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Advanced Drug Delivery Reviews (v.60, #1)
Peptide and protein PEGylation III: advances in chemistry and clinical applications
by Francesco M. Veronese (Theme Editors); J. Milton Harris (Theme Editor) (pp. 1-2).
Disulfide bridge based PEGylation of proteins by Steve Brocchini; Antony Godwin; Sibu Balan; Ji-won Choi; Mire Zloh; Sunil Shaunak (pp. 3-12).
PEGylation is a clinically proven strategy for increasing the therapeutic efficacy of protein-based medicines. Our approach to site-specific PEGylation exploits the thiol selective chemistry of the two cysteine sulfur atoms from an accessible disulfide. It involves two key steps: (1) disulfide reduction to release the two cystine thiols, and (2) bis-alkylation to give a three-carbon bridge to which PEG is covalently attached. During this process, irreversible denaturation of the protein does not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using α,β-unsaturated-β′-mono-sulfone functionalized PEG reagents. The combination of: — (a) maintaining the protein's tertiary structure after reduction of a disulfide, (b) bis-thiol selectivity of the PEG reagent, and (c) PEG associated steric shielding ensure that only one PEG molecule is conjugated at each disulfide. Our studies have shown that peptides, proteins, enzymes and antibody fragments can be site-specifically PEGylated using a native and accessible disulfide without destroying the molecules' tertiary structure or abolishing its biological activity. As the stoichiometric efficiency of our approach also enables recycling of any unreacted protein, it offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines.
Keywords: Site-specific; PEGylation; Protein disulfide bonds; Therapeutic proteins
Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase by Angelo Fontana; Barbara Spolaore; Anna Mero; Francesco M. Veronese (pp. 13-28).
Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the γ-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new γ-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH2) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them α-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor ( B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.
Keywords: Transglutaminase; Protein modification; PEGylation; Proteolysis; Protein drugs; Disordered proteins; B; -factor; Protein recognition; Enzymes
Releasable PEGylation of proteins with customized linkers by David Filpula; Hong Zhao (pp. 29-49).
Releasable PEGylation employs customized linkers that reversibly tether a therapeutic moiety with polyethylene glycol polymers. The choice of releasable PEG linkers may have numerous applications that are insufficiently addressed by stable polymer attachment. Releasable PEGylation provides regeneration of authentic and fully active drug and allows tailored design of critical pharmacological parameters such as the maximal drug concentration and total drug exposure. This provides a prodrug format that combines beneficial attributes of PEGylation with controlled release. The linker release mechanisms are shown to be kinetically controlled by the design of a hydrolytically labile center and side chains for the steric modulation of the intramolecular elimination reactions and linker self-immolation. Recent reports have described both aromatic and aliphatic based customized linkers that release the unaltered original drug under physiological conditions and at therapeutically useful release rates. These studies have examined bioconjugates of cytokines, peptide hormones, immunotoxins, enzymes, and reporter proteins.
Keywords: Reversible PEGylation; Customized PEG; Prodrug; Peptide; Protein therapeutics; Poly(ethylene glycol)
Formulation of Neulasta® (pegfilgrastim) by Deirdre Murphy Piedmonte; Michael J. Treuheit (pp. 50-58).
Neulasta® (pegfilgrastim) is a PEGylated version of its parent molecule NEUPOGEN® (Filgrastim). This work describes the formulation development for Neulasta® (pegfilgrastim), and the analytical techniques used to monitor degradation during these studies. Stability was assessed as a function of pH, protein concentration, buffer type, tonicity modifiers and surfactant concentration under both accelerated conditions and quiescent long-term storage. The stability of Neulasta® (pegfilgrastim) to agitation and successive freeze–thaw cycles was also assessed. Neulasta® (pegfilgrastim), at a protein concentration of 10 mg/mL formulated in 10 mM acetate, pH 4.0 with 5% sorbitol and 0.004% polysorbate 20, is stable for two years stored at 2–8 °C.
Keywords: Abbreviations; Recombinant; methionyl human G-CSF (r-metHuG-CSF); poly(ethylene glycol) (PEG); sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); Brain-Derived Neurotrophic Factor (BDNF); Megakaryocyte Growth and Development Factor (MGDF); size exclusion high performance liquid chromatography (SE-HPLC); cation exchange chromatography (CEX); RP-HPLC (reversed-phase liquid chromatography)r-metHuG-CSF; Neulasta®; (pegfilgrastim); Formulation; PEGylation
PEG-uricase in the management of treatment-resistant gout and hyperuricemia by Merry R. Sherman; Mark G.P. Saifer; Fernando Perez-Ruiz (pp. 59-68).
Hyperuricemia results from an imbalance between the rates of production and excretion of uric acid. Longstanding hyperuricemia can lead to gout, which is characterized by the deposition of monosodium urate monohydrate crystals in the joints and periarticular structures. Because such deposits are resolved very slowly by lowering plasma urate with available drugs or other measures, the symptoms of gout may become chronic. Persistent hyperuricemia may also increase the risk of renal and cardiovascular diseases. Unlike most mammals, humans lack the enzyme uricase (urate oxidase) that catalyzes the oxidation of uric acid to a more soluble product. This review describes the development of a poly(ethylene glycol) (PEG) conjugate of recombinant porcine-like uricase with which a substantial and persistent reduction of plasma urate concentrations has been demonstrated in a Phase 2 clinical trial. Two ongoing Phase 3 clinical trials include systematic assessments of gout symptoms, tophus resolution and quality of life, in addition to the primary endpoint of reduced plasma urate concentration.
Keywords: Poly(ethylene glycol); Urate oxidase; Therapeutic enzyme; Gout; Hyperuricemia
Anti-cancer PEG-enzymes: 30 years old, but still a current approach by Gianfranco Pasut; Mauro Sergi; Francesco M. Veronese (pp. 69-78).
PEGylation (i.e. the covalent link of PEG strands) is a well known technique used to improve pharmaceutical properties of bioactive proteins and peptides. Even in cancer therapy some proteins, in particular enzymes, can find many applications, because of their antiproliferative action or ability to reduce side effects of chemotherapies, but to do so they need to be properly formulated. Unfortunately, formulation alone can not fulfil all the requirements to yield a safe and successful protein preparation for therapeutic applications. In particular, for many proteins fast clearance from the body and potential immunogenicity are severe limitations, which can not be easily overcome without taking into consideration a purposely designed drug delivery system. Among the approaches in the field of drug delivery, PEGylation has so far been the best choice for protein delivery.Here, we describe some examples of PEGylated enzymes useful in antitumoral therapies and the most recent advances in this field.
Keywords: Anti-cancer enzymes; Asparaginase; Methioninase; Arginine deiminase; Arginase; Uricase
PEGylation of cyanovirin–N, an entry inhibitor of HIV by H. Zappe; M.E. Snell; M.J. Bossard (pp. 79-87).
Cyanovirin–N (CV–N) is a potent inhibitor of human immunodeficiency virus and many other viruses. It has a high potential for use as a systemic compound to control viral load or in the development of microbicides to prevent primary viral infection. Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pharmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Several strategies were used to covalently bond poly(ethylene glycol) (PEGylate) to CV–N. Random PEGylation at lysine residues resulted in poor retention of antiviral activity. Many site-directed mutants were made to test site-specific PEGylation. One mutant, where glutamine 62 was replaced with cysteine (CV–N(Q62C)) and PEGylated with maleimide activated PEG, retained significant anti-HIV activity in vitro.
Keywords: Abbreviations; IPTG; isopropyl-β-D-thiogalactopyranoside; SDS–PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; Na-acetate; sodium acetate; AZT; azidothymidine; EDTA; ethylenediaminetetra-acetic acid; HPLC; high pressure liquid chromatography; MALDI-TOF-MS; matrix-assisted laser desorption ionization – time of flight mass spectrometry; DC-SIGN; dendritic cell-specific ICAM-3 receptor/HIV-1-binding protein; MMR; macrophage mannose receptor; PEG; polyethylene glycol; HIV; human immunodeficiency virus; SIV; simian immunodeficiency virus; FIV; feline immunodeficiency virus.PEG; CV–N; PEGylated-CV–N; Cyanovirin–N