The amino group of
doxorubicin (DXR) was found to be bound to the carboxyl group of
carboxymethylpullulan (CMPul) either directly or through tetrapeptide spacers, including Gly-
Gly-Phe-Gly,
Gly-Phe-Gly-Gly and
Gly-Gly-Gly-Gly. These conjugates had DXR contents of 6.1-7.1%, with the degree of substitution of carboxymethyl groups being 0.6 per
sugar moiety. These conjugates associate in
phosphate-buffered saline (PBS) (pH 7.4), forming
micelles with hydrophobic DXR inside and hydrophilic CMPul on the outside. The amounts of DXR released from the conjugates in the presence of rat liver lysosomal
enzymes were determined by HPLC. The rate of the drug release differed among the conjugates tested. CMPul-DXR conjugate bound through Gly-
Gly-Phe-Gly released 35% of its DXR over 24 h. On the other hand, CMPul-DXR conjugate without spacer released no free DXR. The antitumor effect of each conjugate in rats bearing Walker 256 was studied by monitoring the
tumor weights after a single
intravenous injection. Compared with DXR, CMPul-DXR conjugates bound through Gly-
Gly-Phe-Gly and
Gly-Phe-Gly-Gly spacers significantly suppressed the
tumor growth, while CMPul-DXR conjugate bound through
Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate bound through
Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate without spacer showed no in vivo antitumor effect even at a dose equivalent to as much as 20 mg/kg of DXR.