In our earlier work [lOI on aminor~bosyl-bound prodrugs of adriamy~in (ADR) using poly(a-L-glutamic acid) (PGA) grafted in high yield (90-l 00 mol.%) with various peptide spacers as a plasma-soluble macromolecular carrier we observed rather low cytotoxie activities in L1210 leukemia and B16 melanoma in vitro assays. These results may be tentatively explained by a decreased susceptibility of the spacer-bound adriamycin moiety to hydrolysis by lysosomal enzymes due to the high spacer load. This hypothesis was tested by the study of two conjugates prepared by a different route. Peptide conjugates of adriamycin (Gly-GlyLeu-ADR and Gly-Gly-Gly-Leu-ADR) were synthesized using the trityl N-protecting group and were coupled to PGA in 4.5 mol.% load according to the method described earlier (I1 f. Howeuer, these conjugates were almost totally devoid of cell ~owth-inhibiting activity in L1210 and B16 in vitro tests. The data suggest that either the uptake of the polymeric prodrugs into the cell by pinocytosis is highly dependent on spacer load or molecular weight, or that lysosomal digestion is too slow for efficient release of ADR. Possibly, enzymatic de~adation of PGA which is known to occur only between pH 4 and 6 is rate-limiting for release of the drug. Current studies include the enzymatic degradation of PGA-peptide spacer-drug systems using ~-nitroaniline as a model drug and papain as the enzyme. By variation of the length and load of spacer it can be estimated under which conditions the release of drug (using UV spectrometry) is faster than de~adation of the polymer (as determined by viscometry). In addition, the uptake of PGA and derivatives with a fluorescent label into tumor cells is studied using laser flow cytometry and laser microscopy.

The synthesis and purification of radiolabelled immunoconjugates, composed of a human IgM monoclonal antibody directed against an int~cellular tumour-as~ciated antigen and either poly ( CY-L&I-tamic acid) (PGA) or poly [ N5-( 2-hydroxyethyl)-L-~utamine] (PHEG) is described. Coupling of polymers to the antibody was performed through disulfide bond formation involving a single thiol group at the C-terminus of the polymer chain and 2-pyridyldisullide groups introduced onto the antibody. The antibody was iodinated with 13'1 before conjugation. The polymers contained tyrosinamide in a low degree of substitution and were radiolabelled with 12% '2SI-labelled PGA and PHEG were found to be stable for at least 3 days in murine and human plasma. The bio~st~bution in mice of the doubly labelled immunoconjugates was studied and was compared with the pharmacokinetics of the individual components.PHEG showed a relatively slow blood clearance, the half-life being approximately 10 h with low uptake in liver, kidneys and spleen. PGA was rapidly cleared from the circulation and was significantly taken up in liver, kidneys and spleen. The biodist~bution of both immunoconju~tes was indistinguishable from that of the IgM proper, with plasma half-lives of approximately 6 h, indicating that the pharmacokinetic properties of the immunoconjugates are largely determined by the antibody part.

No abstract

The synthesis of a trisgalactoside-terminated cholesterol derivative is described. Tris(galactosyloxymethyl)-aminomethane is coupled to cholesterol by using glycyl and succinyl as intermediate hydrophilic spacer moieties. The resulting cholesteryl ester dissolves easily in water, forming monodisperse micelles. When added to dispersions of liposomes or plasma lipoproteins in water, the substance becomes incorporated rapidly into these structures, causing an increase of their buoyant density. Liposomes or low-density lipoproteins, preloaded with the substance, are rapidly cleared from the circulation and taken up by the liver after intravenous injection in rats. This uptake is inhibited by N-acetylgalactosamine but not by N-acetylglucosamine, indicating the specificity of this process.

Poly(a-L-glutamic acid) (PGA) was grafted with amino acid and ofigopeptide spacers up to 5 amino acids with the use of ~,~I-~arbony~diimidazo~e and 2~3-dihydro-1,2-bentisoth~azole-3~n-l~l-dioxide (saecharin~ as an additive, and these polypeptides were characterized. The antitumor antibiotic adriamycin was covalently coupled via an amide bond onto PGA and onto the grafted polymers with the use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ); these conjugates were characterized. The conjugates containing Gly-Gly--L-Leu spacer arms did yield free adriamycin upon digestion with papain. Adriamy~in gave fairly stable complexes with PGA-~driamy~in and branched po~ypeptide-adriamycin conjugates; these complexes were characterized.