Protein and peptide delivery system

Protein and Peptide
Delivery
Under the guidance of
Dr. R.R. Shah
Prepared by
Nikita Gangwani
M.Pharm 1st year
1

Sr No. Contents
1 Introduction
2 Protein structure
3 Protein functions
4 Protein and peptide delivery
5 Advantages of the delivery
6 Delivery challenges
7 Barriers for protein delivery
8 Physicochemical properties of proteins and peptides
9 Routes of drug delivery
10 Evaluation of proteins and peptides
11 Recent advances
2

Introduction
• PROTEINS:
Proteins are the natural polymer molecules made of
amino acids units and joined together by
polypeptide bonds (Protein > 50 amino acids)
• PEPTIDES:
These are short polymers formed by linking in a
defined order of amino acids (peptide < 50 amino
acids)
3

Structure of protein
 Primary structure- Array of number and specific sequence of amino
acids in protein structure
 Secondary structure- Regularly repeating local structures stabilized
by hydrogen bond.
 Tertiary structure-Three dimensional structure of functional
protein
 Quaternary structure- Contains two or more polypeptide chains
associated by non-covalent forces
4

Role of proteins
1. STRUCTURAL FUNCTIONS:
Responsible for strength of body including
collagen, elastin found in bone matrix,
vascular system and other organs
2. DYNAMIC FUNCTIONS:
Acts as enzymes, hormones, blood clotting
factors, immunoglobulins, membrane
receptors, besides their function in genetic
control, muscle contraction.
6

Protein and Peptide Drug Delivery
• Comes under novel drug delivery system.
• Most abundant material of living system and biological cells
• Proteins and peptides are the main building blocks of life and
are now evolving as a very promising brand of therapeutic
entities. Therapeutic proteins have increased dramatically in
number and frequency of use since the introduction of first
recombinant protein viz, human insulin, 25 years ago.
• Delivery of proteins in body have limited its use
• Due to rapid progress in biotechnology, as well as gene
technology, production of potential therapeutic peptides and
proteins in commercial quantities possible
7

Advantages
• The mode of delivery is convenient, i.e., eye drops.
• Systemic absorption is extremely rapid.
• Avoid first-pass metabolism.
• The formulation can be designed to prolong drug
action and/or reduce drug concentrations to achieve
consistent drug action with least side effects.
• The drug delivery can be controlled precisely
8

Delivery Challenges
• Low permeability due to large molecular size
• Susceptibility to enzyme degradation
• Short plasma half life
• Immunogenicity
• Aggregation
• Denaturation
• Protein binding
9

Barriers for protein delivery
• Enzymatic barriers
• Intestinal Epithelial Barrier
• Capillary Endothelial Barrier
• Blood Brain Barrier(BBB)
11

ENZYMATIC BARRIERS
• The enzymatic degradation is brought about mainly via
two ways:
1.Hydrolytic cleavage of peptide bonds by processes,
such as insulin-degrading enzyme, angiotensin-
converting enzymes and renin. Proteolysis is an
irreversible reaction and hence potentially causes the
of damage of the peptide and protein drug.
2.Chemical modification of protein such as
phosphorylation by kinases, oxidation by xanthine
oxidase or glucose oxidase.
• It limits absorption of protein drugs from G.I tract
12

INTESTINAL EPITHELIAL
BARRIER
• Serves as a barrier for transport of protein
drugs across the intestinal epithelium
• Several mechanisms that are involved in the
transport of peptide /protein drugs across the
intestinal epithelium are
A. Passive & carrier mediated transport
B. Endocytosis & Transcytosis
C. Paracellular Movement.
13

A. Passive and carrier mediated transport:
• Active transport appears to be the predominant
mechanism. Accounts for the extensive
absorption of di-and tripeptides from small
intestine
B. Endocytosis and transcytosis
• Cellular internalization of peptides/proteins
may occur by Endocytosis whereby peptide
/proteins, which are too large to be absorbed
by carrier mediated transport, are taken up.
• The Different pathways of Endocytosis
involve Phagocytosis(cell eating),
Pinocytosis(cell drinking)
14

C. Paracellular movement:
• The transport of drugs through the junction
between the GI epithelial cells.
• Two mechanism involved in drug absorption
are-1.Permeation through tight junction of
epithelial cells (insulin) 2.Persorption
The small intestine epithelial mucosa serves as
a barrier to the permeation of macromolecules
15

CAPILLARY ENDOTHELIAL
BARRIER
• To cross the capillary endothelium the
peptides/proteins must pass between the cells or
alternatively transverse across the endothelial cells
themselves.
• Solutes that transverse the endothelial cell
membrane may get modified or metabolized by
cytoplasmic enzymes.
• Thus, the endothelial passage poses metabolic or
enzymatic barrier to the solution passage.
16

BLOOD BRAIN BARRIER (BBB)
• The blood-brain barrier (BBB) represents a major obstacle to the delivery of
proteins to the brain compartment. It consists of several barriers with the two
that are best described being the vascular BBB, and the blood-cerebrospinal
fluid (blood-CSF) barrier
• At both sites, the BBB is formed by a monolayer of cells that are cemented
together by tight junctions and have other mechanisms that control or retard
leakage of plasma into the CNS.
• Allows passage of small, lipophilic, uncharged molecules and gases. Large
molecules like proteins do not pass the BBB easily
17

Physicochemical properties of
proteins and peptides
1. Solubility and partition coefficient:
• Aqueous solubility of peptide is strongly dependent upo
n pH, ionic strength and temperature. At isoelectric poin
t the aqueous solubility of peptide is minimal
• Unless the C and N terminals are blocked, peptides are
very hydrophilic
• Low octanol-water partition coefficient
2.Aggregation, self association and hydrogen bonding:
• Human insulin was found to be more self‐aggregating
than bovine insulin. Additions of additives like non ioni
c surfactants (Pluronic F 68) stabilize the peptide again
st self aggregation 18

3. Molecular size and shape:
• Influence diffusion of drugs through epithelial layers
• Low diffusivities
4. Conformation, stereospecificity and immunogenicity:
• Needs to preserved as change during formulation and
sterilisation will affect membrane permeability
• Peptides are often recognised as immunogenic
• Use of inert polymers like PEG and albumin
5. Electrostatic charges:
Zwitterionic peptide- negative effect on membrane
permeability even though the effective partition
coefficient is high.
19

DRUGDELIVERYCLASSIFICATION
Pulmonary Parenteral Transdermal
Implants Ocular Nasal
Miscellaneous Oral
RouteofAdministration
PEGylation Pro-drug Polymerdepot
DrugModification
DrugDelivery
Routes of drug delivery in protein and peptide
delivery system
20

Parenteral drug delivery systems
• Proteins and peptides are mostly delivered by parenteral
route as it provides the best choice for efficient delivery of
these drugs
• Types of administrations used:
1. Intravascular route:
IV or even intrarterial route preferred
2. Intramuscular route
3. Subcutaneous tissue:
• Drug is administered under the fold of skin in the
subcutaneous tissue.
21

• Controlled release is obtained from
implantable biodegradable polymeric
devices. i.e Polylactic acid, poly (d, l-
lactide co-glycoside)
• Vasopressin,a nonapeptide with a molecula
r weight of 1084 daltons, is an antidiuretic
hormone. Using a device prepared by cove
ring a section of microporous polypropyle
ne (Accurel) tubing with collodion, a long
lasting and constant in-vitro release of
vasopressin was achieved.
22

A. Liposome based drug delivery
• Liposomes are microscopic vesicles composed of one or
more aqueous compartments.
• Liposomes in Proteins delivery :
• Lecithin is used for controlled drug release.
• Bleomycin encapsulated in thermosensitive liposomes
enhanced antitumor activity and reduced normal tissue
toxicity
• Negatively charged liposomes containing Insulin produced a
prolonged hypoglycemic effect in diabetic drugs, which are
injected by subcutaneous injection.
24

B. Hydrogel based drug delivery system
• Hydrogels are three dimensional network of polymers
that are insoluble and have the ability to swell in water.
• Biodegradable hydrogels are used.
Eg, Hydroxymethylacrylate used to minimize mechanical
irritation to surrounding tissue.
• Swelling occurs as a result of change in pH, ionic
strength, solvent composition, pressure, etc
• Drug incorporated in hydrogel- Insulin
25

C. Emulsions based delivery
• Emulsions can be used for parenteral drug delivery of proteins
and peptides used to prolong the release of drug.
E.g. subcutaneous administration of muramyl dipeptide in a w/o
emulsion. It is used to potentiate immune system.
D. PUMPS
• Types of pumps:
1) Implantable infusion pumps: Drug is implanted
subcutaneously,
• Pumps are filled with drug through a septum with a needle.
• Pumps deliver drugs to central vein for 7-14 days at a constant
rate.
2) Mechanical pumps: technically simple, rugged and can be
easily manipulated to deliver peptidal drugs in several
different wave form.
• Example: insulin has been successfully delivered by portable
syringe.
26

3) Osmotic pumps:
• These pumps can be implanted subcutaneously.
• Some of the representative examples of drugs that have been
delivered in osmotic pumps include ACTH, calcitonin, LHRH,
growth hormone, and vasopressin.
E. Peptide carriers
1. Cellular carriers
• Peptide/protein pharmaceuticals can be encapsulated in
erythrocytes to achieve their prolonged release or targeting.
• Advantages: Biodegradability , Non immunogenic profile, Offers
enzymatic and immunological protection to the entrapped drug.
2. Soluble carriers (MACROMOLECULES)
• The peptide/proteins drug can be conjugated with a
polymer/macromolecule.
• Advantages: improves the stability and the plasma half-life of the
protein is increased significantly
• PEG is one of the most widely employed polymers for protein
conjugation.
Eg- Asparaginase with PEG - prolong plasma half life 27

Characters of polymers:
It should be biodegradable.
It should be bio compatible.
Two types of polymers are used:
1)Natural polymers- Collagen , hemoglobin and gelatin.
2)Synthetic polymers- polyesters like polylactides, polyanhydrides,
polyglycolides, polyphosphoesters
Peptides: Calcitonin, somatostatin
Drug release by the polymer:
1. Diffusion of drug out of the polymer:
Governed by ficks law of diffusion
2. Polymer degradation:
Polymer degradation takes place by hydrolysis, enzymatic action
(phosphatases, proteases) after which the drug is released
3. Polymer scaffolds:
• Drug is protected from enzymatic degradation by incorporating it in
polymer matrix at a predetermined rate for a prolonged duration of
action 28

Non parenteral routes of drug
delivery:
• Oral route
• Rectal route
• Nasal route
• Pulmonary route
• Buccal route
• Transdermal route
• Ocular route.
29

Oral route of administration
• Oral route is the most popular route of delivery from the
patient’s point of view
• Main advantages of this route are convenience,
acceptability and high patient compliance.
• The main barriers to effective oral delivery are more
pronounced in case of peptide moieties than traditional drug
candidates, which are as follows:
1. Poor intrinsic permeability of peptides/proteins across
biological membranes
2. Susceptibility to enzymatic attack by intestinal proteases
and peptidases.
3. Rapid post-absorptive clearance.
4. Physical instability like aggregation and adsorption.
30

• Various approaches that are developed for oral delivery of proteins and
peptide drugs to overcome the problems by oral route:
A. Modification by chemical synthesis of prodrugs and analogues
• Done to Improve the Enzymatic Stability as well as Membrane Permeations.
• It is Applicable for the reducing the Immunogenicity.
• Involves two approaches:
1. Amino acid Modifications: The Modification of amino acid is one of the
important approach in which the Substitution of the D- amino acid and L-
amino acid is important to alter the Physiological Properties of Protein and
Peptides.
2. Hydrophobization:
• NOBEX TECHNOLOGY which Involves bonding of
polyethylene glycol (PEG) and alkyl groups or fatty acid radicals to produ
ce desired amphiphilic oligomers. These oligomers are conjugated to prot
eins or peptides to obtain desired amphiphilic products that can transverse
the aqueous and lipid layer of the mucosa and resist degradation
31

B. Enzyme inhibitors
• The Protease inhibitors are Co-administered with
Protein and Peptide to supress the Proteolytic
activity and prevent degradation of proteins.
• Eg: aprotinin (trypsin /chymotrypsin inhibitor),
amastatin, bestatin, boroleucine, puromycin
(aminopeptidase inhibitors), camostatt mesilate
C. Bioadhesive systems:
• By virtue of their mucoadhesive properties,
intensify the contact between dosage form and
the intestinal mucosa and assist the drug to exert
its effects at locally high concentrations within a
restricted area 32

• Due to increased contact to the absorbing mucosa a
steep concentration is maintained and increase in the
absorption and local delivery of drug is seen.
• Ex: Vasopressin Hydroxy propyl methacrylate
nanoparticle cross linked by divinyl glycol in vivo
absorption through rat intestine is increased
D. Carrier systems:
• This strategy is particularly applicable in the case of
poorly absorbed peptides/proteins, which are unstable in
the Gastro intestinal (GI) lumen and their targeting to a
specific tissue or organ is to be affected.
• Lipid carriers and emulsions :
Liposomes comprises of bilayers with an aqueous core,
both lipid soluble and water soluble drugs can be
encapsulated. Solid lipid nanospheres and fat emulsions
can also be used
33

• Emulsomes
• Emulsomes are colloidal drug carrier units.
• A lipoidal drug delivery vehicle and prepared using relatively higher
concentration of lecithin (5-10%).
• The internal phase remains to be in solid or quasi-solid state at
ambient temperatures.
• External phase- w/o/w palmitic acid in octylDecyl triglyceride and
the internal phase- macromolecule (Insulin) for oral administration.
• Particulate Carriers
• Can be replicating and non-replicating in nature.
• The replicating systems comprise of attenuated or genetically
modified strains of viruses and bacteria, e.g. genetically engineered
Vaccine virus and attenuated strains of Salmonella.
• The non-replicating particulate systems are polymeric particles and
lipid containing particles. They encapsulate the drugs within the
particles and thereby lend a protective cover.
34

Recent advances in oral delivery
• Biosante pharma developed a system based on calcium
phosphate to administer an oral form of insulin called
CAPIC
• Nanoparticulate technology; calcium phosphate
particles containing Insulin synthesized in presence of
PEG-3350 and modified by aggregating the particles
with casein to obtain the calcium phosphate-PEG-
insulin-casein oral delivery system
• Oral insulin administration in diabetic mice showed
reduction and maintenance of normal BSL
35

Rectal route of administration
• Rectum is a highly vascularised body cavity and is devoid of
villi.
• Drugs are administered in form of suppositories, gel, dry
powders.
• EX: Insulin, calcitonin
• Advantages:
1. Reduced proteolytic degradation.
2. Improved systemic bioavailability with co-administration of
absorption enhancers. EX: surfactants
3. Large dose can be administered.
• Challenges in delivery:
1. Elimination of drug during bowel movement
2. Low patient compliance
3. Limited surface area available
36

Nasal route of administration
• The nasal route has been employed for producing local action on the
mucosa which is more permeable compared to oral mucosa.
• large surface area, highly vascularized mucosa, porous endothelial
membrane
• Advantages:
1. Rapid onset of action
2. First pass metabolism can be avoided
3. Direct nose to brain delivery of drug can be achieved as it can bypass
blood-brain barrier.
• Disadvantages:
1. Long-term usage causes nasal mucosal irritation and pathological changes
therein
2. Low systemic bioavailability and variable absorption
3. Peptidases and proteases present in the mucus or associated with nasal
membrane serve as enzymatic barrier in protein/peptide absorption
37

• Various approaches for Nasal Delivery of peptide/protein
drugs are:
1. Viscosity modification
The clearance time from the nasal cavity can be delayed by
using solutions with higher viscosity. For example the half
time of clearance could be increased significantly with 0.6 %
of hydroxypropyl methylcellulose.
2. pH Modification
Peptides and proteins usually exhibit the lowest solubility at
their isoelectric point. Thus, by adjusting the pH farther away
from the isoelectric point of a particular peptide, its solubility
can be increased. Satisfactory nasal absorption of insulin was
observed with sodium deoxycholate, that insulin is capable of
crossing the nasal membrane in an acidic medium.
• Synarel spray- Nafarelin, is a gonadotropin-releasing
hormone agonist (GnRH agonist) medication which is used in
the treatment of endometriosis 38

Pulmonary route of administration
• Lungs are attractive site for systemic delivery of proteins and
peptides because of their enormous surface area(70 sq.m) and high
vascularization. Alveoli and lungs are the absorption sites and
drugs are absorbed through lungs by carrier mediated transport
• Advantages:
1. Decrease in dose requirement.
2. Fast absorption owing to thin alveolar epithelial membrane
3. Avoidance of FPM and harsh conditions of the gut
• Disadvantages:
1. Inflammation may be observed in lungs.
2. Degree of bioavailability was less due to hydrolytic enzymes
present in lungs
3. Central airway epithelium is largely constituted of ciliated
columnar cells which expresses tight intercellular junctions-
significantly limit paracellular transport of proteins and peptides
39

• Particle characteristics such as aerodynamic diameter can be
engineered to deliver particles to different areas of the lung.
The aerodynamic diameter da is derived from Stoke's Law
and is defined by:
da=(ρp/ρ0)0.5dg
Where ρp is the particle density, ρ0 is standard particle density
(1 g/cm3) and dg is the geometric diameter of the particle.
• Particle characteristics such as aerodynamic diameter can be
engineered to deliver particles to different areas of the lung.
The deposition of particles according to their aerodynamic
diameter is
a) 10-30 micrometer: oropharyngeal region
b) 2-10 micrometer: trachea, bronchi
c) Less than 2 micrometer- alveolar region
• Mechanofusion™- imparts remarkable inhalation properties
to lyophilized nanoparticulate systems. Nanocomposites of
salmon calcitonin developed.
40

Buccal route of administration
• Oral mucosa, including the lining of the cheek (buccal mucosa), floor
of mouth gingival mucosa,
offers excellent accessibility and avoids first‐pass hepatic metabolism.
• Advantages:
• Less sensitive to irreversible irritation even on long term treatment.
• Well acceptable to the patients.
• Easy accessibility administration as dosage forms
• Disadvantages:
• Barriers for diffusion of drug across the buccal membrane:
1. Epithelial barriers.
2. Numerous elastic fibers in the dermis,
3. Peptidases in the saliva and the mucus layer and microbial flora.
• Various strategies employed for buccal delivery are:
• Adhesive promoters:
• Absorption promoters/permeabilizers :
e.g. Sodium lauryl sulphate, sodium myristate, bile salt, sodium glycocholate
41

1. Adhesive tablets
• Based on hydroxypropyl cellulose polymer
2. Adhesive Gels
• Viscous adhesive gels for local therapy using polyacrylic acid
and polymethacrylate as gel forming polymers.
• Gels are reported to prolong residence time on the oral mucosa
to a significant level
3. Adhesive Patches
• the adhesive polymer may act as the drug carrier itself, and as an
adhesive link between a drug loaded layer and the mucosa.
• Alternatively, a drug containing disk may be fixed to the
mucosa. In this approach drug loss to the saliva is decreased
• Eg:Thyrotropin‐releasing hormone, oxytocin, vasopressin,
LHRH analogs, calcitonin, insulin have been easily applied
through this route
42

Transdermal route of administration
• The drug is formulated in form of a patch that is applied over the skin and the absorption of
drug takes place through the skin
• Advantages:
1. Better and improved patient compliance
2. Controlled administration is possible and thereby avoidance of toxic effects.
3. Drugs with shorter half-life can be administered
• Disadvantages:
1. A low rate of permeation for most protein drugs due to their large molecular weight and
hydrophilicity
2. High intra and inter patient variability
• Various approaches for tdds:
1. Iontophoresis
Iontophoresis is a method that induces migration of ions or charged molecules when an electric
current is allowed to flow through an electrolyte medium. Protein/and peptide (charged
molecules) are repelled by the same charge on electrode and penetrate through the skin
under the influence of electric current. Drug molecule flow through epidermis →→→
dermis→ papillary layer →→ subdermal tissue→→blood circulation.
Example: Insulin, TRH, Vasopressin, Leuprolide are successfully delivered by this technique.
43

2. Phonophoresis
• In this method, ultrasound is applied via a coupling contact
agent to the skin.
3. Penetration enhancers
• Penetration enhancers have the properties of reversibly
reducing the barrier resistance of the horny layer.
• Eg: Oleic acid, DMSO, surfactants, azone
4. Prodrugs:
The enzymes present in the skin selectively regenerate the active
drug. Prodrug with modeled physico-chemical characteristics
are permeated well across the skin than drug.
• Macroflux® patch technology is a patch application system to
ensure consistent dosing and ease of use. Drug coating on the
microprojection array allows for rapid, direct delivery of high-
molecular-weight drugs through the skin barrier layer,
optimizing bioavailability and efficient drug utilization. 44

• Suitable candidates:
1. Polypeptide antibiotics: cyclosporin, bacitracin, polymixin
2. Immunomodulating activities: Cyclosporine, interferons.
3. Act on inflammation : Substance P, enkephalins.
4. Affect wound healing: Epidermal growth factor, fibronectin.
• Disadvantages:
• Systemic bioavailability is low
• Ocular tissues are sensitive to the presence of foreign substances
• Strategies for ocular delivery:
1. Prodrug approach
2. Mucoadhesive particulate carriers
3. Nanoparticles
4. Penetration enhancers
Ocular route of administration
45

Evaluation of protein and peptide
drug formulations
• Stability testing
• The capability of a particular formulation in a specific
container/closure system to remain within its physical, chemical,
microbiological, toxicological and protective specifications.
• Evaluates the effect of environmental factors on the quality of the
a drug substance or a formulated product which is utilized for
prediction of its shelf life and proper storage conditions.
• Bioassay
• Due to complexity of proteins, bioassay are required to assess
potency of the formulation. Bioassay are of two types : in vitro
and in vivo. In case of in vitro bioassays response of cells to
hormones and growth factors is monitored. In case of in vivo
bioassay pharmacological response of animals to proteins is
monitored. Eg: post injection BSL in rabbits is measured for
bioassay of insulin
46

• UV spectroscopy
• Proteins containing aromatic amino acid residues such
as phenyl alanine, tyrosine, tryptophan can be detected
by UV spectroscopy. It can be used as an analytical
determination method
• Bradford assay
• This assay employs the principle that in the presence of
proteins in an acidic medium, absorption maximum of
coomassie brilliant blue G-250 dye changes. If there is
no protein to bind, the solution remains brown. The dye
forms a complex with carboxyl end of proteins by Van
der Waals forces to form a blue colored solution. The
intensity of the colored solution can be measured using
a spectrophotometer to determine the concentration of
the protein in the sample. 47

• Differential scanning calorimetry
• Used as a tool for investigating transitions of
conformation as a function of temperature and, more
importantly, the effect of potential stabilizing
excipients in a protein solution.
• Chromatography
• To study stability of proteins and peptides. Various
modes used are
1. Normal Phase HPLC
2. Reverse Phase HPLC
3. Ion Exchange
4. Chromatofocusing 48

• Electrophoresis
• Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) used. Proteins are
denatured by boiling in the SDS solution. All
charges of protein are masked by negative charge of
dodecyl sulphate. Thus protein moves on
polyacrylamide gel strictly on basis of size of
protein molecule. This technique is useful for
determining molecular weight of proteins. For
visualization of proteins on the gel, reagents used
are silver nitrate, coomassie brilliant blue dye
49

Recent advances
• Smart polymer based delivery system:
• biodegradable polymeric systems delivering protein and
peptide drugs
• smart polymer‐based injectable drug delivery systems
• Smart polymers are classified according to the external
stimulus they respond to:
1. Temperature sensitive:
Poly (ethylene oxide)‐poly (propylene oxide)‐poly(eth
ylene oxide) triblock copolymers
2. Phase sensitive polymers: Poly (D,L‐lactide), Poly (D
,L‐lactide‐co glycolide)
3. pH sensitive polymers: Poly(methacrylic acid g‐ethyle
ne glycol
4. Photosensitive polymers: polyvinyl alcohol 50

• Hybrid protein delivery systems:
• Heterologous hybrid proteins can be designed bearin
g the combined or reordered features of one or more
proteins that display effector functions, protection a
bilities and recognition properties.
• Site specific hybrid proteins may be produced:
1. Ligated gene fusion hybrid delivery systems: Hybr
id protein between interferon γ and TNF β have be
en developed.
2. Synthetically linked hybrid conjugates
51

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