Akira Horinouchi

Identification of metabolomic biomarkers for drug-induced acute kidney injury in rats.

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. Copyright

MiRNA expression atlas in male rat

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.

Relationship between in vitro phospholipidosis assay using HepG2 cells and 2-week toxicity studies in rats

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.

Similar compounds searching system by using the gene expression microarray database

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.

Gene expression profiling in rat liver treated with compounds inducing elevation of bilirubin

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.

Secondary Polycythemia in Male B6C3F1 Mice with Spontaneously Occurring Hepatocellular Carcinomas

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.

Erythropoietin mRNA in hepatocellular carcinomas and kidney in male B6C3F1 mice with secondary polycythemia

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 non-tumor-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.

Design and synthesis of a novel series of orally active, selective somatostatin receptor 2 agonists for the treatment of type 2 diabetes

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.

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers

Abstract 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.