Long-term metabolic analyses

 

The 3D spheroid system has been optimized for functional stability and primary human hepatocytes retain expression of phase 1 and phase 2 drug metabolizing enzymes and drug transporters for multiple weeks in culture (Fig. 1a-c).

As a consequence, metabolic profiles remain temporally stable, as demonstrated by maintained metabolic profiles of dextromethorphan (Vorrink et al., 2017). Dextromethorphan is metabolized by CYP2D6 to dextrorphan and by CYP3A4 to 3-methoxymorphinan, which can be metabolized further to 3-hydroxymorphinan. Three donors were analyzed, which classified phenotypically as extensive (donors 1 and 2) or poor (donor 3) CYP2D6 metabolizers. In both extensive metabolizers, the vast majority of dextromethorphan was demethylated in freshly isolated cells as well as in spheroids after three weeks in culture (Fig. 1d). Over culture time, the metabolic spectrum slightly tilted from CYP2D6-mediated O-demethylation to N-demethylation catalyzed by CYP3A4. Notably, dextromethorphan metabolism in the poor CYP2D6 metabolizer (donor 3) was strongly biased towards 3-methoxymorphinan in freshly isolated cells as well as after long-term spheroid culture in agreement with in vivo data [1]. Combined, the presented data indicates that metabolic profiles are stable in 3D spheroid culture and phenotypic differences observed in vivo can be successfully translated into an in vitro setting. This constitutes a key prerequisite for metabolic profiling, pharmacokinetic analyses and drug clearance assessments.


Figure 1:Inter-individual differences in metabolic patterns are preserved in the 3D spheroid culture. Expression of phase I (CYP1A2, CYP2C8 , CYP2C9, CYP2D6 , CYP3A4; a ) and phase II drug metabolizing enzymes ( UGT1A1, UGT2B15 , GSTP1 ; b ) and drug transporters ( ABCC2 , ABCC3 , SLCO1B1 ; c ) remains at physiological levels in the 3D culture system (red), whereas expression is mostly lost in 2D culture of the same donors (n=3). Note that expression of GSTP1, a marker for non-mature hepatocytes, rapidly increased in 2D monolayers whereas it remained at physiological levels in 3D spheroid culture. The data is presented on semi-log plots showing fold change expression changes compared to freshly isolated cells. Dashed lines depict the evolution of fold changes between 2D and 3D culture over time. Error bars indicate SEM. d, Metabolic profiles of dextromethorphan metabolism in PHH from three different donors in freshly isolated cells (FIC) and spheroids after three weeks in culture. CYP2D6 and CYP3A4 were phenotyped directly after isolation by rate of formation of dextrorphan and 6β-hydroxytestosterone, respectively. Values indicate pmol produced per minute per million cells. EM = extensive metabolizer, PM = poor metabolizer. Figure from Vorrink et al., FASEB J, 2017.

 

References:

  • 1. Jones et al., CPT, 1996