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Gentronix Reformats GreenScreen HC for Higher Throughput

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Scientists at Manchester, UK-based genotoxicity screening shop Gentronix this week said they have developed and validated a higher-throughput version of the company’s GreenScreen HC genotoxicity assay.

The new GADD45a-GFP human cell-based assay was reformatted from four compounds per 96-well microplate to test 12 compounds per plate from a starting concentration of 100 µM over three serial dilutions in a higher-throughput, automated screening mode, a company official told CBA News this week.

The Gentronix researchers, whose work was published this month in the Journal of Biomolecular Screening, found that the rate of positive genotoxicity results was 7.3 percent and the rate of cytotoxicity was 33 percent, which corresponded to what can be seen in proprietary collections of commercial customers of the GreenScreen HC assay, Andrew Knight, commercial services manager for Gentronix, told CBA News an e-mail.

The GreenScreen assay also demonstrated a higher specificity compared to an in silico genotoxicity assessment method using Derek for Windows software, and identified chemicals that were not highlighted for genotoxic potential.

Although higher-throughput screening from a fixed, low concentration reduces sensitivity to less potent genotoxins, previously reported high specificity (i.e., a very low rate of false positives) was maintained, said Knight. This is essential in early hazard assessment, where misclassification of a hazard can lead to the unnecessary rejection of useful compounds in drug development.

The theoretical, moderate throughput was approximately 750 compounds per week using manual or robotic protocols.

The protocol was used to screen 1,266 diverse compounds comprising the Sigma Library of Pharmacologically Active Compounds.

“We knew that the data would increase confidence in the assay in the drug development field, at the hit-to-lead and lead optimization stages,” Knight said.

This screening campaign would have been a very large endeavor for a small biotech company without the high-throughput screening resources available to a large pharma, however. Knight pointed out that using the normal nine dilutions per compound of the standard assay would have required over 300 96-well plates to be set up.

“However, by testing from a lower top concentration than usual and restricting the testing to three dilutions, it became a readily achievable goal,” he said.

In comparison to the other in vitro mammalian genotoxicity assays, such as the Mouse Lymphoma Assay and Chromosome Aberration test, GreenScreen HC is fast, high-throughput, less labor intensive, and requires less compound — just a few milligrams, Knight said.

However, in a bid to streamline R&D activities and reduce overhead costs, pharmaceutical companies are increasingly turning to contract research organizations to conduct their routine genotoxicity testing. According to Knight, Gentronix has seen an upturn in its own service offering for GreenScreen HC in the last 12 months.

“This has also been helped by the development of GreenScreen HC to incorporate metabolic activation using S9 liver extracts, and the ability to screen relatively high numbers of compounds and report on results within a couple of weeks rather than months,” he said.

An early and reliable indication of genotoxic liability can equate to significant cost savings for pharmaceutical companies. If a wide choice of chemistry is available, candidate compounds with positive toxicity results may be dropped.

“These compounds are very likely to fail later in in vitro and in vivo genotoxicity assays, and they should not remain in the expensive drug development process longer than is necessary,” said Knight. He continued by saying that when less choice in terms of chemistry is available, “compounds might be returned to medicinal chemistry for modifications that might allow separation of genotoxic hazard from useful pharmacology.”

If the disease that the compounds are intended to treat is life threatening, they might be prioritized for follow-up studies for more comprehensive safety assessment, where dosing, et cetera, may be taken into account.

This allows valuable resources to be redirected to more promising chemistries and candidates. “GreenScreen HC results can also be applied in weight of evidence assessments where conflicting in vitro data arises, reducing the number of confirmatory rodent bioassays required,” Knight said.

This reformatted version of the GreenScreen HC assay will be available on request for applications in high-volume, high-throughput genotoxic liability screening of library compounds, said Knight. Gentronix continues to supply reagent kits for the standard GreenScreen HC assay that more fully profiles test chemicals over a wider concentration range.

In a new field of application, the reformatted GreenScreen HC has already been used in Phase 1 of the US Environmental Protection Agency’s ToxCast program to profile 320 pesticide compounds, Knight said. He added that, “the speed of testing and data processing, the clear presentation of results, and high reproducibility have been essential features of the assay for its successful involvement in this project.”

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