Vincenza Andrisano

A Development of new drugs:

(i) development of new drugs for the treatment of degenerative diseases (Alzheimer's disease and cancer) : structural characterization of protein targets (i.e. amyloid peptides) and candidate leads (circular dicroism in solution, HPLC-MS),enzyme kinetics, determination of mechanism of action of new potential acetylcholinesterase inhibitors with dual function, inhibition studies of b-amyloid fibril formation by circular dichroism and  fluorescence spectroscopy (ii) characterization of the ligand/target peptide/protein/enzyme interactions (classical and multiwells spectroscopic kinetic methods, biochromatography, analysis through optical biosensor, HPLC-MS) (iii) Immobilisation of target enzymes on solid matrices which are then inserted in fluidic and chromatographic systems for binding studies.

B) Development of analytical methods for the determination of drugs and biologically active compounds in complex matrices (pharmaceutical formulations, cosmetics, natural products, biological samples) by : liquid chromatography coupled with mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with photodiode array detector (HPLC-DAD), liquid chromatography coupled with  circular dichroism , UPLC (ultra-pressure liquid chromatography).

Stability and photostability studies of drugs and biologically active compounds. Validation of analitycal methods.

A1.Innovative analytical methodologies for drug discovery. The goal will be the development of screening methods for the selection of potential drugs, coming from both synthetic and natural sources, for the treatment of Alzheimer's disease (AD), cancer and obesity.

One of the characteristics of AD that hinders the discovery of effective disease-modifying therapies is the multi-factorial nature of its etiopathology. AD is an age-related neurodegenerative disorder clinically characterized by the loss of memory and progressive deficits in different cognitive domains. Great effort has been devoted to elucidate the relationships between the hallmarks of the disease, that is, i) amyloid-β (Aβ) plaques, a massive deposit of amyloid peptide generated by successive cleavage of the amyloid precursor protein (APP) by β-(BACE-1) and γ-secretase, ii) neurofibrillary tangles, composed of hyperphosphorylated tau protein, and iii) loss of neurons in the hippocampus and nucleus basalis of Maynart. AD is characterized by a pronounced degradation of the cholinergic system and by alterations in other neurotransmitter systems such as the glutamatergic and serotoninergic ones.

To circumvent this drawback, the use of multi-target-directed ligands (MTDLs) has recently been proposed as a means of simultaneously hitting several targets involved in the development of the AD syndrome. With these concepts in mind, a more successful research program is aimed at identifying new MTDLs possessing different activities toward AD relevant targets, such as AChE, Ab processing and aggregation, and oxidative stress.

On these premises, β-secretase, also called BACE-1 (β-site APP-cleaving enzyme), the enzyme involved in the generation of the amyloid peptide, BChE (butyrylcholinesterase or pseudo-cholinesterase) which, following recent publications, might replace AChE as  acetylcholine degradating enzyme in patients with moderate AD, and amyloid peptides self-assembly will be the focus of this project as new targets for MTDLs screening for lead selection in AD treatment.   

The project is aimed to optimize online HPLC systems with the development of immobilized enzyme reactors (IMER) coupled with MS and/or electrochemical and/or spectroscopic detection for the high throughput screening of new inhibitors as potential new drugs for Alzheimer's disease.

By following our previous work on AChE, IMERs will be used to investigate the properties of selected potent inhibitors, including a qualitative assessment of the mechanism of enzyme inhibition (i.e. competitive, non-competitive, etc.) and a quantitative evaluation of inhibition constants (Ki), as previously described. The new systems will allow us to further exploit the selected targets and to gain more information on new potent inhibitors. More in details,  the screening of D-carnosine analogues will be performed on AChE-IMER.

IMERs preparation and characterisation involve essentially three steps: the immobilization of a target enzyme, the assessment of unchanged protein functions after immobilization, and the determination of ligand binding parameters, after the insertion of the immobilized protein in a separation system. Concerning BChE and BACE-1 immobilization, micro-IMERs will be prepared by using an in situ immobilization procedure on an ethylendiamine monolithic Convective Interaction Media (EDA-CIM) disk. The activity and kinetic parameters of the micro-IMERs will be investigated by insertion in a HPLC system with fluorescent, UV or mass detection. 

BACE1 micro-IMER will be characterized in terms of units of immobilised enzyme and optimal mobile phase conditions for activity, by using as substrate Casein-FITC and JMV2236 a peptide mimicking the Swedish-mutated APP (amyloid precursor protein) sequence. In the case of  BChE-IMER butyrylthiocholine will be used as substrate and Ellman's Reagent will be added to the mobile phase in order to obtain a UV-Vis detectable product.

Inhibition studies will be carried out by simultaneous injecting the substrate and the tested compounds. The relative IC50 value will be obtained by evaluating the percentage reduction of product peak area at increasing inhibitor concentration.

Concerning the development of efficient methodologies for lead selection in cancer treatment, in recent years, an increasing interest in HDACs as target for new anti-neoplastic drugs has been registered, due to the ability of HDACs inhibitors to arrest tumour cell growth in vitro and in animal models at doses that cause little or no toxicity.

HDACs inhibitors (short chain compounds i.e., butyrate and valproate, and hydroxamic acids such as thrichostatin A) have been described as potential anticancer drugs in a variety of preclinical studies. In fact, treatment of cells with these inhibitors results in the increase of highly acetylated forms of histones, which affect gene expression, cell differentiation, and apoptosis.

Analytical methodologies for the determination of the activity of new HDACs inhibitors are therefore required for the selection of new anti-tumour leads in more complex biological systems.  

In this respect, a HPLC-ESI-MS method has been already developed to determine intact histone proteins after extraction from nuclei of HT29 colon cancer cell lines. However, besides the acetylation level of histone proteins, the determination of the specific aminoacid involved in the acetylation is a critical information to correlate histone post-translational modifications and cell cycle evolution. A HPLC-MS method with  immobilized trypsin reactor will be optimised to analyse the tryptic map and the acetylation sites of histone proteins after inhibition of HDAC.

In this field capillary electrophoresis will be also applied. To this end new permanently coated capillaries, able to reduce the adsorption of proteins as well as the electroosmotic flow, will be prepared. The coating procedures based on different polymers (hydroxypropylmethyl cellulose and polyvinyl alcohol) will be optimized to obtain highly efficient separations of human histones.

Considering the important biological activity recently described for the lipophilic constituents (alkamides and polyacetylenes) of Echinacea, HPLC and CE methods will be optimized and validated for the separation, identification and quantitation of these compounds in complex matrices, such as plant material, extracts and natural products.

Regarding Ephedra and Citrus species, fully validated methods for a simple, fast and reliable analysis of Ephedra and Citrus alkaloids, without the use of ion-pairing, will be developed to ensure the quality and safety of commercial products. In the case of Ephedra, validated methods will be optimized and applied to monitor the quality of Ephedra plant material and extracts, to determine the composition of commercial products and to verify their label claims, including the absence of ephedrine alkaloids in Ephedra-free products. Since Ephedra and Citrus alkaloids are chiral compounds, enantioselective techniques will be developed to separate and quantify the enantiomers, and evaluate their distribution in crude extracts and natural products.

Analytical protocols will be optimized for the extraction, derivatization and quantitation of these analytes by GC/MS and HPLC/UV techniques.

A2 Innovative analytical methodologies for ADME studies Selected active compounds from RA1 will be characterised for their affinity to plasma proteins (human serum albumin; HSA, and alpha-1-acid-glycoprotein, AGP) by means of biochromatography and optical biosensor methodologies. The proteins will be anchored to monolithic silica matrix, or to the biosensor surface chip. The immobilization procedure will be selected in order to guarantee that the anchored protein maintain the same binding properties as in solution. The different classes of compounds will be screened for their bound fraction and their binding parameters (KA, ka, kd) will be determined. In particular, optical biosensors result well suited for the kinetic parameters determination. This aspect is particularly important in the selection of possible hits because of the significant contribution of the kinetics to the drug distribution. Further characterization of distribution parameters of drug candidates will be obtained by studying the binding site/s on the plasma carriers by displacements experiments with selected markers of the single sites. This will allow determining the nature of the binding sites for each class of compounds, and then to evaluate possible interactions with metabolites and potential co-administered. This interaction between drugs represents a hot topic because of the significant influence of the free fraction and then on the drug activity and toxicity. An alternative approach to the immobilized protein column is given by capillary electrophoresis in free solution; HSA is used as a pseudo-stationary phase and information on drug-protein and drug-drug interactions can be achieved. Phase I of the project will concern the development of liposome and protein immobilization procedures to the silica matrix and biosensor surfaces; Phase II will concern the application of the methodologies to get information on absorption and distribution parameters of the studied drugs.

The research activity will be extended to the analysis of drugs in plasma after administration of innovative or experimental formulations. To this end, highly sensitive and selective methods will be developed, based on liquid chromatography (HPLC) and  capillary electrophoresis (CE) combined with appropriate detectors. The studies will be carried out in collaboration with Clinics and  Health Centres.

A3  Innovative analytical methodologies for quality control, pre-formulation and formulation. The determination of drug related impurities in complex matrices requires for rapid and selective analytical methods; to this regard, electrokinetic techniques will be applied to develop highly selective and enantioselective methods for synthetic and herbal drugs. Mixed electrokinetic systems based on double pseudostationary phases (cyclodextrins and ionic surfactants) and/or microemulsions, will be firstly characterized from the physical and chemical point of view. To this regard, capillary electrophoresis experiments will be directed to the estimation of the binding of surfactant to cyclodextrins and the obtained information will be integrated with the results from other independent techniques (dynamic light scattering and electron paramagnetic resonance spectroscopy). In particular, the extent as well as the stoichiometry of the interaction between cyclodextrins and surfactant monomers, which are the competing components of the pseudostationary phase in CE, have to be considered in order to clarify the mechanism of the separation processes. Successively, the application of the characterized proposed systems, will be directed to the determination of chiral phytomarkers in a variety of edible plant extracts. Precisely, ( -)-catechin and ( -)-gallocatechin have been found to be useful as markers of degradation in plant extracts because their presence is only related to the epimerisation of ( -)-epicatechin and ( -)-epigallocatechin, two of the most widespread secondary metabolites in plant kingdom. Since the kinetic of the considered epimerisation has shown to be rather fast, the presence of these exogenous compounds in food and plant extracts can be considered as the evidence of product deterioration. Furthermore, recent evidences have demonstrated the lower bioavailability of ( -)-catechin compared to that of natural (+)-catechin. For these reasons, the analysis of the selected chiral phytomarkers will be proposed as a useful tool in the quality assessment of food and herbal drugs as well as to provide information about their manufacture processing and to assure their compliance with the trade laws.