In vitro and in vivo activity of melflufen (J1)in lymphoma

Abstract

Background: Melphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen (melphalan flufenamide ethyl ester, previously called J1) is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma.

Methods: Cytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice.

Results: Melflufen showed activity with cytotoxic IC50-values in the submicromolar range (0.011-0.92 μM) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 μM) and an increased ratio vs. melphalan (range 13-455, average 108, p < 0.001). Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals.

Conclusion: This study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases.

Keywords: Alkylating agents; Cancer therapeutics; J1; Melflufen; Prodrug.

Fig. 1

Chemical structure of melflufen (a) and melphalan (b)

Fig. 2

Activity of melflufen in primary lymphoma cells. The cytotoxicity of melflufen in human primary lymphoma cells, after incubation for 72 h, was tested by the Fluorometric Cytotoxicity Assay. Each dose response curve is one patient cell culture, plotted as survival index (%) as a function of concentration

Fig. 3

Effects of melflufen on cell cycle phase distribution. KM-H2 (a) and SU-DHL-10 (b) cell lines were incubated for 40 h during basal conditions before treatment with melflufen for 12, 24 and 48 h. The analyses were performed by flow cytometry and show distribution of the cell cycle phases G0/G1, S and G2/M resulting from treatment with melflufen 0.1-0.4 μM

Fig. 4

Effects of melflufen in subcutaneous DOHH-2 xenografted mice. Presented as tumor volume (a), survival (b) and body weight change (c). Mice in control group (n = 5) gained weight and showed no signs of toxicity, but individuals were prematurely sacrificed due to tumor size on day 33 (n = 2) and 35 (n = 2). Animals in vincristine control group had excellent tumor control but lost weight and one animal was sacrificed on day 25, one on day 33 due to >20 % weight reduction. Animals treated with melflufen had no weight loss but significant tumor growth reduction on day 33 (p < 0.05). Log-rank test showed a significant difference in survival between the control group and the melflufen treated group (p = 0.0144). In A and C mean values with SEM are displayed