Reversible PEGylation

Reversible PEGylation reactions involving 1,4- or 1,6- benzyl elimination (BE) and bicine linkers are well-established strategies for releasable PEGylation of small drugs, 

As expected, the rate of release can be modulated by modification of the heteroatom that brings together the polymers and the linker (X in compounds 53 and 54), by introduction of additional side chains, or by increasing the steric hindrance. Related to mPEG 3, bicine linkers 53 are particularly interesting and useful structures, due to the well-known advantages that have been described previously for branched mPEG polymers. 

Thioester 59 has recently been introduced for reversible protein PEGylation [221], Carbamate linkage formation with the target protein affords conjugate 60 (Equation 3.30), which imder reductive conditions decomposes to 61, ultimately giving the free protein (Equation 3.31). 

Disulfide bridges can be cleaved under reductive conditions or by sulfide exchange reaction. Thus, this simple concept has successfully been exploited for reversible PEGylation. The chemistry that supports this approach, using conjugate 

62 as an example, is presented in Equation 3.32. Upon reduction of the disulfide bridge of 62 (by a cysteine amino acid, or any other entity containing a -SH group, such as glutatione), intermediate sulfide 63 is obtained. 1,6-Elimination from 63 delivers the unmodified native protein and sulfur product 64 (Equation 3.32) [222,223]. 

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Fig. 1 PEGylation technologies. Nonspecific and irreversible PEGylation is associated with several limitations, such as altered drug properties. To overcome this problem, a new generation of PEGylation technologies that enable highly specific and reversible PEGylation have been developed...

Peleg-Shulman T, Tsubery H, Mironchik M, Fridkin M, Schreiber G, Shechter Y (2004) Reversible PEGylation a novel technology to release native interferon alpha2 over a prolonged time period. J Med Chem 47 4897 1904... 

Scheme 3.10 Bicine linkers used in reversible PEGylation.

Fluorenyl-based reagent 58 has been used for the attachment of mPEG of up to 40 kDa to proteins by means of urethane bond formation (Scheme 3.11). The fluorenyl moiety releases the native unmodified protein at a slow controlled rate by hydrolysis reaction, as is shown in Equation 3.29 [216]. Reagent 58 has been used in the reversible PEGylation of exendine-4 [216], human growth hormone, interferon a2 [217], enkephalin [218], atrial natriuretic peptide [219] and insulin [220]. 

Scheme 3.11 Fluorenyl-based reagent for reversible PEGylation.

M. Nesher, Y. Vachutinsky, G. Fridkin, Y. Schwarz, M. Fridkin, Y. Shechter, D. Lichtstein, Reversible PEGylation prolongs the hypotensive effect of atrial natriuretic peptide. Bioconjugate Chem., 19 (1) 342-348, 2008. 

Y. Shechter, M. Mironchik, S. Rubinraut, H. Tsubery, K. Sasson, Y. Marcus, M. Fridkin, Reversible PEGylation of insulin facilitates its prolonged action in vivo. Eur. J. Pharm. 

S. Zalipsky, N. Mullah, C. Engbers, M.U. Hutchins, R. Kiwan, Thiolytically cleavable dithiobenzyl urethane-linked polymer-protein conjugates as macromolecular prodrugs reversible PEGylation of proteins. Bioconjugate Chem., 18 (6) 1869-1878, 2007. 

Neutropenia associated with interferon or pegylated interferon therapy is defined as an absolute neutrophil count (ANC) of less than 1000 cells/mm3 in rare cases, an ANC less than 500 cells/mm3 maybe observed. The neutropenia is more common and in some cases more severe with pegylated interferon than with unmodified interferon. Neutropenia usually occurs within the first 2 weeks after initiating either formulation of interferon, with the WBC count stabilizing by week four or six. The neutropenia is reversible upon discontinuing therapy. Granulocyte colony-stimulating factor has been used as an adjunctive therapy for interferon-induced neutropenia in hepatitis patients.44... 

Nie Y, Gunther M, Gu Z, Wagner E (2011) Pyridylhydrazone-based PEGylation for pH-reversible lipopolyplex shielding. Biomaterials 32 858-869... 

The formation of liposomes [or better arsonoliposomes (ARSL)], composed solely of arsonolipids (Ars with R=lauric acid (C12) myristic acid (C14) palmitic acid (C16) and stearic acid (C18) (Fig. 1) have been used for ARSL construction), mixed or not with cholesterol (Choi) (plain ARSL), or composed of mixtures of Ars and phospholipids (as phosphatidylcholine [PC] or l,2-distearoyl- -glyceroyl-PC [DSPC]) and containing or not Choi (mixed ARSL), was not an easy task. Several liposome preparation techniques (thin-film hydration, sonication, reversed phase evaporation, etc.) were initially tested, but were not successful to form vesicles. Thereby a modification of the so called one step or bubble technique (8), in which the lipids (in powder form) are mixed at high temperature with the aqueous medium, for an extended period of time, was developed. This technique was successfiil for the preparation of arsonoliposomes (plain and mixed) (9). If followed by probe sonication, smaller vesicles (compared to those formed without any sonication [non-sonicated]) could be formed [sonicated ARSL] (9). Additionally, sonicated PEGylated ARSL (ARSL that contain polyethyleneglycol [PEG]-conjugated phospholipids in their lipid bilayers) were prepared by the same modified one-step technique followed by sonication (10). 

J. Chen, M. Zhao, F. Feng, A. Sizovs, and J. Wang, Tunable thioesters as reduction responsive functionality for traceless reversible protein PEGylation, J Am Chem Soc, 135 (30), 10938-41,2013. 

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