title: Activity of hydrolytic enzymes in tumour cells is a determinant for anti-tumour efficacy of the melphalan containing prodrug J1.
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.
authors.affiliation: Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden. joachim.gullbo@medsci.uu.se
authors.forename: Joachim
authors.initials: J
authors.lastname: Gullbo
authors.forename: Malin
authors.initials: M
authors.lastname: Wickström
authors.forename: Marcus
authors.initials: M
authors.lastname: Tullberg
authors.forename: Hans
authors.initials: H
authors.lastname: Ehrsson
authors.forename: Rolf
authors.initials: R
authors.lastname: Lewensohn
authors.forename: Peter
authors.initials: P
authors.lastname: Nygren
authors.forename: Kristina
authors.initials: K
authors.lastname: Luthman
authors.forename: Rolf
authors.initials: R
authors.lastname: Larsson
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides; D005470:Fluorometry; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008558:Melphalan / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000627:therapeutic use*; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology*; D009842:Oligopeptides / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000627:therapeutic use*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011355:Prodrugs; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D018906:Antineoplastic Agents, Alkylating; D004151:Dipeptides; D007202:Indicators and Reagents; C482580:L-melphalanyl-p-L-fluorophenylalanine ethyl ester; D009842:Oligopeptides; D011355:Prodrugs; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D007930:Leucine; C012211:ubenimex; D008558:Melphalan