Ritonavir - A Novel Multidrug Resistance Modulator in Cancer Chemotherapy and Ocular Neovascular Diseases

Title from PDF of title page, viewed on July 15, 2015Dissertation advisor: Ashim K. MitraVitaIncludes bibliographic references (pages 195-222)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2014Multidrug resistance (MDR), a clinical outcome characterized by subtherapeutic intracellular drug concentration, is one of the predominant factors limiting effective cancer chemotherapy. Several possible mechanisms and molecular alterations have been implicated in the development of MDR, including activation of efflux transporters and metabolizing enzymes in response to therapeutic agents. Therefore, the primary objective of my dissertation project is to develop strategies for overcoming drug resistance in cancer chemotherapy. Human adenocarcinoma cells (LS-180) were treated for 72 hours with vinblastine alone and in the presence of ritonavir. The expression of efflux transporters (MDR1 and MRP2), metabolizing enzyme (CYP3A4) and nuclear hormone receptor (PXR) was induced in response to vinblastine. This overexpression was completely neutralized when cells were cotreated with ritonavir. Uptake of [3H] lopinavir and Vivid™ assay further confirmed the functional activity of transcribed genes upon cotreatment. Reduced cell proliferation, migration and increased apoptosis of cancer cells were further indicative of enhanced activity of chemotherapeutics (doxorubicin, paclitaxel, tamoxifen and vinblastine) in the presence of ritonavir. Combination therapy of anticancer drug with ritonavir may overcome drug resistance by neutralizing overexpression of efflux transporters and metabolizing enzymes. Hypoxia leading to neovascularization has also been implicated in the development of MDR and ocular neovascular diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in retinal pigment epithelial (RPE) cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising antiangiogenic role with fewer side effects. Our studies (quantitative PCR, immunoblot analysis, ELISA and angiogenic assay) have demonstrated that ritonavir inhibits the expression of hypoxia-inducible factor-1α (HIF-1α) mediated vascular endothelial growth factor (VEGF) expression in RPE cells probably via inhibition of PI3K/AKT pathway. This inhibition may reduce retinal neovascularization. These findings shed new light on the possibility of incorporating ritonavir in the treatment regimen of ocular angiogenic diseases. Although many inhibitors of HIF-1α are in clinical trials, additional benefit of using ritonavir is that it has been given to HIV patients with relatively low toxicity. The process of traditional drug development could be fast tracked since ritonavir is clinically approved for human use. However, further preclinical and clinical experiments are necessary to determine the repositioning of ritonavir in the treatment of ocular neovascular diseasesMechanisms of drug resistance in cancer chemotherapy: Coordinated role and regulation of efflux transporters and metabolizing enzymes -- Differential effect of MDR1 and MRP2 in cellular translocation of gemifloxacin -- PXR mediated induction of efflux transporters by fluoroquinolones: a possible mechanism for development of multidrug resistance -- Rotonavir: a novel therapeutic for overcoming drug resistance in cancer chemotherapy -- Hypoxia-inducible factor 1 (HIF-1): a potential target intervention in ocular neovascular diseases -- Molecular expression and functional activity of efflux and influx transporters in hypoxia induced retinal pigment epithelial cells -- Ritonavir inhibits HIF-1a mediated VEGF expression in retinal pigment epithelial cells -- Summary and recommendations -- Appendi

Novel biotinylated lipid prodrugs of acyclovir for the treatment of herpetic keratitis (HK): Transporter recognition, tissue stability and antiviral activity

Purpose: Biotinylated lipid prodrugs of acyclovir (ACV) were designed to target the sodium dependent multivitamin transporter (SMVT) on the cornea to facilitate enhanced cellular absorption of ACV. Methods: All the prodrugs were screened for in vitro cellular uptake, interaction with SMVT, docking analysis, cytotoxicity, enzymatic stability and antiviral activity. Results: Uptake of biotinylated lipid prodrugs of ACV (B-R-ACV and B-12HS-ACV) was significantly higher than biotinylated prodrug (B-ACV), lipid prodrugs (R-ACV and 12HS-ACV) and ACV in corneal cells. Transepithelial transport across rabbit corneas indicated the recognition of the prodrugs by SMVT. Average Vina scores obtained from docking studies further confirmed that biotinylated lipid prodrugs possess enhanced affinity towards SMVT. All the prodrugs studied did not cause any cytotoxicity and were found to be safe and non-toxic. B-R-ACV and B-12HS-ACV were found to be relatively more stable in ocular tissue homogenates and exhibited excellent antiviral activity. Conclusions: Biotinylated lipid prodrugs demonstrated synergistic improvement in cellular uptake due to recognition of the prodrugs by SMVT on the cornea and lipid mediated transcellular diffusion. These biotinylated lipid prodrugs appear to be promising drug candidates for the treatment of herpetic keratitis (HK) and may lower ACV resistance in patients with poor clinical response. © 2013 Springer Science+Business Media New York