2. POINTS TO BE CONSIDERED...
• Definition of proteins.
• Classification of proteins.
• Properties of proteins.
• Various routes of administration of proteins and peptides.
• Development of delivery system for peptide based
pharmaceuticals.
• Barriers to protein drug delivery.
3. PROTEINS
Proteins are biochemical compounds consisting of one or
more polypeptides, which is a single linear polymer chain
of amino acids bonded together by peptide bonds between
the carboxyl and amino groups of adjacent amino acid
residues. Like other biological macromolecules, proteins are
essential parts of organisms and participate in virtually
every process within cells, making them ideal for various
testing procedures to identify effective disease prevention
techniques.
4. EXAMPLES OF PROTEINS,
Casein from milk, collagen , keratin, polymerase,actin ,
myosin,haemoglobin,antibodies,insulin, snakevenom.
CLASSIFICATION OF PROTEINS
Proteins can be classified:
1. On the basis of structure.
2. On the basis of functions.
5. 1.ON THE BASIS OF STRUCTURE...
• PRIMARY STRUCTURE:
Protein primary structure is the linear sequence of amino acids
in a peptide or protein.
Amino acid, eg- glycine, alanine.
Peptide bonds,covalently
attached.
6. • SECONDARY STRUCTURE:
Protein secondary structure is
the three dimensional form of
local segments of proteins.
The two most common
secondary structural elements
are alpha helics and beta
sheets, though beta turns and
omega loops occur as well.
7. • TERTIARY STRUCTURE:
Protein tertiary structure is the three
dimensional shape of a protein. The
tertiary structure will have a single
polypeptide chain "backbone" with
one or more protein secondary
structures, the protein domains.
Amino acid side chains may interact
and bond in a number of ways. The
interactions and bonds of side chains
within a particular protein determine
its tertiary structure.
8. • QUATERNARY STRUCTURE:
Protein quaternary structure is
the number and arrangement
of multiple folded protein
subunits in a multi-subunit
complex. The quaternary
structure refers to the number
and arrangement of the protein
subunits with respect to one
another. Examples of proteins
with quaternary structure include
hemoglobin, DNA polymerase,
and ion channels.
9. 2. ON THE BASIS OF FUNCTIONS...
• ENZYMES: For example, DNA polymerases are enzymes that
synthesize DNA molecules from deoxyribonucleotides, the
building blocks of DNA. These enzymes are essential for DNA
replication and usually work in pairs to create two identical DNA
strands from a single original DNA molecule. During this process,
DNA polymerase "reads" the existing DNA strands to create two
new strands that match the existing ones.
• HORMONES: Examples of peptide hormones include
antidiuretic hormone (ADH), a pituitary hormone important in
fluid balance, and atrial-natriuretic peptide, which is produced
by the heart and helps to decrease blood pressure.
10. • ANTIBODIES:An antibody (Ab), also known as an
immunoglobulin (Ig), is a large, Y-shaped protein
produced mainly by plasma cells that is used by the
immune system to neutralize pathogens such as
pathogenic bacteria and viruses.
• MOTOR PROTEINS: Motor proteins are class of
molecular motors that are able to move along the surface
of a suitable substrate. They convert chemical energy
into mechanical work by the hydrolysis of ATP.
Flagellar rotation, however, is powered by proton pump.
For example, actin and myosin,present in muscle fibers
in animals.
11. PROPERTIES OF PROTEINS
Solubility and partition coefficient: Aqueous
solubility of peptide is strongly dependent upon pH,
presence of metallic ion, ionic strength and
temperature. At isoelectric point the aqueous solubility of
peptide is minimal where the drug is neutral or has no net
charge. Peptides are very hydrophilic with a very low
octanol-water partition coefficient, so to improve the
absorption of peptides by passive diffusion, their lipophilicity
should be increased.
12. Aggregation, self association and hydrogen
bonding: Self-aggregation tendency of peptides modifies
their intrinsic properties. Human insulin was found to be
more self-aggregating than bovine insulin. Additions of
additive like non ionic surfactants (Pluronic F 68) stabilize
the peptide formulation against self aggregation.
13. Various routes of administration of proteins and
peptides.
1. Parenteral systemic delivery
2. Non-parenteral systemic delivery
a) Oral route
b) Nasal route
c) Buccal route
d) Ocular route
e) Rectal route
f) Transdermal route
14. 1. PARENTRAL SYSTEMIC DELIVERY-
Parenteral delivery consist of three major routes, IV,IM and SC.
Intravenous administration is currently the method of choice for systemic
delivery of proteins and peptides eg. erythropoietin injection. Insulin,
interferons, and gammaglobulins have been reportedly metabolized
and/or bound to tissue at injection sites following IM administration, and
as a result, the systemic bioavailability of these protein drugs following IM
administration is often less than that obtained by IV injection. For SC
administration, insulin is best example for the treatment of
diabetes,The controlled delivery of peptide or protein based
pharmaceutical from subcutaneously implanted polymeric devices in
which gel formulation of cross linked
polyacrylamide‐polyvinylpyrrolidone is used to achieve the prolonged
release of immunoglobulin, luteinizing hormone, bovine serum albumin,
insulin, and prostaglandin.
15. 2. Non-parentral systemic delivery-
a)Oral route: The ease of administration and higher degree
of patient compliance with oral dosage forms are the major
reasons for preferring to deliver proteins and peptides by
mouth. Strategies for oral delivery:
• Alter the GIT environment for maximum solubility and enzyme
stability of the protein by using formulation excipients such as
buffers, surfactants and protease inhibitors.
• If the enzyme attack can be defeated or delayed, the proteins can
be presented for absorption.Proteins and peptides could be
derivatised with polyethylene glycol (PEG) to achieve properties
such as retention of activity, prevention of immunogenicity and
prevention of excessive enzymatic degradation.
16. • Another strategy for oral delivery is to promote absorption
through the intestinal epithelium.A typical transport mechanism
for proteins across the epithelial boundary is paracellular
transport. There are tight junctions between each of the cells in
the epithelium that prevent water and aqueous soluble
compounds from moving past these cells.A number of absorption
enhancers are available that will cause these tight junctions to
open transiently, allowing water‐soluble proteins to pass. Fatty
acids, surface‐active agents, EDTA, glycerides and bile salts
have all been shown to be effective in opening these tight
junctions.
17. Potential problem associated with oral protein
delivery:
The oral administration of peptide and protein drugs faces two
problems.
• The first is protection against the metabolic barrier in GIT. The
whole GIT and liver tend to metabolize proteins and peptides into
smaller fragments of 2-10 amino acids with the help of a variety
of proteolytic enzyme (proteases).
• The second problem is the absence of a carrier system for
absorption of peptides with more than three amino acids.
18. Prodrug approach:
Proteins are labile due to susceptibility of the peptide
backbone to proteolytic cleavage, as well as their
molecular size and complex secondary, tertiary and
sometimes even quaternary structures. Therefore proteins
can be modified chemically to give more stable prodrugs
with increased plasma half-lives. Some strategies for
prodrug formation include olefenic substitution, d-amino
acid substitution, dehydro amino acid substitution,
carboxyl reduction, retro inversion modification,
polyethylene glycol (PEG) attachment to amino group and
thio‐methylene modification.
19. b) Nasal route:
Pulmonary protein delivery offers both local targeting for the
treatment of respiratory diseases and increasingly appears to be a
viable option for the delivery of proteins systemically. The lung is
easy to access, has decreased proteolytic activity compared
with the gut, and allows rapid absorption and avoidance of first-
pass metabolism for systemically delivered drugs.For those being
investigated for delivery via inhalation, the ultimate site of action
may be the airway surface (e.g. DNase), the airway cells (e.g.
cyclosporin), or the systemic circulation (e.g. insulin). Careful choice
of carrier and device can facilitate delivery to a specific area of the
lungs. Once delivered, a carrier can further influence the distribution
and rate of clearance from the site of action. Eg, Nonapeptides
Vasopressin, Oxytocin.
20. Advantages of nasal route:
• Convenient, simple, practical way of drug administration
• The high vascularization permits better absorption.
• First pass metabolism can be avoided.
• Rapid onset of action.
Disadvantages of nasal route:
• Long term use may lead to toxicity to mucosa.
• During disease states (e.g. common cold) some
alteration in the nasal environment may occur.
21. c) Buccal route:
This route offers excellent accessibility and avoids
degradation of proteins and peptides that occurs as a
result of oral administration, gastrointestinal absorption and
first-pass hepatic metabolism. Peptide absorption occurs
across oral mucosa by passive diffusion and it is unlikely
that there is a carrier-mediated transport mechanism.
22. Various strategies employed for buccal delivery:
• Adhesive tablets, polymer used can be
hydroxypropylcellulose.
• Adhesive gels, using polyacrylic acid and
polymethacrylate as gel forming polymers.
• Adhesive patches.
• Addition of penetration enhancers such as fatty acids,
cyclodextrins,surfactants etc.
Thyrotropin-releasing hormone, tripeptide, oxytocin,
vasopressin ,calcitonin, insulin have been applied to this
route.
23. Advantages of buccal route:
• It is robust, much less sensitive to irreversible
irritation even on long term treatment.Prevention from
enzymatic attack. Well acceptable by patients.
• Easy to administer, and it is painless.
Disadvantages of buccal route:
• Surface area is less.
• Difficult to handle in case of having food and drinks.
24. d) Ocular route :
The ocular Route is the site of choice for the localized delivery
of opthalmologically active peptides and proteins for the
treatment of ocular disease that affect the anterior segment
tissues of eye. The use of nanoparticles, liposomes,
gels,ocular inserts, bioadhesives or surfactants are
necessary to enhance Ocular absorption of proteins or
peptides. The polypeptide antibiotics like tyrothricin,
gramicidin, tyrocidine, bacitracin and polymyxins have often
been considered potential candidates for achieving local
pharmacological actions in the eyes.
25. Proteins or peptides with opthalmological
activities:
• Affect aqueous humor dynamics: Calcitonin gene
related factors.
• Immunomodulating activities: Cyclosporine, interferons.
• Act on inflammation : Substance P, enkephalins.
• Affect wound healing: Epidermal growth factor,
fibronectin.
26. Advantages of ocular route:
• Localised action can be obtained.
• Painless approaches can be formulated.
Disavantages of ocular route:
• The systemic bioavailability achieved by this route is
very low.
• Ocular tissues are sensitive to the presence of foreign
substances and patient acceptance could be rather
low.
27. e) Rectal route:
The coadministration of an absorption promoting
adjuvants such as sodium glycocholate, has been
reported to enhance the rectal absorption of insulin. Bile
salts, such as sodium salts of cholic, deoxycholic and
glycocholic acids, have also been shown to enhance the
rectal absorption of insulin in rats and human volunteers.
Vasopressin and its analogs, pentagastrin and gastrin,
calcitonin analogs,and human albumin have been
investigated for rectal delivery of protein or peptide
based pharmaceuticals.
28. Advantages of rectal delivery:
• It is highly vascularized.
• It avoids first pass or presystemic metabolism.
• It is suitable for drugs that cause nausea/vomiting and
irritate GI mucosa on oral administration.
• A large dose of drugs can be administered.
29. f) Transdermal route:
Transdermal delivery of peptides and proteins avoids the
disadvantages associated with the invasive parenteral
route of administration and other alternative routes such
as the pulmonary and nasal routes. Since proteins have a
large size and are hydrophilic in nature, they cannot
permeate passively across the skin due to the stratum
corneum which allows the transport of only small
lipophilic drug molecules. Enhancement techniques such
as chemical enhancers, iontophoresis, microneedles,
sonophoresis aid in the delivery of proteins by overcoming
the skin barrier in different ways.
30. Approaches for transdermal delivery:
• Iontophoresis : It is use of electric current to drive
charged drug molecules into skin by placing them under
an electrode of like charged. For eg, DC Iontophoresis,
Pulse DC iontophoresis.
• Phonophoresis: In this method ultrasound is applied via
a coupling contact agent to the skin. Insulin, IFN γ,
erythropoietin can be delivered by this method.
• Penetration enhancers: Penetration enhancers like
oleic acid, dimethylsulphoxide. Surfactants have been
used for topical delivery of peptide or proteins.
31. • Prodrugs: Prodrug with modified physicochemical
characteristic permeated well across the skin. LHRH, TRH,
neurotensin can be delivered by this method.
Advantages of transdermal route:
• Avoids the hepatic first‐pass effect and gastrointestinal
breakdown.
• Provides controlled and sustained administration, particularly
suitable for the treatment of chronic disease.
• Enables self-administration and improves patient compliance,
due to its convenience and ease of use.
32. Disadvantages of transdermal route:
• A low rate of permeation for most of protein drugs due
to their large molecular weight.
• Because the skin has a relatively low proteolytic
activity, the peptide drugs have poor skin permeability.
33. Paracellular delivery of peptides—a rational approach:
Currently, a new route-the paracellular pathway,is being
explored for delivery of peptides. As opposed to the
transcellular pathway, the paracellular pathway is a water-
filled pathway, which is amicable to the delivery of polar
molecules like peptides and proteins. Another advantage is
that by traversing through the area between the two cells
the peptide also circumvents the intracellular lysosomal
enzymes. Eg,Polysaccharide hydrogels useful in the
development of controlled release formulations for protein
drugs. Polysaccharide microspheres, Microencapsulation
of protein drugs etc.
35. Development of delivery system for peptide
based pharmaceuticals
1. FORMULATION CONSIDERATIONS-
• Preformulation studies.
• Surface adsoption behaviour.
• Aggregation behaviour of protiens and peptide molecules.
2. PHARMACOKINETIC CONSIDERATIONS-
• Metabolic degradation of proteins and poeptides in liver,
kidney, non-targeted tissues and even at the site of
administration by peptidases and proteinases.
36. 3. ANALYTICAL CONSIDERATIONS-
• Bioassays
• Chromatography - HPLC
• Electrophoretic methods
• Fast atom bombardment mass spectroscopy
• Immunoassays
4. REGULATORY CONSIDERATIONS-
• FDA,EPA(environmental protection
agency),OSHA(occupational safety and health
administration, and U.S.Department of agriculture.
37. Barriers to protein drug delivery.
• Enzymatic barriers
• Intestinal epithelial barrier.
• Cappilary endothelial barrier.
• Blood brain barrier.
38. REFERENCE
• A.Semalty, Mona Semalty, R. Singh, S. K. Saraf 1 & Shubhini Saraf 2
“Properties and Formulation of Oral Drug Delivery Systems of Protein and
Peptides”,Indian Journal of Pharmaceutical Sciences,Department of
Pharmaceutical Sciences, H.N.B. Garhwal University, Srinagar,India,November -
December 2007.
• Abdul Muheem a, Faiyaz Shakeel b, Mohammad Asadullah Jahangir c,
Mohammed Anwar a, Neha Mallick a, Gaurav Kumar Jain a, Musarrat Husain
Warsi a, Farhan Jalees Ahmad a, “ A review on the strategies for oral
delivery of proteins and peptides and their clinical perspectives”,Saudi
Pharmaceutical Journal,Received 20 March 2014; accepted 6 June
2014,Available online 16 June 2014.
• Ratnaparkhi M.P.,* Chaudhari S.P., Pandya V.A.,Department of Pharmaceutics,
“Peptides And Proteins In Pharmaceuticals” ,International Journal of Current
Pharmaceutical Research,ISSN- 0975-7066,Vol 3, Issue 2, 2011,Marathwada
Mitra Mandal’s College of Pharmacy, Thergaon, Pune.
