Many new molecules in drug pipelines are short-lived proteins. Gary Hu, American Peptide Company, explains how PEGylation can make the molecules more effective by granting them more time in the body to do their work
Peptides seem so promising as drug candidates. However, their small size makes delivery difficult and gives them an extremely short half-life; they are often cleared by the kidneys or reticulo-endothelial system only minutes after being administered, and they are susceptible to degradation by proteolytic enzymes. These problems can be solved by linking them to polyethylene glycol (PEG).
PEG consists of a repeating chain of ethylene oxide (CH2CH2O)x. PEG molecules can be long or short, straight or branched. PEG groups are linked to a reactive group on the peptide, which is usually lysine but can also be aspartic acid, glutamic acid, free cysteine, serine, threonine, the N-terminal amine or the C-terminal carboxylic acid.
Once linked to a peptide, each ethylene glycol subunit becomes tightly associated with two or three water molecules. This has the dual function of rendering the peptide more soluble in water and increasing its size. Since the kidneys filter substances according to their size, the addition of PEG’s molecular weight alleviates the renal clearance undergone by small peptides.
PEG’s globular structure acts as a shield to protect the peptide from proteolytic degradation, and reduces the immunogenicity of foreign peptides by reducing uptake by dendritic cells.
PEG itself is not immunogenic or toxic. By increasing the circulating half-life of peptide drugs, sometimes by factors in the order of 100, PEGylation allows for lower doses to be used and administrations to be less frequent. This saves money and resources, promotes patient compliance and reduces the development of toxicity, tolerance or allergic reactions.
In addition to improving the pharmacokinetic and pharmacodynamic properties of peptide drugs once inside the body, PEGylation can also aid in drug delivery.
The first PEGylated drug to be approved by the FDA was Adagen, in March 1990.1 It is the first successful enzyme replacement therapy for an inherited disease. Adagen is PEGylated bovine adenosine deaminase, and is used to treat people with the type of X-linked Severe Combined Immunodeficiency Disease (SCID) brought to public attention by the ‘Bubble Boy’. It is marketed by Enzon Pharmaceuticals, which was founded in the 1970s by Dr Abraham Abuchowski for the stated purpose of bringing PEGylated drugs to market.
Another drug using Enzon’s proprietary PEG platform is Oncaspar, the PEGylated version of L-asparaginase.1 L-asparaginase is the enzyme that degrades the amino acid asparagine. In contrast to normal cells, leukemic cells are unable to produce asparagine on their own and therefore must get it from the diet. Oncaspar is given to people with leukemia to kill leukemic cells by starving them of asparagine. PEGylation extends the half-life of this enzyme from 20 hours to 357 hours and reduces adverse immune reactions.
Amgen’s Neulasta, or pegfilgrastim, is a drug currently being used to fight chemotherapy induced neutropenia in cancer patients.1 It is a PEGylated form of recombinant granulocyte colony-stimulating factor, a cytokine that stimulates the survival of the neutrophils that are so vital to fighting infection but are unfortunately destroyed by chemotherapy. Pegfilgrastim requires only one injection per chemotherapy trial, whereas the original drug, filgrastim, had to be injected daily for two weeks.
A recent trend in cancer therapeutics focuses on metastases and the angiogenesis that enables their occurrence. The urokinase-mediated plasminogen activation system plays a central role in breaking down the extracellular matrix, and inhibiting this process might be a valuable anticancer strategy. Although an inhibitor, DX-1000, was identified using phage display, it was cleared so rapidly by the kidneys because of its low molecular weight that it was clinically useless. Site-specific PEGylation, however, enhanced its in vivo stability and slowed its clearance but did not significantly diminish its antiangiogenic capabilities.2
Sometimes the lysine residues are in an inaccessible region of the peptide, or in the active or binding site. In these cases, site directed mutagenesis can engineer free cysteine residues into a chosen place in the peptide to link with PEG. This approach was used successfully to PEGylate recombinant GM-CSF, a cytokine that stimulates the proliferation of macrophages and is used clinically to treat melanoma, Crohn’s disease, and myeloid and hematopoietic disorders including neutropenia.3 Because of its short circulating half-life GM-CSF must be injected daily. PEGylation increased this half-life from only one hour to twenty-two.
The hepatitis C and hepatitis B viruses are being successfully treated with PEGylated interferon α-2a,4 which goes by the rather cute name of Pegasys when made by Hoffman-LaRoche and the more pedestrian Pegintron when made by Schering-Plough. PEGylation prolongs the serum half-life of interferon α-2a tenfold, from 6–9 hours to 72–96 hours, and that of interferon α-2b from 6–9 hours to 40. Once weekly dosing with any of these PEGylated interferon as produced higher rates of viral eradication than the standard thrice weekly dosing of interferon α, and had a comparable safety profile.
There are a number of other PEGylated peptides in various stages of development for a range of maladies. Somavert is a PEGylated human growth hormone antagonist marketed by Pfizer to treat acromegaly, a rare hormonal disease in which excessive insulin-like growth factor-1 causes soft-tissue enlargement.1
Researchers at Bayer Healthcare Pharma-ceuticals developed a reproducible method for PEGylating a peptide they hope to use to treat type 2 diabetes.5 Branched PEGs were linked to lysostaphin, an antibacterial endopeptidase that can be used to fight multidrug-resistant strains of Staphylococcus aureus, to increase its serum half-life from less than one hour to 24 hours. PEGylation also reduced the generation of antibodies against lysostaphin.6
PEGylation can clearly make drugs more effective by granting them more time in the body to work. But it can also help by delivering these drugs more efficiently where they need to go. Intranasal delivery is a very attractive route for administering biologically active peptides, and scientists at MDRNA, formerly Nastech Pharmaceutical and now Marina Biotech, demonstrated that PEGylated peptides can act as permeation enhancers for nasal drug delivery. Furthermore, synthetic PEGylated glycoproteins have been used in lieu of viruses for targeted gene delivery.7 Genes delivered in this manner were able to be expressed in vivo.
cell growth inhibition
PEG can not only help peptides achieve their goals; the converse is also true. PEG has been shown to act as a cell repellant, preventing mammalian and bacterial cell growth on plastics and metals used as medical implants. However, grafting it to these materials has proven to be a challenge. Linking PEG to a hydrophilic molecule created a coating on polystyrene that reduced the attachment of both human umbilical vein endothelial cells and Staphylococcus aureus.8 Other PEGylated peptides had affinities for other implant materials like titanium. And these cytophobic coatings may find other uses, for example in proteomic studies and cell culture technologies.
Sometimes PEGylation can cause a decrease in the binding affinity or activity compared with the unconjugated peptide. However, in all cases, the extended half-life more than compensates for this effect. So too the slight increase in manufacturing cost incurred by PEGylation will certainly be deemed worthwhile.
references:
1. JM Harris, RB Chess. Nat Rev Drug Discov. 2003 Mar; 2(3):214-21.
2. L Devy et al. Neoplasia. 2007 Nov; 9(11):927-37.
3. DH Doherty et al. Bioconjug Chem. 2005 Sep-Oct; 16(5):1291-8.
4. J Shepherd, et al. Health Technol Assess. 2004 Oct; 8(39):iii-iv, 1-125.
5. I Tom et al. AAPS J. 2007 Jun 22; 9(2):
E227-34.
6. S Walsh, A Shah, J Mond. Antimicrob Agents Chemother. 2003 Feb; 47(2):554-8.
7. CP Chen et al. Bioconjug Chem. 2007 Mar-Apr; 18(2): 371-8.
8. Kenan DJ et al. Chem Biol. 2006 Jul; 13(7):695-700.