Guided by :

Mrs. M.R.P. RAO

(Pharmaceutics department)

Presented by :
Mr.Dhanesh H. Sali.

AISSMS COLLEGE OF PHARMACY, PUNE
CONTENTS
Introduction
Protein and peptide drugs
Parenteral drug delivery systems
Non Parenteral drug delivery systems
Development of drug delivery systems
Insulin
Conclusion
References
2
INTRODUCTION
Proteins are the most abundant macromolecules
in the living cells, occurring in all cells and all parts
of cells.
Cells can produce proteins that have strikingly
different properties and activities, by joining same
20 amino acids in many different combinations and
sequences.
The term protein is used for molecules composed
of over 50 amino acids, and peptide for molecules
composed of less than 50 amino acids.
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4
Scientific advances in molecular and cell biology
have resulted in the development of two new
biotechnologies. The first utilizes recombinant DNA
to produce protein products.
The second technology is hybridoma technology.
Various proteins and peptides drugs are epidermal
growth factor, tissue plasminogen activator.
PROTEIN AND PEPTIDE DRUGS
Management of illness through medication is
entering a new era in which a growing number of
biotechnology produced peptide and protein drugs
are available for therapeutic use.
Ailments that can be treated effectively by this new
class of therapeutic agents include cancers,
memory impairment, mental disorders,
hypertension.
5
MARKETED PROTEINS IN FREEZE DRIED
FORMULATIONS
Product Formulation Route Indication
Metrodin FSH 75 IU i.m. Induction of
ovulation
Pergonal FSH and LH i.m. infertility
Profasi HCG i.m. Infertility
Elspar Asparginase i.m. i.v. Leukemia
Glucagon Glucagon i.m. i.v. s.c. Hypoglycemia
Acthar Corticotropin i.m. i.v. s.c. Hormone
Deficiency
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MARKETED PEPTIDES IN READY TO USE
FORMULATIONS
Product Formulation Route Indication
Pitressin 8-Arginine
Vasopressin
i.m. s.c. Post operative
abdominal
distension

Lupron Leuprolide s.c. Prostatic cancer

Syntocinon Oxytocin i.m. i.v. Labour induction

Sandostatin Octreotide s.c. Intestinal tumour
Calcimar Salmon calcitonin s.c. hypercalcemia

7
SUSTAINED RELEASE DOSAGE FORMS
Product
Formulation
Route Indication
Lupron

Leuprolide i.m. Prostatic
cancer
H.P.Acthar
gel
ACTH i.m. s.c. Antidiuretic
Pitrressin
tannate in
oil
Vasopressin
tannate
i.m. Endocrine
cancer

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PROTEIN AND PEPTIDE DRUGS
They are therapeutically effective only by parenteral
route.
Repeated injections are required.
Therapeutic applications of these drugs rely on
successful development of viable delivery systems
to improve their stability and bioavailability.
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PARENTERAL ROUTE
Most efficient route.
Extremely short duration of action.
Hence, viable drug delivery techniques are to be
developed such as controlled drug delivery systems
for prolongation of biological activity.
Judicious choice of route of administration should
be done.
Complications arising from this route are :
Thrombophlebitis
Tissue necrosis
immunogenicity

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PARENTERAL ROUTE
BIO DEGRADABLE POLYMERS BASED DRUG
DELIVERY SYSTEMS :
Microspheres are used as drug carriers which are
made of natural or synthetic polymers.
Natural polymers have advantage that they are
biocompatible and inexpensive. But they are
lacking purity. Synthetic polymers are PLA, PGA,
PLGA.
Mechanism of degradation are : firstly random
chain scission occurs. Then soluble oligomeric
products are formed which then gets converted to
soluble monomers.
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PLGA biodegrades into lactic and glycolic
acids. These acids enter into TCA cycle and
then eliminated as carbon dioxide and
water. Injectable controlled release
formulations of certain drugs are formulated
using lactide/glycolide copolymers. Such
drugs are LHRH, calcitonin, insulin.
Nanoparticles made of PLGA, albumin
polystyrene have potential for targeted drug
delivery.

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LIPOSOMES BASED DRUG
DELIVERY SYSTEMS

Liposomes are microscopic vesicles composed of
one or more lipid layers that enclose aqueous
compartments. Liposome membranes are semi
permeable and can thus be used as controlled
release systems. Liver is natural target for
liposomes.
Disadvantage is low stability of liposomes.
13
HYDROGEL BASED DRUG
DELIVERY SYSTEMS
Hydrogels have advantage of biocompatibility.
Insulin has been incorporated into hydrogels and
widely investigated.
Emulsions , multiple emulsions, micro emulsions,
resealed erythrocytes can also be used to deliver
protein and peptide drugs.
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SELF REGULATED DEVICES:

These are capable of receiving the
physiological feedback information and
adjusting drug output from delivery systems
in response to feedback information.
These devices are of two types :
feedback signal modulates rate of drug release.
Feedback signal triggers drug release.
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BASIC PRINCIPLES OF SELF
REGULATED DEVICES
COMPETITIVE DESORPTION :

Insulin molecules with covalently attached sugar
molecules are used that is also known as
glycosylated insulin.
These are complementary to major binding site of
conA. It is carbohydrate binding protein.
Glycosylated insulin can be bound to conA and
reversibly displaced from conA by glucose in direct
proportion of glucose concentration.
16
MEMBRANE CONTROLLED
DEVICES
Glucose oxidase is immobilized in cross linked
polymers. In absence of external glucose amine
groups are unprotonated and membrane porosity is
such that insulin molecules are unable to diffuse
out.
Glucose when diffused into membranes gets
oxidized by glucose oxidase to gluconic acid which
protonates amino groups due to which membrane
porosity increases and now insulin can diffuse out.
17
EROSION CONTROLLED
DEVICES

Reaction between glucose and glucose oxidase
generates gluconic acid. Poly(ortho esters)
polymers erodes as pH decreases. Release of
insulin can be modulated using this approach.
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Non parenteral systemic delivery

These routes are useful for long term therapy.
Without permeation enhancers lower bioavailability
is achieved when these routes are used.
Lower bioavailability is due to poor mucosal
permeability.
Sodium tauroglycocholate is commonly used
penetration enhancer.

Nasal route :
Poor permeability is common problem.
Proteolytic enzymes in nasal mucosa degrades the
administered drugs.

Pulmonary route :
Monodisperse aerosol with a mass median aerodynamic
diameter of 3 m was reported to achieve alveolar
deposition of 50% or more drug.

20
Ocular route :
Ocular absorption can be enhanced by use of
nanoparticles, liposomes, gels, ocular inserts.

Buccal route :
Mucoadhesive dosage forms can be used.

Rectal route :
solid dispersion of insulin with mannitol can produce rapid
release of insulin from suppositories.
21
Transdermal route :
Skin has very low proteolytic activity.
Two types of iontophoresis are used :
DIRECT CURRENT MODE
PULSE CURRENT MODE

Vaginal route :
Especially useful to deliver hormones.
Not much accepted in developing countries.
22
DEVELOPMENT OF DELIVERY SYSTEMS FOR PEPTIDE AND
PROTEIN BASED PHARMACEUTICALS
Considerations are to be given for following aspects
:

Preformulation and Formulation considerations
Pharmacokinetic considerations
Analytical considerations
Regulatory considerations

23
PREFORMULATION AND FORMULATION
CONSIDERATIONS


Preformulation data is to be generated for following
aspects :

Isoelectric point
pH solubility profile
pH stability profile
Excipient compatibilities
24
pH :
Solution pH is important for stability purpose. For
simple peptides pH of minimum degradation should be
identified. Peptides are usually formulated at slightly
acidic pH (3-5). For proteins pH is set away from
isoelectric pH to avoid aggregation.
Insulin is more stable at pH 5.4. However for solubility
reasons insulin injection pH are 2.5-3.5 or 7-7.8.

SALTS :
Ammonium sulphate is a strong stabilizer. Hence
saturated solution of ammonium sulphate is used in
protein purification process.
25
Surface adsorption :
Glass and plastic surfaces adsorbs proteins and
peptides.
To avoid surface adsorption albumin, gelatin, sodium
chloride can be used.

Aggregation behaviour :
To prevent aggregation additives are used such as : urea,
glycerol, EDTA, lysine, poloxamer 188.

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PHARMACOKINETIC CONSIDERATIONS
Basal insulin secretion in healthy subjects shows
circadian rhythm with peak time at 15:00 hrs.
It has been suggested that larger amount of insulin
is needed in afternoon and night.
Hence delivery systems could be designed by
considering such aspects.
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ANALYTICAL CONSIDERATIONS
Many tests are required for stability of protein
products to assure identity, purity, potency and
stability of formulation.
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 : e.g., post
injection blood sugar in rabbits is monitored for
bioassay of insulin.


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U.V. SPECTROSCOPY
Proteins containing aromatic amino acid residues
such as phenyl alanine, tyrosine, tryptophan can be
detected by u.v. spectroscopy.
Ultraviolet spectroscopy can be used for in process
quality control.
Protein aggregates scatter u.v. light and
absorbance increases. Hence u.v. spectroscopy
can be used to monitor protein aggregation.

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30
BRADFORD ASSAY :
This assay employs the principle that in the presence of
proteins absorption maximum of coomassie brilliant blue
dye changes from 465nm to 595nm.

BIURET TEST :
Structure of biuret and proteins are similar. Biuret in
presence of proteins or peptides reduces copper to
cuprous ions in alkaline solutions and colour complex is
developed.

THERMAL ANALYSIS
Differential scanning calorimetry (DSC) is gaining
widespread use as a tool for investigating
transitions of confirmation as a function of
temperature and, more importantly, the effect of
potential stabilizing excipients in a protein solution.
The apex of the endothermic peak is the transition
temperature between native and partially unfolded
confirmations.



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ELECTROPHORESIS
Most often used technique for protein products is
sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE).
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.

32
LIQUID CHROMATOGRAPHY

To study stability of proteins and peptides HPLC is
useful technique. Various modes used are
Normal Phase HPLC
Reverse Phase HPLC
Ion Exchange
Chromatofocusing


33
REGULATORY CONSIDERATIONS
Four federal agencies regulates biotechnology
products :
1. US Food and drugs administration (USFDA)
2. Environmental protection agency (EPA)
3. Occupational safety and health administration
(OSHA)
4. US Department of agriculture (USDA)
34
PROTEIN INSTABILITIES
The degradation of proteins and peptides can be
divided into two main categories :
1. Those that involve a covalent bond.
2. Those involving a conformational change. This
process is often referred to as denaturation.

35
PEPTIDE FRAGMENTATION

The peptide bond (RNH-CO-R) is succeptible to
hydrolysis.
Peptide bonds are considered stable unless hydrolysis is
assisted by neighbouring group. Hydrolysis rate is
affected by solution pH.

DEAMIDATION
It means removal of ammonia from amide moiety.
Deamidation is the major factor for instability of insulin,
ACTH, Human Growth Hormone. In acidic media
peptides deamidate by direct hydrolysis.


36
37
OXIDATION
Sulphur containing amino acids are prone to oxidation.

MAILLARD REACTION
In the maillard reaction the carbonyl group (RCH=O)
from glucose can react with the free amino group in a
pepide to form a Schiff base. This reaction is acid
catalysed.

DIMERISATION AND POLYMERIZATION
Insulin forms a small amount (about 1%) of covalent
dimer and polymer during two years cold storage.
Production of these species increases as temperature
increases.



DENATURATION

o Specific confirmation is required for proteins to exert
pharmacological and physiological activities.
Denaturation is a process of altering protein confirmation.
Heat, organic solvents, high salt concentration,
lyophilization can denature proteins.
Protein confirmation refers to the specific tertiary
structure, which is determined by the primary and
secondary structures and the disulfide bonds and is held
together by three forces : hydrogen bonding, salt bridges,
and hydrophobic interactions.

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COMMON STABILIZERS

SERUM ALBUMIN :
It can withstand heating to 60
o
C for 10 hours.
At pH 2 albumin molecule expands and elongates but
can return to native confirmation reversibly. Also, it shows
good solubility.
Mechanism for such behaviour may be one of the
following :
Inhibition of surface adsorption or cryoprotection.


39
40
AMINO ACIDS

Glycine is most commonly used stabilizer.
Mechanism of action of amino acids as stabilizers may
be one of the following :
Reduce surface adsorption.
Inhibit aggregate formation.
Stabilize proteins against heat denaturation.

SURFACTANTS


They cause denaturation of proteins by hydrophobic
disruption. However judicious use of surfactants can
protect proteins from other denaturants. Proteins have
tendency to concentrate at liquid/liquid or liquid/air
interface. Due to this proteins may adopt non native
confirmation and such confirmation is having less
solubility.
Optimal concentration of surfactants for stabilization
should be greater than cmc. Ionic surfactants are more
effective stabilizers than non ionic surfactants.
Various surfactants used are : poloxamer 188,
polysorbate.

41
POLYHYDRIC ALCOHOLS AND
CARBOHYDRATES :
They contain CHOH-CHOH- groups which are
responsible for stabilizing proteins. They stabilize
proteins against denaturation caused by elevated
temperature or by freeze drying or by freeze thaw
cycles.
Many important therapeutic proteins and peptides
are derived from blood such as immune globulin,
coagulation factors. For viral destruction
pasteurization at 60
o
C for 10 hours is needed.
Hence thermal stability is needed. Long chain
polyhydric alcohols are more effective as
stabilizers. e.g. sorbitol, xylitol.

42


Mechanism of action as stabilizers for polyhydric alcohols
is that they have effect on structure of surrounding water
molecules which strengthens hydrophobic interactions in
protein molecules.
Mechanism of action as stabilizers for carbohydrates is
that they provide dry network that provides significant
support for protection.
Polyhydric alcohols used are sorbitol, mannitol, glycerol,
PEG.
Carbohydrates used are glucose, mannose, sucrose,
ribose.

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ANTI-OXIDANTS
Thiol compounds such as thioacetic acid, triethanolamine,
reduced glutathione and metal chelants such as EDTA are
used as antioxidants.

MISCELLANEOUS
Certain enzymes can be stabilized by using compounds
having similar structures of enzymes. e.g. Glucose stabilizes
glucoamylase while aspargine stabilizes asparginase.
Compounds forming stable complex through ionic interaction
with proteins can stabilize proteins.
Calcium is essential for thermal stability of certain amylases or
proteases.


INSULIN
Insulin was the first protein for which an amino acid
sequence was determined (by sangers group in
Cambridge in 1955)
It consists of two peptide chains (A and B of 21 and 30
amino acid residues respectively).
Insulin suspensions may be prepared by pH change
method. Insulin has an isoelectric point at approximately
pH 5. when it is mixxed with basic protein such as
protamine, it is readily precipitated when pH is between
isoelectric points of insulin and protamine i.e. pH 6.9 to
7.3.

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46

Protamine zinc insulin (PZI) contain an excess quantity of
zinc to retard absorption. According to BRITISH
PHARMACOPOEIA of 1958, a phosphate buffer is added
to each individual vial containing acidified solution of
insulin, protamine and zinc so that pH is between 6.9 to
7.3. The preparation is compounded in final container by
mixing PZI and buffer in filling operations.
Main problem in using insulin is to avoid plasma
concentration fluctuations. To solve this problem various
formulations are available varying in onset and duration
of action.

47

Insulin Lispro : it is insulin analogue in which lysine and
proline residues are switched. It is rapidly acting hence
patient can inject immediately before start of meal.
Insulin Glargine : it is modified insulin analogue. Its intent
is to provide constant basal insulin supply. It is clear
solution, forms microprecipitate at physiological pH of
subcutaneous tissue and absorption from subcutaneous
site is prolonged. It avoids risk of night time
hypoglycemia when used in conjunction with short acting
insulin.

Name Particle size
(m)
Action Composition pH Duration
(hours)
Insulin
injection USP
prompt Insulin + zinc
chloride
2.5-3.5 5-7
Prompt insulin
zinc
suspension
USP

2
rapid Insulin + zinc
chloride +
buffer
7.2-7.5 12
Insulin zinc
suspension
USP
10-40(70%)
2(30%)
INTERMEDIATE Insulin + zinc
chloride +
buffer
7.2-7.5 18-24
Extended
insulin zinc
suspension
10-40 Long-Acting Insulin + zinc
chloride +
buffer
7.2-7.5 24-36
Globin zinc
insulin
injection
INTERMEDIATE
Globin +
Insulin + zinc
chloride
3.4-3.8 12-18
Protamine zinc
insulin
suspension
Long-Acting Protamine +
insulin +zinc
7.1-7.4 24-36
Isophane
insulin
suspension
USP
30
INTERMEDIATE
Protamine
+zinc chloride
+insulin +
buffer
7.1-7.4 18-24
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USP INSULIN TYPE STRENGTHS
Insulin injection (regular insulin) U-40 mixed,
U-100 mixed, U-500
Isophane insulin suspension (NPH
Insulin)
U-40 mixed,
U-400 mixed
Isophane insulin suspension
(70%) and insulin injection (30%)
U-100
Insulin zinc suspension (Lente
insulin)
U-40 mixed,
U-100 mixed
Extended insulin zinc suspension
(Ultra lente insulin)
U-40 mixed,
U-100 mixed
Prompt insulin zinc suspension
(semilente insulin)
U-40 mixed,
U-100 mixed
Protamine zinc insulin suspension
(PZI Insulin)
U-40 mixed,
U-100 mixed
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CONCLUSION
Protein and peptide based pharmaceuticals are rapidly
becoming a very important class of therapeutic agents
and are likely to replace many existing organic based
pharmaceuticals in the very near future.
Peptide and protein drugs will be produced on a large
scale by biotechnology processes and will become
commercially available for therapeutic use.
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This poses an urgent challengeto the pharmaceutical
industry to develop viable delivery systems for the
efficient delivery of these complex therapeutic in
biologically active form.
Much work needs to be done on the development of
viable delivery systems for non parenteral administration
to make peptide and protein pharmaceuticals
commercially viable and therapeutically useful.
REFERENCES
1) Agrawal S, Udupa N, Protein and peptide drug delivery :
recent advances. In : Jain NK, editor. Progress in controlled
and novel drug delivery systems. 1
st
ed. Delhi : CBS
Publishers; 2004.p.184-204.
2) Chien YW : Novel drug delivery systems. 2
nd
ed. New York :
Marcel Dekker Inc; 2005.p.631-745.
3) Yu Chang John Wang : Parenteral products of proteins and
peptides. In : Lieberman HA, Avis KE, editors. Pharmaceutical
dosage forms : Parenteral medications, volume 1. 2
nd
ed. New
York Marcel Dekker Inc; 2005.p.283-320.
4) Block JH, Beale JM. Wilson and Gisvolds textbook of organic
medicinal and pharmaceutical chemistry. 11
th
ed. Philadelphia
: Lippincott Williams and wilkins; 2005.p.851-852.



52
53
5) Patel NK, Pharmaceutical suspensios. In :
Lachman l, Lieberman HA, Kanig JL, editor. The
theory and practice of pharmacy. 3
rd
ed. Mumbai :
Varghese Publishing House; 1987.P.488-489.
6) Aulton ME : Pharmaceutics : The science of
dosage form design. 2
nd
ed. Toronto : Churchill
livingstone; 2006.p.544-553.
7) Poon CY

: Clinical Analysis. In : Troy DB, editor.
Remington : The Science of Dosage form Design.
21
st
ed. Volume 1. Philadelphia : Lippincott
Williams and wilkins; 22005.p.577-578.
8) www.ida.lib (accessed on 15/4/2010.)
9) Rang HP, Dale MM : Pharmacology. 5
th
ed. Toronto
: Churchill livingstone; 2003.p.386-388.



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10) Massey FH, Sheliga TA : Development of
aggregation resistant insulin formulations. Pharm
Res; 3 : 26S (1986).
11) www.wikipedia.org (accessed on 16/4/2010.)
12) Agharkar SN, Motola S. Preformulation research
of parenteral medications.In : Lieberman HA, Avis,
KE, editors. Pharmaceutical dosage forms :
parenteral medications; volume 1. 2
nd
ed. New York
: Marcel Dekker Inc; 2005.p.150-155.

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