Cell cultures as tools in biopharmacy

European Journal of Pharmaceutical Sciences 11 Suppl. 2 (2000) S51–S60 www.elsevier.nl / locate / ejps

Cell cultures as tools in biopharmacy ¨ ¨ Annette Braun, Sibylle Hammerle, Kayoshi Suda, Barbara Rothen-Rutishauser, Maja Gunthert, ¨ Stefanie D. Kramer, Heidi Wunderli-Allenspach* Biopharmacy, Department of Applied BioSciences, Federal Institute of Technology ETH, CH-8057 Zurich, Switzerland Received 13 July 2000; accepted 31 July 2000

Abstract A survey is given on a few selected cell culture models that are used for transport studies. They are characterised for growth, transcellular electrical resistance and cytoarchitecture. The importance of standardisation in view of their use as transport models is documented. Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data. Transport studies are presented that were performed in a two-chamber set-up, the Costar  ‘‘vertical diffusion system’’. A series of non-homologous compounds showed similar permeability data (Papp ) in the different cell cultures. The origin of the cell type had no remarkable influence on passive transcellular permeation. MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation. They have low expression of transporter proteins and low metabolic activity. In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition. MDCK cells are easy to handle. Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached. To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1 -transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor. Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies.  2000 Elsevier Science B.V. All rights reserved. Keywords: Cell cultures; Standardisation; Characterisation; Passive permeation, P-glycoprotein; Efflux

1. Introduction In drug design and drug development adequate model systems have to be introduced at an early moment to avoid loss of promising compounds at an advanced stage due to insufficient absorption into, and distribution throughout the body. Beside toxicity, the pharmacokinetic characteristics were a major reason for failures of compounds in clinical studies in the past. Several strategies are pursued to establish tools for the prediction of in vivo barrier passage Abbreviations: BBB, Blood–brain barrier; Caco-2, Human colon adenocarcinoma cell line; D 7.4 octanol , Distribution coefficient in the noctanol / buffer system at pH 7.4; EBSS, Earle’s balanced salt solution; ECV304, Human umbilical cord endothelial cell line; FCS, Foetal calf serum; GI, Gastro-intestinal; GLP Good laboratory practice; HEPES, N-[2-Hydroxyethyl]piperazine-N9-[2-ethanesulfonic acid]; MDCK, Madin Darby canine kidney epithelial cell line; MDR1-MDCK, MDCK cells transfected with the mdr1 (multidrug resistance, P-gp) gene; PET, Polyethylene terephthalate; P-gp, P-glycoprotein; rho123, Rhodamine 123; SDS, Sodium dodecyl sulfate; T24, Bladder carcinoma epithelial cell line; TJ, Tight junction(s) *Corresponding author. Tel.: 141-1-635-6040; Fax: 141-1-635-68-82. E-mail address: [email protected] (H. Wunderli-Allenspach).

¨ of compounds (Kramer, 1999). Among them are computational approaches based on physicochemical parameters (e.g. Young et al., 1998; Crivori et al., 2000), as well as the characterisation of the partitioning behaviour of substances in biphasic systems (e.g. n-octanol water, liposome / buffer, Dlog P). For permeation studies, model systems have been established at various levels of complexity (Fig. 1), reaching from animals down to isotropic lipophilic phases (e.g. Kansy et al., 1998). When screening for permeation characteristics, the choice of a test system always represents a compromise between high throughput with low predictive potential and low throughput with high predictive potential. Cell cultures take an intermediate position within the pyramid of complexity for permeation studies. They permit low to medium throughput, and, as typical ‘‘added value assays’’, represent an important tool in drug development (Audus et al., 1990; Artursson and Borchardt, 1997). In the following, a few defined cell culture model systems are reviewed which have been used for transport studies in our laboratory. Their characteristics are summarised with respect to growth curves, transcellular electrical

0928-0987 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 00 )00164-0

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The relevance of transport studies with multilayered cells was analysed with MDR1-MDCK cells, which originate from transfection of Madin Darby canine kidney (MDCK) cells with the mdr1 gene, the gene coding for the Pglycoprotein, P-gp (Pastan et al., 1988). As found for other transfected cells, MDR1-MDCK cells have lost contact inhibition and show only limited polarisation (see Section 3.4). The same cells were used for comparison with the Caco-2 cells with respect to their potential to screen for P-gp substrates and inhibitors.

2. Standardisation of cell cultures

Fig. 1. The pyramid of complexity. Cell cultures represent an intermediate level of complexity between animals and isotropic lipophilic phases.

resistances (TEER), cytoarchitecture (e.g. monolayers vs. multilayers), tight junction (TJ) formation, and tightness of cell layers (mannitol permeation test) (Table 1). Transport studies are presented, which have been performed under controlled conditions with a two-chamber vertical diffusion system (Costar  ) using a set of non homologous drugs with log D 7.4 octanol values between ,0 and 13.5. The kinetics of transport was studied in various cell cultures in the apical-to-basal and basal-to-apical direction, and apparent permeation coefficients (Papp ) were calculated. Information was collected about the reproducibility of Papp .

Successful use of cell culture models is closely related to standardisation (Fig. 2). A comprehensive review with ample references was recently published dealing with cell culture techniques and standardisation procedures (Wunderli-Allenspach, 2000). Here, we will restrict ourselves to a brief survey of the most important issues. GLP includes a close track of the origin of cells and of passage numbers. Growth media should not be changed without need, and if changes are made, the growth characteristics of cells have to be checked under the new conditions. Batches of foetal calf serum, FCS, or any serum should be tested on the very cells before being used for cell culture, as toxins and hormones can be present that influence cell growth differentially. For special studies it may be necessary to adapt cells to serum-free media (Reid and Luntz, 1997). The support membrane is another important component influencing cell growth and differen-

Table 1 a Cell type

Seeding density [310 24 cells / cm 2 ]

Time to reach confluence [d]

Time to reach stationary phase [d]

Cell density in stationary phase [310 25 cells / cm 2 ]

Caco-2 b

10

2–3

|10

4

750–800

6.961.6

180–250

3.963.4

MDCK type II c

5

2

|7

5

MDR1-MDCK d MDCK parents

5 5

2 2

|10 |7

5 5

ECV304 e

5

1

15–21

8

a

TEER in stationary phase [V cm 2 ]

1000–10,000 140–190 250–350

Papp mannitol in stationary phase [310 7 cells / cm 2 ]

33.067.8 N.D. 18.560.8

All media contained 100 units penicillin / ml and 100 mg streptomycin / ml. Incubations were in a 5% CO 2 atmosphere at 378C. Cells were grown on polycarbonate membranes (Costar TranswellE [3407) except for ECV304 which were cultivated on PET membranes (Falcon [3090). Medium was changed twice weekly and 24 h before experiments. Cultures were regularly tested for mycoplasm infections. b Caco-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and studied between passage [28–60. Cells were grown in Minimum Essential Medium with Earle’s Salts (EMEM) containing 20% foetal calf serum and 0.15% NaHCO 3 (Rothen-Rutishauser et al., 2000). c ` MDCK cells were a gift from Michel Paccaud (Institut d’hygiene, Geneva, Switzerland). They were used between passage 216–270. Cells were propagated in Eagle’s minimum essential medium (MEM) with Earl’s salts supplemented with 20 mM HEPES and 0.15% NaHCO 3 containing 10% foetal calf serum (Rothen-Rutishauser et al., 1998). d Wild type MDCK cells (MDCK parent) and mdr1 -transfected MDCK cells (MDR1-MDCK) were a gift from Ira Pastan (Pastan et al., 1988). Cells were propagated in Dulbecco’s modified Eagle’s medium with Glutamax-I (DMEM) containing 10% foetal calf serum and 0.15% NaHCO 3 . In all experiments with MDR1-MDCK cells, the passage number was below 50. MDR1-MDCK cells were grown in the same medium with additional colchicine (80 ng / ml) to maintain a continuous selection pressure to express P-gp (Pastan et al., 1988). e The ECV304 cell line (American Type Culture Collection CRL-1998) was cultured in medium M199 with 10 mM HEPES containing 10% foetal calf serum (Suda et al., submitted). Cells were used between passage [133–172.

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apical localisation can be distinguished from a patchy distribution (see Sections 3.2 and 3.4).

3. Selected cell culture models for transport studies

3.1. General

Fig. 2. Standardisation of cell cultures. A comprehensive review on cell culture techniques and standardisation was published (Wunderli-Allenspach, 2000). Details on growth conditions and on the characteristics of the respective cell cultures used for transport studies are summarised in Table 1.

tiation of cells. It is important to keep standard supports for cells or, if this is not possible due to the experimental design (e.g. studies on living cells in the microscope, which need special insert membranes), characteristics under the chosen conditions have to be verified (see Section 3.2). Furthermore, confluence of cells should not be the only criterion when performing transport studies, because significant differences exist between confluent cells in the exponential growth phase and confluent cells in the stationary growth phase. Qualitative and quantitative differences are encountered in protein expression which is relevant in particular for transport proteins (Anderle et al., 1998; Rothen-Rutishauser et al., 1998). Beside protein expression, the TEER value can also change in dependence of the growth phase. In ECV304 cells, for instance, the TEER value increases during the exponential phase and only stabilises in the stationary growth phase, several days after reaching confluence (manuscript in preparation). In MDCK cells, however, TEER values, after an initial peak, remain constant during the exponential phase and into the stationary growth phase (see Section 3.3; RothenRutishauser et al., 1998). For the characterisation of cell cultures under various growth conditions, the confocal laser scanning microscope (CLSM) is of great value (for review see Wunderli-Allenspach, 2000). Optical scans through the cell layer(s) with subsequent image restoration permit to gain information on the z-axis, which corresponds to the direction of transport. For instance, the cytoarchitecture can be studied with z-scans through the cell layer(s), allowing a distinction between mono- and multilayer arrangements (see Section 3.2). Tight junctions (TJ) can be explored, e.g. formation of a complete regular network such as in MDCK cells vs. an incomplete TJ barrier formation in primary endothelial cultures (unpublished). In addition, transporters such as P-gp can be localised by fluorescence immunocytochemistry with specific antibodies, and a homogeneous

Various cell types of epithelial and endothelial origin have been used for transport studies in search of in vitro models for in vivo barriers such as epithelia of the GI tract, nasal mucosa, skin, and the blood–brain barrier, BBB (Audus et al., 1990). This survey concentrates on a few selected cell culture models, the characteristics of which are summarised in Table 1.

3.2. Caco-2 cells Caco-2, an epithelial human colon adenocarcinoma cell line, has been widely used to predict intestinal absorption of potential drug candidates (Hilgers et al., 1990; Artursson and Karlsson, 1991). The ample publication list on Caco-2 cells illustrates not only the interest in this cell line, but, at a closer look, also reveals the problems associated with suboptimal standardisation in various laboratories (Artursson and Borchardt, 1997). Cells used in our studies have been purchased from the American Type Culture Collection (ATCC). They are cultured on polycarbonate membranes. With a seeding density of 10 5 cells / cm 2 , Caco-2 cells grow confluent within 2 to 3 days. They reach the stationary growth phase after |10 days in culture, and the cell density at that stage is about 4310 5 cells / cm 2 . On polycarbonate insert membranes, Caco-2 cells form monolayers (Fig. 3). F-actin labelling with rhodamine-phalloidin reveals certain heterogeneity of the apical surface structures of cell layers (Fig. 3a), and labelling of P-gp with specific antibodies shows localisation at the apical surface. Not all of the cells are positive for P-gp, which leads to a patchy appearance (Fig. 3b). Fig. 3c (F-actin labelled) and Fig. 3d (P-gp labelled) show the contact region of a P-gp expressing and a non-expressing cell. Cell layers are tight, the Papp value for mannitol is about 10 26 cm / s. This is in the same range as previously published (Irvine et al., 1999). On polyethylene terephthalate (PET) membranes multilayers occur (Rothen-Rutishauser et al., 2000). Monolayers exhibit a regular TJ network, whereas in multilayers TJ are not only found between the uppermost cells, but also in between cells in lower layers. Polarisation of cells in multilayers is partially lost. Transport studies are usually done after 21 days in culture, when expression of transporters, e.g. P-gp, reaches its maximum (Anderle et al., 1998). The fact that not all cells express P-gp (see above, Fig. 3) may explain why in studies on directed transport with Caco-2 cells (comparison of apical to basal and basal to apical, respectively), differences between the transport rate in the two directions

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Fig. 3. Localisation of P-gp in Caco-2 cells by CLSM. Caco-2 cells were cultured under the conditions indicated in Table 1. They were fixed and prepared for CLSM as described (Rothen-Rutishauser et al., 2000). All micrographs represent optical sections (x, y-level) through the apical area of the cell monolayer with projections of the x, z-axis and y, z-axis, respectively (Wunderli-Allenspach, 2000). (a) F-actin labelled with rhodamine-phalloidin (Molecular Probes), 27 days in culture; (b) cells labelled with anti-P-gp antibody, 27 days in culture; (c) F-actin labelled with rhodamine-phalloidin, 18 days in culture; (d) same region as (c) labelled with anti-P-gp antibody (mouse; Dako [4E3; secondary antibody: goat anti-mouse cyanine 5, Chemicon).

are smaller than expected for a cell layer with a homogeneous distribution of efflux proteins (Section 4.2).

3.3. MDCK cells The Madin Darby canine kidney (MDCK) cell line is among the best studied epithelial cell lines with respect to genetics, lipid composition, expression of proteins and other parameters (for review see McRoberts et al., 1981). More recently, MDCK cells have also been used for transport studies (Irvine et al., 1999). The cell line has been established in the 1950s, when cryopreservation was not yet available, which explains the fact that relatively high passage numbers are being used (passage [216–270 in our study). Subclones exist that show characteristic TEER values: about 4000 V cm 2 for type I, and about 200–300 Vcm 2 for type II cells (Richardson et al., 1981). For transport studies, we used type II MDCK cells

(Rothen-Rutishauser et al., 1998). If seeded at a density of 5310 4 cells / cm 2 on polycarbonate membranes, they grow confluent within 2 days and reach the stationary growth phase after about 7 days with a cell density of 5310 5 cells / cm 2 . In MDCK cell cultures, a steep increase in the TEER value is observed as soon as confluence is reached. This is followed by a drop in TEER that has been associated with the installation of channels in the plasma membrane (Cereijido et al., 1978). TEER then stabilises around 180–250 V cm 2 , where it remains throughout the exponential and stationary phases. MDCK cells form polarised monolayers, which are sealed with a TJ network close to the apical surface of the cells. This network is established as soon as cell–cell contacts are made (RothenRutishauser et al., 1998). A Papp value for mannitol of about 4310 27 cm / s was determined. Up to 20 days after seeding no P-gp activity can be observed, although some ¨ of the protein is expressed in a few cells (Hammerle et al.,

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2000). Only at later times (.25 days) low activity could be detected with the rhodamine 123 (rho 123) assay (see Section 4.3).

3.4. MDR1 -MDCK cells MDCK cells type II have been used for transfection with the mdr1 -gene, the gene coding for the P-gp (Pastan et al., 1988). The goal was to construct a useful tool for the screening of potential P-gp substrates and inhibitors, however, transport studies remain scarce (Horio et al., 1989). We have characterised the MDR1-MDCK cells by various methods. Under identical conditions, MDR1MDCK cells exhibit similar growth curves as the MDCK wild type reaching the stationary phase later (|10 days after seeding) than the parent cells. MDR1-MDCK cells, as other transfected cells, are not contact inhibited and form irregular multilayers, which have partly lost their ¨ characteristic polarity (Hammerle et al., 2000). TJ are found not only close to the apical surface, but also within the cell layers. MDR1-MDCK cells reproducibly show 2 TEER values .1,000 V cm with fluctuation up to 10,000 2 V cm . A Papp value for mannitol of about 3310 26 cm / s was determined, i.e. about ten times higher than for the MDCK parents (see Section 4.2). As a result of the transfection of the mdr1 -gene, MDR1-MDCK cells express large amounts of P-gp. CLSM studies revealed that strong, homogeneous P-gp expression is found at the apical side of the uppermost cell layer, but some is also found throughout the lower layers within the cytoplasm of ¨ the cells (Hammerle et al., 2000).

3.5. ECV304 cells Efforts have been made over the years to define an adequate BBB model. Commonly used cell culture models for BBB are based on primary brain microvessel endothelial cell cultures in co-culture with astrocytes (Engelbertz et al., 2000). However, cell lines have definite advantages over primary cultures with respect to easy handling and reproducibility of the starting material despite their draw¨ backs, i.e. loss of typical BBB characteristics (Kramer et al., 2000). Human endothelial cell lines are still scarce, and the ECV304 cells, a cell line reported to originate from human umbilical cord vein, has been used for various applications (Hughes, 1996; Hurst and Fritz, 1996; Dobbie et al., 1999; Vinals et al., 1999). When seeded at 5310 4 cells / cm 2 , ECV304 cells (ATCC strain) are confluent after 1 day in culture and grow to a cell density of 8310 5 cells / cm 2 within 15–21 days on PET membranes. At this stage the stationary growth phase is reached with TEER values of 250–350 V cm 2 . ECV304 cells form monolayers, which display a fairly complete TJ network after confluence. A Papp value for mannitol of 1.8310 26 cm / s was determined. Little P-gp is expressed and activity (rho123 assay;

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see Section 4.3.) can only be measured |50 days after ¨ seeding (Hammerle et al., 2000). The identity of the ECV304 cell line has recently been questioned (Dirks et al., 1999). Fingerprinting revealed an identical genotype of ECV304 and T24, an epithelial urinary bladder carcinoma cell line. Still, the phenotype of the ECV304 comprises some typically endothelial features (manuscript in preparation). LDL-uptake was demonstrated in ECV304 cells, but was not found in T24 cells. Von Willebrand factor was expressed in ECV304 cells, whereas in T24 cells only low levels could be detected. As no alternative endothelial cell line with high TEER and typical endothelial features is available at the time, ECV304 cells are still of interest as a BBB model system for specific purposes.

4. Transport studies with various cell culture models

4.1. General The apparent permeability coefficient (Papp ) is used to quantify transport in a two-chamber diffusion system. Drug transfer from the donor to the receiver chamber through the cell layer of interest is measured as increase in drug concentration in the receiver chamber over time, and the differential increase in the amount of drug is calculated. Papp [cm / s] values are determined as follows: dQ 1 Papp 5 ] ]] dt c 0 A

(1)

where dQ / dt [mol / s] is the increase in the amount of drug in the receiver chamber per time interval, A [cm 2 ] the growth area of the cell culture insert, and c 0 [mol / ml] the initial drug concentration in the donor chamber. Conditions are chosen such that sink conditions are maintained throughout, which means that the kinetics of transfer is followed up to a maximum in the receiver chamber of about 10% of the total amount of drug applied. Control experiments were run with membranes without cells for all compounds. Correction for transport through the control membranes did not significantly change the Papp values for the drugs tested (manuscript in preparation). The experimental set-up has to be standardised for transport studies. In our laboratory we used the twochamber Costar  ‘‘vertical diffusion system’’, in which the membrane supporting the cell layer is vertically inserted between donor and receiver chamber (Fig. 4). For comparison, the Falcon  horizontal insert system in multiwell plates mounted on a rocking plate was applied. As we could show (manuscript in preparation), significant differences in Papp values were encountered in the two transport systems. Tenfold lower Papp values were obtained in the Falcon  insert system as compared to the Costar  system. This means that care has to be taken when comparing published Papp values. Quantitative comparison can only

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complete balance of uptake into cells as well as into the insert membrane and to estimate the possible loss through non-specific adsorption.

4.2. Comparative permeation studies The standardised cell culture models were used in our laboratory to gain more information on similarities and dissimilarities, respectively, between the different cell types with respect to drug permeation. Studies were performed with a set of non-homologous drug compounds with log D 7.4 octanol values between ,0 and 13.5 (Table 2). The corresponding molecular weights were between 130 and 500. Papp values were calculated from data obtained with apical-to-basal transport in the various cell culture models, and correlations were made between pairs of cell types, e.g. MDCK parents vs. MDR1-MDCK cells, and MDCK vs. Caco-2 cells. To start out, a small set of compounds was studied in MDCK parents and MDR1-MDCK cells, respectively (Fig. 5). As already listed in Table 1, MDR1-MDCK cells are more permeable to inannitol by a factor of ten as compared to MDCK type II cells. This is particularly well illustrated with the log–log plot (Fig. 5A). Enhanced permeability for mannitol in the transfected cells is found despite the fact that TEER values are significantly higher in these cell cultures as compared to the MDCK type II cell cultures (Table 1). For diazepam, warfarin and hydroxyacefamide Papp , values are similar in both cell types. This is evident from the log–log plot as well as from the linear plot (Fig. 5B), although in the latter differences can be better appreciated. At least for passive permeation multilayers ¨ (Hammerle et al., 2000) do not seem to have a major impact. Comparative studies were also made between the MDCK type II cells and the ECV304 cells. Again no

Fig. 4. Experimental set-up for transport studies with the Costar  vertical diffusion system. Media used in transport studies were the same as listed in Table 1 for cell culture except that the amount of FCS was reduced from 10 to 1%. Apparent permeation coefficients (Papp ) were calculated as described (see Section 4.1; manuscript in preparation).

be made safely if data were produced under identical conditions. To monitor drug permeation in a two-chamber system, a sensitive detection method has to be applied. With radiolabelled compounds, the sensitivity is high and drug concentrations can be kept low. Experiments run with MDCK cells in the Costar  system revealed concentration independent Papp values for the tested compounds within a range of 10 nM to 6 mM (manuscript in preparation). Problems may arise in transport studies because of the poor solubility of compounds. As a consequence, our transport medium was supplied with 1% FCS in the donor as well as in the receiver chamber to enhance solubility and reduce sticking of compounds to surfaces. Recoveries determined for each transport experiment permit to make a Table 2 Compounds used for transport studies No.

Model drug (radioactive label)

MW

pKa

log P a

log DpH

1 2 3 4 5 6 7 8 9 10 11 12 13 14

R,R-Chloramphcnicol ( 14 C) Chlordiazepoxide ( 14 C) Diazepam ( 14 C) Domperidone ( 3 H) Glibenclamide ( 3 H) Hydroxyacetanilide ( 3 H) Lidocaine ( 14 C) Mannitol ( 14 C) R,S-Propranolol ( 3 H) Salicylic acid ( 14 C) Theophylline ( 14 C) Tolbutamide ( 14 C) Warfarin ( 14 C) R,S-Verapamil ( 3 H)

323.1 299.8 284.7 425.9 494.0 151.2 234.3 182.2 259.3 138.1 180.2 270.4 308.2 454.6

5.50 (base) 4.60 (base) 3.31 (base) 7.90 (base) 5.30 (acid) 9.50 (acid) 7.78 (base) – 9.22 (base) 3.00 (acid) 8.57 (acid) 5.30 (acid) 4.90 (acid) 8.60 (base)

1.00–1.64 1.45–2.50 2.49–2.99 4.05 3.08 1.16 1.28–2.26 – 0.73–3.54 22.11–2.26 20.02 2.34–2.52 1.83–3.79 1.04–2.7

1.00–1.63 1.45–2.50 2.49–2.99 3.43 0.98 1.16 0.75–1.73 – (21.10)–1.71 (26.51)–(22.14) (20.05) 0.24–0.42 (20.67)–1.29 (20.19)–1.47

a

Hansch and Leo, 1979.

P P Values calculated from (1): D 5 ]]]] (acid); D 5 ]]]] (base). (1 1 10 pH – pK a ) (1 1 10 pK a – pH ) c Number of transport experiments apical-to-basal. b

b 7.4

n (a→b)c MDCK

n (a→b)c Caco-2

4 8 7 3 4 6 25 13 33 3 12 6 3 3

9 9 6 3 6 6 6 5 12 6 9 9 6 9

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Fig. 5. Comparison of Papp values in MDR1-MDCK and MDCK parent cells. Transport was studied in the apical to basal direction. Experiments were performed as indicated in Fig. 4, and Papp values were calculated (see Section 4.1). Data points represent mean values and standard deviations of $3 independent experiments. (. . . .) line of identity. (A) log–log plot; (B) linear plot.

Fig. 6. Comparison of Papp values of a set of non-homologous compounds in MDCK and Caco-2 cells. The numbers of the tested compounds correspond to the ones listed in Table 2. Transport was studied in the apical to basal direction. Experiments were performed as indicated in Fig. 4, an Papp values were calculated (see Section 4.1). Data points represent mean values and standard deviations of n independent experiments (n indicated in Table 2). (? ? ?) line of identity. (A) log–log plot; (B) linear plot.

significant difference was found between the Papp values determined in the two systems (manuscript in preparation). Comparison of drug permeation in Caco-2 cells and MDCK type II cells, respectively, was made for the complete set of compounds listed in Table 2. All substances were chemically stable under the incubation conditions (data not shown). Data from apical-to-basal transport experiments are shown in Fig. 6. The differences in Papp values between the two cell types are relatively small. The correlation coefficient is r 2 50.86 (n514) as compared to the value of r 2 50.79 obtained by Irvine et al. (1999) for a set of 55 compounds. The set of Irvine et al. (1999) also comprised propranolol, salicylic acid and warfarin. The Papp values of these compounds were in the same range as those determined in our laboratory (within a factor of two). No trend is found for domperidone ([4), glibenclamide ([5), theophylline ([11) and verapamil

([14), which have been described as P-gp substrates (Stein, 1997). This is in accordance with the data from Irvine et al. (1999). The authors hypothesised that P-gpmediated transport was saturated under their experimental conditions. However, in our experiments drug concentrations were between 15 to 10 4 times lower than those of Irvine et al. (1999), i.e. in the pM to nM range, and we neither find a difference between Papp values determined in MDCK and Caco-2 cells. Thus saturation of the transporter can be excluded. From these data we conclude that P-gp substrates cannot be spotted by comparative studies of apical-to-basal permeation in Caco-2 and MDCK cells. This will be further substantiated in the next section. Another aspect to be considered in transport studies is the possible metabolic activity of CYP enzymes in the MDCK and Caco-2 cell cultures, respectively. Of particular interest is the CYP3A4 isoform, which is involved in drug

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metabolism (Guengerich, 1999). Western blot analysis of the cell cultures in our laboratory revealed high CYP3A4 expression for the MDR1-MDCK cells, less for the Caco-2 cells and not detectable levels in MDCK parent cells (data unpublished). Preliminary experiments indicate that a low enzyme activity is present in MDR1-MDCK cells and no activity is measurable in Caco-2 cells. This is in accordance with Crespi et al. (1996), who found a low CYP3A4 activity in nontransfected Caco-2 cells.

4.3. Screening for P-gp substrates Traditionally, P-gp substrates are identified by comparison of apical-to-basal and basal-to-apical transport in a two-chamber system, and by inhibition of the carriermediated component, e.g. by verapamil or cyclosporin A. Transport studies in the apical-to-basal and basal-to-apical direction were thus performed in Caco-2 cells with a subset of eight compounds. As a control the same drugs were tested in MDCK cells with the same protocol. As can be seen in Fig. 7, no significant differences between apical-to-basal and basal-to-apical permeation were found for any of the substances tested in the MDCK cells,

Fig. 7. Comparison of apical-to-basal (a→b) and basal-to-apical (b→a) transport in MDCK and Caco-2 cells, respectively. The numbers of the tested compounds correspond to the ones listed in Table 2. Experiments were performed as indicated in Fig. 4, and Papp values were calculated (see Section 4.1). Data represent mean values and standard deviations of $3 independent experiments.

whereas with Caco-2 cells, differences were observed for several compounds. For domperidone, glibenclamide, theophyllin and verapamil, Papp values of the basal-toapical permeation were significantly higher than from apical-to-basal permeation. All these drugs have been reported to act as substrates for the P-gp transporter (Stein, 1997). The same protocol, namely apical-to-basal and basal-toapical transport in a the two-chamber vertical diffusion system, was also applied to the MDR1-MDCK cells, which express high levels of P-gp at the apical surface (Pastan et ¨ al., 1988; Hammerle et al., 2000). Vinblastin was tested as a typical P-gp substrate in the presence and absence of verapamil as an inhibitor. No significant difference was found between the apical-to-basal and basal-to-apical transport, which means that P-gp activity could not be ¨ shown with this experimental set-up (Hammerle, unpublished). In contrast to passive permeation, where multilayers do not interfere (see Section 4.2), P-gp-mediated transport is possibly influenced by the presence of several cell layers. Most likely, the loss of polarisation in the ¨ lower cell layers (Hammerle et al., 2000) precludes directed transport in these cells. The P-gp activity in the uppermost cell layer does not appear to produce enough net efflux to show up as a difference in the apical-to-basal and basal-to-apical transport balance. This may explain the fact that despite the successful transfection of the mdr1 gene, the MDR1-MDCK cells have not been established as a transport model and publications remain scarce (Horio et al., 1989). As a consequence, we tested a one-chamber transport system to screen for P-gp substrates and inhibitors. Such a system, which concentrates on the efflux out of cells, has previously been established for microscopic studies with the fluorescent compound rho 123 as a P-gp substrate (Shapiro and Ling, 1997). We used this approach for the MDR1-MDCK cells. Rho123 efflux was monitored in the CLSM in living cells that had been preloaded with the ¨ fluorescent compound (Hammerle et al., 2000). It could be abolished by inhibition with verapamil. Efflux of rho123 out of MDR1-MDCK cells and its partial inhibition by verapamil and cyclosporin A can also be monitored by fluorescence spectrophotometric analysis (Fig. 8). In a different approach, using a one-chamber system as well, P-gp activity in MDR1-MDCK cells was assessed by an uptake assay. Radiolabelled vinblastin was incubated with cells in the presence or absence of inhibitors (verapamil or cyclosporin A). After washing of the cells, the amount of vinblastin in the cells was determined by liquid scintillation counting (Fig. 9). The amount of vinblastin was lowest in the control culture due to P-gp activity (Fig. 9A). In the presence of verapamil (Fig. 9B) and cyclosporin A (Fig. 9C), more vinblastin remained in the cells. From these experiments, we conclude that MDR1-MDCK cells are useful for screening for P-gp substrates and inhibitors

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5. Conclusions and perspectives

Fig. 8. Efflux kinetics of rho123 from MDR1-MDCK cells. For kinetic studies cultures were grown in TPP  6-well plates. Cells were incubated for 30 min at 378C with rho123 (100 mg / ml in assay buffer; Fontaine et al., 1996). After washing twice with ice cold assay buffer, efflux was determined by incubation at 378C with 3 ml prewarmed assay buffer on a rocking plate. At the times indicated, samples were taken for measurement in the fluorescence spectrophotometer (excitation: 480 nm, emission 535 nm, slit width 4 nm). For inhibition experiments 0.1 mM verapamil or 0.1 mM cyclosporin A (final concentrations) were added 5 min prior to washing. The same respective inhibitor concentrations were maintained throughout the efflux experiment. ♦, efflux without inhibitor; m, with verapamil; s, with cyclosporin A. Data points represent mean values and standard deviations of $3 independent experiments.

under the condition that efflux or uptake studies are performed in a one-chamber system. The lack of polarisation of cells within the multilayers precludes determination of Papp , values in a two-chamber diffusion system.

Provided that cell cultures are cultivated under controlled standardised conditions, they show cell type-specific characteristics with respect to growth, expression of proteins and cyoarchitecture. Papp values are reproducible. However, depending on the experimental set-up used for transport studies, calculated Papp values can differ widely even with standardised cell cultures. All tested cell culture models, independently of their origin, yield similar results for transcellular passive permeation under identical conditions; multilayers do not appear to interfere. For the screening of potential P-gp substrates, transport has to be compared between the apical-to-basal and basal-to-apical direction across P-gp-expressing cell monolayers in the presence and absence of P-gp inhibitors to test for specificity. In turn, P-gp substrates are not necessarily detected if transport is compared between MDCK cells, which express little P-gp, and Caco-2 cells, which at 21 days after seeding show high P-gp expression. Although MDR1-MDCK cells have ample P-gp expression at the apical surface of the uppermost cell layer, transport studies in a two-chamber vertical diffusion system do not provide interpretable data with respect to P-gp-mediated transport. This is possibly due to loss of polarity encountered in the lower layers of multilayer cell cultures typical for transfected cells. In these cases, a one-chamber system can be used for efflux and / or uptake studies in the presence and absence of inhibitors. If cell cultures are to be used for screening of passive transcellular permeation, MDCK cells provide a potential permeation model (Irvine et al., 1999). Despite the origin from kidney they lend themselves as an easy to handle system with low expression of transporters and little metabolic activity. Caco-2 cells are more prone to external influences, which risk to alter the expression of transporters such as P-gp, and to change growth characteristics (monolayers / multilayers). For the screening of P-gp substrates, better model systems have to be developed. The problem that contact inhibition and polarisation of cells are lost in transfected cells remains to be solved, before vertical diffusion systems can routinely be used.

Acknowledgements Fig. 9. Vinblastin uptake in MDR1-MDCK cells. Cells were grown in 24 well plates. They were incubated at 378C on a rocking plate with 200 nM radiolabelled vinblastin (specific activity: 3000 Bq / nmol) in growth medium containing 1% FCS. For inhibition, 0.1 mM verapamil and 0.1 mM cyclosporin A, respectively, were added together with vinblastin. After 2 h, the cell layer was washed twice with ice cold EBSS and the cells lysed in 0.5 ml 1N NaOH containing 1% SDS. The radioactivity of the samples was determined by liquid scintillation counting. Data points represent mean values and standard deviations of ?3 independent experiments. (A) uptake without inhibitor; (B) uptake in the presence of verapamil; (C) uptake in the presence of cyclosporin A.

Projects were supported by the Roche Research Foundation, Basel, Switzerland (S.H.), by a grant of ETHZ (K.S.), and by a travelling research fellowship from The Wellcome Trust, UK (S.K.).

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