• 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.
    • Evaluations of a human kidney (HK2 cells) model for drug-induced cytotoxicity and BK virus infection

      Kinjo, Minori; Dowling, Thomas C. (2008)
      Renal transplant patients receive drugs such as tacrolimus (TAC), sirolimus (SRL), cyclosporin (CSA) for immunosuppression, and low-dose cidofovir (CID) for treatment of BK virus associated nephropathy (BKVN). In this series of studies, we evaluated the effects of clinically achievable concentrations of drug combination therapy on p-glycoprotein (P-GP) and organic anion (OA) transporters (OAT-1,-3 and MRP2) expression and function in human renal proximal tubule (HK2) cells. HK2 cells were further evaluated as a host for BK virus and a model for drug screening for the treatment of BKVN. HK2 cells were exposed to drug combinations over 5 days with determination of P-GP and OAT-1 expressions and functions in HK2 cells. P-GP function was assessed by rhodamine 123 (R123) uptake and OA transport activity was evaluated by para-aminohippurate transport (PAH) during acute and chronic exposures. Exposure to supraclinical concentrations of TAC, SRL and CSA resulted in significant reductions in cell viability whereas expression of P-GP, and OA transporters remained unchanged. A significant concentration-dependent toxicity was demonstrated by trypan blue exclusion but not by XTT assay, suggesting the importance of drug-uptake mechanism in designing experiments. R123 accumulation was significantly increased during TAC, SRL and CSA exposure. PAH transport remained unchanged in the presence of TAC, SRL, and CSA; however a trend towards decreased transport in the presence of CID was observed. HK2 cells were shown to express Caveolin-1, and were permeable to BK virus. Viral growth was gradual after 20 days post-infection, and stabilized after 55 days. A possible dormant stage of BK viral cycle was observed. The efficacy of CID was tested on the BK virus-infected HK2 cell model, and the quantitative PCR data showed that 24-hour CID exposure suppressed viral replications after 4 days incubation in a concentration-dependent manner. The key finding in these studies suggest that immunosuppressants used in renal patients may impair renal P-GP function. Exposure to drug combinations including CID may increase the risk of nephrotoxicity in patients receiving immunosuppressants. A BK virus infected HK2 cell model was developed that could be further utilized to study BK virus infection and new treatments for BKVN.

      Jin, Runyan; Dowling, Thomas C. (2010)
      The hepatitis C virus (HCV) has infected at least 4 million people in the United States, and is one of the leading causes of liver cirrhosis, end–stage liver disease and hepatocellular carcinoma. HCV genotype 1 is responsible for the majority of HCV infections in the U.S. and is highly resistant to anti–HCV therapy. The current standard of care for treating HCV is a pegylated interferon alpha (PEGIFN) 2a or 2b combined with ribavirin (RBV). African American (AA) patients have lower rates of sustained virologic response (SVR) to combination therapy compared to Caucasians (CA) in part due to a higher incidence of HCV genotype 1. However, other mechanisms of impaired viral response in AA are yet to be identified. In a previous study, no racial difference was found in the pharmacokinetics (PK) of PEGIFN. Thus, it is possible that RBV PK may be altered in AA. Moreover, the anti–viral activity of RBV was shown to be concentration–dependent. Therefore we hypothesized that altered RBV PK is a significant factor contributing to impaired virologic response to PEGIFN combination therapy in AA and CA with HCV genotype 1. To test this hypothesis, we pursued 4 aims. First, a sensitive and specific method using high performance liquid chromatography with tandem mass spectrometry was established to quantify RBV plasma concentrations in human plasma. Next, a population PK model of RBV based on 144 patients enrolled in the VIRAHEP–C study evaluated the impact of race on RBV PK. A population pharmacodynamic (PD) model was then developed to identify host factors associated with SVR. Lastly, PK simulations were performed to develop novel high–dose regimens of RBV in AA. The results showed that body weight was a significant covariate for apparent clearance of RBV. Both body weight and race were significant covariates on apparent peripheral volume (Vp/F), with Vp/F nearly 50% greater in AA compared to CA. Insulin resistance, RBV exposure during the first week of therapy, and IL28B–related single nucleotide polymorphism (SNP) rs12979860 predicted SVR in the PD model. A series of high-dose RBV regimens were developed based on simulation data to maximize drug exposure and SVR. In conclusion, the results of this population PK/PD study strongly support future studies of alternative dosing strategies to optimize RBV exposure and SVR in AA following treatment with PEGIFN and RBV for HCV genotype 1.
    • The Role of the Beta-1,2-adrenergic and GRK5 Receptor Single-Nucleotide-Polymorphisms (SNPs) on the Treatment of Heart Failure with Beta-blockers

      Rogers, Hobart Lee; Dowling, Thomas C. (2010)
      Despite recent advances in medical treatment, heart failure still remains a primary cause of mortality in the U.S. Pharmacologic β-blockade using carvedilol, metoprolol succinate, or bisoprolol in heart failure is associated with reduced mortality and morbidity and considered a class I recommendation in the consensus guidelines for the treatment of systolic heart failure. Nevertheless, β-blockers are still underused in this patient population. Additionally, significant interpatient variability in response to these agents exists. The differential response to β-blockade may be due to genetic heterogeneity of the β-adrenergic receptor (β-AR), in the form of single-nucleotide-polymorphisms (SNPs). SNPs with functional differences have been identified on the β1-AR at positions 49 (Ser/Gly) and 389 (Arg/Gly) and β2-AR at positions 16 (Gly/Arg) and 27(Gln/Glu). Furthermore, a functional SNP on the G-protein coupled receptor kinase (GRK) has also been identified. These SNPs have all been linked to significant differences in receptor expression and function in experimental models. The objective of this study was to characterize the clinical implications of the β1,2-AR and GRK SNPs on the treatment of heart failure with β-blockers. Eighty-six NYHA class II-III heart failure patients receiving maximum titrated doses of either carvedilol or metoprolol succinate were enrolled in this study. Heart rate (HR) was measured at rest and immediately following a 6-minute walk test, and a blood sample was obtained for genotyping. Genotypes were determined by Taqman genotyping assays. To account for plasma β-blocker concentrations, two novel enantioselective HPLC assays with fluorescence detection were developed. Our primary finding was that metoprolol-treated patients who were homozygous for Arg at position 389 on the β1-AR had both an enhanced negative chronotropic and antihypertensive response to β-blockade when corrected for plasma S-metoprolol concentrations. When evaluating the β2-AR SNPs, both the carvedilol-treated Gly16 homozygotes and the Glu27 carriers had a significantly enhanced antihypertensive response compared to their subsequent counterparts. These findings suggest that SNPs in both the β1,2-ARs are associated with clinical response to either carvedilol or metoprolol succinate. Moreover, our findings emphasize the importance of accounting for pharmacokinetic differences when evaluating pharmacodynamic relationships.