PROTEIN AND

PEPTIDE DRUG
DELIVERY SYSTEMS
Guided by :
Mrs. M.R.P. RAO
(Pharmaceutics
department)

Presented by :
Mr.Dhanesh H. Sali.

AISSMS COLLEGE OF
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.
3
 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.

4
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 6
 MARKETED PEPTIDES IN READY TO USE
FORMULATIONS
Product Formulation Route Indication

Pitressin 8-Arginine i.m. s.c. Post operative

Vasopressin 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 ACTH i.m. s.c. Antidiuretic

gel

Pitrressin Vasopressin i.m. Endocrine

tannate in oil tannate cancer
8
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.

9
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
10
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.

11
 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.
12
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.

14
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.
15
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.

18
 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.

19
 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 µm 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.

26
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.

27
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.
28
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.

29
 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.

30
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.
 
 
31
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
 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.

37
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.

38
 COMMON STABILIZERS

SERUM ALBUMIN :
 It can withstand heating to 60o 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
 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.

40
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 60o 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.

43
 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.
 

44
INSULIN
 Insulin was the first protein for which an amino
acid sequence was determined (by sangerʾs 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.

45
 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.

46
 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.

47
 Name Particle size (µm) Action Composition pH Duration
(hours)
Insulin injection prompt Insulin + zinc 2.5-3.5 5-7
USP chloride
Prompt insulin rapid Insulin + zinc 7.2-7.5 12
zinc suspension 2 chloride + buffer
USP

Insulin zinc 10-40(70%) INTERMEDIATE Insulin + zinc 7.2-7.5 18-24

suspension USP 2(30%) chloride + buffer

Extended insulin 10-40 Long-Acting Insulin + zinc 7.2-7.5 24-36

zinc suspension chloride + buffer

Globin zinc INTERMEDIATE Globin + 3.4-3.8 12-18

insulin injection Insulin + zinc
chloride

Protamine zinc Long-Acting Protamine + 7.1-7.4 24-36

insulin insulin +zinc
suspension

Isophane insulin 30 INTERMEDIATE Protamine +zinc 7.1-7.4 18-24

48
suspension USP chloride +insulin
+ buffer
USP INSULIN TYPE STRENGTHS
Insulin injection (regular insulin) U-40 mixed,
U-100 mixed, U-500
Isophane insulin suspension (NPH U-40 mixed,
Insulin) U-400 mixed
Isophane insulin suspension (70%) U-100
and insulin injection (30%)
Insulin zinc suspension (Lente U-40 mixed,
insulin) U-100 mixed
Extended insulin zinc suspension U-40 mixed,
(Ultra lente insulin) U-100 mixed
Prompt insulin zinc suspension U-40 mixed,
(semilente insulin) U-100 mixed
Protamine zinc insulin suspension U-40 mixed,
(PZI Insulin) U-100 mixed 49
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.

50
 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.

51
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