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European Biopharmaceutical Review

Tackling Toxicity

Marc Conti, Corine Matar, Alexandre Ismail and Josua Franck at BioQuanta examine the problems facing new technologies in early toxicity assessment

Drug development is becoming more costly and time consuming as each year passes. A major part of this is the cost of compounds that are carried late into the development process, but which fail at or after critical junctures (such as clinical testing and market approval). Toxicity is the principal cause of these late stage failures, but curiously it is still tested late in the process. Therefore, the early elimination of toxic leads is key to reducing overall cost.

Currently, drug development starts by searching for active compounds with highthroughput screening (HTS). Toxicity evaluation of hits resulting from this primary screening is a strategic point for dropping toxic molecules from the development process. “With greater demands for proof of safety, the demands for preclinical data will only go up,” explains Steve Usdin (1). HTS is a valuable approach for drug discovery; now we must ask the question: what are the requirements of a post-HTS toxicity screening system?

HIGH-THROUGHPUT SCREENING

HTS is currently the most widely used technique for finding new compounds with therapeutic activity. It is the fastest way to screen the large compound libraries of pharmaceutical companies, and they have invested heavily in this technology. Currently, an HTS campaign consists of several runs requiring specific preparations. Each run measures the effect of a molecule on one parameter of cell status. After each run, read-out technologies are needed to analyse the data and determine which parameter the next HTS run will measure. Molecules yielding the desired effect (or combination of effects) on the cell lines being tested are called hits. After this step, some hits are retained as leads and moved to the next development step, hit confirmation. It is imperative to confirm all hits resulting from an HTS campaign using a complementary assay.

With the growing need for better characterisation of molecules from the pharmaceutical industry, HTS is giving way to more enabling technologies, such as high content screening (HCS) and micro HTS (μHTS). HCS uses multiparametric HTS runs, typically relying on high resolution cellular imaging. This gives much more information, at the expense of creating an enormous volume of data which must be stored and analysed. μHTS is an HTS measurement with smaller sample volume and higher precision. However, this also means that μHTS assays take longer to develop.

Bottlenecks and Late Stage Attrition

It is important to understand that the main difficulty with HTS is in data interpretation. Indeed, the results depend on the model of interpretation used. The same experiment can yield very different results. Due to this variation, HTS campaigns often contain bottlenecks in processing due to the need to confirm HTS results by other assays. Another problem with HTS screening is the false hit rate. “HTS generates as much misinformation as information”, according to Frank Brown of Accelrys (2); false hits are one of the reasons for the increasing costs of development.

HTS is well suited for activity screening but should not be used to characterise a compound as a lead because the system lacks the necessary precision. Thus, there is a clear gap to be filled in terms of complementary and confirmatory technologies to be implemented after HTS hit selection, such as performing toxicity assays.

AFTER HTS – TOXICITY TESTING

HTS measurements show short to midterm toxicity effects. However, most of the failures in drug development arise from long-term toxicity effects that are only detected in clinical trials or after market release. This clearly shows an unmet need for predictive toxicity testing starting from the very beginning of drug development. Despite their quality, current toxicity assays are not comprehensive enough to eliminate molecules with long-term or chronic exposure toxicity. Many of these molecules have latent effects on mitochondria or oxidative stress mechanisms. When one of these systems is disturbed, ATP production is affected and the cell enters a metabolically disregulated state, inducing long-term toxicity. Mitochondrial toxicity and induced oxidative stress are not widely evaluated, but some technologies for early assessment of those parameters recently appeared on the market.

Do we have the Tools for Predictive Toxicity?

Currently two main assay types are used: manual assays and microplate assays for HTS. Unfortunately, both have their drawbacks. The manual assays are precise, with a coefficient of variation (CV) of five to eight per cent, but only one parameter is measured on a single compound per sample. This introduces variations between the samples, and is slow. Manual methods can give useful results, but generally only add to the cost of development.

HTS brings the automation needed to deal with the number of hits resulting from a primary screening, but HTS assays have an average CV of 13 per cent. Thus, only large responses from assays can be detected, which can only reveal acute or short-term toxicity. Lower level responses cannot be distinguished, and so long-term toxicity remains undetected. This also requires the tests to be run in triplicate, adding substantial delays and costs for each parameter tested.

Existing technologies cannot meet the technical and cost requirements of early-stage toxicity assessment. This lack of performance explains why long-term toxicities still cannot be detected before large-scale clinical trials or market release. It remains a major problem in drug development.

CHARACTERISTICS OF AN EARLYSTAGE TOXICITY ASSESSMENT SYSTEM

To deal with the volume of hits that HTS can generate, it is clear that a high level of automation is also required for an earlystage toxicity assessment system. μHTS has the advantage of working with reduced sample sizes, and of collecting data on multiple cellular pathways with multiparametric measurements. However, collecting data on multiple cellular pathways requires the cell to remain intact. Current assay techniques often require extraction of sub-cellular components, which introduces a lot of variation in measurements. These key characteristics must be assembled into one unique technology, which operates on the smallest unit of life – the cell.

A Comprehensive Panel for Mitochondrial and Oxidative Stress

A large panel of assays allows a comprehensive vision of cellular metabolism and can efficiently detect toxicity by measuring a large number of parameters. Recent studies have shown that most toxicity (such as cardio-, neuro-, hepato-toxicity and cancer) have mechanisms involving mitochondrial or oxidative-stress pathways (3). By testing a large panel of parameters on these two pathways, it is possible to detect most problems leading to chronic toxicities. To extend the assay panel to all metabolic pathways of mitochondrial activity, one could also measure the cellular energetic status, inflammation and cell death. A truly global vision of metabolic changes resulting from exogenous compounds can enable the detection of toxicity more accurately by identifying its exact mechanism. With this technology in hand, toxicity can be assessed at early stages in drug discovery. A technology with these capabilities could also be used at other points of the drug development process as a guidance tool.

A GUIDANCE TOOL

A guidance tool offers decision support from the beginning to the end of a project by providing information to make the go/no-go decision at each step. For drug development, it should take the form of complementary assays for toxicity performed throughout the process. This can eliminate the unnecessary development of compounds that could never have been drugs because of toxicity.

This guidance tool can deliver results as a toxicity score that feeds an adaptive classification of compounds during three key stages of drug development. The first point of implementation would be just after the primary HTS screening and would consist of scoring the toxicity of each hit. The second timepoint would be the preclinical phase, where compounds could be categorised by their level of toxicity on cells from various animal models. The third stage of application would be during Phases I, II and III.

Integrating a toxicity assessment system would enable the best compounds to be chosen for development and market release while saving time. It would accelerate each development step by adding detailed information on the mechanisms of action and toxicity. It is even conceivable to screen for toxicity on cells from patients. Thus the adaptive categorisation of compounds would also make it possible to determine personalised dosages for different patient groups based on their genotype and phenotype.

CONCLUSION

HTS is likely to remain a key technology for drug discovery, but it needs to be complemented by toxicity assessments at various time points during drug development. Existing methods do not have the breadth, speed or accuracy to detect long-term toxicity. A novel technology combining these characteristics would be of high value for improving the safety and profitability of the drug development process.

References

  1. Usdin S, PDUFA blizzard, BioCentury, 15th February 2010
  2. Glaser V, High Throughput Screening Retools for the Future, Bio-IT World, www.bio-itworld.com/BioIT_Article.aspx?id=86836, 8th March 2010
  3. Dykens JA and Will Y, The significance of mitochondrial toxicity testing in drug development, Drug Discovery Today 12, 17/18, September 2007

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Marc Conti, PharmD, PhD, is a Biologist in the Department of Biochemistry, University Hospital, CHU de Bicętre. He is also the Senior Scientific Advisor, Biochemistry, for BioQuanta. Marc is a specialist in the exploration of metabolic disorders due to oxidative stress or electron transport chain dysfunction, and in the automation of biological methods. 

Corine Matar is a Biochemical Engineer at BioQuanta and a third-year PhD student in Biochemistry and Molecular Biology. She has developed and manages a highly specific analysis platform for assays of mitochondrial and oxidative stress in cells undergoing metabolic changes. This platform is currently used for medical, pharmaceutical and cosmetics research.

Alexandre Ismail is a Masters student at Sup’Biotech in Paris and is in business development for in silico services at BioQuanta. He holds a BA in Biochemistry from Hunter College, and has participated in computational chemistry research programmes at Hunter College, Rutgers University, Wolfram Research, Duquesne University, the University of Pittsburgh, and the City College of New York.

Josua Franck is a Masters student at Sup’Biotech, a biotechnology school in Paris. He is currently an intern at BioQuanta. He works on marketing for the Mitoxis platform of specialised in vitro assays.

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Marc Conti
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Corine Matar
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Alexandre Ismail
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Josua Franck
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