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Copolymers of camptothecin (CPT) and [N-(2-hydroxypropyl) methacrylamide] (HPMA) are novel anticancer drugs that show improved pharmacological profile in animal models as compared to the free drug CPT. We investigate here the aggregation properties of a HPNIA-glycyl-6-aminohexanoyl-glycyl-CPT copolymer (similar to20,000 Da). The molecular size of HPMA-copolymer CPT is followed over 5 orders of magnitudes of concentration in isotonic buffer by measuring either the time resolved fluorescence anisotropy (FA) of CPT or the autocorrelation function of the light scattered by the copolymer. A detailed analysis of these data suggests the presence of elongated structures with axial ratio similar to3 in the range 0.1-0.5 mug/ml and aggregates with association number higher than 2 in more concentrated solutions (up to 10 mg/ml). The binding affinity of HPMA-copolymer CPT for serum albumin is inversely dependent on the degree of aggregation of the copolymer. We also show that the copolymer concentration in plasma from mice treated with an active, non-toxic, dose of HPMA-copolymer CPT, decreases from 3 to 0.01 mg/ml in 72 h. In the same range of concentrations in vitro, we do not detect hydrophobic aggregates of polymers with high (>3) association number. Our study indicates that the circulating HPMA-copolymer CPT in mice should not undergo extensive aggregation and should interact with serum albumin more weakly than free CPT. (C) 2004 Elsevier B.V. All rights reserved.

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C 8 analytical column, using a mixture of acetonitrile–25 m M phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.

Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N -(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its α-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2–0.4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were ∼5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [ 3 H]CPT-conjugate and free [ 3 H]CPT. Radioactivity uptake in tumour was evident only after [ 3 H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.