Another option is to use the thiol groups of
Another option is to use the thiol groups of cysteine residues, which can be modified by use of PEG-maleimide and vinyl sulfone. However, changes in PEGylation interactions or reaction conditions can result in changes in the functional properties of the therapeutic proteins [, , ].
A study was conducted to optimize site-specific PEGylation of Exendin-4 (Ex4-Cys), an analogue of glucagon-like peptide-1 (GLP-1) with anti-diabetic properties, using a high-molecular-weight trimeric PEG. PEGylation of the C-terminus (C40-tPEG-Ex4-Cys) was carried out using Ex4-Cys and activated trimeric PEG. The resulting C40-tPEG50K-Ex4-Cys derivative had a better t1/2 in circulating blood (7.53-fold increase) and its AUCinf (a measure of total exposure to the drug) relative to Ex4-Cys was increased over 45-fold. Further, its hypoglycemic duration, a measure of its pharmacologic activity, was increased 8-fold relative to that of native Ex4-Cys, with a dose of 25 nM/kg .
Fusion to human serum albumin Human serum albumin (HSA) is one of the best-characterized proteins in the pharmaceutical field. It is responsible for transporting endogenous and exogenous compounds and has a long average half-life (around 19 days). In part, this is due to its size – it is around 66 kDa, which is almost at the boundary of the kidney’s lasofoxifene capacity – and also the fact that it is the most abundant protein in plasma. It tends to accumulate around tumors and inflamed tissues sites, and this feature opens the potential of fusing albumin to a target protein to aid targeting to the therapeutic site of interest . It is widely used as an excipient, especially for biotechnology products. Recombinant versions of the protein are available, which alleviate any potential concerns about the transmission of infectious agents associated with the human plasma-derived protein . Many researchers have developed methods to improve novel albumin-based drug carriers and these can generally be categorized into three main categories: (1) low-molecular-weight proteins fused with albumin; (2) polymerization; (3) surface modification (Fig. 2) . I has recently emerged as an adaptable carrier for drug delivery to transport therapeutic peptides and proteins against diabetes, cancer, and infectious diseases . Therapeutic compounds have been pharmaceutically enhanced by multiple techniques using albumin to improve their distribution, bioavailability and the half-life. For example, non-covalent interactions allow the binding of the albumin to a broad range of endogenous and exogenous ligands. Albumin dimerization in particular has significant potential and advantages for clinical applications, as both a plasma expander and as a drug carrier. Such dimers are present at elevated levels in the circulating blood of patients with chronic renal disease and also result from oxidative damage in the blood . Many molecules of therapeutic interest bind to endogenous albumin in the blood through its fatty acid binding sites, thereby prolonging their half-life and bioavailability. For example, the human insulin analogue, Detemir (marketed by Novo Nordisk as Levemir), is long-acting due to the myristic acid moiety bound to the Lys residue at position B29 of insulin. The attached fatty acid facilitates binding to albumin thereby prolonging the circulatory half-life of this insulin derivative in blood [57,58]. Covalent binding of a drug to albumin can be achieved either through direct chemical conjugation or via the use of a small molecule to link the two components. Alternatively, it can be achieved through gene fusion to create a chimeric protein that is expressed in a suitable host, resulting in the production of a single polypeptide . The gene fusion approach has been used to attach albumin to the N- and or C-termini of several proteins of therapeutic interest, to extend their half-life. Examples of therapeutic proteins that have been attached to HSA include interferons , growth factors , hormones, cytokines, coagulation factors , and antibody fragments .