PRECLINICAL STUDIES OF PIXANTRONE AS IMMUNOSUPPRESSIVE DRUG IN THE ANIMAL MODEL OF MYASTHENIA GRAVIS

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 treated 24 h post immunization (p.i.) with a single dose of PIX (16.25 mg/Kg x1, 8.12 mg/Kg x1, 4.06 mg/Kg x1) and sacrificed after 10 days. A single dose of PIX was able to reduce the number of total live cell count of LNCs from PIX treated rats compared to vehicle, accordingly with a decrease of ex vivo TAChR specific T cell proliferation (90%). In parallel we evaluated the effect of the drug in physiological conditions, administered a single dosage of PIX (16.25 mg/Kg) in HD rats or HD rats immunized only with CFA and compared to HD controls, but the drug had no effect on the number of total live cells on LNCs. These data demonstrated that lower PIX doses were able to modulate selectively the TAChR specific proliferation in vivo and allowed us to tested alternative schedules, on the base of previous studies [1], in the therapeutic treatments. We studied in vivo the effect of long-term treatments with PIX in EAMG rats, immunized with TAChR/CFA and treated 4 weeks p.i with PIX, at two cumulative dosage 48.75 mg/Kg (16.25 mg/Kg q14dx3 as positive control, 8.12 mg/Kg q7dx6) and 24.36 mg/Kg (4.06 mg/Kg q7dx6, 8.12 mg/Kg q14dx3). Vehicle treated rats was administered with a sterile saline solution. In parallel we evaluated the effect of a chronic administration of PIX in HD rats (16.25 mg/Kg q14dx3) compared to HD controls, but PIX treatments did not affected in vivo the body weight of the HD animals and had no effect on the number of total live cells of LNCs. At the end of the experiments (11 weeks p.i.) only PIX cumulative dosage 48.75 mg/Kg shown a pharmacological effect, modulating clinical signs of EAMG (gain of body weight and decrease in the clinical score), affecting the number of total live cells of LNCs and decreasing the TAChR specific proliferation in the same organ. Moreover, qPCR revealed a reduction in IFNγ mRNA transcription levels in SPNs, accordingly with the decrease in anti-Rat Abs titre in the sera of the PIX treated rats. On the contrary the cumulative dosage 24.36 mg/Kg resulted ineffective in the modulation of classical monitoring parameters of EAMG. Although we observed an effect on TAChR specific proliferation of LNCs, these treatments resulted ineffective to inhibit the production of IFNγ and, as consequence, of pathogenic Anti Rat AChR Abs. We also tested in vivo the chronic toxicity of the drug on MPCs and both of treatments at the cumulative dosage 48.75 mg/Kg resulted myelotoxic in HD and EAMG rats compared to HD controls, but the protocol 8.12 mg/Kg q7dx6 presented a lower toxicity compared to the 16.25 mg/Kg q14dx3. Thus following the protocol 8.12 mg/Kg q7dx6 we observed major efficacy in the disease modulation with fewer myelotoxic effects. Conversely the cumulative dosage 24.36 mg/Kg did not interfere on cell viability compared to HD. In conclusion we speculated that though both of treatments for the cumulative dosage 48.75 mg/Kg were able to improve EAMG, PIX pharmacokinetic profile in the schedule 8.12mg/Kg q7dx6 overlaps well with its clearance (8 days), thus allowing the achievement of a systemic steady-state, with the consequent improvement of the efficacy of this treatment compared to 16.25mg/Kg q14dx3 schedule. The cumulative dosage of 24.36 mg/Kg instead resulted ineffective to modulate EAMG because it didn’t reach the systemic effective dose in rats. On the basis of our results we proposed PIX as promising immunomodulatory agent suitable for clinical investigation in MG patients, that present severe form of the disease or major contraindications to the classical therapies.