PDEPT: polymer-directed enzyme prodrug therapy

Abstract

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...

Prodrug Strategies in Anticancer Chemotherapy

“…[124] A recent extension of the basic passive targeting strategy is polymer-directed enzyme prodrug therapy (PDEPT). [125,126] Conceptually similar to ADEPT (Section 2.1.1), PDEPT is a two-step antitumor approach combining two polymer conjugates: an enzymatically cleavable macromolecular prodrug and the respective polymer-enzyme conjugate. Passive accumulation of both components should enable a controlled release of the cytotoxic drug at the tumor site.…”

“…Initial preclinical evidence for this prodrug concept has been obtained with HPMA-enzyme conjugates and HPMA conjugates of doxorubicin. [125,126] Biodistribution experiments that support the EPR concept by demonstrating an enhanced tumor uptake of macromolecules have usually been carried out in animals, mostly rodent xenograft models. Less information is available from studies using orthotopic tumor models, and little is known about the EPR activity of native tumors in humans.…”

“…Satchi et al developed HPMA copolymer-bound cathepsin B that was co-administered with PK1. [126] The PDEPT combination proved to be more efficacious than PK1 alone in a B16F10 melanoma model and showed antitumor activity against a COR-L23 xenograft, whereas PK1 was not active. The examples mentioned above show that the development of non-targeted HMPA-based prodrugs of doxorubicin did not come to an end with PK1 entering clinical trials.…”

Microarrays of 41 Human Tumor Cell Lines for the Characterization of New Molecular Targets: Expression Patterns of Cathepsin B and the Transferrin Receptor

“…Cathepsin B, a lysosomal protease, is overexpressed in numerous cancers and has been implicated in increased tumor metastasis [6]. In addition to specific, potentially anti-metastatic inhibitors [7], several cathepsin B-sensitive prodrugs have been designed [8, 9]. The transferrin receptor is a transmembrane glycoprotein that mediates the cellular uptake of transferrin, thereby providing for the increased iron demand in proliferating cells.…”

Functionalization of Cationic Polymers for Drug Delivery Applications

“…The drug molecules can be of the same type or several drugs can be conjugated to the carrier at the same time to achieve a synergistic effect for a more efficient combination treatment. 141,142 Carrier systems consisting of the three above-mentioned components can be complemented by targeting ligands in order to achieve cell-specific targeting; however, no specific tumour markers have been found so far. This is the reason why modern approaches include targeting of overexpressed proteins on the cell surface or in the cytosol (active targeting) as well as exploiting the enhanced permeability and retention effect (EPR) 143 for passive targeting.…”