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

Guided by:Mr. M. R. DabhiAsst. Prof. Pharmaceutics

Mr. M. K. Raval

Asst. Prof. Pharmaceutics

Department of Pharmaceutical Sciences, Saurashtra University, Rajkot 360005.

PEGylation definitionModification 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

IntroductionPegylation 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 nonimmunogenicity

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 conjugatesConjugate Properties and applications Small molecule Improved solubility, controlled type drugs permeability through biological barriers, Affinity Used in aqueous 2-phase partitioning longevity in bloodstream, controlled ligands and systems for purification and analysis of release cofactors biological macromolecules and cells. Peptides Improved solubility, conformational Enzymatic biologically active conjugates analysis, reactors Proteins Resistance to proteolysis, reduced immunogenicity and antigenicity, longevity in bloodstream, tolerance induction. Uses: therapeutics, organic soluble reagents, bioreactors

Potential types of PEG conjugatesConjugate type Oligonucleotid es Lipids Liposomes and Biomaterials particulates Properties and applications Improved solubility, resistance to nucleases, cell membrane permeability Used for preparation of PEG-grafted liposomes Longevity in bloodstream, RES-evasion 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

As biodegradable polymeric matrices used in

Use of PEG

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 ProcessA 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 of ) a PE nd G (6 )

. les ecu mol ] PEG [ l) o ar lyc g e (7) s ne va ucci thyle ri PEG en d at ly( imidyl o etho io carb ate m f p o ns n o form nates o io u f vat si olyl i z t c ng imida e a h ) (4ph r t fo en od yl eth M ca rb on at es

Application of PEGylation

The bloodbrain 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 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. 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

PEGylated nanoparticles for brain delivery

PEG-based hydrogelsPEG 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

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 -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

Prevent Immune Reaction

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 1020 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 nonPEGylated 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

IFN-2a and IFN-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-Hodgkins lymphoma and myeloma.

* Pharmacokinetic profiles for IFN - 2 a and 40 kDa PEG - IFN - 2 a . IFN-2a 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) IFN2a 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 antibodyPEGradiometal 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.

These are just a few of the biomedical applications

Other PEGylated products

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.

PEG conjugates PEGasparaginase (Oncaspar) PEGadenosine deaminase (Adagen) PEGinterferon 2a (Pegasys) PEGinterferon 2b (PEG Intron) PEGG-CSF (pegfilgrastim, Neulasta) PEGgrowth hormone receptor antagonist (Pegvisomant, Branched) Somavert PEGanti-VEGF aptamer (Pegaptanib, Macugen)

Approved PEG ConjugatesDisease Acute lymphoblastic leukemia Severe combined immunodeficiency disease Hepatitis C (SCID) Hepatitis C and clinical trials for cancer, multiple sclerosis, Treating of neutropenia during HIV/AIDS chemotherapy Acromegaly Macular degeneration (agerelated)

Limitations in the use of PEGThe 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 conjugates characteristics. excretion from the body at high molecular weights PEG can accumulate in the liver, leading to macromolecular syndrome.

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