Genotoxicity assessment in a changing world

The graph of Doom:
The cost of bringing a new therapeutic agent to market has continued to rise, with current estimates anywhere between $800M and $1.2B per successful launch. If this were not bad enough, the number of New Drug Applications (NDAs) has remained worryingly low, with late stage withdrawal of clinical and launched products a continuing factor contributing to unacceptable attrition rates across the pharmaceutical industry, a situation that has often been referred to as the ‘graph of Doom’.

In order to address poor attrition rates, those involved in the discovery and development of new pharmaceutical products are charged with critically assessing current operations to find cost effective ways of increasing productivity. Further, the expectation is that through fundamental modifications to the process, not only will more products be progressed to clinical evaluation and beyond but they will also be devoid of the liabilities historically observed.

Genetic Toxicology: Where are we now?
One such factor that contributes to overall attrition rates concerns the ability of a compound to cause damage to DNA. Collectively referred to as genotoxicity, there are several mechanistic causes for such damage.

In order to guard against genotoxic compounds being developed into drugs (with the exception of certain drug classes e.g. cancer chemotherapeutics where damage to DNA is a core component of the drugs mode of action) it is a regulatory requirement that all pharmaceutical compounds undergo pre-clinical safety testing.

A range of tests comprise the battery of regulatory tests because there is currently no single test that can detect all relevant genotoxic compounds. The current regulations require:

Current regulations (modified in the new recommendations Option 1, (see below))

i. A test for gene mutation in bacteria;

ii. A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma tk gene mutation assay;

iii. An in vivo test for genotoxicity, generally a test for chromosomal damage using rodent hematopoietic cells, either for micronuclei or for chromosomal aberrations in metaphase cells.

Probably the best known of all the genetic toxicology tests is the Ames bacterial reverse mutation test. However it misses approximately 40% of geneotoxins due to its lack of eukaryotic targets! Hence the need for mammalian in vitro tests.

There are several tests routinely used and sensitivity for the detection of genotoxins is increased by using tests such as the in vitro micronucleus test (MNT) or the Mouse Lymphoma Assay (MLA) in combination with Ames. However these have their own shortcomings, particularly in the generation of false positives.

So there is often a need for additional in vitro and in vivo testing which delays development of a drug candidate until such time as weight of evidence justifies progression. Not an easy problem to unravel when you consider the permutations of false positive and false negative rates for the various assays.  This leads to costly delay in both time and money.

These tests have historically been conducted relatively late in the pre-clinical phase of drug development due to high sample requirement, relatively low assay throughput and the expense of time consuming animal studies. It is therefore a critical component of both time and cost reduction that better ways are found to accurately detect genotoxins prior to later stage expensive development.

Genetic Toxicology: Where are we going?
The ICH has published (http://www.ich.org/LOB/media/MEDIA4474.pdf) recommendations for a change to the regulatory requirements that, if approved, would give pharmaceutical companies two options for the standard battery. Option 1 remains as in the current regulations (see above) but with changes to the test protocol to reduce the top concentration of testing from 10mM to 1mM and the level of cytotoxicity at which the tests are performed. Additionally there will no longer be the requirement to test into the precipitating range for insoluble compounds. Option 2 would allow progression directly from Ames to in vivo studies without the need for a mammalian in vitro assay, although information from other tests used to rule out genotoxins missed by Ames is not ruled out and there would be the requirement for two in vivo endpoints in the standard battery:

Option 2

 i.  A test for gene mutation in bacteria;

ii. An in vivo assessment of genotoxicity with two tissues, usually an assay for micronuclei using rodent hematopoietic cells and a second in vivo assay.

This change is suggested in recognition that existing mammalian in vitro assays exhibit poor specificity and the addition of a second in vivo test (potentially incorporated into general toxicity tests) should allow a reduction in the number of animals required thus reducing animal testing.

However, the fundamental problem remains that there is an urgent need for a better higher throughput mammalian cell based system that allows for the rapid and accurate detection of genotoxic compounds earlier in the drug discovery process. A new assay from Gentronix Ltd, Manchester UK, GreenScreen HC is an important new assay that combines the attributes of early screening capability with the high sensitivity and specificity required to make reliable predictions of genotoxicity. 

GreenScreen HC: Changing the way we do genetic toxicology
GreenScreen HC is a cell based reporter assay and has several features that make it particularly useful for the early detection of genotoxic hazard. Human TK6 cells have been transfected with the GADD45a regulatory element (which is involved in the cellular response to DNA damage) coupled to a gene encoding for Green Fluorescent Protein (GFP). As a result, when GADD45a transcription is upregulated the cells produce GFP. A further important component of the technology recognizes that the p53 tumour suppressor protein is a key regulator of GADD45a regulation, hence the selection of the human lymphoblastoid TK6 cell line which is p53 competent. GreenScreen HC can detect all known classes of genotoxin.

The assay was conceived as a screening tool and therefore has been developed in 96 well microplate format. It is configured to test 4 compounds per plate over 9 dilutions against two strains; the test strain and a control strain. The test strain is capable of producing GFP in response to DNA damage while the control strain is frame shifted and therefore unable to produce the fluorescent protein. Any increase in fluorescence in the control strain must be due to factors other than the production of GFP, such as fluorescence from the test compound or an increase in cellular autofluorescence. The control strain data can therefore be used to subtract these effects from the test strain data. Each plate also carries positive genotoxic controls at two concentrations. Performance of the controls is used as an indicator of proper performance of the test.

The assay is straightforward to set up and is based on a mix-wait-read protocol. It is scalable with routine laboratory automation. This means that hazard assessment can now be performed much earlier in the drug discovery process which allows chemists to concentrate effort on drug series without genotoxic liability and halting the progression of genotoxic candidates prior to expensive and time consuming studies.

Genotoxins exert their effect by different mechanisms and at different times during cell growth and division. So in order to detect all known classes, the cells must proceed through a full cell cycle. Because DNA damage causes cell cycle delay, GreenScreen data is collected at both 24hr and 48hr time points. The fluorescence is measured by plate reader, and the data normalized to culture density. The normalized GFP signal is recorded and plotted as a dose response curve. The culture density is used also used as an indication of toxicity.

If the GFP signal crosses a predefined statistically significant threshold a compound is classified as genotoxic because it will have triggered an increase in GADD45a over base levels which is indicative of DNA damage. Much validation work has been carried out to confirm the sensitivity and specificity of the GreenScreen assay by Gentronix and others (See Hastwell et al 2006) as well as its robustness and reproducibility on transfer to other laboratories.

GreenScreen HC-S9: Metabolic activation and detection of pro-genotoxins
Many compounds are metabolized in vivo, and the metabolites can be genotoxic. In order to assess this in vitro, it is usual to employ S9 liver extracts (which can be derived from various species, and often chemically treated rodents) which contain many of the metabolizing enzymes found in vivo. However, S9 liver extracts are coloured, fluorescent and particulate, all attributes that interfere with normal GFP detection.

Gentronix has overcome this issue with the launch of GreenScreen HC-S9. The assay uses the same GFP technology but under a standardized S9 protocol. It is still microplate based so the throughput of compounds is maintained. Test compounds are incubated in the presence of S9 for three hours, followed by a simple wash cycle to remove S9. The cells are then incubated and read at 24 and 48 hrs using Flow cytometry. Gentronix has demonstrated that the protocol with S9 reproduces the results of the GreenScreen HC assay, and in addition detects pro-genotoxins in the presence of S9.

GreenScreen HC and HC-S9 now provide comprehensive solutions that can be used in early screening and for more complex problem resolution.  

Bringing the pieces together:
It is clear that there are issues with the way in which regulatory safety assessment is currently conducted with respect to genotoxicity. There are also clear indications that the industry and regulatory authorities are fully aware that change is required and are collectively driving that change forward. 

The way in which drug discovery is conducted is changing with the desire to identify problem compounds as early as possible. This will reduce the money wasted on compounds with undesirable attributes and concentrate effort on candidates that have the right profile for advancement to the clinic.

In answer to these needs drug discovery screening is increasingly carried out in a matrix format with much more information gathered upfront to inform decisions on compound generation with hazard assessment moving into discovery more so than ever before. This is a fundamental process change.

Now with the advent of enabling technologies such as GreenScreen HC that provides the accuracy and scalability in genotoxicity assessment, companies have the tools to achieve the required process changes necessary to genuinely identify problem compounds early.

Reference:
P.W.Hastwell, L. Chai, K.J. Roberts, T.W. Webster, J.S. Harvey, R.W. Rees and R.M. Walmsley. High-specificity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay.  Mutat. Res.  607 (2006): 160-175.

About Gentronix
Gentronix is an innovative biotechnology company helping to accelerate the pace of drug development in the pharmaceutical industry. Gentronix is developing a range of productivity-enhancing tools for scientists in the drug discovery field, designed to ease or remove current bottlenecks in preclinical research and development. 

Contact
For further information please contact:
Steve Beasley, Commercial Director
Gentronix Limited
CTF Building, 46 Grafton Street, Manchester, M13 9NT UK
T: +44 (0)161 603 7676
E: steve.beasley@gentronix.co.uk
www.gentronix.co.uk