Comparison of the binding kinetics of antibody-targeted N-(2-hydroxypropyl) methacrylamide (HPMA)-bound doxorubicin in vitro and in vivo

Antibody-targeted conjugates have been previously shown to have antitumour and immunosuppressive activity both in vitro and in vivo. In this study we have examined the binding kinetics of antibody-targeted doxorubicin (DOX) based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers following i.v. administration to mice (DOX concentration 5mg/kg) or after in vitro incubation with the cells for different time intervals using fluorescence analysis. We have found that binding of the conjugate to the target cell subpopulation is more effective and much faster in vivo than in vitro. In vivo, 1 min was long enough for reproducible binding of the conjugate to peripheral blood leukocytes (PBL), while 15 min was necessary for significant binding to splenocytes. To reach the same situation in vitro it was necessary to incubate the cells with the conjugate for at least 1 h. By fluorescence analyses it was shown that in vitro these antibody-targeted drugs are bound to PBL, splenocytes and thymocytes. After i.v. administration the conjugates were bound to PBL and splenocytes but were not present on the surface of thymocytes. These alterations in pharmacokinetics may account for the decreased toxicity and improved efficacy reported previously.