about protein and peptide drug delivery system

1. PROTEIN AND
PEPTIDE DRUG
DELIVERY
SYSTEM
Presented by: Rajat Singh
M.Pharm Ist year
Drug Delivery system

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
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. PROTEIN AND PEPTIDE DRUGS
– They are therapeutically effective only by parenteral routes.
– Repeated injections are required.
– Therapeutic applications of these drugs rely on successful development of
viable delivery systems to improve their stability and bioavalibility.

8. 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

9. 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.

10. – PLGA biodegradables 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.

11. LIPOSOME 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.

12. 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.

13. 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.

14. 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 conc A. It is carbohydrate
binding protein.
– Glycosylated Insulin can be bound to conc A and reversibly displaced from conc
A by glucose in direct proportion of glucose concentration.

15. 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.

16. 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.

17. NON PARENTERAL
SYSTEMIC DELIVERY
– These routes are useful for long term therapy.
– Without permeation enhancers lower bioavailability is due to poor mucosal
permeability.
– Sodium tauroglycocholate is commonly used penetration enhancer.

18. – 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 3µm was
reported to achieve alveolar deposition of 50% or more drug.

19. – 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.

20. – 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.

21. 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

22. PREFORMULATION AND
FORMULATION CONSIDERATION
– Preformulation data is to be generated for following aspects :
– Isoelectric point
– pH solubility profile
– pH stability profile
– Excipient compatibilities

23. 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 sulfate is a strong stabilizer. Hence saturated solution of ammonium
sulfate is used in protein purification process.

24. Surface adsorption:
– Glass and plastic surfaces adsorbs proteins and peptides.
– To avoid surface adsorption albumin, gelatin, sodium chloride can be used.
Aggregation behavior :
– To prevent aggregation additives are used such as : urea, glycerol, EDTA, Lysine,
poloxamer 188.

25. 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.

26. 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 bioassay 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.

27. 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.

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

29. 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 peak is the
transition temperature between native and partially unfolded confirmations.

30. 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.

31. 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

32. REGULATORY
CONSIDERATIONS
– Four federal agencies regulates biotechnology products:
1. US Food and Drug administration (USFDA)
2. Environmental protection agency (EPA)
3. Occupational Safety and Health administration (OSHA)
4. US Department of agriculture (USDA)

33. PROTEIN INSTABILITY
– 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 reffered to as
denaturation.

34. 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 major factor
for instability of insulin, ACTH, Human Growth Hormone. In acidic media
peptides deamidate by direct hydrolysis.

35. 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 peptide to form a Schiff base . This reaction acid catalyzed.
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.

36. DENATURATION
– 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 disulphide bonds
and is held together by three forces: hydrogen bonding, salts bridges, and
hydrophobic interactions.

37. COMMON STABILIZERS
SERUM ALBUMIN:
– It can withstand heating to 60 0C for 10 hours.
– At pH 2 albumin molecule expends and elongates but can return to native
confirmation reversibly. Also, it shows good solubility.
– Mechanism for such behavoiur may be one of the following:
Inhibition of surface adsorption or cryoprotection.

38. 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 aggregation formulation
– Stabilize proteins against heat denaturation.

39. SURFACTANTS
– They causes 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.

40. 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 peptide are derived form blood such
as immune globulin, coagulation factors. For viral destruction pasteurization at
60 0C for 10 hours is needed. Hence thermal stability is needed. Long chain
polyhydric alcohols are more effective as stabilizers. E.g. sorbitol, xylitol.

41. – Mechanism of action as stabilizers for polyhydric alcohols is that 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.

42. ANTI- OXIDANTS
– Thiol compounds such as thioacetic acid, triethanolamine, reduced glutathione and
metal chelants such as EDTA are used as antioxidants.
MISCELLANEOUS
– Certain enzyme 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 amylase or proteases.

43. 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.
– This poses an urgent challenge to 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.

44. THANK YOU