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  • Introduction

The use of proteins like cytokines as human therapeutics is harnessing a lot of attention and has expanded in the past few years. There are several biopharmaceutical products undergoing clinical trials and constant improvements are being made to deliver products with a better efficacy and pharmacodynamic (PD) properties. However many of these products have certain limitations like low solubility, short half-life and proteolytic degradation. (1) Cytokines are small proteins responsible for regulating inflammation, hematopoesis and immunity. These proteins also have the same drawbacks which forms a major obstruction in the development of therapeutic cytokines. (2) Pegylation is one of the most common techniques utilised to overcome these problems. Pegylation is the conjugation of poly ethylene glycol (PEG) molecule to proteins. Poly ethylene glycol is a linear or branched polymer having hydroxyl groups at its termini. These end groups can readily be modified or substituted with other functional groups. The pegylated protein now has improved properties and a better efficacy. (1)

  • Chemistry of Pegylation

In order to couple PEG to biomolecules, it is essential to activate it by derivitising PEG with functional groups at its termini. The selection of functional group depends on the reactive group present on the molecule which will be coupled to PEG. Since lysine is one of the most common amino acid present in proteins, PEG are usually activated with functional groups that are capable of reacting with lysine. Certain factors which affect the unique properties of pegylated biomolecules are the chemistry used for PEG conjugation, the molecular weight of PEG and the number of PEG molecules attached to the polypeptide.(1)

  • Advantages of Pegylation

Conjugation with PEG imparts unique properties to the protein/drug. PEG molecule is soluble in aqueous and organic solvents, and thus is a good candidate for the conjugation with biological molecules. The conjugation of PEG limits the diffusion across membranes, confining the drug in the plasma compartment, thus reducing the volume of distribution.

Some of the benefits of pegylation are:

  • Prolonged circulation time and stability

The characteristic feature of an extended half-life is mainly due to two effects- reduction in the rate of clearance and protection from degradation. PEG has a large molecular size and hydrodynamic radii because it is highly hydrated.(2) This increased hydrodynamic radii reduces their renal clearance thus extending its half-life in the plasma.(3) PEG of lower molecular weight gets cleared mainly by the kidneys whereas PEG of higher molecular weight are cleared by the liver. For several proteins the main challenge in producing therapeutics is their rapid degradation by enzymes. Conjugation with PEG protects the proteins by inhibiting the access of proteolytic enzymes. Pegylation imparts a greater stability to the protein and at the same time also maintains its efficacy.

  • Reduced Immunogenicity and Toxicity

The PEG molecule on the protein minimizes the display of antigenic epitopes thus preventing the formation of neutralizing antibodies. This not only increases its circulatory half-life but also decreases the toxicity of the drug. By concealing the antigenic epitopes it not only prevents their recognition by the immune system but also delays their clearance. The use of pegylated drugs in cosmetics and food has also been permitted by the FDA. (2)

  • Pegylated interleukin-10 (IL-10) : Use in Neuropathic pain.

Neuropathic pain is a condition caused by any disease, physical injury or chemotherapy. Pro inflammatory cytokines like tumor necrosis factor (TNF) and IL-1 are released by the spinal cord glia which in turn increase the pain processing. The release of these cytokines can however be suppressed by IL-10. The attachment of PEG to IL-10 improves its biodistribution and makes this protein a useful candidate for treatment of neuropathic pain. The IL-10 can be pegylated either by acylation or reductive amination. The technique of acylation is targeted on all the available amine groups whereas reductive amination targets the N-terminal amine group. Pegylation by reductive amination retained the activity of IL-10 and acylation on the other hand reduced its activity by upto 3 fold. The efficiency of IL-10 in reversing allodynia was studied and the magnitude of reversal was higher in animals which were given pegylated IL-10. The animals which received the unmodified IL-10 again started suffering from allodynia 3 hours after its administration whereas the duration of reversal was 8 hours in case of animals treated with pegylated IL-10. The potential of this pegylated IL-10 is mainly because of its ability to down regulate the pro-inflammatory cytokines.(4)

  • Pegylated Granulocyte colony stimulating factor (G-CSF) : Use in Neutropenia.

G-CSF is a 19.6 KDa cytokine molecule which is formed by monocytes and functions by triggering the differentiation and growth of neutrophils.(3) Filgrastim (Neupogen) is a recombinant G-CSF obtained from E.coli which is used to treat neutropenia, a reduction in the neutrophil count.(5) However this molecule has a very short half-life because of its rapid clearance by receptor mediated endocytosis and renal filtration.(2) It is therefore conjugated with PEG to form a longer acting pegfilgrastim. (Neulasta) PEG is an inert group and doesn’t affect the activity of the molecule. It increases the half-life of the drug from 3.5 hours to 42 hours.(5)

  • Pegylated IFN-beta-1a: Use in Multiple Sclerosis

The current treatment of MS with IFNs has limited efficacy and several side-effects because of its frequent dosage. The molecule is thus pegylated in order to enhance its solubility, stability and efficacy. PEG-IFN-beta-1a is produced by the addition of a 20KDa methoxy-PEG-O-2-methyl propionaldehyde to the N- terminus of IFN-beta-1a. The pegylated molecule increases the efficiency of the treatment and also helps reduce the rate of relapses. (6)

  • Pegylated IFN alpha-2b (PEG-Intron) : Use in Cancer and Hepatitis C.

Pegylation is extensively being used to modify several therapeutic proteins like TNF-α, G-CSF and IL-2. These pegylated proteins are more efficient than the native ones and have enhanced pharmacologic properties. Peg Intron is IFN alpha-2b with a molecule of PEG attached to it via a covalent linkage to histidine-34. Histidine -34 is a site which maintains the biological activity of the molecule. This modification increases its half-life by reducing clearance and thus can be administered once a week as compared to the trice weekly dosage of native IFN alpha-2b. A high molecular weight PEG could result in a greater half-life but would adversely affect the biological activity of the drug. The potency of the molecule is affected if the amino acid to which PEG is attached modifies the protein structure or hampers receptor binding. Data has indicated that there is an inverse interrelation between the antiviral activity of pegylated IFNs and their half-life and hence it is important to maintain a correct balance.

PEG-Intron is currently used for the treatment of hepatitis C virus (HCV) infection. HCV infection is a very common blood borne disease affecting millions of people world wide and is the major cause of liver diseases. A phase III clinical trial of IFN-α-2b versus PEG IFN-α-2b in patients suffering from hepatitis c showed that PEG-Intron had a better antiviral efficacy. The weekly dosage of PEG-Intron greatly improved the sustained virologic response rate. PEG-Intron was also better tolerated than IFN-α-2b. (7)

PEG-Intron has also been employed for the treatment of cancers like melanoma. Interferon regulatory factor-1 (IRF 1) is a transcriptional regulator of the IFN signalling pathway. IRF 1 has a tumor suppressing activity and is involved in inducing apoptosis. The induction of IRF 1 was analysed in response to pIFNα and the anti cancer drug vinblastine (VBL) in melanoma cells. IRF-1 was induced in 3-6 hours when treated with pIFNα , however there was no induction when treated with VBL alone. pIFNα makes the melanoma cells more susceptible to death by drugs like VBL. The combined treatment of VBL and pIFNα also increased the levels of active Bak, a protein involved in apoptosis, resulting in an overall increased cell death. The melanoma cells have a defective p53 gene and the upregulation of IRF-1 helps activate the p21 gene whose protein product is involved in cell cycle regulation and apoptosis. (8)

  • Pegylated IFN alpha-2a (PEGASYS) : Use in Hepatitis C

The current formulations of PEGASYS consist of a mixture of different forms of monopegylated IFN-α-2a. A 40 KDa PEG moiety is attached via an amide linkage to a lysine residue.(2) These PEG molecules can be incorporated at single or diverse sites on the protein molecule. However excessive pegylation is found to reduce the drug activity. During the treatment of hepatitis c, the therapeutic response is examined by estimating the number of patients who have complete absence of the virus, 6 months after the therapy ends. This is known as the sustained virological response (SVR). Trials have shown that a weekly incorporation of pegylated IFN-α-2a is more efficient than the administration of standard IFN-α-2a thrice a week.(9)

On administration IFN-α-2a brings about an antiviral state in the infected cells and prohibits HCV replication(10). These IFNs induce all the interferon-stimulated genes (ISGs) via the JAK/STAT pathway. These genes prevent the production of viral proteins and reduce the stability of viral RNA.(2) It also stimulates the host cell mediated immune response by activating NK cells, macrophages and cytotoxic T lymphocytes. Randomised trials were carried out to determine the therapeutic efficacy of pIFN-α-2a. The pegylated form not only had a higher virological response but also brought about great improvements in the liver histology. Pegylated IFN-α-2a also brought about an increased synthesis of 2’,5’-oligoadenylate synthetase (OAS) , a key protein involved in IFN mediated prohibition of viral activity. (10) Pegylation extended the half-life of the molecule from 3.8 hours to 65 hours and also reduced renal clearance by 100 fold. PEG limits the distribution of the drug to the plasma only, where it acts on the virus and brings about an increase in the period of exposure of the virus to the drug.(2)

  • Summary of the FDA approved drugs. (2, 3)








Therapy induced neutropenia




( Neulasta)


Hepatitis B/C






Hepatitis C






Pegylation of proteins like cytokines has advanced over the past few years and the emergence of these pegylated drugs will keep growing as the field of therapeutics expands. A variety of drugs with increased efficiency and properties can be developed by employing this technique. A lot of work has been carried out for the development of therapeutic cytokines for the treatment of several diseases. However drawbacks like renal clearance and a small half-life have reduced their potential and efficacy for treatment diseases like hepatitis C. Pegylation has greatly contributed to overcome these problems and is undoubtedly a more productive method of interferon administration. As research in this field continues, the development of newer optimally designed cytokines will also continue to deliver drugs with improved parameters.