Developing in vitro assays to quantitatively evaluate the interactions of dressings with wounds.

Evaluating interactions between dressing and wound is important for understanding wound management. This study quantitatively compared four polyurethane foam-based wound dressings for their absorption profile, cell penetration, and adherence using two novel in vitro assays. The dressing with uniform pore sizes varying from 25~75 µm showed the highest absorption of both culture media and serum. The same dressing showed a 1.2- to 3.6-fold lower cell adherence (3 hours) than the other dressings, and ~20-fold lower cell penetration (5 days) than dressings with pore sizes varying from 55 to 343 µm. Additionally, cell and dressing interactions using a 3-dimensional wound healing assay showed that the dressings with the smallest pore size of 25~75 µm maintained the highest cell viability (76.3%) and promoted cell migration into the wound site. This data suggest that polyurethane foam dressing with smaller and evenly distributed pores promotes wound healing with less cellular adhesion and penetration.

Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model.

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants.

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds-chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates.

Hepatic Bioactivation of Skin-Sensitizing Drugs to Immunogenic Reactive Metabolites.

The clinical use of some drugs, such as carbamazepine, phenytoin, and allopurinol, is often associated with adverse cutaneous reactions. The bioactivation of drugs into immunologically reactive metabolites by the liver is postulated to be the first step in initiating a downstream cascade of pathological immune responses. Current mechanistic understanding and the ability to predict such adverse drug cutaneous responses have been partly limited by the lack of appropriate cutaneous drug bioactivation experimental models. Although in vitro human liver models have been extensively investigated for predicting hepatotoxicity and drug-drug interactions, their ability to model the generation of antigenic reactive drug metabolites that are capable of eliciting immunological reactions is not well understood. Here, we employed a human progenitor cell (HepaRG)-derived hepatocyte model and established highly sensitive liquid chromatography-mass spectrometry analytical assays to generate and quantify different reactive metabolite species of three paradigm skin sensitizers, namely, carbamazepine, phenytoin, and allopurinol. We found that the generation of reactive drug metabolites by the HepaRG-hepatocytes was sensitive to the medium composition. In addition, a functional assay based on the activation of U937 myeloid cells into the antigen-presenting cell (APC) phenotype was established to evaluate the immunogenicity potential of the reactive drug metabolites produced by HepaRG-derived hepatocytes. We showed that the reactive drug metabolites of known skin sensitizers could significantly upregulate IL8, IL1ß, and CD86 expressions in U937 cells compared to the metabolites from a nonskin sensitizer (i.e., acetaminophen). Thus, the extent of APC activation by HepaRG-hepatocytes conditioned medium containing reactive drug metabolites can potentially be used to predict their skin sensitization potential.

Hepatic spheroids used as an in vitro model to study malaria relapse.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

TGFß1-mediated suppression of cytochrome P450(CYP) induction responses in rat hepatocyte-fibroblast co-cultures.

Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFß1 production in the co-cultures. Addition of 100 pg/ml TGFß1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFß1 as one of the important factors impairing drug induced CYP induction in co-cultures and suggests that caution needs to be exercised in the use of hepatocyte-fibroblast co-cultures for CYP induction studies.

Application of genomics in preclinical drug safety evaluation.

Understanding the response of biological systems to xenobiotics is fundamental to the evaluation of drug safety. Toxicologists have traditionally gathered pathological, morphological, chemical and biochemical information from in vivo studies of preclinical species in order to assess drug safety and to determine how new drugs can be safely administered to the human patient population. In recent years the emerging "-omics" technologies have been developed and integrated into preclinical studies in order to better assess drug safety by gaining information on the cellular and molecular events underlying adverse drug reactions. Genomics approaches in particular have become readily available and are being applied in several stages of drug development. The burgeoning literature on what has become known as "toxicogenomics" has for the most part highlighted successful applications of gene expression profiling in predictive toxicology, enabling decisions to be made on the developability of a compound early in the drug development process. It is also becoming apparent that toxicogenomic approaches are good starting points to develop experiments designed to gain a mechanistic insight into drug toxicities within and across species. Gene expression arrays permit the measurement of responses of essentially all the genes in the entire genome to be monitored, and knowledge of the function of the genes affected can identify the potential mechanisms to then be confirmed using conventional biochemical, toxicological and pathological approaches. As toxicologists put these technologies into practice they build up a knowledge base to better characterize toxicities at the molecular level and to make the search for much needed, novel biomarkers of toxicity more achievable.

The evolution of gene expression studies in drug safety assessment.

Since the identification in the 1950s of deoxyribonucleic acid as the building block of life, the impact of molecular biology has been far-reaching. Understanding the processes of how DNA is replicated, transcribed into RNA and then translated into protein products has not only provided a fundamental knowledge of life but has also spawned a plethora of applications. Molecular biology has been high profile and widespread in research into the biology of disease and in drug discovery. It has additionally found application in understanding the adverse effects, or toxicity, of candidate drugs and how they interfere with biochemical and biological processes. In recent times the biggest impact of molecular biology in toxicology has been through the study of differential gene expression, largely as a result of the advent of genomics. This review seeks to describe how toxicogenomics strategies have been implemented and integrated into nonclinical studies of drug safety.

ABC gene-ranking for prediction of drug-induced cholestasis in rats.

As legacy toxicogenomics databases have become available, improved data mining approaches are now key to extracting and visualizing subtle relationships between toxicants and gene expression. In the present study, a novel "aggregating bundles of clusters" (ABC) procedure was applied to separate cholestatic from non-cholestatic drugs and model toxicants in the Johnson & Johnson (Janssen) rat liver toxicogenomics database [3]. Drug-induced cholestasis is an important issue, particularly when a new compound enters the market with this liability, with standard preclinical models often mispredicting this toxicity. Three well-characterized cholestasis-responsive genes (Cyp7a1, Mrp3 and Bsep) were chosen from a previous in-house Janssen gene expression signature; these three genes show differing, non-redundant responses across the 90+ paradigm compounds in our database. Using the ABC procedure, extraneous contributions were minimized in comparisons of compound gene responses. All genes were assigned weights proportional to their correlations with Cyp7a1, Mrp3 and Bsep, and a resampling technique was used to derive a stable measure of compound similarity. The compounds that were known to be associated with rat cholestasis generally had small values of this measure relative to each other but also had large values of this measure relative to non-cholestatic compounds. Visualization of the data with the ABC-derived signature showed a very tight, essentially identically behaving cluster of robust human cholestatic drugs and experimental cholestatic toxicants (ethinyl estradiol, LPS, ANIT and methylene dianiline, disulfiram, naltrexone, methapyrilene, phenacetin, alpha-methyl dopa, flutamide, the NSAIDs--indomethacin, flurbiprofen, diclofenac, flufenamic acid, sulindac, and nimesulide, butylated hydroxytoluene, piperonyl butoxide, and bromobenzene), some slightly less active compounds (3'-acetamidofluorene, amsacrine, hydralazine, tannic acid), some drugs that behaved very differently, and were distinct from both non-cholestatic and cholestatic drugs (ketoconazole, dipyridamole, cyproheptadine and aniline), and many postulated human cholestatic drugs that in rat showed no evidence of cholestasis (chlorpromazine, erythromycin, niacin, captopril, dapsone, rifampicin, glibenclamide, simvastatin, furosemide, tamoxifen, and sulfamethoxazole). Most of these latter drugs were noted previously by other groups as showing cholestasis only in humans. The results of this work suggest that the ABC procedure and similar statistical approaches can be instrumental in combining data to compare toxicants across toxicogenomics databases, extract similarities among responses and reduce unexplained data varation.

Drug-induced oxidative stress in rat liver from a toxicogenomics perspective.

Macrophage activators (MA), peroxisome proliferators (PP), and oxidative stressors/reactive metabolites (OS/RM) all produce oxidative stress and hepatotoxicity in rats. However, these three classes of hepatotoxicants give three distinct gene transcriptional profiles on cDNA microarrays, an indication that rat hepatocytes respond/adapt quite differently to these three classes of oxidative stressors. The differential gene responses largely reflect differential activation of transcription factors: MA activate Stat-3 and NFkB, PP activate PPARa, and OS/RM activate Nrf2. We have used gene signature profiles for each of these three classes of hepatotoxicants to categorize over 100 paradigm (and 50+ in-house proprietary) compounds as to their oxidative stress potential in rat liver. In addition to a role for microarrays in predictive toxicology, analyses of small subsets of these signature profiles, genes within a specific pathway, or even single genes often provide important insights into possible mechanisms involved in the toxicities of these compounds.

Release of (and lessons learned from mining) a pioneering large toxicogenomics database.

AIM: We release the Janssen Toxicogenomics database. This rat liver gene-expression database was generated using Codelink microarrays, and has been used over the past years within Janssen to derive signatures for multiple end points and to classify proprietary compounds. MATERIALS & METHODS: The release consists of gene-expression responses to 124 compounds, selected to give a broad coverage of liver-active compounds. A selection of the compounds were also analyzed on Affymetrix microarrays. RESULTS: The release includes results of an in-house reannotation pipeline to Entrez gene annotations, to classify probes into different confidence classes. High confidence unambiguously annotated probes were used to create gene-level data which served as starting point for cross-platform comparisons. Connectivity map-based similarity methods show excellent agreement between Codelink and Affymetrix runs of the same samples. We also compared our dataset with the Japanese Toxicogenomics Project and observed reasonable agreement, especially for compounds with stronger gene signatures. We describe an R-package containing the gene-level data and show how it can be used for expression-based similarity searches. CONCLUSION: Comparing the same biological samples run on the Affymetrix and the Codelink platform, good correspondence is observed using connectivity mapping approaches. As expected, this correspondence is smaller when the data are compared with an independent dataset such as TG-GATE. We hope that this collection of gene-expression profiles will be incorporated in toxicogenomics pipelines of users.

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes.

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.

Inverse gene expression patterns for macrophage activating hepatotoxicants and peroxisome proliferators in rat liver.

Macrophage activation contributes to adverse effects produced by a number of hepatotoxic compounds. Transcriptional profiles elicited by two macrophage activators, LPS and zymosan A, were compared to those produced by 100 paradigm compounds (mostly hepatotoxicants) using cDNA microarrays. Several hepatotoxicants previously reported to activate liver macrophages produced transcriptional responses similar to LPS and zymosan, and these were used to construct a gene signature profile for macrophage activators in the liver. Measurement of cytokine mRNAs in the same liver samples by RT-PCR independently confirmed that these compounds are associated with macrophage activation. In addition to expected effects on acute phase proteins and metabolic pathways that are regulated by LPS and inflammation, a strong induction was observed for many endoplasmic reticulum-associated stress/chaperone proteins. Additionally, many genes in our macrophage activator signature profile were well-characterized PPARalpha-induced genes which were repressed by macrophage activators. A shared gene signature profile for peroxisome proliferators was determined using a training set of clofibrate, WY 14643, diethylhexylphthalate, diisononylphthalate, perfluorodecanoic acid, perfluoroheptanoic acid, and perfluorooctanoic acid. The signature profile included macrophage activator-induced genes that were repressed by peroxisome proliferators. NSAIDs comprised an interesting pharmacological class in that some compounds, notably diflunisal, co-clustered with peroxisome proliferators whereas several others co-clustered with macrophage activators, possibly due to endotoxin exposure secondary to their adverse effects on the gastrointestinal system. While much of these data confirmed findings from the literature, the transcriptional patterns detected using this toxicogenomics approach showed relationships between genes and biological pathways requiring complex analysis to be discerned.

A gene expression signature for oxidant stress/reactive metabolites in rat liver.

Formation of free radicals and other reactive molecules is responsible for the adverse effects produced by a number of hepatotoxic compounds. cDNA microarray technology was used to compare transcriptional profiles elicited by training and testing sets of 15 oxidant stressors/reactive metabolite treatments to those produced by approximately 85 other paradigm compounds (mostly hepatotoxicants) to determine a shared signature profile for oxidant stress-associated hepatotoxicity. Initially, 100 genes were chosen that responded significantly different to oxidant stressors/reactive metabolites (OS/RM) compared to other samples in the database, then a 25-gene subset was selected by multivariate analysis. Many of the selected genes (e.g., aflatoxin aldehyde reductase, diaphorase, epoxide hydrolase, heme oxgenase and several glutathione transferases) are well-characterized oxidant stress/Nrf-2-responsive genes. Less than 10 other compounds co-cluster with our training and testing set compounds and these are known to generate OS/RMs as part of their mechanisms of toxicity. Using OS/RM signature gene sets, compounds previously associated with macrophage activation formed a distinct cluster separate from OS/RM and other compounds. A 69-gene set was chosen to maximally separate compounds in control, macrophage activator, peroxisome proliferator and OS/RM classes. The ease with which these 'oxidative stressor' classes can be separated indicates a role for microarray technology in early prediction and classification of hepatotoxicants. The ability to rapidly screen the oxidant stress potential of compounds may aid in avoidance of some idiosyncratic drug reactions as well as overtly toxic compounds.

Tethered spheroids as an in vitro hepatocyte model for drug safety screening.

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 µm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.

Purpose-driven biomaterials research in liver-tissue engineering.

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.

A robust high-throughput sandwich cell-based drug screening platform.

Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatocytes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.

Galactosylated cellulosic sponge for multi-well drug safety testing.

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 µm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell-cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to ß-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.

Diabetogenic effect of a series of tricyclic delta opioid agonists structurally related to cyproheptadine.

The unexpected observation of a hyperglycemic effect of some tricycle-based delta opioid receptor (DOR) agonists led to a series of studies to better understand the finding. Single administration of two novel tricyclic DOR agonists dose dependently elevated rat plasma glucose levels; 4-week toxicology studies confirmed the hyperglycemic finding and further revealed pancreatic ß-cell hypertrophy, including vacuole formation, as well as bone dysplasia and Harderian gland degeneration with regeneration. Similar diabetogenic effects were observed in dog. A review of the literature on the antiserotonergic and antihistaminergic drug cyproheptadine (CPH) and its metabolites revealed shared structural features as well as similar hyperglycemic effects to the present series of DOR agonists. To further evaluate these effects, we established an assay measuring insulin levels in the rat pancreatic ß-cell-derived RINm5F cell line, extensively used to study CPH and its metabolites. Like CPH, the initial DOR agonists studied reduced RINm5F cell insulin levels in a concentration-dependent manner. Importantly, compound DOR potency did not correlate with the insulin-reducing potency. Furthermore, the RINm5F cell insulin results correlated with the diabetogenic effect of the compounds in a 5-day mouse study. The RINm5F cell insulin assay enabled the identification of aryl-aryl-amine DOR agonists that lacked an insulin-reducing effect and did not elevate blood glucose in repeated dosing studies conducted over a suprapharmacologic dose range. Thus, not only did the RINm5F cell assay open a path for the further discovery of DOR agonists lacking diabetogenic potential but also it established a reliable, economical, and high-throughput screen for such potential, regardless of chemotype or target pharmacology. The present findings also suggest a mechanistic link between the toxicity observed here and that underlying Wolcott-Rallison Syndrome.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.