• Effect of the tight junction modulator Zonula occludens toxin (Zot) and the P-glycoprotein inhibitor itraconazole on brain transport enhancement and their potential for renal drug interactions

      Karyekar, Chetan S.; Eddington, Natalie D.; Dowling, Thomas C. (2002)
      Drug delivery to the brain presents many challenges to the pharmaceutical scientist in part due to limited drug transport across the blood brain barrier (BBB). This dissertation focused on evaluation of a novel protein, Zonula occludens toxin (Zot) and a P-glycoprotien (P-gp) modulator, itraconazole, to enhance brain drug delivery, and to investigate their potential for drug interactions in the kidney. Zot, a protein elaborated by vibrio cholerae, is known to modulate tight junctions in intestinal epithelial cell models and enhance oral bioavailability. We initially evaluated the ability of Zot to modulate tight junctions in a bovine brain microvessel endothelial cell (BBMEC) model. The results from this study indicate that Zot transiently and reversibly enhances paracellular transport of selected marker and chemotherapeutic compounds. Subsequent in vivo studies in rats were consistent with these findings, where Zot caused a four-fold enhancement in brain uptake of paclitaxel, a poorly permeable compound. Truncation studies on Zot isolated its active fragment (DeltaG). Preliminary studies in rats indicated that DeltaG was susceptible to metabolism by proteases. Use of the protease inhibitors (leupeptin and captopril) with DeltaG improved its activity as measured by brain uptake of sucrose. However, DeltaG did not significantly enhance brain uptake of paclitaxel in rats. Itraconazole, a known P-gp inhibitor has been safely used for many years to treat mycotic infections. We assessed the effect of itraconazole on the brain uptake of paclitaxel in rats. The dosing strategy applied in this study did not significantly enhance brain uptake of paclitaxel suggesting that either higher concentrations (>1 mug/ml) are required or itraconazole may not effectively inhibit P-gp at the BBB. Next, we evaluated the effect of DeltaG and itraconazole on the renal handling of a P-gp substrate, cimetidine, in vitro. In the kidney, P-gp is known to be located in high concentrations in mesangium and proximal tubule where active secretion of drugs occurs. Studies in the renal tubular epithelial cell line MDR1-MDCK indicated that itraconazole decreased P-gp mediated cimetidine efflux. However, DeltaG did not alter cimetidine transport in this model. To further evaluate the effect of itraconazole on renal P-gP in vivo, a Phase I pharmacokinetic drug interaction study was conducted in healthy volunteers. Cimetidine and iothalamate (GFR) clearances were measured at baseline and following itraconazole dosing (200 mg bid x 4 days). Here, itraconazole reduced the tubular secretory clearance of cimetidine by 25%, resulting in a 21% increase in the plasma cimetidine AUC0--4 hrs. This data suggests that the observed renal P-gp interaction may be clinically significant. In conclusion, we anticipate that this research will provide a basis for newer strategies to effectively deliver drugs to the brain and increase awareness of potential drug interactions when using such approaches.