Cytotoxicity

Purpose

Only one in ten drug candidates make it through the development process. One-third of these failures are due to unacceptable toxicity levels. A cytotoxicity assay is a rapid and cost-effective tool to sort out the likely failures before a compound is entered into the costly development process and to help choose the optimal candidate.

Ricerca's in vitro cytotoxicity assays are designed based on our development experience. Using a selection of cell lines tailored to our client's drug leads we can evaluate a group of qualified lead compounds and identify those most likely to fail.

Assay Protocol

Lead compounds are dissolved in DMSO and serially diluted to a final concentration range of 0.5-1,000 mM (DMSO ≤0.7%). The diluted compounds are incubated with plated human cells at 37 °C in humidified 5% CO₂ atmosphere for 4 hours. Cellular toxicity is measured using the absorbance or fluorescence changes of tetrazolium dyes, Alamar Blue or MTT, that occur when enzymatically reduced by metabolically active cells. Toxicity is reported as the LC₅₀, the concentration of compound that is lethal to 50% of the cells.

Cell types that have been used at Ricerca to determine cytotoxicity include human hepatocytes, rat hepatocytes, HepG2 cells (human hepatoma cell line), HL-60 cells (human lymphoma cell line) and NIH/3T3 cells (murine embryonic fibroblast cell line). The choice of which cell type to use is based on the number of compounds submitted and the needs of the Sponsor.

Confirmation Data

The cytotoxicity assay was performed on eight drug compounds. The results are tabulated and compared to published toxicity data (Li et. al., Chemico-Biol. Interact. 1999, 121, 17-35.) in Figure 1.

Figure 1: Toxicity in Human Hepatocytes

Figure 2 shows an example of representative data obtained from a cytotoxicity assay in rat hepatocytes.

Figure 2: Toxicity in Rat Hepatocytes