A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per µg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.

Characterization of indoleamine-2,3-dioxygenase 1, tryptophan-2,3-dioxygenase, and Ido1/Tdo2 knockout mice.

Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) degrade tryptophan (Trp) to kynurenine (Kyn), and these enzymes have promise as therapeutic targets. A comprehensive characterization of potential safety liabilities of IDO1 and TDO2 inhibitors using knockout (KO) mice has not been assessed, nor has the dual Ido1/Tdo2 KO been reported. Here we characterized male and female mice with KOs for Ido1, Tdo2, and Ido1/Tdo2 and compared findings to the wild type (WT) mouse strain, evaluated for 14 days, using metabolomics, transcriptional profiling, behavioral analysis, spleen immunophenotyping, comprehensive histopathological analysis, and serum clinical chemistry. Multiple metabolomic changes were seen in KO mice. For catabolism of Trp to Kyn and anthranilic acid, both substrates were decreased in liver of Tdo2 and dual KO mice. Metabolism of Trp to serotonin and its metabolites resulted in an increase in 5-Hydroxyindole-3-acetic acid in the Tdo2 and dual KO mice. Ido1 and dual KO mice displayed a Kyn reduction in plasma but not in liver. Nicotinamide synthesis and conversion of glucose to lactic acid were not impacted. A slight decrease in serum alkaline phosphatase was seen in all KOs, and small changes in liver gene expression of genes unrelated to tryptophan metabolism were observed. Regarding other parameters, no genotype-specific changes were observed. In summary, this work shows metabolomic pathway changes for metabolites downstream of tryptophan in these KO mice, and suggests that inhibition of the IDO1 and TDO2 enzymes would be well tolerated whether inhibited individually or in combination since no safety liabilities were uncovered.

Development and Application of a Transcriptomic Signature of Bioactivation in an Advanced In Vitro Liver Model to Reduce Drug-induced Liver Injury Risk Early in the Pharmaceutical Pipeline.

Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection.

AhR Activation in Pharmaceutical Development: Applying Liver Gene Expression Biomarker Thresholds to Identify Doses Associated With Tumorigenic Risks in Rats.

Aryl hydrocarbon receptor (AhR) activation is associated with carcinogenicity of non-genotoxic AhR-activating carcinogens such as 2,3,7,8-tetrachlorodibenzodioxin (TCDD), and is often observed with drug candidate molecules in development and raises safety concerns. As downstream effectors of AhR signaling, the expression and activity of Cyp1a1 and Cyp1a2 genes are commonly monitored as evidence of AhR activation to inform carcinogenic risk of compounds in question. However, many marketed drugs and phytochemicals are reported to induce these Cyps modestly and are not associated with dioxin-like toxicity or carcinogenicity. We hypothesized that a threshold of AhR activation needs to be surpassed in a sustained manner in order for the dioxin-like toxicity to manifest, and a simple liver gene expression signature based on Cyp1a1 and Cyp1a2 from a short-term rat study could be used to assess AhR activation strength and differentiate tumorigenic dose levels from non-tumorigenic ones. To test this hypothesis, short-term studies were conducted in Wistar Han rats with 2 AhR-activating carcinogens (TCDD and PCB126) at minimally carcinogenic and noncarcinogenic dose levels, and 3 AhR-activating noncarcinogens (omeprazole, mexiletine, and canagliflozin) at the top doses used in their reported 2-year rat carcinogenicity studies. A threshold of AhR activation was identified in rat liver that separated a meaningful "tumorigenic-strength AhR signal" from a statistically significant AhR activation signal that was not associated with dioxin-like carcinogenicity. These studies also confirmed the importance of the sustainability of AhR activation for carcinogenic potential. A sustained activation of AhR above the threshold could thus be used in early pharmaceutical development to identify dose levels of drug candidates expected to exhibit dioxin-like carcinogenic potential.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

A multi-species assay for siRNA-mediated mRNA knockdown analysis without the need for RNA purification.

INTRODUCTION: Various animal models are routinely used to evaluate the efficacy and toxicity of small interfering RNA (siRNA) therapeutics. Given that the most common measure of efficacy with siRNA therapeutics is mRNA knockdown, the development of a single assay for quantification of siRNA-mediated mRNA knockdown in multiple species would provide significant time and cost-savings during preclinical development. METHODS AND RESULTS: We have developed an assay targeting short consensus sequences of a particular mRNA in multiple species using the principles of a recently-reported stem-loop RT-qPCR method (Chen et al., 2005). The multi-species RT-qPCR assay is highly sensitive, reproducible, has a dynamic range of seven orders of magnitude, and it can be used to quantify a specific mRNA in crude tissue homogenates without the need for RNA purification. Compared to the limitations of conventional RT-qPCR assays, this assay provides a simple and robust tool for mRNA quantification to evaluate siRNA-mediated mRNA knockdown. DISCUSSION: This assay can potentially become a routine method for mRNA quantification to evaluate siRNA-mediated mRNA knockdown.

Intracisternal A particle genes: Distribution in the mouse genome, active subtypes, and potential roles as species-specific mediators of susceptibility to cancer.

Rodents, mice and rats in particular, are the species of choice for evaluating chemical carcinogenesis. However, different species and strains often respond very differently, undermining the logic of extrapolation of animal results to humans and complicating risk assessment. Intracisternal A particles (IAPs), endogenous retroviral sequences, are an important class of transposable elements that induce genomic mutations and cell transformation by disrupting gene expression. Several lines of evidence support a role of IAPs as mouse-specific genetic factors in responses to toxicity and expression of disease phenotypes. Since multiple subtypes and copies of IAPs are present in the mouse genome, their activity and locations relative to functional genes are of critical importance. This study identified the major "active" subtypes of IAPs (subtype 1/1a) that are responsible for newly transposed IAP insertions described in the literature, and confirmed that (1) polymorphisms for IAP insertions exist among different mouse strains and (2) promoter activity of the LTRs can be modulated by chemicals. This study further identified all the genes in the C57BL/6 mouse genome with IAP subtype 1 and 1a sequences inserted in their proximity, and the major biofunctional categories and cellular signaling networks of those genes. Since many "IAP-associated genes" play important roles in the regulation of cell proliferation, cell cycle, and cell death, the associated IAPs, upon activation, can affect cellular responses to xenobiotics and disease processes, especially carcinogenesis. This systemic analysis provides a solid foundation for further investigations of the role of IAPs as species- and strain-specific disease susceptibility factors.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Monoclonal antibodies reactive with the BAF155 (SMARCC1) and BAF170 (SMARCC2) components of human SWI/SNF-related complexes.

Mammalian SWI/SNF-related chromatin remodeling complexes are required for transcription controls that underlie differentiation, development, and tumor suppression. The complexes each consist of an ATPase of the SWI2/SNF2 family and approximately seven stably associated non-catalytic subunits. In spite of the importance of these complexes to biological processes, monoclonal antibodies to the various subunits have not been readily available. Mammalian complexes can vary in subunit composition, but the BAF155 (SMARCC1) subunit and a closely related protein, BAF170 (SMARCC2), appear to be ubiquitous components. Here we report the development of antibodies raised against a BAF155-derived peptide. The antibodies were raised against a single peptide of 18 amino acids. However, hybridomas expressing antibodies of two different specificities were isolated. One, designated DXD7, is specific for BAF155. The other, designated DXD12, is reactive with both BAF155 and BAF170. The antibodies are reactive against both native and denatured proteins, and are suitable for immunoprecipitation and Western blots. The DXD7 antibody is suitable additionally for immunofluorescence assays.

Two related ARID family proteins are alternative subunits of human SWI/SNF complexes.

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.