Chemicals.

Astemizole, irinotecan hydrochloride, dextrorphan tartrate, diclofenac sodium salt, 7-ethyl-10-hydroxycamptothecin (SN38), 4-hydroxydiclofenac, S-mephenytoin, 4-hydroxymephenytoin, paclitaxel, and testosterone were purchased from Cayman Chemical (Ann Arbor, MI). The 7-hydroxycoumarin was purchased from Chem Service (West Chester, PA). Bupropion hydrochloride was obtained from AK Scientific (Union City, CA). The 7-hydroxycoumarin sulfate potassium salt was obtained from Santa Cruz Biotechnology (Dallas, TX). Chlorzoxazone, coumarin, dextromethorphan hydrobromide, 6β-hydroxytestosterone, 7-hydroxycoumarin β-D-glucuronide sodium salt, (2S, 3S)-hydroxy bupropion hydrochloride, 7-ethoxycoumarin, and phenacetin were purchased from Sigma-Aldrich (St. Louis, MO). The 6-hydroxychlorzoxazone, 6α-hydroxypaclitaxel, midazolam, 1′-hydroxymidazolam, O-desmethyl astemizole, and 4-hydroxy-S-mephenytoin were obtained from Toronto Research Chemicals (Toronto, Canada).

Human Intestine.

Human intestines from multiple donors were obtained from the International Institute for the Advancement of Medicine (IIAM, Exton, PA).

Enterocyte Isolation and Cryopreservation.

Isolation of enterocytes from human intestines was performed via enzymatic digestion of the intestinal lumen based on procedures previously reported for porcine intestines (Bader et al., 2000; Hansen et al., 2000). The following lengths (postpyloric sphincter) were used to aid the identification of the various regions of the small intestine for enterocyte isolation: duodenum, 26 cm (9.84 inches); jejunum, 2.5 m (8.2 feet); and ileum, 3.5 m. The intestines were recovered with a warm ischemic time of less than 15 minutes and shipped to our laboratory on wet ice in University of Wisconsin Preservation Solution, with a cold ischemic time of less than 24 hours.

Upon receipt of the small intestines, adipose tissue associated with intestines was removed by dissection. The intestinal lumen was washed rapidly with cold calcium and magnesium-free Hank’s balanced salt solution to remove intestinal contents, followed by digestion with an isotonic buffer containing 0.25 mg/mL type I collagenase (Sigma-Aldrich). The cells released from the intestinal lumen were sieved to remove relatively large cell clusters, followed by partial purification by differential centrifugation (100g, 20 minutes). The enterocytes were quantified and cryopreserved immediately after isolation using a programmable liquid nitrogen cell freezer and stored in the vapor phase of liquid nitrogen maintained at <−150°C.

Recovery of Cryopreserved Enterocytes.

Cryopreserved enterocytes (In Vitro ADMET Laboratories, Columbia, MD) were thawed in a 37°C water bath for approximately 2 minutes and transferred by pouring into a 50 ml conical of cryopreserved enterocyte recovery medium (In Vitro ADMET Laboratories) that was prewarmed in a 37°C water bath. The thawed enterocytes were recovered by centrifugation at 100g for 10 minutes at room temperature. After centrifugation, the supernatant was removed by decanting. A volume of 250 µl 4°C hepatocyte/enterocyte incubation medium (HQM; In Vitro ADMET Laboratories) was added to the intact pellet of enterocytes at the bottom of the conical tube, followed by gentle agitation to reconstitute an enterocyte suspension. Viability and yield were quantified in a hemacytometer based on trypan blue dye exclusion (Sigma-Aldrich).

Measurement of Enterocyte Diameter.

Photomicrographs of the enterocytes were taken using a phase-contrast photomicroscope. The photomicrographs were printed, and the diameters were measured and corrected for the magnification factor. Results (µm) are expressed as mean and standard deviation values of 50 randomly chosen cells from each enterocyte lot.

Incubation of Enterocytes with DME Substrates.

DME substrate incubations were performed in a cell culture incubator maintained at 37°C with a humidified atmosphere of 5% CO₂. Enterocyte cell density was adjusted to 3 × 10⁶ cells/ml in HQM. Aliquots of 50 µl cell suspension were added to individual wells (150,000 viable enterocytes/well) of a 96-well plate for the evaluation of drug metabolism activities. After cell addition, the 96-well plate was prewarmed in the incubator for 15 minutes, followed by the addition of 50 µl prewarmed (37°C) HQM containing DME substrates at 2× final concentration and incubated for 2 hours. The final incubation mixture in each well therefore had a volume of 100 μl, with a cell density of 1.5 × 10⁶ cells/ml. Substrates for the multiple drug metabolism pathways evaluated are shown in Table 1. Metabolism was terminated in each well by the addition of 100 µl acetonitrile. The final incubation samples were stored at −80°C for the subsequent LC/MS-MS analysis.

LC/MS-MS Quantification of Metabolite Formation.

Upon thawing, an aliquot of 100 µl acetonitrile containing an internal standard of 250 nM tolbutamide was added to each sample. All samples were centrifuged at 13,000 rpm for 5 minutes. An aliquot of 100 µL supernatant from each was transferred to a 96-well plate and was diluted with 200 µL deionized water for LC/MS-MS analysis using an API 4000 QTRAP mass spectrometer with an electrospray ionization source (AB SCIEX, Framingham, MA) connected to Agilent 1200 series high-pressure liquid chromatography (Agilent Technologies, Santa Clara, CA). An Agilent Zorbax Eclipse Plus C18 column (4.6 × 75 mm i.d., 3.5 μm; Agilent Technologies) at a flow rate of 1 mL/min was used for the chromatography separation. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). The gradient for the positive ion mode operation was programmed as follows: 0 to 2.5 minutes, increase B from 5 to 95%; 2.5 to 3.5 minutes, 95% B; 3.5 to 3.6 minutes, decrease B to 5%; run time 5 minutes. The gradient program for the negative ion mode was as follows: 0 to 3 minutes, increase B from 5 to 95%; 3 to 4 minutes, 95% B; 4 to 4.2 minutes, decrease B to 5%; run time 6 minutes. Data acquisition and data procession were performed with the software Analyst 1.6.2 (AB SCIEX). Standard assays of the metabolites were performed in LC/MS-MS mass transition monitoring mode, monitoring the mass transitions (parent to daughter ion). The metabolism substrates used, identities of the metabolites quantified, and LC/MS-MS parameters are shown in Table 1.