The cardioprotector dexrazoxane augments therapeutic efficacy of mitoxantrone in experimental autoimmune encephalomyelitis

Abstract

The present study investigates the immunological effects of a combination treatment of mitoxantrone and the cardioprotector dexrazoxane in experimental autoimmune encephalomyelitis (EAE). Mitoxantrone, an anthracycline-derived immunosuppressive drug has been approved recently for treatment of very active multiple sclerosis (MS). Its prolonged use is limited due to its cardiotoxic properties. Dexrazoxane (DZR (S)-(+)-1,2-bis (3,5.dioxopiperazinyl)propane, ICRF-187) is an iron III chelator which in animal models and in cancer patients reduces anthracycline and mitoxantrone induced cardiotoxicity when given immediately before these agents. We examined the immunological effects of dexrazoxane in combination with mitoxantrone in experimental autoimmune encephalomyelitis (EAE) in Lewis rats. EAE was induced by active immunization with myelin basic protein (MBP) or by adoptive transfer of MBP specific T cells (AT-EAE). The clinical course, spinal cord pathology, activity of metalloproteinases (MMP-2 and MMP-9) and T cell apoptosis were assessed. Monotherapy with DZR ameliorated slightly the course of actively induced EAE and AT-EAE. The combination of DZR and mitoxantrone was superior to mitoxantrone given alone. Clinical amelioration ran in parallel with the marked reduction of inflammatory infiltration which was nearly abolished by the combination treatment. DZR did not affect the activity of metalloproteinase 9 and did not increase the proportion of apoptotic lymph node cells ex vivo or T cells in situ. We conclude that in addition to its cardioprotective role, DZR augments mitoxantrone-mediated immunosuppressive effects in animal models of human central nervous system (CNS) autoimmune disease. Clinical trials in MS patients are warranted to evaluate the unexpected immunosuppressive efficacy of DZR as add-on treatment.

Figures

Fig. 1

Clinical course of adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE; a) and actively induced EAE (b) in the Lewis rat. Statistical significant different disease courses (anova, P < 0·05) were calculated in PBS versus Mitox, Mitox versus DZR, DZR versus PBS and DZR versus Mitox/DZR both in AT-EAE and EAE. Each experiment has been repeated at least twice with identical results.

Fig. 2

Immunohistochemical analysis of T cell (a,b) and macrophage (c,d) infiltration in PBS (a,c) versus DZR (b,d)-treated AT-EAE animals. Note marked reduction in perivascular infiltrates after DZR alone.

Fig. 3

Number of infiltrating T cells (a) and proportion of apoptotic T cells (b) in spinal cord sections (right ordinate) from animals with severe AT-EAE (day 7 p.i.) after treatment with DZR on days 1, 3, 5 (three animals/group, *P< 0·05, Mann–Whitney U-test).

Fig. 4

Proliferation of T lymphocytes isolated from lymph nodes of Lewis rats treated previously with DZR versus treatment with PBS (two animals each, proliferation in triplicates, differences not significant by using Mann–Whitney U-test).

Fig. 5

Proliferation of T lymphocytes isolated ex vivo from lymph nodes of Lewis rats immunized with MBP/CFA after addition of the indicated concentrations of DZR and FeCl3 in ordinate scales. (proliferation in triplicate).

Fig. 6

Detection of MMP-9 and MMP-2 proteinase activity in spinal cord from Lewis rats by zymography (SDS–substrate gel analysis). Zone of clearing represents proteinase activity. Each lane represents spinal cord tissue from one animal 5 days postinjection of encephalitogenic T cells, treated either with DZR, MITOX or DRZ/MITOX. White areas represent gelatin digestion at levels of 92 kDa and 72 kDa (corresponding to MMP-9 and MMP-2). In contrast, clearing appears to be unchanged at the level of 72-kDa.

Fig. 7

Flow cytometric analysis of apoptotic lymph node cells ex vivo (annexin staining). DZR, MITOX, DZR/MITOX represent results from lymph nodes of six animals at day 13 of EAE (Mann–Whitney U-test).