Biochemical Pharmaco/ogy. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Vol. 40, No. 6. pp. 1193-1200. 1990. Printed in Great Britain. 0006-2952/90 $3.00+ 0.00 0 1990. Pergamon Press plc CYTOTOXICITY OF CHLORAMBUCIL AND CHLORAMBUCIL-FATTY ACID CONJUGATES AGAINST HUMAN LYMPHOMAS AND NORMAL HUMAN PERIPHERAL BLOOD LYMPHOCYTES A. ANEL,* T. HALMOS, J. M. TORRES, A. PIAEIRO,t K. ANTONAKIS and J. URIEL~ Laboratoires de Chimie des ProtCines et de Chemie Organique Biologique, Institut de Recherches Scientifiques sur le Cancer, B.P. N”. 8, 94802 Villejuif, France and tDepartamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias. Universidad de Zaragoza, Zaragoza, Spain zyxwvutsrqponm (Received 17 November 1989; accepted 30 March 1990) Abstract-The cytotoxic activity of chlorambucil (Chl) and of chlorambucil-fatty acid conjugates of different degree of unsaturation have been assayed in vitro upon two human lymphoma cell lines and comparatively, upon quiescent and mitogen-activated lymphocytes from healthy blood donors. The cell toxicity observed with Chl-arachidonic acid and Chl-docosahexaenoic acid against lymphoma cells was, at any experimental condition used, equal or higher than the individual toxic potential of either chlorambucil or fatty acids. The two conjugates, like chlorambucil alone, were toxic against mitogen- activated lymphocytes. Contrary to chlorambucil, Chl-arachidonic at any concentration tested, lacked of toxicity towards normal non-activated lymphocytes. Chl-oleic acid conjugate was, whatever the cell species tested, much less toxic than Chl alone. In conclusion, the coupling of chlorambucil with polyunsaturated fatty acids increases: (a) the selectivity against neoplastic versus quiescent lymphocytes and (b) the toxicity for B-lymphoma cells. The selective effect of Chl-fatty acid conjugates is discussed in relation with the expression of an AFP/AFP-receptor autocrine system in malignant lymphoblastoid cells and in mitogen-activated lymphocytks. Chemotherapeutic agents currently used for anti- tumor therapy are selected for their toxicity upon rapidly proliferating cells. A limitation of their use lies on the deleterious effects that they exert upon normal tissues and especially on inmunocompetent cells [l-3]. Many attempts have been made to increase the selectivity of anticancer drugs towards malignant cells and/or to decrease their adverse effects on normal cells. The coupling of drugs with enzymes, radioisotopes, DNA, toxins and mitogens has been tested with this purpose [4,5]. A great deal of work has also been made by conjugating drugs with antibodies directed against specific tumor anti- gens [6-111. An alternate approach, though limited to the chemotherapy of hepatomas and other alpha- fetoprotein (AFP) secreting tumors, was followed by Deutsch and coworkers [12] using daunomycin-fatty acid derivatives. The potent antitumor activity of these conjugates was supposed to depend upon the high affinity of AFP for fatty acids and the fact that these tumors synthesize AFP. Work from our laboratory has provided evidence that, contrary to normal resting cells, many tumor cells of varied origin express, in vitro and in uiuo, specific cell surface receptors to AFP [13,14] and that these receptors, through their interaction with AFP, can be involved in the transfer of fatty acids into the cells [15]. We have explored the possibility of improving the selectivity of antitumor drugs by * On leave from: Departamento de Bioquimica y Biol- ogia Molecular y Celular, Facultad de Ciencias, Univ- ersidad de Zaragoza, Zaragoza, Spain. $ To whom correspondence should be addressed. the use of their fatty acid conjugates on neoplastic cells expressing AFP-receptors. Covalent conjugates of chlorambucil and fatty acids of different degree of unsaturation have been prepared and assayed to check their cytotoxic activities in vitro upon two human lymphoma cell lines (RAJI, a B-cell-derived lymphoma and CEM, a T-cell-derived one) and, comparatively, upon quiescent and mitogen acti- vated lymphocytes from normal donors. The choice of this model was determined by previous studies [14] showing that these human T and B lympho- blastoid cell lines expressed both AFP and AFP- receptors. MATERIALS AND METHODS General chemical methods. All reactions were con- ducted under nitrogen with exclusion of the light. Solutions were concentrated to dryness under reduced pressure below 30”. Thin layer chroma- tography was performed on Silica Gel 60 F254 (Merck, Darmstadt, F.R.G.) and Silica Gel 60 (230- 400 mesh) was used for flash chromatography [16]. Chlorambucil derivatives were detected by exam- ination under UV light and unsaturated fatty acids were located by exposure to iodine vapours. Rfvalues are given for hexane-ethylacetate (7/3, v/v) as the eluent. ‘H-NMR spectra were recorded for solutions in CDC13 with tetramethylsilane as the internal standard, using a Bruker MSL 300 spectrometer. Preparation of chlorambucil hydroxyethylester (ChEtOH). To a stirred solution of chlorambucil (304 mg, 1 mmol) in dry dichloromethane were added dimethylaminopyridine (6.1 mg, 0.05 mmol), 1193 BP W6-c