The alkylating prodrug J1 can be activated by aminopeptidase N, leading to a possible target directed release of melphalan

Wickström, Malin; Viktorsson, Kristina; Lundholm, Lovisa; Aesöy, Reidun; Nygren, Helen; Sooman, Linda; Fryknäs, Mårten; Vogel, Lotte K.; Lewensohn, Rolf; Larsson, Rolf; Gullbo, Joachim

doi:10.1016/j.bcp.2009.12.022

Cited by 50 publications

(74 citation statements)

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“…Mel-flufen is rapidly incorporated into the tumor cells, followed by intracellular hydrolysis, which in part is mediated by aminopeptidase N (ANPEP), an enzyme overexpressed in several tumor cell malignancies (18). Studies using solid tumor cell models showed that treatment with mel-flufen causes at least a 10-fold higher loading of melphalan, which explain its higher tumor cell cytotoxicity (15)(16)(17)19). To date, the mel-flufen activity against multiple myeloma cells is undefined.…”

Section: Introductionmentioning

confidence: 99%

In Vitro and In Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells

Chauhan

Ray

Viktorsson

et al. 2013

Clinical Cancer Research

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126

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Purpose The alkylating agent melphalan prolongs survival in multiple myeloma (MM) patients; however, it is associated with toxicities and development of drug-resistance. Here, we evaluated the efficacy of melphalan-flufenamide (Mel-flufen), a novel dipeptide prodrug of melphalan in MM. Experimental Design MM cell lines, primary patient cells, and the human MM xenograft animal model were utilized to study the antitumor activity of mel-flufen. Results Low doses of mel-flufen triggers a more rapid and higher intracellular concentrations of melphalan in MM cells than is achievable by free melphalan. Cytotoxicity analysis showed significantly lower IC50 of mel-flufen than melphalan in MM cells. Importantly, mel-flufen induces apoptosis even in melphalan-, and bortezomib-resistant MM cells. Mechanistic studies show that siRNA knockdown of aminopeptidase N, a key enzyme mediating intracellular conversion of mel-flufen to melphalan, attenuates anti-MM activity of mel-flufen. Furthermore, mel-flufen-induced apoptosis was associated with: 1) activation of caspases and PARP cleavage; 2) ROS generation; 3) mitochondrial dysfunction and release of cytochrome-c; and 4) induction of DNA damage. Moreover, mel-flufen inhibits MM cell migration and tumor-associated angiogenesis. Human MM xenograft studies showed a more potent inhibition of tumor growth in mice treated with mel-flufen than mice receiving equimolar doses of melphalan. Finally, combining mel-flufen with lenalidomide, bortezomib, or dexamethasone triggers synergistic anti-MM activity. Conclusion Our preclinical study supports clinical evaluation of mel-flufen to enhance therapeutic potential of melphalan, overcome drug-resistance, and improve MM patient outcome.

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Section: Introductionmentioning

confidence: 99%

In Vitro and In Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells

Chauhan

Ray

Viktorsson

et al. 2013

Clinical Cancer Research

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126

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“…Mel‐flufen biotransformation and cell killing effects has in other tumour types been shown to depend on aminopeptidases (Chauhan et al., 2013; Wickstrom et al., 2010). The effect of bestatin‐mediated aminopeptidase blockade on mel‐flufen effects in UC was therefore evaluated (Figure 6A).…”

Section: Resultsmentioning

confidence: 99%

“…One may speculate that the intact mel‐flufen or the de‐esterified form of mel‐flufen could possibly function as a reservoir of mel‐flufen which gradually can be converted to the active melphalan and contribute to the observed increased cytotoxicity after mel‐flufen exposure in these cells. The conversion of mel‐flufen to melphalan has in other solid tumour cells been shown to rely on aminopeptidases, which are inhibited by bestatin (Chauhan et al., 2013; Wickstrom et al., 2010). Also in UC cells, we found evidence of aminopeptidases to be critically involved as bestatin blocked intracellular melphalan accumulation after mel‐flufen treatment and, accordingly, reduced mel‐flufen‐induced cell death.…”

Section: Discussionmentioning

confidence: 99%

Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

et al. 2016

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Background: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide‐based alkylating agent melphalan‐flufenamide (mel‐flufen) for UC. Methods: UC cell lines J82, RT4, TCCsup and 5637 were treated with mel‐flufen, alone or combined with cisplatin, gemcitabine, dasatinib or bestatin. Cell viability (MTT assay), intracellular drug accumulation (liquid chromatography) apoptosis induction (apoptotic cell nuclei morphology, western blot analysis of PARP‐1/caspase‐9 cleavage and Bak/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho‐Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome. Results: In J82, RT4, TCCsup and 5637 UC cells, mel‐flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel‐flufen induced apoptosis seen as activation of Bak/Bax, cleavage of caspase‐9/PARP‐1 and induction of apoptotic cell nuclei morphology. Combining mel‐flufen with cisplatin or gemcitabine in J82 cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of mel‐flufen‐induced kinome alterations in J82 cells revealed that mel‐flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for mel‐flufen cytotoxicity. Immunohistochemical analysis of the putative mel‐flufen biomarker ANPEP demonstrated prominent expression levels in tumours from 82 of 83 cystectomy patients. Significantly longer median overall survival was found in patients with high ANPEP expression (P = 0.02). Conclusion: Mel‐flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC.

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“…Under the action of aminopeptidases, like aminopeptidase N [4], melflufen is hydrolyzed, leading to high intracellular concentrations of alkylating moieties (e.g. melphalan), able to interact with nucleic acids within the tumor cells [5].…”

Section: Introductionmentioning

confidence: 99%

“…In AML, APN is and is expressed on stem cells and leukemic blasts [6, 10], on the cell surface [11, 12] and in the cytoplasm [12], and recently occurrence of APN was also demonstrated in microvesicles from patients with myeloid tumors [13]. Since APN mediates cleavage of melflufen to its parental drug melphalan in the tumor cell cytoplasm, or in environment with high concentration of APN, a clinical usability of melflufen in AML is suggested [4]. …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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The alkylating prodrug J1 can be activated by aminopeptidase N, leading to a possible target directed release of melphalan

Cited by 50 publications

References 36 publications

In Vitro and In Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells

In Vitro and In Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells

Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

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

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