The long-term goal of this research is to improve the ocular bioavailability of peptidomimetics in particular and polar drugs in general by targeting dipeptide transport mechanisms in the cornea and conjunctiva. The central hypothesis of the present 3-year proposal is that the corneal and conjunctival epithelial penetration of cidofovir could be improved through derivatization into prodrugs that are substrates for the H+-dipeptide transporters (PepT1/PepT2). Cidofovir is a hydrophilic nucleoside shown to be effective in the treatment of cytomegalovirus retinitis following i.v. and intravitreal administration. Thus, there are three specific aims: (l) To kinetically characterize the H+-dipeptide transport mechanism in the cornea and conjunctiva, (2) To determine whether it is PepT1, PepT2, or both, that contributes to dipeptide transport in these tissues, and (3) To determine the extent to which dipeptide prodrugs would improve the corneal and conjunctival epithelial penetration of cidofovir. H+-dipeptide transport in the pigmented rabbit cornea and conjunctiva will be measured using 3H-carnosine as a model substrate. Experiments will be performed to obtain evidence for H+-dipeptide transport: electrogenicity; directionality; dependency on energy, pH, and concentration; and substrate specificity. Moreover, Northern blot analysis will be conducted to crosscheck the recent preliminary RT-PCR results on the possible existence of PepTI but not PepT2 mRNA in corneal epithelial cells and of both PepT1 and PepT2 mRNAs in conjunctival epithelial cells. Western blot analysis and immunostaining will be performed to further delineate the cellular distribution of PepT1/PepT2 in the corneal and conjunctival epithelia. Two dipeptide prodrugs of cidofovir will then be synthesized and tested with respect to corneal and conjunctival penetration. The amino acid linker that affords prodrugs with a high affinity for the H+-dipeptide transporter will next be applied to two other nucleosides: uridine and zidovudine. Finally, the resulting prodrugs as well as the one for cidofovir will be tested for possible selectivity towards a particular dipeptide transporter isoform. The significance of this research is that it will set the stage for future characterization of the molecular identity, regulation, and subcellular trafficking of the H+-dipeptide transporter isoform in the corneal and conjunctival epithelial cells. From the drug delivery point of view, the results may be applicable for improving the ocular absorption of other topically applied low m.w. hydrophilic drugs.
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