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Timestamp: 2019-04-20 19:11:01+00:00

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PEGylation or covalent attachment of poly(ethylene glycol) improves the pharmacokinetic properties of protein drugs. In vivo circulation lifetimes are increased and dosages are decreased, resulting in improved patient quality of life. PEG may be attached to proteins using a variety of different chemical reactions. This review discusses currently available FDA-approved PEGylated protein drugs, their intended use and target, and the PEG attachment chemistry utilized.
Steevens N. S. Alconcel received a BS in Chemistry in 2007 from the University of Southern California, where he worked with Professor G. K. Surya Prakash on modification of Strecker and Mitsunobu reactions to synthesize fluorinated analogues. He is currently working toward his PhD in organic chemistry at the University of California, Los Angeles. Steevens is a trainee in the NIH-sponsored Chemistry–Biology Interface Training Program. His research focuses on the synthesis of protein–polymer conjugates for drug delivery.
Arnold S. Baas received his BA from Baylor University in 1985 and his MD from UT Southwestern in 1989. He was resident, followed by chief resident at the University of Michigan before training as a fellow in cardiology at the University of Washington. Baas was a clinical assistant professor there before moving to UCLA to the David Geffen School of Medicine and Division of Cardiology. Baas' interests are focused on general cardiology, cardiac transplantation, mechanical circulatory support, cardiomyopathy, atherosclerosis, cardiac rehabilitation, and cardiac imaging.
Heather D. Maynard received her BS degree from UNC-Chapel Hill in chemistry and an MS from UCSB in Materials Science. Maynard's PhD in chemistry was awarded in 2000 from Caltech, and she was an American Chemical Society postdoctoral fellow at the ETH Switzerland from 2000–2002. In 2002, Maynard moved to UCLA where she is now an Associate Professor in the Department of Chemistry and Biochemistry. Her interests include polymer synthesis, biohybrid materials, and surface modification.
PEGylation, or covalent attachment of poly(ethylene glycol) (PEG) to proteins, was introduced by Abuchowski and coworkers in 1977.1 They reported on the superior immunogenic properties of bovine serum albumin (BSA)–PEG conjugates compared to the unmodified biomolecule. In later work Abuchowski and coworkers were able to demonstrate that PEGylation resulted in enhanced circulation lifetimes, as well as reduced immunogenicities relative to native proteins.2 Since that time, many researchers have demonstrated that PEGylation improves pharmacological properties of proteins. As a result, several FDA-approved protein–polymer drugs currently exist, and many others are currently being studied in clinical trials.3 This review focuses on the synthesis and mode of action of protein–PEG conjugates that are already FDA-approved for use to treat diseases in humans.
The main objective of using polymers in drug delivery is to stabilize and improve the therapeutic activity of the standalone drug.4–6 The non-ionic, hydrophilic PEG provides a steric shield for conjugates from recognition by the patient's immune system and effectively increases the size of the biomolecule,7 thereby reducing clearance from the bloodstream.8 By increasing the half-life of the protein drug, the dosage frequency is reduced.9 Since most protein drugs need to be injected, this result is significant. Increased duration of pharmacological activity, reduction of toxic side effects, and increased quality of life due to controlled, timed release are several positive results attributed to PEGylation. The detailed pharmacokinetics of PEGylated drugs have been recently reviewed.10 The dosage schedule and biological activity for each of the FDA-approved protein-PEG drugs are described in this review.
PEGylation may also decrease parent drug activity by changing conformation, sterically interfering, and altering electrostatic binding properties.3 Notably, the shape and number of PEG chains conjugated to a protein affects the stability and efficacy of the drug conjugate. Chain location is also critical to activity, as nonspecific PEGylation near an active site or to a region of a protein that causes a conformational change deleteriously affects the activity of the intended drug.9 As of today, there are well-established methods of PEGylation available,7 with a large array of different linking chemistries used.3,4,6,7,11,12 There has been a lot of recent effort placed on strategies that result in the site-specific conjugation.13 However, for many FDA-approved drugs, non-specific attachment strategies are used; these are described below and summarized in Table 1.
Initially, PEG was synthesized by reacting mPEG (monomethoxy-PEG) of molecular weight (Mw) 5000 with cyanuric chloride, forming PEG dichlorotriazine. In this approach, one of the two remaining chlorines on PEG dichlorotriazine is displaced by nucleophilic amino acid units such as lysine, serine, tyrosine, cysteine, and histidine. The remaining chlorine is not as electrophilic, but may react to cause crosslinking of the protein. This is a nonspecific attachment process so multiple units of PEG are attached to the protein. Depending on the pH of the buffer used in the reaction, a small measure of control can be obtained to conjugate to specific nucleophilic amino acid units.1,14 This PEG was utilized at first by the company to study the drug, but is not what is used in the FDA-approved formulation.
Oncaspar® (mPEG-L-asparaginase). Oncaspar (pegaspargase) by Enzon is classified as an antineoplastic drug used to treat acute lymphoblastic leukemia and was approved in 1994.17–20 It consists of a PEGylated L-asparaginase which is responsible for the hydrolysis of asparagine to aspartic acid. L-Asparaginase depletes asparagine, an amino acid essential for tumor growth. Native L-asparaginase18,21 has been shown to cause hypersensitivity reactions and shock. Oncaspar is an improvement upon the native enzyme. Phase I studies with PEGylated L-asparaginase showed increased serum half-life (357 h) relative to the native enzyme (20 h) with fewer cases of hypersensitivity.22 The drug can be delivered either intramuscularly or intravenously. The recommended dose is 2500 IU m−2 and should be administered at the most every 14 days. If the volume administered is greater than 2 mL for intramuscular administration, multiple injection sites must be used.
Fig. 1 Synthesis of the branched PEG used to prepare PEGASYS.
Fig. 2 Synthesis of Neulasta by imine formation and reductive amination with sodium cyanoborohydride.
Covalent attachment of PEG to proteins produces conjugates with significantly improved pharmacokinetic properties compared to the unmodified proteins. Specifically, PEG reduces clearance, slows enzymatic degradation and provides an effective shield from the immune system. Proteins must be administered by subcutaneous or intravenous injection, and longer half-lives lead to fewer injections. As a result there are a number of FDA-approved PEG–protein conjugates that have been reviewed herein with many other PEGylated drugs currently in phase I, II, and III trials that were not described.3,6,43 There are several chemistries that are utilized in FDA-approved PEG drugs, including amidation, reductive amination, and Michael addition, although most target multiple residues.
Nonspecific reaction of PEG with proteins leads to heterogeneous mixtures of multiple PEGs attached at different sites. This can lead to reductions in protein bioactivity. As a result, there has been a lot of effort placed on developing specific modification chemistries. An obvious choice is covalent attachment to free cysteines, as described above for Cimzia. The free cysteines can be naturally occurring or recombinantly placed away from the active site of a protein. PEGs with activated disulfide, maleimide, and vinyl sulfone end groups have been utilized for this purpose. In addition, proteins have either been modified chemically or produced recombinantly with artificial amino acids to present groups that react chemoselectively with PEGs.67,68 The field of incorporating a non-canonical amino acid and subsequent modification with PEG is still in the early stages of investigation. However, examples of chemistries that have been utilized are oxime bond formation and azide/alkyne click chemistries.69–71 These strategies produce homogeneous conjugates that retain bioactivity and are likely to play an increasing role in the protein–drug area.
PEG is classified to be Generally Regarded As Safe (GRAS) by the FDA and has been shown to be removed by renal clearance. Human studies show urinary excretion of PEG units up to 20 kDa, with up to 190 kDa PEG seen excreted in mouse and rat studies.3,66 However, there are potential limitations of PEG including its non-degradability in the body, and these have been recently reviewed.3 It has been discussed that PEG of molecular weights below 40–60 kDa are required to minimize accumulation in the liver72 making it advantageous to utilize smaller branched PEGs, cleavable by hydrolysis, instead of larger, nondegradable linear PEGs to form the conjugates. Branched PEG chains may provide improved shielding from the immune system, reducing immunogenicity and antigenicity further, thus minimizing the size of the PEG required. In recent years controlled radical polymerization (CRP) techniques have been increasingly utilized to synthesize these branched PEG polymers. In particular, atom transfer radical polymerization (ATRP)73–75 and reversible addition–fragmentation chain transfer (RAFT) polymerization76–78 have been employed to make end chain reactive branched PEGs12,79,80 and to form bioconjugates directly by polymerization from modified proteins.81,82 In fact, recent studies have exploited the latter approach to polymerize branched poly(ethylene glycol) methyl ether methacrylate (PEGMA) in situ from therapeutic proteins such as recombinant human growth hormone (rh-GH)83 and an intein fusion protein.84 Importantly, this grafting from approach retains protein activity in many cases and eliminates the need for postpolymerization conjugation. These strategies lead to conjugates that in some cases are superior to their PEGylated analogs.85 Recent reviews have summarized CRP approaches to bioconjugation.3,79,80,86 It is anticipated that CRPs will be increasingly utilized in the future to synthesize protein–polymer drugs.
The authors appreciate the National Institutes of Health (NCI R21 CA 137506-02) for funding. S.N.S.A. thanks the NIH sponsored Chemistry and Biology Interface (CBI) Training Program (2T32GM008496-16) for a fellowship.
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