A generation 5 PEGylated (PEG 1100)
polylysine dendrimer, conjugated via a stable
amide linker to OtBu protected
methotrexate (MTX), was previously shown to have a circulatory half-life of 2 days and to target solid
tumors in both rats and mice. Here, we show that deprotection of MTX and substitution of the stable linker with a
matrix metalloproteinase (
MMP) 2 and 9 cleavable linker (PVGLIG) dramatically increased plasma clearance and promoted deposition in the liver and spleen (50-80% of the dose recovered in the liver 3 days post dose). Similar rapid clearance was also seen using a scrambled
peptide suggesting that clearance was not dependent on the cleavable nature of the linker. Surprisingly,
dendrimers where OtBu capped MTX was linked to the
dendrimer surface via the hexapeptide linker showed equivalent in vitro cytotoxicity against HT1080 cells when compared to the uncapped
dendrimer and also retained the long circulating characteristics of the stable constructs. The OtBu capped MTX conjugated
dendrimer was subsequently shown to significantly reduce
tumor growth in HT1080
tumor bearing mice compared to control. In contrast the equivalent
dendrimer comprising uncapped MTX conjugated to the
dendrimer via the same hexapeptide linker did not reduce
tumor growth, presumably reflecting very rapid clearance of the construct. The results are consistent with the suggestion that protection of the α-carboxyl group of
methotrexate may be used to improve the circulatory half-life and reduce the liver accumulation of similar MTX-conjugated
dendrimers, while still retaining antitumor activity in vivo.