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Summary Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumour approach using a combination of a polymeric prodrug and polymer-enzyme conjugate to generate cytotoxic drug selectively at the tumour site. In this study the polymeric prodrug N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Gly-Phe-Leu-Gly-doxorubicin conjugate PK1 (currently under Phase II clinical evaluation) was selected as the model prodrug, and HPMA copolymer-cathepsin B as a model for the activating enzyme conjugate. Following polymer conjugation (yield of 30-35%) HPMA copolymer-cathepsin B retained ~20-25% enzymatic activity in vitro. To investigate pharmacokinetics in vivo, 125 I-labelled HPMA copolymer-cathepsin B was administered intravenously (i.v.) to B16F10 tumour-bearing mice. HPMA copolymercathespin B exhibited a longer plasma half-life (free cathepsin B t 1/2α = 2.8 h; bound cathepsin B t 1/2α = 3.2 h) and a 4.2-fold increase in tumour accumulation compared to the free enzyme. When PK1 (10 mg kg -1 dox-equiv.) was injected i.v. into C57 mice bearing subcutaneously (s.c.) palpable B16F10 tumours followed after 5 h by HPMA copolymer-cathepsin B there was a rapid increase in the rate of dox release within the tumour (3.6-fold increase in the AUC compared to that seen for PK1 alone). When PK1 and the PDEPT combination were used to treat established B16F10 melanoma tumour (single dose; 10 mg kg -1 dox-equiv.), the antitumour activity (T/C%) seen for the combination PDEPT was 168% compared to 152% seen for PK1 alone, and 144% for free dox. Also, the PDEPT combination showed activity against a COR-L23 xenograft whereas PK1 did not. PDEPT has certain advantages compared to ADEPT and GDEPT. The relatively short plasma residence time of the polymeric prodrug allows subsequent administration of polymer-enzyme without fear of prodrug activation in the circulation and polymer-enzyme conjugates have reduced immunogenicity. This study proves the concept of PDEPT and further optimisation is warranted. PDEPT concept using PK1 and HPMA copolymer-cathepsin B as a model combination. PK1 has proven ability to target solid tumours by the EPR effect (Seymour et al, 1994) with concomitant renal elimination resulting in low blood levels within 1-5 h in animals and man (Seymour et al, 1990;Vasey et al, 1999). HPMA copolymer-cathepsin B ( Figure 2B) was selected for PK1 activation as the PK1 Gly-Phe-Leu-Gly polymer-dox linker was designed to permit intralysosomal dox liberation due to action of the lysosomal cysteine proteases (Duncan et al, 1984). First it was necessary to prepare an HPMA copolymer-cathepsin B conjugate that would retain sufficient enzyme activity after conjugation. Activity was monitored in vitro using a low molecular weight substrate N-Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (Bz-Phe-Val-Arg-NAp) and the polymeric substrate PK1. The biodistribution of 125 I-labelled HPMA copolymer-cathepsin B and 125 I-labelled cathepsin B was assessed in mice bearing subcutaneous (s.c.) B16F10 tumours and this model wa...

Summary Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumour approach using a combination of a polymeric prodrug and polymer-enzyme conjugate to generate cytotoxic drug selectively at the tumour site. In this study the polymeric prodrug N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Gly-Phe-Leu-Gly-doxorubicin conjugate PK1 (currently under Phase II clinical evaluation) was selected as the model prodrug, and HPMA copolymer-cathepsin B as a model for the activating enzyme conjugate. Following polymer conjugation (yield of 30-35%) HPMA copolymer-cathepsin B retained ~20-25% enzymatic activity in vitro. To investigate pharmacokinetics in vivo, 125 I-labelled HPMA copolymer-cathepsin B was administered intravenously (i.v.) to B16F10 tumour-bearing mice. HPMA copolymercathespin B exhibited a longer plasma half-life (free cathepsin B t 1/2α = 2.8 h; bound cathepsin B t 1/2α = 3.2 h) and a 4.2-fold increase in tumour accumulation compared to the free enzyme. When PK1 (10 mg kg -1 dox-equiv.) was injected i.v. into C57 mice bearing subcutaneously (s.c.) palpable B16F10 tumours followed after 5 h by HPMA copolymer-cathepsin B there was a rapid increase in the rate of dox release within the tumour (3.6-fold increase in the AUC compared to that seen for PK1 alone). When PK1 and the PDEPT combination were used to treat established B16F10 melanoma tumour (single dose; 10 mg kg -1 dox-equiv.), the antitumour activity (T/C%) seen for the combination PDEPT was 168% compared to 152% seen for PK1 alone, and 144% for free dox. Also, the PDEPT combination showed activity against a COR-L23 xenograft whereas PK1 did not. PDEPT has certain advantages compared to ADEPT and GDEPT. The relatively short plasma residence time of the polymeric prodrug allows subsequent administration of polymer-enzyme without fear of prodrug activation in the circulation and polymer-enzyme conjugates have reduced immunogenicity. This study proves the concept of PDEPT and further optimisation is warranted. PDEPT concept using PK1 and HPMA copolymer-cathepsin B as a model combination. PK1 has proven ability to target solid tumours by the EPR effect (Seymour et al, 1994) with concomitant renal elimination resulting in low blood levels within 1-5 h in animals and man (Seymour et al, 1990;Vasey et al, 1999). HPMA copolymer-cathepsin B ( Figure 2B) was selected for PK1 activation as the PK1 Gly-Phe-Leu-Gly polymer-dox linker was designed to permit intralysosomal dox liberation due to action of the lysosomal cysteine proteases (Duncan et al, 1984). First it was necessary to prepare an HPMA copolymer-cathepsin B conjugate that would retain sufficient enzyme activity after conjugation. Activity was monitored in vitro using a low molecular weight substrate N-Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride (Bz-Phe-Val-Arg-NAp) and the polymeric substrate PK1. The biodistribution of 125 I-labelled HPMA copolymer-cathepsin B and 125 I-labelled cathepsin B was assessed in mice bearing subcutaneous (s.c.) B16F10 tumours and this model was a...

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