Limits...
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.

Show MeSH

Related in: MedlinePlus

Importance of multiple endpoints, time and concentration-response curves. The toxicity of rotenone was evaluated with the rat hepatoma (H4IIE) cell line. Cells were seeded into a 96-well culture plate at a density of 10,000 cells per 100 µL of culture medium containing 20% serum. Following a 48 h equilibration period, rotenone was added at concentrations ranging from 0 to 100 µM and allowed to incubate at 37°C with 5% CO2 for 6 h. These data illustrate the importance of time, concentration-response, and multiple endpoint analysis for interpreting in vitro toxicity data. After 24 h, all of the biochemical endpoints respond in a similar manner, and resolution between response profiles is difficult. The addition of a shorter exposure time allows separation between the different endpoints. It is clear that mitochondrial markers (ATP and MTT) are most sensitive to rotenone. Each point on the graph represents a mean of 4-5 wells. The coefficient of variation ranged from 10-15%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908937&req=5

Figure 5: Importance of multiple endpoints, time and concentration-response curves. The toxicity of rotenone was evaluated with the rat hepatoma (H4IIE) cell line. Cells were seeded into a 96-well culture plate at a density of 10,000 cells per 100 µL of culture medium containing 20% serum. Following a 48 h equilibration period, rotenone was added at concentrations ranging from 0 to 100 µM and allowed to incubate at 37°C with 5% CO2 for 6 h. These data illustrate the importance of time, concentration-response, and multiple endpoint analysis for interpreting in vitro toxicity data. After 24 h, all of the biochemical endpoints respond in a similar manner, and resolution between response profiles is difficult. The addition of a shorter exposure time allows separation between the different endpoints. It is clear that mitochondrial markers (ATP and MTT) are most sensitive to rotenone. Each point on the graph represents a mean of 4-5 wells. The coefficient of variation ranged from 10-15%.

Mentions: Drugs and chemicals can affect cell health at multiple levels at varying exposure concentrations and after different times of exposure. This means that certain subcellular targets such as mitochondrial function may be significantly affected before the cell dies and releases marker enzymes for viability into the media. This is an important concept when in vitro cell based biochemical or molecular data are intended to predict toxicity in either animals or humans. An example of this type of response profile occurs following a 6 h in vitro exposure to the pesticide rotenone, a known mitochondrial poison, at concentrations ranging from 0.1 to 100 µM (Fig. 5).


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)

Importance of multiple endpoints, time and concentration-response curves. The toxicity of rotenone was evaluated with the rat hepatoma (H4IIE) cell line. Cells were seeded into a 96-well culture plate at a density of 10,000 cells per 100 µL of culture medium containing 20% serum. Following a 48 h equilibration period, rotenone was added at concentrations ranging from 0 to 100 µM and allowed to incubate at 37°C with 5% CO2 for 6 h. These data illustrate the importance of time, concentration-response, and multiple endpoint analysis for interpreting in vitro toxicity data. After 24 h, all of the biochemical endpoints respond in a similar manner, and resolution between response profiles is difficult. The addition of a shorter exposure time allows separation between the different endpoints. It is clear that mitochondrial markers (ATP and MTT) are most sensitive to rotenone. Each point on the graph represents a mean of 4-5 wells. The coefficient of variation ranged from 10-15%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Importance of multiple endpoints, time and concentration-response curves. The toxicity of rotenone was evaluated with the rat hepatoma (H4IIE) cell line. Cells were seeded into a 96-well culture plate at a density of 10,000 cells per 100 µL of culture medium containing 20% serum. Following a 48 h equilibration period, rotenone was added at concentrations ranging from 0 to 100 µM and allowed to incubate at 37°C with 5% CO2 for 6 h. These data illustrate the importance of time, concentration-response, and multiple endpoint analysis for interpreting in vitro toxicity data. After 24 h, all of the biochemical endpoints respond in a similar manner, and resolution between response profiles is difficult. The addition of a shorter exposure time allows separation between the different endpoints. It is clear that mitochondrial markers (ATP and MTT) are most sensitive to rotenone. Each point on the graph represents a mean of 4-5 wells. The coefficient of variation ranged from 10-15%.
Mentions: Drugs and chemicals can affect cell health at multiple levels at varying exposure concentrations and after different times of exposure. This means that certain subcellular targets such as mitochondrial function may be significantly affected before the cell dies and releases marker enzymes for viability into the media. This is an important concept when in vitro cell based biochemical or molecular data are intended to predict toxicity in either animals or humans. An example of this type of response profile occurs following a 6 h in vitro exposure to the pesticide rotenone, a known mitochondrial poison, at concentrations ranging from 0.1 to 100 µM (Fig. 5).

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