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1 wz (2008), 38«3y J. Kor. Pharm. Sci., Vol. 38, No. 3, (2008) In-Vitro p Caco-2 MDCK s p sƒ š Á qáwz w w w ( Á ) Comparison of Caco-2 and MDCK Cells As an In-Vitro ADME Screening Model Woon-Jung Go, Eun-pa Cheon and Hyo-Kyung Han BK21 Project team, College of Pharmacy, Chosun University, Seosuk-dong, Gwangju, Korea (Received May 20, 2008ÁAccepted June 10, 2008) ABSTRACT The present study compared the feasibility of Caco-2 and MDCK cells as an efficient in-vitro model for the drug classification based on Biopharmaceutics Classification System (BCS) as well as an in-vitro model for drug interactions mediated by P-gp inhibition or P-gp induction. Thirteen model drugs were selected to cover BCS Class I ~ IV and their membrane permeability values were evaluated in both Caco-2 and MDCK cells. P-gp inhibition studies were conducted by using vinblastine and verapamil in MDCK cells. P-gp induction studies were also performed in MDCK cells using rifampin and the P-gp expression level was determined by western blot analysis. Compared to Caco-2 cells, MDCK cells required shorter period of time to culture cells before running the transport study. Both Caco-2 and MDCK cells exhibited the same rank order relationship between in-vitro permeability values and human permeability values of all tested model compounds, implying that those in-vitro models may be useful in the prediction of human permeability (rank order) of new chemical entities at the early drug discovery stage. However, in the case of BCS drug classification, Caco-2 cells appeared to be more suitable than MDCK cells. P-gp induction by rifampin was negligible in MDCK-cells while MDCK cells appeared to be feasible for P-gp inhibition studies. Taken all together, the present study suggests that Caco-2 cells might be more applicable to the BCS drug classification than MDCK-cells, although MDCK cells may provide some advantage in terms of capacity and speed in early ADME screening process. Key words Caco-2 cell, MDCK cell, In-vitro model, Permeability, BCS genomics, bioinformatics, combinatorial chemistry ü z š»w ùš., x Caco-2, MDCK, HeLa, HEK ƒ transfected cell line w ƒ in-vitro» z» š ù, ( y, x, z, )» {w wš xr., z in-vitro ü k š sƒ y» n ƒ š. w w y w ¾ w, q w Tel : 062) , hkhan@chosun.ac.kr w y w, ü ü k z w in vitro ü k sƒ» y ƒ ü y w v w. Caco-2 cell mw s n n š, Caco-2 cell n dw in-vitro x š. 1) w MDCK cell Caco-2 cell epithelial cell ƒ p œm ùküš š. p, MDCK cell Caco-2 cell n ƒ, Caco-2 cell MDCK cell w w ƒ w. 2) w Caco-2 cell d n n w 4ƒ ù BCS 3) (Biopharmaceutics Classification System) w. US FDA ƒ, BCS ƒƒ w n p 183

2 184 š Á qáwz 4ƒ w. Class I High Solubility High Permeability, Class II Low Solubility High Permeability, Class III High Solubility Low Permeability, Class IV Low Solubility Low Permeability p ƒ ù., BCS w w ü s n v w, ü n w dw ý in-vitro sƒ BCS y ƒ j w w š w. w ù ww ü s» š. ww w w ù P-glycoprotein (P-gp) w ü l. P-gp 1976 s š ü x ƒ x. 4), P-gpƒ,,,, s š. P-gp v s 5) polarized cell membrane apical side x w P-gp polarized expression, w y l» w, w P-gp mw n ww w t. 2), n P-gp y w w ü k p dw w w w.» w s P-gp x y d wš P- gp inhibition/induction study mw ƒ š P-gp w y ƒ w dw in-vitro š sƒ MDCK cell z sƒw. w Caco-2 ù MDCK cell s d w in-vitro s n ƒ BCS 6) ù w sƒw w p sƒ w w š w œwš w. x Caco-2 cell MDCK cell w s w l w, antipyrine, caffeine, phenylalanine, metoprolol, theophylline, piroxicam, naproxen, atenolol, ranitidine, fexofenadine, tetracycline, furosemide, ofloxacin Sigma Chemical Co.(St Louis, MO, US) w. yw HPLC w. Antiβactin w Sigma (Saint Louis, MO, US) w š, anti P-gp mouse w Calbiochem (Darmstadt, Germany), alkaline phosphatase anti Mouse Ig G Jackson Immuno Research (West Grove, PA) w. HPLC w pump (LC-10AD), automatic injector (SIL-10A), UV detector (SPD-10A) chromatographic system (Shimadzu Scientific Instruments, Tokyo, Japan) w. Octadecylsilane column Gemini C18( mm, 5 um, Phenomenex, Torrance, CA) w. s Caco-2 cell MDCK cell 10% FBS, 1% nonessential amino acids, 1 mm sodium pyruvate, 1% L-glutamine penicillin(100 U/mL)/streptomycin(100 mg/ml) w w Dulbecco s modified Eagle s medium(dmem) w w. s 5% CO 2, 90%, 37 o C CO 2 Incubator MCO-175(Sanyo, Moriguchi City, Japan) w. s d w s n d BCS Class I~IV sww 13 w. 2) Class I: Antipyrine, Caffeine, Phenylalanine, metoprolol, Theophylline Class II: Piroxicam, Naproxen Class III: Atenolol, Ranitidine, Fexofenadine, Tetracycline Class IV: Furosemide, Ofloxacin xw ph 6.5 (ù 0.2% w ethanol DMSO ƒ), paracellular marker 3 H-mannitol (0.1 uci/ml) ƒw w. Transwell (24 mm Diameter Inserts, 0.4 um Pore Size, Corning Inc., NY, USA) w s w z, ƒƒ well l s w z, ph 6.5 s 2. ƒƒ well (1.5 ml) donor side (apical side), š ph 7.4 (2.5 ml) receiver side (basolateral side), plate shaker g. w (0, 15, 30, 60, 90, 180 min), 200 ul v receiver side l w, (200 ul) ph 7.4

3 In-Vitro p Caco-2 MDCK s p sƒ 185 receiver side w, receiver side w w. Donor side l (0, 60, 180 min) ƒƒ 200 ul v w z, ƒƒ v 3 H-mannitol scintillation counter w w. ƒƒ v HPLC w w. P-gp x y d MDCK cell P-gp x y w šƒ», MDCK cell P-gp x ƒ yw d w. MDCK cell ƒƒ 6-well plate seedingw z, seedingw ú l 3, 7, 10, 14, 21 w ƒƒ well l wš sd ice-cold PBS 3. s lp z v w western blot w P-gp x d w. P-gp inhibition study Vinblastine ph 7.4 (ù 0.2% w ethanol DMSO ƒ), paracellular marker 3 H-mannitol (0.1 uci/ml) ƒw w. Transwell (24 mm Diameter Inserts, 0.4 um Pore Size, Corning Inc., NY, USA) w s w z, ƒƒ well l s w z, ph 6.5 sd 2. ƒƒ well (2.5 ml) donor side (basolateral side), š ph 6.5 (1.5 ml) receiver side (apical side), plate shaker g. w (0, 15, 30, 60, 90, 180 min), 100 ul v receiver side l w, (100 ul) ph 6.5 receiver side w, receiver side w w. Donor side l (0, 60, 180 min) ƒƒ 100 ul v wš, ƒƒ v 3 H-mannitol scintillation counter w w. ƒƒ v HPLC w w. t P-gp inhibitor verapamil apical side ƒw z,» vinblastine B to A permeability d w z,» (equation) w verapamil w vinblastine s n w % inhibition w. % I=100 ({P app(b-a) i/p app(b-a) } 100) P app(b-a) :P-gp substrates B-A s n P app(b-a) i:inhibitor w P-gp substrate B to A s n P-gp induction study MDCK cell ƒƒ 6-well plate seedingw z, p z rifampin ƒw cell w. z ƒƒ 3, 7, 14 ƒƒ well l w z sd ice-cold PBS 3. s lp z v w western blot w P-gp x d w. Table I Apparent Permeability of 13 Model Drugs in Caco-2 and MDCK Cells (Mean ± SD, n = 6) BCS Class Drug Caco-2 P app ( 10 6 cm/s) MDCK P app ( 10 6 cm/s) Class I Antipyrine 6.3± ±0.74 Class I Caffeine 9.1± ±2.5 Class I Metoprolol 5.0± ±1.7 Class I Phenylalanine 11± ±1.4 Class I Theophylline 5.7± ±0.17 Class II Naproxen 11±1.2 15±3.2 Class II Piroxicam 42±6.6 62±16 Class III Atenolol 1.5± ±0.43 Class III Fexofenadine 0.08 ± ±0.04 Class III Ranitidine 0.55 ± ±0.63 Class III Tetracycline 0.09 ± ±0.01 Class IV Furosemide 0.49 ± ±0.46 Class IV Ofloxacin 0.86 ± ±0.02

4 186 š Á qáwz Figure 1 Correlation between in-vitro permeability and human permeability. š In-vitro cell growth Caco-2 cell seeding z 2 ƒ ù TEER w, MDCK cell 170,000 cells/cm 2 density seedingw 3, 60,000 cells/cm 2 density seedingw 5-6 w z w TEER ùkü, tight junction network x s {. w, TEER value MDCK cell Ωcm 2 Caco-2 cell Ωcm 2 w û ùk ù, in-vivo w Caco-2 cell ƒ w sƒ w ƒ. s n d BCS BCS I-IV š sww 13 w, Caco-2 MDCK cell w s n d w z <Table I>, <Figure 1> <Figure 2> w. <Figure 1>, MDCK cell Caco-2 cell permeability human permeability (rank order relationship), in-vitro x ƒ human permeability d (rank order) w w w. ù, s x w yw human permeability dw» w x w w in-vitro permeability human permeability sƒw v w., x w human permeability w in-vitro permeability human permeability w w ƒ. Caco-2 cell MDCK cell s n (Table I) w 13 BCS w z, ü BCS w. <Table I> <Figure 2>, metoprolol( ü ƒ90% )» High permeability Low permeability group w, Caco-2 cell 13 w Class w, MDCK cell antipyrine

5 In-Vitro p Caco-2 MDCK s p sƒ 187 Figure 2 Classification of 13 model drugs based on BCS. theophylline w 11. x BCS w, Caco-2 cell MDCK cell w y w. P-gp inhibition study <Figure 3>, w MDCK cell P-gp x ƒw w ¾ P-gp x 2 ƒw, ƒw., P-gp x w, MDCK cell Caco-2 cell ƒ seeding z 3 w, inhibition study ww z. MDCK cell vinblastine s n d w P app(a-b) 0.60±0.3, P app(b-a) 3.02 ±0.94. t P-gp substrate vinblastine P app(b-a) /P app(a-b) ratioƒ 5 MDCK cell vinblastine effluxƒ x w d. P- gp x w western blot data (Figure 3) vinblastine s n š w, MDCK cell P-gp w efflux x w. w, t P-gp inhibitor

6 188 š Á qáwz ww, 48 hr MDR1 ƒ LS174T cell w,» w P-gp w ƒ MDCK cell P-gp induction sƒw» w invitro x ww. Figure 3 P-gp expression in MDCK cells. verapamil wì w, vinblastine P app(b-a) ƒ 1.87±0.36 ùkù, vinblastine effluxƒ 38% d., MDCK cell Caco-2 cell ƒ P-gp inhibition study w in-vitro x. P-gp induction study <Figure 3>, MDCK cell rifampin 3, 7, 14 w z, P-gp x rifampin w s w ƒ.(1 vs 2) David R. Foster et al. 7) š w rifampin w Caco-2 cell LS174T cell w carcinoma cell line induction test ww, z mrna level d w LS174T cell x w ABCB1(P-gp) ƒ Caco-2 down-regulation ù ù (24 hr e) z w (48 hr e). w Oliver Burk et al. 8) LS174T cell rifampin 48 hr¾ w, MDR1 ƒ šw., x š k k BCS w Caco- 2 cell MDCK cell w y. w MDCK cell P-gp inhibition sƒ w in-vitro x wù, w P-gp induction sƒw» w in-vitro x ww t t,. š x 1) Shah P, Jogani V, Bagchi T and Misra A, Role of Caco-2 cell monolayers in prediction of intestinal drug absorption, Biotechnol. Prog., 22, (2006). 2) Tang F, Horie K and Borchardt RT, Are MDCK cells transfected with the human MDR1 gene a good model of the human intestinal mucosa?, Pharm. Res., 19, (2002). 3) G.L. Amidon, H. Lennernas, V.P. Shah and J.R. Crison, A theoretical basis for a biopharmaceutics drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability, Pharm. Res., 12, (1995). 4) R. L. Juliano and V. Ling., A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants, Biochem. Biophys. Acta., 455, (1976). 5) A. H. Schinkel, C. A. Mol, E. Wagenaar, L. van Deemter, J. J. Smit and P. Borst. Multidrug resistance and the role of P- glycoprotein knockout mice, Eur. J. Cancer, 31A, (1995). 6) C-Y. Wu and L.Z. Benet, Predicting drug disposition via application of BCS Transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system, Pharm. Res., 22, (2005). 7) R. Huang, D.J. Murry, D. Kolwankar, S.D. Hall and D.R. Foster, Vincristine transcriptional regulation of efflux drug transporters in carcinoma cell lines, Biochem. Pharmacol., 71, (2006).

7 In-Vitro p Caco-2 MDCK s p sƒ 189 8) A. Geick, M. Eichelbaum and O. Burk, Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin, J. Biol. Chem., 276, (2001). 9) Löbenberg R and Amidon GL., Modern bioavailability, bioequivalence and biopharmaceutics classification system. New scientific approaches to international regulatory standards, Eur. J. Pharm. Biopharm., 50, 3-12 (2000). 10) Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H and Tokuda H, Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells, Eur. J. Pharm. Sci., 10, (2000). 11) Volpe DA., Variability in Caco-2 and MDCK cell-based intestinal permeability assays, J. Pharm. Sci., 97, , (2008). 12) Takamatsu N, Kim ON, Welage LS, Idkaidek NM, Hayashi Y, Barnett J, Yamamoto R, Lipka E, Lennernäs H, Hussain A, Lesko L and Amidon GL., Human jejunal permeability of two polar drugs: cimetidine and ranitidine, Pharm. Res., 18, (2001). 13) Winiwarter S, Bonham NM, Ax F, Hallberg A, Lennernäs H and Karlén A., Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach, J. Med. Chem., 41, (1998). 14) Takamatsu N, Welage LS, Idkaidek NM, Liu DY, Lee PI, Hayashi Y, Rhie JK, Lennernäs H, Barnett JL, Shah VP, Lesko L and Amidon GL, Human intestinal permeability of piroxicam, propranolol, phenylalanine, and PEG 400 determined by jejunal perfusion, Pharm. Res., 14, (1997).

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14.531~539(08-037).fm G Journal of the Korea Concrete Institute Vol. 20, No. 4, pp. 531~539, August, 2008 š x y w m š gj p { sƒ z 1) * 1) w w Evaluation of Flexural Strength for Normal and High Strength Concrete with Hooked