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Venlafaxine has potential as a therapeutic option for patients with depressive disorder, migraine, and pruritus unresponsive to antihistamines.

Context: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable.Objective: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach.Method: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs.Results: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation.Conclusion: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.

Urinary hippuric acid (HA) and phenylacetylglycine (PAG) are biomarker candidates for drug-induced phospholipidosis (PLD). To confirm their utility in preclinical and clinical settings, it is essential to develop and validate their quantification method in advance. In this study, we have applied liquid chromatography-tandem mass spectrometry (LC/MS/MS) for simultaneous quantification of HA and PAG in rat urine, and matrix based ion suppression was assessed by post-column infusion assay. Effective sample dilution reduced matrix effect of urine to be negligible level and calibration curves showed good correlation between those in urine diluent and buffer alone. Reliability of this assay was confirmed by the assessments for intra- and inter-day precisions and accuracies of quality control samples. The method was applied to rat urine after multiple oral administrations of PLD-inducing drugs, and the changes in HA and PAG concentrations and their ratio were successfully detected as rat plasma in previous report. This is the first report to quantify HA and PAG easily and accurately as potential biomarkers to monitor PLD status. This assay would be useful tool for monitoring PLD in toxicological studies by non-invasive sampling.

The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis-time-of-flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2-bromoethylamine or n-phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3-methylhistidine (3-MH), 3-indoxyl sulfate (3-IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3-MH and 3-IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3-MH, 3-IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats.

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for the diagnosis of elevated total bilirubin (TBIL) and direct bilirubin (DBIL), we extracted 59 probe sets of rat hepatic genes from the data for seven typical drugs, gemfibrozil, phalloidin, colchicine, bendazac, rifampicin, cyclosporine A, and chlorpromazine, which induced this phenotype from 3 to 28 days of repeated administration in the present study. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters in the treated groups from their controls. Eighteen more drugs in the database, reported to elevate TBIL and DBIL, were estimated by PCA using these probe sets. Of these, 12 drugs, that is methapyrilene, thioacetamide, ticlopidine, ethinyl estradiol, alpha-naphthylisothiocyanate, indomethacin, methyltestosterone, penicillamine, allyl alcohol, aspirin, iproniazid, and isoniazid were also separated from the control clusters, as were the seven typical drugs causing elevation of TBIL and DBIL. The principal component 1 (PC1) value showed high correlation with TBIL and DBIL. In the cases of colchicine, bendazac, chlorpromazine, gemfibrozil, and phalloidin, the possible elevation of TBIL and DBIL could be predicted by expression of these genes 24 h after single administration. We conclude that these identified 59 probe sets could be useful to diagnose the cause of elevation of TBIL and DBIL, and that toxicogenomics would be a promising approach for prediction of this type of toxicity.

MicroRNAs (miRNAs) are small (~22 nucleotide) noncoding RNAs that play pivotal roles in regulation of gene expression. The value of miRNAs as circulating biomarkers is now broadly recognized; such tissue-specific biomarkers can be used to monitor tissue injury and several pathophysiological conditions in organs. In addition, miRNA profiles of normal organs and tissues are important for obtaining a better understanding of the source of modulated miRNAs in blood and how those modulations reflect various physiological and toxicological conditions. This work was aimed at creating an miRNA atlas in rats, as part of a collaborative effort with the Toxicogenomics Informatics Project in Japan (TGP2). We analyzed genome-wide miRNA profiles of 55 different organs and tissues obtained from normal male rats using miRNA arrays. The work presented herein represents a comprehensive dataset derived from normal samples profiled in a single study. Here we present the whole dataset with miRNA profiles of multiple organs, as well as precise information on experimental procedures and organ-specific miRNAs identified in this dataset.

The purpose of this study was to investigate the cause of polycythemia occurring in control male B6C3F, mice with hepatocellular carcinomas from 2-yr carcinogenicity studies. Erythrocyte counts and plasma erythropoietin levels in these mice were significantly increased compared to those in nontumor-bearing mice. Hepatocellular carcinomas in the mice were well differentiated, and the neoplastic hepatocytes contained either or both of 2 types of intracytoplasmic inclusion bodies; one was relatively large and weakly eosinophilic (pale inclusion body), while the other was relatively small and strongly eosinophilic (globular inclusion body). The pale eosinophilic inclusions but not the globular ones were immunohistochemically positive for erythropoietin. Ultrastructurally, the erythropoietin-positive inclusions were characterized by granular materials in dilated cisternae of rough endoplasmic reticulum, suggesting increased protein synthesis. Erythropoietin-negative inclusions were dense bodies that were not surrounded by a delimiting membrane. These findings indicate that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis and secretion of erythropoietin by neoplastic hepatocytes.

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis-time-of-flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2-bromoethylamine or n-phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3-methylhistidine (3-MH), 3-indoxyl sulfate (3-IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3-MH and 3-IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3-MH, 3-IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats.

Secondary polycythemia was observed in hepatocellular carcinoma-bearing B6C3F1 mice which were used as control group animals in 2-year carcinogenicity studies. Statistically significant increases in erythrocyte count, hematocrit value, hemoglobin concentration, and absolute reticulocyte count were noted in the peripheral blood of hepatocellular carcinoma-bearing mice as compared with non-tumor-bearing mice. A decrease in mean corpuscular volume and mean corpuscular hemoglobin value was also noted in hepatocellular carcinoma-bearing mice. Mean corpuscular hemoglobin concentration and relative reticulocyte count in hepatocellular carcinoma-bearing mice were comparable to those in non-tumor-bearing mice. In addition, the plasma erythropoietin level in hepatocellular carcinoma-bearing mice was significantly higher than that in hepatocellular adenoma-bearing mice and that in non-tumor-bearing mice. Therefore, the hematological changes observed in hepatocellular carcinoma-bearing mice were diagnosed as secondary polycythemia. At necropsy, the spleen in hepatocellular carcinoma-bearing mice was dark-red and enlarged 2 to 4 times compared with that in non-tumor-bearing mice. Histopathologic examination revealed proliferation of the erythroid cells and megakaryocytes in the spleen and bone marrow in hepatocellular carcinoma-bearing mice, suggesting an increase in erythropoietic activity. These findings indicate that increased levels of plasma erythropoietin in hepatocellular carcinoma-bearing mice stimulate erythropoiesis in the hematopoietic organs and result in proliferation of microcytic and hypochromic erythrocytes. Possible mechanisms for the induction of secondary polycythemia in hepatocellular carcinoma-bearing mice are discussed.

Localization of erythropoietin messenger RNA was studied by an in situ hybridization procedure using anti-sense oligonucleotide probes of murine erythropoietin in the kidneys of mice with acute anemia. Acute anemia was induced by depletion of blood. Five hundred microliters of blood were withdrawn form orbital plexus 6, 24, and 30 hours before necropsy. Animals were sacrificed 6 hours after the last bleeding. Erythropoietin levels in the plasma, renal cortex, and medulla were 797.0mU/ml, 216.7, and 11.3mU/g protein, respectively, and these values were 50, 16, and 4.5times higher than those in the untreated control mice. Silver grains indicating erythropoietin messenger RNA expression were observed in the interstitial cells of the renal cortex and outer stripe of the outer medulla in the mice with acute anemia, but not in the renal tubules or glomeruli. Silver grains were not detected in the kidneys of the untreated control mice. From these results, it was concluded that the main area of erythropoietin synthesis in the kidneys was the interstitial cells in the renal cortex and outer stripe ofthe outer medulla in the mice with acute anemia.

We investigated the presence of α2u-globulin, which has generally been thought to be a protein specific to male rats, in the kidneys of female rats. Immunohistochemical, immunoelectron microscopic and immunochemical studies were performed on the kidneys of male and female F344 rats using rabbit anti-serum prepared with α2u-globulin purified from male rat urine. In male rats, hyaline droplets containing granules positive for immunohistochemical staining of α2u-globulin were observed in the proximal convoluted tubular epithelium. In females, fine granules positive for staining of α2u-globulin were observed in the same region, although so-called hyaline droplets were not observed in HE-stained specimens. Upon immunoelectron microscopic examination, gold particles indicating the presence of α2u-globulin were found localized in the lysosomes of proximal convoluted tubular epithelial cells in both sexes. Furthermore, immunohistochemistry revealed that α2u-globulin was localized in the submaxillary glands in both sexes and in the liver in males. In the immunochemical study, a double immunodiffusion test, precipitin lines were formed between the anti-serum and the kidney preparation and between the anti-serum and the submaxillary gland preparation from female rats. Spur formation was observed between the precipitin lines for the anti-serum and the submaxillary gland preparation and the precipitin lines for the anti-serum and urine in female rats; therefore, a partial difference in the antigenicity of α2u-globulin between males and females was suggested. From these results, we concluded that α2u-globulin exists in the kidneys in the female rats, and it is considered to originate mainly from the submaxillary glands, not the liver.

Drug candidates under development by industry frequently show phospholipidosis as a side-effect in pre-clinical toxicity studies. This study sets up a cell-based assay for drug-induced phospholipidosis (PLD) and its performance was evaluated based on the in vivo PLD potential of compounds in 2-week toxicity studies in rats. When HepG2 cells were exposed simultaneously to PLD-inducing chemicals and a phospholipid having a fluorophore, an accumulation of phospholipids was detected as an increasing fluorescent intensity. Amiodarone, amitriptyline, fluoxetine, AY-9944, and perhexiline, which are common PLD-inducing chemicals, increased the fluorescent intensity, but acetaminophen, ampicillin, cimetidine, famotidine, or valproic acid, which are non-PLD-inducing chemicals, did not. The fluorescent intensity showed concordance with the pathological observations of phospholipid lamellar bodies in the cells. Then to confirm the predictive performance of the in vitro PLD assay, the 32 proprietary compounds characterized in 2-week toxicity studies in rats were evaluated with this in vitro assay. Because this in vitro assay was vulnerable to cytotoxicity, the innate PLD potential was calculated for each compound. A statistically significant increase in the in vitro PLD potential was seen for the compounds having in vivo PLD-inducing potential in the rat toxicity studies. The results suggest that the in vitro PLD potential could be appropriate to detect the appearance of PLD as a side effect in pre-clinical toxicity studies in rats.

Numbers of microarrays have been examined and several public and commercial databases have been developed. However, it is not easy to compare in-house microarray data with those in a database because of insufficient reproducibility due to differences in the experimental conditions. As one of the approach to use these databases, we developed the similar compounds searching system (SCSS) on a toxicogenomics database. The datasets of 55 compounds administered to rats in the Toxicogenomics Project (TGP) database in Japan were used in this study. Using the fold-change ranking method developed by Lamb et al. [Lamb, J., Crawford, E.D., Peck, D., Modell, J.W., Blat, I.C., Wrobel, M.J., Lerner, J., Brunet, J.P., Subramanian, A., Ross, K.N., Reich, M., Hieronymus, H., Wei, G., Armstrong, S.A., Haggarty, S.J., Clemons, P.A., Wei, R., Carr, S.A., Lander, E.S., Golub, T.R., 2006. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929-1935] and criteria called hit ratio, the system let us compare in-house microarray data and those in the database. In-house generated data for clofibrate, phenobarbital, and a proprietary compound were tested to evaluate the performance of the SCSS method. Phenobarbital and clofibrate, which were included in the TGP database, scored highest by the SCSS method. Other high scoring compounds had effects similar to either phenobarbital (a cytochrome P450s inducer) or clofibrate (a peroxisome proliferator). Some of high scoring compounds identified using the proprietary compound-administered rats have been known to cause similar toxicological changes in different species. Our results suggest that the SCSS method could be used in drug discovery and development. Moreover, this method may be a powerful tool to understand the mechanisms by which biological systems respond to various chemical compounds and may also predict adverse effects of new compounds.

The purpose of this study was to investigate the cause of polycythemia occurring in hepatocellular carcinoma-bearing control male B6C3F, mice from 2-yr carcinogenicity studies. Erythrocyte counts and plasma levels of erythropoietin in mice with hepatocellular carcinomas were significantly increased compared with the values in non-tumor-bearing mice. Erythropoietin mRNA in 4 of 5 nontumor-bearing mice was detected in the kidney, but no visible signals for hepatic erythropoietin mRNA in 5 of 5 non-tumor-bearing mice were detected by the reverse transcriptase competitive polymerase chain reaction method. Erythropoietin mRNA was expressed in neoplastic hepatocytes from 8 of 9 hepatocellular carcinoma-bearing mice, and this expression was accompanied by decreased expression of erythropoietin mRNA in the kidneys from these mice. The present findings show that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis of erythropoietin mRNA by neoplastic hepatocytes. Keyvords. Erythropoietin messenger RNA; hepatocellular carcinoma; kidney; polycythemia; RT-PCR; B6C3F, mice

The present study was conducted to evaluate the variations in the normal auditory brainstem response (ABR) between the F344 and Wistar rats. It was also investigated whether strain differences exist in the ABR profiles of each strain of rat given furosemide, which induced ototoxicity. The typical waveform of ABRs in the F344 rats were similar to those observed in the Wistar rats. There were no significant differences between the strains in almost all parameters, but peak latencies from P1 to P3 in the F344 rats tended to be longer than those in the Wistar rats. After a single intravenous injection of furosemide, the minimum effective dosage levels, at which each peak of the ABR disappeared, were 53.3 +/- 8.2 and 70.0 +/- 8.9 mg/kg in the F344 and Wistar rats, respectively. A single intravenous injection of 100 mg/kg furosemide delayed the reversal of decrease in amplitude, prolongation of peak latency and the reappearance time in F344 rats. Thus, the normal waveform of ABR was comparable in the F344 and Wistar rats, but a tendency towards a prolongation in each latency was noted in the F344 rat. The results also suggested that the ABR of F344 rats were more vulnerable to furosemide ototoxicity than that of Wistar rats.