Chimeric Mice With Humanized Livers Demonstrate Human-Specific Hepatotoxicity Caused by a Therapeutic Antibody Against TRAIL-Receptor 2/Death Receptor 5.

The activation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2)/death receptor 5 (DR5) induces apoptosis in various tumor cells but not in normal human cells. Because some therapeutic antibodies targeting TRAIL-R2 have demonstrated severe hepatotoxicity in clinical applications, novel in vivo models reflecting clinical hepatotoxicity are now required. In this study, we investigated the hepatotoxicity caused by KMTR2, an anti-human TRAIL-R2 monoclonal antibody, in chimeric mice with humanized livers (PXB-mice). PXB-mice were exposed to KMTR2 by single or repeated (weekly for 4 weeks) intravenous administrations, and the analyses of blood chemistry, liver histopathology, hepatic gene expression, and toxicokinetics were performed. Treatment with 1 or 10 mg/kg of KMTR2 increased alanine transaminase (ALT) activity and human ALT1 levels in blood. Histopathological analysis revealed that cell death and degeneration with the infiltration of inflammatory cells in human but not mouse hepatocytes were increased in a time-dependent manner after KMTR2 administration. Furthermore, increases in TdT-mediated dUTP nick end labeling (TUNEL)-positive human hepatocytes and serum concentration of cleaved cytokeratin 18, a human-specific apoptosis marker, were observed. RNA sequence analysis showed that the gene expression profile changed in different manners between human and mouse hepatocytes and the up-regulation of TRAIL-R2-related genes was observed only in human hepatocytes. Taken together, these results indicate that KMTR2-mediated TRAIL-R2 activation induces apoptosis of human hepatocytes and hepatotoxicity in PXB-mice and suggest that chimeric mice with humanized liver can be novel tools for the evaluation of in vivo human-specific hepatotoxicity induced by therapeutic antibodies in pre-clinical studies.

Effect of antigen-dependent clearance on pharmacokinetics of anti-heparin-binding EGF-like growth factor (HB-EGF) monoclonal antibody.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance.

Soluble heparin-binding EGF-like growth factor (HB-EGF) detected by newly developed immuno-PCR method is a clear-cut serological biomarker for ovarian cancer.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF plays pivotal roles in pathophysiological processes such as development and cell proliferation. In this study, we developed an immuno-PCR system for the determination of soluble HB-EGF concentrations in human serum. Utilizing a monoclonal antibody with neutralizing activity against HB-EGF as a capture antibody resulted in increasing selectivity for an active form of HB-EGF in serum, and immuno-PCR system improved its sensitivity compared to the currently available methods. As a result of measurement of HB-EGF in 20 ovarian cancer patients and 20 healthy volunteers, ovarian cancer patients showed significantly higher concentrations than healthy volunteers (P< 0.05), which indicates that soluble HB-EGF detected by newly developed immuno-PCR system can be useful serological biomarkers such as a diagnostic biomarker for ovarian cancer, and a predictive and pharmacodynamic biomarker for anti-HB-EGF targeted therapies under development.

Mechanisms of the hepatoprotective effects of tamoxifen against drug-induced and chemical-induced acute liver injuries.

Although estrogen receptor (ER)α agonists, such as estradiol and ethinylestradiol (EE2), cause cholestasis in mice, they also reduce the degree of liver injury caused by hepatotoxicants as well as ischemia-reperfusion. The functional mechanisms of ERα have yet to be elucidated in drug-induced or chemical-induced liver injury. The present study investigated the effects of an ERα agonist, selective ER modulators (SERMs) and an ER antagonist on drug-induced and chemical-induced liver injuries caused by acetaminophen, bromobenzene, diclofenac, and thioacetamide (TA). We observed hepatoprotective effects of EE2, tamoxifen (TAM) and raloxifene pretreatment in female mice that were exposed to a variety of hepatotoxic compounds. In contrast, the ER antagonist did not show any hepatoprotective effects. DNA microarray analyses suggested that monocyte to macrophage differentiation-associated 2 (Mmd2) protein, which has an unknown function, is commonly increased by TAM and RAL pretreatment, but not by pretreatment with the ER antagonist. In ERα-knockout mice, the hepatoprotective effects of TAM and the increased expression of Mmd2 mRNA were not observed in TA-induced liver injury. To investigate the function of Mmd2, the expression level of Mmd2 mRNA was significantly knocked down to approximately 30% in mice by injection of siRNA for Mmd2 (siMmd2). Mmd2 knockdown resulted in a reduction of the protective effects of TAM on TA-induced liver injury in mice. This is the first report of the involvement of ERα in drug-induced or chemical-induced liver injury. Upregulation of Mmd2 protein in the liver was suggested as the mechanism of the hepatoprotective effects of EE2 and SERMs.

IL-4 mediates dicloxacillin-induced liver injury in mice.

Drug-induced liver injury (DILI) is a major problem in drug development and clinical drug therapy. In most cases, the mechanisms are still unknown. It is difficult to predict DILI in humans due to the lack of experimental animal models. Dicloxacillin, penicillinase-sensitive penicillin, rarely causes cholestatic or mixed liver injury, and there is some evidence for immunoallergic idiosyncratic reaction in human. In this study, we investigated the mechanisms of dicloxacillin-induced liver injury. Plasma ALT and total-bilirubin (T-Bil) levels were significantly increased in dicloxacillin-administered (600 mg/kg, i.p.) mice. Dicloxacillin administration induced Th2 (helper T cells)-mediated factors and increased the plasma interleukin (IL)-4 level. Neutralization of IL-4 suppressed the hepatotoxicity of dicloxacillin, and recombinant mouse IL-4 administration (0.5 or 2.0 µg/mouse, i.p.) exacerbated it. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is a cognate receptor for prostaglandin (PG) D(2), and is suggested to be involved in Th2-dependent allergic inflammation. We investigated the effect of 13,14-Dihydro-15-keto-PGD(2) (DK-PGD(2); 10 µg/mouse, i.p.) administration on dicloxacillin-induced liver injury. DK-PGD(2)/dicloxacillin coadministration resulted in a significant increase of alanine aminotransferases and a remarkable increase of macrophage inflammatory protein 2 expression. In conclusion, to the best of our knowledge, this is the first report to demonstrate that dicloxacillin-induced liver injury is mediated by a Th2-type immune reaction and exacerbated by DK-PGD(2).

An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and gamma-glutamylcysteine synthetase knockdown cells.

Drug-induced hepatotoxicity is often caused by CYP-dependent activation of the drugs into reactive metabolites. CYP3A4 is one of the main isoforms involved in the metabolic activation of drugs. In this study, an adenovirus vector expressing CYP3A4 (AdCYP3A4) was constructed. After 3days infection of AdCYP3A4, the testosterone 6beta-hydroxylase activity reached to 325pmol/min/mg protein in H4IIE (rat hepatoma) cells. To knockdown the gamma-glutamylcysteine synthetase heavy chain subunit (GCSh) and decrease the intrinsic glutathione (GSH) level, we used an adenovirus vector with short hairpin RNA against rat GCSh (AdGCSh-shRNA). Three days infection of AdGCSh-shRNA and AdCYP3A4 simultaneously with H4IIE cells decreased the intracellular GSH level by 50-60% without affecting the expression level of CYP3A4. Using this cell-based system sensitive to the cytotoxicity of reactive metabolites, drugs known for their hepatotoxicity were evaluated. As a result, troglitazone, flutamide, and acetaminophen caused significant decreases of cell viability in AdCYP3A4/AdGCSh-shRNA group compared to the other groups (AdGFP, AdCYP3A4, AdGFP/AdGCSh-shRNA groups), indicating that reactive metabolite(s) produced by CYP3A4 and subsequently conjugated by GSH would be involved in the cytotoxicity. These results suggest that this cell-based assay system expressing CYP3A4 with GCSh knockdown would be useful for the prediction of CYP3A4-mediated cytotoxicity in preclinical drug development.

Knockdown of superoxide dismutase 2 enhances acetaminophen-induced hepatotoxicity in rat.

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. Previously, we reported the establishment of an adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA), and applied this to evaluate drug-induced cytotoxicity. In this study, infection of AdSOD2-shRNA to Fisher 344 rats resulted in a significant decrease of SOD2 mRNA, protein expression, and SOD2 enzyme activity to 28%, 35%, and 39%, respectively, 7 days after infection. Serum AST and ALT were significantly increased by single oral administration of acetaminophen (1000 mg/kg) to these SOD2-knockdown rats without fasting compared with the control adenovirus infected groups. Heme oxygenase-1 protein, known to be induced by oxidative stress, was detected in SOD2-knockdown rats administered acetaminophen. In addition, protein carbonyl and lipid peroxidation, also known to be induced by oxidative stress, were significantly increased in SOD2 knockdown rats. This is the first report of a SOD2-knockdown rat model that could be useful to evaluate the drug-induced hepatotoxicity with high sensitivity.

Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity.

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. In this study, we established SOD2-knockdown cells. An adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA) was constructed, and infection of AdSOD2-shRNA to rat hepatic BRL3A cells resulted in significant decreases of SOD2 mRNA and protein by 60%, and SOD2 activity by 50% after 3 days infection. We previously constructed an adenovirus expressing cytochrome P450 3A4 (AdCYP3A4). Co-infection of AdSOD2-shRNA and AdCYP3A4 to BRL3A cells was carried out to evaluate the superoxide- and CYP3A4-mediated formation of active metabolites, and mitochondrial toxicity, ROS and superoxide radical production and lipid peroxidation were selected to assess the cell viability. Albendazole, carbamazepine, dapsone, flutamide, isoniazid, nifedipine, sulfamethoxazole, trazodone, troglitazone, and zidovudine demonstrated significant increases of SOD2- and CYP3A4-mediated cytotoxicity. In conclusion, we constructed a highly sensitive cell system to evaluate oxidative stress and CYP3A4 mediated cytotoxicity that could be useful in preclinical drug development.