Detailed information about proteins and peptides and barriers for protein delivery

1. PROTEIN AND PEPTIDE DELIVERY
Presented By- Ashwini K. Bawankule
M. Pharm I year
(Department of Pharmaceutics)
Smt. Kishoritai Bhoyar College of Pharmacy, Kamptee

2. CONTENTS-
Introduction
Peptide and Protein structure
Physicochemical properties of peptides and proteins
Instability of proteins and peptides
Need of protein and peptide DDS
Advantages of protein and peptide DDS
Function of protein and peptide DDS
Barriers for protein delivery
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3. INTRODUCTION-
 Protein and Peptide drug delivery system are the Novel drug Delivery
System.
 Proteins and peptides are the most abundant components of biological
cells.
 They act as enzymes, hormones, structural element and
immunoglobulin.
 The term Protein is derived from a Greek word Proteios Means
Holding the first Place
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 Proteins are the high molecular weight mixed polymer of Alpha amino
acids joined together the Peptide Linkages.
 In Protein mainly contain Carbon, Nitrogen, Oxygen and Sulphur
Molecule
 Peptides are the Condensation Product of Alpha Amino acids
 The twenty different naturally occurring amino acids join with each
other by peptide bonds and build polymers referred to peptides and
proteins.
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5.  Although the distinction between peptides and proteins are - peptide
contains less than 20 amino acids, having a molecular weight less than
5000, while a protein possesses 50 or more amino acids and its
molecular weight lies above this value.
 In case of Peptide-
 the two amino acids are condensed to from dipeptides,
three are condensed to forms Tripeptides,
Four are condensed to from Tetra peptide and
2-20 amino acids are condensed to form Polypeptides.
 The Polymers of 100 and more than 100 Amino acid called has Proteins
…..continue
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6. Proteins are classified into two types
Depending on
Solubility of protein Complexity in structure
Globular protein Fibrous protein
Soluble in water or
common solvents
Insoluble in water and
common solvents
Simple protein Conjugated
protein
Derived
protein
Contains
only one
amino acids
Contains amino
acids and non
protein parts
Hydrolysis
product formed
by the action of
the physiological
agents like heat,
chemical agent,
and enzymatic
actions
Ex. Keratin,
collagen and elastin
Polypeptides arranged
in parallel position
Polypeptide chains are
tightly folded into
compact globular or
spherical shapes
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7.  The most of pharmaceutical proteins and peptides are absorbed IM, IV
and SC route of absorption, but the oral route is more convenient for
absorption of protein as compared to other.
 Various problems associated with administration of protein and peptide
drugs are needed to overcome by different pharmaceutical approaches.
 Several approaches available for maximizing pharmacokinetic and
pharmacodynamics properties are-
 chemical modification,
formulation vehicles,
mucoadhesive polymeric system,
use of enzyme inhibitors,
absorption enhancers,
penetration enhancers etc.
…..continue
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8. PEPTIDE AND PROTEIN STRUCTURE-
 All peptides and proteins are polymers of amino acids connected via
amide linkages referred to as peptide bonds
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9. PROTEIN
STRUCTURE
Primary Structure Quaternary StructureSecondary Structure
Specific sequence
of amino acids in it
Respective arrangement of
individual amino acids
along the polypeptide
backbone
Determined
genetically by the
sequence of
nucleotides in DNA
Results in very specific and
well-ordered structures as
helices, loops, β-strands
and β-turns
Tertiary Structure
Three dimensional
arrangement of a single
protein molecule
Form of protein as it
exists in solid state
or in solution state
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10. Peptide Structure-
 The peptide chain usually consists of – Polyamide backbone
 At one end of the peptide chain is a free amino group
And at the other end a free carboxylic acid group
 The side chains along the backbone, if any, are derived from the
amino acids that contribute the chain
containing
Tetrahedral carbon atoms
between
Amide groups
NH2-C-C-C-C-C-COOH
Fig. General structure of peptide
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11. Physicochemical Properties of proteins and peptides-
 Physical Properties-
 Dissociation
 Optical activity
 Solubility, hydration and swelling power
 Foam formation and foam stabilization
 Emulsifying effect
 Chemical Properties-
 Arginine residue
 Glutamic and aspartic acid residue
 Cysteine residue
 Methionine residue
 Histidine residue
 Tryptophan residue
 Tyrosine residue
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12. Stability of Protein and Peptides-
 Physical Stability-
Denaturation
Adsorption
Aggregation and precipitation
 Chemical Stability-
Deamidation
Oxidation and reduction
Proteolysis
Disulphide exchange
Recemization
β-Elimination
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13. Denaturation-
 The denaturation of Protein Molecule refers to the loss or damage of the
globular structure of protein molecule leads to protein unfolding.
 The physical denaturation is may be caused by the changes in the
environment of protein molecules such as temperature, pH,
introduction of hydrophobic surfaces or by introduction of interfaces by
the addition of organic solvents.
 Conditions that denature proteins includes-
 Solvent changes from an aqueous to organic solvents(e.g. alcohols,
acetone) or to a mixed solvent as alcohol and water
 pH change- alters the ionization of the carboxylic acid and amino
groups of amino acids and thereby the changes carried by the
molecules
PHYSICAL STABILITY
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 Alteration in ionic strength affect the charge carried by molecules as
well.
 Temperature rise leads to an increment in the thermal energy of the
molecules, which may break the hydrogen bonds that otherwise
stabilize the secondary, tertiary and quaternary structure of these
entities. Even chemical bonds are broken at very high temperature
 With a rearrangement in structure the inherent biological activity may
be lost with an emergence of totally new range of activity.
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15.  Denaturation can either be reversible or irreversible
In reversible denaturation
 However in the case of irreversible denaturation
…..continue
the unfolding of the polypeptide to their original state reverses the
conformational changes in the molecules
For example, a denatured protein is restored on the denaturant
the unfolded proteins fail to restore their original structure
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16.  This may be due to the fact that the protein may undergo some physical
or chemical process that inhibits the original pattern of folding, or even
the proteins may be misfolded that disallows their proper renaturation
 Denaturation may lead to-
decrease in solubility,
alteration in surface tension,
loss of crystallizing ability,
changes in constituent group reactivity and molecular profile,
vulnerability to enzymatic degradation,
loss or alteration of antigenicity
 loss of specific biological activity
…..continue
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17. Adsorption-
 Peptides and proteins posses both polar and nonpolar residues and are
thereby amphiphilic in nature hence they tend to adsorb at interfaces
such as air-water and air-solid
 A conformational rearrangement leading to denaturation can be induce
by their interfacial adsorption
 Once the protein molecules are adsorbed, they form some short range
bonds with the surface resulting into further denaturation of
polypeptide moieties
 As the adsorption of peptides and proteins at the interface is rapid, the
rate of conformational changes will be relatively slower
 On adsorption there may be a loss or change in biological activity as the
molecular structure is rearranged
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18. Aggregation and Precipitation-
 The denatured, unfolded protein may rearrange in such a manner that
hydrophobic amino acid residue of various molecules associate together
to form the aggregates
 If the aggregation is on a macroscopic scale, precipitation occurs
 Interfacial adsorption may be followed by aggregation and precipitation
 The extent to which aggregation and precipitation occur is defined by
the relative hydrophilicity of the surfaces in contact with the
polypeptide or protein solution
 Insulin form finely divided precipitates on the walls of the containers,
referred to as frosting
 Agitation of polypeptide and protein solutions introduces air bubbles,
thereby increasing the hydrophobic air interface
 In this process, aggregation and precipitation of polypeptides or
proteins is augmented
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19. Deamidation-
 This reaction involves the hydrolysis of the side chain amide linkage of an
amino acid residue leading to the formation of a free carboxylic acid
 Asparagine (Asn) and Glutamine (Glu) are particularly susceptible to
deamidation especially when the C-amino acid is Glycine (Gly) or Serine (Ser)
 The deamidation of Asn residue is accelerated at neutral and alkaline pH
 In-vivo deamidation is observed with-
 Human growth hormone (hGH)
 Bovine growth hormone (bGH)
 Prolactin
 Adrenocorticotropic hormone (ACTH)
 Insulin
 Lysozyme
 Secretin
CHEMICAL STABILITY
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20. Oxidation and Reduction-
 Oxidation of susceptible amino acids is one of the major degradation
pathways for peptides and proteins
 Oxidation commonly occurs during isolation, synthesis and storage of
proteins
 Mild atmospheric oxygen, temperature, pH, trace amount of metal ions
and buffers influence the oxidation reaction
 The oxidation of amino acid residue is followed by a significant loss of
biological activity
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21. Proteolysis-
 The hydrolysis of peptide bonds within the polypeptide or protein
destroys or at least reduces its activity
 Proteolysis may occur on exposing the proteins to harsh conditions,
such as prolonged exposure to extremes of pH or high temperature or
proteolytic enzymes
 Bacterial contamination is the most common source of proteases
 This can be avoided by storing the protein in the cold under sterile
conditions
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22. Disulphide Exchange-
 Disulphide bonds may break and reform with incorrect pairings
This results in an alteration in the 3D structure followed by resultant
change in biological activity
This reaction can be catalysed in neutral as well as in alkaline media by
thiols, which may arise as a result of hydrolytic cleavage of disulphides.
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23. Racemization-
 Racemization is the alteration of L-amino acids to D,L-mixtures
 With the exception of Glycine, all the mammalian amino acids are chiral
at the carbon bearing chain and are susceptible to base-catalysed
racemization
 Racemization may form peptide bonds that are sensitive to proteolytic
enzymes
 This reaction can be catalysed in neutral and alkaline media by thiols,
which may arise as a result of hydrolytic cleavage of disulphides
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24. β-Elimination
 The mechanism involved in the β-elimination is similar to the
racemization, i.e. it proceeds through a carbanion intermediate
 Higher elimination rate prevails under alkaline conditions which
ultimately lead to loss of biological activity
 Protein residues susceptible to β-elimination under alkaline conditions
include Cysteine, Lysine, Phenylalanine and Serine
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25. NEED OF PROTEIN AND PEPTIDE DRUG
DELIVERY SYSTEM-
 The protein and peptides are very important in biological cells
and Organic Molecules
 In the Absence of proteins and peptides causes diseases like
Diabetes mellitus. (Caused due to the lack of protein called
INSULIN)
 Now a days R-DNA technology and hybridoma techniques also
used in protein and peptide based pharmaceuticals
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26. ADVANTAGES OF PROTEIN AND
PEPTIDE DRUG DELIVERY SYSTEM
 Erythropoietin is mainly used for production of RBC
 The protein Tissue plasminogen activator is used for Heart
attack, Stroke
 Oxytocin is used in management of labor pain
 Bradykinin increases the peripheral circulation
 Somatostatin decrease bleeding in gastric ulcer
 Gonadotropin induce ovulation
 Insulin maintain blood sugar level
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27. FUNCTIONS OF PROTEIN AND
PEPTIDE DRUG DELIVERY SYSTEM
 Transport and storage of small molecules and biological molecules
 Coordinated motion via muscle contraction
 The Mechanical support from fibrous protein
 Generation and transmission of nerve impulses
 Enzymatic catalysis in biochemical reactions
 The Immune protection through antibodies
 The Control of growth and differentiation via hormones
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28. BARRIERS TO PEPTIDE AND PROTEIN
DELIVERY
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29. BARRIERS TO PEPTIDE AND PROTEIN DELIVERY-
Enzymatic Barriers
Intestinal Epithelial Barriers
Capillary Endothelial Barriers
Blood Brain Barriers
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30. Enzymatic Barriers
 Enzymatic barrier is the most important barrier that limits the
absorption of protein/peptide drugs from gastrointestinal tract
 The enzymatic degradation is brought about by mainly via two ways-
of peptide bonds
• Insulin-degrading enzyme
• Enkephalinase
• Angiotensin-converting enzyme
• Renin
by
HYDROLYTIC CLEAVAGE CHEMICAL MODIFICATION
of protein such as
• Phosphorylation by kinases
• Oxidation by xanthine oxidase or glucose
oxidase
• Carbamylation by pronase,
chymotrypsin and trypsin
• Denaturation
• Ubiquitization 30

31. .....continue
The enzymatic barrier predominantly has three essential features-
The proteolytic enzymes
are ubiquitous and hence
the peptides/proteins are
prone to degradation in
multiple anatomical sites
viz.
The site of administration,
blood, vascular endothelium,
kidney, liver etc.
Thus they have to be
guarded against degradation
in all the anatomical sites to
ensure that they reach the
site of action in intact form 31
The anatomical site where
peptide/ protein is located is
likely to have the presence of all
the proteases capable of
degrading the same
Thus, the peptide/ protein has to
be protected from all the enzymes
before it can elicit the
pharmacological action
A particular peptide/
protein may be prone to the
degradation at more than
one linkage within the
backbone, each locus being
mediated by a specific
protease
Thus all the vulnerable
linkages entail for
protection or modification

32. Intestinal Epithelial Barriers
 There are several mechanisms that are involved in transport
of peptide/ protein drugs across the intestinal epithelium and
are as follows-
 Passive and carrier mediated transport
 Endocytosis and transcytosis
 Paracellular movement
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33. Passive and Carrier Mediated Transport-
 Active transport appears to be predominant mechanism
 Peptides with more than three or four amino acid residues are
transported across the intestinal mucosa by the peptide transport
system
 Stereoisomerism, side-chain length and N- and C- terminal
substitution are reported to affect dipeptide absorption
 L-Ala-L-Phe and L-Leu-L-Leu were found to absorb more rapidly
than their D isomers
 Acidic or basic dipeptides have lower affinity compared to neutral
dipeptides for peptide transport
Decreases the affinity of dipeptide transport system
Methylation and acylation of N- terminal amino acid groups
Esterification of C- terminal carboxyl groups
Presence of - linkage
Incorporation of β-amino acids
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34. Endocytosis and Transcytosis-
 Cellular internalization of peptides/ proteins may occur by
endocytosis whereby
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peptides/ proteins which are too large to be absorbed by carrier
mediated transport, are taken up
The different pathways of endocytosis
Fluid-phase
Endocytosis
Receptor Mediated
Endocytosis

35. Fluid-phase Endocytosis (FPE)
Also referred to as nonspecific endocytosis or pinocytosis
The macromolecules dissolved in the extracellular fluid are incorporated by bulk
transport into the fluid phase of endocytosis vesicles
Receptor-mediated Endocytosis (RME)
Also referred to as specific endocytosis
The macromolecules are first bound to the plasma membrane before they are
incorporated into endocytic vesicles
When the membrane binding site is a specific receptor for the macromolecule
involved, the process is referred to as receptor-mediated endocytosis (RME)
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36. Transcytosis
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The process of transcytosis categorically combines elements of
membrane protein sorting and endocytosis
In polarized cell, such as the intestinal epithelial cells, some endosomes
carrying the ligands or the receptor-ligand complexes bypass the
lysosomes and migrate towards the basolateral membrane
Thereby the ligand is released in the extracellular space bound by the
basolateral membrane
This process is known as transcytosis
This pathway presents an important route for mucosal transport of peptides and proteins
The small intestine epithelial mucosa serves as a barrier to the permeation of macromolecules

37. Paracellular Movement-
•This involves the movement between the tight junctions between cells
and/or across the spaces formed when cells are extruded into the intestinal
lumen
•Paracellular movement plays an important role in the absorption of water
from the intestinal lumen
•The passage of water across the tight junction is capable of carrying the
dissolved drug or facilitates the transport of macromolecules, which otherwise
cannot travel across the apical membrane
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39. Capillary Endothelial Barrier-
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The luminal surface of capillaries consists of the plasma membrane surface
monolayer of endothelial cells
joined together by more or less continuous tight or occluding junctions
of a
Capillaries can be broadly classified into three major classes
Sinusoidal or discontinuous Fenestrated Continuous

40. …..continue
•To cross the capillary endothelium the peptides/ proteins must pass between
the cells or alternatively travels across the endothelium cells themselves.
•Solutes that travels across the endothelial cells membrane may get modified
or metabolized by cytoplasmic enzymes.
•Thus, the endothelial passage poses metabolic or enzymatic barrier to the
solute passage.
•The tight junction of endothelium serve as the major extracellular barrier to
solute exchange.
•These junctions, hamper the transcapillary movement of macromolecular
tracers injected into plasma or interstitial fluid.
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41. The endothelial barrier is modulated by several physiological
parameters
Glucocorticoids tighten
the blood-brain barrier
by decreasing vesicular
transport
Angiotensin, bradykinin,
histamine and serotonin
increase vascular
permeability by opening
large gaps in the
endothelial junctions or
post capillary venules
Inflammatory agents,
vasopressive agents,
certain hormones and
hypertensive agents,
relax the endothelial
barrier
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42. Blood Brain Barrier (BBB)-
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•The blood brain barrier (BBB) is a collection of cells that press together to
block many substances from entering the brain while allowing others to pass
•BBB is formed by a single layer of tile-like endothelial cells that line the inner
surfaces of capillaries in the brain
•In capillaries outside the brain, these cells have gaps between them
•Through these intercellular spaces the water, ions and some molecules diffuse
•On the other hand, the endothelial cell linings of capillaries in the brain are
tightly packed, allowing no diffusion except water
•BBB prevents toxins from entering the blood stream

43. 43
The circulatory molecules use a number of different mechanisms for their
transport across BBB. These includes-
Lipid mediated
transport of
small but
lipophilic
molecules
Carrier mediated
transport of
hydrophilic
nutrients and their
drug analogues
Plasma protein
mediated transport
of acidic drugs,
peptides and
highly lipophilic
drugs
Bulk flow
transcytosis could
be pinocytosis or
tubulocanalecular
molecular transport

44. Delivery of Peptide and Protein Drugs
 Oral Route
 Buccal Route
 Nasal Route
 Transdermal Route
 Pulmonary Route
 Rectal Route
 Parenteral Route
 Ocular Route
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