Resúmenes








Summary

        Proteins are macromolecules very abundant in the organism. Their main physiological functions are to act as a vehicle for different agents, to act as enzymes, etc. In particular, the importance of transport proteins (HSA and BSA) is to carry different endogenous and exogenous agents (including drugs) through the blood-stream.
      Binding of drugs to SA in biological systems is a key process that can modulate a number of properties of the carried agent, such as increased solubility in plasma, decreased toxicity, protection against oxidation or prolongation of the in vivo half-life; hence, binding is essential for understanding biodistribution, metabolism, elimination or pharmacological effect of drugs in the body. 
        Several techniques have been used to investigate drug-protein binding processes. The aim of this work has been to develop alternative methodologies in order to get further insight into the relevant interactions taking place between flurbiprofen and the above mentioned serum albumins. For this purpose, the transient species generated by irradiation were used as probes to study the nature of these interactions, as their properties serve as quantitative parameters sensitive to the microenvironment. Thus, fluorescence and laser flash photolysis techniques were used.
        First, the first excited states of flurbiprofen were fully characterised and the main processes leading to their deactivation were identified. The excited state lifetimes and the quantum yields of the involved photophysical processes have been determined, and found to be very sensitive to the medium. Specially, triplet excited state is highly sensitive to the microenvironment, which can be used as a reporter to study the interactions between the drug and HSA or BSA.
        Next, several model dyads containing the FBP, Trp and Tyr chromophores were studied. This should give information about the operating mechanisms in the excited state interactions using chemically well-defined systems, where the non-covalent supramolecular drug-protein interactions are modeled by the analogous intramolecular processes in covalently linked dyads. Thus, different dyads were synthesised between the FBP pure enantiomers and tryptophan or tyrosine amino acid residues. The singlet excited state was highly sensitive to the drug-amino acid interaction. In FBP-Trp systems, most of the energy provided by the incident radiation at 266 nm reaches the 1Trp*, either via direct absorption by this chromophore or by SSET from 1FBP*. The main process observed is intramolecular 1Trp* quenching, that could take place by an electron transfer mechanism. On the other hand, a minor, yet stereoselective deactivation of 1FBP* leads to detectable exciplexes and/or radical ion pairs. Finally, the first triplet excited state of FBP can be reached by TTET from 3Trp* or by BET from the charge separated states. By contrast, in FBP-Tyr systems a SSET process from 1FBP* to 1Tyr* is not observed, but instead stereoselective quenching of the singlet excited state of the drug takes place. In polar solvents this quenching is favoured either via electron transfer or exciplex formation, while in non-polar solvents, only exciplex formation is allowed.
        For further progress in the understanding of drug/protein interactions intermolecular FBPMe/SA and FBP/SA systems were studied. In these cases, the first triplet excited state of the drug was highly sensitive to the interaction with the protein. Triplet lifetimes of FBPMe and FBP were longer in the presence of protein. This can be attributed to a slower deactivation of the excited states inside the SA binding sites, where an exceptional microenvironment protecting the triplet excited state from attack by a second drug molecule, oxygen or other reagents is provided. First, FBPMe/SA system was studied. The choice of FBPMe was done for experimental convenience, since its hydrophobic character should favour inclusion within SA (preferentially in site I). Both (R)- and (S)-FBPMe have been investigated in order to obtain valuable information about the possible stereodifferentiation in the interaction with proteins. Regression analysis of the decay kinetics of the transient absorption spectra corresponding to the triplet-triplet absorption provided precise information on the number of binding sites and their degree of occupancy. In FBPMe/HSA system, a remarkable stereodifferentiation in the triplet lifetimes of FBPMe within the protein was observed. For the same FBPMe/HSA ratio, inclusion within HSA is slightly favored for the (S)- isomer. Besides, a significant stereodifferentiation was observed when comparing the occupation level of the two sites, with higher preference for site I in the case of (S)-FBPMe. In general, when the FBPMe/SA ratio was low (far from saturation) all the drug was protein-bound and the high affinity site I was selectively populated. Progressive increase of the FBPMe/SA ratio resulted in saturation of site I and (subsequently) site II. This led to a decrease of site selectivity and the appearance of increasing amounts of free FBPMe. Finally, it was demonstrated that FBPMe preferably interacts to HSA than to BSA.
        The last problems studied were the FBP/SA interactions, because FBP is the real drug prescribed for the treatment of several diseases. Again, regression analysis of the triplet decay kinetics provided information on the number of binding sites and their degree of occupancy. Moreover, the assignment of FBP bound in site I and site II in SA was confirmed by displacement of the drug by selective site II probes, as (S)-ibuprofen and capric acid. On the other hand, the triplet lifetimes and the degree of occupancy of both sites were dependent on the nature of the albumin. Finally, a slight stereodifferentiation in the interaction of (S)- or (R)-FBP with BSA was observed.

        
      






Resúmenes


222