Investigation of in vitro and in vivo pharmacokinetics and biological evaluation of pharmacologically active compounds

The first part of this thesis deals with the evaluation of in vitro and in vivo pharmacokinetics of novel broad-spectrum antiviral compounds active against enveloped viruses. The design and synthesis of new series of antiviral compounds with 1H-pyrrol-methylene thioxodihydropyrimidine structure, has been realized by Professor Maurizio Botta's research group, at the University of Siena. Compounds’ antiviral activity has been evaluated on several enveloped viruses, such as ZIKAV, DENV-2 and five influenza strains including the pandemic strain H7N9. The selectivity against enveloped viruses, time of addition and binding experiments confirmed their ability to intercalate in the viral envelope membrane, oxidize phospholipids and alter the fluidity of the lipid bilayer, compromising the efficacy of the virus-cell fusion step and preventing viral entry. With the aim of investigating the in vitro ADME properties, the most active compounds were selected to assay their chemical-physical properties and early select the most promising lead candidate. Thus, membrane permeability, binding to human serum albumin, and stability in human plasma and microsomes have been assayed. Finally, the lead candidate was selected to evaluate preliminary in vivo pharmacokinetic parameters; after formulation studies, the compound was administrated intravenously (iv) at the dose of 25 mg/kg and 12.5 mg/kg. The second chapter of this Ph.D. thesis concerns the investigation of the in vitro biological profile of nitric oxide-donor largazole prodrugs. Two hybrid analogues of largazole, as dual HDAC inhibitor and nitric oxide (NO) donors potentially useful as anticancer agents, have been designed and synthesized thanks to the collaboration between Professor Maurizio Botta’s research group and IRBM. Largazole is a natural product identified as the most potent and selective Class-I deacetylase (HDAC) inhibitor, that showed a broad-spectrum growth-inhibitory activity against epithelial and fibroblastic tumor cell lines and a low cytotoxicity profile. Over the last decades, dual nitric oxide (NO) donors/HDAC inhibitors have been developed as novel anticancer chemical entities, potentially more efficacious than selective HDAC inhibitors, owing to the capability of NO to specifically modulate the function of some HDAC isoforms and to overcome tumor cell resistance to conventional treatments. Thus, after the synthesis, the characterization of derivatives compounds and the in vitro NO release assay performed by Professor Maria Frosini using the Griess method, biological evaluation of their antiproliferative activities against U-2OS, Caco-2, and IMR-32 cell lines have been conducted. To further explain the additive antiproliferative effect of NO-donor compounds vs largazole, their stabilities both in human plasma and in cell culture medium were assessed. The third and last chapter of this Ph.D. thesis deals with the project I participated in during my exchange period at the research group of Professor Per Artursson, who hosted me for 5 months, at Uppsala University. In collaboration with AstraZeneca, a series of antisense oligonucleotide (ASO) conjugates, targeting MALAT1 chosen as a model target, were used to assess and validate their silencing efficiency and enhance/overcome endosomal escape. The MALAT1 silencing efficiencies of lipophilic ASO-conjugates and a peptide-ASO have been determined in the presence and absence of a cyclic cell permeation peptide (CPP) in two human embryonic kidney (HEK293) cell lines of which one overexpressing the target G-protein coupled receptor selected for the study. For this purpose, the expression levels of the MALAT1 gene mRNA were measured using qPCR in a time (0-24-48 hrs) and concentration (0.5-2.5-5 µM of ASOs and 5-10-15 µM of CPP)-dependent manner.