Derivatives of melphalan designed to enhance drug accumulation in cancer cells.
The objective of this study was to develop chemical strategies to improve the uptake and accumulation of melphalan (L-Mel and D-Mel), a cytotoxic agent, into cancer cells. Dipeptides synthesized from L- (or D-) Mel and L-glutamic acid (L-Glu) or L-valine (L-Val) and their methyl or ethyl esters (all compounds were trifluoroacetic acid salts) were evaluated for cytotoxicity and cellular uptake using Caco-2 cells, a human colon carcinoma cell line, and RT-2 cells, a rat brain glioma cell line. Treatment of Caco-2 cells with L-Mel or D-Mel (0.5 mg/ml equivalent of melphalan) for 48 h resulted in approximately 50% cell survival. Treatment of the Caco-2 cells with dipeptide derivatives of L-Mel (or D-Mel) (11c-d, 12c-d and 13) caused similar cytotoxicity effects (approximately 50-70% of cell survival). When the cytotoxicities of the esters of L-Mel, D-Mel and their dipeptide derivatives (11a-b, 12a-b and 14) in Caco-2 cells were determined, less than 10% cell survival was observed. Similar results were observed in RT-2 cells. When the cellular uptake properties of these compounds were determined in Caco-2 cell monolayers, L-Glu-L-Mel (12c), L-Glu-D-Mel (12d), and L-Mel-L-Glu (11c) generated slightly lower intracellular levels of L-Mel or D-Mel than when the cell monolayer was treated with the amino acids (L-Mel or D-Mel). In Caco-2 cells treated with 11c, 12c or 12d, low levels of the dipeptides were also detected. Caco-2 cell monolayers treated with D-Mel-L-Glu (11d) or D-Mel-L-Val (13) showed very low levels of the amino acids (L-Mel or D-Mel), but generally higher levels of the dipeptides. In contrast to the amino acids (L-Mel, D-Mel) or the dipeptide derivatives (11c-d, 12c-d and 13), the ester derivatives of the amino acids [L-Mel(OEt), D-Mel(OEt)] or the dipeptides (11a-b, 12a-b and 14) produced 5-20 times higher intracellular concentrations of potentially cytotoxic metabolites (e.g., L-Mel, D-Mel, Mel-containing dipeptides or Mel-containing dipeptide monoesters). L-Mel(OEt), D-Mel(OEt), L-Glu(OEt)-L-Mel(OEt) (12a), L-Glu(OEt)-D-Mel(OEt) (12b), and L-Mel-L-Glu(OEt)2 (11a) accumulated mainly as either L-Mel or D-Mel, and the percentages of L-Mel or D-Mel were 99%, 99%, 90%, 75% and 98% of the total intracellular concentration of potentially cytotoxic agents, respectively. D-Mel-L-Glu(OEt)2 (11b) accumulated as its monoester (> 95%) and D-Mel-L-Val(OMe) (14) accumulated as its dipeptide metabolite (> 98%). Inclusion of Gly-Pro, carnosine, L-Phe or L-Glu did not inhibit uptake of the dipeptide derivatives of L-Mel (or D-Mel) or their esters. These results suggest that the cellular uptake of the dipeptide derivatives of melphalan and their esters is probably via passive diffusion rather than being facilitated by an amino acid transporter or a di/tripeptide transporter. The higher intracellular levels of cytotoxic agents generated from the ester derivatives of the amino acids and the dipeptides are probably due to their higher lipophilicity and the overall neutral charge of the esters and subsequent intracellular formation of the more polar amino acids (L- or D-Mel) and/or Mel-containing dipeptides. Finally, these studies suggest that dipeptides of D-Mel [11b, 11d, 13] have inherent cytotoxicity properties.
Kupczyk-Subotkowska
L
L
Department of Pharmaceutical Chemistry, Simons Research Laboratories, University of Kansas, Lawrence 66047, USA.
Tamura
K
K
Pal
D
D
Sakaeda
T
T
Siahaan
T J
TJ
Stella
V J
VJ
Borchardt
R T
RT
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000138:chemical synthesis / Q000378:metabolism* / Q000627:therapeutic use; D018938:Caco-2 Cells / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005910:Glioma / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008558:Melphalan / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000627:therapeutic use; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D018906:Antineoplastic Agents, Alkylating; D008558:Melphalan