As we enter the twenty-first century, research at the interface of polymer chemistry and the biomedical sciences has given rise to the first nano-sized (5-100 nm) polymer-based pharmaceuticals, the 'polymer therapeutics'. Polymer therapeutics include rationally designed macromolecular drugs, polymer-drug and polymer-protein conjugates, polymeric micelles containing covalently bound drug, and polyplexes for DNA delivery. The successful clinical application of polymer-protein conjugates, and promising clinical results arising from trials with polymer-anticancer-drug conjugates, bode well for the future design and development of the ever more sophisticated bio-nanotechnologies that are needed to realize the full potential of the post-genomic age.

The dawning era of polymer therapeutics

Alloxan and streptozotocin are widely used to induce experimental diabetes in animals. The mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood. The cytotoxic action of both these diabetogenic agents is mediated by reactive oxygen species, however, the source of their generation is different in the case of alloxan and streptozotocin. Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed by the Fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis.

/pdf/the-mechanism-of-alloxan-and-streptozotocin-action-in-b-331fni3xal.pdf

The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas.

Review article: free radicals, antioxidants and human disease: where are we now?

Free radicals, antioxidants, and human disease: where are we now?

https://www.farm.ucl.ac.be/FARM2129/2008-2009/Tulkens/anemie-neutropenie/Veronese-Pegylation-Drug-Discovery-Today-2005-10-1451-1458.pdf

PEGylation, successful approach to drug delivery.

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-nitroanilide 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 amino-acyl-p-nitroanilides; 3) the cleavage of all side-chains displays a broad pH optimum pH 5 to pH 7; 4) the Michaelis-Menten constant Km for side-chain cleavage varied between 26,1 and 143,2 mg/ml, depending on the amino acid sequence of the side-chain.

Polymers containing enzymatically degradable bonds, 7. Design of oligopeptide side-chains in poly[N-(2-hydroxypropyl)methacrylamide] copolymers to promote efficient degradation by lysosomal enzymes†

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

Evidence that desferrioxamine cannot enter cells by passive diffusion.

Desferrioxamine and CP94 (1,2-diethyl-3-hydroxypyridin-4-one) are metal chelators used or proposed for use in the clinical treatment of iron overload. Recent data on their capacity to deplete intracellular iron led to the conjecture that the differences observed arose from the different membrane-penetration properties of the two compounds. The time-course of accumulation and subsequent release of [14C]CP94 by the rat visceral yolk sac in-vitro was compared with that of [14C]desferrioxamine and for 125I-labelled poly(vinylpyrrolidone), a marker for fluid-phase endocytosis. The results indicate that [14C]CP94 crosses the plasma and lysosome membranes rapidly whereas [14C]desferrioxamine and 125I-labelled poly(vinylpyrrolidone) are effectively incapable of crossing these membranes, entering cells only by endocytosis. It is concluded that although CP94 readily enters and leaves cells, desferrioxamine has the potential to accumulate to high concentration in the lysosomes and complex with intralysosomal iron. The results support and extend the proposed correlation between pharmacological activity and capacity for membrane penetration.

Cellular Uptake and Release of Two Contrasting Iron Chelators

N-(2-Hydroxypropyl)methacrylamide copolymers bearing oligopeptide side-chains terminating in p-nitroaniline (NAp) were incubated with rat liver lysosomal enzymes in the presence of the thiol glutathione, and the rate of p-nitroaniline release was measured. Twelve of the 16 side-chains investigated were hydrolysed to release p-nitroaniline and in all but one case degradation was partially or totally inhibited by leupeptin. The effect of substrate concentration on the degradation of the most readily cleaved side-chain, -Ala-Gly-Val-Phe-NAp, was measured.

H.C. Cable

Papers

Polymers containing enzymatically degradable bonds, 7. Design of oligopeptide side-chains in poly[N-(2-hydroxypropyl)methacrylamide] copolymers to promote efficient degradation by lysosomal enzymes†

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro

Evidence that desferrioxamine cannot enter cells by passive diffusion.

Cellular Uptake and Release of Two Contrasting Iron Chelators

Degradation of side-chains of N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal thiol-proteinases