PEGylation Technique and scope of it

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PEGylation Technique and scope of its
Applications in Drug Delivery Systems

• Presented
• By
• P.Shyam
• M.Pharm II sem

Department of Pharmaceutics,
University College Of Pharmaceutical Sciences,
Kakatiya University, Warangal.
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Basic Concepts..

• What is PEG?
• How it is formed?
• What are different types?
• Why it is chosen?

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Contents

• Introduction
• Chemistry of PEGylation
• PEGylation process
• PEGylation Technology
• Applications of PEGylation technique in NDDS
• Novel Applications
• Conclusion
• References

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INTRODUCTION
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What is PEGylation ?
PEGylation is the covalent coupling of
Non-Toxic, Hydrophilic Poly ethylene glycol
(PEG) to active Pharmaceutical ingredients Such
as Proteins , Peptides , Antibodies, colloids
etc.
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Who are the Pioneers ?

• The Technology was developed from the pioneering
• work carried out in the 1950s and 1960s on
• the coupling of polymers to proteins, and by
  the 1970s,
• Frank .F.Davis, Dr. Abraham Abuchowski and
  colleagues were using PEG for protein
  modification.
• The first PEG-Protein company was Enzon founded
  in 1981.
• The first approved PEG-Drug Product was
• PEG-Adenosine deaminase,Approved in 1990 by
• US-FDA.

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Why is PEGylation a Hot Topic

• Non-toxic, non-immunogenic, highly soluble in
  water and FDA approved
• Since 1990 many PEGylated drugs have been
  synthesized and approved including drugs for
  cancer, Hepatitis, HIV, and MS
• Low cost of manufacturing
• Part of a multi-billion dollar molecular
  medicines market

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The need for PEGylation

• The Novel Proteins and Peptides have become
  important new drugs with advent of a revolution
  in Biotechnology.
• More than 80 Poly Peptide Drugs are marketed in
  The U.S.
• More than 350 Proteins and Peptides are
• undergoing clinical trails right now.
• About a third of Drug candidates in clinical
  trails are
• Poly peptides.

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The purpose of PEGylation..

• To Improve drug solubility
• To Reduce dosage frequency, without diminished
  efficacy with potentially reduced toxicity
• To Extend circulating life
• To Increase drug stability
• To Enhance protection from proteolytic
  degradation
• Opportunities for new delivery formats and dosing
  regimens
• To Extend patent life of previously approved
  drugs

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How do the PEGs Work
PEGylation increases the half-life of the
biomolecule in the body via
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Reducing Kidney Filtration

• PEGylation significantly increases the apparent
  size of the conjugated drug compound

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Chemistry of PEGylation
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Structure of PEG
Molecular formula C2n2H4n6On2

• Synthesized from the polymerization of ethylene
  oxide
• Using chemical tools to link PEG molecules to
  native proteins can yield conjugates with more
  favorable behavior

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PEG is not ready for conjugation
reactions by itself..
1.Needs a capped terminus with unreactive
moiety 2. Other end has reactive moiety that is
covalently with reactive partner (protein,
peptide, other compounds)
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Method for the activation of PEG molecules.
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Conjugation Chemistry
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Conjugation Chemistry
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Derivatives
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PEGylation process
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functionalization of the PEG polymer at one or
both terminals
PEGs that are activated at each terminus with
the same reactive moiety are known as
homobifunctional,
If the functional groups present are different,
then the PEG derivative is referred as
heterobifunctional or heterofunctional.
The chemically activated derivatives of the PEG
polymers are prepared to attach the PEG to the
desired molecule.
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The first generation PEGylation Process

• PEG polymerichydroxyl groups are reacted with,
  anhydrides,
• acid chlorides, chloroformates and carbonates to
  form PEG Derivative

• The most common reactive sites on polypeptides
  for attaching PEG polymers are the a or e amino
  groups of lysine or the N-terminal amino-acid
  groups of other Amino acids.

• Mainly used linear PEG polymers with molecular
  masses of 12 k Da or less
• Unstable bonds between the drug and PEG were also
  sometimes used, which leads to degradation of the
  PEGdrug conjugate during manufacturing and
  injection.

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Limitations

• Isomerization of polymer
• Early PEGylation was performed with Methoxy PEG
  (mPEG), which was contaminated with PEG DIOL and
  which resulted in the cross linking of proteins
  to form inactive aggregates.
• Diol contamination Can reach up to 10-15

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The second generation PEGylation Process

• Second-generation PEGylation strives to avoid
  the pitfalls associated with mixtures of isomers,
  diol contamination, unstable bonds and
  low-molecular mass mPEG.
• PEGylating site-specifically can minimize the
  loss of biological activity and reduce
  Immunogenicity.
• For instance, because there are far fewer
  cysteine residues
• than lysine groups on polypeptides, the THIOL
  groups of
• cysteine are ideal for specific modifications.

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• PEG derivatives include the incorporation of
  degradable linkages to release drugs at targeted
  sites as well as the synthesis and use of
  HETEROBIFUNCTIONAL PEGs.
• One method (of the many under investigation) for
  releasing drugs from PEG employs a Para- or ortho
  -disulfide of benzyl urethane.
• When subjected to mild reducing conditions, such
  as inside the endosomes of cells, the drug breaks
  free .
• Heterobifunctional PEGs contain dissimilar
  terminal groups, which are advantageous for
  applications in immunoassays, biosensors and
  probes to link macromolecules to surfaces, as
  well as for the targeting of drugs, liposomes or
  viruses to specific tissues.
• Another improvement in second-generation PEG-
  polymers is the use of branched structures, in
  contrast to the solely linear structures found in
  first-generationPEGs20. Branched PEGs of greatly
  increased molecular mass up to 60kda.

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Quality control considerations

• PEG quality is important to achieve reproducible
  PEGylation.
• Traditional PEG systems are polydispersed.
• The starting material for activated PEGs is
  mPEG-OH. The mPEG-OH contains small amounts of
  PEG diol. When the mPEG-OH is activated for
  conjugation, several PEGs can be formed
• The desired activated mPEG-X
• Di-activated PEG that comes from PEG diol
• Any mPEG-OH that has not been activated
• It is important to understand the concentration
  of these various PEGs as they have a direct
  impact on the quality of your conjugate. 

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• The industry typically utilizes NMR to determine
  functionality, but this technique does not allow
  measurement of the various PEGs.
• Advanced analytical techniques such as LC-MS
  allow us to separateand quantify the various
  PEGs.
• This is illustrated by the different elusion
  times in the LC of each of these PEGs as shown in
  the accompanying chart.

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Traditional PEGylation Vs CelaSYS
Traditional PEGylation chain-like structure
polydisperse cross-links possible
structurally determined fluctuations in
quality at any time limited optimization possibi
lities
CelaSYS branched structure monodisperse
cross-links impossible consistently high,
reproducible quality various drug-specific Optim
ization possibilities
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• The PEGylation process was further developed to
  determine the optimal PEG/Protein ratio.
• Optimization of the PEG gave good PEGylation
  efficiency with no residual un modified protein.
• High reproducibility of PEGylation achieved by
  performing the PEGylation for 2 hrs at a pH of
  9.5 and subsequently performing one step
  chromatographic purification.

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PEGylation Technology

• Three different strategies of PEGylation
  Technology
• Chemical PEGylation Technology
• Enzymatic PEGylation Technology
• Genetic PEGylation Technology

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Chemical PEGylation Technology

• Use of established chemistry procedures.
• Reactions occur in high yields.
• Broad applicability.
• Disadvantages
• Reactions are not highly specific.
• Side reactions can occur and PEGylation can be
  incomplete.

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Enzymatic/Genetic PEGylation Technology.

• Highly specific
• Few side-reactions
• Disadvantages
• Restricted to a limited number of applications
• Process requires a recognition site
• Enzyme has to be separated at the end of the
  process.

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Applications of
PEGylation techniques in NDDS

• In Protein Drug Delivery
• In Brain Drug Delivery
• In Colloidal Drug Delivery
• In Gene Drug Delivery

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In Protein Drug Delivery

• PEGASYS PEGylated alpha-interferons for use in
  the treatment of chronic hepatitis C and
  hepatitis-B(Hoffman-La Rochen)
• ADAGEN receivedapproval for the treatment of
  severe combined immunodeficiency(SCID), a
  disease associated with an inheriteddeficiency of
  adenosine deaminase36. Before the availability
• PEG-Intron PEGylated alpha-interferons for use
  in the treatment of chronic hepatitis C and
  hepatit B(Schering-Plough / Enzon)
• Oncaspar PEGylated L-asparaginase for the
  treatment of acute lymphoblastic leukemia in
  patients who are hypersensitive to the native
  unmodified form of L-asparaginase (Enzon).
• This drug was recently approved for front line
  use.
• Neulasta PEGylated recombinant methionyl human
  granulocyte colony stimulating factor for severe
  cancer chemotherapyinduced neutropenia(Amgen)

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• Pegfilgrastim (Neulasta), which was approved in
  2002, is a pegylated form of the earlier drug
  filgrastim (Neupogen). Both contain recombinant
  methionyl human G-CSF,which is known as
  filgrastim.
• The drugs stimulate the production of the
  infection fighting white blood cells
  (neutrophils) that are depleted by cancer
  chemotherapy. Whereas filgrastim requires daily
  injections for about 14 days, pegfilgrastim
  requires one injection per chemotherapy cycle.
• A pegylated form of human growth hormone
  antagonist called pegvisomant (Somavert) is being
  developed for the treatment of ACROMEGALY.
• Pegvisomant has been approved in Europe, and is
  awaiting FDA approval in the US.

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Pharmacokinetic profiles for interferon (IFN)-a2a
and 40 kDa polyethylene glycol (PEG)IFN-a2a.
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• PEGylated Nanoparticles for brain delivery
• The bloodbrain barrier (BBB) is formed by
  special endothelial cells sealed with tight
  junctions.
• Blocks many compounds that might be of
  therapeutic value disorders.
• Disrupting the BBB carries high risks for
  patients.
• Polymer nanoparticles, such as n-hexadecylcyanoacr
  ylate (PHDCA), show promise as a way to transport
  drugs across the BBB.Animal studies show that
  PEGPHDCA penetrates into the brain to a
  significantly greater extent than PHDCA alone.
• PEGPHDCA distributes into deep areas of the
  brain, including the striatum ,hippocampus, and
  hypothalamus.
• movement occurs without damage to the BBB

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PEGylated liposomes

• LIPOSOME is a Phospholipid capsule that protect
  enclosed drug from degradation.
• Liposomes are pegylated to prolong their blood
  circulation time.
• Compared with classical liposomes, pegylated
  counterparts show increased half-life, decreased
  plasma clearance, and a shift in distribution in
  favour of diseased tissues.
• PEG is incorporated into the lipid bilayer of the
  liposome, forming a hydrated shell that protects
  it from destruction by proteins.
• For the antitumour drug doxorubicin, peglyation
  of the
• liposome brings an eightfold increase in plasma
  half-life of the liposome compared to an
  unmodified liposome.
• Pegylated liposomes are also less extensively
  taken up by the
• Reticulo endothelial system and are less likely
  to leak drug while in circulation.

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PEGylated targeted nanoparticles for drug/gene
delivery and imaging in pancreatic cancer
AREA 1
AREA 2
AREA 3
Biodegradable calcium phosphate nanoparticles for
gene delivery
Quantum dots for optical imaging and drug delivery
Biodegradable polymeric nanoparticles for drug
delivery
Genetic material
Targeting molecule
Anticancer drug
PE-mPEG
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PEG-based hydrogels

• PEG can be chemically cross linked to form
  polymer networks that swell and form gels.
• The biocompatibility ideal for wound-healing
  applications.
• In 2000, the FDA approved surgical sealant Focal
  Seal to prevent air leaks in the lungs following
  the removal of lung tumors and other chest
  surgeries.
• FocaSeal uses a PEG that is applied as a liquid,
  and then transformed into a water proof hydrogel
  seal by irradiation.
• The sealant protects wound sites from leaking
  during tissue healing, and then naturally
  degrades and dissolves.
• .

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Spray Gel

• Prevents post-operative adhesion formation.
• Internal wounds often develop adhesionsa type of
  scar tissuethat cause severe pain
• Spray Gel is sprayed onto the wound site and acts
  as a protective barrier during healing.
• This material also degrades and dissolves at a
  programmed rate.
• Other PEG-based hydro gels under development
  deliver encapsulated drugs as implants.
• Degradable linkages between hydro gels and
  incorporated drugs allow drugs to be slowly and
  specifically released in the body.

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In Gene Drug Delivery

• Gene delivery vectors do not possess the basic
  pharmacokinetic properties required for systemic
  applications.
• Polyplexes, lipoplexes and lipopolyplexes all
  have potential for gene delivery to organs such
  as lung, liver and spleen.
• PEGylation, to enhance the circulation lifetimes
  of these particles.
• SPLP, which possesses long circulation lifetimes
  and which preferentially delivers plasmid to
  distal tumor sites following intravenous
  injection, with associated gene expression.
• Enhanced levels of gene expression may be
  achieved by
• modifying the lipid composition.
• The use of PEG-Cer molecules with optimized
  dissociation rates may result in enhanced in vivo
  activity

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Novel Applications

• These are just a few of the biomedical
  applications of pegylation undergoing
  investigation.
• Other molecules including small-molecule drugs,
  cofactors, oligonucleotides, lipids, saccharides
  and biomaterials, can also be pegylated as well.
• Other candidates include
• PEGylated insulin with a lengthened circulation
  time and reduced immunogenicity.
• PEGylated antibody fragments for immunotherapy or
  tumor targeting.

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PEGylatedN 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. PEGylated Nan
particles to cross the bloodbrain barrier or
using pegylated DNA-containing liposomes with
tethered antibodies to provide targeted gene
therapy.
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Conclusion
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References
EFFECT OF PEGYLATION ON PHARMACEUTICALS J.Milton
Harris Robert B. Chess Long-circulating
vectors for the systemic delivery of genes David
B Fenske1, Ian MacLachlan2 Pieter R
Cullis www..nature.com/reviews/drugdisc http//tr
anspeg.pbwiki.com http//www.celares.com http//ph
arma.dow.com http//www.nektar.com www.cuil.com w
ww.wikipedia.org
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Thank you!
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