Interacción de los fármacos antineoplásicos tamoxifeno y 4-hidroxitamoxifeno con membranas fosfolipídicas

Resum

Objectives On the light of the highly lipophylic nature of antineoplastic drugs tamoxifen (TMX) and 4-hydroxytamoxifen (HTMX), widely used in the treatmen of patients with ER-positive breast cancer, the general objective of this thesis is to deepen into the effect of those compounds on the structure and functionality of phospholipid membranes. This is translated into the following specific objectives: - To study the interaction of TMX and HTMX with model phospholipid membranes composed of the phospholipid 1,2 dipalmitoylphosphatidylcholine (DPPC). - To analyze the influence of TMX and HTMX on the structure and functionality of systems composed of the phospholipid 1,2 dielaidoylphosphatidylethanolamine (DEPE). - To characterize the molecular mechanism of the permeabilization of phospholipid membranes of various compositions by TMX and HTMX. Methodology These studies have been carried out under a biophysical approach, using experimental techniques and molecular dynamics (MD) simulations widely used in this field. The main experimental techniques are briefly described as follows. - The model membranes have been constituted by multilamellar vesicles (MLV) or unilamellar vesicles (LUV). MLV have been essentially prepared by the thin film hydration method, whereas LUV have been manufactured by polycarbonate filter extrusion. - Differential scanning calorimetry (DSC), has allowed analyzing the effect of the drugs under study on the thermotropic phase behaviour of the phospholipids. From the experimental thermograms phase diagrams were constructed, which allowed to analyze the influence of these compounds on the phase behaviour of the various phospholipids. - Fourier-transform infrared spectroscopy (FTIR), which provided information on drug-phospholipid interactions from the effect observed on the characteristic absorption bands arising from the phospholipid polar headgroup (phosphate, carbonyl), as well as from acyl chains (methylene, terminal methyl). - X-Ray diffraction (XRD), allowing measurements both in the small and the wide angle. Our equipment provided electron density profiles of the phospholipids in the absence and presence of TMX and HTMX, analyzing the effect on bilayer thickness, acyl chain organization, and lipid polymorphism. - Fluorescence spectroscopy. This technique has been used to monitor the effect of TMX and HTMX on membrane fluidity, using fluorescent probes such as DPH and TMA-DPH. In addition, the quantitative analysis of membrane permeabilization was carried out using a fluorescent assay with carboxyfluorescein entrapped in LUV. - Complementary to the experimental studies, MD simulations were carried out. In these simulations a lipid bilayer was constructed using the selected phospholipids, and incorporated the drugs under study, always at molar ratios similar to those used in the experimental studies. Simulations were performed using GROMACS, with force field parameters CHARMM36. Initial membrane structures were constructed using Packmol, with productive runs of ca. 120 ns. Graphical plots were done using PyMOL. Results TMX and HTMX affect the phase behaviour of systems composed of the phospholipid DPPC, giving rise to drug-enriched domains within the bilayer (lateral phase separation), but both compounds locate in different regions within the membrane. MD data support the tendency of these compounds to aggregate inside the membrane, and locate TMX all along the bilayer, whereas HTMX is placed closer to the lipid/water interface. Incorporation of TMX into systems composed of DEPE gives rise to a widening of the lamellar-Lβ to lamellar-Lα phase transition, decreasing phase transition temperature. The Lβ/Lα phase transition shows multiple endotherms, indicating a lateral seggregation of TMX/DEPE domains within the plane of the bilayer. TMX and HTMX also displace and widen the lamellar-to-hexagonal-HII phase transition. Phase diagrams show that both drugs facilitate formation of the hexagonal-HII phase. FTIR spectroscopy shows that neither TMX nor HTMX induce a significant perturbation of DEPE bilayer hydration. MD simulations indicate that both compounds do not affect bilayer thickness, area per lipid, or the conformation of DEPE molecules, but do show a different location, and a different aggregation behaviour, of both compounds. TMX provokes rapid and extensive membrane permeabilization in 1-palmitoyl-2-oleoyl-sn-glicero-3-phosphocholine (POPC) systems, the effect of HTMX being much weaker. Liposomal contents leakage curves are adjusted to two components, yielding rate constants corresponding to a slow and a fast processes. Interestingly, incorporation of phosphatidylglycerol or phosphatidylethanolamine protects the membrane against drug-induced permeabilization, MD providing data for a feasible explanation. FTIR shows an increase in gauche conformers, and a dehydration of the polar region of the membrane by effect of TMX or HTMX. Conclusions - The results shown in this thesis allow to conclude that TMX, as well as HTMX, incorporate into phospholipid membranes, forming domains segregated with the plane of the bilayer. Whereas TMX can locate all along the bilayer, HTMX is mostly found closer to the lipid/water interface. - Both drugs perturb the region of the phospholipid acyl chains, and dehydrate the polar headgroups region, the effects of TMX being quantitatively more significant. - TMX and HTMX can regulate membrane curvature, and thus to promote formation of non-lamellar phases. - It is shown that TMX, and HTMX to a lesser extent, permeabilize phosphatidylcholine membranes, allowing release of liposome aqueous contents. This action correlates with their distinct capacity to form aggregates, and their different location with the lipid bilayer. - Drug-induced membrane permeabilization is modulated by lipid composition, with phosphatidylglycerol and phosphatidylethanolamine having a protecting role. This is due to increased electrostatic interactions in the region of the polar headgroups, and the possibility of forming hydrogen bonds, what results in a more compact membrane. - Our data could provide a molecular basis for some of the pharmacological actions of TMX, and HTMX, not related to estrogen receptor binding, or even explain some the various side effects concomitant to therapy with these two drugs.