Research Articles
The Effect of Cell Culture Conditions on Saquinavir Transport Through, and Interactions with, MDCKII Cells Overexpressing hMDR1

https://doi.org/10.1002/jps.10458Get rights and content

Abstract

MDCK cells are cultured using wide‐ranging conditions and can produce variable results. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII‐PGP) and MDCKII wild‐type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 × 105 or 5 × 104 cells/cm2), substratum (polycarbonate ± collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport. The saquinavir efflux ratio (ratio of BL → AP/AP → BL permeability) for MDCKII‐PGP cells (6.9 × 105 cells/cm2) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 × 104 cells/cm2 on polycarbonate (78) or collagen‐coated polycarbonate (126). The MDCKII/wt saquinavir efflux ratio was 9. Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen‐coated membranes. Collagen coating caused increased Pgp expression and saquinavir efflux ratios correlated (r2 = 0.96) with Pgp expression levels [MDCKII‐PGP (on collagen‐coated polycarbonate) > MDCKII‐PGP (on polycarbonate) > MDCKII/wt (on collagen‐coated polycarbonate)]. These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1957–1967, 2003

Introduction

Saquinavir mesylate (saquinavir) was the first HIV protease inhibitor that was approved for sale in the United States.1 However, saquinavir absorption is highly variable as indicated by AUC coefficients of variation that are ≥30%,2 and the two marketed saquinavir capsule formulations have mean oral bioavailabilities that range from only 4 to 16%.1 Recent investigations have demonstrated that saquinavir is a substrate for the P‐glycoprotein (Pgp, ABCB1) efflux pump,3 and correlated its transport by Pgp with its reduced bioavailability and limited CNS penetration.4

To further investigate saquinavir's transport mechanisms, and the roles of putative transporters, a series of MDCKII (Madin‐Darby canine kidney cells, strain II) cell lines stably transfected with human Pgp, multidrug resistance‐associated protein (MRP1), or canalicular multispecific organic anion transporter (MRP2) were selected as models of epithelial drug transport.5., 6., 7., 8., 9., 10.

MDCKII cells are a commonly used model of drug transport.11 However, there are potential model specific and drug specific limitations to the use of MDCK cell monolayers for probing transport mechanisms. For example, MDCK wild‐type cells are a heterogeneous cell line and can produce variable results.12,13 In addition, the potential variability of MDCK cell transport study results seems to be exacerbated by the current use of a wide range of seeding densities (from 5 × 103 to 6.6 × 105 cells/cm2)11,12 and substrata [including polycarbonate, polyester, collagen‐coated polytetrafluoroethylene (PTFE), polystyrene and a variety of extracellular matrix component preparations to facilitate cell growth and differentiation].11,14,15 Because cell culture conditions have been shown to effect gene expression,16,17 the use of multiple substrata also suggests the possibility of cultured cell expressional differences that need to be assessed. Closely related to the issue of gene expression is the potential role of endogenous transporters in MDCK cells. Historically, MDCK cells were accepted as being free of endogenous transporters such as the organic cation transporter (OCT).18 Although MDCK cells were shown to express endogenous canine Pgp,14,18 this does not rule out the potential contributions of other endogenous transporters whose expression may vary with substrata. Furthermore, saquinavir reportedly impairs the barrier function of a model human intestinal cell line (HT‐29/B6), causing increased mannitol (paracellular) flux.19 Saquinavir's disruption of epithelial cell barrier function could potentially increase any variability associated with the MDCK cell model.

The hypothesis driving this study was that the combined effects of MDCKII cell seeding density (6.9 × 105 or 5 × 104 cells/cm2), substratum (polycarbonate ± collagen coating) and saquinavir presence would significantly alter monolayer integrity, Pgp expression, and saquinavir transport. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII‐PGP) and MDCKII wild‐type cells (MDCKII/wt) were chosen to evaluate the impact of these changes.

Section snippets

Chemicals

Saquinavir mesylate (saquinavir) and [14C]saquinavir were provided by Roche Laboratories (Nutley, NJ).20 The radiochemical purity of [14C]saquinavir was >98% with a specific activity of 26.5 μCi/mg. Saquinavir was shown to be stable in solution for the duration of the studies described herein. [3H]mannitol, [14C]mannitol, and [3H]propranolol were obtained from Sigma Chemicals (St. Louis, MO). Medium, fetal bovine serum (FBS), nonessential amino acids, trypsin, and transport buffer components

Effect of Seeding Density and Substratum on Saquinavir Transport through MDCKII‐PGP Cell Monolayers

To investigate the effects of seeding density and substratum on saquinavir transport through MDCKII‐PGP monolayers, the apical to basolateral (AP → BL) and basolateral to apical (BL → AP) permeabilities of saquinavir and mannitol were determined using MDCKII‐PGP cell monolayers that were prepared by seeding 6.9 × 105 cells/cm2 or 5 × 104 cells/cm2 onto polycarbonate membranes and also by seeding 5 × 104 cells/cm2 onto collagen‐coated polycarbonate membranes. The permeabilities and efflux ratios

Discussion

To our knowledge, this one of the few studies that directly and quantitatively links differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport. The importance of these findings is that they highlight the interrelatedness of cell culture conditions with changes in transport and transporter expression. They also emphasize the critical application of controls in cell culture models.

One goal of this investigation was to develop a standard

Acknowledgements

Financial support has been provided by NIH Grant AI 42007. This work has also been supported by Roche Laboratories, GlaxoSmithKline and Merck Laboratories. We thank Raymond Evers and Piet Borst for providing us with the MDCKII cell lines. We additionally thank Drs. Kurt Amsler, Joseph Polli, and Hugh Wiltshire for their assistance.

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