Hepatotoxicants induce cytokine imbalance in response to innate immune system.

In recent years, attention has been paid to innate immune systems as mechanisms to initiate or promote drug-induced liver injury (DILI). Kupffer cells are hepatic resident macrophages and might be involved in the pathogenesis of DILI by release of pro- and anti-inflammatory mediators such as cytokines, chemokines, reactive oxygen species, and/or nitric oxides. The purpose of this study was to investigate alterations in mediator levels induced by hepatotoxic compounds in isolated Kupffer cells and discuss the relation between balance of each cytokine or chemokine and potential of innate immune-mediated DILI. Primary cultured rat Kupffer cells were treated with hepatotoxic (acetaminophen, troglitazone, trovafloxacin) or non-hepatotoxic (pioglitazone, levofloxacin) compounds with or without lipopolysaccharide (LPS). After 24 hr treatment, cell supernatants were collected and various levels of mediators released by Kupffer cells were examined. Although hepatotoxicants had no effect on the LPS-induced tumor necrosis factor-alpha (TNF-α) secretion, they enhanced the release of pro-inflammatory cytokine interleukin-1 beta (IL-1ß) and suppressed the anti-inflammatory cytokines interleukin-6 (IL-6) and interleukin-10 (IL-10) induced by LPS. These cytokine shifts were not associated with switching the phenotypes of M1 and M2 macrophages in Kupffer cells. In conclusion, the present study suggested that the levels of some specific cytokines are affected by DILI-related drugs with LPS stimulation, and imbalance between pro- and anti-inflammatory cytokines, induced by the up-regulation of IL-1ß and the down-regulation of IL-6 or IL-10, plays a key role in innate immune-mediated DILI.

Identification of 2,3-disubstituted pyridines as potent, non-emetic PDE4 inhibitors.

A series of 2,3-disubstituted pyridines were synthesized as potential non-emetic PDE4 inhibitors. To decrease brain exposure and minimize emesis, we modified the lipophilic moiety of a series of emetic PDE4 inhibitors and found that introduction of a hydroxy group into the pyridine moiety of the side chain led to non-emetic compounds with preserved PDE4 inhibitory activity. Following optimization at the phenoxy group, we identified compound 1 as a potent non-emetic PDE4 inhibitor. Compound 1 showed significant efficacy in an animal model of asthma without inducing emesis.

Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs.

1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3. Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4. In conclusion, dogs showed the most similar metabolic activity to humans.

Significance of reductive metabolism in human intestine and quantitative prediction of intestinal first-pass metabolism by cytosolic reductive enzymes.

The number of new drug candidates that are cleared via non-cytochrome P450 (P450) enzymes has increased. However, unlike oxidation by P450, the roles of reductive enzymes are less understood. The metabolism in intestine is especially not well known. The purposes of this study were to investigate the significance of reductive metabolism in human intestine, and to establish a quantitative prediction method of intestinal first-pass metabolism by cytosolic reductive enzymes, using haloperidol, mebendazole, and ziprasidone. First, we estimated the metabolic activities for these compounds in intestine and liver using subcellular fractions. Metabolic activities were detected in human intestinal cytosol (HIC) for all three compounds, and the intrinsic clearance values were higher than those in human liver cytosol for haloperidol and mebendazole. These metabolic activities in HIC were NADPH- and/or NADH-dependent. Furthermore, the metabolic activities for all three compounds in HIC were largely inhibited by menadione, which has been used as a carbonyl reductase (CBR)-selective chemical inhibitor. Therefore, considering subcellular location, cofactor requirement, and chemical inhibition, these compounds might be metabolized by CBRs in human intestine. Subsequently, we tried to quantitatively predict intestinal availability (F(g)) for these compounds using human intestinal S9 (HIS9). Our prediction model using apparent permeability of parallel artificial membrane permeability assay and metabolic activities in HIS9 could predict F(g) in humans for the three compounds well. In conclusion, CBRs might have higher metabolic activities in human intestine than in human liver. Furthermore, our prediction method of human F(g) using HIS9 is applicable to substrates of cytosolic reductive enzymes.

A new methodology for simultaneous quantification of total-Aß, Aßx-38, Aßx-40, and Aßx-42 by column-switching LC/MS/MS.

This article details the development of a novel method that overcomes the drawbacks of sandwich ELISA (sELISA) and allows reliable evaluation of simultaneous quantification of the amyloid (Aß)-peptides, total-Aß, Aßx-38, Aßx-40, and Aßx-42, in rat brain by optimized sample purification and column-switching liquid chromatographic-tandem mass spectrometry (LC/MS/MS). This method provides accurate analyses of total-Aß, Aßx-38, Aßx-40, and Aßx-42 with a linear calibration range between 0.05 and 45 ng/mL. Verification for accuracy and precision of biological samples were determined by a standard addition and recovery test, spiked with synthetic Aß1-38, Aß1-40, and Aß1-42 into the rat brain homogenate. This method showed <20% relative error and relative standard deviation, indicating high reproducibility and reliability. The brain concentrations of total-Aß, Aßx-38, Aßx-40, and Aßx-42 after oral administration of flurbiprofen in rats were measured by this method. Aßx-42 concentrations (4.57 ± 0.69 ng/g) in rats administered flurbiprofen were lower than those in untreated rats (6.48 ± 0.93 ng/g). This was consistent with several reports demonstrating that NSAIDs reduced the generation of Aß. We report here a method that allows not only the quantification of specific molecular species of Aß but also simultaneous quantification of total-Aß, Aßx-38, Aßx-40, and Aßx-42, thus overcoming the drawbacks of sELISA.

Disposition of exogenous urea and effects of diet in rats.

Although breath test using 13C-labeled urea (CAS 57-13-6, UBT) is becoming popular for the diagnosis of Helicobacter pylori (H. pylori) infection, disposition of exogenously given urea is not fully understood. The purpose of the present study is to elucidate the disposition of exogenous urea and to consider its relation with the UBT safety and biobehavior of endogenous urea. With 14C-labeled urea ([14C]urea), the absorption, distribution, metabolism and excretion including that into breathed air after its administration in trace to large doses in rats were investigated. [14C]Urea was given to fasted and non-fasted rats through intravenous and oral routes. It was found that the disposition of exogenous [14C]urea behaves in a similar way as endogenous urea, and a sufficiently large capacity for disposing urea in rats was suggested from the linear pharmacokinetics within the wide dose range of [14C]urea (2-1000 mg/kg). The safety of urea in UBT was also revealed by consideration of its dose and human urea body pool. It was also suggested that diet stimulates both systemic (as observed after the intravenous dose) and pre-systemic (as with the oral route) decompositions of urea into carbon dioxide and ammonia, but does not affect the renal elimination and distribution pattern in rat tissues. The findings in this study provide us with the quantitative information concerning not only the safety and disposition of urea as a diagnostic agent, but also the biobehavior of endogenous urea in ureotelism.