This thesis describes an investigation of the permeability of three antiretroviral (ARV) drugs (that are under development as vaginal microbicides) using the human endometrial epithelial cell line, HEC-1A, as an in vitro model. Barrier properties of HEC-1A cells in transwell cultures were determined by measurement of transepithelial electrical resistance (TEER), immunofluorescent staining of tight junctions and determination of the bi-directional permeability of, the paracellular marker, mannitol. Expression of specific uptake and efflux transporters were demonstrated by western blotting and compared with human tissue. Findings indicate that HEC-1A cells provide a physiologically relevant model to investigate permeability of candidate vaginal microbicides. In contrast, the commercially available EpiVagina™ model showed sub-optimal barrier properties. Permeabilities of three ARV drugs Tenofovir (TFV), Darunavir (DRV) and Dapivirine (DPV) were investigated in the HEC-1A model. Efflux ratios of all drugs were approximately 1 indicating transporter-independent permeability across the epithelium. Apparent permeability (Papp) values for TFV were consistent with paracellular diffusion while those for DRV and DPV were indicative of transcellular diffusion. No drug-drug interactions were observed when drugs were coadministered in double combinations. To model the effects of inflammation on drug uptake, HEC-1A cultures were stimulated with a variety of toll-like receptor (TLR) ligands. The TLR-3 ligand Poly I:C stimulated pro-inflammatory cytokine production by HEC-1A cells but did not affect permeability of the drugs and no effect on TEER values was evident. Permeability of DRV was also assessed using ex vivo rat and macaque cervicovaginal epithelial tissue. Consistent with findings from the HEC-1A model, DRV transfer across rat epithelium was transporter-independent although the Papp values were significantly lower. Preliminary data from macaque tissue indicate DRV may be effluxed. Development of a robust and physiologically-relevant in vitro model will contribute to development of vaginal microbicides by allowing rapid measurement of drug transporter dependency, drug-drug interactions and testing of formulations aimed at optimising tissue distribution. In this study, all of the ARVs showed transporterindependent transfer across the epithelium with no drug-drug interactions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:724287 |
| Date | January 2017 |
| Creators | Carserides, Constandinos Andreas |
| Contributors | Kelly, Charles George ; Forbes, Benjamin John |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/in-vitro-models-to-measure-antiretroviral-drug-permeability-at-vaginal-mucosa(b703bfcc-4af2-4ff1-b672-a47109d5fc67).html |
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