The TC7 Cell Monolayer is a Valuable in vitro Model for Intestinal Permeability Screening

Transcription

1 The TC7 Cell Monolayer is a Valuable in vitro Model for Intestinal Permeability Screening X.C. Wu, M.R. Davis, S. Gokhin, S. Lee, J.R. Williford, X. Wang, and M.-C. Bodinier Cerep, Inc., Redmond, WA 9852 USA Poster presented at the 6th Annual Meeting of Society for Biomolecular Screening, Vancouver, B.C., Canada, September 6-9, 2

2 OBJECTIVE To evaluate TC7 cells, a subclone of the Caco-2 cell line, as an in vitro intestinal epithelial model for permeability screening of drug discovery compounds.

3 INTRODUCTION The Caco-2 cell line has been used widely as an in vitro intestinal epithelial model for drug transport studies and permeability screening of discovery compounds. Recently a subclone of the Caco-2 cell line, TC7, has been reported as an alternative model for permeability assessment of drug candidates (Ref.1). In the present study, the TC7 subclone was compared with the parental Caco-2 cell line. Permeability of reference compounds was determined and culture conditions were optimized. In addition, activities mediated by various transporters were evaluated, and CYP3A-mediated terfenadine hydroxylation was examined. The results from this study demonstrated that TC7 is a valuable in vitro intestinal epithelial model for permeability screening.

4 METHODS Both Caco-2 and TC7 cells were maintained in high glucose Dulbecco s modified Eagle medium supplemented with 1% fetal bovine serum, 1 mm non-essential amino acids and penicillin/streptomycin/fungizone at 1 U/ml, 1 µg/ml, and.25 µg/ml, respectively. Cells were typically passaged weekly. For permeability experiments, cells were seeded at cells/cm 2 on polycarbonate membrane inserts in 24-well Transwell TM plates (Costar) and used at days post seeding.

5 Permeability experiments were performed in the apical-tobasolateral (A-B) or B-A directions at 37 C with shaking (7 rpm). Compounds were tested at 5 µm in HBSS with 1% DMSO. Buffer A (HBSS-Mes, ph 6.5) was used on the apical side, and Buffer B (HBSS-Hepes, ph 7.4) was used on the basolateral side unless otherwise stated. Samples were taken by a MultiProbe II (Packard) at two time points (time and 12 min for the A-B direction and time and 6 min for the B-A direction). Samples were analyzed by HPLC-MS (HP11 MSD, Hewlett Packard) and/or liquid scintillation counting (TopCount NXT, Packard). 14 C-Mannitol or Lucifer Yellow was used as a cell monolayer integrity marker.

6 The apparent permeability coefficient (P app ) was calculated from the following equation: P app ( cm / s) dm dt R A ( C 1 C D, mid R, mid ) where dm R /dt is the appearance rate of the test compound in the receiver chamber, A is the surface area of the cell monolayer, C D,mid is the mid-point concentration of the test compound in the donor chamber, and C R,mid is the mid-point concentration of the test compound in the receiver chamber during the course of the assay.

7 Correlation of TC7 Permeability with Caco-2 Permeability 2 TC7 P app (1-6 cm/s) R 2 =.968 n = 24 compounds Caco-2 P app (1-6 cm/s)

8 Figure 1. TC7 permeability vs. Caco-2 permeability. Permeability was determined in the A-B direction with the 24 compounds listed below. These data indicate that the TC7 permeability correlated well with the Caco-2 permeability for this set of reference compounds. acebutolol cimetidine methyl scopolamine piroxicam alprenolol dexamethasone metoprolol progesterone atenolol griseofulvin nadolol propranolol bremazocine hydrocortisone phenytoin sulfasalazine caffeine indomethacin pindolol terbutaline chlorpromazine labetalol pirenzepine warfarin

9 Polarized Permeation of P-glycoprotein Substrates Apparent Permeability (1-6 cm/s) TC7 A-B B-A Vinb Colch Eryth Caco-2 A-B B-A Vinb Colch Eryth Figure 2. Permeability of vinblastine (Vinb), colchicine (Colch), and erythromycin (Eryth). Compounds were tested at 5 µm at ph 7.4 in both the apical and basolateral sides. These data indicate that an efflux system (most likely the P-glycoprotein) that was expressed in Caco-2 cells was present in TC7 cells as well.

10 Effect of P-glycoprotein Modulators on Vinblastine Permeability 25 TC7 25 Caco-2 Vinblastine Papp (1-6 cm/s) A-B B-A A-B B-A Control Verap CsA Control Verap CsA Figure 3. The permeability of vinblastine (5 µm) was determined in the absence (Control) and presence of 1 µm verapamil (verap) or 1 µm cyclosporin A (CsA) at ph 7.4 on the apical (A) and basolateral (B) sides. Inhibitors were added along with vinblastine to the A and B sides for the A-to-B and B-to-A fluxes, respectively. These data further support that P-glycoprotein is expressed in TC7 cells.

11 Optimization of Cell Culture Conditions: 2 week culture vs. 3 week culture 35 7 A-B Papp (1-6 cm/s) wk 3 wk B-A Papp (1-6 cm/s) wk 3 wk 5 1 Prop Labe Rani Verap Vinb Colch Eryth Quin Prop Labe Rani Verap Vinb Colch Eryth Quin Figure 4. The permeability of the reference compounds (5 µm) was determined with TC7 cells. These data indicate that the 2 week culture differentiated as well as the 3 week culture. Therefore TC7 cells at 2 to 3 weeks in culture (days 13 to 26) are routinely used for permeability screening at Cerep. Abbreviations: Prop, propranolol; Labe, labetalol; Rani, ranitidine; Verap, verapamil; Vinb, vinblastine; Colch, colchicine; Eryth, erythromycin; Quin, quinidine.

12 Transporter activity: TC7 vs. Caco TC7, d13/p2 Caco-2, d13/p24 5 TC7, d2/p2 Caco-2, d2/p24 A-B Papp (1-6 cm/s) A-B Papp (1-6 cm/s) BA Prop Phe Gly-Sar Vinb Dig BA Prop Phe Gly-Sar Vinb Dig Figure 5. Compounds were tested at either 1 µm (for benzoic acid, L-phenylalanine, and L-glycyl-sarcosine) or 5 µm (for propranolol, vinblastine, and digoxin). Compounds were either 3 H- or 14 C-labeled and detected by liquid scintillation counting on Packard TopCount NXT. Abbreviations: BA, benzoic acid; Prop, propranolol; Phe, L-phenylalanine; Gly-Sar, L-glycyl-sarcosine; Vinb, vinblastine; Dig, digoxin.

13 CYP 3A Activity Terfenadine Hydroxylation: TC7 vs. Caco-2 Hydroxyterfenadine Produced (%) TC7 Caco-2 Day 15 Day 22 Figure 6. Terfenadine (2 µm) was added to the apical (A) side. Incubations were performed for 3 hour. Hydroxyterfenadine was detected on both A and B sides with the amount on the A side being 3-6 fold of that on the B side. The data are expressed as the percentage of the total peak area of hydroxyterfenadine on both the A and B sides over the initial peak area of terfenadine on the A side.

14 CONCLUSION The TC7 permeability correlated well with the Caco-2 permeability for compounds that are typically transported by passive diffusion. The transporter activity mediated by P-glycoprotein and monocarboxylic acid transporter (MCT) in TC7 cells was similar to those in Caco-2 cells. However, the transporter activity mediated by the large neutral amino acid transport system (LNAA) and the dipeptide transporter (Pept1) appeared to be higher in TC7 cells than in Caco-2 cells. It appeared that CYP3A activity is 4-5 fold higher in TC7 cells than in Caco-2 cells (2-3 week TC7 culture vs. 3 week Caco-2 culture). The TC7 cells differentiated well at day 13 in culture and were suitable for permeability screening from day 13 through day 26 post-seeding. The Caco-2 cells appeared to be suitable for permeability screening from day 2 through day 26.

15 REFERENCES 1. Gres, M-C. et al. (1998) Pharm. Res., 15: