Active pharmaceutical ingredients (APIs) based on synthetic peptides are recognized as potent and selective drugs. Endogenous peptides such as peptide hormones are involved in complex biological processes in a specific and precise manner. The in vivo bioavailability of a typical peptide drug is limited by its low circulating half-life, which could measure a few hours or, even more likely, a few minutes. This is due to enzymatic digestion and other endogenous factors, such as renal clearance. Due to their low toxicity relative to many other classes of drugs, the short half-life of peptides can often be compensated for by a higher than therapeutic level of dosing. However, the administration of peptide drugs by high-dose injection can lead to wide fluctuations in blood levels that can be detrimental to a sustained therapeutic response. This problem is usually mitigated by more frequent dosing of the peptide. In the treatment of chronic diseases in particular, more frequent dosing of peptide drugs translates into reduced patient compliance, higher costs, and a greater chance for adverse events and side effects.
Chemical modification of the peptide using polyethylene glycol (PEG) can improve drug performance with minimal increase in manufacturing cost. PEG is a highly investigated polymer that is used in the covalent modification of biopolymers like proteins and peptides. It is incorporated into the manufacturing process of the bulk API in a technique known as PEGylation. The effects of PEGylation on peptide pharmacokinetics include avoidance of reticuloendothelial (RES) clearance, mitigation of immunogenicity, and reduction of enzymatic proteolysis and of losses by renal filtration, with potentially beneficial changes in biodistribution. These effects can dramatically increase the half-life of a peptide in vivo, with potential collateral improvement in bioavailability but without adversely affecting binding and activity of the peptide ligand.
Figure: Advantages in PEGylation
The most common form of PEG is a linear or branched polyether (HO-(CH2CH2O)n-CH2CH2-OH) with terminal hydroxyl groups synthesized by anionic ring opening polymerization. Monofunctional methoxy-PEG (mPEG; CH3O-(CH2CH2O)n-CH2CH2-OH) is preferred for peptide modification, as it can be derivatized with a number of linkage moieties, yielding methoxy-PEG-amines,-maleimides, or -carboxylic acids.
Four general factors affect the performance of PEGylated peptides:
PEGylation chemistry is the most important factor determining the yield of the PEGylation process and the scalability of the manufacturing protocol. Peptide and linker have to be very pure and very stable during the conjugation reaction to yield a pure conjugate with high efficiency.
The first step in coupling PEG monomethyl ether to a peptide is to activate mPEG with a functional group. Its nature depends on the available reactive groups on the peptide, such as lysine, aspartic acid, cysteine, glutamic acid, serine, threonine, the N-terminal amine, and the C-terminal carboxylic acid or other specific sites. Examples of PEG chemistry include modifications of free cysteine. These are possible with selective reagents, if sufficient care is taken to prevent dimerization during the coupling. A variety of PEG derivatives, such as PEG-maleimide, are readily available. The conjugation of a peptide to many of these derivatives can be rate- and site-controlled by monitoring and adjusting the pH during the coupling. Carbohydrates can also be oxidized and conjugated to hydrazide PEG derivatives, but glycopeptides are not very common, and a discussion on them would exceed the scope of this article.
Both weak and relatively strong linkages to the peptide can be achieved. Thereby, strong linkage is consistent with the objective of increasing the circulating half-life of the peptide ligand and its stability in solution. A weak PEG-peptide linkage is desired in peptide prodrugs. For example, the imidazole of a histidine residue is reacted with mPEG succinimidyl carbonate under slightly acidic conditions. The resulting base-labile peptide-PEG carbamate linkage is hydrolyzed in a controlled fashion in vivo. This slow release can be chemically fine-tuned to occur in endogenous circulation over hours, days or weeks.
Although the effect has not been demonstrated with peptides, some protein PEG conjugates show lower activity as a conjugate but, due to pharmacokinetics, a higher relative in vivo activity. For instance, PEGIntron (PEGylated interferon alpha-2b) has a degradable linkage, which improves its half-life. PEG is coupled to the imidazole ring of histidine. The carbamate linkage is stable at pH 5. At physiologic pH, PEG is slowly released from the protein with concomitant increase of the protein’s biological activity.
Apart from linear single-arm PEG, various more complex PEG structures exist. Branched PEG is generated by attaching two linear PEGs to the alpha and epsilon groups of a lysine core, yielding a well-defined PEG containing a reactive carboxyl group. Subsequently, the lysine-based linker can be converted into a wide range of derivatives, such as thiols and maleimides. Due to the adhered water molecules (PEG can bind 2-3 water molecules per ethylene oxide unit), the dimer PEG behaves like a much larger monomer PEG in vitro, with the biological effect of reducing renal clearance. In heterobifunctional PEG, different reactive groups are attached to the ends of a single (or dimer) PEG chain. As a result, the conjugates contain two peptide chains in close proximity, which may improve affinity, for instance, with dimerized cell surface receptors.
PEGylation is an extremely valuable tool to improve the pharmacologic properties of peptide drugs in vivo. Diverging linear, branched, or more complex types of PEGs can be conjugated by application of appropriate chemistry. The strength of the PEG-peptide linkage can be varied in order to achieve a resilient conjugate, or, alternatively slow release of the active peptide, the latter for example in the case of prodrugs. The power of the method becomes obvious from several reported cases. For example, PEGylation with mPEG2-NHS 40 Kd decreases clearance of certain proteins, for instance interferon alfa (marketed by Roche as Pegasys®) from 9 hours for the native molecule to 77 hours for the conjugate. In peptide conjugates, DAC (Drug Affinity Construct) technology by ConjuChem Inc. uses site-specific conjugation of peptides to albumin to retain a therapeutic potency and duration of activity in circulation comparable to native albumin. This construct increases the circulating half-life of the peptide from hours to weeks.