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Digital archive of theses discussed at the University of Pisa


Thesis etd-01282010-111422

Thesis type
Tesi di dottorato di ricerca
Thesis title
Investigation of drug-biomacromolecules interaction phenomena by NMR spectroscopy
Academic discipline
Course of study
tutor Prof. Uccello Barretta, Gloria
  • albumina
  • interazione
  • NMR
  • polisaccaridi
Graduation session start date
Release date
NMR methods were exploited in the investigation of drug-protein or drug-polysaccharide interaction processes. Great usefulness of proton selective relaxation rates, cross-relaxation parameters and diffusion coefficients was pointed out. For the determination of binding parameters (association constant, number of binding sites) different approaches, involving linear or non-linear fitting of experimental data, were compared. In the analysis of stereochemical aspects of drug-biomacromolecule binding interactions, proton selective relaxation rate measurements were mainly employed. The interaction of three non-steroidal anti-inflammatory drugs (ketorolac, diclofenac and flurbiprofen) with human serum albumin was investigated, putting in light great relevance of NMR spectroscopy in the analysis of binding processes involving low-affinity sites of the protein.
Polysaccharides (tamarind-seed polysaccharide, TSP; hydroxyethylcellulose, HEC; and hyaluronic acid, HA) employed in ophthalmic formulations containing ketotifen fumarate (KT) were taken into consideration and investigated by relaxation rate measurements. Among the three polysaccharides, TSP showed enhanced affinity for the drug. Low stability of HA-KT formulation was also evidenced. Drug-macromolecule interaction data have been confirmed by using a different analytical technique, i.e. the dynamic dialysis.
NMR potentialities were also probed in the field of polysaccharide formulations employed as artificial tears for the therapy of dry eye syndrome. Information on their mucoadhesive properties were obtained by analyzing polysaccharide-mucin interaction, which required the use of suitable low-molecular weight probes. In particular eye drops based on TSP and another vegetal polysaccharide (arabinogalactan AG) were compared by using KT as interaction probe and by measuring KT proton selective relaxation rates and cross-relaxation parameters in order to detect polysaccharide-mucin interactions. In this way enhanced affinity of TSP to mucin with respect to AG was clearly demonstrated. Moreover, the affinities TSP-HA mixtures to mucin were investigated in comparison with TSP or HA, by using ketotifen and diclofenac as interaction probes. The mixture TSP-HA showed superior mucin affinity with respect to the single polysaccharides: a strong dependence on the TSP/HA ratio was pointed out, which constituted the basis for the development of new artificial tears. Ability of TSP-HA mixture to bind water, which is fundamental in formulations for ophthalmic uses, was compared to that of TSP or HA. In consideration of the strong synergic interactions occurring between the two polysaccharides, information about the nature of TSP-HA interactions were extracted from relaxation rate measurements of selected protons of the two polysaccharides. Finally, underivatized and derivatized chitosans were explored in view of their pharmaceutical applications in formulations with ketorolac.