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.

A Novel In Vitro Experimental System for the Evaluation of Drug Metabolism: Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Cryopreserved Human Hepatocytes).

We report here a novel experimental system, cryopreserved MetMax human hepatocytes (MMHHs), for in vitro drug metabolism studies. MMHHs consist of cofactor-supplemented permeabilized cryopreserved human hepatocytes. The use procedures for MMHHs are significantly simplified from that for conventional cryopreserved human hepatocytes (CCHHs): 1) storage at -80°C instead of in liquid nitrogen and 2) usage directly after thawing without centrifugation and microscopic evaluation of cell density and viability and cell density adjustment. In this study, we compared MMHHs and CCHHs in CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP2J2, monoamine oxidase A, aldehyde oxidase, flavin-containing monooxygenase, UDP-glucuronyl transferase, SULT, N-acetyltransferase 1, and acetaminophen glutathione (GSH) conjugation activities based on liquid chromatography-tandem mass spectrometry quantification of substrate metabolism. MMHHs were prepared from CCHHs consisting of hepatocytes pooled from 10 individual donors. The drug metabolizing enzyme activities of both CCHHs and MMHHs were cell concentration and time dependent, with specific activities of MMHHs ranging from 27.2% (carboxylesterase 2) to 234.2% (acetaminophen GSH conjugation) of that for CCHHs. As observed in CCHHs, sequential oxidation and conjugation was observed in MMHHs for coumarin, 7-ethoxycoumarin, and acetaminophen. 7-Hydroxycoumarin conjugation results showed that metabolic pathways in MMHHs could be selected via the choice of cofactors, with glucuronidation but not sulfation observed in the presence of UDP-glucuronic acid and not 3-phosphoadenosine-5-phosphosulfate, and vice versa. Results with noncytotoxic and cytotoxic concentrations of acetaminophen showed that drug metabolism was compromised in CCHHs but not in MMHHs. Our results suggest that the MMHHs system represents a convenient and robust in vitro experimental system for the evaluation of drug metabolism.

Human Enterocytes as an In Vitro Model for the Evaluation of Intestinal Drug Metabolism: Characterization of Drug-Metabolizing Enzyme Activities of Cryopreserved Human Enterocytes from Twenty-Four Donors.

We report in this work successful isolation and cryopreservation of enterocytes from human small intestine. The enterocytes were isolated by enzyme digestion of the intestinal lumen, followed by partial purification via differential centrifugation. The enterocytes were cryopreserved directly after isolation without culturing to maximize retention of in vivo drug-metabolizing enzyme activities. Post-thaw viability of the cryopreserved enterocytes was consistently over 80% based on trypan blue exclusion. Cryopreserved enterocytes pooled from eight donors (four male and four female) were evaluated for their metabolism of 14 pathway-selective substrates: CYP1A2 (phenacetin hydroxylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (bupropion hydroxylation), CYP2C8 (paclitaxel 6α-hydroxylation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19 (S-mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation), CYP2J2 (astemizole O-demethylation), UDP-glucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7-hydroxycoumarin sulfation), and carboxylesterase 2 (CES2; irinotecan hydrolysis) activities. Quantifiable activities were observed for CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, CYPJ2, CES2, UGT, and SULT, but not for CYP1A2, CYP2A6, CYP2B6, and CYP2D6. Enterocytes from all 24 donors were then individually evaluated for the quantifiable drug metabolism pathways. All demonstrated quantifiable activities with the expected individual variations. Our results suggest that cryopreserved human enterocytes represent a physiologically relevant and convenient in vitro experimental system for the evaluation of intestinal metabolism, akin to cryopreserved human hepatocytes for hepatic metabolism.

Utilizing In Vitro Dissolution-Permeation Chamber for the Quantitative Prediction of pH-Dependent Drug-Drug Interactions with Acid-Reducing Agents: a Comparison with Physiologically Based Pharmacokinetic Modeling.

For many orally administered basic drugs with pH-dependent solubility, concurrent administration with acid-reducing agents (ARAs) can significantly impair their absorption and exposure. In this study, pH-dependent drug-drug interaction (DDI) prediction methods, including in vitro dissolution-permeation chamber (IVDP) and physiologically based pharmacokinetic (PBPK) modeling, were evaluated for their ability to quantitatively predict the clinical DDI observations using 11 drugs with known clinical pH-dependent DDI data. The data generated by IVDP, which consists of a gastrointestinal compartment and a systemic compartment separated by a biomimic membrane, significantly correlated with the clinical DDI observations. The gastrointestinal compartment AUC ratio showed strong correlation with clinical AUC ratio (R=0.72 and P=0.0056), and systemic compartment AUC ratio showed strong correlation with clinical Cmax ratio (R=0.91 and P=0.0003). PBPK models were also developed for the 11 test compounds. The simulations showed that the predictions from PBPK model with experimentally measured parameters significantly correlated with the clinical DDI observations. Future studies are needed to evaluate predictability of Z-factor-based PBPK models for pH-dependent DDI. Overall, these data suggested that the severity of pH-dependent DDI can be predicted by in vitro and in silico methods. Proper utilization of these methods before clinical DDI studies could allow adequate anticipation of pH-dependent DDI, which helps with minimizing pharmacokinetic variation in clinical studies and ensuring every patient with life-threatening diseases receives full benefit of the therapy.