Application of Cryopreserved Human Intestinal Mucosa and Cryopreserved Human Enterocytes in the Evaluation of Herb-Drug Interactions: Evaluation of CYP3A Inhibitory Potential of Grapefruit Juice and Commercial Formulations of Twenty-Nine Herbal Supplements.

Commercial formulations of 29 commonly used herbal supplements (HSs) and grapefruit juice were evaluated for drug interaction potential via quantification of their CYP3A inhibitory potential in two in vitro experimental models of human small intestine, cryopreserved human intestinal mucosa (CHIM), and cryopreserved human enterocytes (CHEs). Two CYP3A substrates were used-in the studies with CHIM, CYP3A activity was quantified via liquid chromatography tandem mass spectrometry quantification of midazolam 1'-hydroxylation, whereas in CHE, luciferin-IPA metabolism to luciferin was quantified by luminescence. Upon treatment of CHIM with the estimated lumen concentration of the HS upon each oral administration (manufacturers' recommended dosage dissolved in 200 ml of culture medium), >80% CYP3A inhibition was observed for green tea extract, St. John's wort, valerian root, horehound, and grapefruit juice. Less than 50% inhibition was observed for fenugreek, aloe vera, guarana, soy isoflavone, maca, echinacea, spirulina, evening primrose, milk thistle, cranberry, red yeast rice, rhodiola, ginkgo biloba, turmeric, curcumin, white kidney bean, garlic, cinnamon, saw palmetto berries, panax ginseng, black elderberry, wheat grass juice, flaxseed oil, black cohosh, and ginger root. The results were confirmed in a a dose-response study with HSs obtained from three suppliers for the four inhibitory HSs (green tea extract, horehound, St. John's wort, valerian root) and three representative noninhibitory HSs (black cohosh, black elderberry, echinacea). Similar results were obtained with the inhibitory HSs in CHE. The results illustrate that CHIM and CHE represent physiologically relevant in vitro experimental models for the evaluation of drug interaction potential of herbal supplements. Based on the results, green tea extract, horehound, St. John's wort, and valerian root may cause drug interactions with orally administered drugs that are CYP3A substrates, as was observed for grapefruit juice. SIGNIFICANCE STATEMENT: In vitro evaluation of 29 popular herbal supplements in cryopreserved human intestinal mucosa identified green tea extract, horehound, St. John's wort, and valerian root to have CYP3A inhibitory potential similar to that for grapefruit juice, suggesting their potential to have clinically significant pharmacokinetic interaction with orally administered drugs that are CYP3A substrates. The results suggest that cryopreserved human intestinal mucosa can be used for in vitro evaluation of drug interactions involving enteric drug metabolism.

Cryopreserved Human Intestinal Mucosal Epithelium: A Novel In Vitro Experimental System for the Evaluation of Enteric Drug Metabolism, Cytochrome P450 Induction, and Enterotoxicity.

We report here a novel in vitro enteric experimental system, cryopreserved human intestinal mucosa (CHIM), for the evaluation of enteric drug metabolism, drug-drug interaction, drug toxicity, and pharmacology. CHIM was isolated from the small intestines of four human donors. The small intestines were first dissected into the duodenum, jejunum, and ileum, followed by collagenase digestion of the intestinal lumen. The isolated mucosa was gently homogenized to yield multiple cellular fragments, which were then cryopreserved in a programmable liquid cell freezer and stored in liquid nitrogen. After thawing and recovery, CHIM retained robust cytochrome P450 (P450) and non-P450 drug-metabolizing enzyme activities and demonstrated dose-dependent induction of transcription of CYP24A1 (approximately 300-fold) and CYP3A4 (approximately 3-fold) by vitamin D3 as well as induction of CYP3A4 (approximately 3-fold) by rifampin after 24 hours of treatment. Dose-dependent decreases in cell viability quantified by cellular ATP content were observed for naproxen and acetaminophen, with higher enterotoxicity observed for naproxen, consistent with that observed in humans in vivo. These results suggest that CHIM may be a useful in vitro experimental model for the evaluation of enteric drug properties, including drug metabolism, drug-drug interactions, and drug toxicity.

Inter-individual and inter-regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donors.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Development of a high-speed, multiplexed sample-delivery instrument for LC-MS/MS bioanalysis.

BACKGROUND: The number of new chemical entities and types of in vitro and in vivo samples that require bioanalysis in drug discovery is large and diverse. In addition, method development time is limited as data turnaround is the highest priority. These circumstances require that a well-defined set of bioanalysis options be available in short timeframes to triage samples for analysis. METHOD: The Apricot Designs Dual Arm (ADDA) instrument is an LC-MS/MS sample delivery system that features a flexible hardware design coupled with software automation to enhance throughput in LC-MS/MS bioanalysis drug discovery. The instrument can perform high-throughput LC-MS/MS (8-10 s/sample) for screening and in vitro bioanalysis, as well as multiplexed LC for traditional gradient or isocratic LC approaches. The instrument control software is designed to integrate with DiscoveryQuant™ software (AB Sciex) and a global database of MS/MS conditions. CONCLUSION: Development of the sample delivery platform and its application in high-throughput and gradient LC will be described.