INFLUENCE OF CARRIER ON BIODISTRIBUTION AND INVITRO CYTOTOXICITY OF METHOTREXATE BRANCHED POLYPEPTIDE CONJUGATES

Methotrexate (MTX) has been conjugated to various structurally related, synthetic, branched polypeptides containing a poly(L-Lys) backbone by the aid of water-soluble carbodiimide. The average degree of MTX incorporated was found to be dependent on the size of the polymer and on the identity of the terminal amino acid residue of the side chains. Consequently the average molar substitution ratio was in the range of 4.9-72.0 MTX per carrier molecule. CD spectra of conjugates showed significant differences in solution conformation correlating with the identity of the side-chain-terminating amino acid. Polycationic conjugates XAK-MTX (X = Leu or D-Leu) assumed essentially ordered (helical) secondary structure, while the CD spectrum of the amphoteric conjugate (X = Glu) corresponded to only a partially ordered conformation in PBS. The covalent attachment of MTX to branched polypeptides results in a reduction of drug in vitro cytotoxicity influenced by the carrier structure. Conjugation to amphoteric polymers, depending on the configuration and position of glutamic acid (XAK-MTX vs AXK-MTX type conjugates) resulted in a decrease of anti-791T cell activity. However polycationic conjugates bearing L-Leu at the side chain terminal position (LAK-MTX) produced a compound with cytotoxicity only about 60 times less effective than free MTX. The biodistribution in mice has been characterized by blood clearance, whole-body retention, and tissue distribution 24 h after iv administration. Blood clearance of MTX-branched polypeptides could be significantly prolonged by incorporation of glutamic acid into the side chain. The presence of a D-amino acid in the terminal position of the side chain (D-LAK-MTX vs LAK-MTX) or adjacent to the polylysine backbone (a-D-ek-MTX vs aek-MTX) resulted in an elevated whole-body survival. Interestingly enough, the retarded blood survival of amphoteric or polycationic conjugates did not implicate similar tissue distribution. Polycationic carriers were directed predominantly to spleen, liver, and kidney, while conjugates with Glu in the side chain were taken up by the lung, kidney, and liver. It was demonstrated that branched polypeptide-MTX conjugates constructed from a polycationic or even amphoteric carrier can (a) sustain the cytotoxic activity of MTX at a level comparable to that of the frequently used HSA-MTX and (b) be present in the circulation (amphoteric conjugates) and/or in the body (both amphoteric and polycationic conjugates) for a much longer period of time than HSA-MTX or free drug. This study suggests that it is feasible to alter beneficially the body distribution and in vitro toxicity of MTX by logical combination of side-chain sequence, configuration, and identity of amino acid X in the branches of the carrier molecule.