Characterization of Lipophilicity and Blood Partitioning of Pyrrolizidine Alkaloids and Their N-Oxides In Vitro and In Silico for Toxicokinetic Modeling.

Lipophilicity and blood partitioning are important determinants for predicting toxicokinetics using physiologically-based toxicokinetic modeling. In this study, the logarithm of the n-octanol : water partition coefficient and the blood-to-plasma concentration ratio were, for the first time, experimentally determined for the pyrrolizidine alkaloids intermedine, lasiocarpine, monocrotaline, retrorsine, and their N-oxides. Validated in vitro assays for determination of the n-octanol : water partition coefficient (miniaturized shake-flask method) and the blood-to-plasma conentration ratio (LC-MS/MS-based depletion assay) were compared to an ensemble of in silico models. The experimentally determined octanol : water partition coefficient indicates a higher affinity of pyrrolizidine alkaloids and their N-oxides to the aqueous compared to the organic phase. Depending on the method, in silico determined n-octanol : water partition coefficients overpredicted the experimental values by ≥ 1 log unit for three out of four pyrrolizidine alkaloids (SPARC), four out of six (CLOGP), five out of eight (KowWIN), and three out of eight (S+logP) pyrrolizidine alkaloids and their N-oxides. The blood-to-plasma concentration ratio obtained in vitro suggested a low binding affinity of pyrrolizidine alkaloids and their N-oxides towards red blood cells. For all eight pyrrolizidine alkaloids and their N-oxides, in silico predicted blood-to-plasma ratios deviated from experimental values by less than 50%. In conclusion, for physiologically-based toxicokinetic modeling of pyrrolizidine alkaloids and their N-oxides, the experimental octanol : water partition coefficient should be preferred, while the blood-to-plasma concentration ratio predicted by the acid/base classification model is a suitable surrogate for experimental data.