Investigations of Combined Strategies to reverse P-glycoprotein- and BCRP-mediated Multidrug Resistance in Human Ovarian Cancer Cells and Xenograft Tumors

Purpose: The ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp) and BCRP are implicated in the multidrug resistance (MDR) of many tumors. Pgp-mediated MDR can be functionally inhibited using small molecule inhibitors or transcriptionally downregulated by MDR1 antisense oligodeoxynucleotides (ODNs). Interestingly, simultaneous inhibition of several ABC transporters including Pgp and BCRP by cyclosporin A has been shown to circumvent MDR in clinical trials. In this thesis, the novel multi-targeted tetrahydroisoquinolin-ethyl-phenyl-amine-based MDR inhibitors XR9577, WK-X-34, WK-X-50 and WK-X-84 were thoroughly in vitro and in vivo characterized for interaction with Pgp, BCRP and MRP-transporters and compared to treatments with MDR1 antisense ODNs. Methods: The novel MDR inhibitors and cyclosporin A were examined for cellular toxicity in several cell lines. Inhibition of BCRP-mediated mitoxantrone efflux was studied in BCRP-overexpressing MCF7/mx cells. Inhibition of Pgp function was assessed in 99mTc-Sestamibi and daunorubicin transport assays. Reversal of Pgp- and BCRP-mediated resistance towards daunorubicin and mitoxantrone, respectively, were investigated in A2780/Adr and MCF7/mx cells. Potential MRP-interactions were evaluated in 5-CFDA efflux assays using selectively transfected MRP-1, -2 and -3 cell lines. Daunorubicin transport and Pgp surface expression in resistant A2780/Adr cells treated with MDR1 antisense ODNs were determined using flow cytometry, fluorescence microscopy and protein staining techniques. The in vivo performance and toxicity of WK-X-34 was evaluated by 99mTc-Sestamibi imaging experiments using multidrug resistant human ovarian cancer (A2780/Adr) xenograft models. To study antisense treatments in vivo, A2780/Adr xenograft models were dosed intratumorally with MDR1 antisense ODNs for three days followed by either WK-X-34 or vehicle treatment. 99mTc-Sestamibi distribution and accumulation were analyzed by imaging of the animals and gamma-counting of the isolated tissues. Tumor xenografts were analyzed histologically and Pgp expression was monitored by immunohistochemistry and RT-PCR.Results: WK-X-34 and XR9577 were the most potent MDR inhibitors and completely reversed 99mTc-Sestamibi and daunorubicin accumulation at concentrations of 10 and 1 mM. Daunorubicin chemosensitivity was increased by 7-8-fold after 10 mM XR9577 or WK-X-34 pretreatment. All WK-X-compounds showed significant BCRP inhibition comparable to novobiocin in both mitoxantrone transport and chemosensitivity assays. Compared to cyclosporin A, the in vitro toxicity was reduced for all WK-X-compounds. Moreover, XR9577 or WK-X-34 showed increased specificity for Pgp and BCRP as only resistant cells were targeted and significant MRP interactions were not detected. In immunocomprimised mice dosed with WK-X-34 (20 mg/kg; i.p.), uptake of 99mTc-Sestamibi was significantly increased in A2780/Adr xenograft tumors (AUCs0-4h 136%; p 99mTc-Sestamibii were detected in the brain, liver and intestine of WK-X-34 treated animals. MDR1 antisense treatments significantly increased daunorubicin levels in A2780/Adr cells. In vivo, 99mTc-Sestamibi retention was significantly increased by over two fold in the antisense pretreated tumors. Upon additional WK-X-34 treatment, tissue/muscle ratios of 99mTc-Sestamibi were significantly increased by 9-fold in the untreated but only by 2-fold in the antisense pretreated tumors and correlated with results from the 1-hour 99mTc-Sestamibi images. 99mTc-Sestamibi levels in the A2780/Adr tumors could not be further increased by a combined treatment with antisense ODNs and WK-X-34 as compared to WK-X-34 alone. MDR1 mRNA levels and Pgp expression were down-regulated in the antisense treated tumors.Conclusion: Among the novel MDR inhibitors, WK-X-34 and XR9577 were most effective in modulating Pgp and BCRP and demonstrated an increased in vitro tolerance and specificity over cyclosporin A. In vivo, WK-X-34 potently inhibited the Pgp-mediated MDR phenotype in resistant human ovarian cancer xenograft models. In comparison to WK-X-34, MDR1 antisense ODNs were less effective while associated with more complications. Based on these in vitro and in vivo characterizations, WK-X-34 may have potential utility in the treatment of multidrug resistant tumors

Modelling the risk-benefit impact of H1N1 influenza vaccines

BACKGROUND: Shortly after the H1N1 influenza virus reached pandemic status in June 2009, the benefit-risk project team at the European Medicines Agency recognized this presented a research opportunity for testing the usefulness of a decision analysis model in deliberations about approving vaccines soon based on limited data or waiting for more data. Undertaken purely as a research exercise, the model was not connected to the ongoing assessment by the European Medicines Agency, which approved the H1N1 vaccines on 25 September 2009. METHODS: A decision tree model constructed initially on 1 September 2009, and slightly revised subsequently as new data were obtained, represented an end-of-September or end-of-October approval of vaccines. The model showed combinations of uncertain events, the severity of the disease and the vaccines' efficacy and safety, leading to estimates of numbers of deaths and serious disabilities. The group based their probability assessments on available information and background knowledge about vaccines and similar pandemics in the past. RESULTS: Weighting the numbers by their joint probabilities for all paths through the decision tree gave a weighted average for a September decision of 216 500 deaths and serious disabilities, and for a decision delayed to October of 291 547, showing that an early decision was preferable. CONCLUSIONS: The process of constructing the model facilitated communications among the group's members and led to new insights for several participants, while its robustness built confidence in the decision. These findings suggest that models might be helpful to regulators, as they form their preferences during the process of deliberation and debate, and more generally, for public health issues when decision makers face considerable uncertainty