| HIV/AIDS has become one of the greatest threats to human health. Although inclusion of protease inhibitors (PIs) in HAART has greatly slowed the disease progression and improved the outcome of clinical treatment, the eradication of HIV/AIDS remains as a major medical challenge. Saquinavir (Saq) is the first anti-HIV PI approved by FDA. Despite of its high anti-HIV potency, its therapeutic efficacy is very limited due to its low oral absorption and bioavailability, which is attributed to extensive cellular efflux mediated by P-gp and first-pass metabolism mediated by CYP3A4. The objective of this dissertation is to develop novel Saq vitamin prodrugs targeting influx nutrient transporters and circumvent the P-gp mediated efflux as well as CYP3A4 mediated metabolism thus improving the absorption and bioavailability of Saq. First, two vitamin transport systems including sodium-dependent multivitamin transporter (SMVT) and sodium-dependent vitamin C transporter (SVCT), which are responsible for the uptake and transport of biotin and ascorbic acid (AA), were investigated using in vitro cell culture models. The carrier-mediated vitamin uptake and transport mechanisms were delineated. It was found that the uptake of biotin and AA was saturable with Km of 13.0 muM and 83.2 muM, respectively. The uptake processes were sodium, pH and temperature dependent. Both SMVT and SVCT dominantly located at apical membrane of the polarized cells. Subsequently, two vitamin prodrugs of Saq, Biotin-Saq and ascorbic-succinic-saquinavir (AA-Su-Saq), were synthesized and evaluated. The stability, cytotoxicity and metabolic kinetics of the prodrugs, and the interactions between the prodrugs and influx/efflux transport proteins were studied. The results indicated that the vitamin prodrugs were nontoxic and more metabolically stable than Saq. The prodrugs were recognized by vitamin transporters and showed less affinity to P-gp. The prodrug modification significantly decreased efflux and metabolism and improved the absorptive permeability and oral bioavailability of Saq. In conclusion, the influx nutrient transporters i.e. SMVT and SVCT can be targeted to improve the delivery of poorly absorbed drugs such as anti-HIV PIs. Finally, the effect of common buffer component HEPES on the uptake and transport of P-gp substrates was carefully examined. It was found out that HEPES in the cell growth medium and experimental buffer may affect the ATP-dependent cellular influx and efflux processes, which may provide some mechanistic insight into possible reasons for inconsistencies reported from various laboratories. |
