NDDS

1. PROTEIN AND PEPTIDE DRUG DELIVERY.
1
Presented by:
Chinchole Pravin Sonu
(M.PHARM 2nd
SEM)
DEPARTMENT OF PHARMACEUTICS & QUALITY
ASSURANCE
R. C. Patel Institute of Pharmaceutical
Education and Research, shirpur.

2.  INTRODUCTION TO PROTEINS AND PEPTIDES
 ROUTES OF ABSORPTION
 PROPERTIES OF PROTEINS AND PEPTIDES
 CONSTRAINTS (POTENTIAL PROBLEMS) IN DELIVERY OF PROTEINS AND
PEPTIDES
 PHARMACEUTICAL APPROACHES
 INCORPORATION INTO DRUG DELIVERY MATRIX
 STABILITY ASPECTS OF PROTEINS AND PEPTIDES
 RECENT ADVANCES IN DELIVERY OF THERAPEUTIC PROTEINS AND PEPTIDES
 REFERENCES
2
PRESENTATION OVERVIEW :

3.  The word PROTEIN was derived from a Greek word “proteios” means
holding the first place.
 Proteins are the most abundant organic molecules of the living system.
 They occur in every part of the cell and constitute about 50% of the cellular
dry weight.
 Mulder , a Dutch chemist in 1838 used the term PROTEINS for the high
molecular weight , nitrogen rich and most abundant substances present in
animals and plants.
3
INTRODUCTION :

4.  ENDOGENOUS PROTEINS AND PEPTIDES :
 These plays an important role in the regulation and integration of life
processes.
e.g. 1) Haemoglobin ,myoglobin, etc. affect the transport of oxygen within the
body.
2) In the form of skin ,hairs, muscles , etc. proteins hold together ,protect
and provide structure to the body of a multi-cellular organism.
3) Enzymes ,hormones and antibodies, etc. catalyze ,regulate and protect
the body chemistry.
4
INTRODUCTION :

5.  Most pharmaceutical proteins and peptides are formulated as solution
or
suspensions and delivered by invasive route such as IM, IV, or SC
Injections but these routes have their own DEMERITS like
i.Poor patient compliance .
ii.Pain and discomfort associated with these routes.
iii.Inconvenience to treat the pediatric patients.
5
ROUTES OF ABSORPTION :

6.  The oral route presents a series of advantages towards other drug delivery
systems like ..
i.It is most convenient route of drug administration.
ii.Avoidance of pain and discomfort associated with injections.
iii.Higher patient compliance.
iv.Elimination of possible infections caused by the use or reuse of needles.
However still designing oral protein and peptide drug delivery have
poses a persistent challenge to the formulation scientists because of their
several unfavourable physicochemical properties like …
i.Large molecular size.
ii.Susceptibility to enzymatic degradation.
iii.Short plasma half-life. 6
ROUTES OF ABSORPTION :

7. IV. High immunogenicity.
V. Tendency to undergo aggregation. Adsorption and denaturation .
VI. Less bioavailability..(less than 1%)
The challenge is here to improve the oral bioavailability from less than
1% to at least 30-40 %.
 The other routes that have been tried with varying degrees of success
are :
Oral, Buccal, Intranasal, Pulmonary, Transdermal, Ocular and Rectal..
7
ROUTES OF ABSORPTION :

8. A) ABSORPTION PROPERTIES :
1) Molecular weight and size.
2) Conformation and Stereospecificity .
3) Immunogenicity.
B) PHYSICOCHEMICAL PROPERTIES :
1) Solubility and partition coefficient.
2) Aggregation and hydrogen bonding.
8
PROPERTIES OF PROTEINS AND PEPTIDES :

9. The oral administration of protein and peptide drugs faces two formidable
potential problems during their oral absorption :
1)Metabolic degradation of therapeutic proteins and peptides by variety of
proteolytic enzymes.
2) Poor membrane permeability.
9
CONSTRAINTS (POTENTIAL PROBLEMS) :

10. A) Chemical Modification : (Prodrug approach)
a) Amino acid modification.
b) Hydro phobization.
B) Use of enzyme (protease ) inhibitors.
C) Use of absorption enhancers.
D) Formulation vehicles.
E) Mucoadhesive polymeric systems.
10
PHARMACEUTICAL APPROACHES :

11.  A chemical modification of protein and peptide drugs improves their
enzymatic stability and membrane permeation. Also , it can be used for
minimizing the immunogenicity.
 Prodrug approach includes :
1) Amino acid modification.
2) Hydrophobization.
11
PRODRUG APPROACH (CHEMICAL MODI.) :

12. A ) AMINO ACID MODIFICATION :
• Modification of individual amino acids by the substitution of D- amino
acid with the L-amino acid can significantly alter physiological properties
of proteins and peptides.
e.g. Desmopressin and Deaminovasopressin are the two analogs of
vasopressin, former involves deamination of first amino acid and
replacement of last L-arginine with D-arginine to give
Deaminovasopressin.
It contributes to enhanced membrane permeability and enzymatic
stability.
12
PRODRUG APPROACH (CHEMICAL MODI.) :

13. B ) HYDROPHOBIZATION :
• By surface modification using the lipophilic moieties.
e.g. NOBEX INSULIN by PALMITOYLATION.
Conjugation of insulin to 1,3-dipalmitoylglycerol at free amino acid
groups of glycine , phenylalanine and lysine to form mono and
dipalmitoyl insulin having facilitated transfer of insulin across mucosal
membrane of large intestine and improved stability against intestinal
enzymatic degradation.
13
PRODRUG APPROACH (CHEMICAL MODI.) :

14.  The whole GIT and liver tend to metabolize proteins and peptides into
smaller fragments of 2-10 amino acids with the help of variety of proteolytic
enzymes (proteases ).
 So protease inhibitors are co-administered with proteins and peptides to
alter the environment for maximum enzyme stability to supress proteolytic
activity.
 They are of 4 major types :
i.Aspartic proteases ( pepsin,rennin ,etc. )
ii.Cystinyl proteases ( papain, endopeptidase )
iii.Serinyl proteases ( thrombin, trypsin )
iv.Metallo proteases ( carboxypeptidase )
14
USE OF ENZYME (PROTEASE) INHIBITORS :

15. 15
USE OF ENZYME (PROTEASE) INHIBITORS :
Sr. No. Protein Protease inhibitor Results
1. Insulin Aprotinin. Significant reduction in insulin
digestion.
2. Insulin Camostat mesilate. Improvementin activity profile
of drug.

16.  Penetration enhancers are the formulation components that temporarily
disrupt the mucosal barrier to improve the permeation of large
macromolecular substances like proteins and peptides.
 following classes of compounds are commonly used :
a.Surfactants- Polysorbate, SLS, Pluronic F-68.
b.Chelating agents- EDTA.
c.Fatty acids-Sodium caprate.
d.Mucoadhesive polymers- Thiomers, cellulose derivatives.
e.Phospholipids- PC.
16
USE OF PENETRATION ENHANCERS :

17.  MECHANISMS OF ACTION OF PENETRATION ENHANCERS :
I.Detergents and surfactants increases the transcellular transport of drugs by
disrupting the structure of lipid bilayer rendering the cell membrane more
permeable.
II. Calcium Chelators believe to exert their action by complex formation with
calcium ions ,thus passing through the tight junctions (TJ’s) and facilitate
paracellular transport of hydrophilic drugs.
III. Fatty acids have been shown to improve paracellular absorption by
phospholipase C activation and upregulation of intracellular Ca+
,
leading to contraction of actin-myosin filaments and opening of TJ’s.
17
USE OF PENETRATION ENHANCERS :

18.  The oral delivery of therapeutic proteins and peptides can be successfully
achieved by using various carrier systems like :
 Dry emulsions
Microspheres
Liposomes
Nanoparticles
18
FORMULATION VEHICLES :

19.  S/O/W multiple emulsion of Insulin :
i.Solubilization into oil phase can avoid degradation of proteins.
ii.Coating of insulin molecule with a llipophilic surfactant enhance permeation
through intestinal mucosa.
Drawbacks :
i.Physical-chemical instability on long term storage.
ii.Reqirement for storage at low temperature.
19
FORMULATION VEHICLES :

20.  DRY EMULSIONS :
To overcome the problem of instability on long term storage, multiple
emulsions are now-a days replaced by dry emulsions.
Dry emulsions can be prepared by :
 Spray drying
 Lyophillization
 Evaporation
Using pH –responsive polymers like HPMCP, dry emulsions can be enteric
coated and site specificity is achieved.
20
FORMULATION VEHICLES :

21.  MICROSPHERES :
pH responsive microspheres can be used as oral delivery vehicles for proteins
and peptides thereby protecting them from proteolytic degradation in the
stomach and upper portion of small intestine.
In the basic environment of the intestine , the complex dissociate resulting in
swelling of microspheres and rapid drug (insulin) release.
 NANOPARTICLES :
Particles in the nanometric size range are absorbed intact by the intestinal
epithelium and they are less prone to enzymatic degradation.
21
FORMULATION VEHICLES :

22. Particle size ,surface charge and influence of surface ligand affect the uptake of
nanoparticles in the GI tract..
 LIPOSOMES :
ENCAPSULATION of insulin with the sugar chain portion of mucin and of PEG
completely suppressed the degradation of insulin in the intestinal fluid whereas
UNCOATED LIPOSOMES suppressed it only partially.
Surface coating of liposomes with PEG or mucin gained resistance against
dagestion by bile salts and increased stability in the GI tract.
22
FORMULATION VEHICLES :

23. • These systems prevent the presystemic metabolism of the therapeutic
proteins and peptides.
• Residence time of drug delivery system at the site of action is also increased
by decreasing the drug clearance rate.
e.g. Thiomers, polyacrylic acid derivatives and cellulose derivatives.
The stronger mucoadhesive properties of thiomers are believed to be based
on covalent bonds between thiol groups of the thiomer and cystein- rich
domains of mucus glycoproteins.
( Higher amount of thiol groups =Stronger mucoadhesive properties.)
23
MUCOADHESIVE POLYMERIC SYSTEMS :

24. There are four methods for incorporaton of drugs into the delivery matrix :
EMULSIFICATION
EXTRUSION AND SPRAY DRYING
POLYMERIZATION
24
INCORPORATION INTO DRUG DELIVERY MATRIX:

25.  EMULSIFICATION :
• Water souble drug is first dissolved in aqueous solution and the polymer is
dissolved in organic solvent. The two solutions are then mixed with in an
appropriate proportion to produce w/o emulsion.
• this primary emulsion is then emulsified into an aqueous solution
containing an emulsifier to produce w/o/w emulsion.
• finally, the organic solvent is removed from emulson by evaporation under
reduced pressure ,by filtration or by increasing the temperature .
25
INCORPORATION INTO DRUG DELIVERY MATRIX:

26.  EXTRUSION :
• When extrusion or spraying is employed to form microspheres, the core
material or matrix containing drug ,incorporated as solution or particulate
is ejected from the orifice of a fine tube , syringe or nozzle to form
microdroplets.
The size of droplets depends upon :
1.Properties of liquid. (melt, solution or suspension )
2.Orifice diameter.
3.Jet velocity.
26
INCORPORATION INTO DRUG DELIVERY MATRIX:

27.  POLYMERIZATION :
HYDROGELS -polymeric drug delivery systems.
Preparation – by mixing monomer with the drug, an initiator and a cross-
linking agent.
e.g. Intravascular delivery of proteins via hydrogel system that is photo
polymerized in-situ on the inner surface of blood vessels.
Drawback : -radiation may have deleterious effect on the integrity of
proteins.
27
INCORPORATION INTO DRUG DELIVERY MATRIX:

28.  PROTEIN DEGRADATION PATHWAYS :
Two basic mechanisms :
1. Physical
2.Chemical
•In chemical degradation, the native structure of the protein is changed by
modifications into the primary structure.
•In physical degradation, the native structure of the protein is changed by
modifications into the higher order structures of proteins i.e. secondary,
tertiary or quaternary structure.
28
STABILITY ASPECTS :

29.  PHYSICAL INSTABILITY :
Denaturation is the primary sign of physical instability of proteins.
In globular proteins, hydrophobic residues are buried in the interior and
hydrophilic residues are available for interaction with the aqueous solvents.
Denaturation refers to the loss of this globular structure which leads to
protein unfolding.
Denaturation may be caused by changes in the environment of the proteins
such as temperature, pH , introduction of hydrophobic surfaces or by
introduction of interfaces by the addition of organic solvents.
29
STABILITY ASPECTS :

30.  CHEMICAL INSTABILITY :
The chemical instability of proteins and peptides is a result of following four
types of reactions :
a)Oxidation
b)Deamination
c)Peptide bond hydrolysis
d)Disulphide exchange
30
STABILITY ASPECTS :

31.  OXIDATION :
Amino acid side chains of proteins and peptides are susceptible to oxidation
by either
i.atmospheric oxygen
ii.metal ions like copper or iron
iii.reagents such as hydrogen peroxide
e.g. Methionine residues under acidic conditions are especially prone to
oxidation by reagents like hydrogen peroxide ,producing methionine
sulfoxide.
It is an issue when hydrogen peroxide is used to sterilize formulation
vessels or the formulation area.
31
STABILITY ASPECTS :

32.  DEAMINATION :
The hydrolysis of an amide side chain of certain amino acid residues (like
glutamine and aspargine ) is called as deamination.
Some conditions like change in temperature and pH are shown to facilitate
deamination of therapeutic proteins and peptides.
 PEPTIDE BOND HYDROLYSIS :
e.g. When aspartic acid residues are heated to 90-1000
C ,at pH 4 ,the
hydrolysis of Asp-X bond leads to the loss of biological activity .
32
STABILITY ASPECTS :

33.  DISULFIDE EXCHANGE :
Many therapeutic proteins contain cystein residues that form disulfide bonds.
These bonds are important components of the structural integrity of proteins.
Incorrect linkages of these disulfide bonds often leads to a change in the
three –dimensional structure of proteins and thereby it’s biological activity.
33
STABILITY ASPECTS :

34. PEGylation :
PEGylation, is a process of attaching the strands of the polymer PEG to most
typical peptides fragments, that can help to meet the challenges of improving the
safety and efficiency of many therapeutic macromolecules.
It is widely used for the modification of proteins ,peptides ,antibody fragments
and oligonucleotides.
 Properties of PEG:
1.Non-toxic. and non –immunogenic.
2. Hydrophilicity.
3. High flexibiity.
34
RECENT ADVANCES :

35.  ADVANTAGES OF PEGylation :
1.Increased bioavailability.
2.Optimized pharmacokinetics.
3.Decreased immunogenicity.
4.Decreased frequency of administration.
MECHANISM OF PEGylation :
1.Increases molecular weight of molecule.
2.Increases drug solubility.
3.Enhanced protection from proteolytic degradation.
4.Reduced dosing .
35
RECENT ADVANCES :

36.  MATKETED PREPARATIONS AND APPLICATIONS :
1.PEGylated interferon alpha -2a : In Hepatitis B
1.PEGylated interferon alpha-2b : In Hepatitis C
3. PEGylated liposome containing doxorubicin : in Cancer.
36
RECENT ADVANCES :

37. Therapeutic proteins and peptides administered IV or SC are often cleared
rapidly from the circulation and therefore need to be injected frequently in
order to maintain therapeutic level in the blood.
Various types of liposomal formulations have been utilized as drug delivery
vehicles for sustained release of proteins and peptides like unilamellar or
multilamellar vesicle systems but few deals with the mulltivesicular liposomes ,
called as “DepoFoam particles.”
The DepoFoam technology is capable of :
1)Accomodating high drug loading and high recovery.
2)High encapsulation efficiency
3)Controlling the sustained delivery of macromolecular drugs.
37
Depo-Foam TECHNOLOGY :

38. A unique feature of DepoFoam system is that inside each DepoFoam particle,
discontinous internal aqueous chambers ,bounded by a continuous network of
lipid membranes render a higher aqueous volume to lipid ratio and much larger
particle diameter as compared to SUV’s or MLV’s.
38
Depo-Foam TECHNOLOGY :
SUV. MLV DepoFoam.

39. 1. N.K. JAIN; Advances in Controlled And Novel Drug Delivery; Ist edition-
2001; CBS Publishers And Distributors; 232-260.
2. U. SATYANARAYANA, U. CHAKRAPANI; Biochemistry ; IIIrd edition -2006;
Uppala Authors And Publishers Interlinks ; 43-68.
3. JAMES SWARBRICK ;ENCYCLOPEDIA OF P’CEUTICAL TECHNOLOGY ; IIIrd
edition ; Vol. -1 ; 2713-2730.
4. E. J. McNally ; Protein Formulation and Delivery ; Marcel-Dekker (P) ;
Vol.99 ; 1-4 , 71-110, 239-243.
39
REFERENCES :

40. 1. www,ijpsonline.com
2. www.pharmainfo.net
3. www.asiapharmaceutics.info
4. www.academicjournals.org
40
WEB REFERENCES :

41. 41