Activity of Hydrolytic Enzymes in Tumour Cells is a Determinant for Anti-tumour Efficacy of the Melphalan Containing ProdrugJ1

Abstract

Recently, we presented a series of melphalan containing di- and tripeptides with high cytotoxic activity and J1 (l-melphalanyl-p-l-fluorophenylalanine ethyl ester) was identified as one of the most interesting compounds. It was speculated that the increased activity compared to melphalan itself, demonstrated both in vitro and in vivo, resided in increased transport over the tumour cell membrane and/or hydrolytic cleavage and liberation of melphalan inside the cells. Indeed, overexpression of hydrolytic enzymes like peptidases, esterases and proteases has been described in several types of human malignancies, thus providing a target for selective chemotherapy. In this work, the details of the increased activity was further investigated and potential tumour selectivity is discussed. The intracellular delivery of melphalan is investigated in detail using peptidase resistant dipeptide derivatives, by enzyme inhibitors and probes for enzymatic activity and by studying the time dependency of drug effect as well as intracellular drug concentrations (cellular pharmacokinetics). The results show that the activity of the dipeptide mustards is highly dependent on intracellular hydrolysis, which result in rapid intracellular release of the alkylating unit (i.e. free melphalan) in cells with high enzymatic activity. The maximum intracellular melphalan concentration following J1 exposure was reached already after 15 min, thereafter declining with a half-life of approximately 1 h. This rapid intracellular loading resulted in less reduction of activity for J1 than for melphalan and six other standard drugs when human tumour cell lines were exposed to the drugs for a limited time (simulating short half-life in vivo). Peptidase inhibitors inhibited the activity and intracellular release of melphalan, and dipeptide derivatives designed to resist the action of peptidases was less active than the corresponding normal dipeptide.

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

“…Previous studies showed that lipophilicity and an early intracellular hydrolysis of mel-flufen by peptidases inside the cells to release melphalan contributes to achieving rapid and high intracellular concentrations of melphalan (19,27,28). Earlier findings also showed that in tumor cells a limited exposure time, which simulate short half-life in vivo, proved more favorable for mel-flufen than for melphalan indicating a trapping mechanism through the enzymatic activation (29). Together, these results suggest that mel-flufen administration in patients with multiple myeloma is a more efficient therapeutic strategy for delivering higher concentrations of intracellular melphalan than by directly exposing cells to free melphalan.…”

The novel alkylating prodrug J1: diagnosis directed activity profile ex vivo and combination analyses in vitro

“…The prodrug J1 has advantage over melphalan in that it provides more efficient intracellular delivery of melphalan, depending on the activity of hydrolytic enzymes [10]. Thus it is anticipated that the activity profile of J1 and melphalan in various tumor and normal cells is different, in part mediated through different levels of activating enzymes like aminopeptidases.…”

“…It has previously been shown that J1 is a prodrug of melphalan relying on hydrolytic activation by aminopeptidases [10]. In 1999 Martínez et al [13] studied the aminopeptidase activity in surgical specimens from nine breast cancer specimens, and found significantly increased activity of some studied aminopeptidases (for example soluble alanyl, arginyl and cystinyl aminopeptidases) in comparison to normal tissue controls.…”

“…In vitro studies show that J1 is rapidly incorporated into the cytoplasm of tumor cell lines followed by intracellular hydrolysis, which results in release of melphalan. J1 may thus be considered as a prodrug of melphalan, activated through the action of aminopeptidases [10], which may provide an attractive cancer target since the activity of several aminopeptidases is elevated in plasma and effusions from cancer patients [11][12][13]. A clinical phase I study of J1 in adults with disseminated solid tumor malignancies is currently ongoing.…”

DNA Alkylating Agents

“…Melphalan-flufenamide may thus be considered as a prodrug of melphalan, and its enzymatic activation ( Figure 5.7) is performed by aminopeptidases such as aminopeptidase N (APN). 18 Interestingly, this enzyme is overexpressed in certain malignancies, which provides an opportunity to achieve some tumor selectivity. 19 Estramustine (Estracyt ® , Emcyt ® ), which consists of a β-estradiol unit linked to a nitrogen mustard portion via a carbamate bridge, was designed as a prodrug because the electron-withdrawing effect of the carbonyl group makes the electron density of its nitrogen atom insufficient to trigger aziridinium formation.…”