The primary purpose of this study was to comprehensively delineate specificity of the
peptide spacer sequence to
tumor-expressed
proteases for the design of macromolecular carrier-
peptide spacer-
drug conjugate system. 225 conjugates of
carboxymethyldextran polyalcohol (CM-Dex-PA) as water-soluble carrier and a
dansyl derivative (N-(4-aminobutyl)-5-(dimethylamino)-1-naphthalenesulfonamide, DNS) as the model
drug linked with different tetrapeptide spacers (Gly-Gly-
P(2)-P(1),
P(2), P(1): Ala, Asn, Gly,
Cit, Gln, Ile,
Leu, Met, Phe,
Pro, Ser, Thr,
Trp, Tyr, and Val) were combinatorially synthesized. First, the drug release assay of all of the fluorogenic model conjugates was performed in murine Meth A solid
tumor homogenates. The drug release rate was higher with conjugates having hydrophobic
amino acids at
P(2). It was also found that conjugates with Asn release the
drug rapidly and, in contrast, those with Pro does not. Second, we selected three
peptide spacers (Gly-
Gly-Phe-Gly, Gly-Gly-Ile-
Gly, Gly-Gly-
Pro-Leu), which release only DNS at different rates, and applied them to
doxorubicin (DXR) conjugates. These three DXR conjugates were used for investigating relationships with drug release, pharmacokinetics, and antitumor activity against Meth A bearing mice of these conjugates. The release of DXR from the conjugates corresponded well with that of DNS conjugates in
tumor homogenates. CM-Dex-PA-Gly-
Gly-Phe-Gly-DXR and CM-Dex-PA-Gly-Gly-Ile-Gly-DXR indicated strong antitumor activity, with the comparable pharmacokinetic profile of released DXR in
tumor. Taken with the fact that the drug release rate in
tumor homogenates was approximately 10-fold different between these two DXR conjugates, it is likely that cellular uptake of the conjugate would be rate-limiting, rather than the drug release process under the in vivo situation. However, much weaker antitumor activity was observed with CM-Dex-PA-
Gly-Gly-Pro-Leu-DXR, of which the drug release was extremely slow.