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Romosozumab (EVENITY™ [romosozumab-aqqg in the US]) is a humanized monoclonal antibody that inhibits sclerostin and has been approved in several countries for the treatment of osteoporosis in postmenopausal women at high risk of fracture. Sclerostin is expressed in bone and aortic vascular smooth muscle (AVSM). Its function in AVSM is unclear but it has been proposed to inhibit vascular calcification, atheroprogression, and inflammation. An increased incidence of positively adjudicated serious cardiovascular adverse events driven by an increase in myocardial infarction and stroke was observed in romosozumab-treated subjects in a clinical trial comparing alendronate with romosozumab (ARCH; NCT01631214) but not in a placebo-controlled trial (FRAME; NCT01575834). To investigate the effects of sclerostin inhibition with sclerostin antibody on the cardiovascular system, a comprehensive nonclinical toxicology package with additional cardiovascular studies was conducted. Although pharmacodynamic effects were observed in the bone, there were no functional, morphological, or transcriptional effects on the cardiovascular system in animal models in the presence or absence of atherosclerosis. These nonclinical studies did not identify evidence that proves the association between sclerostin inhibition and adverse cardiovascular function, increased cardiovascular calcification, and atheroprogression.

Drug induced liver injury (DILI) is one of the key safety concerns in drug development. To assess the likelihood of drug candidates with potential adverse reactions of liver, we propose a compound attributes-based approach to predicting hepatobiliary disorders that are routinely reported to US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS). Specifically, we developed a support vector machine (SVM) model with recursive feature extraction, based on physicochemical and structural properties of compounds as model input. Cross validation demonstrates that the predictive model has a robust performance with averaged 70% of both sensitivity and specificity over 500 trials. An independent validation was performed on public benchmark drugs and the results suggest potential utility of our model for identifying safety alerts. This in silico approach, upon further validation, would ultimately be implemented, together with other in vitro safety assays, for screening compounds early in drug development.

Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting genome instability including chromosomal structural rearrangements in normal cells are not currently assessed in the genetic toxicology battery. Here, we show that drug-induced replication stress leads to increased genome instability in vitro using proliferating primary human cells as well as in vivo in rat bone marrow (BM) and duodenum (DD). p53-binding protein 1 (53BP1, biomarker of DNA damage repair) nuclear bodies were increased in a dose-dependent manner in normal proliferating human mammary epithelial fibroblasts following treatment with compounds traditionally classified as either genotoxic (hydralazine) and nongenotoxic (low-dose aphidicolin, duvelisib, idelalisib, and amiodarone). Comparatively, no increases in 53BP1 nuclear bodies were observed in nonproliferating cells. Negative control compounds (mannitol, alosteron, diclofenac, and zonisamide) not associated with cancer risk did not induce 53BP1 nuclear bodies in any cell type. Finally, we studied the in vivo genomic consequences of drug-induced replication stress in rats treated with 10 mg/kg of cyclophosphamide for up to 14 days followed by polymerase chain reaction-free whole genome sequencing (30X coverage) of BM and DD cells. Cyclophosphamide induced chromosomal structural rearrangements at an average of 90 genes, including 40 interchromosomal/intrachromosomal translocations, within 2 days of treatment. Collectively, these data demonstrate that this drug-induced genome instability test (DiGIT) can reveal potential adverse effects of drugs not otherwise informed by standard genetic toxicology testing batteries. These efforts are aligned with the food and drug administration's (FDA's) predictive toxicology roadmap initiative.

Inhibition of sclerostin with sclerostin antibody (Scl-Ab) has been shown to stimulate bone formation, decrease bone resorption, and increase bone mass in both animals and humans. To obtain insight into the temporal cellular and transcriptional changes in the osteoblast (OB) lineage associated with long-term Scl-Ab treatment, stereological and transcriptional analyses of the OB lineage were performed on lumbar vertebrae from aged ovariectomized rats. Animals were administered Scl-Ab 3 or 50mg/kg/wk or vehicle (VEH) for up to 26weeks (d183), followed by a treatment-free period (TFP). At 50mg/kg/wk, bone volume (BV/total volume [TV]) increased through d183 and declined during the TFP. Bone formation rate (BFR/bone surface [BS]) and total OB number increased through d29, then progressively declined, coincident with a decrease in total osteoprogenitor (OP) numbers from d29 through d183. Analysis of differentially expressed genes (DEGs) from microarray analysis of mRNA isolated from laser capture microdissection samples enriched for OB, lining cells, and osteocytes (OCy) revealed modules of genes that correlated with BFR/BS, BV/TV, and osteoblastic surface (Ob.S)/BS. Expression change of canonical Wnt target genes was similar in all three cell types at d8, including upregulation of Twist1 and Wisp1. At d29, the pattern of Wnt target gene expression changed in the OCy, with Twist1 returning to VEH level, sustained upregulation of Wisp1, and upregulation of several other Wnt targets that continued into the TFP. Predicted activation of pathways recognized to integrate with and regulate canonical Wnt signaling were also activated at d29 in the OCy. The most significantly affected pathways represented transcription factor signaling known to inhibit cell cycle progression (notably p53) and mitogenesis (notably c-Myc). These changes occurred at the time of peak BFR/BS and continued as BFR/BS declined during treatment, then trended toward VEH level in the TFP. Concurrent with this transcriptional switch was a reduction in OP numbers, an effect that would ultimately limit bone formation. This study confirms that the initial transcriptional response in response to Scl-Ab is activation of canonical Wnt signaling and the data demonstrate that there is induction of additional regulatory pathways in OCy with long-term treatment. The interactions between Wnt and p53/c-Myc signaling may be key in limiting OP populations, thus contributing to self-regulation of bone formation with continued Scl-Ab administration.

Sclerostin antibody (Scl-Ab) increases bone formation through a process dependent on the activation of canonical Wnt signaling, although the specific signaling in the osteoblast lineage in vivo is largely unknown. To gain insight into the signaling pathways acutely modulated by Scl-Ab, the transcriptional response of subpopulations of the osteoblast lineage was assessed by TaqMan and microarray analyses of mRNA isolated from laser capture microdissection (LCM)–enriched samples from the vertebrae of ovariectomized rats during the first week after Scl-Ab administration. Briefly, 6-month-old Sprague-Dawley rats were ovariectomized and, after 2 months, received a single dose of vehicle (VEH) or 100 mg/kg Scl-Ab (n = 20/group). Lumbar vertebrae were collected at 6, 24, 72, and 168 hours postdose and cryosectioned for LCM. Osteocytes were captured from bone matrix, and osteoblasts and lining cells were captured from bone surfaces based on fluorochrome labeling. mRNA was isolated, amplified, and profiled by TaqMan and microarray. Expression analysis revealed that Scl-Ab caused strikingly similar transcriptional profiles across all three cell types. Only 13 known canonical Wnt target genes, the majority with known functions in bone, showed a significant change in expression by microarray in response to Scl-Ab, with Wisp1 and Twist1 being the most responsive. Coincident with increased expression of Wnt target genes was the upregulation of numerous extracellular matrix (ECM) genes. The acute and progressive upregulation of ECM genes in lining cells supports their activation into matrix-producing osteoblasts, consistent with modeling-based bone formation. A similar transcriptional profile in osteocytes may indicate that Scl-Ab stimulates perilacunar/pericanalicular matrix deposition. Pathway analyses indicated that Scl-Ab regulated a limited number of genes related to cell cycle arrest and B-cell development. These data describe the acute downstream signaling in response to Scl-Ab in vivo and demonstrate selected canonical Wnt target gene activation associated with increased bone formation in all mature osteoblast subpopulations. © 2015 American Society for Bone and Mineral Research.

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.

A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect. Controlled exposures of microarrays to ozone confirmed this factor as the root cause, and we present data that show susceptibility of a class of cyanine dyes (e.g., Cy5, Alexa 647) to ozone levels as low as 5−10 ppb for periods as short as 10−30 s. Other cyanine dyes (e.g., Cy3, Alexa 555) were not significantly affected until higher ozone levels (>100 ppb). To address this environmental effect, laboratory ozone levels should be kept below 2 ppb (e.g., with filters in HVAC) to achieve high quality microarray data.

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution. Our method for determining differential expression consists of two steps: 1) We apply a nominal threshold on fold change and platform p-value to designate whether a gene is differentially expressed in each treated and control sample relative to the averaged control pool, and 2) We compared the number of samples satisfying criteria in step 1 between the treated and control groups to estimate the statistical significance based on a null distribution established by sample permutations. The method captures group effect without being too sensitive to anomalies as it allows tolerance for potential non-responders in the treatment group and outliers in the control group. Performance and results of this method were compared with the Significant Analysis of Microarrays (SAM) method. These two methods were applied to investigate hepatic transcriptional responses of wild-type (PXR(+/+)) and pregnane X receptor-knockout (PXR(-/-)) mice after 96 h exposure to CMP013, an inhibitor of β-secretase (β-site of amyloid precursor protein cleaving enzyme 1 or BACE1). Our results showed that CMP013 led to transcriptional changes in hallmark PXR-regulated genes and induced a cascade of gene expression changes that explained the hepatomegaly observed only in PXR(+/+) animals. Comparison of concordant expression changes between PXR(+/+) and PXR(-/-) mice also suggested a PXR-independent association between CMP013 and perturbations to cellular stress, lipid metabolism, and biliary transport.

The molecular mechanisms of action of a HIV protease inhibitor, ritonavir, on hepatic function were explored on a genomic scale using microarrays comprising genes expressed in the liver of Sprague–Dawley rats (Rattus norvegicus). Analyses of hepatic transcriptional fingerprints led to the identification of several key cellular pathways affected by ritonavir treatment. These effects were compared to a compendium of gene expression responses for 52 unrelated compounds and to other protease inhibitors, including atazanavir and two experimental compounds. We identified genes involved in cholesterol and fatty acid biosynthesis, as well as genes involved in fatty acid and cholesterol breakdown, whose expressions were regulated in opposite manners by ritonavir and bezafibrate, a hypolipidemic agonist of the peroxisome proliferator-activated receptor α. Ritonavir also upregulated multiple proteasomal subunit transcripts as well as genes involved in ubiquitination, consistent with its known inhibitory effect on proteasomal activity. We also tested three other protease inhibitors in addition to ritonavir. Atazanavir did not impact ubiquitin or proteasomal gene expression, although the two other experimental protease inhibitors impacted both proteasomal gene expression and sterol regulatory element-binding protein-activated genes, similar to ritonavir. Identification of key metabolic pathways that are affected by ritonavir and other protease inhibitors will enable us to understand better the downstream effects of protease inhibitors, thus leading to better drug design and an effective method to mitigate the side effects of this important class of HIV therapeutics.

The antiandrogen flutamide (FLU) is used primarily for prostate cancer and is an idiosyncratic hepatotoxicant that sometimes causes severe liver problems. To investigate FLU's overt hepatic effects, especially on inducible drug clearance-related gene networks, FLU's hepatic gene expression profile was examined in female Sprague-Dawley rats using approximately 22,500 oligonucleotide microarrays. Rats were dosed daily for 3 days with FLU at 500, 250, 62.5, 31.3, and 15.6 mg/kg/day, and hepatic RNA was isolated. FLU resulted in the dose-dependent regulation of approximately 350 genes. Employing a gene-response compendium, FLU was compared with three classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone, and four atypical CYP1A inducers, indole-3-carbinol (I3C), omeprazole (OME), chlorpromazine (CPZ), and clotrimazole (CLO). The FLU gene response was comparable with classical AhR ligands across a signature AhR ligand gene set that included CYP1A1 and other members of the AhR gene battery. Dose-related responses of CYP1 genes established a maximum response ceiling and discerned potency differences in atypical inducers. FLU had a sharp down-regulation of c-fos that was comparable with all the compounds except CPZ and CLO. FLU absorption, distribution, metabolism, and excretion (ADME) gene expression analysis revealed that FLU, as well as I3C and OME, induced CYP2B and CYP3A, distinguishing them from the classical AhR ligands. By using a compendium of gene expression profiles, FLU was shown to signal in rats similar to an AhR activator with additional CYP2B and CYP3A effects that most resembled the ADME gene expression pattern of the atypical CYP1A inducers I3C and OME.

Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.