Improving carcinogenicity assessment.

The ICH initiated talks in June 2012 to revise regulatory guidance for carcinogenicity assessment of pharmaceutical products, stimulated in part by a proposal called Negative for Endocrine, Genotoxicity, and Chronic Study Associated Histopathologic Risk Factors for Carcinogenicity in the Rat (NEGCARC) from the Pharmaceutical Research and Manufacturing Association (PhRMA). The 2012 STP Town Hall Meeting focused on the need for change in carcinogenicity assessment strategies for pharmaceuticals. Dr. Todd Bourcier from the Division of Endocrine and Metabolic Products, U.S. FDA and a member of the FDA's Alternative Carcinogenicity Assessment Committee, was the guest speaker and a panelist. Dr. Bourcier is also one of FDA's representatives to the ICH S1 Expert Working Group that is discussing changes to regulatory guidelines for carcinogenicity assessment. Drs. Carl Alden and Dan Morton also participated in the panel discussion.

An analysis of pharmaceutical experience with decades of rat carcinogenicity testing: support for a proposal to modify current regulatory guidelines.

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Effect of an experimental proteasome inhibitor on the cytoskeleton, cytosolic protein turnover, and induction in the neuronal cells in vitro.

GT-1 murine neuronal cells exposed to an experimental proteasome inhibitor (EPI) for 24h showed increased cell death via a non-apoptotic mechanism, as assessed by TUNEL and DNA fragmentation assays. Immunofluorescence staining demonstrated that EPI induced reorganization and relocation of non-ubiquinated actin microfilaments and microtubules to the perinuclear region in EPI treated cells. Immunohistochemistry analysis also demonstrated that other non-cytoskeletal proteins became ubiquitinated and/or upregulated including ubiquitin and other stress proteins. Perinuclear-centrosomal accumulation of gamma-tubulin and vimentin, key components of aggresomes, was observed in the EPI treated cells. Biochemical analysis indicated that EPI-induced accumulation of ubiquitinated protein aggregates in GT-1 cells was detergent - and mechanical - disruption resistant, a feature of aggresomes. Similar results were observed in GT-1 cells treated with lactacystin, a prototypical proteasome inhibitor, which is structurally dissimilar to EPI indicating a pharmacologic effect. In conclusion, EPI causes cytoskeletal reorganization and accumulation of diverse ubiquitinated and non-ubiquitinated proteins in the perinuclear region and potentially overloads the endoplasmic reticulum-dependent quality control mechanism. These processes acting alone, or in combination, are hypothesized to affect axonal transport or other aspects of cellular homeostasis and thus, represent events potentially relevant to the development of peripheral neuropathy associated with administration of proteasome inhibitors in nonclinical studies.

Toxicological protein biomarker analysis--an investigative one-week single dose intravenous infusion toxicity and toxicokinetic study in cynomolgus monkeys using an antibody-cytotoxic conjugate against ovarian cancer.

INTRODUCTION: Antibody-cytotoxic conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify toxicological markers in serum that correlate with MLN8866 (an antibody-cytotoxic conjugate) exposure and related pathological events in monkeys. MATERIALS AND METHODS: Cynomolgus monkeys were treated once with 5, 15, or 30 mg/kg MLN8866 via a 20 min intravenous infusion. MLN8866 exposure (Cmax and AUCO-4 day) was determined by quantifying MLN8866 levels in serum. RESULTS: The increase in MLN8866 exposure was approximately dose proportional. Two acute phase proteins in serum (serum amyloid A and haptoglobin) were correlated with MLN8866 exposure and toxicological outcomes (e.g., erythropoiesis and leucopoiesis).

Biomarkers to monitor drug-induced phospholipidosis.

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) was identified as a promising phospholipidosis (PL) biomarker in rats treated with either amiodarone, gentamicin, or azithromycin. Sprague-Dawley rats received either amiodarone (150 mg/kg), gentamicin (100 mg/kg) or azithromycin (30 mg/kg) once daily for ten consecutive days. Histopathological examination of tissues by transmission electron microscopy (TEM) indicated different degrees of accumulation of phospholipidosis in liver, lung, mesenteric lymph node, and kidney of drug-treated rats but not controls. Liquid chromatography coupled to mass spectrometry (LC/MS) was used to identify levels of endogenous biochemical profiles in rat urine. Urinary levels of di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) correlated with induction of phospholipidosis for amiodarone, gentamicin and azithromycin. Rats treated with gentamicin also had increased urinary levels of several phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) species.

Drug safety evaluation through biomarker analysis--a toxicity study in the cynomolgus monkey using an antibody-cytotoxic conjugate against ovarian cancer.

Antibody-cytotoxin conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify serum biomarkers that correlate with MLN8866 (an Antibody-Cytotoxic Conjugate, mAb8866-CT) pathological events in monkeys and to predict the maximal tolerated dose (MTD) level using biomarkers. Cynomolgus monkeys were administered a single dose MLN8666 (5, 15 or 30 mg/kg) by intravenous infusion and evaluated over a 7-day period. Exposure levels were determined by quantifying MLN8866 levels (Cmax and AUC(0-96 h)) in serum. The increase in MLN8866 Cmax and AUC(0-96 h) was approximately dose proportional. Two biomarkers in serum (m/z 316 and m/z 368) were identified to be correlated with MLN8866 toxicological outcomes. The predicted MTD, 11.4 mg/kg, was within the MTD range set by pathology results (5-15 mg/kg). Administration of MLN8866 at 15 mg/kg and 30 mg/kg dose levels resulted in changes in hematology parameters associated with impaired hematopoiesis and bone marrow toxicity. The projected MLN8866 MTD exposure level was integrated with toxicokinetic analysis and showed Cmax=236 microg/mL and AUC(0-96 h)=7246 h mg/mL. The safety of three different MLN8866 dosing regimens with three dosing schedules was explored with pharmacokinetic modeling.

Differential display in rat livers treated for 13 weeks with phenobarbital implicates a role for metabolic and oxidative stress in nongenotoxic carcinogenicity.

Hepatic enzyme inducers such as phenobarbital are often nongenotoxic rodent hepatocarcinogens. Currently, nongenotoxic hepatocarcinogens can only be definitively identified through costly and extensive long-term, repeat-dose studies (e.g., 2-year rodent carcinogenicity assays). Although liver tumors caused by these compounds are often not found to be relevant to human health, the mechanism(s) by which they cause carcinogenesis are not well understood. Toxicogenomic technologies represent a new approach to understanding the molecular bases of toxicological liabilities such asnongenotoxic carcinogenicity early in the drug discovery/development process. Microarrays have been used to identify mechanistic molecular markers of nongenotoxic rodent hepatocarcinogenesis in short-term, repeat-dose preclinical safety studies. However, the initial "noise" of early adaptive changes may confound mechanistic interpretation of transcription profiling data from short-term studies, and the molecular processes triggered by treatment with a xenobiotic agent are likely to change over the course of long-term treatment. Here, we describe the use of a differential display technology to understand the molecular mechanisms related to 13 weeks of dosing with the prototype rodent nongenotoxic hepatocarcinogen, phenobarbital. These findings implicate a continuing role for oxidative stress in nongenotoxic carcinogenicity.An Excel data file containing raw data is available in full at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. Click on the issue link for 33(1), then select this article. A download option appears at the bottom of this abstract. The file contains raw data for all gene changes detected by AFLP, including novel genes and genes of unknown function; sequences of detected genes; and animal body and liver weight ratios. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Application of genetically altered models as replacement for the lifetime mouse bioassay in pharmaceutical development.

The international pharmaceutical regulatory academic and industrial toxicology communities are collaborating to improve the efficiency and effectiveness of cancer hazard identification based on dramatic improvements in our understanding of the cancer process. Guidelines emanating from the International Conference on Harmonization provide for use of in vivo alternatives. Standard practices utilizing lifetime rat and mouse studies are recognized as seriously flawed with over 80% false positive rates. Furthermore, tobacco, the most important human carcinogen commercialized by industry, is negative in these traditional lifetime studies. The lifetime mouse bioassay is generally recognized in pharmaceutical development as not adding value in safety assessment. An international consortium under the aegis of ILSI has recently completed an evaluation of alternative mouse cancer models. Transgenic models are less expensive, use fewer animals and take less time than traditional lifetime bioassays. These alternative models have now been sufficiently evaluated to be considered useful in the safety assessment plan for pharmaceuticals in development. Specifically for example, the rasH2 appears useful in detecting nongenotoxic as well as genotoxic rodent tumorigens with improved concordance with human response. The p53+/- heterozygous mouse apparently identifies hormonal carcinogenic mechanisms, immunosuppressive carcinogens, and genotoxic carcinogens. The TG:AC predicts for rodent tumorigens applied topically. Recent experiences at FDA, CPMP, and MHW indicate that with good planning and agency interactions, regulatory acceptability can be anticipated.

The Tg rasH2 mouse in cancer hazard identification.

The Tg rasH2 transgenic mouse has been developed as an altemative to the lifetime mouse bioassay to predict the carcinogenic potential of chemicals. Unlike the p53+/- mouse, the Tg rasH2 mouse is sensitive to both genotoxic and nongenotoxic carcinogens. The Tg rasH2 mouse, officially designated CB6F1-TgN (RasH2), contains multiple copies of the human c-Ha-ras oncogene and promoter within its genome. These mice develop spontaneous andchemically inducedneoplasms earlierin life and in greaternumbersthan wild-type mice, reflectingtheirenhanced sensitivity to neoplasia. The most common spontaneous neoplasms in control Tg rasH2 mice 8 to 9 months of age are lung adenomas and carcinomas (7.4% incidence), splenic hemangiomas and hemangiosarcomas (5.4%), forestomach squamous cell papillomas and carcinomas (2.4%), and skin neoplasms (1.2%). Simulations have demonstrated that 20 to 25 mice/sex/treatment group are required to provide the assay with adequate statistical power. Four of 6 known or suspected human carcinogens tested in Tg rasH2 mice were positive in this assay. For 19 nonmutagenic agents testing positive in conventional rodent bioassays, 7 chemicals were positive, 10 chemicals were negative, and 2 were equivocal. None of the 10 nonmutagenic rodent carcinogens that were negative in the Tg rasH2 mouse model are considered to be human carcinogens. All nonmutagenic chemicals that were negative in the conventional rodent bioassays were also negative in the Tg rasH2 model. Results for 15 of 18 mutagenic chemicals tested in Tg rasH2 mice agreed with the results of conventional rodent bioassays, and 3 results were equivocal. The Tg rasH2 mouse model appears to predict known or suspected human carcinogens as well as the traditional mouse bioassay, but with fewer positive results for nongenotoxic compounds that are not considered human carcinogens. The Tg rasH2 mouse model is the most thoroughly tested in vivo altemative to the lifetime mouse bioassay for nongenotoxic compounds administered by oral or parenteral routes. The U.S. FDA Carcinogenicity Assessment Committee has determined that the Tg rasH2 model has been adequately evaluated for consideration for carcinogenicity testing of pharmaceutical candidates and its use could contribute to the weight of evidence for carcinogenicity assessment. The FDA will consider proposals to replace lifetime mouse carcinogenicity studies with 6-month Tg rasH2 mouse studies to support pharmaceutical registration on a case-by-case basis.

Acute molecular markers of rodent hepatic carcinogenesis identified by transcription profiling.

Currently, the only way to identify nongenotoxic hepatocarcinogens is through long-term repeat dose studies such as the 2 year rodent carcinogenicity assay. Such assays are both time consuming and expensive and require large amounts of active pharmaceutical or chemical ingredients. Thus, the results of the 2 year assay are not known until very late in the discovery and development process for new pharmaceutical entities. Although in many cases nongenotoxic carcinogenicity in rodents is considered to be irrelevant for humans, a positive finding in a 2 year carcinogenicity assay may increase the number of studies to demonstrate the lack of relevance to humans, delay final submission and subsequent registration of a product, and may result in a "black box" carcinogenicity warning on the label. To develop early identifiers of carcinogenicity, we applied transcription profiling using several prototype rodent genotoxic and nongenotoxic carcinogens, as well as two noncarcinogenic hepatotoxicants, in a 5 day repeat dose in vivo toxicology study. Fluorescent-labeled probes generated from liver mRNA prepared from male Sprague-Dawley rats treated with one of three dose levels of bemitradine, clofibrate, doxylamine, methapyrilene, phenobarbital, tamoxifen, 2-acetylaminofluorene, 4-acetylaminofluorene, or isoniazid were hybridized against rat cDNA microarrays. Correlation of the resulting data with an estimated carcinogenic potential of each compound and dose level identified several candidate molecular markers of rodent nongenotoxic carcinogenicity, including transforming growth factor-beta stimulated clone 22 and NAD(P)H cytochrome P450 oxidoreductase.