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Melphalan flufenamide (melflufen) is a novel lipophilic peptide-drug conjugate recently approved in the European Union and the United Kingdom for the treatment of relapsed refractory multiple myeloma. Melflufen rapidly crosses the cell membrane, and inside tumor cells, melflufen utilizes peptidases and esterases to release entrapped hydrophilic metabolites with alkylating activity. In vitro, in whole blood, melflufen was rapidly distributed into blood cells and quickly converted to its main metabolite melphalan, with maximum cellular concentrations of noncovalently bound melflufen and melphalan after 1 and 6 minutes, respectively. Melphalan outflow from blood cells was slow, with peak concentrations in plasma after 25 minutes. The pharmacokinetics of melflufen was best described by a 2-compartment model. Following a 30-minutes intravenous infusion of 40 mg in 27 patients with relapsed refactory multiple myeloma, mean half-life in the α phase of the curve was 1.24 minutes, half-life in the β phase of the curve 26.7 minutes, and clearance 13.4 L/min. Desethyl-melflufen exposure was below 20% compared to melflufen. Based on population analysis (298 patients with relapsed refactory multiple myeloma), the melphalan pharmacokinetics were well characterized by a 3-compartment model with melflufen dosing into a peripheral compartment, assuming instantaneous distribution of melflufen into cells and subsequent rapid metabolism to melphalan. Mean clearance and central and deep peripheral volumes of distribution were 22.4 L/h, 2.70 L, and 51.3 L, respectively. Clearance increased and maximum concentration decreased with increasing body weight and estimated glomerular filtration rate. In conclusion, melflufen administration differs from melphalan administration by a more rapid distribution into cells, which, in conjunction with a rapid intracellular metabolism, allows for higher maximum concentrations of alkylating agents, and by a more extensive distribution of melphalan to peripheral tissues., (© 2023, The American College of Clinical Pharmacology.)

Introduction: The overall survival of patients with multiple myeloma has improved with the advent of novel agents; however, multiple myeloma remains incurable. Combinations of standard-of-care agents such as immunomodulators, proteasome inhibitors, and anti-CD38 monoclonal antibodies are increasingly used in earlier lines of therapy. Patients with disease that is refractory to multiple novel agents represent a population with high unmet medical need and for whom therapies with new mechanisms of action could be beneficial. Melphalan flufenamide (melflufen) has demonstrated encouraging activity in patients with relapsed and refractory multiple myeloma., Areas Covered: This review provides an overview of the mechanism of action of melflufen, a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly delivers alkylating agents into tumor cells. It reviews key Phase I and II clinical trial data for melflufen in combination with dexamethasone as well as in triplet combinations with daratumumab or bortezomib. The safety profile of melflufen, which is characterized primarily by clinically manageable hematologic adverse events, is described., Expert Opinion: Melflufen has potential to fill a gap in the myeloma treatment landscape by providing a new mechanism of action with clinically meaningful efficacy and a favorable safety profile in patients refractory to multiple novel agents.

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.

Ovarian cancer carries a significant mortality. Since symptoms tend to be minimal, the disease is often diagnosed when peritoneal metastases are already present. The standard of care in advanced ovarian cancer consists of platinum-based chemotherapy combined with cytoreductive surgery. Unfortunately, even after optimal cytoreduction and adjuvant chemotherapy, most patients with stage III disease will develop a recurrence. Intraperitoneal administration of chemotherapy is an alternative treatment for patients with localized disease. The pharmacological and physiochemical properties of melflufen, a peptidase potentiated alkylator, raised the hypothesis that this drug could be useful in ovarian cancer and particularily against peritoneal carcinomatosis. In this study the preclinical effects of melflufen were investigated in different ovarian cancer models. Melflufen was active against ovarian cancer cell lines, primary cultures of patient-derived ovarian cancer cells, and inhibited the growth of subcutaneous A2780 ovarian cancer xenografts alone and when combined with gemcitabine or liposomal doxorubicin when administered intravenously. In addition, an intra- and subperitoneal xenograft model showed activity of intraperitoneal administered melflufen for peritoneal carcinomatosis, with minimal side effects and modest systemic exposure. In conclusion, results from this study support further investigations of melflufen for the treatment of peritoneal carcinomatosis from ovarian cancer, both for intravenous and intraperitoneal administration.

Our prior study utilized both in vitro and in vivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells., (© 2016 John Wiley & Sons Ltd.)

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.

Purpose: Melflufen (melphalan flufenamide, previously designated J1) is an optimized and targeted derivative of melphalan, hydrolyzed by aminopeptidases overexpressed in tumor cells resulting in selective release and trapping of melphalan, and enhanced activity in preclinical models., Methods: This was a prospective, single-armed, open-label, first-in-human, dose-finding phase I/IIa study in 45 adult patients with advanced and progressive solid tumors without standard treatment options. Most common tumor types were ovarian carcinoma (n = 20) and non-small-cell lung cancer (NSCLC, n = 11)., Results: In the dose-escalating phase I part of the study, seven patients were treated with increasing fixed doses of melflufen (25-130 mg) Q3W. In the subsequent phase IIa part, 38 patients received in total 115 cycles of therapy at doses of 30-75 mg. No dose-limiting toxicities (DLTs) were observed at 25 and 50 mg; at higher doses DLTs were reversible neutropenias and thrombocytopenias, particularly evident in heavily pretreated patients, and the recommended phase II dose (RPTD) was set to 50 mg. Response Evaluation Criteria In Solid Tumors (RECIST) evaluation after 3 cycles of therapy (27 patients) showed partial response in one (ovarian cancer), and stable disease in 18 patients. One NSCLC patient received nine cycles of melflufen and progressed after 7 months of therapy., Conclusions: In conclusion, melflufen can safely be given to cancer patients, and the toxicity profile was as expected for alkylating agents; RPTD is 50 mg Q3W. Reversible and manageable bone marrow suppression was identified as a DLT. Clinical activity is suggested in ovarian cancer, but modest activity in treatment of refractory NSCLC.

Aminopeptidase N (APN) has been reported to have a functional role in tumor angiogenesis and repeatedly reported to be over-expressed in human tumors. The melphalan-derived prodrug melphalan-flufenamide (melflufen, previously designated J1) can be activated by APN. This suggests that this alkylating prodrug may exert anti-angiogenic properties, which will possibly contribute to the anti-tumoral activity in vivo. This work presents a series of experiments designed to investigate this effect of melflufen. In a cytotoxicity assay we show that bovine endothelial cells were more than 200 times more sensitive to melflufen than to melphalan, in HUVEC cells the difference was more than 30-fold and accompanied by aminopetidase-mediated accumulation of intracellular melphalan. In the chicken embryo chorioallantoic membrane (CAM) assay it is indicated that both melflufen and melphalan inhibit vessel ingrowth. Two commercially available assays with human endothelial cells co-cultured with fibroblasts (TCS Cellworks AngioKit, and Essen GFP-AngioKit) also illustrate the superior anti-angiogenic effect of melflufen compared to melphalan. Finally, in a commercially available in vivo assay in mice (Cultrex DIVAA angio-reactor assay) melflufen displayed an anti-angiogenic effect, comparable to bevacizumab. In conclusion, this study demonstrates through all methods used, that melphalan-flufenamide besides being an alkylating agent also reveals anti-angiogenic effects in different preclinical models in vitro and in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)