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

Transcription

1 The TC7 Cell Monolayer is a Valuable in vitro Intestinal Epithelial Model for Membrane Permeability Screening X.C. Wu, J.R. Williford, S. Gokhin, S. Lee, M.R. Davis, P. Sheffels, X. Wang, J. Migeon, and M.C. Bodinier Cerep, Inc., Redmond, WA USA

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

3 INTRODUCTION The Caco-2 cell line has been widely used 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 the P- glycoprotein-mediated efflux was examined. In addition, a correlation between TC7 permeability and the percentage human absorption in vivo was also reported.

4 METHODS Both Caco-2 and TC7 cells were maintained in Dulbecco s modified Eagle medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and penicillin/streptomycin/fungizone at 100 U/ml, 100 µg/ml, and 0.25 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 the basolateral-to-apical (B-A) directions at 37 C with shaking (70 rpm). Compounds were tested at 50 µm in HBSS (Life Technology, Cat.# 11201) with 1% DMSO. Buffer A (HBSS-Mes, ph 6.5) was used in the apical side, and Buffer B (HBSS-Hepes, ph 7.4) was used in the basolateral side unless otherwise stated. Samples were taken by a MultiProbe II (Packard) at two time points (time 0 and 120 min for A-B direction and time 0 and 60 min for B-A direction). Samples were analyzed by HPLC-MS (HP1100 MSD, Hewlett Packard). 14 C-Mannitol was used as a cell monolayer integrity marker, and the radioactivity was determined using a TopCount NXT (Packard).

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 200 TC7 P app (10-6 cm/s) R 2 = n = 24 compounds Caco-2 P app (10-6 cm/s)

8 Figure 1. TC7 permeability vs. Caco-2 permeability. Permeability was determined in the A-B direction with 24 compounds as 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 (10-6 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 50 µm at ph 7.4 in both the apical and basolateral sides. These data indicated 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 (10-6 cm/s) A-B B-A A-B B-A 0 Control Verap CsA 0 Control Verap CsA Figure 3. The permeability of vinblastine (5 µm) was determined in the absence (Control) and presence of 100 µm verapamil (verap) or 10 µm cyclosporin A (CsA) at ph 7.4 in 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 TC7 Permeability vs. Percent Human Absorption 100 Human Absorption (%) LOG (TC7 Papp) R 2 = n = 18 compounds Figure 4. The permeability was determined in the A-to-B direction at ph 6.5 in the A side and ph 7.4 in the B side. Compounds (listed in Table 1) were tested at 50 µm. The human absorption data were obtained from the literature as listed in the Reference section.

12 Table 1. TC7 Permeability vs. Percent Absorption in Human Compound Name P app ± S.D. References for % Absorption 10-6 cm/s (n*) Absorption Data Nadolol 1.02 ± 1.14 (2 ) 34 Ref. 2 Atenolol 1.00 ± 0.56 (6 ) 50 Ref. 2 Ranitidine 0.68 ± 0.53 (20 ) 50 Ref. 3 Pimozide 0.60 ± 0.32 (3 ) 55 Ref. 4 Hydrocortisone 16.1 ± 0.62 (3 ) 89 Ref. 5 Oxprenolol 21.9± 2.49 (3 ) 90 Ref. 2 Phenytoin 61.6± 3.61 (3 ) 90 Ref. 4 Propranolol 28.3 ± 3.48 (24 ) 90 Ref. 5 Alprenolol 30.1 ± 1.85 (3 ) 93 Ref. 5 Metoprolol 24.6 ± 4.35 (5 ) 95 Ref. 5 Warfarin 57.0 ± 6.72 (3 ) 98 Ref. 5 Caffeine 76.2 ± 8.00 (3 ) 100 Ref. 4 Dexamethasone 10.1 ± 0.55 (3 ) 100 Ref. 4 Imipramine 23.9 ± 1.10 (3 ) 100 Ref. 4 Ketoprofen 61.0 ± 2.44 (3 ) 100 Ref. 4 Naproxen 89.9 ± 9.22 (3 ) 100 Ref. 4 Probenecid 30.6 ± 2.89 (3 ) 100 Ref. 4 Yohimbine 21.2 ± 1.56 (3 ) 100 Ref. 4 *: n represents the number of replicate cell monolayers.

13 CONCLUSION The TC7 cell monolayer is a valuable in vitro intestinal epithelial model for membrane permeability assessment.

14 REFERENCES 1. Gres, M-C. et al. (1998) Pharm. Res., 15: Meier, J. (1982) Am Heart J. 104: Physicians Desk Reference, 53 rd ed., (1999) Medical Economics, Montvale, NY. 4. Hardman, J.G., Goodman, A., and Limbird, L.E. (1996) Goodman & Gilman s The Pharmacological Basis of Therapeutics. 9 th ed., McGraw-Hill. 5. Artursson, P. and Karlsson, J. (1991). Biochim. Biophys. Res. Commun. 175: