November 15, 2015 (Vol. 35, No. 20)

Early-Drug Screening Is Due For Review

Before a new prescription medicine gains marketing approval, it typically goes through a development process that lasts at least a decade. Moreover, according to the Tufts Center for the Study of Drug Development, the costs can exceed $2.5 billion. To raise the odds that time and money will be lavished on only the most promising drug candidates, developers need to improve early-stage drug screening. It is here that ADME/Tox activities play a key role.

GEN recently interviewed a number of experts in the field to gain some insights on some of the best current practices.

GEN: What are some of the best methods for accurately detecting genotoxic carcinogens?

Dr. Taylor: The ability to confidently detect, identify, and quantify genotoxic compounds is critically important for the safety of pharmaceuticals. These compounds include a range of volatile and nonvolatile compounds and trace metals. Mass spectrometry is the ideal technique for the high-sensitivity detection and measurement of such compounds. Depending on the type of compounds being analyzed, the techniques of GC/MS, LC/MS, and ICP/MS together with software processing tools provide a comprehensive set of analytical tools for such qualitative and quantitative analysis.

Dr. Bulera: There is no single assay that can definitively detect genotoxic carcinogens. Instead, it is best to employ a tiered approach using in vitro and in vivo assays.

The BlueScreen assay using TK6 cells and the flow cytometry micronucleus with CREST staining in human lymphocytes are two highly accurate in vitro assays. In addition to rodent bioassays using mice/rats/transgenic mice, the Pig-a-Gene Mutation Assay and the Comet/Micronucleus assay are in vivo assays used to detect genotoxic carcinogens. Furthermore, assays can be designed to identify compounds that directly interact with DNA or cause mutations by interfering with proteins involved in cell replication.

Dr. Dilworth: Regulations for genotoxicity testing differ between the various industries. For instance, in the cosmetics industry, where animal testing is banned, in vitro and QSAR-based testing are commonly used in combination to determine potential genotoxicity through a weight-of-evidence approach. Incorporating metabolizing systems and the use of 3D reconstructed skin models are now of interest in improving the prediction of genotoxic carcinogens for cosmetic products.

Within the pharmaceutical industry, both in vitro and in vivo testing are required to ensure safety of new drugs prior to human studies; however, high-throughput testing for particular mechanisms or signaling pathways are often employed at an early stage to identify potential liability. Phosphorylation of biomarkers such as H2AX histone using high-content screening, or reporter systems to monitor gene regulation in response to exposure to genotoxic substances are two screening techniques often used in drug discovery.

Dr. Cali: Methods for detecting genotoxins are prescribed by the FDA in accordance with a 2012 guidance document, which is  available on the FDA’s website. Because of the potential for false positives and negatives from any one test, a battery of assays is recommended with in vitro and in vivo tests.

A bacterial reverse mutagenesis assay (Ames test) is used in combination with mammalian cell tests including the metaphase chromosome aberration, micronucleus, and mouse lymphoma thymidine kinase mutation assays. A higher throughput method called GreenScreen (from Gentronix), which is gaining popularity, uses a reporter-based system that exploits a DNA-damage-sensitive promoter.

Mr. Hickman: There are many assays that will detect genotoxicity, but the best approach is to combine them into a battery to investigate the mode of action (MOA) with the right mechanism. An Ames assay will detect most carcinogens; however, if the assay is positive, the possibility for cytogenicity and other mutagens remains open.

The next step is to explore clastogenicity and aneugenicity with either an in vitro chromosome aberration assay or preferably an in vitro micronucleus assay. The best assays that will explore mutagenicity at the in vitro level are either a mouse lymphoma or HPRT assay.

If any of the in vitro assays are positive, you have your result, but if negative, you can follow on with in vivo assays—the in vivo chromosome aberration; in vivo micronucleus for a cytogenetic MOA; or a comet assay for DNA damage. At the in vivo level, it is recommended to perform a transgenic rodent mutation assay (Big Blue) with either somatic or germ cells to look for mutagens.

Dr. Thyagarajan: Most genotoxic substances are found to be carcinogenic, and common tests for genotoxic carcinogens include the Ames test, the micronucleus assay, and the comet assay. These assays are performed in bacteria or mammalian cell lines.

However, genotoxicity may be caused by a reactive metabolic intermediate of a drug candidate and such metabolites may be missed in tests using bacteria or mammalian cell lines. To overcome this limitation, animal models can be used. Some exemplary models include transgenic models such as the Big Blue® Mouse, p53+/−, and the rasH2 mouse (for both genotoxic and nongenotoxic compounds).

Dr. Witek: The erythrocyte-based micronucleus assay can be used in vitro to detect carcinogenic chemicals and is required by safety organizations for registration of new industrial and pharmaceutical compounds. Simple antibody-based systems to detect mitotic arrest or phosphorylated H2AX that indicates double-strand breaks in DNA can also be used to screen for carcinogenic compounds.

A second test, which typically uses liver since it is the major organ of drug metabolism, is required for validation of the first assay. The current state-of-the-art techniques that address this need are the in vivo comet, transgenic rodent mutation and repeated-dose liver micronucleus assays.

GEN: Which techniques in drug development should be considered to determine early hepatotoxicity?

Dr. Taylor: The characterization of active/reactive metabolites is a critical factor in drug discovery and development. The technique of choice for high-sensitivity detection and identification of these compounds is LC/MS, in which LC provides effective chromatographic separation of analytes prior to high-resolution mass spectrometry.

The use of 2D LC provides an additional dimension of separation prior to MS analysis for such metabolites in complex matrices, which are not effectively resolved by single LC separation. This includes chiral compounds and their metabolites.

Dr. Bulera: While histopathology and evaluation of clinical pathology parameters should remain the gold standard for determination of hepatotoxicity in drug development, emphasis should be placed upon techniques that allow earlier detection of hepatotoxicity. Techniques in the fields of genomics, proteomics, and metabolomics—such as mass spectrometry and protein and gene-array analyses—should be considered and combined with other techniques that are utilized in drug development, such as immunohistochemistry and ELISA.

The use of these techniques may allow for the identification of biomarkers that are predictive of hepatotoxicity and that can be used to detect hepatotoxicity prior to the onset of irreversible injury.

Dr. Dilworth: Hepatotoxicity is notoriously difficult to predict, mainly because preclinical animal testing often fails to detect this type of toxicity. Furthermore, there are numerous mechanisms by which hepatotoxicity manifests itself, the effects of which may be time-dependent.

New in vitro approaches, many of which utilize high-content screening, evaluate panels of different mechanisms within varying cell types and over several time points. 3D microtissue models, which are more physiologically relevant and can be dosed over extended time periods, are now showing promise, and are expected to take a greater role in routine screening for hepatotoxicity. Incorporating the data from these in vitro assays with expected exposure will assist in increasing the predictive capability and will facilitate extrapolation to the in vivo setting.

Dr. Cali: Cultured primary hepatocytes are the gold standard for predicting in vivo hepatotoxicity of drugs and other xenobiotics. Advances in hepatocyte culture technology, which have enabled their routine use, coupled with advances in optical chemistries for cell-based assays now enable rapid in vitro hepatotoxicity profiling.

For example, Promega exploits bioluminescence and fluorescence to measure cell viability, cytotoxicity, oxidative stress (via the detection of reactive oxygen species and glutathione), and apoptosis. Many of the assays can be mixed and matched in multiplex formats to support an internally controlled approach that greatly reduces the amount of costly hepatocytes required for analysis.

Mr. Mitchell: Hepatocytes represent the major site in the human body for drug metabolism and elimination. As such, drug-induced liver injury remains a significant concern during drug development. Early testing of compounds for toxicity, CYP induction and inhibition, as well as transporter interactions in a holistic, well-characterized human hepatic model, can aid in the identification of potential drug liabilities.

The HepaRG cell line has been demonstrated to express major xenobiotic sensors, uptake and efflux transporters, phase I and II metabolizing enzymes, and key transcription factors, making this cell line ideal for early hepatotoxicity assessment during drug development.

Dr. Thyagarajan: Evaluating hepatotoxicity involves measuring cytotoxic markers in human hepatocytes (or cell lines such as HepG2) in culture treated with a compound of interest. Recently, human hepatocyte models derived from induced pluripotent stem cells (iPSCs) have also been used. Additionally, several in silico methods such as DILI-sim are used for predicting hepatotoxicity.

However, hepatotoxicity can be affected by drug metabolism and disposition in physiological systems. To address this, several models have been developed. These include transgenic models where key components of drug metabolism pathways have been humanized (e.g., humanized PXR/CAR) or animal liver cells have been replaced with human analogues (e.g., FRG and TK-NOG).

Dr. Witek: High-content screening with quantitative cell-based imaging is an in vitro technique providing rapid phenotypic identification of hepatotoxic compounds. The method allows for fast and accurate assessment of cytotoxicity and genotoxicity by utilizing various fluorescent probes that enable users to look at cell morphologies, viability, proliferation, DNA damage, mitotic index, mitochondrial health, and other cellular functions.

As the hepatotoxicity of compounds cannot be assessed by a single in vitro assay, and current methods are not fully predictive, the ability of high-content screening to simultaneously analyze multiple parameters offers a powerful tool for early safety evaluation and selection of drug candidates.

GEN: What is one particularly promising technology that is emerging in the ADME/Tox field?

Dr. Taylor: The ability to rapidly assess potential drug compounds in ADME/Tox has driven the need for high-throughput screening and analysis. Use of the automated RapidFire sample preparation system together with MS has provided a highly effective solution for early compound screening and assessment.

Dr. Bulera: Two emerging technologies are the development of 3D tissue culture models and the organ-on-a-chip technologies. These technologies have the potential to be better representations of organ/tissue physiologically, which will provide greater translational relevance to human clinical outcomes than many of the current in vitro cell culture models.

Dr. Dilworth: Stem cells are already showing promise in the field of cardiotoxicity where the cells have the potential for beating behavior. Co-culture models that combine iPSC-derived cells with other cell types in 3D tissue models are also showing value. Although iPSC-derived hepatocytes still require further development, their value in drug metabolism and hepatotoxicity studies is expected to be recognized in the future as better differentiation techniques become available.

Dr. Cali: Promega recently released a complete cell-based bioluminescent assay system for measuring xenobiotic induction of the major cytochrome P450s involved in induction-based adverse drug interactions. The cell-based P450-Glo™ assays for CYP1A2, -2B6, and -3A4 enable rapid microplate hepatocyte analysis that preserves cells for downstream analysis.

Mr. Mitchell: Traditional ADME/Tox testing in cell lines rely upon chemical modulators to assess drug safety and metabolism liabilities. Recent advances in genetic engineering technologies, such as zinc finger nuclease and CRISPR systems, have enabled development of novel targeted knock-out and knock-in cell lines for more predictive, early ADME/Tox testing.

Dr. Thyagarajan: The “humanization” of drug metabolism pathways is one promising technology. This includes genetic replacement of mouse drug metabolism genes with human ones or tissue humanization in which liver/immune cells in mice are replaced with human counterparts to create physiologic systems that can recapitulate human-like drug metabolism and disposition.

Dr. Witek: 3D organotypic spheroids, organoids, and microtissues are becoming the next-generation in vitro culture systems for modeling liver function. Those systems are able to incorporate co-cultures of liver parenchymal and nonparenchymal cells to model complex cellular behaviors and provide metabolic functions that more closely mimic those found in vivo.

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