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Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance.

McKim JM - Comb. Chem. High Throughput Screen. (2010)

Bottom Line: It is generally recognized that early evaluation of new drug candidates is necessary to improve the process.In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity.This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

View Article: PubMed Central - PubMed

Affiliation: CeeTox Inc., 4717 Campus Dr., Kalamazoo, MI 49008, USA. jmckim@ceetox.com

ABSTRACT
One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

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Related in: MedlinePlus

In vitro toxicity profiles produced by methotrexate. Rat hepatoma (H4IIE) cells were used as the test system under the conditions described under Fig. (5). Concentration response data, measured with several markers of cell health, show a pronounced reduction in cell proliferation. A 50% response is a maximum response given that the exposure time was 24 h and the doubling time for these cells is 22 h. Cell viability remains high in the face of inhibited cell proliferation. MTT is dependent on cell number; however, the observed reduction in cellular ATP was independent of cell number. If MTT had been the only assay used to assess toxicity, the compound would have appeared toxic. If membrane leakage had been used to measure viability, the compound would have shown low toxicity. By combining several key endpoints that measure cell health, it is possible to determine the primary effect, most sensitive subcellular target, and intended effect versus unintended toxicity.
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Figure 7: In vitro toxicity profiles produced by methotrexate. Rat hepatoma (H4IIE) cells were used as the test system under the conditions described under Fig. (5). Concentration response data, measured with several markers of cell health, show a pronounced reduction in cell proliferation. A 50% response is a maximum response given that the exposure time was 24 h and the doubling time for these cells is 22 h. Cell viability remains high in the face of inhibited cell proliferation. MTT is dependent on cell number; however, the observed reduction in cellular ATP was independent of cell number. If MTT had been the only assay used to assess toxicity, the compound would have appeared toxic. If membrane leakage had been used to measure viability, the compound would have shown low toxicity. By combining several key endpoints that measure cell health, it is possible to determine the primary effect, most sensitive subcellular target, and intended effect versus unintended toxicity.

Mentions: Another example of the importance of multiple endpoint analysis for correct interpretation of in vitro toxicity data can be seen with the drug methotrexate, which was intended to be used as an antiproliferative for the treatment of cancer. The in vitro cytotoxicity data shown in Fig. (7) demonstrate that the drug potently inhibits cell proliferation to a maximum of 50% without acute cell death. The drug also effectively induces apoptosis as determined by caspase 3 activation. An undesired side effect of this drug is mitochondrial damage and oxidative stress leading to liver toxicity during therapy [51-53]. In Fig. (7) there is a potent concentration-dependent loss in cellular ATP that is independent of the reduction in cell number. Although methotrexate is an effective antiproliferative drug used to treat cancer it may also cause undesired toxicity. Multi-parametric in vitro assays can provide important clues about potential undesired effects.


Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance.

McKim JM - Comb. Chem. High Throughput Screen. (2010)

In vitro toxicity profiles produced by methotrexate. Rat hepatoma (H4IIE) cells were used as the test system under the conditions described under Fig. (5). Concentration response data, measured with several markers of cell health, show a pronounced reduction in cell proliferation. A 50% response is a maximum response given that the exposure time was 24 h and the doubling time for these cells is 22 h. Cell viability remains high in the face of inhibited cell proliferation. MTT is dependent on cell number; however, the observed reduction in cellular ATP was independent of cell number. If MTT had been the only assay used to assess toxicity, the compound would have appeared toxic. If membrane leakage had been used to measure viability, the compound would have shown low toxicity. By combining several key endpoints that measure cell health, it is possible to determine the primary effect, most sensitive subcellular target, and intended effect versus unintended toxicity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908937&req=5

Figure 7: In vitro toxicity profiles produced by methotrexate. Rat hepatoma (H4IIE) cells were used as the test system under the conditions described under Fig. (5). Concentration response data, measured with several markers of cell health, show a pronounced reduction in cell proliferation. A 50% response is a maximum response given that the exposure time was 24 h and the doubling time for these cells is 22 h. Cell viability remains high in the face of inhibited cell proliferation. MTT is dependent on cell number; however, the observed reduction in cellular ATP was independent of cell number. If MTT had been the only assay used to assess toxicity, the compound would have appeared toxic. If membrane leakage had been used to measure viability, the compound would have shown low toxicity. By combining several key endpoints that measure cell health, it is possible to determine the primary effect, most sensitive subcellular target, and intended effect versus unintended toxicity.
Mentions: Another example of the importance of multiple endpoint analysis for correct interpretation of in vitro toxicity data can be seen with the drug methotrexate, which was intended to be used as an antiproliferative for the treatment of cancer. The in vitro cytotoxicity data shown in Fig. (7) demonstrate that the drug potently inhibits cell proliferation to a maximum of 50% without acute cell death. The drug also effectively induces apoptosis as determined by caspase 3 activation. An undesired side effect of this drug is mitochondrial damage and oxidative stress leading to liver toxicity during therapy [51-53]. In Fig. (7) there is a potent concentration-dependent loss in cellular ATP that is independent of the reduction in cell number. Although methotrexate is an effective antiproliferative drug used to treat cancer it may also cause undesired toxicity. Multi-parametric in vitro assays can provide important clues about potential undesired effects.

Bottom Line: It is generally recognized that early evaluation of new drug candidates is necessary to improve the process.In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity.This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

View Article: PubMed Central - PubMed

Affiliation: CeeTox Inc., 4717 Campus Dr., Kalamazoo, MI 49008, USA. jmckim@ceetox.com

ABSTRACT
One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation.

Show MeSH
Related in: MedlinePlus