In vitro and in vivo anti-leukemic activity of the peptidase-potentiated alkylator melflufen in acute myeloid leukemia

Abstract

The novel aminopeptidase potentiated alkylating agent melflufen, was evaluated for activity in acute myeloid leukemia in a range of in vitro models, as well as in a patient derived xenograft study. All tested AML cell lines were highly sensitive to melflufen while melphalan was considerably less potent. In the HL-60 cell line model, synergy was observed for the combination of melflufen and cytarabine, an interaction that appeared sequence dependent with increased synergy when melflufen was added before cytarabine. Also, in primary cultures of AML cells from patients melflufen was highly active, while normal PBMC cultures appeared less sensitive, indicating a 7-fold in vitro therapeutic index. Melphalan, on the other hand, was only 2-fold more potent in the AML patient samples compared with PBMCs. Melflufen was equally active against non-malignant, immature CD34+ progenitor cells and a more differentiated CD34+ derived cell population (GM14), whereas the stem cell like cells were less sensitive to melphalan. Finally, melflufen treatment showed significant anti-leukemia activity and increased survival in a patient derived xenograft of AML in mice. In conclusion, melflufen demonstrates high and significant preclinical activity in AML and further clinical evaluation seem warranted in this disease.

Keywords: acute myeloid leukemia; alkylator; drug development; melflufen; pre-clinical.

Figure 1. The cytotoxic activity (as concentration vs survival measured by FMCA) of melflufen

A. and melphalan B. in AML cell lines MV-4-11, HL60, Kasumi and KGI-A. Mean values with SD of three independent experiments.

Figure 2. Combination effects of melflufen and cytarabine or daunorubicin in HL-60 cells

The MacSynergy II program (Prichard) was also used to analyze the whole concentration matrix according to the Bliss independence model. The three-dimensional differential surface plot demonstrates synergy as peaks above a theoretical additive plane (positive volume of synergy), and antagonism as depressions below it (negative volume of synergy).

Figure 3. Time course for melflufen's accumulation and cytotoxic effect in CCRF-CEM leukemia cells

The peptidase-potentiated effect results in a rapid accumulation of the active drug, and with decreasing cell densities A-C-E. there are more drug molecules per cell, and activity increases (curves shifts to the left). The rapid accumulation results in a competition for melflufen, and with high cell densities (A) maximum effect is obtained already after 15 minutes, i.e. cells have taken up all melflufen from the medium. When the cell number is lower (C and E) there is a time course for the accumulation, and longer exposure yields higher activity (i.e. lower IC50). For melphalan the situation is completely different as the absorption of the drug is slow; longer exposure time results in higher activity regardless if the cell density is high or low, suggesting rather slow absorption and a concentration equilibrium B-D-F.

Figure 4. The cytotoxic activity (as concentration vs survival measured by FMCA) and mean IC50 (95% confidence intervals) of cytarabine, daunorubicin, melflufen and melphalan in AML patient samples

Error bars denote SEM.

Figure 5. In vivo effects of cytarabine and melflufen

Effect presented as percent of HLAABC positive AML-ps cells in blood (determined by FACS) on day 30 A., proportion of animals free of leukemia at end of experiment on day 104 B., change in mean body weight of remaining live animals C. and median Kaplan-Meier survival curve D. Error bars denote SD.