Cell cultures as tools in biopharmacy
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 of compounds (Krämer, 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, Δlog 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 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 Doctanol7.4 values between <0 and +3.5. 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. 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 P-glycoprotein, 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.
Section snippets
Standardisation of cell cultures
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
General
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.
Caco-2 cells
Caco-2, an epithelial human colon adenocarcinoma cell line, has been widely used to predict intestinal absorption of potential
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:where dQ/dt [mol/s] is the increase in the amount of drug in the receiver chamber per time
Conclusions and perspectives
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
Acknowledgements
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.).
References (33)
- et al.
P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels
J. Pharm. Sci.
(1998) - et al.
Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial Caco-2 cells
Biochem. Biophys. Res. Comm.
(1991) - et al.
Upregulation of intercellular adhesion molecule-1 expression on human endothelial cells by tumour necrosis factor-α in an in vitro model of the blood–brain barrier
Brain Res.
(1999) - et al.
Use of rhodamine 123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers
Life Sci.
(1996) - et al.
Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia
J. Biol. Chem.
(1989) Functional characterization of the spontaneously transformed human umbilical vein endothelial cell line ECV304: Use in an in vitro model of angiogenesis
Exp. Cell Res.
(1996)- et al.
MDCK (Madin-Darby canine kidney) cells: a tool for membrane permeability screening
J. Pharm. Sci.
(1999) Absorption prediction from physicochemical parameters
Pharm. Sci. Tech. today
(1999)- et al.
Identification of two strains of MDCK cells which resemble separate nephron tubule segments
Biochim. Biophys. Acta
(1981) - et al.
High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells
Biochim. Biophys. Acta
(1999)
Intestinal drug absorption and metabolism in cell cultures: Caco-2 and beyond
Pharm. Res.
The use of cultured epithelial and endothelial cells for drug transport and metabolism studies
Pharm. Res.
Polarized monolayers formed by epithelial cells on a permeable and translucent support
J. Cell Biol.
Development of Caco-2 cells expressing high levels of cDNA-derived cytochrome P4503A4
Pharm. Res.
Predicting blood–brain barrier permeation from three-dimensional molecular structure
J. Med. Chem.
ECV304 (endothelial) is really T24 (bladder carcinoma): Cell line cross-contamination at source
In Vitro Cell Dev. Biol. Anim.
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