Cancer is a multifactorial disease characterized by a very complex etiology. Basing on its complex nature, a promising therapeutic strategy could be based by the “Multi-Target-Directed Ligand” (MTDL) approach, based on the assumption that a single molecule could hit several targets responsible for the pathology. Several agents acting on DNA are clinically used, but the severe deriving side effects limit their therapeutic application. G-quadruplex structures are DNA secondary structures located in key zones of human genome; targeting quadruplex structures could allow obtaining an anticancer therapy more free from side effects. In the last years it has been proved that epigenetic modulation can control the expression of human genes, playing a crucial role in carcinogenesis and, in particular, an abnormal expression of histone deacetylase enzymes are related to tumor onset and progression. This thesis deals with the design and synthesis of new naphthalene diimide (NDI) derivatives endowed with anticancer activity, interacting with DNA together with other targets implicated in cancer development, such as HDACs. NDI-polyamine and NDI-polyamine-hydroxamic acid conjugates have been designed with the aim to provide potential MTDLs, in order to create molecules able simultaneously to interact with different targets involved in this pathology, specifically the G-quadruplex structures and HDAC, and to exploit the polyamine transport system to get selectively into cancer cells. Macrocyclic NDIs have been designed with the aim to improve the quadruplex targeting profile of the disubstituted NDIs. These compounds proved the ability to induce a high and selective stabilization of the quadruplex structures, together with cytotoxic activities in the micromolar range. Finally, trisubstituted NDIs have been developed as G-quadruplex-binders, potentially effective against pancreatic adenocarcinoma. In conclusion, all these studies may represent a promising starting point for the development of new interesting molecules useful for the treatment of cancer, underlining the versatility of the NDI scaffold.

In this thesis is described the design and synthesis of potential agents for the treatment of the multifactorial Alzheimer’s disease (AD). Our multi-target approach was to consider cannabinoid system involved in AD, together with classic targets. In the first project, designed modifications were performed on lead molecule in order to increase potency and obtain balanced activities on fatty acid amide hydrolase and cholinesterases. A small library of compounds was synthesized and biological results showed increased inhibitory activity (nanomolar range) related to selected target. The second project was focused on the benzofuran framework, a privileged structure being a common moiety found in many biologically active natural products and therapeutics. Hybrid molecules were designed and synthesized, focusing on the inhibition of cholinesterases, Aβ aggregation, FAAH and on the interaction with CB receptors. Preliminary results showed that several compounds are potent CB ligands, in particular the high affinity for CB2 receptors, could open new opportunities to modulate neuroinflammation. The third and the fourth project were carried out at the IMS, Aberdeen, under the supervision of Prof. Matteo Zanda. The role of the cannabinoid system in the brain is still largely unexplored and the relationship between the CB1 receptors functional modification, density and distribution and the onset of a pathological state is not well understood. For this reasons, Rimonabant analogues suitable as radioligands were synthesized. The latter, through PET, could provide reliable measurements of density and distribution of CB1 receptors in the brain. In the fifth project, in collaboration with CHyM of York, the goal was to develop arginine analogues that are target specific due to their exclusively location into NOS enzymes and could work as MRI contrasting agents. Synthesized analogues could be suitable substrate for the transfer of polarization by p-H2 molecules through SABRE technique transforming MRI a more sensitive and faster technique.

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.

The present work aimed to synthesizing new biologically active small molecules as innovative antitumor lead candidates and to the total synthesis of a natural compound with selectivity towards cancer hypoxia. In this context, a first project involved the design and synthesis of N-acylhydrazone based inhibitors of lactate dehydrogenase A (LDH-A). The structures of the new molecules where designed by means of virtual screening and synthesized to obtain a library of analogs which were evaluated on the enzyme. Active compounds were also screened on cells of non-Hodgkins lymphoma and one of them proved to be a promising inhibitor, suggesting that the N-acylhydrazone as suitable scaffolds for LDH-A inhibitors. The second project aimed to the synthesis of Galloflavin (GF) analogs and to the study of the compound’s SAR. GF is an LDH-A inhibitor which was previously identified and synthesized by our group. Its poor solubility and stability prevented us from studying its SAR maintaining the core structure. Therefore, the synthesis of three potential classes of structural analogs was devised and carried out. One compound was found to reproduce GF’s behaviour on the enzyme and in cell, therefore being a good starting point for the study. A small library of analogs was synthesized and biological tests are ongoing to acquire in-depth knowledge about the key pharmacophores of this interesting inhibitor. The third project was carried out at Aarhus University in the group of Prof. Thomas Poulsen. The work focused on the total synthesis of Rakicidin A, a macrolide of natural origin which was identified and isolated from soil samples and is known for its interesting properties in selectively inducing cell death in hypoxic environments and being also active on cancer stem cells. The total synthesis involved several steps including key enantioselective reactions to build the 5 stereocenters on the molecule.

Plants, animals and micro-organisms represent a reservoir of natural products, the so called “natural source-derived compounds”. This is particularly true for the plant kingdom, as it offers a variety of species still used as remedies for several diseases in many parts of the world. Nevertheless, the bioactive potential of many plant species remains largely unexplored. Thus, biodiversity represents an unlimited source of chemical entities with potential beneficial effects on human health. These compounds are usually secondary metabolites often present in low quantity in plant material and their extraction, purification and quantitation still remain a great challenge for analytical scientists. The research activity carried out during these three years of PhD Programme was focused on the development, validation and application of original methods aimed at the quali-quantitative analysis of compounds with potential bioactive interest in plant matrices, foods, drinks and related products, as well as the analytical screening of plant by-products from cosmetic manufacture. Bioactive substances, belonging to the classes of polyphenols, aminoacids, coumarins, triterpenes and phytohormones, have been investigated as authenticity markers, in order to identify high quality products and to valorise niche products. The study regarded herbs (Argania spinosa), fruits (Citrus × myrtifolia, Punica granatum) and berries (Myrtus communis) mainly used as folk medicines for their broad spectrum of supposed pharmacological and therapeutic effects. The analytical methods developed within this study are based on high performance liquid chromatography and ultra-high performance liquid chromatography coupled to spectrofluorometric detection, triple quadrupole and high-resolution triple quadrupole mass spectrometry (HPLC-F, LC-MS/MS and UHPLC-HRMS). Significant efforts have been put also into the development and optimisation of miniaturised sample pretreatment strategies, such as micro-solid phase extraction (µSPE) and micro-extraction by packed sorbent (MEPS), able to purify complex matrices of natural origin (whole fruits, fruit parts, leaves and their extracts) and derived commercial products.

The work described in this thesis was performed in order to develop advanced analytical methods suitable to select and characterize GS- 3β inhibitors in vitro. GSK-3β is recognized as a key target for the development of new therapeutic agents for Alzheimer disease (AD). We validated an UHPLC-UV-Vis diode arrays detector (DAD) method for the very fast identification (resolution in less than 2 min) and determination of ADP and ATP in enzyme-based assay containing GSM-S syntetic peptide, ATP and GSK-3β. By using this method, selected inhibition hits will be characterized by defining their competitive mode of action with the substrate rather than with the ATP cofactor, in view of the discovery of compounds endowed of an increased GSK-3β selectivity over other protein-kinases. Next we hypothesized that GSK-3β could be directly involved in the regulation of histone acetylation through HDAC protein. Our hypothesis accounts that inhibition of GSK-3β, which leads to reduced HDAC activity, could restores the acetylation level in histones, protecting against neurodegeneration as both aging and AD pathology are associated with loss of histone acetylation (H4/H3) at N-terminus. Therefore, quantification of histone modifications on individual lysine residues is of crucial importance to understand their role in cell biology. We developed a targeted liquid chromatography mass spectrometry (LC-MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 from murine macrophage-like cell line RAW 264.7, with the perspective to apply the method on neuronal cells upon administration of GSK-3β inhibitors. The analytical strategy we developed shows that careful optimization of chemical derivatization steps at the protein and at the peptide level, combined with a more extensive digestion using chymotrypsin and trypsin, allows to differentiate between acetylation levels of each lysine residues.

Polypharmacology-based strategies are gaining ever-increasing attention as useful approaches to develop disease-modifying drug candidates for effective Alzheimer’s disease (AD) treatment. In this scenario, multitarget-directed ligands could increase efficiency by simultaneous modulation of several targets involved in AD pathogenesis. In drug discovery, natural products (NPs) represent an excellent source of evolutionary-chosen “privileged structures”. In this thesis, the polyphenol curcumin, found in Curcuma longa L, encompassing the essential structural elements for the concurrent inhibition of two validated AD targets, BACE-1 and GSK-3β, was rationally identified as lead compound. Aimed at developing well-balanced dual BACE-1/GSK-3β modulators with good BBB permeability, different series of curcumin-based derivatives were designed and synthetized by introducing suitable chemical modifications on the side aryl ring(s) and in the 4-position of the main scaffold. Furthermore, considering the pivotal role of the intramolecular H-bond network of curcumin’s central fragment in establishing appropriate interactions with target binding sites, several complexation and bioisosteric cyclization strategies were performed. Thanks to its strong Michael acceptor reactivity toward critical cysteine residues, curcumin exerts neuroprotection by additional activation of the Keap1-Nrf2-ARE signaling pathway. Thus, aimed at affecting the electrophilicity of its α,β-unsaturated carbonyl fragment, allowing a fine-tuning of its reactivity, diverse electrophilic functions were inserted in different positions of the curcumin scaffold. Furthermore, considering the neuroprotective and antioxidant potentials of simple coumarins, several curcumin-coumarin hybrids were also prepared. Recently, the inhibition of additional AD-correlated protein kinases (PKs), such as CK1 and LRRK2, could offer promises to achieve a successful treatment and indole was envisaged as useful scaffold for both PKs’ inhibition. Thus, a small library of indole-based derivatives was designed and synthetized as valuable BBB permeable pharmacological tools. Finally, chitosan (CS), a natural, nontoxic, biocompatible and biodegradable polysaccharide, was selected to develop CS-based bioconjugates for nanoparticles’ preparation as innovative drug delivery and targeting systems.

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

No description available.

Negli ultimi anni, un crescente numero di studiosi ha focalizzato la propria attenzione sullo sviluppo di strategie che permettessero di caratterizzare le proprietà ADMET dei farmaci in via di sviluppo, il più rapidamente possibile. Questa tendenza origina dalla consapevolezza che circa la metà dei farmaci in via di sviluppo non viene commercializzato perché ha carenze nelle caratteristiche ADME, e che almeno la metà delle molecole che riescono ad essere commercializzate, hanno comunque qualche problema tossicologico o ADME [1]. Infatti, poco importa quanto una molecola possa essere attiva o specifica: perché possa diventare farmaco è necessario che venga ben assorbita, distribuita nell’organismo, metabolizzata non troppo rapidamente, ne troppo lentamente e completamente eliminata. Inoltre la molecola e i suoi metaboliti non dovrebbero essere tossici per l’organismo. Quindi è chiaro come una rapida determinazione dei parametri ADMET in fasi precoci dello sviluppo del farmaco, consenta di risparmiare tempo e denaro, permettendo di selezionare da subito i composti più promettenti e di lasciar perdere quelli con caratteristiche negative. Questa tesi si colloca in questo contesto, e mostra l’applicazione di una tecnica semplice, la biocromatografia, per caratterizzare rapidamente il legame di librerie di composti alla sieroalbumina umana (HSA). Inoltre mostra l’utilizzo di un’altra tecnica indipendente, il dicroismo circolare, che permette di studiare gli stessi sistemi farmaco-proteina, in soluzione, dando informazioni supplementari riguardo alla stereochimica del processo di legame. La HSA è la proteina più abbondante presente nel sangue. Questa proteina funziona da carrier per un gran numero di molecole, sia endogene, come ad esempio bilirubina, tiroxina, ormoni steroidei, acidi grassi, che xenobiotici. Inoltre aumenta la solubilità di molecole lipofile poco solubili in ambiente acquoso, come ad esempio i tassani. Il legame alla HSA è generalmente stereoselettivo e ad avviene a livello di siti di legame ad alta affinità. Inoltre è ben noto che la competizione tra farmaci o tra un farmaco e metaboliti endogeni, possa variare in maniera significativa la loro frazione libera, modificandone l’attività e la tossicità. Per queste sue proprietà la HSA può influenzare sia le proprietà farmacocinetiche che farmacodinamiche dei farmaci. Non è inusuale che un intero progetto di sviluppo di un farmaco possa venire abbandonato a causa di un’affinità troppo elevata alla HSA, o a un tempo di emivita troppo corto, o a una scarsa distribuzione dovuta ad un debole legame alla HSA. Dal punto di vista farmacocinetico, quindi, la HSA è la proteina di trasporto del plasma più importante. Un gran numero di pubblicazioni dimostra l’affidabilità della tecnica biocromatografica nello studio dei fenomeni di bioriconoscimento tra proteine e piccole molecole [2-6]. Il mio lavoro si è focalizzato principalmente sull’uso della biocromatografia come metodo per valutare le caratteristiche di legame di alcune serie di composti di interesse farmaceutico alla HSA, e sul miglioramento di tale tecnica. Per ottenere una miglior comprensione dei meccanismi di legame delle molecole studiate, gli stessi sistemi farmaco-HSA sono stati studiati anche con il dicroismo circolare (CD). Inizialmente, la HSA è stata immobilizzata su una colonna di silice epossidica impaccata 50 x 4.6 mm di diametro interno, utilizzando una procedura precedentemente riportata in letteratura [7], con alcune piccole modifiche. In breve, l’immobilizzazione è stata effettuata ponendo a ricircolo, attraverso una colonna precedentemente impaccata, una soluzione di HSA in determinate condizioni di pH e forza ionica. La colonna è stata quindi caratterizzata per quanto riguarda la quantità di proteina correttamente immobilizzata, attraverso l’analisi frontale di L-triptofano [8]. Di seguito, sono stati iniettati in colonna alcune soluzioni raceme di molecole note legare la HSA in maniera enantioselettiva, per controllare che la procedura di immobilizzazione non avesse modificato le proprietà di legame della proteina. Dopo essere stata caratterizzata, la colonna è stata utilizzata per determinare la percentuale di legame di una piccola serie di inibitori della proteasi HIV (IPs), e per individuarne il sito(i) di legame. La percentuale di legame è stata calcolata attraverso il fattore di capacità (k) dei campioni. Questo parametro in fase acquosa è stato estrapolato linearmente dal grafico log k contro la percentuale (v/v) di 1-propanolo presente nella fase mobile. Solamente per due dei cinque composti analizzati è stato possibile misurare direttamente il valore di k in assenza di solvente organico. Tutti gli IPs analizzati hanno mostrato un’elevata percentuale di legame alla HSA: in particolare, il valore per ritonavir, lopinavir e saquinavir è risultato maggiore del 95%. Questi risultati sono in accordo con dati presenti in letteratura, ottenuti attraverso il biosensore ottico [9]. Inoltre, questi risultati sono coerenti con la significativa riduzione di attività inibitoria di questi composti osservata in presenza di HSA. Questa riduzione sembra essere maggiore per i composti che legano maggiormente la proteina [10]. Successivamente sono stati eseguiti degli studi di competizione tramite cromatografia zonale. Questo metodo prevede di utilizzare una soluzione a concentrazione nota di un competitore come fase mobile, mentre piccole quantità di analita vengono iniettate nella colonna funzionalizzata con HSA. I competitori sono stati selezionati in base al loro legame selettivo ad uno dei principali siti di legame sulla proteina. In particolare, sono stati utilizzati salicilato di sodio, ibuprofene e valproato di sodio come marker dei siti I, II e sito della bilirubina, rispettivamente. Questi studi hanno mostrato un legame indipendente dei PIs ai siti I e II, mentre è stata osservata una debole anticooperatività per il sito della bilirubina. Lo stesso sistema farmaco-proteina è stato infine investigato in soluzione attraverso l’uso del dicroismo circolare. In particolare, è stato monitorata la variazione del segnale CD indotto di un complesso equimolare [HSA]/[bilirubina], a seguito dell’aggiunta di aliquote di ritonavir, scelto come rappresentante della serie. I risultati confermano la lieve anticooperatività per il sito della bilirubina osservato precedentemente negli studi biocromatografici. Successivamente, lo stesso protocollo descritto precedentemente è stato applicato a una colonna di silice epossidica monolitica 50 x 4.6 mm, per valutare l’affidabilità del supporto monolitico per applicazioni biocromatografiche. Il supporto monolitico monolitico ha mostrato buone caratteristiche cromatografiche in termini di contropressione, efficienza e stabilità, oltre che affidabilità nella determinazione dei parametri di legame alla HSA. Questa colonna è stata utilizzata per la determinazione della percentuale di legame alla HSA di una serie di poliamminochinoni sviluppati nell’ambito di una ricerca sulla malattia di Alzheimer. Tutti i composti hanno mostrato una percentuale di legame superiore al 95%. Inoltre, è stata osservata una correlazione tra percentuale di legame è caratteristiche della catena laterale (lunghezza e numero di gruppi amminici). Successivamente sono stati effettuati studi di competizione dei composti in esame tramite il dicroismo circolare in cui è stato evidenziato un effetto anticooperativo dei poliamminochinoni ai siti I e II, mentre rispetto al sito della bilirubina il legame si è dimostrato indipendente. Le conoscenze acquisite con il supporto monolitico precedentemente descritto, sono state applicate a una colonna di silice epossidica più corta (10 x 4.6 mm). Il metodo di determinazione della percentuale di legame utilizzato negli studi precedenti si basa su dati ottenuti con più esperimenti, quindi è necessario molto tempo prima di ottenere il dato finale. L’uso di una colonna più corta permette di ridurre i tempi di ritenzione degli analiti, per cui la determinazione della percentuale di legame alla HSA diventa molto più rapida. Si passa quindi da una analisi a medio rendimento a una analisi di screening ad alto rendimento (highthroughput- screening, HTS). Inoltre, la riduzione dei tempi di analisi, permette di evitare l’uso di soventi organici nella fase mobile. Dopo aver caratterizzato la colonna da 10 mm con lo stesso metodo precedentemente descritto per le altre colonne, sono stati iniettati una serie di standard variando il flusso della fase mobile, per valutare la possibilità di utilizzare flussi elevati. La colonna è stata quindi impiegata per stimare la percentuale di legame di una serie di molecole con differenti caratteristiche chimiche. Successivamente è stata valutata la possibilità di utilizzare una colonna così corta, anche per studi di competizione, ed è stata indagato il legame di una serie di composti al sito I. Infine è stata effettuata una valutazione della stabilità della colonna in seguito ad un uso estensivo. L’uso di supporti cromatografici funzionalizzati con albumine di diversa origine (ratto, cane, guinea pig, hamster, topo, coniglio), può essere proposto come applicazione futura di queste colonne HTS. Infatti, la possibilità di ottenere informazioni del legame dei farmaci in via di sviluppo alle diverse albumine, permetterebbe un migliore paragone tra i dati ottenuti tramite esperimenti in vitro e i dati ottenuti con esperimenti sull’animale, facilitando la successiva estrapolazione all’uomo, con la velocità di un metodo HTS. Inoltre, verrebbe ridotto anche il numero di animali utilizzati nelle sperimentazioni. Alcuni lavori presenti in letteratura dimostrano l’affidabilita di colonne funzionalizzate con albumine di diversa origine [11-13]: l’utilizzo di colonne più corte potrebbe aumentarne le applicazioni. / Lately, an increasing number of scientists, academics as well as pharmaceutical industries, have focused their attention on the development of strategies to characterise the ADMET properties of the candidate drugs as early as possible. This trend is due to the awareness that about half of all drugs in development fail to make it to the market because of ADME deficiencies and that at least half of the ones that do make it to market still have some ADME or toxicological problems [1]. No matter how active nor specific is a chemical: to turn it into drug it needs to be well absorbed, distributed throughout the body, metabolised in a not too rapid nor too slow way and completely eliminated. Moreover, it and its metabolites should not be toxic for the body. Thus, it is clear how a rapid determination of ADMET parameters in early stages of drug discovery would save money and time, allowing to choose the better compounds and to eliminate any losers, early and cheaply. This thesis is set in this context, showing the application of a simple technique, biochromatography, to quickly evaluate candidate drugs as far as binding to human serum albumin (HSA) is concerned. Furthermore it shows another suitable independent technique, namely circular dichroism, able to study the same drug-protein system, allowing a deeper insight into the stereochemistry of the binding process. HSA is the most abundant protein in the blood. It acts as a carrier for a wide range of molecules either endogenous, such as bilirubin, tiroxine, steroid hormones and fatty acids, or xenobiotics. Furthermore, it allows the solubilisation of hydrophobic compounds (e.g. taxanes), characterized by very low solubility. The binding to albumin is usually stereoselective and occurs at high-affinity binding sites level. It is also well known that competition of drugs for the same sites on HSA can meaningfully alter their free fraction affecting their activity and toxicity. Thus, by its binding properties, HSA can affect the pharmacokinetics as well as the pharmacodynamic properties of drugs. It is not unusual that even whole drug discovery projects have been abandoned due to very strong binding to HSA or short lifetime or poor distribution due to weak binding. This makes HSA the most important serum protein from a pharmacokinetics point of view. A large body of literature has showed the reliability of the biochromatographic technique for the study of the biorecognition processes between proteins and small molecules [2-6]. My work was mainly finalised to use this technique to evaluate the binding characteristics of series of compounds and to improve such technique. To obtain a better comprehension of the binding mechanisms of the molecules investigated, circular dichroism was also employed. First, HSA was immobilised onto a classic packed epoxy silica-based column 50 x 4.6 mm i.d. using a slightly modified procedure previously reported [7]. In brief, the immobilisation was achieved by overnight recirculation of a solution of HSA through the column previously packed with epoxy silica particles, at set pH and ionic strength. Then, the column was characterised in terms of amount of HSA correctly immobilised by the frontal analysis of L-tryptophan [8]. By injecting some racemates known to bind the protein in a stereoselective manner, we also checked that the immobilisation procedure would preserve the binding properties of the free protein. The column so characterised was employed to determine the binding percentage of a small series of five HIV protease inhibitors (PIs), and also it was attempted to identify their binding site(s). The bound drug percentage was calculated from the capacity factor (k) of the samples. This parameter in only aqueous phase was extrapolated by linearly plotting the log k values against the percentage (v/v) of 1-propanol in the eluent mixtures. Only for two of the five compounds, it has been possible to measure the k value without organic modifier. All of the IPs analysed proved to strongly bind HSA; in particular the percentage of binding for ritonavir, lopinavir and saquinavir was found to be higher than 95%. The results are in agreement with data achieved by optical biosensor technique previously published [9]. In addition, these results are consistent with the significant reduction of their inhibitor activity observed in the presence of HSA. This effect seems to be greater for the inhibitors strongly bound to the protein [10]. Displacement studies were also performed by zonal elution approach. By this method, a known concentration of a competitive agent is continuously applied in the mobile phase to the HSA-based column, while small amounts of the studied drugs are injected. The competitors employed were chosen for their selective binding in the main binding areas of HSA. In particular salicylate, ibuprofen, and valproate were employed as markers of Sudlow’s site I, of Sudlow’s site II, and of bilirubin site, respectively. The displacement studies have shown an independent binding of PIs to sites I and II, while a slight anticooperativity was observed for the bilirubin site. The same system, drug – target protein, was finally investigated in solution using circular dichroism spectroscopy. The change in the induced CD spectrum of an equimolar complex HSA/bilirubin was monitored once increasing amounts of ritonavir, chosen as representative of the series, were added. The results confirmed a slight anticooperativity for the bilirubin binding site observed by the biochromatographic approach. Subsequently, the validation protocol previously described was applied to a novel 50 mm epoxy silica-based monolithic column to evaluate the reliability of using such support for biochromatographic studies. That monolithic column showed good chromatographic characteristics in terms of backpressure, efficiency and stability as well as reliability in drug binding parameters determination. Such column was applied in the determination of the binding percentage to HSA of a series of poliaminoquinones developed within a project on Alzheimer’s disease. All samples showed a binding percentage higher than 95%. Furthermore, the data obtained showed a good correlation between binding percentage and side chain chemical features (length and number of amine groups). Also in this case circular dichroism provided useful information about the binding sites on HSA of the chemicals studied: displacement studies indicated an anticooperative binding of these poliaminoquinones to sites I and II, while an independent binding with respect to bilirubin site was observed. The knowledge built up with the monolithic support previously described was applied to a shorter monolithic column (10 x 4.6 mm). The method previously described for the binding percentage determination is based on the data of several analyses, so it is time consuming. The use of a shorter column allows reducing the retention times of analytes. As a consequence the time needed to determine the binding percentage to HSA of a series of molecules undergo a tremendous decrease, turning such biochromatography from medium to highthroughput screening technique. Furthermore, the significant retention time shortness makes unnecessary the use of organic modifier, like 1-propanol, in the mobile phase. After the characterisation of the short column as previously described for the other columns, a series of standards were injected, also by changing the flow rate in order to evaluate the possibility to use high flows. The column was than employed to investigate the binding percentage to HSA and the main binding sites of a series of molecules with different moieties. Finally, the stability of the column was evaluated, in terms of reliability of results after repeated analysis. The development of chromatographic supports based on albumins from other mammalian species (i.e. rat, dog, guinea pig, hamster, mouse, rabbit) may be proposed as a future application of these short columns. In fact, this would allow a better comparison between data achieved by in-vitro experiments and data collected by experiments on animals, making easier the following data extrapolation to human, with the speed of an high-throughput screening method. Moreover it may reduce the number of animals used for pharmacokinetics investigations. Some papers previously published proved the reliability of these albumin-based supports [11-13]: using a shorter column may enhance its applications.