Mitoxantrone and cytotoxic drugs' mechanisms of action.

Evidence has suggested that early, aggressive intervention may improve both short- and long-term outcomes in patients with multiple sclerosis (MS). Cytotoxic agents may offer advantages in this setting, particularly when used as an induction or add-on therapy with immunomodulators. Immunosuppression is the mechanism of action common to all cytotoxic drugs; individual subtleties in immunoregulatory actions are likely of minor importance. In the United States, mitoxantrone is currently the only cytotoxic agent approved for the treatment of MS (secondary-progressive, progressive-relapsing, and worsening relapsing-remitting forms). Therapies in phase III development include cyclophosphamide, mycophenolate mofetil, cladribine, and teriflunomide. All these drugs have exhibited efficacy in controlled clinical trials, although the degree of benefit with respect to MRI and clinical endpoints has varied both within and among the various agents. Further investigations are needed to determine whether cytotoxic drugs represent a substantial improvement over treatments that have a more targeted impact on the immune system.

Mitoxantrone treatment in multiple sclerosis induces TH2-type cytokines.

OBJECTIVES: Mitoxantrone is a cytotoxic drug with immune modulatory properties used in the treatment of progressive forms of multiple sclerosis (MS). We explored the effect of mitoxantrone treatment in MS patients on cytokine patterns induced in peripheral blood mononuclear cells (PBMC) and T-cell subsets ex vivo. MATERIALS AND METHODS: Blood was obtained before mitoxantrone infusion and 6, 12 and 18 days thereafter. Proliferation and prototypic TH1-, TH17- and TH2-type cytokines were determined following in vitro stimulation of PBMC, CD4+ and CD8+ T cells. In addition, a patient cohort receiving its first mitoxantrone treatment was cross-sectionally compared with a cohort of patients with more than 1 year of treatment. RESULTS: Mitoxantrone treatment increased the ex vivo production of the TH2 cytokines interleukin-4 (IL-4; P < 0.05) and IL-5 (P < 0.001) in phytohemagglutinin-stimulated CD4+ T cells within 18 days of treatment. The cross-sectional study revealed that long-term treatment with mitoxantrone increased the inducibility of IL-4 and IL-5 secretion by PBMCs and CD4+ T cells even further. No significant changes were observed for interferon-γ, tumour necrosis factor-α, IL-17 and IL-10. Mitoxantrone did not alter the proliferative capacity of ex vivo-stimulated T cells. CONCLUSION: Mitoxantrone treatment in MS enhances the inducibility of TH2-type cytokines, which may contribute to its beneficial effects in MS.

Mitoxantrone induces cell death in peripheral blood leucocytes of multiple sclerosis patients.

Mitoxantrone (MX) is a cytotoxic drug with proven clinical efficacy in active multiple sclerosis (MS). In this ex vivo study we investigated the immunological effects of MX on peripheral blood leucocytes (PBL) from MS patients. PBL were isolated from 46 patients with active MS (mean age 42 years, female : male 1.4 : 1) before and immediately after 1 h MX infusion. Isolated PBL were cultured and stimulated with phytohaemagglutinin (PHA), T cell receptor stimulating monoclonal antibody (MoAb) X35 or kept in culture medium alone. Proliferation was measured by [3H]-thymidine incorporation. MX-uptake and cell death in PBL subpopulations was analysed by flow cytometry using antibodies against cluster of differentiation (CD)-surface antigens, annexin V (AnnV) and propidium iodide (PI). MX was incorporated rapidly into PBL. After only a 1-h in vivo exposure, MX reduced proliferative responses in unstimulated and stimulated PBL (PHA: - 17%, MoAb X35: -13%). MX-exposed PBL showed an increase of AnnV+/PI+ cells (unstimulated: 12%, PHA: 15%), which was even more pronounced 2 weeks after infusion. No difference was observed between de novo MX-treated patients and those on long-term MX treatment. In T cell receptor stimulated PBL, cell death was induced preferentially in CD19-positive B cells and to a lesser extent in CD8-positive T cells. MX is incorporated rapidly in circulating PBL of MS patients and induces a pronounced suppression of proliferative responses. This suppression appears to be mediated at least partly by the induction of late apoptotic/necrotic cell death with a preferential susceptibility of B cells.

The effect of mitoxantrone on anti-pig immunization in baboons.

Besides virological and physiological concerns, the success of xenotransplantation (Xt) is still dependent on the prevention of delayed xenograft rejection (DXR). Although multifactorial, DXR is mainly due to xenonatural antibody (Ab) recognizing their xenogenic antigen (Ag) followed by complement activation. Despite the use of intensive treatments capable of inhibiting the humoral response, DXR can still not be avoided and always occurs within weeks following transplantation. Moreover, these latter treatments currently used in Xt could not be used clinically in humans because of their high risk of over-immunosuppressing the patients. Mitoxantrone (Mx) is a drug well known for its antiproliferative properties and is used clinically in oncology and in the treatment of relapsing multiple sclerosis. In models of arthritis in rats, it has been shown to be 10 to 20 times more powerful than cyclophosphamide (CyP) at blocking both inflammatory and B-cell responses. Because of its B-cell inhibitory capacity and considering the implication of the humoral response in xenograft rejection, we have compared Mx with CyP for its ability to block in vivo anti-pig immunization induced via subcutaneous injections of pig red blood cells into baboons. Neither drug was able to inhibit the anti-pig responses following the first and second immunizations, emphasizing the particularity of preformed Ab responses. However, the rise in Ab in the Mx treated animals was significantly delayed as compared with the non-treated as well as the CyP treated animals and was mainly because of a profound depletion of circulating B-cells. Mx displays an interesting antihumoral effect that we now intend to test in a pig kidney to baboon Xt model, with anticipated administration of the drug allowing an early B-cell depletion.

Immunopharmacologic modulation of experimental allergic encephalomyelitis: low-dose cyclosporin-A treatment causes disease relapse and increased systemic T and B cell-mediated myelin-directed autoimmunity.

Therapies with immunosuppressive drugs in autoimmune experimental diseases often down-regulate disease but sometimes may lead to paradoxical disease exacerbation. To elucidate possible mechanisms behind such phenomena the effects were studied of mitoxantrone (Mx) and cyclosporin A (CsA) given at high and low doses on clinical course, and on autoreactive T- and B-cell responses in actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats. Treatment with Mx and high dose CsA abrogated EAE and decreased dramatically the measured immune responses compared to vehicle-treated control EAE rats. Low-dose CsA treatment caused a disease relapse 20-30 days post immunization (p.i.). This relapse was accompanied by increased numbers of cells spontaneously producing IFN-gamma in the CNS and regional lymph nodes. Furthermore, anti-myelin and anti-MBP secreting cells were increased as were numbers of primed T cells that produced IFN-gamma in response to myelin antigens. It was concluded that these aspects of the myelin autoreactive immune response correlated well with clinical disease and are useful in evaluating immunotherapeutic intervention. Low-dose CsA treatment may interfere with systemic down-regulatory mechanisms acting on both T- and B-cell myelin-directed autoimmunity.

Treatment of multiple sclerosis with mitoxantrone.

Ten multiple sclerosis patients, all with a rapid deteriorating disease profile, were treated with 12 mg/m2 of the cytostatic agent mitoxantrone, administered every 3 months. This dosage is only 25% of what a patient with a solid tumour would normally receive during the same time period. In all treated patients the deterioration was stopped following the initial dosage; in four out of ten patients there was even an immediate improvement of the neurological status. Eight out of nine patients showed an improvement after 1 year as compared with their enrollment status; the other one remained stabile. In correlation with the clinical improvement, the mean P100 latencies of visual evoked potentials showed a reduction after 1 year. However, the changes identified through magnetic resonance imaging were even clearer than those seen clinically. At admission, this group of patients presented with a total of 169 gadolinium (Gd)-enhancing lesions. Only 10 lesions were enhancing in nine patients 12 months after the initiation of treatment. It appears that mitoxantrone accelerates the disappearance of Gd-enhancing lesions and prevents the development of new ones. Minimal side effects such as mild nausea and a slight faintness were evident in six patients and then for only 1-2 days.

The anti-mitozantrone monoclonal antibody NO-1, protects acute leukaemia cell lines from the cytotoxic effects of mitozantrone.

The monoclonal antibody NO-1 was raised against the potent anti-cancer drug mitozantrone by immunization of a BALB/c mouse with a mitozantrone-keyhole limpet haemocyanin conjugate in Freund's complete adjuvant. This antibody was shown to be highly effective in vitro at neutralizing the cytotoxic effects of mitozantrone for the acute leukaemia cell lines ALL-1 and MOLT4. In order to achieve complete protection, a drug to antibody molar ratio of 1.5:1 was required. The neutralizing effect was specific for mitozantrone, as NO-1 antibody offered no protection of the MOLT4 cell line to the cytotoxic effects of the anthracycline drug daunorubicin when used at a near identical molar ratio. NO-1 antibody has already proven a highly successful monoclonal reagent for use in a competitive enzyme-linked immunosorbent assay for the accurate and sensitive quantitation of mitozantrone in serum. The neutralizing properties of NO-1 suggest other possible applications for this antibody. These could include a use in the rapid clearance of pharmacologically active mitozantrone from the circulation following very high dose administration prior to bone marrow transplantation and for the construction of bispecific antibodies for targeting mitozantrone to tumour cell populations.

Suppression of cytolytic function in murine lymphocytes exposed to 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD): inhibition of proliferation by cytotoxic T-lymphocyte precursors.

Previous reports have shown that exposure of murine splenic lymphocytes to the substituted anthraquinone 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD) results in a persistent, strong immunosuppression which is limited to induction of cytotoxic T-lymphocytes (CTL). In the present study the cellular mechanism of this specificity was examined in detail. Proliferation of T-lymphocytes is inhibited by in vitro exposure to AEAD, with suggestive evidence that this inhibition may be selective for the CTL precursor cells. Activation and differentiation of CTL precursors into functional CTL, as well as the function of T-helper lymphocytes, was unaffected by AEAD exposure. Rather, the committed CTL precursor cells are prevented from clonal proliferation, resulting in a much smaller population of antigen-induced CTL effectors. Finally, it was shown that AEAD is unable to prevent the anamnestic response of CTL memory cells to eliciting antigen. Taken together these data provide strong evidence that AEAD-induced CTL suppression results from its antiproliferative effect, directed primarily toward the CTL precursor subpopulation. This effect is manifested as decreased CTL function due to lower absolute number of specific CTL, since AEAD has no significant direct effect on expression of either specific or polyclonal cytolysis by T-lymphocytes.