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. 2007 May 4;336(1):12-21.
doi: 10.1016/j.ijpharm.2006.11.031. Epub 2006 Nov 15.

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

Pradeep K Karla  1 , Dhananjay Pal, Tim Quinn, Ashim K Mitra
Affiliations

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

Pradeep K Karla et al. Int J Pharm. .

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. [(3)H]-cyclosporine-A and [(14)C]-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 approximately 272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band approximately 181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at approximately 190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [(3)H]-cyclosporine-A and [(14)C]-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 [(3)H]-cyclosporine-A and [(14)C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A-->B transport of [(3)H]-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.

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Figures

Figure 1
Figure 1
Sigmoidal dose response curve (variable slope) for SV40-HCEC obtained from Graph pad Prism 4.0.
Figure 2
Figure 2
Sigmoidal dose response curve (variable slope) for rPCEC obtained from Graph pad Prism 4.0.
Figure 3
Figure 3
Uptake of [14C]-erythromycin and [3H]-cyclosporine-A in the presence of MK-571 in MDCKII-MRP2, rPCEC and SV40-HCEC. Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 4
Figure 4
Uptake of [14C]-erythromycin in the presence of unlabeled cyclosporine-A (25 and 50 μM) in rPCEC and SV40-HCEC. Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 5
Figure 5
(A). Polarized transport of [3H]-cyclosporine-A in SV40-HCEC from A→B as compared from B→A direction. (B). A→B and B→A permeability of [3H]-cyclosporine-A in SV40-HCEC in the presence of MK-571 (75 μM). Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 5
Figure 5
(A). Polarized transport of [3H]-cyclosporine-A in SV40-HCEC from A→B as compared from B→A direction. (B). A→B and B→A permeability of [3H]-cyclosporine-A in SV40-HCEC in the presence of MK-571 (75 μM). Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 6
Figure 6
(A). Polarized transport of [14C]-erythromycin in SV40-HCEC from A→B as compared from B→A direction. (B). A→B and B→A permeability of [14C]-erythromycin in SV40-HCEC in the presence of MK-571 (75 μM). Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 6
Figure 6
(A). Polarized transport of [14C]-erythromycin in SV40-HCEC from A→B as compared from B→A direction. (B). A→B and B→A permeability of [14C]-erythromycin in SV40-HCEC in the presence of MK-571 (75 μM). Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 7
Figure 7
Polarized transport of [3H]-cyclosporine-A in rabbit cornea from A→B as compared from B→A direction. Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 8
Figure 8
Transport of [H3]-cyclosporine-A in rabbit cornea from A→B and B→A in the presence of MK-571 (125 μM). Each data point represents the mean ± SD of three determinations. Statistical significance was tested by two-factor ANOVA. *P ≤ 0.05; **P ≤ 0.01.
Figure 9
Figure 9
Expression of ABCC2 in HCEC, rPCEC, rabbit cornea along with MDCKII-MRP2 positive control. RT-PCR products ∼272 bp confirms the expression of ABCC2 in various corneal epithelial cells.
Figure 10
Figure 10
Expression of ABCC2 in SV40-HCEC along with MDCKII-MRP2 positive control. RT-PCR products ∼272 bp confirms the expression of ABCC2.
Figure 11
Figure 11
Expression of ABCC1 in SV40-HCEC and HCEC. RT-PCR products ∼181 bp confirms the expression of ABCC1.
Figure 12
Figure 12
Immunoprecipitation followed by western blot indicating the presence of ABCC2 in membrane fraction of human corneal epithelial cells along with membrane fraction of MDCKII-MRP2 (positive control) with cytosolic fraction of SV40-HCEC as negative control.
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