An integrated approach for early in vitro seizure prediction utilizing hiPSC neurons and human ion channel assays.

Seizure liability remains a significant cause of attrition throughout drug development. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. We assessed the activity of 15 pro-seizurogenic compounds (7 CNS active therapies, 4 GABA receptor antagonists, and 4 other reported seizurogenic compounds) using automated electrophysiology against a panel of 14 ion channels (Nav1.1, Nav1.2, Nav1.6, Kv7.2/7.3, Kv7.3/7.5, Kv1.1, Kv4.2, KCa4.1, Kv2.1, Kv3.1, KCa1.1, GABA α1ß2γ2, nicotinic α4ß2, NMDA 1/2A). These were selected based on linkage to seizure in genetic/pharmacological studies. Fourteen compounds demonstrated at least one "hit" against the seizure panel and 11 compounds inhibited 2 or more ion channels. Next, we assessed the impact of the 15 compounds on electrical signaling using human-induced pluripotent stem cell neurons in microelectrode array (MEA). The CNS active therapies (amoxapine, bupropion, chlorpromazine, clozapine, diphenhydramine, paroxetine, quetiapine) all caused characteristic changes to electrical activity in key parameters indicative of seizure such as network burst frequency and duration. The GABA antagonist picrotoxin increased all parameters, but the antibiotics amoxicillin and enoxacin only showed minimal changes. Acetaminophen, included as a negative control, caused no changes in any of the parameters assessed. Overall, pro-seizurogenic compounds showed a distinct fingerprint in the ion channel/MEA panel. These studies highlight the potential utility of an integrated in vitro approach for early seizure prediction to provide mechanistic information and to support optimal drug design in early development, saving time and resources.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

Mechanism of hepatobiliary toxicity of the LPA1 antagonist BMS-986020 developed to treat idiopathic pulmonary fibrosis: Contrasts with BMS-986234 and BMS-986278.

In a Phase 2 clinical trial, BMS-986020, a lysophosphatidic acid receptor-1 (LPA1) antagonist, produced hepatobiliary toxicity (increased ALT, AST, and ALP; cholecystitis) and increases in plasma bile acids (BA). Nonclinical investigations conducted to identify a potential mechanism(s) for this toxicity examined BMS-986020 and two LPA1 antagonists structurally distinct from BMS-986020 (BMS-986234 and BMS-986278). BMS-986020 inhibited hepatic BA efflux transporters BSEP (IC50 1.8 µM), MRP3 (IC50 22 µM), and MRP4 (IC50 6.2 µM) and inhibited BA canalicular efflux in human hepatocytes (68% at 10 µM). BMS-986020 inhibited mitochondrial function (basal and maximal respiration, ATP production, and spare capacity) in human hepatocytes and cholangiocytes at ≥10 µM and inhibited phospholipid efflux in human hepatocytes (MDR3 IC50 7.5 µM). A quantitative systems toxicology analysis (DILIsym®), considering pharmacokinetics, BA homeostasis, mitochondrial function, oxidative phosphorylation, and reactive intermediates performed for BMS-986020 recapitulated clinical findings ascribing the effects to BA transporter and mitochondrial electron transport chain inhibition. BMS-986234 and BMS-986278 minimally inhibited hepatic BA transporters (IC50 ≥20 µM) and did not inhibit MDR3 activity (IC50 >100 µM), nor did BMS-986234 inhibit BA efflux (≤50 µM) or mitochondrial function (≤30 µM) (BMS-986278 not evaluated). Multiple mechanisms may be involved in the clinical toxicity observed with BMS-986020. The data indicate that this toxicity was unrelated to LPA1 antagonism since the mechanisms that likely influenced the adverse clinical outcome of BMS-986020 were not observed with equipotent LPA1 antagonists BMS-986234 and BMS-986278. This conclusion is consistent with the lack of hepatobiliary toxicity in nonclinical and clinical safety studies with BMS-986278.

Cardiovascular microphysiological systems (CVMPS) for safety studies - a pharma perspective.

The integrative responses of the cardiovascular (CV) system are essential for maintaining blood flow to provide oxygenation, nutrients, and waste removal for the entire body. Progress has been made in independently developing simple in vitro models of two primary components of the CV system, namely the heart (using induced pluripotent stem-cell derived cardiomyocytes) and the vasculature (using endothelial cells and smooth muscle cells). These two in vitro biomimics are often described as immature and simplistic, and typically lack the structural complexity of native tissues. Despite these limitations, they have proven useful for specific "fit for purpose" applications, including early safety screening. More complex in vitro models offer the tantalizing prospect of greater refinement in risk assessments. To this end, efforts to physically link cardiac and vascular components to mimic a true CV microphysiological system (CVMPS) are ongoing, with the goal of providing a more holistic and integrated CV response model. The challenges of building and implementing CVMPS in future pharmacological safety studies are many, and include a) the need for more complex (and hence mature) cell types and tissues, b) the need for more realistic vasculature (within and across co-modeled tissues), and c) the need to meaningfully couple these two components to allow for integrated CV responses. Initial success will likely come with simple, bioengineered tissue models coupled with fluidics intended to mirror a vascular component. While the development of more complex integrated CVMPS models that are capable of differentiating safe compounds and providing mechanistic evaluations of CV liabilities may be feasible, adoption by pharma will ultimately hinge on model efficiency, experimental reproducibility, and added value above current strategies.

Development and characterization of rat duodenal organoids for ADME and toxicology applications.

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

A pharmaceutical industry perspective on microphysiological kidney systems for evaluation of safety for new therapies.

The human kidney contains approximately one million nephrons. As the functional unit of the kidney, the nephron affords an opportunity to approximate the kidney at a microphysiological scale. Recent emergence of physiologically accurate human tissue models has radically advanced the possibilities of mimicking organ biology and multi-organ combinations in vitro. Anatomically, the nephron is one of the most complex, sequentially integrated microfluidic units in the body making the miniaturized microfluidic systems excellent candidates for capturing the kidney biology in vitro. While these models are promising, there are a number of considerations for practical implementation into a drug development paradigm. Opportunities for pharmaceutical industry applications of new MPS models often start with drug safety testing. As such, the intent of this article is to focus on safety and ADME applications. This article reviews biological functions of the kidney and options for characterizing known roles in nephrotoxicity. The concept of "context-of-use" is introduced as a framework for describing and verifying the specific features of an MPS platform for use in drug development. Overall, we present a perspective on key attributes of microphysiological kidney models, which the pharmaceutical industry could leverage to improve confident safety and ADME evaluations of experimental therapies.

Microphysiological lung models to evaluate the safety of new pharmaceutical modalities: a biopharmaceutical perspective.

The lung is a complex organ; it is both the initial barrier for inhaled agents and the site of metabolism and therapeutic effect for a subset of systemically administered drugs. Comprised of more than 40 cell types that are responsible for various important functions, the lung's complexity contributes to the subsequent challenges in developing complex in vitro co-culture models (also called microphysiological systems (MPS), complex in vitro models or organs-on-a-chip). Although there are multiple considerations and limitations in the development and qualification of such in vitro systems, MPS exhibit great promise in the fields of pharmacology and toxicology. Successful development and implementation of MPS models may enable mechanistic bridging between non-clinical species and humans, and increase clinical relevance of safety endpoints, while decreasing overall animal use. This article summarizes, from a biopharmaceutical industry perspective, essential elements for the development and qualification of lung MPS models. Its purpose is to guide MPS developers and manufacturers to expedite MPS utilization for safety assessment in the biopharmaceutical industry.

Transcriptomic profiles of tissues from rats treated with anticancer drug combinations.

To achieve therapeutic goals, many cancer chemotherapeutics are used at doses close to their maximally tolerated doses. Thus, it may be expected that when therapies are combined at therapeutic doses, toxicity profiles may change. In many ways, prediction of synergistic toxicities for drug combinations is similar to predicting synergistic efficacy, and is dependent upon building hypotheses from molecular mechanisms of drug toxicity. The key objective of this initiative was to generate and make publicly available key high-content data sets for mechanistic hypothesis generation as it pertains to a unique toxicity profile of a drug pair for several anticancer drug combinations. The expectation is that tissue-based genomic information that are derived from target tissues will also facilitate the generation and testing of mechanistic hypotheses. The view is that availability of these data sets for bioinformaticians and other scientists will contribute to analysis of these data and evaluation of the approach.

The Evolving Design of NIH-Funded Cardio-Oncology Studies to Address Cancer Treatment-Related Cardiovascular Toxicity.

Cardiovascular (CV) toxicity from cancer therapy is a significant and growing concern. Conventional oncology clinical trial designs focused singularly on cancer treatment efficacy have not provided sufficient information on both CV risk factors and outcomes. Similarly, traditional CV trials evaluating standard interventions typically exclude cancer patients, particularly those actively receiving cancer therapy. Neither trial type simultaneously evaluates the balance between CV toxicity and cancer outcomes. However, there is increasing collaboration among oncologists and cardiologists to design new cardio-oncology trials that address this important need. In this review, we detail five ongoing, oncology-based trials with integrated CV endpoints. Key design features include: 1) a careful assessment of baseline risk factors for CV disease; 2) an introduction of cardioprotective interventions at various timepoints in cancer therapy; 3) a balance of the risk of subclinical CV injury with the need for ongoing cancer treatment; and 4) an understanding of the time profile for development of clinically apparent CV toxicity. Additional critical priorities in cardio-oncology clinical research include harmonization of data collection and definitions for all physician- and patient-reported exposures and outcomes.

Erythrofordins D and E, two new cassaine-type diterpenes from Erythrophleum suaveolens.

Two new cassaine-type diterpenoids, namely erythrofordins D (1) and E (2), sourced from a Cameroon collection of Erythrophleum suaveolens were isolated and assessed for anti-tumor activity. In the NCI-60 cancer cell assay, erythrofordins D (1) and E (2) were found to be cytotoxic in the low micro molar ranges with a mean GI50 value of 2.45 and 0.71 µM, mean TGI value of 9.77 and 2.29 µM, and a mean LC50 of 26.92 and 11.48 µM for 1 and 2 respectively. Using the COMPARE algorithm, the new compounds were found to have similar NCI-60 response profiles to the known cardiac glycosides hyrcanoside and strophanthin. In addition, in an assay examining the viability and contractile function in human cardiomyocytes derived from induced pluripotent stem-cells, erythrofordins showed cardiotoxicity effects at concentrations as low as 0.03 µg/mL.

Role of Mcl-1 in regulation of cell death in human induced pluripotent stem cell-derived cardiomyocytes in vitro.

Targeting the anti-apoptotic protein Mcl-1 holds a promise to improve therapy of multiple types of Mcl-1 dependent cancers but raises concerns of on-target cardiotoxicity due to the presence and reported role of Mcl-1 in heart. Herein, we investigated the importance of Mcl-1 in the survival and contractile function of human pluripotent stem cell-derived cardiomyocytes in culture. Effective knockdown of Mcl-1 with siRNAs reproducibly resulted in early (measured at Day 3) marginal alterations in caspase 3/7 activity, LDH leakage, ATP content and cellular impedance. After 14 days of Mcl-1 knockdown, loss of mitochondrial membrane potential, deteriorating effects on mitochondrial ultrastructure, and alterations in beat rate and amplitude were revealed. Inhibition of Bcl-xL by siRNA-mediated knockdown or selective inhibitors did not cause any overt cellular responses except for a minimal increase in caspase 3/7 activity; however, loss of Mcl-1 concomitant with down-regulated Bcl-xL activated apoptosis and caused extensive cell death as reflected by an 80% loss in cell index, activation of caspase-3 with associated PARP cleavage, and a decrease in beat amplitude and mitochondrial membrane potential after 3 days of Mcl-1/Bcl-xL knockdown., Together, these findings suggest that Mcl-1 and Bcl-xL provide duplicate safeguard measures in maintaining structural and functional integrity of cardiomyocytes. Hence, BH3-mimetic drugs targeting Mcl-1 may be well tolerated in the presence of intact Bcl-xL.

Clinically Relevant Concentrations of Anticancer Drugs: A Guide for Nonclinical Studies.

Approved and marketed drugs are frequently studied in nonclinical models to evaluate the potential application to additional disease indications or to gain insight about molecular mechanisms of action. A survey of the literature reveals that nonclinical experimental designs (in vitro or in vivo) often include evaluation of drug concentrations or doses that are much higher than what can be achieved in patients (i.e., above the maximally tolerated dose or much higher than the clinically relevant exposures). The results obtained with these high concentrations may be particularly helpful in elucidating off-target effects and toxicities, but it is critical to have a dose-response curve that includes the minimally effective or clinically effective concentration for comparison. We have reviewed the clinical literature and drug product labels for all small molecules and biological agents approved by the FDA for use in oncology to identify and compile the available pharmacokinetic parameters. The data summarized here can serve as a guide for selection of in vitro concentrations and in vivo plasma exposures for evaluation of drug effects in nonclinical studies. Inclusion of drug concentrations or exposures that are relevant to those observed in clinical practice can improve translation of nonclinical mechanism of action findings into potentially relevant clinical effects. Clin Cancer Res; 23(14); 3489-98. ©2017 AACR.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (ß-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

Multi-parametric assessment of cardiomyocyte excitation-contraction coupling using impedance and field potential recording: A tool for cardiac safety assessment.

INTRODUCTION: The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization screening due to its association with life-threatening "Torsades de Pointes" (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative to examine the possible modification and refinement of the ICH E14/S7B guidelines. One of the main components of the CiPA initiative is to utilize a predictive assay system together with human cardiomyocytes for risk assessment of arrhythmia. METHOD: In this manuscript we utilize the xCELLigence® CardioECR system which simultaneously measures excitation-contraction coupling together with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to assess the effect of 8 reference compounds across 3 different independent sites. These 8 compounds were part of Phase I CiPA validation study. RESULTS: Our data demonstrate that hERG channel blockers, such as E4031 and moxifloxacin, prolonged field potential duration (FPD) at low concentration and induced arrhythmic beating activity as measured by field potential (FP) recording and impedance (IMP) recordings at higher concentrations. On the contrary, nifedipine, an inhibitor of calcium channel, didn't disrupt the periodicity of cell beating and weakened cell contractile activity and shortened FPD. Multichannel inhibitors, such as flecainide, quinidine and mexiletine, not only increased FPD and induced arrhythmia but also significantly reduced the amplitude of FP spike. JNJ303, an IKs inhibitor, only affected FPD. Comparison of the compound effect on FPD across the 3 different sites is consistent in terms of trend of the effect with observed 3-10 fold differences in minimal effective concentration at which a minimum of 10% response is detected. In addition, pentamidine, a hERG trafficking inhibitor which induced irregular beating activity over a more prolonged duration of time was readily flagged in this assay system. Taken together, this multi-parameter assay using hiPSC-CMs in conjunction with simultaneous measurement of ion channel activity and contractility can be a reliable approach for risk assessment of proarrhythmic compounds.

Enhancing quality of life as a goal for anticancer therapeutics.

Translational cancer research requires a quality-of-life-driven agenda.

Human CD34(+) progenitor hematopoiesis in liquid culture for in vitro assessment of drug-induced myelotoxicity.

Utilization of validated CFU-GM assays for myelotoxicity screening is hampered by its labor-intensive and low-throughput nature. Herein, we transformed the defined CFU-GM assay conditions and IC90 endpoint into a higher throughput format. Human CD34(+) hematopoietic progenitors were cultured in a 96-well plate for 14 days with the same cytokine (rhGM-CSF) used in the CFU-GM assay. Expansion and differentiation toward myeloid lineages were manifested by characteristic changes in nuclear and cytoplasmic morphology and by temporal expression patterns of CD34, CD11b and CD13 markers. Inhibition of CD34(+) cell myelopoiesis by 12 anticancer drugs known to induce myelotoxicity in the clinic was quantifiable using either general cytotoxicity endpoints (cell growth area or total nucleus count) or lineage specific readouts (count of cells expressing CD11b and/or CD13). The IC50 and IC90 values derived from the concentration-response curves of 14-day drug exposure in CD34(+) cell culture were highly correlated with those from the international validation study of the CFU-GM assay, demonstrating capability to assess general cytotoxicity, cell proliferation and myelopoiesis simultaneously. These results suggest that this human CD34(+) hematopoietic progenitor cell assay can be used as a direct replacement for the validated, low throughput CFU-GM assay, and could expand application of in vitro myelotoxicity testing.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes.

Oral and intravenous pharmacokinetics of 5-fluoro-2'-deoxycytidine and THU in cynomolgus monkeys and humans.

INTRODUCTION: 5-Fluoro-2'-deoxycytidine (FdCyd; NSC48006), a fluoropyrimidine nucleoside inhibitor of DNA methylation, is degraded by cytidine deaminase (CD). Pharmacokinetic evaluation was carried out in cynomolgus monkeys in support of an ongoing phase I study of the PO combination of FdCyd and the CD inhibitor tetrahydrouridine (THU; NSC112907). METHODS: Animals were dosed intravenously (IV) or per os (PO). Plasma samples were analyzed by LC-MS/MS for FdCyd, metabolites, and THU. Clinical chemistry and hematology were performed at various times after dosing. A pilot pharmacokinetic study was performed in humans to assess FdCyd bioavailability. RESULTS: After IV FdCyd and THU administration, FdCyd C(max) and AUC increased with dose. FdCyd half-life ranged between 22 and 56 min, and clearance was approximately 15 mL/min/kg. FdCyd PO bioavailability after THU ranged between 9 and 25 % and increased with increasing THU dose. PO bioavailability of THU was less than 5 %, but did result in plasma concentrations associated with inhibition of its target CD. Human pilot studies showed comparable bioavailability for FdCyd (10 %) and THU (4.1 %). CONCLUSION: Administration of THU with FdCyd increased the exposure to FdCyd and improved PO FdCyd bioavailability from <1 to 24 %. Concentrations of THU and FdCyd achieved after PO administration are associated with CD inhibition and hypomethylation, respectively. The schedule currently studied in phase I studies of PO FdCyd and THU is daily times three at the beginning of the first and second weeks of a 28-day cycle.

Toxicogenomics profiling of bone marrow from rats treated with topotecan in combination with oxaliplatin: a mechanistic strategy to inform combination toxicity.

Combinations of anticancer agents may have synergistic anti-tumor effects, but enhanced hematological toxicity often limit their clinical use. We examined whether "microarray profiles" could be used to compare early molecular responses following a single dose of agents administered individually with that of the agents administered in a combination. We compared the mRNA responses within bone marrow of Sprague-Dawley rats after a single 30 min treatment with topotecan at 4.7 mg/kg or oxaliplatin at 15 mg/kg alone to that of sequentially administered combination therapy or vehicle control for 1, 6, and 24 h. We also examined the histopathology of the bone marrow following all treatments. Drug-related histopathological lesions were limited to bone marrow hypocellularity for animals dosed with either agent alone or in combination. Lesions had an earlier onset and higher incidence for animals given topotecan alone or in combination with oxaliplatin. Severity increased from mild to moderate when topotecan was administered prior to oxaliplatin compared with administering oxaliplatin first. Notably, six patterns of co-expressed genes were detected at the 1 h time point that indicate regulatory expression of genes that are dependent on the order of the administration. These results suggest alterations in histone biology, chromatin remodeling, DNA repair, bone regeneration, and respiratory and oxidative phosphorylation are among the prominent pathways modulated in bone marrow from animals treated with an oxaliplatin/topotecan combination. These data also demonstrate the potential for early mRNA patterns derived from target organs of toxicity to inform toxicological risk and molecular mechanisms for agents given in combination.

Characterization of Batracylin-induced Renal and Bladder Toxicity in Rats.

Batracylin (NSC-320846) is a dual inhibitor of DNA topoisomerases I and II. Batracylin advanced as an anticancer agent to Phase I clinical trials where dose limiting hemorrhagic cystitis (bladder inflammation and bleeding) was observed. To further investigate batracylin's mechanism of toxicity, studies were conducted in Fischer 344 rats. Once daily oral administration of 16 or 32 mg/kg batracylin to rats for 4 days caused overt toxicity. Abnormal clinical observations and adverse effects on clinical pathology, urinalysis, and histology indicated acute renal damage and urothelial damage and bone marrow dysfunction. Scanning electron microscopy revealed sloughing of the superficial and intermediate urothelial layers. DNA damage was evident in kidney and bone marrow as indicated by histone γ-H2AX immunofluorescence. After a single oral administration of 16 or 32 mg/kg, the majority of batracylin was converted to N-acetylbatracylin (NAB) with a half-life of 4 hr to 11 hr. Mesna (Mesnex™), a drug known to reduce the incidence of hemorrhagic cystitis induced by ifosfamide or cyclophosphamide, was administered to rats prior to batracylin, but did not alleviate batracylin-induced bladder and renal toxicity. These findings suggest that batracylin results in DNA damage-based mechanisms of toxicity and not an acrolein-based mechanism of toxicity as occurs after ifosfamide or cyclophosphamide administration.