Three water soluble copolymers based on N-(2-hydroxypropyl)methacrylamide were prepared. Copolymer I contains adriamycin, a chemotherapeutic agent, attached via enzymatically degradable oligopeptide (glycylphenylalanylleucylglycine; G-F-L-G) side chains. The other two copolymers contained the photosensitizer, meso-chlorin e6 monoethylene diamine disodium salt (Mce6). In Copolymer II, the chlorin is attached via the degradable G-F-L-G sequence, and it was bound by the nondegradable glycyl spacer in Copolymer III. Initially, the copolymers were characterized separately in vitro and in vivo. Combinations of the copolymer bound chemotherapeutic agent and each of the copolymer bound photosensitizers were then assessed for antitumor effect in vivo. Localization/retention studies (A/J mice; Neuro 2A neuroblastoma solid tumor) were performed with the two copolymers containing Mce6 as well as the free drug. Results of these experiments demonstrated a very different tumor uptake profile for the two copolymers. While the free drug was rapidly cleared from tumor tissue, the copolymer containing Mce6 attached via the non-degradable bond was retained for an extended period; drug concentrations in the tumor were high even after 5 days. On the other hand, a high concentration of the copolymer containing Mce6 bound via the degradable sequence was taken up by the tumor, yet its concentration in the tumor was substantially diminished at 48 h after administration. This shows indirect evidence of in vivo cleavage of Mce6 from the copolymer in the lysosomal compartment which is supported by direct evidence of cleavage by cathepsin B (a lysosomal enzyme) in vitro. Antitumor effects were assessed on Neuro 2A neuroblastoma induced in A/J mice for all three copolymers. Photodynamic therapy (PDT) proved the copolymer with Mce6 bound via the degradable oligopeptide sequence to be a more effective photosensitizer in vivo than the other chlorin containing copolymer. The difference in activity was consistent with the results obtained by photophysical analyses in which the free drug had a higher quantum yield of singlet oxygen generation than the polymer bound drug in buffer. The quantum yield of singlet oxygen generation increased with the enzymatic cleavage of the chlorin from the copolymer. Conditions were subsequently determined for which chemotherapy or PDT would show some antitumor effect, yet be incapable of curing tumors. Finally, combination therapy experiments were performed in which the copolymer bound adriamycin was mixed with either of the copolymer bound chlorin compounds and injected intravenously (i.v.) into the tail veins of mice.(ABSTRACT TRUNCATED AT 400 WORDS)
k.o N-(2-hydroxypropyl)methacrylamide (HPMA, 1) copolymer-chlorin e6-anti-Thy 1.2 antibody conjugates were synthesized. They differ in the method of antibody binding. In conjugate 11, the polyclonal antiThy 1.2 antibody was bound via P-amino groups of lysine residues. Conjugate 14 contained anti-Thy-1.2 antibodies bound via oxidized carbohydrate groups present near the hinge region in the F, part of the antibody molecule. The photodynamic activity of these conjugates towards mouse splenocytes in vitm was compared with the activity of a nontargetable HPMA copolymer-chlorin e6 conjugate (15) and free chlorin e6. Whereas free chlorin e6 was cytotoxic both when kept in the dark and when splenocytes were irradiated, the binding of the drug to HPMA copolymers considerably decreased its cytotoxicity in the dark. Conjugates 11 and 14 containing targeting polyclonal anti-Thy 1.2 antibodies were more biotogidy active compared to the nontargetable conjugate 15. Conjugate 14, which contained anti-Thy 1.2 antibodies bound at a specific location was the most active both in its photodynamic effect on viability of splenocytes and/or suppression of the primary antibody response of mouse splenocytes towards sheep red blood cells in vitro. The results demonstrate the importance of the chemistry of antibody binding on the biological activity of targetable polymeric drugs. a) Part 1: cf. 18). 0 1990, HIithig & Wepf Verlag, Basel CCC 0025-1 16X/90/$03.00
A copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-methacryloylglycine p-nitrophenyl ester (MA-Gly -ONp) was synthesized by radical precipitation copolymerization. Galactosamine was coupled to the polymer by reaction with the active ester side chains. The remaining p-nitrophenyl groups were converted to amine functionalities via a polymeranalogous reaction with an excess of ethylene diamine. Chlorin e6 was activated using a mixed anhydride method and subsequently bound to the modified side chains of the polymer. The photodynamic activity of the conjugate with galactosamine as the targeting moiety was tested in vitro on a human hepatoma cell line: PLC /PRF /5 (Alexander cells). It appears that the conjugate enters the cell interior by receptor mediated pinocytosis via the asialoglycoprotein receptors present on the surface of the PLC cells. Photoactivation of the chlorin containing conjugate with red light proved cytotoxic to the cells. Structure tests comparing the HPMA copolymer -galactosamine-chlorin e6 conjugate and a nontargetable HPMA copolymer -chlorin e6 conjugate, with comparable chlorin e6 content, indicate that the targeted conjugate is more biologically active.
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