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aCM-Dex-PA was labelled with 3H-glycine. b Parentheses represent AUC ratios.

aCM-Dex-PA was labelled with 3H-glycine. b Parentheses represent AUC ratios.

The peptidyl spacer (GGFG) provided slow releases of both DX-8951 and G-DX-8951 in vitro (tumor homogenates and cathepsins) and in vivo; the slow drug-release in tumor tissue is extremely advantageous to cytotoxicity of the time-dependent antitumor drug, DX-8951. A high steric hindrance of the bulky DX-8951 moiety was found to be responsible for the slow releases of both drugs from CM-Dex-PA-GGFG-DX-8951, while the GGFG spacer showed a fast drug-release with such a less bulky drug as doxorubicin.

The CM-Dex-PA-GGFG-DX-8951 conjugate was optimised on molecular sizes, degrees of carboxymethylation and loadings of DX-8951 to obtain DE-310, as follows. CM-Dex-PA was synthesized by carboxymethylation of Dex-PA which was obtained by periodate oxidation of dextran followed by borohydride reduction. Tetra-peptidyl DX-8951 (GGFG-DX-8951) was covalently linked to CM groups of CM-Dex-PA through acid amide bonds to give CM-Dex-PA-GGFG-DX-8951. The conjugates with various ranges of molecular size, degrees of carboxymethylation and contents of DX-8951

were synthesized, and their antitumor activities and therapeutic indexes were evaluated with murine Meth A fibrosarcoma solid tumor-bearing mice.

As the results, the conjugates with MWs (ca.100K~ca.500K), degrees of carboxymethylation (ca.0.3~ca.0.4 per sugar residue) and DX-8951 contents (less than 8%), although these values depend on analytical methods used, showed the equivalency. Thus, based on these results, DE-310 was specified. The structural feature of DE-310 is shown in Fig. 2, which was determined by the analytical methods such as Controlled Smith Degradation10 (analyses of mild-acid degradation products of DE-310) and NMR spectroscopy.

Figure 2. Partial structural feature of DE-310

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