PRECLINICAL STUDIES OF PIXANTRONE AS IMMUNOSUPPRESSIVE DRUG IN THE ANIMAL MODEL OF MYASTHENIA GRAVIS
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
PRECLINICAL STUDIES OF PIXANTRONE AS IMMUNOSUPPRESSIVE DRUG IN THE ANIMAL MODEL OF MYASTHENIA GRAVIS / C. Ruocco ; tutors: F. Baggi, A. Panerai ; coordinator: A.Panerai. DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2012 Feb 03. 24. ciclo, Anno Accademico 2011. [10.13130/ruocco-chiara_phd2012-02-03].
Abstract:
Myasthenia Gravis (MG) is an antibody-mediated T-dependent autoimmune disease, involving the neuromuscular junction. Experimental autoimmune Myasthenia Gravis (EAMG) is induced in female Lewis rats by immunization with Acetylcholine Receptor from Torpedo Californica (TAChR) in Complete Freund’s Adjuvant (CFA). The injection induces the activation of specific Th1 cells in response to the antigen of immunization, stimulating the production of anti-TAChR antibodies (Abs) that are able to cross-react with self-rat AChR, generating specific anti rat AChR Abs, which activate complement system inducing the receptor and endplate degradation and damaging the transmission from nerve to muscle. Therapeutic strategies for MG include immunosuppressive drugs, that exert an aspecific immune dysfunction. These treatments are effective in a large amount of patients, however, clinical responses to conventional therapies appear ineffective in some patients or the severity of side effects can limit their prolonged administration. Thus, new specific autoimmune therapies are needed for treatment of unresponsive or intolerant MG patients. Pixantrone (PIX), an aza-antracenedionic derivate, is a drug with antitumor activity due to its cytotoxic effect, via DNA intercalation and topoisomerase II inhibition, linked to its cumulative dose. Owing to this mechanism of action, PIX presents also an immunosuppressive activity and was recently tested in the EAMG, in which have demonstrated to improve both preventive and therapeutic treatments when administered at the dose 16.25 mg/Kg q7dx3 for a cumulative dosage of 48.75 mg/Kg [1].
In the present study we have focused our attention to the therapeutic treatment of EAMG; in particular, the aim of our study has been to evaluate different PIX schedules, in order to identify the lowest dosage able to ameliorate ongoing EAMG, thus further lowering the risk of toxic effects.
We first evaluated PIX effect on the in vitro proliferative response of lymphoid cells, to determine the pharmacologic properties of the drug in presence of different cells subpopulations, characterized by different proliferative responses: R97-116 specific T cell line, lymphocytes (LNCs) and splenocytes (SPNs) from R97-116 primed rats, SPNs from HD rats and peripheral blood mononuclear cells (PBMCs) from human healthy donors. For this purpose, by dose-response curves we demonstrated that PIX (0.1 pM - 1 µM) inhibited lymphoid cell responses in a dose dependent manner. Moreover our data demonstrated that a lower concentration of PIX was sufficient to inhibit 50% of maximal response of R97-116 specific T cell line, represented by an homogeneous pool of cells (more than 90%). In mixed populations, such as LNCs and SPNs isolated from R97-116 primed rats, composed to 20-40% of antigen-specific cells, or even in SPNs from HD rats and PBMCs from healthy human donors, the IC50 for PIX is found to be increased. Complete suppression of proliferative response, was achieved with nM PIX concentrations for cells in active proliferation (i.e. R97-116 T cell line) and raised to µM PIX concentrations in presence of SPNs from HD rats and PBMCs from healthy human donors. Our in vitro experiments suggested that PIX efficacy is higher for T cells that are able to actively proliferate.
We also tested in vitro whether PIX (0.1 nM, 1 nM, 10 nM, 10 µM) was able to interfere with differentiation of myeloid precursors cells (MPCs), from bone marrow of HD rats, in immature dendritic cells. The drug resulted toxic (reduction in cell viability ≤ 50%) already at PIX 1 nM and the cell viability was dramatically reduced to 0% when the cells were exposed to PIX concentrations 10 nM and 10 µM.
Then we investigated in vivo PIX efficacy, firstly on TAChR-immunized rats t
Tipologia IRIS:
Tesi di dottorato
Keywords:
EAMG ; Myasthenia Gravis ; Pixantrone
Elenco autori:
C. Ruocco
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