Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

Abstract

Summary During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-PheLeu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184,ugml-1) could be selected to achieve a cytotoxic effect.

Synthesis of Poly(vinyl alcohol)-Doxorubicin Conjugates Containing cis-Aconityl Acid-Cleavable Bond and Its Isomer Dependent Doxorubicin Release

“…Fractions corresponding to the isomers were collected and analyzed by TOF-MS spectrum. Their structure were confirmed by one-and two-dimensional NMR ( 1 H, 13 C, 1 H, 1 H COSY, 1 H, 13 C HMQC). The yields of trans-ADOX and cis-ADOX were 44.8 and 36.3%, respectively.…”

“…These products were separated completely by using HPLC (Fig. 2) and were analyzed by TOF-MS spectroscopy and one-and two-dimensional NMR ( 1 H, 13 spectroscopy and 1 H-and 13 C-NMR experiments. 32) ADOX was bound to PVA according to the synthetic scheme shown in Fig.…”

“…Its structure was confirmed by one-and two-dimensional NMR ( 1 H, 13 Preparation of PVA-ADOX Conjugates Ethylenediamine residues were introduced to the hydroxyl groups of PVA molecules by the 1,1Ј-carbonyldiimidazole (CDI) activation method. [18][19][20] Four milliliters of dimethyl sulfoxide (DMSO) containing CDI (74 mg) was added to 400 mg of PVA dissolved in 60 ml of DMSO, followed by stirring for 1 h at room temperature.…”

“…N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing a tetrapeptide side chain (Gly-Phe-Leu-Gly) terminated in DOX and, optionally, galactosamine, as a targeting moiety, 9) were recently approved in the United Kingdom for phase I/phase II clinical trials to treat primary hepatoma. HPMA copolymer prodrugs produced increased life spans and an increased number of long-term survivors [10][11][12][13] depending on the structure of the conjugate, timing of administration, and number of doses. Similarly, HPMA copolymers containing the anthracycline antibiotics, Daunorubicin (DNR) 12,13) and DOX 11) were modified with fucosylamine, and it was shown that they interact with the receptor on L1210 cells in vitro and in vivo.…”

“…HPMA copolymer prodrugs produced increased life spans and an increased number of long-term survivors [10][11][12][13] depending on the structure of the conjugate, timing of administration, and number of doses. Similarly, HPMA copolymers containing the anthracycline antibiotics, Daunorubicin (DNR) 12,13) and DOX 11) were modified with fucosylamine, and it was shown that they interact with the receptor on L1210 cells in vitro and in vivo. Subsequent experiments were performed to test the pharmacologic activity of these conjugates in vivo against L1210 leukemia in DBA 2 mice.…”