Search

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).

A new approach for covalent coupling diethylenetriaminepentaacetic acid (DTPA) molecules to a partially reduced monoclonal antibody utilizes a malemide modified copolymer of hydroxyethyl methylacrylate and methyl methacrylate (DTPA copolymer) prepared by the group transfer polymerization (GTP) method. An average of 6 DTPA molecules were incorporated per mol maleimeide DTPA copolymer and 1.5 mol maleimide DTPA copolymer per mol antibody. Maleimide DTPA copolymer modified antibody was intramolecularly cross-linked, reduced immunoactivity and had a high in vivo liver uptake.

We have synthesized a long-chain hydrocarbon covalently coupled to diethylene-triaminepenta-acetic acid (stearylamine-DTPA) and have incorporated this compound in liposomes during their preparation. The lipophilic hydrocarbon chain anchors the molecule in the lipid bilayer, exposing the DTPA groups on the surface for chelation. Ethanolic solutions of the lipids are evaporated to dryness under nitrogen in multidose vials; the lipids are suspended in the vial by adding a small volume of distilled water followed by sonication. The liposomes are then labeled by transcomplexation in the case of Ga-67 and by conventional stannous reduction in the case of Tc-99m, by adding the activities directly to the vial. These liposomes bind 95 +/- 5% of Ga-67 and Tc-99m activity, as determined by paper chromatograph assay, eliminating the need for a purification step. The labeled liposomes release about 5% of their Ga-67 activity, and about 30% of their Tc-99m activity after 2 hr of incubation in 50% human plasma at 37 degrees C. Activity released from liposomes labeled with Ga-67 or Tc-99m oxine is much greater under the same conditions. In normal mice the labeled liposomes show biodistributions that are comparable with that obtained with liposomes labeled by conventional techniques.

Five DTPA-amide and ester derivatives have been synthesized and their Gd3+ stability constants have been measured using a simple spectrophotometric method. These results are compared to stability constants measured for Gd3+ binding to two different DTPA-conjugated proteins. Although the thermodynamic constants for Gd3+ binding to DTPA-monopropylamide and DTPA-monopropylester relative to Gd(DTPA)2- decrease by log K = 2.6 and 3.4, respectively, the blood pH conditional constants differ from Gd(DTPA)2- only by log K = 1.2 and 1.9, respectively. The corresponding dipropylamide and ester conjugates of DTPA show considerably lower thermodynamic and conditional constants. This has important implications in the covalent attachment of chelates to macromolecules for use in magnetic resonance imaging. The measured binding constants for Gd(DTPA)-IgG and Gd(DTPA)-BSA suggest that many of the DTPA molecules in these systems, prepared under our experimental conditions, are disconjugated. The model compound results indicate that it is important to use methods in attaching DTPA to macromolecules which preclude dionjugation of the chelate. Otherwise, their affinity for Gd3+ and consequently their usefulness as MRI contrast agents may be severely compromised.

Bifunctional derivatives of the chelating agents ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, in which a p-isothiocyanatobenzyl moiety is attached at the methylene carbon atom of one carboxymethyl arm, was synthesized by reductive alkylation of the relevant polyamine with (p-nitrophenyl)pyruvic acid followed by carboxymethylation, reduction of the nitro group, and reaction with thiophosgene. The resulting isothiocyanate derivatives reacted with monoclonal antibody B72.3 to give antibody-chelator conjugates containing 3 mol of chelator per mole of immunoglobulin, without significant loss of immunological activity. Such conjugates, labeled with the radioisotopic metal indium-111, selectively bound a human colorectal carcinoma implanted in nude mice when given intravenously. Uptake into normal tissues was comparable to or lower than that reported for analogous conjugates with known bifunctional chelators. It is concluded that substitution with a protein reactive group at this position in polyaminopolycarboxylate chelators does not alter the chelating properties of these molecules to a sufficient extent to adversely affect biodistribution and thus provides a general method for the synthesis of such chelators.

A series of bifunctional chelating agents--substituted benzyl derivatives of diethylenetriaminetetra acids--was synthesized. These agents were used to label antibodies with a lanthanide, Eu3+. The stabilities of their antibody conjugates were evaluated under different conditions and the dissociation rates of Eu3+ were measured at pH 3.2, which is used for fluorescence enhancement in time-resolved fluoroimmunoassays. The synthesized complexing agents were also compared to other reagents used for metal labelings, to diethylenetriaminepentaacetic acid-dianhydride, and to p-isothiocyanatophenyl-EDTA. The asymmetric p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid-Eu3+ showed reasonably good stability at neutral pH but released Eu3+ rapidly in the acidic fluorescence enhancement solution. This makes it an optimal choice for chelate labeling in dissociation-based time-resolved fluoroimmunoassays.

The synthesis, binding of radioactive cations, liposomal encapsulation, and biodistribution of the oxidized-inulin reaction product with ethylenediamine and diethylenetriaminepentaacetic acid (4) are described. The four-step synthesis of the inulin derivative proceeded in a good overall yield of 72%. The complex of the inulin derivative with either 67Ga3+ or 111In3+ was stable in vivo and did not readily distribute into tissues, being excreted primarily in urine after intravenous administration to mice. The liposome-entrapped inulin derivative can be loaded with radioactive heavy metal cations by mobile ionophores in high radiochemical yields of 80-91%. Following the intravenous administration of the liposomal encapsulation of the indium-111-labeled inulin derivative, the entrapped compound had a biodistribution characteristic of liposomes and allowed an estimation of the extent of the intracellular uptake of liposomes. The ability of the inulin derivative to chelate many different types of metals will allow the use of this probe for studying subtle differences in tissue distribution resulting from different drug targeting or delivery protocols in the same animal by multiple labeling techniques. Moreover, the chelate-conjugated inulin permits studies of the applications of drug delivery systems in primates or human subjects by noninvasive techniques such as gamma-scintigraphic or nuclear magnetic resonance imaging methods.

Two chemically labile linkages, disulfide and diester, and two stable linkages, thioether and hydrocarbon, were introduced between antibody and 111In-DTPA in order to modify their biodistributions. The biodistributions of the new linkages were evaluated in rats with target antigens localized in lungs. For a comparison purpose, the antibody-DTPA conjugate with a peptide linkage was used as a control conjugate. The antibody conjugates with the stable linkages produced the biodistributions similar to that of the peptide linked conjugate during a 48 h period. The disulfide and diester conjugates, however, cleared from blood much faster and are retained in normal organs much lower than the peptide conjugate. The disulfide and the diester conjugate amplified the lung (target) to blood ratio by 15 and 6 times, respectively at 48 h, as compared to the corresponding target to blood ratio of the control conjugate. Compared to the control conjugate, a 3 times higher target to liver ratio was also obtained by the disulfide conjugate and a 4 times higher target to kidney ratio was obtained by the diester conjugate at 48 h.

An attempt was made to develop a macromolecular paramagnetic contrast agent that would be easily and inexpensively synthesized, low in toxicity, and quickly eliminated from the body after completion of the desired imaging studies. Under more rigorous conditions than attainable with proteins, the bis-anhydride of DTPA was reacted in anhydrous, hot DMSO with dextrans ranging in size from 17,000 molecular weight (MW) to 150,000 MW in various ratios of DTPA to glucose units from 1:1 to 1:40. Up to several hundred DTPA units per saccharide complex could be attached with this technique. Depending on the initial polysaccharide length and ratio of DTPA to glucose units, various degrees of cross-linking occur. Average MW of the polymers ranging from 17,000 to particles several million MW could be synthesized. The ester bond thus formed is stable for prolonged periods in solution. Depending on the polymer size, soluble material, gels, or particles are formed. Relaxation rates of the soluble material are similar or better than Gd-DTPA on a molar basis of Gd3+. The particulate material demonstrates T2 shortening out of proportion to T1, as expected for solid material, from increased susceptibility effects.

Ytterbium-DTPA was evaluated as a potential intravascular contrast agent. Ytterbium-DTPA was synthesized from ytterbium oxide and diethylene triamine penta-acetic acid (DTPA). CT scans of increasing concentrations of ytterbium and iodine showed that at 125 kVp, ytterbium was denser than an equal concentration of iodine. The LD50 of intravenous ytterbium-DTPA was 10 mM/kg (1.73 g ytterbium/kg) in rats. In enhanced CT scans and pulmonary angiography in dogs, ytterbium was visibly denser than iodine, and CT Hounsfield units showed greater enhancement of the aorta and inferior vena cava with ytterbium. The animals showed no sign of acute or delayed toxicity. Ytterbium-DTPA deserves further evaluation as a contrast agent for high kVp techniques.