Future Directions in Polymer Conjugation

Although two of the approved PEGylated proteins described above resulted from random modification, it seems unlikely that this approach will yield viable biopharmaceuticals in many cases. Instead, site-specific PEGylation will be required for many candidate therapeutics. Assuming that structural information can be obtained for the target protein, engineering of surface-accessible cysteine residues is an attractive approach. For antibody fragments retaining the hinge cysteine residue, it has been demonstrated that modification of this residue with PEG-maleimide generates a product that retains high-affinity antigen binding and has prolonged half-life in vivo (180). PEGylation of Fab or scFv antibody fragments can exploit engineered cysteine mutants that have been selected for high expression and reactivity (181). It is expected that further refinements in PEGylation technology will yield additional options for cysteine modification. This will include other reactive functional groups on the PEG chain, greater variety in the length and branching of PEG chains, and less polydispersity in the average chain length. Such developments will enable fine-tuning of the phar-macokinetic/pharmacodynamic properties while ensuring a reliable product.

For proteins where site-specific modification is not possible or losses in activity result with this approach, releasable PEGs (182) may be a viable option. Such PEGs either use chemistry where the PEG is released slowly by hydrolysis under physiological conditions or employ a cleavable linker that is recognized by extracellular or intracellular enzymes. Releasable PEGs are particularly important for small-molecule drugs. Analogous to a prodrug strategy, the PEG is used for extending half-life, but the polymer interferes with activity. With the appropriate cleavable linker and upon uptake into cells, the PEG chain is removed by lysosomal enzymes and the small-molecule drug is rendered active. This approach has been used to increase the antitumor activity of doxorubicin (183).

The goal of improved drug exposure may be attained through slow-release formulations rather than covalent modification of the protein. Much work has been done to design biodegradable microspheres for slow release of therapeutic proteins. For example, Nutropin DepotĀ® consists of hGH contained in microspheres formed from poly-(D,L-lactide-coglycolide) copolymer. When implanted subcutaneously, hGH is slowly released, initially by diffusion followed by both diffusion and polymer degradation. Efficacious doses of hGH could be delivered via this method, which are comparable to what can be achieved with more frequent IV injections (184).

Alternative Polymers

An alternative posttranslational modification to increase half-life is to engineer additional glycosylation sites into a protein that is produced in mammalian cells (185). The serum half-life of human erythropoietin (EPO), which has 3 N-linked and 1 O-linked glycosylation sites, has been found to be proportional to the sialic acid-containing carbohydrate content of the molecule. Addition of 2 N-linked glycosylation sites to EPO resulted in a molecule (AranespĀ®, Amgen, Inc.) with threefold longer half-life than EPO. Less frequent dosing of Aranesp is required to achieve efficacy equal to EPO in anemic patients. The mechanism explaining the longer half-life of Aranesp is unclear; however, it is unlikely to result simply from a change in kidney clearance since the addition of two carbohydrate moieties does not have a significant effect on hydrodynamic volume.

Polymers of N-acetylneuraminic acid, polysialic acids, have shown potential for increasing the half-life of proteins (186). Like PEG, polysialic acids are built from repeating units, are hydrophilic, can be produced in varied chain lengths, and can be activated at one end for coupling to proteins. Since polysialic acids are built from natural modules, they appear to be nonimmunogenic. Unlike PEG, polysialic acids are biodegradable, so conjugates may have improved safety profiles. Polysialylated proteins have been shown to have improved serum half-life; however, the flexibility in chain length and chemistry of attachment is not yet on par with PEGylation. In addition, since sialic acid has a negative charge, polysialylation will add negative charge to the protein. For each target protein, the consequence of increased negative charge will need to be examined.

Results from modification of EPO and G-CSF with hydroxyethyl starch (HESylation) have been recently reported (PEGS meeting, 2008). Hydroxyethyl starch is a blood plasma substitute, is well tolerated in humans, has low immunogenicity, and is biodegradable. HESylated EPO and G-CSF were shown to have pharmacodynamic properties similar to the PEGylated versions of these molecules. Utility of these HESylated molecules and potential for additional applications of HESylation await further research.

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