The Lesson Learned from Pegylated Proteins

The inspiration and motivation to start working with pegylated lipids like PEG-DSPE came to Annie Yau-Yang and others at LTI from pioneering research in the 1970s by Frank Davis, Abraham Abuchowski, and colleagues who foresaw the potential of the conjugation of polyethylene glycol (PEG) to proteins (Abuchowski et al., 1977; Abuchowski founded Enzon, Inc., which brought three pegylated drugs to the market. Various length (>350-50,000 Da) chains of PEG polymer are available. Low-molecular-weight drugs were also pegylated. However, the main pegylated products so far are a few proteins and one liposomal formulation — Doxil® (the topic of this review article). For peptides and proteins (including antibody fragments), mainly relatively large PEG polymers of >5000 Da were used. It was found that pegylation helps improve safety and efficacy as well as reduce the immunogenicity of many therapeutics (Veronese and Harries, 2002; Veronese and Pasut, 2005). The suggested mechanism by which pegylation works is that it is a result of the alterations it produces in the physicochemical properties of the molecule to which the PEG residue is covalently attached. These may include changes in level of hydration, conformation, electrostatic binding, and hydrophobicity/hydrophilicity balance. Covalently attached highly flexible and highly hydratedhydrated PEG [three to four molecules of water per 1 ethylene oxide oxygen (Tirosh et al., 1997,1998)] induce changes in structure and lead to increase in size and bulkiness. All together this results in "steric stabilization,” which reduces nonspecific protein-protein interaction and nonspecific protein-cell interactions. These physical and chemical changes increase systemic retention of the therapeutic agent. Also, it can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.

PEG polymer has only two reactive OH groups (one at each end of the PEG molecule); in order to prevent the PEG from inducing intra- and inter-cross linkages, one of these hydroxyl groups was methylated.

Pegylation, by increasing the molecular weight of a molecule and its level of hydration, can impart several significant pharmacological advantages over the native unmodified molecule, such as

  • • Improved agent solubility
  • • Extended circulating life
  • • Therefore, reducing dosage frequency, without diminished efficacy with potentially reduced toxicity
  • • Increased drug stability
  • • Enhanced protection from proteolytic degradation
  • • Reduced immunogenicity

In addition, pegylated drugs may have the following commercial advantages:

  • • Opportunities for new delivery formats and dosing regimens
  • • Extended patent life of previously approved drugs

This resulted in the development of many pegylated proteins and peptide drugs, (only a few of which made it to the market), so now the clinical value of pegylation is well established. ADAGEN (PEG-bovine adenosine deaminase), manufactured by Enzon Pharmaceuticals,

Inc., was the first pegylated protein (approved by the FDA in March 1990) to enter the market. It is used to treat X-chromosome-linked severe combined immunogenicity syndrome, as an alternative to bone marrow transplantation and enzyme replacement by gene therapy. Now a large number of pegylated protein and peptide pharmaceuticals are in clinical use. The most successful examples are

• PEGASYS: Pegylated interferon alpha for use in the treatment of chronic hepatitis C and hepatitis В (Hoffmann-La Roche)

  • Pegintron: Pegylated interferon alpha for use in the treatment of chronic hepatitis C and hepatitis В (Schering-Plough/ Enzon)
  • Oncaspar: Pegylated L-asparaginase for the treatment of acute lymphoblastic leukemia in patients who are hypersensitive to the native unmodified form of L-asparaginase (Enzon). This drug was recently approved as a first-line treatment
  • Neulasta: Pegylated recombinant methionyl human granulocyte colony-stimulating factor for severe cancer- chemotherapy-induced neutropenia (Amgen)

Many other pegylated peptides and proteins are now in clinical trials or under development.

The success of pegylated proteins was the driving force for the successful development of Doxil® as the first FDA-approved liposomal drug and nano-drug (November 17,1995). In lipids, PEG chains of >350 to 15,000 were tried (equivalent to 8 to 334 ethylene oxide units) and various considerations, such as the metabolism of the pegylated lipids and the rate of secretion via the kidneys were used in the decision which PEG length to select for lipid pegylation.

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