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PEGylation

And
Its Application In
Pharmaceuticals
Prepared by:
Mr. K. R. Chaudhari M. Pharma. II (Pharmaceutics)

Guided by:
Mr. M. R. Dabhi Mr. M. K. Raval
Asst. Prof. Pharmaceutics Asst. Prof. Pharmaceutics

Department of Pharmaceutical Sciences,


Saurashtra University, Rajkot 360005.
PEGylation
definition
Modification of a protein, peptide or non-
peptide molecule by the linking of one or
more polyethylene glycol (PEG) chains.
Background
 Protein and peptide drugs hold great promise as
therapeutic agents. However, many are degraded by
proteolytic enzymes, can be rapidly cleared by the
kidneys, generate neutralizing antibodies and have a
short circulating half-life
 In many instances, proteins must be administered
frequently to be effective, which may increase cost,
inconvenience and the risk of adverse reactions.
 Formulation changes (e.g. liposomes, microspheres,
hydrogels and monoclonal antibodies) have been
investigated to modify the molecular and biochemical
characteristics of proteins
 Pegylation was first developed by Davis, Abuchowski and
colleagues in the 1970s to enhance the delivery of
therapeutic molecules
Limitation of
protein as a drug
Introduction
Pegylation is the conjugation process by
which polyethylene glycol chains are
attached to protein and peptide drugs.
The FDA has approved PEG for use as a
vehicle or base in foods, cosmetics and
pharmaceuticals, including injectable,
topical, rectal and nasal formulations
because PEG is nontoxic and non-
immunogenicity


The polymer, PEG, is shielding the protein surface
from degrading agents by steric hindrance. And the
increased size of the conjugate is at the basis of
the decreased kidney clearance of the PEGylated
protein
Potential types of
PEG conjugates
Conjugate Properties and applications
Small
type molecule Improved solubility, controlled
drugs permeability through biological barriers,
Affinity Used in aqueous
longevity 2-phase partitioning
in bloodstream , controlled
ligands and systems
release for purification and analysis of
cofactors
Peptides biological macromolecules
Improved solubility and cells.
, conformational
Enzymatic reactors
analysis, biologically active conjugates
Proteins Resistance to proteolysis, reduced
immunogenicity and antigenicity, longevity
in bloodstream, tolerance induction. Uses:
therapeutics,
organic soluble reagents, bioreactors
Potential types of
PEG conjugates
Conjugate type Properties and applications
Oligonucleotid Improved solubility, resistance to
es nucleases, cell membrane permeability
Lipids Used for preparation of PEG-grafted
liposomes
Liposomes and Longevity in bloodstream, RES-evasion
Biomaterials
particulates Reduced therombogenicity, reduced
protein and cell adherence
About PEG
PEG has several chemical properties that make

it especially useful in various biological,


chemical and pharmaceutical formulations:
Non-toxic and non-immunogenic – can be added
to media and attached to surfaces and
conjugated to molecules without interfering
with cellular functions or target
immunogenicities.
Hydrophilic (aqueous-soluble) – attachment
to proteins and other biomolecules
decreases aggregation and increases
solubility.
Highly flexible – provides for surface
treatment or bio conjugation without steric
hindrance
Use of PEG
 As biodegradable polymeric matrices used in
controlled-release systems
 Ointment base.
 Suppository bases.
 Suspending agents
 Emulsion stabilizers
 Plasticizer in soft gelatine capsules
 Enhance the effectiveness of tablet binders
 Impart plasticity to granules
 Used for thermoplastic granulations
 Enhance the aqueous solubility or dissolution
characteristics of poorly soluble compounds



Use of PEG
 Film coatings
 Plasticizers in microencapsulated products
 Lubricants, anti-adherent
 Used in insulin-loaded micro particles for the oral
delivery of insulin
 Used in inhalation preparations to improve
aerosolization
 PEG nanoparticles to improve oral bioavailability


PEGylation Process
A PEG polymer is first chemically
activated in order to react with a
polypeptide drug.
A variety of chemical modifications are
used to prepare an active PEG derivative
with a functional group—
active carbonate, active ester,
aldehyde, or tresylate suitable for
coupling to a given target molecule.
The activated PEG derivative is then
covalently linked to a reactive group
on the polypeptide drug.
Pegylation
(5 les.
of ) a ecu
o l
PE nd E G] m
G (6 ) [P
)
ar co l
g l y
e (7 n e
va ) su yl e
ri PEG c c(ientih
at o l y midy
et h od
io f p l ca e m
nson o r t
bmoana
i u f o r tes
v a t l y l of
ti s i z o
e ac ng imida
r th (4p)h
fo en
hod yl
Met ca
rb
on
at
es
Application of
PEGylation
PEGylated nanoparticles for
brain delivery
The blood–brain barrier (BBB) is formed by special
endothelial cells sealed with tight junctions. This
unique membrane blocks many compounds that might be of
therapeutic value in the treatment of neurological or
psychiatric disorders. Injecting drugs directly into the
brain or disrupting the BBB carries high risks for
patients.
Polymer nanoparticles, such as n-hexadecylcyanoacrylate

(PHDCA), show promise as a way to transport drugs across


the BBB. Animal studies show that PEG–PHDCA penetrates
into the brain to a significantly greater extent than
PHDCA alone. PEG–PHDCA distributes into deep areas of
the brain, including the striatum, hippocampus, and
hypothalamus. Furthermore, this movement occurs without
damage to the BBB or other brain structures. The method
seems promising for the development of drug carriers
for brain delivery
PEG-based hydrogels
PEG can be chemically cross-linked to form

polymer networks that swell and form gels. These


swollen, jelly-like materials are called hydrogels,
and are well suited for a range of medical
applications. The biocompatibility of hydrogels
makes them ideal for wound-healing applications
PEGylated Liposomes
 Liposomes can be modified with PEG to prolong
their blood circulation time. PEGylated liposomes
are characterised by an increased half-life and
decreased plasma clearance compared with
classical liposomal preparations.
 The incorporation of PEG into the lipid bilayer
attracts a hydrated shell around the liposome
and protects the bilayer from plasma proteins
and lipoproteins, producing an 8-fold increase in
plasma half-life of the liposome compared with an
unmodified liposome.
 Pegylated liposomes are also less extensively
taken up by cells of the reticuloendothelial
system and are less likely to leak drug while in
circulation.

 Doxorubicin encapsulation in PEG-coated liposomes
decrease toxicities such as nausea/vomiting,
alopecia and cardio toxicity compared with
standard doxorubicin preparations.
 Increase tumour response due to enhanced drug
accumulation in tumour cells




Prevent Immune
Reaction
 Pegylation has the potential to decrease adverse
effects of the therapeutic molecules they are
attached to and, thus, to increase patient compliance
and improve quality of life.
 Pegaspargase, formed by pegylation of the ε-amino
groups on the lysine residues of asparaginase, is
available for use in the US for the treatment of
patients with acute lymphocytic leukaemia, acute
lymphoblastic leukaemia and chronic myelogenous
leukaemia.
 The primary advantages of pegaspargase over the
unmodified compound are that it decreases the
tendency to induce an immune response, allowing the
majority of patients with hypersensitivity to the
native enzyme to tolerate pegaspargase without
further clinical hypersensitivity, and extends the
half-life from the 20 hours seen with the native
compound to 357 hours with the PEG-modified compound
PEGylation of growth
hormone-releasing hormone
(GRF) analogues
 Synthetically produced GRF is Sermorelin. It maintains
bioactivity in vitro and is almost equally
effective in eliciting secretion of endogenous
growth hormone in vivo.
 The main drawbacks associated with the pharmaceutical
use of GRF relate to its short half-life in plasma,
about 10–20 min in humans, which is caused mostly by
renal ultrafiltration and enzymatic degradation at
the N terminus. PEGylation has been considered as
one valid approach to obtain more stable forms of
the peptide, with a longer in vivo half-life and
ultimately with increased pharmacodynamic response.
PEGylated dendrimers
 The dendritic architecture with well-defined size,
shape and controlled exterior, has pharmaceutical
applications in drug delivery, solubilisation, DNA
transfection and diagnosis.
 However reticuloendothelial system (RES) uptake, drug
leakage, immunogenicity, haemolytic toxicity,
cytotoxicity, hydrophobicity restrict the use of
these nanostructures.
 PEGylation of dendrimers can generally overcome
these shortcomings. Haemolytic and different cell
line studies have shown reduced toxicity of
PEGylated dendrimers than cationic dendrimers.
 PEGylated dendrimers have proved capable of forming
stable complex with plasmid DNA and achieved
improved gene transfection as compared to non-
PEGylated dendrimers.
 Attachments of targeting moiety on the surface of
partially PEGylated dendrimer created much
interest as a delivery system for crossing of
biological barriers and deliver the bioactive
agent near the vicinity of target site.
 Recent successes also demonstrate potential of
PEGylated dendrimers as magnetic resonance imaging
contrast agent and in carbonyl metallo-immunoassay.
PEGylated Interferon
 PEGs of varying lengths and shapes can be attached to
IFNα-2a and IFNα-2b to optimise the protein
conjugate and change the pharmacokinetics.
 Vd, Clearance, absorption half life, tmax, and other
kinetic parameters are optimized for batter
sustained therapy.
 Longer plasma half life of INF could be related to
either the sustained rate of absorption or the
reduced clearance.
 Interferon has potential implications in multiple
therapeutic indications, including patients with
hepatitis B and C infections, malignant melanoma,
renal cell carcinoma, chronic myelogenous leukaemia,
non-Hodgkin’s lymphoma and myeloma.

* Pharmacokinetic
profiles for IFN - α2 a
and 40 kDa PEG - IFN - α2 a .
IFN-α2a is injected every

other day and its short


lifetime in circulation
leads to pulsed blood
concentrations levels
which cycle below
efficacious levels. The
branched PEG 40 (kDa) IFN-
α2a has a long
circulating lifetime due
to the presence of the PEG,
and the once weekly
injection leads to near
constant blood
concentrations above the
therapeutic level over the
one-week period.

* Algranati, N. E., et al.,


Hepatology 30, 190A (1999).
PEG oligonucleotides
 Oligonucleotides and aptamers are 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 exo- and endo-nucleases (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 oligomer aptanib,
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.
PEG as a diagnostic
carrier
 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
 Furthermore, the bio-distribution pattern of
radiodiagnostics is profoundly changed, as in
the case of the C225 antibody–PEG–radiometal
chelators in which PEG acts also as linker
between the targeting and diagnostic moieties.
 Radiolabeled-pegylation PEG molecule having the
radio molecule 18 F, is covalently attached to
any ligand and this compound is used as imaging
agent.
Other PEGylated
products
 These are just a few of the biomedical applications
of pegylation either approved by the FDA or
undergoing investigation.
 Although proteins and peptides have been the main
targets for pegylation, other molecules, including
small-molecule drugs, cofactors, oligonucleotides,
lipids, saccharides and biomaterials, can be
PEGylated as well.
 Other candidates include PEGylated insulin with a
lengthened circulation time and reduced
immunogenicity; PEGylated antibody fragments for
immunotherapy or tumour targeting; and PEGylated
superoxide dismutase for the treatment of
ischaemia/reperfusion injury or burns.
 The benefits of PEGylated catalase, uricase, honeybee
venom, haemoglobin, pyrrolidone and dextran are also
under investigation.
Approved PEG Conjugates
PEG conjugates Disease
PEG–asparaginase (Oncaspar®) Acute lymphoblastic leukemia
PEG–adenosine deaminase Severe combined
(Adagen®) immunodeficiency disease
PEG–interferon α2a (Pegasys®) Hepatitis
(SCID) C
PEG–interferon α2b (PEG– Hepatitis C and clinical trials for
Intron®) cancer, multiple sclerosis,
PEG–G-CSF (pegfilgrastim, Treating
HIV/AIDS of neutropenia during
Neulasta®) chemotherapy
PEG–growth hormone receptor Acromegaly
antagonist (Pegvisomant,
Branched
Somavert®PEG–anti-VEGF
) Macular degeneration (age-
aptamer (Pegaptanib, Macugen™) related)
Limitations in the
use of PEG
The polydispersivity problem
 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.
excretion from the body

 at high molecular weights PEG can accumulate in

the liver, leading to macromolecular syndrome.


References
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551
§ Abuchowski, A. et al. (1977) Alteration of immunological properties of
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§
§
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