Transcript PROTEIN AND PEPTIDE DRUG DELIVERY

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Department of Pharmaceutics.
CONTENTS

Protein & Peptides
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Structure of protein
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Classification of protein
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Stability problems
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Formulation Aspects
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Barriers
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Approaches
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Parenteral delivery of protein and peptide
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Ocular delivery of protein & peptide
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Conclusion
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References
Definitions
 Protein: polypeptides which occur naturally and have
a defined sequence of amino acids and a threedimensional structure (e.g. insulin).
 Peptide: a short chain of amino acid residues with a
defined sequence (e.g. leuprolide).
 Proteins - Chains of amino acids, each joined together by
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a specific type of covalent bond
Proteins formed by joining same 20 amino acids in many
different combinations and sequences
Protein > 50 amino acids
peptide < 50 amino acids
Function of a protein determined by its non-covalent 3D
structure
Structure of peptides and proteins
 Proteins have in increasing order of complexity
 Primary structure- The amino acid sequence.
 Secondary
structure-
Regularly
repeating
local
structures stabilized by hydrogen bond.
 Tertiary structure-Three dimensional
structure of
polypeptide
 Quaternary structure-The structure formed by several
protein molecules (polypeptide chains).
Protein Structure
Protein Structure
Protein Structure
Lactate Dehydrogenase:
Mixed α /β
Immunoglobulin
Fold: β
Hemoglobin B
Chain: α
Functions
 Transport and storage of small molecules.
 Coordinated motion via muscle contraction.
 Mechanical support from fibrous protein.
 Generation and transmission of nerve impulses.
 Enzymatic catalysis.
 Immune protection through antibodies.
 Control of growth and differentiation via hormones
Applications of protein and
peptide drug delivery system
 Erythropoietin used for production of RBC.
 Tissue plasminogen activator is used for Heart
attack, Stroke.
 Oxytocin maintain labor pain.
 Bradykinin increases the peripheral circulation.
 Somatostatin decrease bleeding in gastric ulcer.
 Gonadotropin induce ovulation.
 Insulin maintains blood sugar level.
Advantages
 Improved patient compliance.
 Reduced frequency of administration.
 Reduced adverse effect profile.
 Potential to reduce product development
time and costs.
 Possible new and broader therapeutic
applications.
Disadvantages
 Very large and unstable molecules.
 Structure is held together by weak
noncovalent forces.
 Easily destroyed by relatively mild storage
conditions and gastric juices.
 Hard to obtain in large quantities.
Problem with Proteins
(in vivo – in the body)
 Elimination by B and T cells
 Proteolysis by endo/exo peptidases
 Small proteins filtered out by the kidneys
very quickly
 Unwanted allergic reactions may develop
(even toxicity)
 Loss due to insolubility/adsorption
CLASSIFICATION OF PROTEINS
According to their biological roles:
 Enzymes – Catalyses virtually all chemical reactions
i.e. 6GDH
 Transport proteins i.e. Haemoglobin of erythrocytes
 Contractile or Motile proteins i.e. Actin and Myosin
 Structural proteins i.e. Collagen
 Defense proteins i.e. Immunoglobulin's and
Antibodies
 Regulatory proteins i.e. Insulin
 Nutrient and storage proteins i.e. Ovalbumin
Stability Profile
 Protein and peptide drugs have poor stability profile.
 The degradation pathways of this class of drugs are due to
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chemical and physical instability
The high chemical and Physical instability presents peculiar
difficulties in the purification, separation, formulation,
storage and delivery of these compounds.
Physical instability involves transformations in the
secondary, tertiary, or quaternary structure of the molecule.
These changes are manifested as denaturation,
aggregation, precipitation and adsorption onto surfaces.
Chemical instability involves alteration in the molecular
structure producing a new chemical entity, by bond
formation or cleavage
Physical Instability
 Denaturation
 Aggregation
 Precipitation
 Adsorption onto surfaces
Denaturation
 Peptides and proteins are comprised of amino acid residues and
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non-polar amino acid residues.
The hydrophobic, non-polar amino acid residues fold upon
themselves in an aqueous environment to form globular
molecules.
The hydrophilic, polar amino acid residues of these molecules are
exposed to the aqueous environment.
On changing the aqueous environment to non-aqueous, they
start unfolding and thereby exposing their hydrophobic residues
to the hydrophobic environment.
This leads to the rearrangement and loss of quaternary and
tertiary structure. On unfolding hydrophobic and hydrogen
bonds are broken
The term denaturation is used to describe any nonproteolytic
modification of the unique structure of a native protein that
effects
Aggregation
 Some
proteins self-associate in aqueous
solution to form oligomers.
 Insulin, for example, exists in several states:
 The zinc hexamer of insulin is a complex of
insulin and zinc which slowly dissolves into
dimers and eventually monomers following
subcutaneous administration, conferring on it
long acting properties
Surface adsorption and
precipitation
 Adsorption of proteins such as insulin on
surfaces such as glass or plastic in giving sets:
 can reduce the amount of agent reaching the
patient
 can lead to further denaturation, which can then
cause
 precipitation and the physical blocking of
delivery ports in protein pumps.
 Denaturation is facilitated by the presence of a
large head space
 allowing a greater interaction of proteins with
the air–water interface.
Chemical Instability-Oxidation
 Tryptophan, methionine, cysteine, histidine, and tyrosine amino acid
side chains contain functionalities that are susceptible to oxidation.
Methionine and cysteine can be oxidized by atmospheric oxygen and
fluorescent light. Oxidation has been observed both in solution and in
the solid state.
 Oxidation of the methionine residues may cause a loss of bioactivity
and, in the case of cysteine residues, the formation of nonnative
disulphide bonds. Oxidation by atmospheric oxygen or auto-oxidation
can be accelerated in the presence of certain metal ions such as copper
and iron.
 Methionine residues under acidic conditions are especially prone to
oxidation by reagents such as hydrogen peroxide, producing methionine
sulphoxide. Oxidation by peroxide may be a concern if the protein is
processed in a manufacturing area that is sterilized using hydrogen
peroxide vapor or using equipment that is so treated, In this case,
experiments must be performed and procedures put in place to ensure
that the protein is not oxidized during manufacturing.
 Oxidation - conversion RSR’ to RSOR’, RSO2R’ or RSO3R’ (Met &
Cys)
Deamidation
 Deamidation is the hydrolysis of a side chain amide on
glutamine and asparagine residues to yield a
carboxylic acid. The deamidation reaction has been
extensively studied and is widely observed in
therapeutic proteins and peptides. Some protein
delivery system processing and formulation
conditions that result in an increase in temperature
or pH have been shown to facilitate deamidation.
 The deamidation process is important because of
the potential loss in protein activity or function.
Deamidation contributes to the reduction in
catalytic activity of lysozyme and ribonuclease at
high temperatures.
 Deamidation - conversion of Asp-Glu sequences to
a-Asp-Glu or b-Asp-Glu
Peptide Bond Hydrolysis
 Aspartic acid residues have been implicated in
the cleavage of peptide bonds, which have
led to a decrease in biological activity. When
lysozyme was heated to 90-100°C at pH 4,
the loss in biological activity was attributed to
hydrolysis of Asp-X bonds
 Proteolysis
- Asp-Pro, Trypsin (at Lys) or
Chymotrypsin (at Phe/Tyr)
Disulphide Exchange
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Many therapeutic proteins contain cysteine residues that form disulphide
bonds. These bonds are important components of the structural
integrity of proteins. Incorrect linkages of these disulphide bonds often
lead to a change in the three-dimensional structure of the protein and
therefore its biological activity.
The aggregation of lyophilized formulations of bovine serum
albumin, ovalbumin, p-lactoglobulin, and glucose oxidase was
attributed to disulphide interchange.
Racemisation and Beta Elimination
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The reaction proceeds in both acidic and alkaline media, but the
mechanisms are different. In neutral and alkaline media, the reaction is
catalyzed by thiols. Thiols may be introduced during formulation (e.g.,
mercaptoethanol as an antioxidant) or by degradation of existing
disulphide bonds via beta elimination of cysteine residues.
How to Deal with These Problems
 Storage
Formulation
Delivery
Protein Stabilization
 Additives for Protein Stabilization
 Protein Stabilization in the Solid State
 Protein Stability within a Delivery Matrix
 Interactions between the Delivery Matrix and
the Protein
 Internal Environment of the Delivery Matrix
Barriers for Protein and
Peptide drug delivery
 Enzymatic Barrier
 Intestinal Epithelial Barrier
 Capillary Endothelial Barrier
 Blood Brain Barrier
Routes of drug transport across
a mucous cell barrier
Classification
DRUG DELIVERY CLASSIFICATION
Drug Delivery
Route of Administration
Pulmonary
Parenteral
Drug Modification
Transdermal Miscellaneous
Implants Ocular
Oral
Nasal
PEGylation
Pro-drug
Polymer depot
Protein Formulations
1
• Protein sequence modification (site directed
mutagenisis)
• PEGylation
• Proteinylation
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3
• Microspher encapsulation
• Formulating with permeabilizers
Site Directed Mutagenesis
 Allows amino acid substitutions at specific
sites in a protein
 i.e. substituting a Met to a Leu will reduce
likelihood of oxidation
E343H
PEGylation
 PEG is a non-toxic, hydrophilic, FDA
approved, uncharged polymer
 Increases in vivo half life
 Decreases immunogenicity
 Increases protease resistance
 Increases stability
Proteinylation
 Attachment of additional or secondary
(nonimmunogenic) proteins for in vivo protection
 Cross-linking with Serum Albumin
 Increases in vivo half life
 Cross-linking or connecting by protein engineering
with antibody fragments
Proteinylation
+
Protein drug
scfc (antibody)
Parenteral Drug formulations
 Intravenous
 Intramuscular
 Subcutaneous
 Intradermal
Advantages & Disadvantages
Advantages
Disadvantages
• Route of delivery for 95% of
proteins
• Allows rapid and complete
action.
• Avoids first pass metabolism
• Problems with overdosing, necrosis
• Local tissue
reactions/hypersensitivity
• Everyone hates getting a needle
 Polymers should be:
 Biodegradable
 Bio-compatible
 Non-toxic
 Examples:
 Natural- Chitosan, Dextrin
 Synthetic- Polylactides/ glycolide Polyanhydrides
 Polyphosphoesters
Release Mechanism
 Diffusion of drug out of the polymer
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 Drug Release by Polymer Degradation
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Hydrolysis
Enzymatic (Phosphotases;
Proteases etc.)
Microsphere Drug delivery
Two types of microspheres
Nonbiodegradable
e.g, ceramic particles
polyethylene co-vinyl acetate
polymethacrylic acid/PEG
Biodegradable (preferred)
e.g, gelatin
polylactic-co-glycolic acid
(PLGA)
Magnetic Targeted Carriers(MTCs)
Founder of FeRx and pioneer of magnetic
targeted drug delivery is Dr. Kenneth Widder
 Microparticles, composed of
elemental iron and activated carbon
 Drug is adsorbed into the MTCs and
transported
The particles serve as delivery
vehicles to the area of the tumor for
site-specific targeting
Liposomal Drug delivery
Spherical vesicles with a phospholipid bilayer
 E.g, Bleomycin encapsulated in
thermo sensitive liposome enhanced
antitumor activity and reduced normal
tissue toxicity
 Liposome have recently been used
successfully as vehicles for vaccines
Hydrogel Based Drug Delivery
Hydrogels are three dimensional networks of hydrophilic
polymers that are insoluble
Emulsion
& Cellular carriers
 Emulsion :
Emulsion can be used for parenteral delivery of protein
and peptides. Multiple emulsion further prolong the
release of drug.
e.g. subcutaneous administration of muramyl
dipeptide in a w/o emulsion
 Cellular carriers:
Protein and peptides can be incorporated in
erythrocytes to achieve the prolong release or
targeting.
Resealed erythrocytes as delivery system for creactive protein, and mainly used to target liver and
spleen.
Emulsification
Coacervation
Extrusion And Spraying
Ocular Delivery of Peptide &
Protein
 Relevant anatomy and Physiology of the Human eye
 Diameter of 23 mm
 Three layers
 Outermost coat : the clear, transparent cornea and
the white, opaque sclera
 Middle layer : the iris anteriorly, the choroid
posteriorly, and the ciliary body at the intermediate
part
 Inner layer : retina
Peptides Useful in Ocular
Pathology
To treat infections and enzymes used to promote wound healing.
a. Bacitracin
 The drug is applied topically to the eye for a variety of conditions,
including eyelid burns and corneal superficial punctate keratits .It is also
used to treat optic neuritis.
 Commercial bacitracin is a mixture of at least nine bacitracins, which is
used for its antibacterial activity
b. Chymotrypsin
 Chymotrypsin is used clinically in the eye for enzymatic intracarpsular
lens extraction.
IMPLANTS
PROTEINS IN PUMPS :
1.Infusaid Model 400 Implantable Pump
2 .Mechanical Insulin Pumps
 Formulation is the beginning of successful drug delivery
 Multiple potential interactions between the protein and
the pump
 Control of the material interface is most important
 Device design and formulation need to work together
and be regulated together.
ORAL PROTEIN DELIVERY :
 Oral Insulin :
 Buccal aerosol delivery system developed by Generex
 Insulin is absorbed through thin tissue layers in mouth and throat
 Insulin is formulated with a variety of additives and stabilizers to
prevent denaturation on aerosolization and to stabilize aerosol
particles
 PH SENSITIVE MICROSPHERES :
 Gel/Microsphere system with polymethacrylic acid + PEG
 In stomach (pH 2) pores in the polymer shrink and prevent protein
release
 In neutral pH (found in small intestine) the pores swell and release
protein
 Process of shrinking and swelling is called complexation (smart
materials)
NASAL DELIVERY OF PROTEINS :
 Extensive microcirculation network underneath the nasal
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mucosa
Drug absorbed nasally can directly enter the systemic
circulation before passing through the hepatic circulation
The nasal administration of peptides has attracted much
interest now a days due to
- Relatively rapid absorption of drug
- Little metabolic degradation
- Relative ease of administration
- Selective to peptide structure and size
Enhancement of nasal absorption of insulin using polyacrylic
acid as a vehicle
Enhancement in the nasal absorption of insulin entrapped in
liposomes through the nasal mucosa of rabbits
Administration of insulin (1 IU/ kg) via the nasal route caused a
significant decrease in the plasma glucose level
The nasal route appears to be a viable means of systemically
delivering many small peptides
PULMONARY DELIVERY :
 Deep lung, an attractive site of protein delivery due to
- Relatively large surface area (100m2)
- Rapid absorption of drug into the blood stream
through the alveoli
 Dura and Inhale developed dry powder delivery systems
for proteins
 40% of the insulin administered in an aerosol, to the
trachea of anaesthetized rabbit was absorbed
 Albumin was largely absorbed within 48 hours of
instillation into the lungs of guinea pigs and dogs
RECTAL DELIVERY
 The rectal delivery offers many advantages
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- Avoidance of drug dilution prior to
reaching the systemic circulation
- Reduction in first-pass metabolism
- Rapid systemic absorption
- Safe and convenient especially in case of
neonates and infants
- Greater dose may be administered
- Withdrawal of drug is possible in case of
adverse effects
 Administration of insulin using the rectal route
shows systemic absorption
TRANSDERMAL PATCHES :
 Proteins embedded in a simple matrix
with appropriate additives
 Patch is coated with small needles that
penetrate the dermal layer
 Proteins diffuse directly into the blood
stream via capillaries
 Less painful form of parenteral drug
delivery
REFERENCES :
 Advances in controlled and Noval drug delivary by N.K.
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JAIN.
Oral protein and peptide drug delivery. In: Binghe W, Teruna
S, Richard S, editors. Drug delivery: Principles and
applications. New Jersey:Wiley Interscience:p.189.Rick.s.
Adessi C, Sotto C. Converting a peptide into a drug:
Strategies to improve stability and bioavailability. Curr Med
Chem. 2002;9:963–78.
Adessi C, Sotto C. Strategies to improve stability and
bioavailability of peptide drugs. Frontiers Med Chem.
2004;1:513–27.
Sayani AP, Chien YW. Systemic delivery of peptides and
proteins across absorptive mucosae. Crit Rev Ther Drug
Carrier Syst. 1996;13:85–184.