Three types of copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared which contain oligopeptide sequences: a) HPMA copolymers containing oligopeptide sidechains terminated with p-nitroaniline; b) soluble HPMA copolymers containing oligopeptide sequences in crosslinks connecting two poly(HPMA) chains; c) a hydrophilic gel, i. e. a threedimensional copolymer of HPMA containing an oligopeptide sequence in the crosslinks. These polymeric substrates (suitable as drug carriers) containing potentially degradable oligopeptide sequences were incubated with an intracellular proteolytic enzyme, bovine spleen cathepsin B. The degradation process of the substrates made it possible to reveal the relationship between the structure of oligopeptide sequences and their degradability. The results suggest an important role played by cathepsin B in the degradation of polymeric substrates investigated in this study under physiological conditions.
N-(2-Hydroxypropyl)methacrylamide copolymers are considered to be a potential drug delivery system. To fulfil this role the drug-polymer linkage must be susceptible to intralysosomal hydrolysis. Taking p-nitroaniline as a drug analogue, copolymers were synthesized bearing oligopeptidyl-p-nitroanilide side-chains designed to match known specificities of the lysosomal enzymes cathepsin L or cathepsin D. Degradation of side-chains by rat liver lysosomal enzymes (measured by monitoring terminal p-nitroaniline release) occurred only in the presence of reduced glutathione (5 mmol/l) and was effectively inhibited by leupeptin, indicating the involvement of thiol-proteinases in every case. Depending on side-chain composition, between 20 and more than 50% of the terminal p-nitroaniline residues were liberated during a 5 h incubation. It has also been shown that 1) a polymer molecule may contain side-chains of a single type that are nevertheless differentially susceptible to lysosomal hydrolysis; 2) two of the side-chains studied liberate only a p-nitroaniline residue, whereas the others also release aminoacyl-p-nitroanilides; 3) the cleavage of all side-chains displays a broad pH optimum pH 5 to pH 7; 4) the Michaelis-Menten constant K , for side-chain cleavage varied between 26,l and 143,2 mg/ml, depending on the amino acid sequence of the side-chain. a) Part 6, cf. Biomaterials 3, 150 (1982). b,
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