In order to use the LDL receptor pathway to target radionuclides to cancer sites for imaging and diagnostic purposes, a labeling procedure of LDL with 111In using the DTPA-bis(stearylamide) (L) has been developed. This bifunctional ligand is intended to be incorporated into the phospholipid monolayer of LDL and to specifically chelate the In3+ cation at the surface. The ligand was incorporated into LDL in buffered medium with a 65-80% yield. The L-LDL samples are stable over a 24 h period when examined by dialysis, allowing their storage before indium-111 radiolabeling. In vitro studies of In-L-LDL particles show that indium labeling is rapidly achieved (1 h). More than 85% of the indium atoms are bound to the chelating functions of the incorporated DTPA derivatives and less than 10% to the nonspecific complexation sites of LDL (e.g., protein residues). After incubation in human serum, the indium activity recovered in the LDL fraction of In-L-LDL samples (95%) is much higher than in In-LDL samples (35%), pointing out the strong stabilizing chelating effect of the ligand. Competitive binding studies show that In-L-LDL are recognized by LDL receptors of A549 cells like native LDL when the In-L/LDL ratio varies from 5 to 30. All these in vitro experiments demonstrate that the In-L-LDL conjugates possess properties suitable for further work with in vivo experiments.

Potentiometric titrations showed that the sum of the first three protonation constants of diethylenetriaminepentaacetate N,N"-bis( butylamide) ( L1) decreases by 6.6 units when compared to the diethylenetriaminepentaacetate (dtpa), and that the complexes of Ga3+ and In3+ of L1 have stability constants 6.1 -6.3 log units lower than those of dtpa. The decreased basicity of the bis(amide) derivative thus correlates with the lower stability of its complexes. However, at pH 7.4 the lower overall basicity of L1 compared to dtpa partly compensates the lower stability of its complexes, thus causing their conditional stability constants to be comparable to those of the dtpa complexes. The efficacy of L' in binding Ga3+ and In3+ under physiological conditions is discussed by comparing pM values at pH 7.4, showing that this compound can withstand the competition of transferrin for In3+ in biological media relevant to medical applications. The 13C and l H NMR shifts were measured for the complexes of A13+, Ga3+, In3+ and Y3+ with L1 and compared with 13C shifts for the dtpa complexes. The complexes of A13+ and Ga3+ are hexadentate, possibly of octahedral geometry, and form various structural isomers, whereas those of In3+ and Y3+, with a single structural isomer of eight-co-ordination, are very similar to the lanthanide complexes. The complexes of A13+ and Ga3+ of L1 have considerable populations of isomers with bound amide groups, whereas the single structural isomer of the complexes of In3+ and Y3+ with both amide groups co-ordinated yield a variety of enantiomers in solution. studies on complexes of La3+ and Lu3+ with the corresponding bis(propy1amide) H3L2,22*23 as well as with our studies and literature data on complexes of A13+, G a 3 + , In3+, Y 3 + , La3+ and Lu3+ with dtpa.'9,25*30 ExperimentalReagents.-All chemicals were of analytical grade, used as received unless specifically noted, and obtained from the following sources: diethylenetriaminepentacetic acid dianhydride, pentasodium and di-sodium or -potassium salts of H,dtpa (Sigma); butylamine, aluminium nitrate hydrate, gallium nitrate hydrate, indium nitrate pentahydrate, yttrium chloride (Aldrich); solvents (Prolabo). Other materials are cited under specific sections.Synthesis and Characterization of the N,N "-Bis(buty1amide) of H,dtpa, H3L'.-Butylamine (0.327 cm3, 3 mmol) was added dropwise with stirring to a solution of diethylenetriaminepentaacetic acid dianhydride (0.536 g, 1.5 mmol) in dry dimethylformamide (50 cm3, 40 "C). The colourless reaction mixture was stirred (4OoC, 1 h) and filtered. Chloroform (50 cm3) was added and, after cooling at 4 "C for 2 h, the white solid was filtered off, washed with diethyl ether (100 cm3) and dried at 40 "C overnight. The compound was recrystallized, from hot ethanol-hexane (1 10: 110 cm3), after cooling at 4 "C overnight. The white crystals were filtered off, washed with acetone (10 cm3) and diethyl ether (10 cm3) and dried overnight at 40 "C.

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