Pharmacokinetic aspects of drug effects in vitro: effects of serum protein binding on concentration and teratogenicity of valproic acid and 2-en-valproic acid in whole embryos in culture

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Abstract
Pharmacokinetic studies were performed in connection with culture experiments. Using the technique of cultivating whole rat embryos of the early postimplantation stage, we measured the concentration of valproic acid (VPA) and 2-en-VPA in the culture medium (free and protein-bound form) and in embryonic tissue. The following results were obtained: The concentrations of VPA and 2-en-VPA reached in the embryos were lower than corresponding total concentrations added to the culture medium, but exceeded the free concentrations in the medium. The concentrations of 2-en-VPA found in the embryo were lower than the comparable VPA total levels because of the more extensive protein binding of 2-en-VPA in the culture serum. The percentage of binding to serum proteins decreased with increasing total drug concentratrations in the medium; the concentration of the free drug in the medium increased overproportionally with increasing total drug concentrations. Therefore, the free drug concentrations in the medium were not proportional to the dose of the drug dissolved in the medium (for a drug bound to plasma proteins). The concentrations of VPA and 2-en-VPA found in the embryos after incubation in vitro were not proportional to the drug concentrations dissolved in the medium. This result has to be taken into account when dose-response relationships are evaluated. VPA concentrations of 40 μg/g wet weight and above in the embryos clearly induced abnormal development in about 30% of the embryos, while 2-en-VPA concentrations as high as 200 μg/g embryo (wet weight) were inactive. Thus, differences in the embryotoxic potencies — in vivo and in vitro — were not due to pharmacokinetic differences. The results of these investigations show for the first time that pharmacokinetic studies are essential for the interpretation of in vitro experiments.