Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

Clinical Investigation on Endogenous Biomarkers to Predict Strong OAT-Mediated Drug-Drug Interactions.

BACKGROUND: Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions early in humans. METHODS: We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition. RESULTS: PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. CONCLUSIONS: The current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction. CLINICAL TRIAL REGISTRATION: EudraCT number: 2016-003923-49.