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Promising biomarkers were identified in adult male Crl:CD (SD) rats for the screening of new chemical entities for their potential to cause liver injury. We examined the serum biochemistry, liver histopathology, and bile acid profiles by LC-MS/MS, and the mRNA expression of transporters and CYPs by an RT-PCR after the following treatments to male Crl:CD (SD) rats: (a) bile duct ligation (BDL); (b) a single oral dose of 150 mg/kg α-naphthylisothiocyanate (ANIT); and (c) repeated oral doses of a novel pyrrolidinecarboxylic acid derivative (abbreviated as PCA) at 30, 300, and 1000 mg/kg. The serum total bile acid levels and bilirubin concentrations were found to be elevated in all of the groups. However, the bile acid component profiles of the PCA group differed significantly from BDL and ANIT models: deoxycholic acid, lithocholic acid, and sulfated bile acids were upregulated in a dose-dependent manner only in the PCA group. In addition, the PCA group demonstrated high levels of hepatic heme oxygenase-1 expression, whereas the profiles of the mRNA levels of the hepatic transporters and CYPs of all groups were found to be similar. The histopathological findings, for both the BDL and ANIT groups, were of bile duct hyperplasia, hepatocyte degeneration and necrosis. In contrast, only bile duct hyperplasia and hepatocyte degeneration were observed in the PCA group, even at a lethal dose. These results indicated that PCA induced a cholestatic condition and the increase of oxidative stress markers implies that this will also lead hepatocellular injury. In conclusion, the serum bile acid components and sulfated bile acid levels, and the expression of oxidative stress markers could provide information that aids in the diagnosis of liver injury type and helps to elucidate the mechanisms of hepatotoxicity. These findings can be extrapolated into our clinical investigation. The analysis of these crucial biomarkers is likely to be a useful screening tool in the lead optimization phase of drug discovery.

A novel orally bioavailable renin inhibitor, DS-8108b (5), showing potent renin inhibitory activity and excellent in vivo efficacy is described. We report herein the synthesis and pharmacological effects of 5 including renin inhibitory activity in vitro, suppressive effects of ex vivo plasma renin activity (PRA) in cynomolgus monkey, pharmacokinetic data, and blood pressure-lowering effects in an animal model. Compound 5 demonstrated inhibitory activities toward human renin (IC50 = 0.9 nM) and human and monkey PRA (IC50 = 1.9 and 6.3 nM, respectively). Oral administration of single doses of 3 and 10 mg/kg of 5 in cynomolgus monkey on pretreatment with furosemide led to dose-dependent significant reductions in ex vivo PRA and sustained lowering of mean arterial blood pressure for more than 12 h.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P3-sparing S1P1 agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC50 value of 3.4 nM for human S1P1, and over 5800-fold selectivity against S1P3. In rat on host versus graft reaction (HvGR), the ID50 value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Interspecies allometric scaling is a useful tool for calculating human pharmacokinetic (PK) parameters from data in animals. In this study, in order to determine the scaling exponent in a simple allometric equation that can predict human clearance (CL) and distribution volume at steady state (Vss) of monoclonal antibodies (mAbs) from monkey data alone, PK data of 24 mAbs were collected and analyzed according to the types of targeted antigens (soluble or membrane-bound antigens). Based on the observed PK data in humans (at clinical doses) and monkeys (at >1 mg/kg), where the PK is expected to be linear, the mean scaling exponents in the allometric equation for CL and Vss, respectively, against body weight were calculated to be 0.79 and 1.12 [95% confidence intervals (CIs): 0.69-0.89 and 0.96-1.28] for soluble antigens, and 0.96 and 1.00 (95% CIs: 0.83-1.09 and 0.87-1.13) for membrane-bound antigens. Using these exponents and monkey PK data (at >1 mg/kg) alone, both human CL and Vss of mAbs can be predicted with reasonable accuracy, i.e., within 2-fold of the observed values. Compared with traditional allometric scaling using PK data from three or more preclinical species, this approach is simple, quick, resource-saving, and useful in drug discovery and development.

To investigate the hepatotoxic potential of tienilic acid in vivo , we administered a single oral dose of tienilic acid to Sprague–Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S -transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3–6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l -buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.

Clinicians sometimes encounter difficulty in choosing a therapeutic strategy due to the uncertainty regarding the type of liver injury. In particular, cholestasis is difficult to diagnose by conventional markers at an early stage of disease. The aim of this study was to identify promising biomarkers for distinguishing the symptom-based types of liver injury (e.g. hepatocellular injury, cholestasis), which was derived from a rigorously statistical perspective. The associations between diagnostic biomarkers (e.g. bile acid components, oxidative stress markers and liver fibrosis markers) and the liver injury types were assessed by a multiple logistic regression analysis using 304 blood samples from patients with liver disease. As a result, reductions in the lithocholic acid (LCA) and deoxycholic acid (DCA) levels, and elevation of the serum sulfated bile acid (SSBA), liver fibrosis marker IV collagen (type IV collagen), hyaluronic acid (HA) and reactive oxygen species (ROS) levels were all significantly associated with cholestasis. On the other hand, elevations in the LCA and type IV collagen levels, and a reduction in the ursodeoxy cholic acid (UDCA) level, were significantly associated with hepatocellular injury. The receiver operating characteristic (ROC) analyses showed that the largest area under the ROC curve (AUC) was found for ROS, followed by DCA, HA, LCA, SSBA and type IV collagen in the cholestatic-type cases. These results indicated that ROS, the secondary bile acid levels such as DCA and LCA, and SSBA are promising biomarkers for cholestasis and for classifying the type of liver injuries. This comprehensive approach will allow for an accurate diagnosis, which will facilitate the selection of an appropriate therapy at the onset of disease.

Objectives. We determined the serum bile acid (BA) composition in patients with liver diseases and healthy volunteers to investigate the relationship between the etiologies of liver disease and BA metabolism.Material and Methods. Sera from 150 patients with liver diseases and 46 healthy volunteers were obtained. The serum concentrations of the 16 different BAs were determined according to the LC-MS/MS method and were compared between the different liver diseases.Results. A total of 150 subjects, including patients with hepatitis C virus (HCV) (n=44), hepatitis B virus (HBV) (n=23), alcoholic liver disease (ALD) (n=21), biliary tract disease (n=20), nonalcoholic fatty liver disease (NAFLD) (n=13), and other liver diseases (n=29), were recruited. The levels of UDCA and GUDCA were significantly higher in the ALD group, and the levels of DCA and UDCA were significantly lower in the biliary tract diseases group than in viral hepatitis group. In the UDCA therapy (−) subgroup, a significantly lower level of TLCA was observed in the ALD group, with lower levels of CDCA, DCA, and GLCA noted in biliary tract diseases group compared to viral hepatitis group.Conclusions. Analysis of the BA composition may be useful for differential diagnosis in liver disease.

Primary human hepatocytes contain a full complement of human drug-metabolizing enzymes and therefore represent a relevant experimental system for the evaluation of pharmacokinetic drug-drug interaction potential in human. In this study, the cytochrome P450 (CYP) induction potential of pantoprazole (PAN) was evaluated and compared to two other proton pump inhibitors (PPIs), omeprazole (OM) and lansoprazole (LAN). Primary human hepatocytes from three donors were studied. The hepatocytes were cultured for 3 days, followed by treatment for 3 days with the PPIs at 2, 5 and 10 microM. Two other known CYP inducers, 3-methylcholanthrene at 1 microM and rifampin at 50 microM, were also evaluated. Induction potentials of these chemicals for CYP1A and CYP3A were evaluated by isozyme activity and isozyme content. 7-Ethoxyresorufin-O-deethylase and testosterone 6beta-hydroxylase activities were used as endpoints for CYP1A and CYP3A, respectively. Isozyme protein contents of CYP1A and CYP3A were evaluated via Western blotting. The results showed that for CYP1A induction, the rank ordering in induction potential was consistently OM > LAN > PAN. CYP3A induction by the PPI's were observed in two of the three hepatocyte cultures, with no apparent differences in induction potency for the three compounds. Our results on CYP1A induction suggest that PAN has a lower drug-drug interaction potential than OM and LAN.

The effects of proton pump inhibitors on thyroid hormone metabolism in rats were examined. Pantoprazole, omeprazole, and lansoprazole were administered repeatedly to female SD rats at doses of 5, 50, and 300 mg/kg/day for 1 week, and changes in UDP-glucuronyltransferase activities were examined. Increases in o-aminophenol UDP-glucuronyltransferase activity, which was measured as that responsible for the glucuronidation of thyroxine, were evident following 7-day high-dose administration of all the proton pump inhibitors tested. Of the three proton pump inhibitors investigated, o-aminophenol UDP-glucuronyltransferase activity was greatest following the high-dose administration of omeprazole. Androsterone UDP-glucuronyltransferase activity in rats treated with the proton pump inhibitors increased significantly, but these increases were smaller than those of o-aminophenol UDP-glucuronyltransferase. Pantoprazole and omeprazole treatment did not affect plasma T4 or T3 significantly, whereas lansoprazole treatment produced marked reductions in plasma T4 but did not affect plasma T3 significantly. After administration of 125I-labeled thyroid hormone to rats treated with the proton pump inhibitors, biliary excretion of radioactivity increased significantly in omeprazole- and lansoprazole-treated rats; these increases were attributed to induction of liver thyroxine UDP-glucuronyltransferase activities. The order of biliary excretion of radioactivity, as well as the o-aminophenol UDP-glucuronyltransferase activity, in the treated animals was: omeprazole > lansoprazole > pantoprazole. Therefore, repeated administration of the proton pump inhibitors increased thyroxine-metabolizing activity via induction of UDP-glucuronyltransferase, and this induction by pantoprazole was less pronounced than that by omeprazole or lansoprazole.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

The covalent binding of reactive intermediates to macromolecules might have potential involvement in severe adverse drug reactions. Thus, quantification of reactive metabolites is necessary during the early stage of drug discovery to avoid serious toxicity. In this study, the relationship between covalent binding and glutathione (GSH) conjugate formation in rat and human liver microsomes were investigated using 10 representative radioactive compounds that have been reported as hepatotoxic or having other toxicity derived from their reactive intermediates: acetaminophen, amodiaquine, carbamazepine, clozapine, diclofenac, furosemide, imipramine, indomethacin, isoniazid, and tienilic acid, all at a concentration of 10 microM. The GSH conjugate formation rate correlates well with the covalent binding of radioactivity (both rat and human, r2 = 0.93), which suggests that quantification of the GSH conjugate can be used to estimate covalent binding. To quantify the GSH-conjugation rate with non-radiolabeled compounds in vitro, the validation study for the determination of GSH conjugate formation using 35S-GSH by radio-HPLC was useful to predict metabolic activation. Following oral administration of 20 mg/kg of the radiolabeled compounds to rats, radioactivity that covalently bound to plasma and liver proteins was determined. The in vivo maximum covalent binding level in liver based on the free fraction of plasma area under the concentration curve (AUC) and in vitro covalent binding rate was found to correlate well (r2 = 0.79). Therefore, this model for in vitro covalent binding studies in human and rat and in vivo rat studies might be useful in predicting human metabolic activation of compounds.

Purpose: Many promising anticancer drugs are limited by myelosuppression. It is difficult to evaluate human myelotoxicity before a Phase I study because of the susceptibility of humans and animals to hematotoxicity. The purpose of this study was to establish a reliable method to predict the human maximum tolerated dose (MTD) of five camptothecin derivatives: SN-38, DX-8951f, topotecan, 9-aminocamptothecin, and camptothecin. Experimental Design: The myelotoxicity of SN-38 and DX-8951f were evaluated on bone marrow from mice, dogs, and humans using a 14-day colony-forming unit, granulocyte-macrophage (CFU-GM) assay to determine the 50%, 75%, and 90% inhibitory concentration values (IC50, IC75, and IC90, respectively). Results: Species differences in myelotoxicity were observed for SN-38 and DX-8951f. Using human and murine IC90s for myelotoxicity of these compounds and other camptothecin compounds (topotecan, 9-aminocamptothecin, and camptothecin), in vivo toxicological data, and pharmacokinetic parameters (data referred to in the literature), human MTDs were predicted retrospectively. The mechanism-based prediction model that is proposed uses the in vitro camptothecin assay and in vivo parameters on the basis of free fraction of area under the concentration-curve at the MTD (r2 = 0.887) and suggests that the human MTDs were well predicted for the five camptothecin derivatives by this model rather than by other models. Conclusion: The human MTDs of the camptothecin drugs were successfully predicted using the mechanism-based prediction model. The application of this model for in vitro hematotoxicology could play an important role for the development of new anticancer agents.

The pharmacokinetics of DX-8951f were greatly improved by DE-310 with the extremely longer retention in bloodstream, resulting in the preferential tumor-targeting, and with the slow release appropriate to camptothecin analog DX-8951f in tumor tissue. On the basis of the pharmacokinetic improvement, DE-310 exhibited enhanced antitumor effects with reduced toxicities by a single and low dose, compared with DX-8951f. These preclinical results suggest that DE-310 is a promising agent for cancer treatment. The phase I trials are still ongoing and recently, the interim clinical data have been reported15, 16.

The ability of pantoprazole to affect the induction of cytochrome P450 (CYP) 1A subfamily was evaluated and compared with two other proton pump inhibitors, omeprazole and lansoprazole, in primary cultured hepatocytes from female Sprague-Dawley rats. The hepatocytes were cultured for 2 days, followed by treatment for 2 days with the proton pump inhibitors at 2, 5 and 10 μM, concentrations that are similar to plasma concentrations found in rats in vivo. The CYP1A inducer 3-methylcholanthrene (at 1 μM) was also evaluated as a positive control. Induction potentials of these chemicals for CYP1A were determined by 7-ethoxyresorufin O -deethylase activity and isozyme contents. The results showed that for CYP1A induction, the rank ordering in induction potential was consistently lansoprazole > omeprazole > pantoprazole. The results are consistent with the existing rat in vivo data, i.e. pantoprazole has lower CYP1A induction potential than omeprazole and lansoprazole.