Synthesis and Characterization of Her-2 Targetable N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates.

Background: Current Her-2 overexpressing cancer therapies are limited by rapid drug resistance development from Trastuzumab (TRZ) treatment or low selectivity and non specific side effects of hepatotoxicity with small molecular tyrosine kinase inhibitors such as lapatinib. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-drug conjugates with tailored side-chain composition have demonstrated their clinical potential as tumor targetable chemotherapeutic delivery systems. The present study evaluates the potential of novel HPMA copolymer-TRZ conjugates to enhance the therapeutic index of Her-2 targeted therapy.Material and Methods: HPMA copolymer-TRZ conjugates were synthesized in two steps. In the first step, an HPMA copolymer precursor containing reactive p-nitrophenyl ester (ONp) side chains was synthesized by free radical precipitation polymerization. In the second step a series of polymer precursors were conjugated to varying amounts of TRZ by aminolysis. The feed ratio of TRZ to HPMA copolymer was calculated based on the number of primary amine groups available on TRZ surface as determined spectrophotometrically using the 2,3,6-Tri-nitrobenzenesulfonic acid (TNBS) assay. The copolymer conjugates were characterized by correlating the feed ratio with the degree of conjugation (TNBS assay) and molecular weight distribution (size exclusion chromatography). The antiproliferative action of HPMA copolymer-TRZ conjugates was evaluated on a Her-2 overexpressing BT474 breast cancer cell line. Cell proliferation was quantified by MTT assay. Activation of Her-2 after treatment with polymer conjugate was measured by western immunoblotting.Results and Discussion: A series of three HPMA copolymer-TRZ conjugates were successfully synthesized and characterized. It was observed that an increase in the feed ratio of HPMA copolymer precursor to TRZ resulted in a corresponding increase in the degree of TRZ conjugation. The size exclusion chromatography profile of the conjugates was also consistent with the degree of conjugation. As HPMA copolymer precursor contents increased, the elution time decreased suggesting an increase in the hydrodynamic volume of conjugates. The growth inhibition effects of all HPMA copolymer-TRZ conjugates were concentration dependant. The half maximal inhibitory concentrations (IC 50 ) of the conjugates with TRZ/HPMA=1/1, 2/1 and 4/1 were 10, 29, and 20 nM respectively, which were comparable to that of free TRZ (8 nM) suggesting that the antibody retained bioactivity against Her-2 overexpressing cancer cells upon conjugation to the polymer. Western immunoblotting showed that polymer conjugated TRZ effectively downregulated activated Her-2 (phospho-Her2) to a greater extent than free TRZ.Conclusion: Here we report for the first time, the successful synthesis, characterization and in vitro evaluation of HPMA copolymer-anti-Her-2 drug (TRZ) conjugates for active targeting to Her-2 overexpressing breast cancer cells. The conjugate compositions were consistent with their molar feed ratios and inhibited model cancer cell growth comparable to the free drug. Preliminary studies indicate improved efficacy of TRZ against Her-2 positive breast cancer cells when administered as polymeric conjugate. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5065.