Primary Small Intestinal Epithelia Are Physiological Alternatives to the Caco-2 Lymphatic Transport Model

Highly lipophilic (log<i>P</i> > 5) pharmaceutical candidates face oral bioavailability challenges that may be minimized by targeting drug delivery to chylomicrons (CMs), chaperones into lymphatic circulation, potentially reducing first-pass metabolic events. Extant models for CM assembly (i.e., Caco-2) by which lymphatic transport may be interrogated <i>in vitro</i> insufficiently recapitulate the specialized lipoprotein synthesis pathways of the small intestine. Here, we leveraged a pulse-chase approach to investigate the response of differentiated primary human duodenum (<i>hDuo</i>) and murine ileum (<i>mIle</i>) enteroid monolayers to mixed micelle treatment with oleic acid (OA) and 2-monoolein (2-MO) lipid digestion products. Monolayers were assessed for changes in (Ia) lipoprotein secretion, (Ib) re-esterification enzyme expression, and (II) CM transport of the model lipophilic compounds halofantrine (HF; log<i>P</i> 7.34) and navitoclax (NX; log<i>P</i> 7.93). Primary cells were found to favor basal-polarized apolipoprotein B-48 (apoB-48) release that was distinct from the bidirectional apoB-100 output of Caco-2. Primary monolayers stimulated by 2-MO secreted ρ < 1.006 g/mL lipoproteins with more TG mass (∼3.5-fold) and greater diameter (>40-fold) than cells receiving OA alone. While the cell line expressed more <i>mttp</i> at baseline, lipid coincubation upregulated <i>mgat2</i> transcription in <i>hDuo</i> and <i>mIle</i> only, indicating preserved 2-MG re-esterification activity. Drug appearance in secreted lipoproteins was higher in stimulated <i>mIle</i> than Caco-2 (4.6- vs 1.6-fold for HF; 14.9- vs 1.3-fold for NX), with the primary culture model transporting a larger proportion of the dose via CMs than the cell line standard. We concluded that pulse-chase in primary monolayer cultures is a promising New Approach Methodology (NAM) for measuring intestinal lymphatic transport of lipid-formulated drugs and could offer, with refinement and validation, an alternative to the lymph-cannulated animal model.