only bronchodilation but also decreased the severity of elastase-induced pulmonary emphysema; however, compared with the intratracheal route of administration, about 5000 times higher dose was required to achieve this effect. Intravenously or intrarectally administered mepenzolate also showed these pharmacological effects. The intratracheal route of mepenzolate administration, but not other routes, resulted in protective effects against elastase-induced pulmonary damage and bronchodilation at a much lower dose than that which affected defecation and heart rate. These results suggest that the pulmonary route of mepenzolate administration may be superior to other routes (oral, intravenous or intrarectal) to treat COPD patients.

The clinical treatment of chronic obstructive pulmonary disease (COPD) requires not only an improvement of airflow by bronchodilation but also the suppression of emphysema by controlling inflammation. Here we screen a compound library consisting of clinically used drugs for their ability to prevent elastase-induced airspace enlargement in mice. We show that intratracheal administration or inhalation of mepenzolate bromide, a muscarinic antagonist used to treat gastrointestinal disorders, decreases the severity of elastase-induced airspace enlargement and respiratory dysfunction. Although mepenzolate bromide shows bronchodilatory activity, most other muscarinic antagonists do not improve elastase-induced pulmonary disorders. Apart from suppressing elastase-induced pulmonary inflammatory responses and the production of superoxide anions, mepenzolate bromide reduces the level of cigarette smoke-induced airspace enlargement and respiratory dysfunction. Based on these results, we propose that mepenzolate bromide may be an effective therapeutic for the treatment of COPD due to its anti-inflammatory and bronchodilatory activities.

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory cells and reactive oxygen species (ROS), such as superoxide anion radical (O-2(center dot-)). There is currently no effective treatment of IPF. We previously developed a human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an antioxidative and anti-inflammatory protein. In this study, we examined the therapeutic effect of HSA-Trx on an IPF animal model of bleomycin (BLM)-induced pulmonary fibrosis. A pharmacokinetic study of HSA-Trx or Trx in BLM mice showed that the plasma retention and lung distribution of Trxc was markedly improved by fusion with HSA. A weekly intravenous administration of HSA-Trx, but not Trx, ameliorated BLM-induced fibrosis, as evidenced by a histopathological analysis and pulmonary hydroxyproline levels. HSA-Trx suppressed active-transforming growth factor (TGF)-beta levels in the lung and inhibited the increase of inflammatory cells in bronchoalveolar lavage fluid, pulmonary inflammatory cytokines, and oxidative stress markers. An in vitro EPR experiment using phosphate-buffered saline-stimulated neutrophils confirmed the O-2(center dot-) scavenging ability of HSA-Trx. Furthermore, post-treatment of HSA-Trx had a suppressive effect against BLM-induced fibrosis. These results suggest that HSA-Trx has potential as a novel therapeutic agent for IPF, because of its long-acting antioxidative and anti-inflammatory modulation effects.

We recently reported that, compared to loxoprofen (LOX, an non-steroidal anti-inflammatory drug), the LOX derivative fluoro-loxoprofen (F-LOX) is less ulcerogenic but has similar anti-inflammatory activity. Our previous in vitro studies suggested that both LOX and F-LOX are pro-drugs, the active metabolites of which are their trans-alcohol forms. In this study, we compared the pharmacokinetics of F-LOX and LOX in rats. Overall, the pharmacokinetic characteristics of F-LOX, including the formation of metabolites in vivo and in vitro, were comparable to those of LOX. However, F-LOX disappeared from the plasma more rapidly than LOX, which could potentially explain its lower ulcerogenicity. However, we showed that F-LOX produced fewer gastric lesions than LOX, even when a higher plasma concentration of F-LOX was maintained. Similar to LOX, F-LOX was readily metabolized to its trans-and cis-alcohol forms, with a higher level of the trans-alcohol form being observed after oral or intravenous administration of the drug. The preferential formation of the trans-alcohol form was also observed after incubation of F-LOX with rat liver homogenates in vitro. These results suggest that, similar to LOX, F-LOX acts as a pro-drug and that there is a metabolic system that selectively produces its active metabolite. © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX).