Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux

doi:10.1016/j.ijpharm.2006.11.031

International Journal of Pharmaceutics

Volume 336, Issue 1, 4 May 2007, Pages 12-21

https://doi.org/10.1016/j.ijpharm.2006.11.031 Get rights and content

Abstract

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [³H]-cyclosporine-A and [¹⁴C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [³H]-cyclosporine-A and [¹⁴C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A → B) permeability of [³H]-cyclosporine-A and [¹⁴C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A → B transport of [³H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.

Introduction

ATP-binding cassette (ABC) proteins comprise a large super family of transmembrane transporters that utilize ATP hydrolysis to translocate their substrates across biological membranes. Members of ABC proteins such as drug resistance proteins, P-glycoprotein (P-gp; MDR1) (Dey et al., 2003) and multidrug resistance associated proteins (MRPs) (Merino et al., 2004) are believed to be major barriers to drug delivery. MRPs can confer drug resistance and cause reduced accumulation of therapeutic agents. A reduction in intracellular drug concentrations was found to correlate with the expression of P-gp encoded by multidrug resistance (MDR1) gene and a transmembrane phosphoglycoprotein (MRP) (Bredel et al., 2004, Sawicka et al., 2004). Also, collective role of P-gp and MRPs in efflux of drug molecules has been reported (Xiao et al., 2005). Human cornea has been shown to express P-gp, which serves as a drug efflux pump (Dey et al., 2003, Dey et al., 2004). Erythromycin (Gaynor et al., 2005), cyclosporine-A (Hesselink et al., 2005), quinolones (ciprofloxacin, grepafloxacin) (Naruhashi et al., 2002, Seral et al., 2003, Michot et al., 2004), steroids, sulfated steroids, estradiol 17-beta-d-glucuronide (Chen et al., 2005, Chu et al., 2004, Zelcer et al., 2003) are widely employed in ocular therapy. However, these drugs are known to be good substrates for MRP. ABCC2 (a member of MRP family) is expressed on the bile canalicular membrane of hepatocytes, as well as on the brush border membrane of renal and intestinal epithelial cells. ABCC2 excretes structurally diverse organic anions including reduced glutathione, glutathione conjugates, bilirubin glucuronides, sulfated and glucuronidated bile salts and nonconjugated organic anions into bile (Suzuki and Sugiyama, 1998, Keppler and Konig, 2000), which accounts for most of the bile salt-independent bile flow (Lauterburg et al., 1984). Roelofsen et al. (1991) were the first to report the efflux of ABCC2 substrates from isolated rat hepatocytes. ABCC2 belongs to subfamily C of the ABC super family and this protein confers resistance to a wide array of chemotherapeutic agents. Role of P-gp as a barrier to corneal delivery of drugs has been reported earlier (Kawazu et al., 1999, Dey et al., 2004).

Therefore, the main objective of this study is to determine if human cornea expresses a homologue of MRP, ABCC2 that is known to express on the apical side of cell membrane and to examine the role in drug efflux. In vitro uptake experiments were performed with [³H]-cyclosporine-A, [¹⁴C]-erythromycin and specific MRP inhibitor (MK-571) employing human corneal epithelial cells (SV40-HCEC), rabbit primary corneal epithelial cells (rPCEC), and MRP-transfected Madin–Darby canine kidney cells (MDCKII-MRP2). Transport experiments were performed with SV40-HCEC and freshly excised rabbit cornea. Also, RT-PCR immunoprecipitation followed by Western blot analysis were applied to confirm the presence of ABCC2 in corneal epithelium.

Section snippets

Materials and methods

MK-571, a specific inhibitor for MRP family and which does not interact with P-gp (Stephens et al., 2002, Walter et al., 2003, Park and Sinko, 2005) was obtained from Biomol International L.P. (PA, USA). [¹⁴C]-erythromycin (specific activity 48.8 mCi/mmol), a proven substrate for MRP (Terashi et al., 2000) was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). [³H]-cyclosporine-A (specific activity: 8 Ci/mol), also a known substrate for MRP (Qadir et al., 2005), [³H]-mannitol

Transfected human corneal epithelial cells

SV40-immortalized human corneal epithelial cell line was obtained as a gift from Araki-Sasaki et al. (1995). Mycoplasma-free SV40-HCEC were grown at 37 °C, humidified 5% CO₂/95% air atmosphere in a culture medium containing 50% of Dulbecco's modified Eagle's medium (DMEM) and 50% of Ham's nutrient mixture F-12 (from Gibco, Paisley, UK) supplemented with 15% (v/v) fetal bovine serum (FBS, from Gibco), antibiotic, antimycotic solution (penicillin 100 U/ml, streptomycin 100 μg/ml and amphotericin B 25

Uptake experiments

Uptake studies were conducted according to standard protocols, with minor modifications (Dey et al., 2003). Briefly, 10–12 days after seeding, the medium was aspirated, and cells were rinsed with DPBS (composition: 130 mM NaCl, 7.5 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM MgSO₄, 1 mM CaCl₂, 0.03 mM KCl, and 5 mM glucose and pH 7.4) at 37 °C and then equilibrated in 1 ml of DPBS for 30 min at 37 °C for control and inhibition experiments. IC50 of MK-571 was found to be about ∼110 μM in rPCEC (Karla et al., 2006)

Permeability measurements

Steady-state fluxes were determined from the slope of the cumulative amount of drug transported versus time graph and expressed per unit of cross-sectional surface area of the membrane as described by Eq. (1). The cumulative amount of drug transported is the total amount of drug present in the receiver chamber after a defined time interval. $Flux (J) = \frac{d M}{d t} \times \frac{1}{A}$

M is the cumulative amount of drug transported and A is the cross-sectional surface area exposed to permeate. Permeability's of SV40-HCEC

Uptake of [³H]-cyclosporine-A and [¹⁴C]-erythromycin in cell culture models

Dose dependant inhibition of ABCC2 was noticed with increased concentrations of MK-571 in both SV40-HCEC and rPCEC. IC50 value obtained for MK-571 in SV40-HCEC was ∼75 μM (Fig. 1) and rPCEC was ∼110 μM (Fig. 2), respectively. Hence concentrations of ∼75 and ∼100 μM were employed for further studies. In MDCKII-MRP2 cells uptake of both [³H]-cyclosporine-A was elevated by ∼1.5-fold (155.51 ± 19.50)% over control (100 ± 10.57)% and uptake of [¹⁴C]-erythromycin was elevated by ∼2-fold (195.16 ± 22.99)%,

Discussion

Topical delivery into the cul-de-sac is, by far, the most common route of ocular drug administration. Absorption from this site may involve corneal and/or noncorneal pathways. The noncorneal route involves transport of the drug across the sclera and the conjunctiva into the deeper intraocular tissues. This route of drug absorption results in poor ocular bioavailability. As molecules reach beyond the corneal–scleral junction, absorption by the local capillary beds may result in drug dumping into

Acknowledgements

We would like to thank Dr. Claudio Tiribelli, MD PhD, Director Centro Studi Fegato (Liver Research Center) (Trieste, Italy) for his help with culturing the MDCKII-MRP2 cell line. We would like to thank Dr. Allison McDermott, associate professor, University of Houston (College of optometry) for her help with culturing of SV40-HCEC. We would like to thank Budda Balasubrahmanyam, graduate research fellow, UMKC-School of pharmacy for his excellent contribution in RT-PCR studies.

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^☆: This work is supported by NIH Grants R01 EY09171-12 and R01 EY10659-10. Part of this work is presented in a preliminary format at Association of Pharmaceutical Scientists (AAPS) (2005), Nashville, Tennessee, Pharmaceutics Graduate Student Research Meeting (PGSRM) Kansas (2005) and Minnesota (2006).

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Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux☆

Abstract

Introduction

Section snippets

Materials and methods

Transfected human corneal epithelial cells

Uptake experiments

Permeability measurements

Uptake of [3H]-cyclosporine-A and [14C]-erythromycin in cell culture models

Discussion

Acknowledgements

Anal. Biochem.

Lancet Oncol.

Am. J. Transplant.

J. Biol. Chem.

An SV40-immortalized human corneal epithelial cell line and its characterization

Invest. Ophthalmol. Vis. Sci.

Comparison of the effects of continuous and intermittent systemic administration on the penetration of gentamicin into infected rabbit eyes

J. Infect. Dis.

Increasing bacterial resistance in pediatric acute conjunctivitis (1997-1998)

Antimicrob. Agents Chemother.

Transport of bile acids, sulfated steroids, estradiol 17-beta-d-glucuronide, and leukotriene C4 by human multidrug resistance protein 8 (ABCC11)

Mol. Pharmacol.

Transport of ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins MRP1, MRP2, and MRP3

J. Pharmacol. Exp. Ther.

Efficacy and ocular penetration of sparfloxacin in experimental streptococcal endophthalmitis

Antimicrob. Agents Chemother.

Pharmacokinetics of erythromycin in rabbit corneas after single-dose infusion: role of P-glycoprotein as a barrier to in vivo ocular drug absorption

J. Pharmacol. Exp. Ther.

Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines

Invest. Ophthalmol. Vis. Sci.

Topical ocular antibiotics induce bacterial resistance at extraocular sites

Br. J. Ophthalmol.

Multidrug resistance-associated protein-1 functional activity in Calu-3 cells

J. Pharmacol. Exp. Ther.

Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers

Pharm. Res.

Modulation of P-glycoprotein-mediated efflux by prodrug derivatization: an approach involving Peptide transporter-mediated influx across rabbit cornea

J. Ocul. Pharmacol. Ther.

Characterization of cyclosporin A transport in cultured rabbit corneal epithelial cells: P-glycoprotein transport activity and binding to cyclophilin

Invest. Ophthalmol. Vis. Sci.

Uptake of [³H]-cyclosporine-A and [¹⁴C]-erythromycin in cell culture models