Action of polymeric prodrugs based on N-(2-hydroxypropyl)meth- acrylamide copolymers. I. Suppression of the antibody response and proliferation of mouse splenocytes in vitro

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Abstract

Splenocytes were removed from A/J mice 14 days after i.p. injection of 1 × 108 sheep red blood cells and were incubated for 10 min, 3 h and 5 days with free daunomycin, free anti Thy 1.2 antibodies or daunomycin conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates with biodegradable (Gly-Leu-Gly; Gly-Phe-Leu-Gly) or non-biodegradable (Gly-Gly) side-chains containing targeting anti Thy 1.2 antibodies. The effect on the antibody response, represented by decrease in the numbers of IgM and IgG plaque-forming, i.e., antibody-releasing mouse splenocytes (plaque-forming cells), was detected. It was shown that a 10 min contact of immunocompetent cells with daunomycin-HPMA copolymer conjugates is sufficient for suppression of antibody formation. However, 3 hours incubation were necessary to obtain significant decrease in a number of PFC. Lymphocytes (mouse splenocytes) were very sensitive to free daunomycin. Concentration of 50 μg/ml eliminated all lymphocytes from tissue culture. At 1–10 ug/ml, a high proportion of cells remained viable, but the antibody response was completely suppressed. A comparable amount of daunomycin (35–70 μg/ml) bound to copolymer with targeting antibodies did not kill lymphocytes in tissue culture, but the antibody response was substantially suppressed (50–90%). In all experiments IgG antibody formation was more sensitive to suppression than IgM response. Biodegradability of the bond between the HPMA copolymer carrier and daunomycin substantially increased the pharmacological effect although a certain degree of immunosuppression was detected with the drug conjugated to targeted non-biodegradable HPMA copolymer. Free daunomycin inhibited [3H]thymidine incorporation by mouse T lymphocytes (cultivation in presence of Con A) at the concentration of 0.1 μg/ml. To achieve the same effect, 5 times more daunomycin bound to biodegradable HPMA copolymer with targeting anti Thy 1.2 antibodies or 500 times more daunomycin bound to non-targeted biodegradable HPMA copolymer was necessary.

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