HPMA copolymer-drug anticancer conjugates have been successfully transferred into clinical evaluation. Such macromolecular constructs selectively accumulate within solid tumours by the enhanced permeability and retention (EPR) effect. However, lysosomotropically activated compounds such as HPMA copolymer- doxorubicin (PK1) present limitations in that they require enzymatic activation within lysosomes in order to release the active moiety which in turn exerts its cytotoxic effect. Hence this study looked at the development of a second generation, membrane active polymeric anticancer conjugates. Using melittin (MLT) as a model peptide, HPMA copolymer-MLT conjugates were synthesised and characterised using standard biochemical techniques (SDS PAGE, BCA protein assay and FPLC). The effect of MLT content using conjugates with high (38.9 ± 2.5 % w/w), medium (25.8 ± 6.2 % w/w) and low (12.1 ± 8.6 % w/w) MLT loading and the effect of peptidyl linker (-Gly- Gly- (GG) and -Gly-Phe-Leu-Gly- (GFLG)) were investigated in vitro. The MLT conjugate with medium loading and GG spacer showed reduced haemolytic activity (Hb50 19.9 ± 6.2 μg/ml (p < 0.05)) in a rat red blood cell lysis model, and maintained cytotoxic activity against a B16F10 murine melanoma cell line (IC50 7.3 ± 1.5 μg/ml; p = 0.46 (NS)) relative to free MLT. This conjugate was chosen to proceed with preliminary in vivo studies. The MTD of HPMA copolymer-MLT was found to be 4- fold greater than that of free MLT (10 mg/kg MLT-equivalent) and body distribution of MLT conjugate 3 mg/kg (MLT-equivalent) (i.p.) showed 3 - 4-fold increased circulation time. However, no improved tumour targeting by the EPR effect was established 4 h after i.p. administration. Disappointingly, no antitumour activity was observed in vivo following i.p. or i.v. administration. Nevertheless, improved synthetic procedures to prepare more compounds of this new class of anticancer agents is warranted
