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Pharmaceutical and Biopharmaceutical Analysis Unit  

Group members: Prof. Marina Carini, Prof. Giancarlo Aldini, Dr. Marica Orioli, Dr. Giangiacomo Beretta

Postdoc and PhD students: Mara Colzani, Luca Cannizzaro,  Danilo De Maddis, Davide Garzon, Alessandra Altomare

The team, which is organized in two main subunits (Bioanalysis and Proteomics), conducts basic as well as applied research within the area of pharmaceutical analysis. The main research areas involve the identification of biomarkers and of pharmacological targets; the study of interactions between drugs and molecular targets through mass spectrometry; the metabolism of drugs and xenobiotics; the development of biologically active compounds; the quality control of drugs, medical devices, nutritional supplements, plant extracts and cosmetics; the isolation, characterization and in vitro/in vivo evaluation of the biological activity of vegetal active principles. In detail, the team components share a wide range of complementary expertises in the following areas of interest:
1) Protein oxidation and carbonylation: proteomic and mass spectrometric approaches.
This topic is framed in an international project (in collaboration with the Tuft's University, Boston, USA and Nagoya University, Japan) finalized to the identification and characterization of new pharmacological targets and biomarkers of oxidative/carbonyl stress associated to a variety of pathological conditions. The research is focused on the development and application of mass spectrometric and immunochemical techniques for the identification of post-translational modifications of proteins in different biological matrices as early biomarkers of oxidative/carbonyl damage.
Recognition of this activity by the scientific community permitted G. Aldini to be nominated Italian delegate in the Management Committee of the COST Action CM1001 (Chemistry of non-enzymatic protein modification - modulation of protein structure and function)

Yamaguchi S, Aldini G, Ito S, Morishita N, Shibata T, Vistoli G, Carini M, Uchida K. Delta12-prostaglandin J2 as a product and ligand of human serum albumin: formation of an unusual covalent adduct at His146. J Am Chem Soc. 2010 Jan 20;132(2):824-32.

2)   Mass spectrometric strategies for studying biomacromolecules-ligand interaction and covalent binding.
Another important field of interest regards the identification and characterization, by different proteomic and mass spectrometric strategies, including high resolution MS (HRMS), of non-covalent and covalent ligand binding to biomacromolecules, in particular to proteins and nucleic acids.  In collaboration with a Spanish research group, the activity is focused on the elucidation of the covalent binding of amoxicillin with serum proteins, with the final aim to give a novel insight into the mechanisms involved in amoxicillin-elicited allergic reactions. In this context, HRMS strategies are applied as novel analytical tools for the study of protein haptenation. HRMS is also applied in the drug discovery field, and in particular for studying the non-covalent interaction between ligand and macromolecules. Such an application is newly emerging, not only for the rapid identification of hit compounds from non-deconvoluted mixtures but also the retrieval of some important information on the ligand-macromolecule complex, including stoichiometry, association constants and the effect of the ligand on the protein conformation. This approach has been applied for the characterization of small ligands of beta2-microglobulin (in collaboration with the University of Pavia) and of small molecules able to recognize T:G mismatches in DNA targets, an approach useful for the discovery of new antitumor drugs (in collaboration with Nerviano Medical Sciences)
Ariza A, Garzon D, Abánades DR, de Los Ríos V, Vistoli G, Torres MJ, Carini M, Aldini G, Pérez-Sala D Protein haptenation by amoxicillin: High resolution mass spectrometry analysis and identification of target proteins in serum. J Proteomics. 2012;77:504-20.
Riccardi Sirtori F, Aldini G, Colombo M, Colombo N, Malyszko J, Vistoli G, D'Alessio R. Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry. ChemMedChem. 2012;7(6):1112-22

3) Development of new detoxifying agents of cytotoxic reactive carbonyl species (RCS).
By considering the lipoxidation-derived reactive carbonyl species (RCS) as potential drug targets in preventing protein carbonylation and related cellular dysfunction, the research project (in collaboration with academic and industrial partners) focuses on the development of new RCS detoxifying agents, using the natural dipeptide carnosine as active scaffold. The project involves an in vitro screening phase devoted to the evaluation of the biological activity and metabolic stability of the developed compounds, followed by in vivo evaluation of the pharmacokinetic and pharmacodynamic profiles (in experimental animal models of carbonyl stress) of the lead compounds identified in the in vitro approach. The research activities above reported contributed to the applications of three patents.

Orioli M, Vistoli G, Regazzoni L, Pedretti A, Lapolla A, Rossoni G, Canevotti R, Gamberoni L, Previtali M, Carini M, Aldini G. Design, synthesis, ADME properties, and pharmacological activities of β-alanyl-D-histidine (D-carnosine) prodrugs with improved bioavailability. ChemMedChem. 2011;6(7):1269-82.

4) Active principles of vegetal source.
In this topic are collected the studies finalised to the isolation, characterization, quantization and evaluation of the biological activity (in vitro and in vivo) of active principles of vegetal sources. The application of combined chromatographic, spectroscopic and mass spectrometric techniques to crude vegetal extracts (Aesculus hippocastanum L., Hedera helix L., Helichrysum italicum G. Don, Echinacea angustifolia, Krameria triandra, Ilex Paraguayensis, Rous Coriaria) allowed to isolate and identify single components (or homogeneous fractions) with anti-edematous, anti-jaluronidase, antioxidant/radical scavenging, cardioprotective or vasorelaxant activity. Current studies are aimed at the identification and characterization of bioactive compounds in different plants (Achillea millefolium, Sumac) and in honeys from different botanical sources.

Beretta G, Granata P, Ferrero, M, Orioli M, Maffei Facino R, Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics. Anal Chim Acta. 2005; 533(2):185–191.

5) Free radicals, reactive oxygen species and radical scavenging compounds.
This topic deals with the development and application of different bioanalytical methodologies for the study of the free radical-induced oxidative damage and for the definition of the antioxidant profile of synthetic compounds or compounds isolated from crude vegetal extracts to be used in the pharmaceutical, nutraceutical and cosmetic fields. The activity evaluation, carried out in in vitro, ex-vivo and in vivo systems, is based on the determination (through both conventional and innovative methodologies) of the total antioxidant power (ORAC, TRAP, TAP assays) of complex matrices such as plasma, serum, foods, dietary supplements, vegetal extracts, cosmetics.

Aldini G, Regazzoni L, Pedretti A, Carini M, Cho SM, Park KM, Yeum KJ. An integrated high resolution mass spectrometric and informatics approach for the rapid identification of phenolics in plant extract. J Chromatogr A. 2011;1218(20):2856-64.

6) ADMET studies
An important aspect in drug discovery is the assessment of metabolic stability, as well as the early structural identification of the metabolites of potential new drugs. Information on the metabolically labile points will suggest structural modifications to improve metabolic stability, and will allow an early safety assessment via the identification of metabolic activation products. The overall strategy of this topic, focusing on several tasks, enables the identification of potential development challenges and ‘‘show stoppers’’, to have the opportunity to select the best candidates for advancement. In this context, assessment of ADMET properties of drug candidates at early drug discovery stages enable structural changes for better scaffold design and compound selection, thus to optimize the discovery process both in terms of cost and time. Metabolic stability in different primate and rodent matrices of rodents and primates (liver subcellular fractions, plasma, human skin), stability in simulated gastrointestinal fluids, metabolites characterization, cell toxicity, pharmacokinetics (experimental animal and man) represent the main tasks, owing to the expertise gained by the team in the following fields:

a) In vitro and in vivo metabolism of drugs and their interaction with the liver drug-metabolizing enzymes,
b) Study of the hepatic detoxification processes of xenobiotics in physiological and pathological situations;
c)      Development and application of new methodologies in mass spectrometry for the quali-quantitative analysis of drugs and metabolites in complex matrices (cells, tissues, biological fluids)
d) Development and application of LC-MS/MS methods to ADME studies (animals, humans).
Panusa A, Regazzoni L, Aldini G, Orioli M, Giombini A, Minghetti P, Tranquilli C, Carini M. Urinary profile of methylprednisolone acetate metabolites in patients following intra-articular and intramuscular administration. Anal Bioanal Chem. 2011;400(1):255-67.

National and International collaborations

Department of Biosciences (University of Milan)
Department of Drug Sciences (University of Pavia)
Department of Drug Sciences (University of Piemonte Orientale)
Department of Drug Sciences and Technology (University of Turin)
Department of Clinical and Preclinical Pharmacology (University of Florence)
Department of Molecular and Clinical Medicine (University “La Sapienza” of Rome)
Department of Pharmaceutical Sciences (University of Catania)
Oncology Business Unit (Nerviano Medical Sciences, Nerviano, Italy)
Coni (National Olympic Italian Commitee)
Ministery of Health
Department of Pharmaceutical Sciences (National Health Institute, Rome, Italy)
Department of Nephrology and Dialysis, A. Manzoni Hospital, Lecco - Italy.
Graduate School of Bioagricultural Sciences, Nagoya University (Japan)
Jean Mayer USDA Human Nutrition Research Center on Aging, Tuft’s University Boston
Division of Human Nutrition, Department of International Health, Johns Hopkins Bloomberg School of Public Health (Baltimora, USA)
Department of Movement and Sport Sciences, Ghent University (Belgium)
Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas (Madrid, Spain)
Institute for Drug Research - Department of Pharmacology  (The Hebrew University Faculty of Medicine, Jerusalem, Israel)
Abteilung Neuropathologie Neurozentrum Universitätsklinikum Freiburg (Germany)

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