In vitro toxicology screening in drug development
20 March 2018
Prior to IND approval drug candidates must go through a comprehensive battery of in vitro and in vivo toxicology testing to maximise safety in clinical trials. For ethical reasons and the costly nature of the in vivo animal tests the pharmaceutical industry relies more and more on in vitro methods for toxicity testing especially in drug discovery phase. To elucidate this topic further, we interviewed an expert on the subject, Dr Sanna-Mari Aatsinki.
At what stage of drug discovery and development project should you start screening for toxicity?
The current trend is to “fail early, fail cheap” to save resources. Screening for toxicity as early as possible may reveal inappropriate drug candidates that should not proceed in the drug development process. Modern HTS assays enable early toxicity screening at reasonable costs in hit-to-lead phase, along with other ADME screening assays.
What are the most common types of toxicity observed in drug development?
Hepatotoxicity and cardiotoxicity result in the most severe problems in drug development and are often the cause of drug withdrawals. In addition, possible mutagenesis caused by drug candidates, i.e. genotoxicity should also be among the first screens.
What kinds of assays are used for liver toxicity screening?
Liver toxicity caused by drugs, also called drug induced liver injury (DILI) can occur through several mechanisms. Direct cell toxicity can be studied by membrane integrity assay (LDH release), and cell viability by assessing cell metabolic activity using MTT assay or by measuring the ATP levels of the cells. Mitochondria can also play an essential role in DILI since they are key cell organelles in the hepatocytes. Mitochondrial toxicity can be studied in liver cells by replacing the cell’s energy source glucose with galactose in the incubation medium to augment toxicity. By using metabolically active hepatocytes in the screening assays, metabolism-dependent toxicity effect is also taken into account.
How do you screen for cardiotoxicity?
Severe cardiotoxic safety issues are mainly caused by the blockage of a potassium ion channel called hERG (human Ether-à-go-go-related gene) resulting in a prolongation of the QT interval. hERG blockage can be screened utilising HTS 384-plate fluorescence polarisation assay which is less expensive and quicker to perform than the traditional patch-clamp assay. However, development is ongoing for more comprehensive cardiotoxicity screening assays, to take into account also e.g. other ion channels that can cause cardiac toxicity.
Why is genotoxicity also among the first toxicity assays to be screened?
Genotoxic compounds can cause mutations in DNA which predisposes to cancer and reproductive problems. Therefore, these features are naturally under closer scrutiny also by the authorities. According to regulatory guidelines the standard test battery must include bacterial reverse mutation test (AMES) and a mammalian genotoxicity assay. AMES as a HTS 384-plate form and the in vitro micronucleus screening test (MNT) using ChoK1 cell line are excellent methods for testing several compounds, both assays being regulatory-compliant (OECD). AMES reveals whether the compound is causing direct mutations to the DNA. By testing different bacterial strains mechanistic information can be revealed (base pair substitutions, frameshifts etc.). AMES screening can be performed with two tester strains of Salmonella; TA98 and TA100 (mini AMES) but in total five strains are required by the authorities. MNT reveals if the compound causes abnormalities in chromosome distribution (aneugenity) or even chromosome breaks (clastogenity) during cell division. The assay is thus performed using actively dividing mammalian cells. The results may suggest the form of mutagenicity, i.e. if the compound is aneugen or clastogen which could be useful information for future development.
Are there any other tox screens to consider in drug discovery?
Based on the properties of your hit compounds there are plenty of screening assays to consider performing in drug discovery phase that could be labelled as tox assays, such as reactive metabolite formation, time-dependent CYP inhibition or transporter inhibition. Also, 3D cell models can be useful when screening for metabolism-dependent toxicity or delayed toxicity; they allow long-term incubation with the cells which stay metabolically active.
What requirements do the authorities have for tox studies?
For IND approvals toxicity studies are strictly controlled since the safety of a new drug candidate is very important. Currently, tox studies have to be performed in animals and in GLP conditions to allow the selection of a safe starting dose in humans. Thus, generally non-GLP screening assays are not accepted as such. However, they can be very useful for in-house decision making before conducting elaborate tox studies for a selected candidate. Continuous development of more predictive tox screening assays is needed to reduce the amount of unnecessary, laborious and expensive animal studies.
Written by Jouni Jukka and Sanna-Mari Aatsinki
PS. If you are interested in a more comprehensive information package on in vitro tox screening, you might want to check out our ebook: In vitro toxicology.