Tesis doctoral de Gerard Minuesa Dinarés
The present thesis has addressed the question of which mechanisms take part in the entry of the nucleoside reverse transcriptase inhibitors (nbtis) used in hiv therapy, focusing our studies on the expression and activity of antiviral drug transporters in immune cells. Firstly, the uptake mechanisms of azidothymidine (azt) were analysed in t lymphocytes. We found out that human nucleoside transporters (hnts, slc28 and slc29 gene families) play a negligible role in azt uptake. Moreover, human organic anion transporters (from slc22 gene family), were not detected in t cells neither functionally nor at the mrna level. Azt, with a higher lipophylic character than the rest of nrtis, showed a temperature-dependent route of entry and was up-regulated by phytohemagglutinin (pha), what supports the idea of a mediated and regulated transport mechanism, at least in part. Following that, we attempted to address the lack of information regarding the expression and activity of drug uptake transporters from slc28, slc29 and slc22 gene families in immune cells. Regarding hnts, both hents and hcnt2 were abundant in lymphocytes and the same transporters and hcnt3 were expressed in monocyte/macrophage lineage cells with preferential activity for hent1 in t cells and hcnt3 and hent1 in macrophages. All these proteins were highly up-regulated by pha. In immune cells human organic cation transporters (hocts, slc22 gene family) showed a more heterogeneous expression profile and a lower activity than hnts, although up-regulation also ocurred upon lymphocyte activation. As a preliminary approach to the direct effect of hiv-1 infection on the expression of transporters, we also explored the changes in mrna levels of hnts in infected primary lymphocytes and mt4. Moreover, the implication of nucleoside salvage and the possible antiviral effects of dipyridamole (dip) was also analysed in these cells. Hiv-1 infection in vitro only affected hcnt2 mrna expression, down-regulating it > 9-fold in mt4 cells whereas in pbmcs or cd4+ t cells any hnt mrna was affected. Dip showed a good antiviral effect, strinkingly decreasing the percentage of infected cells in mt4 at days 2 and 3 post-infecion and the p24 antigen levels at up to day 9 post-infection in pbmcs. Furthermore, focusing on hocts, we studied the interactions and transportability of all nrtis currently used in highly active antiretroviral therapy (haart) with oct1, 2 and 3. All nrtis interacted with hocts with a high affinity and could be modulators of physiological function of hocts in vivo. Lamivudine (3tc) showed two binding sites with hocts: a high-affinity one (in the pm range) and a low-affinity one (in the mm range) and is a novel substrate for hocts, with hoct1 being the most efficient transporter. Finally, we found that abacavir (abc) and azt, drugs frequently coadministered with 3tc, inhibit the transport of 3tc through hocts at low concentrations, which could lead to consequences for 3tc pharmacokinetics. to conclude, throughout the course of the investigation, we discovered that nrtis do not seem to have a common entry pathway, contrary to what it was initially thought. Importantly, we demonstrated that some protein members of the slc28, slc29 and slc22 gene families are highly expressed and do participate in nrti uptake in immune cells. The regulation of the expression and activity of antiviral drug uptake transporters opens a new therapeutic approach in relation to the possible modulation of pharmacodynamics/kinetics of antiretroviral drugs, drug-drug interactions and drug toxicity.
Datos académicos de la tesis doctoral «Uptake transporters of nucleoside-derived anti-hiv drugs: expression and functional analysis in immune cells«
- Título de la tesis: Uptake transporters of nucleoside-derived anti-hiv drugs: expression and functional analysis in immune cells
- Autor: Gerard Minuesa Dinarés
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 26/06/2009
Dirección y tribunal
- Director de la tesis
- Javier Martinez Picado
- Tribunal
- Presidente del tribunal: buenaventura Clotet sala
- margalida Rotger cerd? (vocal)
- (vocal)
- (vocal)