A Phase I Study of TRK-250, a Novel siRNA-Based Oligonucleotide, in Patients with Idiopathic Pulmonary Fibrosis.

Purpose: TRK-250 is a novel single-stranded oligonucleotide carrying a human Transforming growth factor-beta 1-targeting siRNA motif tethered by two proline linkers. Nonclinical studies have shown that TRK-250 may have potency to prevent the progression of pulmonary fibrosis. Herein, a phase I study was conducted to investigate the safety and pharmacokinetics (PKs) of TRK-250 in patients with idiopathic pulmonary fibrosis (IPF). Method: In the phase I study, 34 IPF patients were partially randomized to receive a placebo or TRK-250 in 4 single doses of 2, 10, 30, and 60 mg or multiple rising doses of 10, 30, and 60 mg once per week for 4 weeks by oral inhalation. For both the single- and multiple-dose studies, the primary endpoint was safety, and the secondary endpoint was PKs. Result: In all IPF patients who orally inhaled TRK-250, no significant drug-related adverse events (AEs) were observed. The AEs were mild or moderate, except for one severe case with acute exacerbation. One of the more common AEs was coughing. One patient discontinued treatment before the last dose because of coughing. There were no medically important findings related to safety endpoints based on clinical laboratory data (clinical chemistry, hematology, or urinalysis), vital signs data, electrocardiogram data, physical examination findings, pulse oximetry data, spirometry data, or diffusing capacity of the lung for carbon monoxide data. All the bioanalytical results of PKs in the blood were below the lower limit of quantification. Conclusions: Both the single and multiple doses of TRK-250 were safe and well tolerated in this first study done in IPF patients. Furthermore, TRK-250 was not detected in the systemic circulation following inhalation, indicating low or virtually nonexistent systemic exposure. This study is registered at ClinicalTrials.gov with identifier number NCT03727802.

Pharmacological effects of a vitamin K1 2,3-epoxide reductase (VKOR) inhibitor, 3-acetyl-5-methyltetronic acid, on cisplatin-induced fibrosis in rats.

Cisplatin (CDDP) is a chemotherapeutic agent that is widely used in the treatment of lymphomas and solid malignancies. However, its clinical usage is limited by its severe side effects in the kidneys. Glomerular and tubular injuries in the kidneys commonly progress to interstitial fibrosis and, ultimately, the end stage of renal failure. We previously reported that 3-acetyl-5-methyltetronic acid (AMT) had inhibitory effects on rat renal vitamin K1 2,3-epoxide reductase (VKOR) in vitro and also suppressed mesangial cell proliferation and, consequently, the formation of fibrosis via the vitamin K-dependent activation of the growth arrest-specific 6 (Gas6)/Axl pathway in anti-Thy-1 glomerulonephritis (Thy-1 GN) in rats. In the present study, we demonstrated that AMT alleviated the progression of renal fibrosis in CDDP-treated rats. The repeated intravenous administration of AMT for 28 days dose-dependently suppressed increases in plasma urea nitrogen and plasma creatinine levels as well as creatinine clearance in CDDP-treated rats. Furthermore, the treatment suppressed the expression of α-smooth muscle actin (SMA)-positive cells and ameliorated the extracellular matrix accumulation of collagen III, indicating an antifibrotic effect. In conclusion, our toxicological and histopathological results demonstrated quantitatively the pharmacological inhibitory effects of AMT on the progression of renal fibrosis in CDDP-treated rats.

Identification of Transporters Involved in Beraprost Sodium Transport In Vitro.

BACKGROUND AND OBJECTIVE: Beraprost sodium (BPS) is a chemically stable and orally active prostacyclin analog that is used in the treatment of chronic arterial occlusive disease since 1992 and primary pulmonary hypertension since 1999 in Japan. Multiple-drug therapy is common in clinical practice, and BPS is co-administered with other drugs. Membrane transporters are known to markedly affect pharmacokinetics, safety and efficacy, and many transporter-based drug-drug interactions have been recently reported. However, information on the transporters involved in the pharmacokinetics of BPS is limited. METHODS: First of all, we have examined 11 transporters, ABCB1 (P-glycoprotein: P-gp), ABCG2 (breast cancer resistance protein: BCRP), SLC22A6 (organic anion transporter 1: OAT1), SLC22A8 (organic anion transporter 3: OAT3), SLCO1B1 (organic anion transporting polypeptide 1B1: OATP1B1), SLCO1B3 (organic anion transporting polypeptide 1B3: OATP1B3), SLCO2B1 (organic anion transporting polypeptide 2B1: OATP2B1), SLC22A1 (organic cation transporter 1: OCT1), SLC22A2 (organic cation transporter 2: OCT2), ABCB11 (bile-salt export pump: BSEP), and ABCC2 (multidrug resistance associated protein 2: MRP2) to clarify which of them would be candidates that might recognize BPS as their substrate in transporter-expressing LLC-PK1, S2, and HEK293 cells as well as in membrane vesicles. Furthermore, we determined whether the transport of BPS was inhibited by the typical inhibitors of each transporter, i.e., verapamil for P-gp, Ko143 for BCRP, probenecid for OAT3, rifampicin for OATP1B1 and OATP1B3, cyclosporine for BSEP, and sulfobromophthalein (BSP) for MRP2. RESULTS: The results obtained showed that P-gp, BCRP, OAT3, OATP1B1, OATP1B3, BSEP and MRP2 might be candidates for BPS transporters. From the further evaluation with the typical inhibitors of each transporter, it was confirmed that BPS is a substrate for P-gp, BCRP, OAT3, OATP1B1, OATP1B3 and MRP2, because the typical inhibitor, cyclosporine, had no effects on BPS transport by BSEP. CONCLUSIONS: BPS is a substrate of 6 transporters: P-gp, BCRP, OAT3, OATP1B1, OATP1B3, and MRP2, because their expressing cells and vesicles transported BPS more than in the controls, and BPS transport activities were reduced by the typical inhibitors of tested transporters. Although there are no reports regarding drug-drug interactions between BPS and possible combination drugs expected due to transporters, it may be necessary to notice that that substrates or inhibitors for the 6 mentioned transporters may have effects on pharmacokinetics of BPS when co-administered.

Evaluation of the repeated-dose liver micronucleus assay using 2,4-dinitrotoluene: a report of a collaborative study by CSGMT/JEMS.MMS.

The liver micronucleus assay using young adult rats has the potential to detect liver carcinogens by repeated dosing, and could be expected to be integrated into repeated-dose toxicity studies using a hepatocyte isolation method without the traditional in situ collagenase perfusion. In this study, to assess the performance of the repeated-dose liver micronucleus assay, 2,4-dinitrotoluene (DNT), which is a rodent liver carcinogen, was administered orally to male rats at doses of 50, 100 and 200 mg/kg/day once daily for 14 or 28 consecutive days, and the frequencies of micronucleated hepatocytes (MNHEPs) and micronucleated immature erythrocytes (MNIMEs) were examined. Significant increases in the MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment, and 50 and 100 mg/kg/day in the 28-day treatment. These increases were dependent on both the dose and the number of administrations, which indicates the possibility that the MNHEPs accumulate as a result of repeated dosing. In contrast, no increase in the MNIMEs was observed. In conclusion, the repeated-dose liver micronucleus assay using young adult rats is sufficiently sensitive to detect the genotoxicity of 2,4-DNT at a low dose.

Hepatic microsomal UDP-glucuronosyltransferase (UGT) activities in the microminipig.

The microminipig, a small minipig, was bred as a novel experimental animal for nonclinical pharmacology/toxicology studies by Fuji Micra Inc. (Shizuoka, Japan). Species differences in drug metabolism between humans and the microminipig need to be elucidated in more detail in order to discuss the results of nonclinical studies. Glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) is an important pathway in the metabolism of a wide variety of compounds. The aim of the present study was to identify the characteristics of hepatic UGT activity in the microminipig and compare them with those in humans and other experimental animals. This study examined in vitro UGT activities using liver microsomes from microminipigs (8 months old and 1 day old), humans, mice, rats, dogs, monkeys and minipigs. The glucuronides of estradiol, imipramine, serotonin, propofol, 3'-azido-3'-deoxythymidine (AZT) and morphine, selective substrates of human UGT1A1, 1A4, 1A6, 1A9 and 2B7 (AZT and morphine), respectively, were measured using LC-MS/MS. Estradiol-3-glucuronidation activity was higher in the microminipig than in humans and the other animals. High levels of estradiol-3-glucuronidation were observed in the microsomes of 1-day-old microminipigs. Imipramine-N-glucuronidation, a distinctive conjugation by human UGT1A4, was catalysed by microminipig liver microsomes, but not by dog liver microsomes. Although AZT-glucuronidation activity was low in the microminipig compared with humans, morphine-3-glucuronidation activity in the microminipig was higher than that in humans. The UGT activities in the microminipig were similar to those in the minipig. The results of the present study have provided useful information for selecting an appropriate animal model for nonclinical studies.

Urinary cystatin C as a renal biomarker and its immunohistochemical localization in anti-GBM glomerulonephritis rats.

The usefulness of urinary cystatin C for the early detection of renal damage in anti-glomerular basement membrane (GBM) glomerulonephritis rats was investigated and compared to other biomarkers (ß2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), alpha-glutathione S-transferase (GST-α), mu-glutathione S-transferase (GST-µ), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, tissue inhibitor of metalloprotease-1 (TIMP-1), and vascular endothelial growth factor (VEGF)). Urinary levels of cystatin C increased in anti-GBM glomerulonephritis rats, whereas the conventional markers, plasma creatinine and UN did not, demonstrating its usefulness for the early detection of renal damage associated with anti-GBM glomerulonephritis. As well as cystatin C, urinary ß2-microglobulin, clusterin, GST-α, GST-µ, KIM-1, and NGAL also had the potential to detect renal damage associated with anti-GBM glomerulonephritis. Furthermore, the immunohistochemical localization of cystatin C in the kidney was examined. Cystatin C expression was mainly observed in the proximal renal tubules in anti-GBM glomerulonephritis rats, and its expression barely changed with the progression of glomerulonephritis. Cystatin C expression was also observed in the tubular lumen of the cortex and medulla when glomerulonephritis was marked, which was considered to be characteristic of renal damage. In conclusion, urinary cystatin C, ß2-microglobulin, clusterin, GST-α, GST-µ, KIM-1, and NGAL could be useful biomarkers of renal damage in anti-GBM glomerulonephritis rats. Immunohistochemical cystatin C expression in the proximal renal tubules was barely changed by the progression of glomerulonephritis, but it was newly observed in the tubular lumen when renal damage was apparent.

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase.

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-ß1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-ß-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.

Phosphoenolpyruvate, a glycolytic intermediate, as a cytoprotectant and antioxidant in ex-vivo cold-preserved mouse liver: a potential application for organ preservation.

OBJECTIVES: The aim of this study was to examine the effect of phosphoenolpyruvate (PEP), a glycolytic intermediate, on organ damage during cold preservation of liver. METHODS: An ex-vivo mouse liver cold-preservation model and an in-vitro liver injury model induced by hydrogen peroxide in HepG2 cells were leveraged. KEY FINDINGS: PEP attenuated the elevation of aminotransferases and lactate dehydrogenase leakage during organ preservation, histological changes and changes in oxidative stress parameters (measured as thiobarbituric acid reactive substance and glutathione content) induced by 72 h of cold preservation of the liver. The effects were comparable with the University of Wisconsin solution, a gold standard organ preservation agent. The decrease in ATP content in liver during the cold preservation was attenuated by PEP treatment. PEP prevented the cellular injury and increases in intracellular reactive oxygen species in HepG2 cells. In addition, PEP scavenged hydroxyl radicals, but had no effect on superoxide anion as evaluated by an electron paramagnetic resonance spin-trapping technique. CONCLUSIONS: PEP significantly attenuated the injury, oxidative stress and ATP depletion in liver during cold preservation. The antioxidative potential of PEP was confirmed by in-vitro examination. We suggest that PEP acts as a glycolytic intermediate and antioxidant, and is particularly useful as an organ preservation agent in clinical transplantation.

Urinary cystatin C as a biomarker for diabetic nephropathy and its immunohistochemical localization in kidney in Zucker diabetic fatty (ZDF) rats.

Cystatin C, a cysteine protease inhibitor, is a novel biomarker of renal damage. In the present study, we examined the usefulness of urinary cystatin C for the detection of diabetic nephropathy in Zucker diabetic fatty (ZDF) rats compared to other biomarkers (ß2-microglobulin, calbindin, clusterin, epidermal growth factor (EGF), alpha-glutathione S-transferase (GST-α), mu-glutathione S-transferase (GST-µ), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, tissue inhibitor of metalloprotease-1 (TIMP-1), and vascular endothelial growth factor (VEGF). Urinary levels of cystatin C were increased in ZDF rats where renal damage was not histopathologically observed, and then further increased with the progression of renal damage, demonstrating the usefulness of early detection and accurate assessment of diabetic nephropathy. Urinary ß2-microglobulin, clusterin, GST-µ, KIM-1, and osteopontin had the potency to detect renal damage in ZDF rats as well as cystatin C. We also investigated immunohistochemical localization of cystatin C in the kidney according to progressive renal damage. Cystatin C expression was mainly observed in the proximal renal tubule in ZDF rats, and hardly changed with progression of nephropathy. When renal damage was remarkable, cystatin C expression was also observed in the tubular lumen of the cortex and medulla, which was considered to be characteristic of renal damage in diabetic nephropathy. In conclusion, urinary cystatin C, ß2-microglobulin, clusterin, GST-µ, KIM-1, and osteopontin could be useful biomarkers of diabetic nephropathy in ZDF rats. Immunohistochemical cystatin C expression in the proximal renal tubule was hardly changed by the progression of diabetic nephropathy, but it was newly observed in the tubular lumen when renal damage was remarkable in ZDF rats.

A uremic toxin, 3-carboxy-4-methyl-5-propyl-2-furanpropionate induces cell damage to proximal tubular cells via the generation of a radical intermediate.

3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a furan fatty acid uremic toxin (UT) and a substrate for organic ion transporters, contributes to the accumulation of CMPF in renal tubular cells. Although oxidative stress induced by UTs has been proposed as a mechanism of its toxicity in chronic kidney disease, little information is available regarding the redox property of CMPF and its relation to renal cell damage. The findings herein show that CMPF enhances the production of reactive oxygen species (ROS) in HK-2 cells in the presence of angiotensin II (A-II), an inducer of O(2)(·-). When iron is also present, CMPF and A-II induce the Fenton reaction, resulting in a further increase in ROS production. Such CMPF-induced oxidative stress increases TGF-ß1 secretion in HK-2 cells, and a positive correlation between CMPF-induced ROS production and the secretion of active TGF-ß1 was observed. CMPF caused a reduction in cell viability which was negatively correlated with intracellular ROS production. These negative effects of CMPF in HK-2 cells were completely suppressed by probenecid, an inhibitor of organic anion transport. Interestingly, in vitro ROS assays indicate that CMPF directly interacts with superoxide anion radicals (O(2)(·-)) and peroxy radicals (LOO) to produce CMPF radicals. The subsequent interaction of CMPF radicals with dissolved oxygen leads to the overproduction of O(2)(·-). Based on these findings, we conclude that CMPF, which accumulates in the renal cells, appears to play a prominent role as a pro-oxidant which subsequently leads to renal cellular damage via the overproduction of O(2)(·-).

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

Identification of human cytochrome P450 enzymes involved in the metabolism of a novel к-opioid receptor agonist, nalfurafine hydrochloride.

Nalfurafine hydrochloride (TRK-820) exhibits strong к-opioid agonistic activity and is a new antipruritic agent for uremic pruritus. This study was performed to identify the human hepatic cytochrome P450 isoforms involved in the metabolic conversion of nalfurafine to the decyclopropylmethylated form, de-CPM, using human liver microsomes and E. coli membrane fractions expressing human P450 isoforms. Samples were analysed by liquid chromatography with a radioactivity detector and liquid chromatography-tandem mass spectrometry. The metabolism of nalfurafine by human liver microsomes exhibited a biphasic kinetic profile. Experiments examining the metabolism by E. coli membrane fractions expressing human P450 isoforms indicated that CYP1A1, 2C8, 2C19 and 3A4 had the ability to produce de-CPM. In experiments with human liver microsomes that examined the inhibition of nalfurafine metabolism by anti-human P450 antibodies, anti-CYP3A4 antibody predominantly, and anti-CYP2C8 and 2C19 antibodies moderately, inhibited de-CPM formation. From these results, CYP3A4 appeared to be the major isoform involved in the metabolic decyclopropylmethylation of nalfurafine, while CYP2C8 and 2C19 most likely play a minor role in the formation of de-CPM.

Urinary cystatin C as a biomarker for acute kidney injury and its immunohistochemical localization in kidney in the CDDP-treated rats.

Cystatin C, a cysteine protease inhibitor, is a novel biomarker of renal damage. In the present study, we examined the urinary and plasma levels of cystatin C and how useful they are for the early detection of acute kidney injury (AKI) in CDDP-treated rats in comparison with other biomarkers (ß2-microglobulin, calbindin, clusterin, EGF, GST-α, GST-µ, KIM-1, NGAL, osteopontin, TIMP-1, and VEGF). The urinary levels of cystatin C, GST-α, KIM-1, and EGF changed prior to proximal tubule damage and increases in plasma urea nitrogen and creatinine levels, suggesting their usefulness for predicting AKI. On the other hand, the plasma cystatin C level hardly changed. We also investigated the localization of cystatin C in the kidney according to the progression of renal damage. Cystatin C was predominantly localized in the proximal tubule of the cortex, and its immunohistochemical expression was not affected by CDDP treatment. In addition, cystatin C was observed in the lumen of the renal tubule in the cortex, cortico-medullary junction, and medulla during the progression of renal damage, although its immunoreactive area ratio was very low. In conclusion, urinary cystatin C measurements can detect CDDP-induced AKI as early as KIM-1, GST-α, and EGF in rats, although the change ratio of the cystatin C was smaller than others. Immunohistochemical cystatin C expression in the proximal tubule of the kidney was hardly changed by the CDDP treatment, but it was newly observed in the renal tubule lumen after CDDP treatment.

A possible mechanism for hepatotoxicity induced by BIRB-796, an orally active p38 mitogen-activated protein kinase inhibitor.

BIRB-796, a selective inhibitor of p38 mitogen-activated protein kinase, has entered clinical trials for the treatment of autoimmune diseases. Levels of alanine transaminase, a biomarker of hepatic toxicity in clinical pathology, were found to be increased in Crohn's disease patients treated with BIRB-796. The purpose of the present study was to clarify the molecular mechanism(s) of this hepatotoxicity. A toxicogenomic analysis using a highly sensitive DNA chip, 3D-Gene™ Mouse Oligo chip 24k, indicated that BIRB-796 treatment activated the nuclear factor (erythroid-derived 2)-like 2 signaling pathway, which plays a key role in the response to oxidative stress. A reactive intermediate of BIRB-796 was detected by the glutathione-trapping method using mouse and human liver microsomes. The production of this reactive metabolite in the liver may be one of the causes of BIRB-796's hepatotoxicity.

Toxicological effect of emodin in mouse testicular gene expression profile.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a herbal medicine extracted from the rhizomes of Rheum palmatum, and is known as an inhibitor of casein kinase II (CK2). The CK2α' knockout mice are known to be male-infertile; however, there have been no reports on the toxicity of emodin in male reproductive organs/tissues. To evaluate the toxicological effects of emodin on differential gene expression profiles of the testis as compared with acrylamide, mice were orally administered emodin and acrylamide for 5 days at a dose of 1000 and 50 mg kg(-1) per day, respectively, and euthanized 24 h after the final administration. Both chemicals induced hypospermatogenesis, eosinophilic change and apoptosis of germ cell. A DNA microarray analysis showed that the IGF-1 receptor signaling was most closely related to the above testicular toxicity induced by emodin, and the RhoA regulation, TGF/WNT and cytoskeletal remodeling, TNFR1 signaling and adenosine A2A receptor signaling were commonly associated with the two chemicals. We selected 36 genes associated with CK2, apoptosis and spermatogenesis and determined their expression by quantitative reverse transcription-polymerase chain reaction (qPCR). Both chemicals perturbed the expression of genes associated with CK2. Genes related to spermatogenesis were also affected, as evidenced by hypospermatogenesis, and eosinophilic change and apoptosis of germ cell. The results suggest that emodin causes testicular toxicity, including apoptosis with related the IGF-1 receptor signaling pathway, and the two chemicals commonly affect CK2, spermatogenesis and sperm motility via four pathways, such as TNFR1 signaling.

Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney.

BACKGROUND: p-Cresyl sulfate (PCS), a recently identified anionic uremic toxin, is the main circulating metabolite of p-cresol. In cases of chronic kidney disease (CKD), it might be associated with cardiovascular outcomes and the progression of CKD. However, the renal excretion pathway of PCS is currently unknown. The objective of the present study was to determine whether organic anion transporters (OATs), which are renal tubular basolateral membrane transporters, play an important role in this process. METHODS: The uptake of PCS was investigated using rat renal cortical slices and human proximal tubular cells (HK-2). The active uptake velocity was calculated by subtracting the uptake velocity at 4°C (nonspecific uptake) from that at 37°C. RESULTS: As evidenced by renal cortical slice experiments, the uptake of PCS was saturable with a mean K(m) of 231.6 µM, indicating that the active transport is involved in the basolateral uptake of PCS. Similar results were also observed in HK-2 cells. The active transport of PCS was significantly suppressed by inhibitors of OATs, such as probenecid, benzylpenicillin, p-aminohippuric acid and estrone sulfate. Similar inhibitions were observed in the presence of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropionate, OATs substrates among uremic toxins. In contrast, digoxin and tetraethylammonium that did not interact with OATs had little inhibitory effect. CONCLUSIONS: The findings of the present study strongly suggest that PCS serves as a substrate for OATs, is preferentially recognized by OAT3 and plays a key role in the renal tubular secretion process.

Reduction of non-specific adsorption of drugs to plastic containers used in bioassays or analyses.

INTRODUCTION: Non-specific adsorption (NSA) of drugs to plastic or glass containers used in clinical use is well known, but methods for reducing NSA have been rarely reported. We assessed the NSA to various containers and then investigated methods to reduce NSA. METHODS: Probe drugs (methotrexate, warfarin, chloroquine, propranolol, verapamil, digoxin and paclitaxel) dissolved in water were incubated in conventional or low-adsorption containers for 4h at 4 degrees C and the NSA was determined by HPLC. They were also dissolved in aqueous methanol or acetonitrile and the NSA to a conventional polypropylene microplate was determined. Finally, tissue culture microplates were coated with silane coupling agents and the effects of the coatings were evaluated. RESULTS: Hydrophobic drugs (paclitaxel, verapamil and digoxin) were highly adsorbed to conventional plastic microplates, but in addition to hydrophobic drugs, positively charged drugs were well adsorbed to the tissue culture microplate. Low-adsorption microplates could reduce NSA below 15%, but positively charged or neutral hydrophobic drugs showed relatively higher adsorption. Acetonitrile showed stronger NSA inhibition than that of methanol, but the peak shapes of methotrexate and chloroquine were broadened and split. Among the silane coupling agents, GPTMS suppressed the NSA below 10%. Also, AATMS resembled the NSA pattern of GPTMS, but it increased the adsorption of methotrexate to 29%. DISCUSSION: On conventional plastic microplates, NSA is mainly driven by hydrophobic interactions, but on tissue culture microplates and low-adsorption microplates, in addition to hydrophobic interactions, ionic interactions play a role in the NSA. Therefore, to reduce the NSA to plastic containers, both hydrophobic and ionic interactions should be reduced using amphiphilic organic solvents or neutral and hydrophilic coatings.

Detection of initiation activity of 1,2-dimethylhydrazine in in vivo medium-term liver initiation assay system using 4-week-old rats without hepatocellular proliferative stimuli during the test chemical treatment period.

We have developed an in vivo medium-term liver initiation assay system to detect initiation activities of chemicals on multi-organ carcinogenesis. However, cell proliferation stimuli during the test chemical treatment period, required in the previously used assay models using adult rats, are laborious; moreover, those cause decrease of hepatic metabolic enzymes and psychological and physical discomfort to animals resulting in inaccurate interpretation. Therefore, we investigated the utility of another in vivo medium-term liver initiation assay model using 4-week-old rats without the cell proliferation stimuli. In this study, we confirmed that 4-week-old and 4.5-week-old male rats have high hepatocyte proliferation activity and similar enzyme activities of hepatic Cytochrome P450 subtypes as compared with 8-week-old male rats. Next, the in vivo medium-term liver initiation assay model using 4-week-old rats without cell proliferation stimuli was evaluated for the detection of the initiation activity of 1,2-dimethylhydrazine (DMH), which is a well-known genotoxic carcinogen. Four-week-old rats were orally administered DMH (single dose, 4 or 16 mg/kg; or 4-day repeat, 1 or 4 mg/kg); subsequently, these rats were treated promotion treatment consisted of administration of 2-acetylaminofluorene and carbon tetrachloride. Four weeks after the first DMH administration, the glutathione S-transferase placental form (GST-P)-positive foci induced by DMH in the liver was measured immunohistochemically. The inductions of GST-P-positive foci in all DMH-treated groups were dose-dependent, duration-dependent and significantly higher than that in non-DMH-treated group. From these results, our assay model was detected the initiation activity of DMH simply, and would be useful to evaluate the carcinogenicity of chemicals.

[Pharmacological effects of nalfurafine hydrochloride, a kappa-opioid receptor agonist].

Nalfurafine hydrochloride, a kappa-opioid receptor agonist, was approved in January 2009 and released to the market on March 2009 for the indication of "Improvement of pruritus in hemodialysis patients (only for cases resistant to conventional treatments)" in Japan (Brand Name: REMITCH CAPSULES 2.5 microg, Marketing Authorization Holder: Toray Industries, Inc., Distributed by Torii Pharmaceutical Co., Ltd., Co-developed by Japan Tobacco Inc.). In addition to antipruritic effect, nalfurafine hydrochloride showed ameliorating effects on pain, neuropathic pain, drug dependence, schizophrenia and dyskinesia in non-clinical studies. Therefore, nalfurafine hydrochloride may become a useful therapeutic agent for their diseases.