Abstract
Efflux pump like P-glycoprotein (P-gp) is known to be a major barrier to drug delivery. Functional P-glycoprotein has been recently identified in cornea and corneal cell lines. Thus, it is probable that P-glycoprotein may restrict in vivo ocular drug absorption, resulting in low ocular bioavailability. Experiments were designed using New Zealand albino (New Zealand White) rabbits to assess inhibitors of P-gp efflux to increase drug absorption. Anesthetized rabbits were given constant topical infusions of [14C]erythromycin in the presence and absence of inhibitors. Testosterone, verapamil, quinidine, and cyclosporine A were selected as P-gp inhibitors. Transport experiments were conducted in Madin-Darby canine kidney cells transfected with the human mdr1 gene (MDCK-MDR1). Erythromycin exhibited significant efflux out of MDCK-MDR1 cells, suggesting that erythromycin is a good substrate for P-gp. Ocular pharmacokinetic studies were conducted using a topical single-dose infusion method. Maximum inhibition of P-gp mediated efflux was observed with 500 μM testosterone. Area under the curve (AUC)0-∞ of erythromycin with 500 μM testosterone was almost 4 times higher than AUC0-∞ without any inhibitor. Rate of elimination (k10) for erythromycin and those with inhibitors was found to be similar (141 ± 23 min), suggesting that elimination pathways were not altered. All the inhibitors were found to be nontoxic. Verapamil also inhibited the efflux pump with moderate change in AUC0-∞ and Cmax compared with control. Thus, P-gp is found to be active in vivo, and it restricts topical erythromycin absorption across the cornea, which can be inhibited by known P-gp inhibitors. Therefore, ocular bioavailability of P-gp substrates can be significantly enhanced by proper selection of P-gp inhibitors.
Footnotes
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This work was supported by National Institutes of Health Grants R01 EY09171-08 and R01 EY10659-07.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.069583.
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ABBREVIATIONS: P-gp, P-glycoprotein; CsA, cyclosporine A; DPBS, Dulbecco's phosphate-buffered saline; MDCK, Madin-Darby canine kidney; MEM, minimal essential medium; rPCEC, primary culture of rabbit corneal epithelial cells; TEER, transepithelial electrical resistance; AP, apical; BL, basolateral; AUC, area under the curve; MTS, 3-(4,5-dimethlythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PMS, phenazine methosulfate; bp, base pair(s); RT-PCR, reverse transcriptase-polymerase chain reaction.
- Received April 6, 2004.
- Accepted June 2, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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