Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

Drug-induced Obstructive and Retrograde Nephropathy Associated with α2u-globulin in Male Rats.

The chemically induced accumulation of α2u-globulin protein in male rats causes specific renal lesions and subsequent nephropathy. Herein, we report additional parallel findings in the kidney of male rats consistent with obstructive and retrograde nephropathy. Kidney and urinary bladder samples were evaluated from Wistar rats treated with RG7129 for 2 week and 8 week and from an 8-week mechanistic study using females, intact and castrated males. Histopathological findings were present in intact males in all studies, including hyaline droplet accumulation and granular casts consistent with α2u-globulin nephropathy. In addition, tubular degeneration and regeneration, tubular changes extending from papilla to cortex, tubular dilation, and interstitial and luminal inflammation were observed consistent with retrograde and obstructive nephropathy. Renal and urinary lesions and their severity increased in a time- and dose-dependent manner. Urinalysis findings, including increases in leukocytes, protein, and in kidney biomarkers, kidney injury molecule 1 and clusterin, were present only in intact males. No treatment-related changes were observed in female rats or in castrated males. These results indicate that RG7129 induces α2u-globulin nephropathy, associated with retrograde and obstructive nephropathy secondary to precipitation in intact male rats only, constituting a species- and sex-specific syndrome that is not expected to occur in humans or other species.

Use of toxicogenomics in drug safety evaluation: Current status and an industry perspective.

Toxicogenomics held great promise as an approach to enable early detection of toxicities induced by xenobiotics; however, there remain questions regarding the impact of the discipline on pharmaceutical nonclinical safety assessment. To understand the current state of toxicogenomics in the sector, an industry group surveyed companies to determine the frequency of toxicogenomics use in in vivo studies at various stages of drug discovery and development and to assess how toxicogenomics use has evolved over time. Survey data were compiled during 2016 from thirteen pharmaceutical companies. Toxicogenomic analyses were infrequently conducted in the development phase and when performed were done to address specific mechanistic questions. Prior to development, toxicogenomics use was more frequent; however, there were significant differences in approaches among companies. Across all phases, gaining mechanistic insight was the most frequent reason cited for pursing toxicogenomics with few companies using toxicogenomics to predict toxicities. These data were consistent with the commentary submitted in response to survey questions asking companies to describe the evolution of their toxicogenomics strategy. Overall, these survey data indicate that toxicogenomics is not widely used as a predictive tool in the pharmaceutical industry but is used regularly by some companies and serves a broader role in mechanistic investigations and as a complement to other technologies.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

A novel orally available small molecule that inhibits hepatitis B virus expression.

BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart ("cardiac spheroids", CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.

Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Epithelial cells as active player in fibrosis: findings from an in vitro model.

Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI) to chronic kidney disease (CKD) challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.

Characterization of post-surgical alterations in the bile duct-cannulated rat.

The bile duct-cannulated (BDC) rat is a standard animal model used in ADME experiments. The aim of this study was to investigate post-surgical alterations that are relevant to ADME investigations in BDC rats compared with sham- and non-operated animals. Water and food intake was reduced in the animals' post-surgery. This led to a lower body weight in operated animals. In BDC animals, aspartate aminotransferase (AST) levels in plasma were transiently elevated and total bile acid levels were reduced. Alpha(1)-acid glycoprotein (AGP) in plasma and the concentration of bile components in bile were elevated. Histopathology showed inflammation in the area of the cannulation between the liver and the small intestine. A microarray-based gene expression and RTq-PCR analysis identified altered expression for several genes involved in drug disposition including the down-regulation of cytochrome P450 enzymes. This led to reduced cytochrome P450 content in the liver and lower metabolic activity in microsomes from BDC and sham-operated rats compared with naïve animals. The results of the study suggest that the post-surgical inflammation leads to physiological changes relevant for drug absorption and disposition. These alterations should be accounted for in the interpretation of ADME studies in BDC animals.

Primary endothelial damage is the mechanism of cardiotoxicity of tubulin-binding drugs.

The use of tubulin binders (TBs) in the treatment of cancer often is associated with cardiotoxicity, the mechanism of which has not been elucidated. To test the hypothesis that interstitial cells of the myocardium are the primary target of TBs, we evaluated the acute effects of a single iv administration of three reference TBs: colchicine (0.2 and 2 mg/kg), vinblastine (0.5 and 3 mg/kg), and vincristine (0.1 and 1 mg/kg) 6 and 24 h after dosing. Mitotic arrest was identified at 24 h in all high-dose groups based on an increase in the number of mitotic figures in the interstitium coupled with a decrease in the number of Ki67-positive interstitial cells. Analysis of the myocardial transcriptomic data further supported G2/M cell cycle arrest 6 h after dosing with the high-dose groups of all three compounds. Apoptotic figures and an increase in the number of cleaved caspase 3-positive cells were identified at 6 and 24 h at the highest dose of each compound predominantly in interstitial cells, whereas a few cardiomyocytes were affected as well. Transcriptomic profiling of the myocardium further suggested that some of the affected interstitial cells were endothelial cells based on the upregulation of genes typically associated with vascular damage and downregulation of endothelial cell-specific molecule 1 and apelin. Taken together, these data identify endothelial cells of the myocardium as the primary target of the cardiotoxicity of TBs and identify cell cycle arrest as the mechanism of this toxicity.

Mechanisms underlying off-target effects of the cholesteryl ester transfer protein inhibitor torcetrapib involve L-type calcium channels.

OBJECTIVE: The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. METHODS: Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. RESULTS: Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1-5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel. CONCLUSION: The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.

Characterization and interlaboratory comparison of a gene expression signature for differentiating genotoxic mechanisms.

The genotoxicity testing battery is highly sensitive for detection of chemical carcinogens. However, it features a low specificity and provides only limited mechanistic information required for risk assessment of positive findings. This is especially important in case of positive findings in the in vitro chromosome damage assays, because chromosome damage may be also induced secondarily to cell death. An increasing body of evidence indicates that toxicogenomic analysis of cellular stress responses provides an insight into mechanisms of action of genotoxicants. To evaluate the utility of such a toxicogenomic analysis we evaluated gene expression profiles of TK6 cells treated with four model genotoxic agents using a targeted high density real-time PCR approach in a multilaboratory project coordinated by the Health and Environmental Sciences Institute Committee on the Application of Genomics in Mechanism-based Risk Assessment. We show that this gene profiling technology produced reproducible data across laboratories allowing us to conclude that expression analysis of a relevant gene set is capable of distinguishing compounds that cause DNA adducts or double strand breaks from those that interfere with mitotic spindle function or that cause chromosome damage as a consequence of cytotoxicity. Furthermore, our data suggest that the gene expression profiles at early time points are most likely to provide information relevant to mechanisms of genotoxic damage and that larger gene expression arrays will likely provide richer information for differentiating molecular mechanisms of action of genotoxicants. Although more compounds need to be tested to identify a robust molecular signature, this study confirms the potential of toxicogenomic analysis for investigation of genotoxic mechanisms.

Comparison of coumarin-induced toxicity between sandwich-cultured primary rat hepatocytes and rats in vivo: a toxicogenomics approach.

Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and beta-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end-points at the molecular level between in vitro and in vivo conditions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium.