Prodrugs of N,N-di-(2-chloroethyl)-4-phenylene diamine (PDM) based on soluble poly[N5-(2-hydroxyethyl)-l-glutamine] (PHEG) have been evaluated as tumour-targeted drugs. These materials are designed to exploit the enhanced permeability of tumour vasculature, combining a passive tumour tropism with systemic liberation of free PDM. Modification of PDM by coupling via oligopeptide spacers onto a polymeric carrier significantly reduced its cytotoxicity towards different cell types in vitro. On the other hand, incubation of the cells with the PHEG-Gly-Phe-Ala-Leu-PDM conjugate in the presence of collagenase IV led to the release of lethal amounts of free drug, resulting in higher cytotoxicity for this derivative. The PHEG-Gly-Phe-Ala-Leu-PDM conjugate, which is rapidly degraded by lysosomal and tumour-associated enzymes also showed a decreased systemic toxicity in vivo and could be administered at a dose of 8 mg PDM/kg body weight intravenously, compared with just 2 mg/kg for free PDM. Furthermore, this derivative also showed better antitumour activity against a C26 colorectal carcinoma tumour model, compared with no activity for the free drug. The results indicate that the PHEG-Gly-Phe-Ala-Leu-PDM conjugate is a promising candidate for cancer treatment.

Polymeric prodrugs of N,N-di(2-chloroethyl)-4-phenylenediamine mustard (PDM) were prepared by coupling the drug via an oligopeptide spacer onto poly[N-5-(2-hydroxyethyl)-L-glutamine] (PHEG). In a first step PDM was linked to the peptide spacer and subsequently the low molecular weight peptidyl-PDM derivatives were coupled to 4-nitrophenyl chloroformate activated PHEG. Hydrolytic stability studies clearly demonstrated an improved stability for the polymeric derivatives compared to the parent drug. Dynamic laser light scattering measurements indicated the formation of aggregates. The influence of the amino acid sequence of the spacer on the enzymatic stability of the macromolecular drug conjugate was investigated under different conditions. Accordingly, the conjugates were incubated in buffers of lysosomal or physiological pH and in the presence of lysosomal or tumor-associated enzymes. The gly-phe-ala-leu based derivative proved to be a promising candidate for further biological evaluations.

Two different conjugates having Mitomycin C (MMC) bound to the side chain hydroxyl groups of poly[N5-(2-hydroxyethyl)-l-glutamine] (PHEG) via Gly-Phe-Gly and Gly-Phe-Ala-Leu spacers were compared to the free drug with respect to bioactivity, biocompatibility and immunogenicity. Qualitative as well as quantitative measurements of intracellular calcium fluctuations demonstrated that the macromolecular prodrugs are non-mitogenic for T-lymphocytes. The cytotoxicity towards mouse splenocytes was evaluated by [3H]thymidine incorporation. It was shown that polymeric MMC derivatives act as prodrugs of MMC: toxicity was due to hydrolytic release of the drug itself. Furthermore, attachment of the drug to the polymer considerably decreased its toxicity against haematopoietic precursors in bone marrow as measured by the in vivo colony-forming unit-spleen assay, as well as against reticulocytes in the peripheral blood. The results could be correlated with the hydrolytic stability of the polymeric derivatives. The conjugate bearing the slower releasing peptide spacer, Gly-Phe-Ala-Leu, had superior biological properties.

Poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG) prodrugs of the cytotoxic agent Mitomycin C were synthesized using peptidyl spacers to link the drug to the polymeric carrier. The influence on the length and detailed structure of the oligopeptide on the rate of drug release was investigated in buffer, in the presence of lysosomal enzymes (tritosomes, cathepsin B and D) and metalloprotease type IV collagenase. It was observed that tetra- and hexapeptide based conjugates generally release Mitomycin C (MMC) more effectively than tripeptide derivatives. The gly-phe-ala-leu conjugate released MMC very rapidly both in presence of lysosomal enzymes and collagenase IV. Only in the presence of the aspartic protease cathepsin D, the gly-phe-leu-gly-phe-leu derivative turned out to be a better substrate. In vivo studies against C26 solid tumour bearing mice suggest that PHEG-spacer-MMC conjugates act as prodrugs of MMC: antitumour efficacy of the macromolecular prodrugs was better than free MMC both in inhibition of tumour growth and increasing survival.

Conventional chemotherapy has been limited by a lack of cell specificity, resulting in unwanted peripheral toxicities and low therapeutic efficiency. In an attempt to overcome these problems, a variety of systems have been designed which are able to deliver the drug more effectively to the target site. Watersoluble macromolecular prodrugs, covalently linking the drug through a degradable spacer, constitute an interesting possibility. The linker plays a crucial role and must fulfil certain requirements in order to release the free parent drug in the vinicity of the tumor. This review will discuss some general principles related to the design of such polymeric drug carriers and an overview of the effect of the type of spacer on the rate of drug release and the correlated in vitro and in vivo results will be outlined.

Prodrugs of mitomycin C (MMC) based on soluble poly-[N-(2-hydroxyethyl)-L-glutamine] (pHEG) polymers have been evaluated as tumour-targeted drugs. These materials are designed to exploit the enhanced permeability of tumour vasculature, combining a passive tumour tropism with decreased systemic liberation of free MMC. A tri- or tetrapeptide linkage (e.g. Gly-Phe-Ala-Leu) between pHEG and the aziridine nitrogen of MMC can combine good hydrolytic stability with rapid cleavage by lysosomal enzymes, releasing free MMC. The conjugates showed decreased systemic toxicity and could be administered to mice at a total MMC dose of 15 mg/kg i.v., compared with just 6 mg/kg for free MMC. Conjugates also showed better activity against animal models of established tumours, achieving up to 77% increased life span (ILS) against solid P388 leukaemia, compared with only 23% for free MMC, and up to 121% ILS against solid C26 colorectal carcinoma, compared with no activity for the free drug. Improving the therapeutic index of anticancer drugs by combining tumour tropism with decreased systemic toxicity is a versatile approach that should produce a new generation of improved anticancer agents.

Poly-[N-(2-hydroxyethyl)-L-glutamine] (PHEG) prodrugs of the antitumour antibiotic mitomycin C (MMC) were synthesised using peptidyl spacers, tri- and tetrapeptides, to link the drug to the macromolecular carrier. The relationship between the length and composition of the oligopeptide spacer and the rate of drug release was studied by incubation in buffers, serum and in the presence of enzymes (lysosomal enzymes and collagenase IV). It was observed that tetrapeptide-based conjugates generally release MMC more effectively than tripeptide derivatives. Conjugates having a terminal glycine in the spacer are less stable to hydrolysis than those with a terminal hydrophobic amino acid both in buffer and in serum. The gly-phe-ala-leu conjugate released MMC very rapidly in the presence of both lysosomal enzymes and collagenase IV. Biological experiments indicate that PHEG-MMC conjugates act as prodrugs of MMC: cytotoxicity was observed after hydrolytic release of the active compound in vitro. In vivo studies of P388 solid tumour-bearing mice suggest that conjugates which release MMC slowly may be more effective in inhibiting tumour growth and prolonging animal lifespan. Preliminary in vivo bone marrow toxicity studies indicate that PHEG-MMC prodrugs are less myelosuppressive than free MMC.

Poly-α,β-[N-(2-hydroxyethyl)-d,l-aspartamide] (PHEA) and poly-α,β-[N-(2,3-dihydroxypropyl)-d,l- aspartamide] (PDHPA) are suitable as macromolecular drug carriers. In order to introduce amine- containing drug moieties onto these polymers, partial conversion of the hydroxyl side groups is required. This paper describes the 4-nitrophenyl chloroformate activation of PHEA and PDHPA. It is shown that, during the course of the activation of PHEA, only linear aromatic carbonate structures were formed. However, during the activation reaction of PDHPA, conversion of 4-nitrophenyl carbonates into cyclic carbonate structures could be observed. The relative amount of the two types of carbonate moieties could be controlled by addition of the appropriate catalyst. During the activation reaction of both polymers, the total content of carbonate groups could, for a given set of reaction conditions, be controlled by the amount of chloroformate added. The 4-nitrophenyl carbonate groups easily reacted with amines. However, the conversion of the cyclic carbonate moieties into urethane-bound derivatives was only possible with highly reactive amines. This could be an interesting property for the introduction of different types of amine- containing derivatives onto the polymer backbone. The results of this study demonstrate the feasibility of the described activation methods to prepare macromolecular prodrugs.

This paper describes the synthesis of poly[N5-(2-hydroxyethyl)-l-glutamine] (PHEG) by the aminolysis of poly(γ-benzyl-l-glutamate) (PBG) with 2-aminoethanol. The effects of the temperature and addition of the bifunctional catalyst, 2-hydroxypyridine (2-HP), on the kinetics of aminolysis were studied. The results suggest that the aminolysis reaction takes place much faster with increasing temperature and an increasing amount of added catalyst. Moreover, chain scission, due to aminolysis of the amide bonds of the polymer backbone, can be minimized by the addition of the appropriate amount of 2-hydroxypyridine. These data demonstrate the feasibility of the described method to prepare PHEG derivatives with well controlled molecular weights.

A series of polymeric prodrugs of Mitomycin C (MMC) were prepared by coupling the drug via a tri- or tetrapeptide spacer onto poly-[5N-(2-hydroxyethyl)-L-glutamine] (PHEG). In the first step MMC was linked to the peptide spacer. For preparing the oligopeptide-MMC derivatives N-Fmoc protected oligopeptide was activated as the pentafluorophenyl ester. After coupling with MMC the N-protective group can be removed under mild conditions. The spacer-MMC derivatives were finally coupled to 4-nitrophenyl chloroformate activated PHEG.