PEGYLATION: AN APROACH FOR PROTEIN AND PEPTIDE DRUG
DELIVERY SYSTEMS
Shailesh T. Prajapati*, Amit N. Patel, Chhagan N. Patel
Shri Sarvajanik Pharmacy College Mehsana- 384 001, India.
Corresponding Author: Dr. Shailesh T. Prajapati, Department of Pharmaceutics and
pharmaceutical technology, Shri Sarvajanik Pharmacy College Mehsana- 384 001, India;
Mob:+91 9924456583 ; Email: stprajapati@gmail.com
1
INTRODUCTION
A number of novel drug-delivery mechanisms have been developed to increase the utility of
drugs that are otherwise limited by suboptimal pharmacokinetic properties, such as poor
absorption, distribution, and elimination. These include continuous-release injectable and
liposomal systems, which alter the formulation of the drug, and PEGylation, which alters the
drug molecule.[1]
PEGylation defines the modification of a protein, peptide or non-peptide molecule by the linking
of one or more polyethylene glycol (PEG) chains. This polymer is nontoxic, non-immunogenic,
non-antigenic, highly soluble in water and FDA approved. The PEG-drug conjugates have
several advantages: a prolonged residence in body, a decreased degradation by metabolic
enzymes and a reduction or elimination of\protein immunogenicity. Thanks to these favorable
properties, PEGylation now plays an important role in drug delivery, enhancing the potentials of
peptides and proteins as therapeutic agents.[2]
PEGylation was first described in the 1970s by Davies and Abuchowsky and reported in two key
papers on albumin and catalase modification. This was an important milestone, because at that
time it was not conceivable to modify an enzyme so extensively and still maintain its activity.
Proteins were in fact considered very delicate entities and only few gentle modifications with
low molecular-weight products were carried out, mainly to study SARs.[2]
PEGylation is a new delivery technology that differs from traditional formulation in a number of
ways. For formulated products such as tablets, liquids and capsules, the formulation process is
reversible, the drug becomes active after its release from the formulation and the API remains
unchanged. In PEGylated products, on the other hand, the API is chemically modified in a
durable fashion, and the drug is not released from a formulation but has a permanent action and
is in fact classed as a new API. Consequently, PEGylation has to be considered early in the drug
development process.[2]
The advantages conferred by PEGylation include an increased molecule weight and
hydrodynamic volume and a masking of the surface of the molecule with highly mobile PEG
chains. PEGylation also reduces the rapid renal clearance of small proteins and makes it possible
for liposomes to evade removal from the plasma by lipolytic enzymes and the reticuloendothelial
2
system. As a result, pegylated agents generally have longer plasma half-lives and durations of
bioactivity than their nonpegylated counterparts and benefits of pegylated product given in table
1.[1]
PROPERTIES OF PEG
Polyethylene glycols are pH-neutral, nontoxic water-soluble polymers that consist of repeating
ethylene oxide subunits, each with a molecular weight of 44, and two terminal hydroxyl groups.
They are either linear (5 to 30 kd) or branched (40 to 60 kd) chain structures.PEG has Polydispersity i.e. Molecular weight distribution is narrow (1.01 – 1.1). The pharmacokinetic
properties of PEGs vary according to their molecular weight and site of injection. The area under
the time-concentration curve and the half-life of PEGs increase with their molecular weight. For
example, after intravenous administration in mice the half-life of 50kd PEG is substantially
longer than that of 6 kd PEG (987vs 17.6 minutes); 50kd PEG is also retained longer at the
injection site after subcutaneous or intramuscular injection than is 6 kd PEG. Polyethylene
glycols appear to undergo oxidation by the cytochrome P450 enzyme system, and lowmolecularweight PEGs are excreted into the bile.[1]
PEGylation – MECHANISM OF ACTION
After administration, when PEG comes in contact of aqueous environment, ethylene glycol subunit gets tightly attached to the water molecule. This binding to water renders them high mobility
and hydration. Hydration and rapid motion causes PEGylated protein to function, as it causes
PEG to sweep out a large volume which acts like a shield to protect the attached drug from
enzymatic degradation and interaction with cell surface proteins. This increased size also helps to
prevent rapid renal filtration and clearance sustaining the drug bioavailability. The high steric
hindrance of branched-chain PEGs generally affords greater protection than do linear chain.[1]
FACTORS AFFECTING PERFORMANCE OF PEGylated PRODUCT
Molecular Weight
3
Molecular weight less than 1000 Da of PEG broken down into sub-units, and have some toxicity,
while Molecular weight greater than 1000 Da of PEG: does not demonstrate any toxicity in vivo.
Molecular Weight upto 40,000 – 50,000 Da: used in clinical and approved pharmaceutical
application.
The molecular weight of PEG has a direct impact on the activity; Higher molecular weight PEGs
tends to have higher in-vivo activity due to the improved pharmacokinetic profile like increasing
half life as earlier discussed.[1]
Structure
Branched structure has more size than same molecular weight linear structure so; it’s also helps
to prevent rapid renal filtration and clearance sustaining the drug bioavailability. The high steric
hindrance of branched-chain PEGs generally affords greater protection than do linear chain.[1,4]
Number of PEG chains
Two or more lower molecular weight chains can be added to increase total molecular weight of
PEG complex
Specific location of PEG site of attachment to the molecule.
Optimal PEGylation is product-specific, and can vary depending on the site of attachment, the
chemistry used to create the conjugate, and the characteristics of the PEG used. Effective
PEGylation of a drug may be achieved by attaching a single large PEG at a single site, a
branched PEG at a single site, or several small PEG chains at several sites.[1]
CHEMISTRY OF PEGYLATION
To couple PEG to a molecule (i.e. polypeptides, polysaccharides, polynucleotide’s and small
organic molecules) as shown in Figure 1, it is necessary to activate the PEG by preparing a
derivative of the PEG having a functional group at one or both termini. The functional group is
chosen based on the type of available reactive group on the molecule that will be coupled to the
PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine,
aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminal amino group and the Cterminal carboxylic acid. In the case of glycoproteins, vicinal hydroxyl groups can be oxidized
4
with periodate to form two reactive formyl moieties.[3]
The most common route for PEG conjugation of proteins has been to activate the PEG with
functional groups suitable for reaction with lysine and N-terminal amino acid groups. Lysine is
one of the most prevalent amino acids in proteins and can be up-wards of 10% of the overall
amino acid sequence. In reactions between electrophilically activated PEG and nucleophilic
amino acids, it is typical that several amines are substituted. When multiple lysines have been
modified, a heterogeneous mixture is produced, which is composed of a population of several
polyethylene glycol molecules attached per protein molecule (‘PEGmers’) ranging from zero to
the number of ´-and a-amine groups in the protein.[3]
PEGylation technology is classified into two types:
1. Early PEGylation technology (First generation PEGylation)
2. Advanced PEGylation technology (Second generation PEGylation)
1. First Generation PEGylation
First generation PEGylation methods were fraught with difficulties.
With first generation PEGylation, the PEG polymer was generally attached to ε amino group of
lysine, and gave mixtures of PEG isomers with different molecular masses. The existence of
these isomers makes it difficult to reproduce drug batches, and can contribute to the antigenecity
of the drug and poor clinical outcomes. In addition, first generation methods mainly used linear
PEG polymers of 12 kDa or less. Unstable bonds between the drug and PEG were also
sometimes used, which leads to degradation of PEG-drug conjugate during manufacturing and
injection Early PEGylation was performed with methoxy-PEG (m-PEG), which was
contaminated with PEG diol and which resulted in the cross-linking of proteins to form inactive
aggregates. Diol contamination can reach upto 10-15 %.[1]
Despite these limitations, several first generation PEGylated drugs receive regulatory approval.
Example: Still in use today are Pegademase (ADAGEN®), a PEGylated form of the enzyme
adenosine de-aminase for the treatment of Severe Combined Immuno-Deficiency (SCID) and
Pegaspargase (ONCASPAR®), a PEGylated form of enzyme asparginase for the treatment of
Leukemia.[5]
5
2.
Second Generation PEGylation
Second generation PEGylation strives to avoid pitfalls associated with the first generation
PEGylation.
Overall goal of this technology is to create larger PEG polymers to improve the
Pharmacokinetics and Pharmacodynamic effects seen with lower molecular mass PEGs.
Newer pegylation methods create conjugates with strong linkages that are resistant to sidereactions and are able to withstand purification to remove dio1 contaminants, thereby making it
possible to use high molecular- weight PEGs . These methods attach an activated PEG to the
drug molecule by incorporating part of the activating group as a link between the two entities.
For example, PEGFILGRASTIM® is formed by covalently attaching a 20 kd PEG chain through
a stable secondary amine bond directly to the terminal amino group of the filgrastim molecule. In
this way, the nitrogen atom to which the PEG chain is attached retains its surface charge, a factor
that has been shown to be crucial in conserving the bioactivity of some molecules.[1]
Amine PEGylation and N-terminal PEGylation
Since most applications of PEG conjugation involve labile molecules, the coupling reaction
generally requires mild chemical conditions. In case of polypeptides, the most common reactive
groups involved in coupling are nucleophiles with the following decreasing rank order of
reactivity: thiol, alpha amino group, epsilon amino group, carboxylate, hydroxylate. However,
this order is not absolute, since it depends also on the reaction pH, furthermore other residues
may react in special conditions, as the imidazole group of histidine. The thiol group is rarely
present in proteins, furthermore it is often involved in active sites. The carboxylic groups cannot
be easily activated without having. reaction with the protein amino groups, to yield intra or inter
molecular cross linking. Therefore, amino groups, namely the alpha amino or the epsilon amino
of lysine, are the usual sites of PEG linking.[4] PEGylating Agents used for amino PEGylation
shown in Table 2.
Carboxyl PEGylation
PEG reagents react with carboxylic acid in the presence of coupling agents such as
DCC
(N,N'-dicyclohexylcarbodiimide)
and
EDIC
(N-(3-dimethylaminopropyl)-N'
ethylcarbodiimide, HCl salt). However, the procedure is successful only when amines are not
present in the compound, as for instance in the case of non-peptide drugs. In peptides and
6
proteins the risk of cross-linking is difficult to avoid.[3] PEGylating Agents used for Carboxyl
PEGylation are shown in Table 3.
PEGylation at the –SH (thiol) groups of Cysteine of polypeptides
PEGylation of free cysteine residues in proteins is the main approach for site-specific
modification because reagents that specifically react with cysteines have been synthesized, and
the number of free cysteines on the surface of a protein is much less than that of lysine residues.
In the absence of a free cysteine in a native protein, one or more free cysteines can be added by
genetic engineering. PEGylating site specifically can minimize the loss of biological activity and
reduce immunogenecity.[3] PEGylating Agents used for thiol PEGylation are shown in Table 3.
Hydroxyl PEGylation
PEG-isocyanate is useful for hydroxyl group conjugation yielding a stable urethane linkage.
However, its reactivity may be best exploited for non-peptide moieties such as drugs or
hydroxyl-containing matrices to yield biocompatible surfaces. PEG-isocyanate is in fact highly
reactive with amines also.[3]PEGylating Agents used for Hydroxyl PEGylation are shown in
Table 3.
Hetero-bifunctional PEGs
As applications of PEG chemistry have become more sophisticated, there has been an increasing
need for heterobifunctional PEGs, which are PEGs bearing dissimilar terminal groups. Such
heterobifunctional PEGs bearing appropriate functional groups may be used to link two entities
where a hydrophilic, flexible, and biocompatible spacer is needed. Heterobifunctional PEG can
be used in a variety of ways that includes linking macromolecules to surfaces (for
immunoassays, biosensors or various probe applications), targeting of drugs, liposomes and
viruses to specific tissues, liquid phase peptide synthesis and many others. Preferred end groups
for hetero-bifunctional PEGs are NHS esters, maleimide, vinyl sulfone, pyridyl disulfide, amine,
and carboxylic acids.[3]
Branched structures
Second generation PEGylation uses branched structures of PEG, in contrast to the solely linear
structures found in first generation PEGs. Branched PEGs of greatly increased molecular masses
– upto 60 kDa or more, as compared with the 12 kDa or less found in the first generation PEGs –
have been prepared. A branched PEG ‘acts’ as if it were much larger than a corresponding
linear PEG of the same molecular mass. Branched PEGs are also better at cloaking the attached
7
polypeptide drug from the immune system and proteolytic enzymes, thereby reducing its
antigenecity and likelihood of destruction.[1,3]
Specific PEGylation by enzymes or by reversible protection
The specific conjugation of PEG to the amide group of glutamines or to the hydroxyl group of
serines and threonines is only possible under mild conditions using enzymes. Sato discovered
that glutamine in proteins can be the substrate of the transglutaminase enzymes, if an amino PEG
is used as the nucleophilic donor. Through a transglutamination reaction the enzyme links PEG
to the protein at the level of the glutamine residue as shown in Figure 2.[2] Now a days, PEG
conjugates with different enzyme like arginines n histaminase are also available.
LIMITATIONS IN TH USE OF PEG
PEG is obtained by chemical synthesis and, like all synthetic polymers, it is polydisperse, which
means that the polymer’s batch is composed of molecules having different number of monomers,
yielding a Gaussian distribution of the molecular weights. This leads to a population of drug
conjugates, which might have different biological properties, mainly in body-residence time and
immunogenicity. Polydispersivity problem must be still taken into consideration, especially
when dealing with low molecular weight drugs, either peptide or non-peptide drugs, where the
mass of linked PEG is more relevant for conveying the conjugate’s characteristics, mainly those
related to the molecular size. A second problem for the use of this polymer relates to the
excretion from the body. As for other polymers, PEGs are usually excreted in urine or feces but
at high molecular weights they can accumulate in the liver, leading to macromolecular
syndrome.[2]
APPLICATION OF PEGYLATION
PEG as Diagnostic Carrier
In vivo non invasive diagnosis is done by using tracers detected through magnetic resonance or
radioactivity. Usually they are administered in a chelated form using compounds that can give
specific biodistribution, stability or targeting. PEGylation increases the body-residence time of
paramagnetic chelates that will be cleared more slowly than the unmodified molecules through
the kidney or liver, thus allowing more detailed images by magnetic resonance. C225 is a
monoclonal antibody directed against the epidermal growth factor receptor, which was
8
conjugated
to
a
heterobifunctional
PEG
bearing
a
radiometal
chelator
(diethylenetriaminepentaacetic acid, DTPA) at one terminus. The conjugate DTPA–PEG–C225
retained 66% of binding affinity, and, more importantly, when labeled with Indium-111 (111In)
it showed narrower steady-state distribution than the non-PEGylated 111In–DTPA–C225,
because of reduced nonspecific binding. Therefore, in case of protein targeted diagnostic, PEG
could help to collect better defined images by limiting the background noise due to nonspecific
protein–protein interaction.[2]
PEG oligonucleotides
Mainly antisense oligonucleotides and are now under active investigation as new potential drugs
because of their extremely high selectivity in target recognition. All of them, however, share the
problems of short half-life in vivo because of either low stability towards the eso- and endonucleases (present in plasma and inside the cells) or their rapid excretion caused by their small
size. Furthermore, their negative charge prevents an easy penetration into the cells. A PEG
molecule, bound to the hydroxyl group of a nucleic acid (directly or through a spacer link), was
found to increase the stability towards enzyme degradation,prolong the plasma permanence and
enhance the penetration into cells by masking the negative charges of oligonucleotides. A
PEGylated aptamer, the 28mer oligomeraptanib, has already been approved by FDA for the
treatment of age-related macular degeneration of retina. In this product, a branched PEG of 40
kDa was attached to the oligonucleotides through a pentamino linker.[2]
PEGylated conjugate as Anticancer agent[7]
PEG conjugates with low molecular weight anticancer drugs
PEG has been successful for protein modification but in the case of low molecular weight drugs
it presents a crucial limit, the low drug payload accompanying the available methoxy or diol
forms of this polymer. This intrinsic limitation had for many years prevented the development of
a small drug-PEG conjugate. A few studies have been conducted to overcome the low PEG
loading by either branching the end chain groups or coupling on them small Dendron structures.
Pegamotecan® (Enzon Pharmaceuticals, Inc.) is a prodrug obtained by coupling two molecules
of camptothecin to a diol PEG of 40 kDa. The drug is linked through an ester bond involving the
9
C-20 hydroxyl group and a carboxylic group of PEG. The aim of this approach was double, to
increase the drug half-life in blood by PEGylation and to stabilize by acylation the active lactone
configuration of camptothecin.
PEG-irinotecan: The architecture of new multi-arm PEGs was also exploited for the preparation
of PEG-irinotecan (NKTR-102) by Nektar Therapeutics. The drug has been covalently bound to
a four arms PEG. In preclinical studies NKTR-102 plasma half-life was evaluated in a mouse
model taking into consideration the active metabolite SN-38, released from irinotecan. The
conjugate showed prolonged pharmacokinetic profiles with a half-life of 15 days when
compared to 4 h with free irinotecan.
PEG-docetaxel : PEGylated docetaxel (NKTR-105) has been prepared with the same multi-arm
PEG technology. The derivative has shown good preclinical activity in colon and lung cancer
xenograft models.This product has just entered phase 1 clinical studies enrolling approximately
30 patients with refractory solid tumours who have failed all prior available therapies.
PEG-Protein conjugates
In PEGylation of protein conjugate two different approaches can be identified based on the type
of protein studied:
Heterologous protein, Usually the main limit of these proteins is the immunogenicity rather than
a short pharmacokinetic. Therefore, both PEG molecular weight and coupling chemistry should
ensure a wide shielding of the protein surface or, at least, the immunogenic sites. Basically, in
these cases low molecular weight PEGs (5– 10 kDa) and random amine coupling are used. It is
important to note that all the enzymes studied possess small substrates; these can cross the PEG
layer, around the protein, and easily reach the active site. Conversely, active site approach of
large and hindered substrates would be prevented this compromising the enzyme activity. This
would suggest that PEGylation may be not a suitable approach for immunogenic enzymes having
big substrates.
Endogenous protein, For these biopharmaceutical drugs the prolongation of body circulation
half-life is the driving force in seeking for a polymer conjugate. Most of the endogenous proteins
act through a receptor-mediated activity. This dictates the strategy for an optimum PEGylation
approach, namely a site specific conjugation to generate monoPEGylated isomers. In particular
10
the site of polymer attachment must be far from the receptor recognition area. In this case, it is
mandatory the use of high molecular weight polymers to reach the PEG mass for the desired
half-life prolongation.
PEG-antibody fragment angiogenesis inhibitor (CDP791)
Vascular endothelial growth factor receptor-2 (VEGFR-2) is involved in the formation of new
blood vessels in tumours (angiogenesis), allowing cancer cells to receive nutrients and to
maintain growth. Therefore, a molecule able to block VEGFR-2 can interfere with the
development of tumour vasculature. CDP791 is a PEGylated diFab antibody that binds the
VEGFR-2, with a Kd of 49pM, preventing the activation by VEGF ligands. The unconjugated
CDP791 antibody fragment is affected by a too fast in vivo clearance, because it has a reduced
mass due to the absence of Fc region. This problem was overcome by PEGylation of the cysteine
present at the C-terminus.
PEG-interferon-alpha conjugates
Several clinical studies are evaluating the effectiveness of PEGinterferon-α2b (PEG-INTRON®),
presently used for the treatment of hepatitis B and C, as adjuvant therapy in certain anticancer
protocols. The native interferon-α2b is one of the most studied agents for adjuvant therapy in
stage IIb and stage III melanoma. Improvements in the recurrence-free survival have been shown
when interferon- α2b therapy was prolonged for 12–15 months. This long therapy, consisting in
a daily drug administration, can particularly compromise the patient compliance. This can be
highly improved using the PEGylated form of interferon-α2b, a monoPEGylated derivative
obtained by conjugating the protein with a linear 12 kDa amino reactive PEGylating agent. The
conjugate maintains the therapeutic level of interferon-α2b by a weekly, self administered, dose
schedule and its safety has been studied in several cancers.
PEG-Interferon-α2a, a conjugate obtained by linking a branched PEG 40 kDa to the protein and
marketed as PEGASYS®, is used in clinic to treat hepatitis as PEG-INTRON®. The higher
polymer molecular weight of PEGASYS® (40 kDa versus 12 kDa of PEG-INTRON®) and the
higher stability of the PEG-protein linkages (i.e. His residues are involved in this case) allowed
for the prolonging of the in vivo half-life to 65 h with respect to the 27–37 h of PEG-INTRON®.
11
PEGylation: the in vitro activity is reduced to about 7% of that of native interferon, this being
the weakness of stable polymer conjugation, but this limitation is more than counterbalanced by
the enhanced in vivo half-life of the conjugate.
PEG-granulocyte colony stimulating factor
Granulocyte colony stimulating factor (G-CSF) is used as adjuvant therapy to treat granulocytes
depletion during chemotherapy. The fast blood clearance of the free drug was addressed by
PEGylation. Different PEG coupling approaches were conducted but the most successful one
consisted of a reductive alkylation with PEG aldehyde performed at acidic pH. Under this
condition, a monoPEGylated conjugate was preferentially obtained in which the polymer was
linked to the protein N-terminal α amino group. The PEG 20 kDa conjugate showed an improved
pharmacokinetic profile as consequence to the reduced kidney excretion.
The PEG-G-CSF conjugate (Pegfilgastrim, Neulasta®) was approved for human use in 2002 for
the first and subsequent cycleadministration against febrile neutropenia in patients with
nonmyeloid malignancies receiving myelosuppressive chemotherapy associated with a 30%–
40% risk of febrile neutropenia.
PEG conjugates with enzymes
PEG conjugated Asparaginase
several leukemic lymphoblasts cells rely on the serum supply of asparagine, for their growth,
because they lack the enzyme asparagine synthetase. Asparaginase, the enzyme that converts
asparagine into aspartate and ammonia, has therefore been proposed as a therapeutic agent for
acute lymphoblastic leukaemia (ALL). FDA approval for PEG-asparaginase (Rhone-Poulenc
Rorer as Oncaspar®) was granted in 1994 for treatment of patients with ALL who are
hypersensitive to the two native isoforms of the enzyme. PEGylated asparaginase has been used
in combination with several traditional anticancer molecules, often in a multiagent regimen,
including for example one or more of the following drugs: cyclophosphamide, daunorubicin,
vincristine, cytarabine, prednisone, etc. In these studies the PEGylated enzyme was well
tolerated, showing hyperbilirubinaemia and hyperglycaemia as the most common adverse
effects.
12
Arginine deiminase and Arginase
In literature two types of arginine degrading enzymes are reported, and both have been
suggested as antitumour agents:
citrulline
i) arginine deiminase (ADI), which degrades arginine in
and ammonia, ii) arginase (ARG) that catalyses the conversion of arginine in
ornithine and urea. This enzyme was shown to be even more powerful than asparaginase in
killing human leukaemia cells. arginine depleting enzymes can be useful in treating these
tumours. Indeed, arginine deficiency inhibits tumour growth, angiogenesis and nitric oxide
synthesis.
Chitosan–PEG nanocapsules as new carriers for oral peptide delivery
Chitosan–PEG nanocapsules and the control PEG-coated nanoemulsions were obtained by the
solvent displacement technique. Their size was in the range of 160–250 nm. Their zeta potential
was greatly affected by the nature of the coating, being positive for chitosan–PEG nanocapsules
and negative in the case of PEG-coated nanoemulsions. The presence of PEG, whether alone or
grafted to chitosan, improved the stability of the nanocapsules in the gastrointestinal fluids.
Using the Caco-2 model cell line it was observed that the pegylation of chitosan reduced the
cytotoxicity of the nanocapsules. Finally, the results of the in vivo studies showed the capacity of
chitosan–PEG nanocapsules to enhance and prolong the intestinal absorption of salmon
calcitonin. Additionally, they indicated that the pegylation degree affected the in vivo
performance of the nanocapsules. Therefore, by modulating the pegylation degree of chitosan, it
was possible to obtain nanocapsules with a good stability, a low cytotoxicity and with absorption
enhancing properties.[8]
Gene Delivery
Polyethyleneglycol
modified
polyethylenimine
for
improved
CNS
gene
transfer
One problem of using polycation DNA complexes, especially in an in vivo study, is their poor
solubility. They may immediately precipitate out of a solution when prepared at a higher
concentration. Polyethylene glycol (PEG) modification (PEGylation) often can improve the
solubility of macromolecules, minimize aggregation of particulates and reduce their interaction
with proteins in the physiological fluid. PEGylation of PEI reduced surface charge of PEI/DNA
13
particles, increased their dispersion ability at high concentrations, decreased plasma protein
binding and erythrocyte aggregation, prolonged blood circulation and reduced systemic toxicity
& increased invivo transgene expression of PEI. The study provides the in vivo evidence that an
appropriate degree of PEG modification is decisive in improving gene transfer mediated by
PEGylated polymers.[9]
Small interfering RNA (siRNA) delivery
Small interfering RNA was conjugated with poly(ethylene glycol) (PEG) at four different
terminal ends (sense 3′, sense 5′, antisense 3′, and antisense 5′) via cleavable disulfide and
noncleavable thioether for gene silencing efficiencies. The PEGylation site at the four siRNA
termini and PEG molecular weight were not critical factors to significantly affect gene silencing
activities. Cleavable siRNA-PEG conjugates showed comparable gene silencing activities to
naked siRNA, and exhibited sequence-specific degradation of a target mRNA. Interestingly,
noncleavable siRNA-PEG conjugates were processed by Dicer, enabling to exert RNAi effect
without showing a target sequence-specific manner. However, only cleavable siRNA-PEG
conjugates significantly reduced the extent of INF-α release as compared to noncleavable
siRNA-PEG conjugates, suggesting that they can be potentially used for therapeutic siRNA
applications.[10]
Dendrimer
Despite the robust structure of polyamidoamine(PAMAM)dendrimers, they are not stable when
complexed with surfactants. Modification of PAMAM dendrimers by grafting PEG chains on the
surface of PAMAM substantially improves its colloidal stability in the presence of
sodiumdodecylsulfate(SDS). Michael addition reaction was employed to synthesize PEGylatedPAMAM by activating MPEG with 4-nitrophenylchloroformate.The PEGylated-PAMAM
dendrimers did not aggregate in the presence of upto 100mM SDS as the complexes were
sterically stabilized by PEGchains. ITC and zetapotential measurements revealed that the binding
mechanism of SDS and PEGylated-PAMAM was induced by electrostatic interaction and
polymer-induced micellization of SDS on PEG chains. The interaction of PEGylated-PAMAM
14
and amphiphilic molecules, such as SDS was elucidated, and this provided a useful basis for the
application PEGylated-PAMAM in drug delivery mostly in antiviral and cancer therapy.[11]
Insulin PEGylation
A novel long-acting insulin based on the following properties: (i) action as a prodrug to preclude
supra physiological concentrations shortly after injection; (ii) maintenance of low-circulating
level of biologically active insulin for prolonged period; and (iii) high solubility in aqueous
solution. A spontaneously hydrolyzable prodrug was thus designed and prepared by conjugating
insulin through its amino side chains to a 40 kDa polyethylene glycol containing sulfhydryl
moiety
(PEG40-SH),
employing
recently
developed
hetero-bifunctional
spacer
9-
hydroxymethyl-7(amino-3-maleimidopropionate)-fluorene-N-hydroxysucinimide (MAL-Fmoc0Su). A conjugate trapped in the circulatory system and capable of releasing insulin by
spontaneous chemical hydrolysis has been created. PEG40-Fmoc-insulin is a water-soluble,
reactivatable prodrug with low biological activity. Upon incubation at physiological conditions,
the covalently linked insulin undergoes spontaneous hydrolysis at a slow rate and in a linear
fashion, releasing the nonmodified immunologically and biologically active insulin with a t1/2
value of 30h. A single subcutaneous administration of PEG40-Fmoc-insulin to healthy and
diabetic rodents facilitates prolonged glucose-lowering effects 4- to 7-fold greater than similar
doses of the native hormone. The beneficial pharmacological features endowed by PEGylation
are thus preserved. In contrast, nonreversible, ‘‘conventional” pegylation of insulin led to
inactivation of the hormone.[12]
PEGylated derivatives of rosin (PD) used as sustained release film forming materials for
controlled release formulation. The mechanism of drug release from these coated systems
however followed class II transport (n>1.0).[13]
Recently approved pegylated products shown in Table 4.
CONCLUSION
PEGylation improves the biopharmaceutical properties of drugs that increase stability, resistant
to proteolytic inactivation, decrease to nonexistent immunogenicity, increase circulatory lives
and low toxicity. These type of alter properties improve the efficacy of protein and peptide drug
delivery.
15
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Shechter Y, Mironchik M, Rubinraut S, Tsubery H, Sasson K, Marcus Y, Fridkin M.
Reversible pegylation of insulin facilitates its prolonged action in vivo. European Journal
of Pharmaceutics and Biopharmaceutics 70 (2008) 19–28.
16
13.
Nande VS, Barabde UV, Morkhade DM, Joshi SB, Patil AT. Investigation of PEGylated
Derivatives of Rosin as Sustained Release Film Formers. AAPS PharmSciTech March
2008, 9(1).
17
TABLE 1. Potential benefits of PEGylated products
Greater biologic activity
Greater passive tumour targeting of liposomes
Longer circulating half-life
Lower peak plasma concentrations
Smaller fluctuations in plasma concentrations
Less enzymatic degradation
Less immunogenicity and antigenicity
Greater solubility
Less-frequent administration
Greater patient adherence and improved quality of life
18
TABLE 2. PEGylating agent for Amino PEGylation[6]
PEG reagents
PEGylation
PEG-NHS
The N-hydroxysuccinimide (NHS) activated ester of PEG
carboxylic acid can react with the amino group of lysine.
The coupling requires only mild conditions, pH 7-9, low
temperature (5-25ºC) for short period of time. The
formed amide bond is physiologically stable.
PEG-aldehyde
Reductive amination with primary amines to produce
secondary amines, in the presence of reducing agents
such
as
sodium
cyanoborohydride.
pH
borohydride
and
sodium
is
for
reductive
important
amination.
PEG-isocyanate
Reaction with amine to produce a stable urethane linkage.
PEG epoxide
Nucleophilic addition
PEG-
React with amine to produce a stable thiourea linkage.
isothiocyanate
PEG-COOH
Usually the acid needs to be activated, such as NHS ester.
PEG-NPC
Amine reacts with NPC functionalized PEG under proper
conditions.
PEG-acrylate
Michael addition between amine and acrylate ester
19
TABLE 3. PEGylating agent for Carboxyl, Thiol and Hydroxyl PEGylation[6]
PEG reagents for Carboxyl PEGylation
PEG-amine
Amide formation under DCC or EDIC coupling conditions
PEG-hydrazide
After activated by EDIC at mild acidic pH, the carboxyl group
of proteins readily react with PEG-hydrazide, while the amino
groups present in all reagents remain inactive in this particular
conditions.
PEG reagents for Thiol (-SH) PEGylation
PEG-Maleimide
Michael addition, thiols react with the C=C bond in the
maleimic ring to form a physiological stable linkage. The best
reaction condition is at pH 8.
PEG-OPSS
Disulfide S-S bond formation, which can be reversed by
reducing
agents
such
as
sodium
borohydride
and
thioethanolamine.
PEG-
Michael addition, thiols react with the C=C bond to form
vinylsulfone
a physiological stable linkage.
PEG-thiol
Oxidative disulfide S-S bond formation.
PEG reagents for Hydroxyl PEGylation
PEG-isocyanate
Hydroxyl groups react with PEG-NCO, however special
considerations are required.
PEG-NPC
Hydroxyl groups react with NPC to from a carbonate linkage.
PEG-epoxide
PEG-epoxide reacts with hydroxyls best at pH 8.5-9.5.
20
TABLE 4. Approved PEGylated Products[5]
Brand name
Product
Company
Indication
PEGasys
PEG-INF α-2a
Hoffmann-La
Hepatitis
(interferon)
Roche
PEG-INF α-2b
Enzon
Hepatitis
Amgen
Neutropenia
Enzon
Immunodeficiency
PEG-Intron
(interferon)
Neulasta
PEGfilgrastim(granulocyte
colony
stimulating
factor)
Adagen
PEG-adenosine
deaminase
Oncaspar
PEG-asparginase
Enzon
Cancer
Somavert
PEG-visomant
Pfizer
Acromegaly
PEG-hirudin
PEG-recombinant
Abbot
Thrombosis(phase
hirudin
PEG-
PEG-CDP 870
III)
Pfizer
monoclonal
Rheumatoid
arthritis (phase III)
antibody
PEG-Axokine
PEG-cilliary
Regeneron
Obesity (phase III)
nurotrophic factor
21
Figure 1. PEGylation process in general
22
Figure 2. Specific PEGylation of Glutamine by transglutaminase enzyme.
23