REPORT PhD Activities

1 REPORT PhD Activities

Ph.D. School Science of Drug and Bioactive Substances (Cycle XXXII) Candidate: Arianna Mancini

Supervisors: Prof. Elena Dreassi, Prof. Tiziano Tuccinardi

Thesis Title: Preclinical Characterization and Liposomal Formulations of Pyrazolo[3,4-d]]Pyrimidine SRC Inhibitors for Cancer Treatment

First Year (2016/2017)

During my first year, several formulations were prepared to encapsulate and deliver in vivo the anticancer drug CPD, a pyrazolo[3,4-d]pyrimidine derivative: albumin nanoparticles, liposomes and Tween80 formulations. The last two of these formulations were administered in vivo to evaluate PK and BD. At several time points (5', 15', 30', 60', 90', 180', 240', 480', and 1440'), after drug administration, mice were sacrificed, and blood and organs were collected for the quantitative analysis. Obtained results suggested that CPD-Tween80 formulation, when administered both iv and per os, showed comparable PK and BD profiles, except for brain distribution that was 6 times higher for oral administration. On the other hand, liposomal formulation showed a higher distribution in liver and spleen. Furthermore, liposomes ensured long blood circulation trend, that ensures an increased plasma exposure to the drug and the possibility of a daily single administration.

WORKSHOPS AND SEMINARS

• The Cambridge Chrystallographic Database: use and application, Prof.ssa Fabrizia Fabrizi De Biani, 16/11/2016 (8h)

• Zebrafish: utilizzo in ricerca. Mantenimento e stabulazione in colonia. Dott. Tommaso Sala, 22/11/17.

• SgK1 a novel metabolic target in metabolic syndrome and cancer. Nicola Perrotti, 27/04/17.

• NMTrypl: an european project to bring new drugs in the pipeline of Chagas, Leishmaniasis and HAT. Multitargeting of specific pathways to kill the parasites, Dott.ssa Maria Paola Costi, 2/12/2016.

• From boron allylation chemistry to gold nanoparticles based nanosystem. Jack Chen. 29/06/17.

2 • Materiali e nanomateriali nelle scienze chimiche, farmaceutiche e biomediche. Agnese

Magnan, 18/05/17.

• XI EWDD, European Workshop in Drug Design, Certosa di Pontignano, 21-26/05/17.

•

Second Year (2017/2018)

During my second year, a new formulation was prepared and characterized for the therapy of neuroblastoma: anti-GD2 liposomes for the administration of the anticancer drug CPD, a pyrazolo[3,4-d]pyrimidine derivative. Indeed, neuroblastoma tumor express abundant amounts of disialo-ganglioside GD2 at the cells surface and this epitope is only minimally expressed by normal tissues, such as the cerebellum and peripheral nerves. Thus, anti-GD2 antibody has been proposed as a selective ligand for targeting liposomal drug towards human NB cells.

Cytotoxicity tests have been performed for CPD and CPD-LP for NB cell line, demonstrating a retention of efficacy when CPD is encapsulating in liposomes. NB cell binding tests showed a three times greater binding for anti-GD2 liposomes in respect to bare liposomes.

WORKSHOPS AND SEMINARS

• 09/10/2018 SEMINAR “Variations on a theme – vinylogous mukaiyama aldol reactions in the total syntheses of natural products”. Prof. MARKUS KALESSE.

• 24/05/2018 CERTIFICATE OF ATTENDANCE - VII EUROPEAN WORKSHOP IN DRUG SYNTHESIS. University of Siena (Italy). Prof. MAURIZIO BOTTA.

• 22/03/2018 SEMINAR “Listening to macromolecular dynamics and interactions”. Prof. LUCASZ JAREMKO.

• 21/03/2018 SEMINAR “The conformational universe of proteins”. Dr. MARIUSZ JAREMKO. • 13-15/02/2018 COMPLEMENTARY SKILLS “Creating value from large archive and big data”.

Dr. Giulio Ghellini and Laura Neri (11h).

• 12/02/2018 SEMINAR “Organic bioelectronics: fundamentals, biosensors, transducers”. Prof. FABIO BISCARINI.

3 • 09/01/2018 SEMINAR WITH FINAL EVALUATION TEST “Modalità di anestesia e di

eutanasia-sacrificio”. ORGANISMO PREPOSTO al BENESSERE degli ANIMALI. University of Siena. Dr. GIACOMO MATTEUCCI and Dr. CARLA GAMBARANA.

• 30/11/2017 COMPLEMENTARY SKILLS, “Ethics and etiquette of publication: some do and don't researchers should be aware of”. Prof. MAURO BARNI (3h).

• 21/11/2017 SEMINAR WITH FINAL EVALUATION TEST “Aggiornamento su legislazione in materia di sperimentazione animale”. ORGANISMO PREPOSTO al BENESSERE degli ANIMALI. University of Siena. Dr. GIACOMO MATTEUCCI and Dr. CARLA GAMBARANA. • 15/11/2017 SEMINAR “Coherent Movement of Charge through Molecules”. Prof. THOMAS

HANSEN.

Third Year (2018/2019)

During my PhD third year, three formulations of the anticancer drug CPD, a pyrazolo[3,4-d]pyrimidine derivative (anti-GD2 liposomes, bare liposomes and Tween80 formulations) have been tested in vitro and in vivo for their PK profile and efficacy. Obtained results suggested that liposomes ensured long blood circulation trend, that ensures an increased plasma exposure to the drug and the possibility of a daily single administration. In IMR32-aluc bearing mice, results showed that anti-GD2-LP allowed for a continuous targeting of the tumour area. Cytotoxicity tests have been performed for CPD and CPD-LP and anti-GD2-LP for NB cell line, demonstrating that antiGD2 on the liposomal surface increased the antiproliferative effect for GD2- expressing NB cell lines. NB cell binding tests showed a 60 times greater binding for anti-GD2 liposomes in respect to bare liposomes for GD2- expressing NB cell lines.

WORKSHOPS AND SEMINARS

• 14/11/2018 SEMINAR: Synthetic lethality. As a new paradigm for the development of anticancer agents. Prof. ANDREA CAVALLI

• 13/12/2018 SEMINAR: Realization and application of an innovative technological platform powered by QM/MM simulations for the development of photoactive molecules. Prof.ssa ANNALISA SANTUCCI.

• From 29/03/19 to 3/04/19 Annual Meeting AACR – Atlanta (Georgia, USA)

4 MAURIZIO BOTTA.

• From 20 to 24/06/2019 SEMINAR: Minicourse in protein physics. Prof. PAOLO CARLONI • 2-3/09/2019 COMPLEMENTERY SKILLS: Scientific Writing and Presentation. JOHN L.

TEFORD

• 10/09/2019 COMPLEMENTERY SKILLS: Comunicare in ricerca. ELENA MELI

• 18/09/2019 COMPLEMENTERY SKILLS: Comunicare la ricerca. AGNES ALLANS DOTTIR • 24/09/2019 COMPLEMENTERY SKILLS: Creating value from large archive and big data.

LAURA NERI

• 26/09/2019 COMPLEMENTERY SKILLS: La ricerca dell’informazione di ambito scientifico su internet. LAURA BIANCIARDI

List of publications

1. E. Dreassi; A. Mancini; G. Corbini; F. Tarchi; V. Francardi. Tolerance and Bioaccumulation of Deltamethrin, Tebuconazole e Chlormequat chloride in T. molitor larvae. Manuscript status: Submitted to Journal of Insects as Food and Feed.

2. A. Molinari; G. Iovenitti; A. Mancini; G. Gravina; M. Chebbi; M. Caruana; G. Vignaroli; F. Orofino; E. Rango; A. Angelucci; E. Dreassi; S. Schenone; M. Botta. AuNPs Pyrazolo[3,4-d]Pyrimidine Nanosystem in Combination with Radiotherapy Against Glioblastoma. Manuscript status: Submitted to ACS Med. Chem. Lett.

3. Tassini, S.; Langron, E.; Delang , L.; Mirabelli, C.; Lanko, K.; Crespan, E.; Kissova, M.; Tagliavini, G.; Fontò, G.; Bertoni, S.; Palese, S.; Giorgio, C.; Ravanetti, F.; Ragionieri, L.; Zamperini, C.; Mancini, A.; Dreassi, E.; Maga, G.; Vergani, P.; Neyts, J.; Radi, M. Multi-Target CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach. Med. Chem. 2019, 62 (23), 10833-10847.

4. Fallacara, A.L.; Zamperini, C.; Podolski-Renic, A.; Dinic, J.; Stankovic, T.; Stepanovic, M.; Mancini, A.; Rango, E.; Iovenitti, G.; Molinari, A.; Bugli, F.; Sanguinetti, M.; Torelli, M.; Martini, M.; Maccari, L.; Valoti, M.; Dreassi, E.; Botta, M.; Pesic, M.; Schenone, S. A New Strategy for Glioblastoma Treatment: In Vitro and In Vivo Preclinical Characterization of Si306, a Pyrazolo[3,4-d]Pyrimidine Dual Src/P-Glycoprotein Inhibitor. Cancers (Basel) 2019, 11 (6), 848. https//doi.org/10.3390/cancers11060848.

5 5. Pasero, C.; D’Agostino, I.; De Luca, F.; Zamperini, C.; Deodato, D.; Truglio, G.I.; Sannio, F.; Del Prete, R.; Ferraro, T.; Visaggio, D.; Mancini, A.; Guglielmi, M. B.; Visca, P.; Doquier, J. D.; Botta, M. Alkyl-guanidine Compounds as Potent Broad-Spectrum Antibacterial Agents: Chemical Library Extension and Biological Characterization. J. Med. Chem. 2018, 61 (20), 9162-9176. https//doi.org/10.1021/acs.jmedchem.8b00619.

6. Calandro, P.; Iovenitti, G.; Zamperini, C.; Candita, F.; Dreassi, E.; Chiariello, M.; Angelucci, A.; Schenone, S.; Botta, M.; Mancini, A. Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4-d]pyrimidines for Targeting Hepatocellular Carcinoma. ACS Med Chem Lett. 2018, 9 (7), 646-651. https://doi.org/10.1021/acsmedchemlett.8b00062.

7. Fallacara, A.L.; Mancini, A.; Zamperini, C.; Dreassi, E.; Marianelli, S.; Chiariello, M.; Pozzi, G.; Santoro, F.; Botta, M.; Schenone, S. Pyrazolo[3,4-d]Pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation, characterization and cytotoxicity evaluation against neuroblastoma cell line. Bioorg. Med. Chem. Lett. 2017, 27 (14), 3196-3200.