Genotoxicity testing also needs to be improved. Kathleen Böhme, Ph.D., biochemist at Merck Serono, described her Ph.D. work on an in vitro system for the toxicological evaluation of genotoxic compounds. Assessing genotoxicity is an important aspect of both drug development and chemical safety testing (particularly under the REACH program). However, the current in vitro test battery is limited by low specificity and a high rate of false positives, while the two-year rodent assay is expensive, time-consuming, and also beset by false positives.
“There is a need for new validated tools with higher specificity and reduced cost,” Dr. Böhme said. Accordingly, she has been looking at omics and mechanism-based approaches to in vitro genotoxicity testing using HepG2 cells. “We are aiming to identify the gene-expression signature of compounds being tested.”
Using etoposide, actinomycin D, and MMS as test compounds, she found 66 genes that are possible markers of genotoxicity, with a range of functions including apoptosis, DNA damage, transporter activity, immune response, and development. In parallel, Dr. Böhme has used an assay for p53 activation (with Active Motif’s TransAM™) in which the test compounds showed significant p53 activation.
So far, she has concluded that most direct genotoxicants do show characteristic gene-expression signatures. The challenge now is that most compounds of concern are actually promutagens, and it needs to be established whether the HepG2 cells were capable of metabolizing them to reveal their genotoxic properties.
In fact, most CYPs are expressed only weakly in HepG2 cells compared to human hepatocytes; however, both CYP1A and CYP3A4 are inducible. Therefore, a metabolic-activating system may need to be added to the HepG2 cells to reveal some genotoxicants.
Although p53 activation may be a surrogate marker, gene-expression profiling with a range of genes is actually more promising for genotoxicity screening. Going forward, Dr. Böhme will test more compounds, in different classes, with the aim of building a predictive classification model for genotoxicity that will lead to better risk assessment. Currently, these approaches are at the research stage at Merck Serono, except for the CYP gene induction assay, which is used routinely.
Willem Schoonen, Ph.D., senior research scientist at MSD, part of the U.S. pharmaceutical company Merck, described his company’s approach to in vitro genotoxicity, carcinogenicity, and nongenotoxic carcinogenicity screening as deselection and/or ranking tools in lead optimization.
“Multiple measures of toxicity are needed to decrease attrition in preclinical research,” he said. These screens include in silico (DEREK, TopCat, MultiCASE, Mutalert), in vitro (mutagenicity, clastogenicity, nongenotoxicity carcinogenicity, cytotoxicity, nuclear receptor activation, Phase I and Phase II enzymes) and in vivo (biosafety testing, liver toxicogenomics, teratogenicity) testing.
Dr. Schoonen expanded on development work he has carried out on VitoTox, RadarScreen, and HepG2 genotoxicity assays. VitoTox is based on prediction of DNA damage in S. typhimurium with a luciferase readout, while RadarScreen measures DNA damage to yeast through the promoter of the RAD54 (recombinatorial repair) gene with a beta-galactosidase readout. The HepG2 assays are all luciferase-linked promoter assays with the advantage that HepG2 is a human cell line and can metabolize genotoxic compounds.
Dr. Schoonen described toxicogenomic data obtained with these systems, with a range of compounds (genotoxic, cholestatic, necrotic, and apoptotic reference compounds). A selected set of genes was upregulated in these assays including cystatin A, a possible tumor progression marker; XPC; RAD51C (involved in repair of double-stranded breaks); MDM2, which keeps tumor suppressor p53 inactive in the absence of DNA damage; CKDNIA (p21); and TP5313.
He also explained that VitoTox and RadarScreen have good predictive value. However, the four HepG2 assays had lower predictive value than VitoTox and RadarScreen when compared with in vitro Ames mutagenicity data and in vitro clastogenicity data obtained with CHO or lung cells. But, when compared with in vivo rat clastogenicity data the correlation was much more predictive, which was most likely due to overprediction of clastogenicity scores in the hamster cells lines, which contained a mutated p53 gene.
Pattern recognition is being used as an alternative tool to identify new correlations between these datasets. These comparisons show that HepG2 cells may become better predictors for human in vivo genotoxicity than the currently used gold standards—Ames and in vitro clastogenicity testing.
Only the potent genotoxicants gave true positives in all of the tests investigated, Dr. Schoonen said, while true negatives were easily identified and hierarchical clustering segregated compounds into different classes.
Genotoxicity is not the only reason why a compound can fail on patient safety. James Dykens, Ph.D., drug safety R&D at Pfizer, noted that there are more than 2.2 million adverse drug reactions (ADRs) each year in hospitalized patients in the U.S., more than 350,000 in nursing home residents, and the number of ADRs among ambulatory patients is unknown. These figures translate into at least 106,000 deaths per year, making ADRs the fourth leading cause of death. “Clearly the pharmaceutical industry has been missing some important aspects of drug toxicity,” he said.
Drug-induced mitochondrial toxicity is fast emerging as a new model for these idiosyncratic ADRs. Until recently, mitochondrial toxicity has been missed simply because the tools to detect it were not available.
"Mitochondria are complex organelles that can fail in many ways,” Dr. Dykens commented. “It should not be surprising that xenobiotics produced by pharma can inhibit electron transport.” Among the 44 drugs withdrawn from the market since 1960 and those that have received black-box warnings, there is evidence that several cause mitochondrial impairment, which may be indicated by elevated liver enzymes and lactic acidosis.
Many drugs with organ toxicity will have some kind of mitochondrial liability. Whether a drug’s mitochondrial toxicity will actually have deleterious consequences depends upon its potency.
Mitochondrial ADRs also tend to be idiosyncratic, depending upon the organ’s history and patient genetics. One problem is that young, healthy animals, which are less prone to mitochondrial toxicity, are used in preclinical screens. “We are looking at the wrong models for in vivo mitochondrial studies,” Dr. Dykens said.
Pfizer is, therefore, looking at some new assays for mitochondrial toxicity. One is based on measuring mitochondrial respiration in 96-well plates with the Luxcel oxygen probe, which allows detection of compounds that uncouple oxidative phosphorylation. This revealed how all the thiozolidinediones (some of which have black-box warnings for chronic heart failure) and some statins have this effect.
Another screen is the Seahorse Bioscience metabolic profiling technology that Pfizer has used to detect lactic acidosis with the biguanides. MitoSciences’ assay assesses the effects of compounds on isolated mitochondrial electron transport protein complexes I, IV, and V, which can identify their site of action. This shows that troglitazone interacts with just complex IV, while simvastatin inhibits all three.
Dr. Dykens noted that cell culture is usually carried out at high glucose concentration, which inhibits respiration (the so-called Crabtree effect) and does not, therefore, detect mitochondrial inhibition. “Instead, at Pfizer we have grown cells on galactose to show mitochondrial inhibition. Our goal is to do all these screens much earlier.”