Proteomics involve the large-scale study of proteins, their structure
and physiological role or functions. The term proteomics first appeared
in 1997. It was very similar to the word genome. The word proteome is
actually a combination of protein and genome. To be precise and
specific, proteome is the entire complement or database or set of
proteins produced by a living organism. The protein synthesis in any
organism is very briefly depicted in three steps:(1) transcription of
mRNA; (2) translation of protein based on mRNA; and (3)
posttranslational protein modifications, such as addition of phosphate
or glucose to render it biologically active.

Proteomics have led
to the opening up of several horizons in the field of drug discovery and
drug delivery. Complete elucidation of protein configuration of a human
biological system enables us to identify a protein that is implicated
in the disease. Drugs that alter, inhibit, or suppress such protein are
being designed as therapeutics for clinical use.

Sound knowledge
of three-dimensional structure and functions of a protein is very
important to develop effective delivery systems for such therapeutics.
Since the inception of the human genome project, the idea of drug design
and delivery has undergone a paradigm shift. The focus is to develop a
more site specific, patient friendly, cost effective and above all
tailor-made or highly individualized therapy to replace the generalized
therapy existing until now.

Proteomics provides a hope in
designing drug delivery systems that can be used to identify and target
such biological sites. The concept of individualized or tailor-made
therapy is gradually receiving impetus owing to the realization that a
patient’s response to a drug is largely mediated at the genetic level.
Differences existing at the level of an individual’s genetic make-up led
to development of pharmacogenomics. The field received a tremendous
boost, thanks to progress in proteomics.

Proteomics have been
widely used as biomarkers for a plethora of diseases ranging from
neurodegenerative to cardiovascular diseases. There has always been a
dearth of suitable biomarkers to trace the origin of these diseases.
Cerebrospinal fluid (CSF) biomarkers are potential tools as diagnostics
for clinical trials on mild cognitive impairment in patients with
incipient AD. New, sensitive and specific biomarkers are the need of
this hour to facilitate clinical diagnosis of neurodegenerative
disorders.

Proteomics technology is widely used in discovering
new biomarkers. Development in the identification of proteomic
biomarkers, profiling, resolution, and analysis has been relatively
slow. Researchers investigated some serum-based proteomic biomarkers for
HIV-associated dementia disorders. The conclusions drawn from this work
stressed the importance of multiple protein profiling approaches and
multiple sample fractionation schemes to assess changes in proteomes due
to pathological conditions.

Changing needs of proteins and peptides delivery
Many
peptides and proteins have been commercially explored since the 1950s.
Initial explorations were for peptide-based hormonal drugs and
analogues. The commencement and completion of the human genome project
is considered an important landmark in areas of protein research because
more than 84% of total human proteins have been sequenced and their
structures predicted. However, the development of proteins and peptide
molecules as drug candidates has taken place at only a snail’s pace
because of various technical constraints.

Proteins and peptide
markets are of immense potential because many vaccines and similar
derivatives are predominantly protein or peptide based. Major
limitations of proteins and peptides are the lack of knowledge of the
effects of the administration route and the physiochemicalproperties of
proteins that influence their pharmacokinetics and in vivo behavioural
profile.

Most proteins are delivered by parenteral routes that
are invasive in nature, for example, insulin by subcutaneous route. The
thermo-labile nature, high molecular weight, solubility, stability,
intestinal permeability, tendency to undergo complexation, and
susceptibility to proteolytic enzymes in the gastrointestinal tract upon
oral administration of proteins are other key factors that constraint
the use of proteins in delivery. However, drug delivery systems by
various routes of administration like buccal, nasal, vaginal and
transdermal (iontophoresis and similar techniques) have been tried. The
utility of proteins and their immense potential as drug candidates
motivate pharmaceutical scientists to develop suitable and commercially
feasible drug delivery systems. This is evident from the fact that more
than 40% of pharmaceutical companies are actively working in this area.
The consolidation of molecular biology, biotechnology and pharmaceutical
sciences is seen as a major hope in this direction.

Barriers to protein and peptide absorption in the GI tract
The
key factor restraining the delivery of protein and peptides
administered by oral route is proteolytic enzymes. Proteolytic enzymes
are responsible for rapid hydrolytic and chemical degradation of protein
and peptides in the gastrointestinal tract which results in the loss of
therapeutic activity of protein and peptides. Hence to overcome the
barriers is major hurdle in the effective delivery of proteins and
peptides.

Mucus barrier
The GI tract is divided into
number of organs like the oesophagus, stomach, small intestine, large
intestine and accessory organs like salivary glands, liver, pancreas and
gall bladder. All these organs are lined by the viscous layer called as
mucus layer. The mucus is secreted by the goblet cells in the GI tract
which consists of mucin glycoproteins, enzymes, electrolytes, water, and
so forth. Due to viscosity, presence of proteolytic enzymes and the
interactive nature of these layers, they offer a certain level of
resistance to the protein absorption.

Enzymatic barrier
The
GI tract comprises of variety of proteolytic enzymes such as
aminopeptidases, diaminopeptidases, post-aprolyl-cleaving enzyme,
angiotensin-converting enzyme(ACE), endopeptidase (a metalloproteina
se), and thiol protease enzyme, commonly considered as extracellular
barriers, which are involved in the degradation of peptides and
proteins. The major sites for the degradation of the proteins by enzyme
inside the body are lumen, brush border, the cytosol of the enterocytes
blood, liver, kidney, and vascular endothelia due to an abundance of
proteases. The variation in the pH along the GI tract from stomach colon
is also hurdle in absorption of proteins. The various protease enzymes
with their major site of action are given in table 1 below.

Absorption pathways
Proteins
are mainly absorbed by two pathways such as transcellular and
paracellular across the epithelial barriers. In the transcellular
pathway, peptides and proteins transfer through a specific uptake
mechanism or follow simple partitioning from the aqueous lumen content
to the lipid membrane and from there to the aqueous blood stream. The
paracellular pathway involves the transfer of peptides and proteins
through the space present between the adjacent cells. This space has
radius 8 Å, so only smaller peptides can pass through the space. The
only hindrance in the paracellular pathway is the tight intracellular
junction of the villus cells. The paracellular pathway avoids
degradation of peptides and proteins by proteases present in the cells.
Use of a penetration enhancer significantly improves the transport of
peptides through paracellular pathways. Penetration enhancers such as
zonula occludens toxin (a protein from vibrio cholera), Pz peptide, and
chitosan reversibly open tight junctions between intestinal cells which
increases the molecules present in the GI tract.

The bile salts
like sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate,
and lysophosphatidylcholine usually improve penetration by chelating
action. The use of conjugated bile salts have been found to
significantly increase solubilization of cyclosporine and thereby its
oral bioavailability.

Apart from these factors the molecular
weight, size, structure, charge distribution, immunogenicity,
solubility, partition coefficient, liphophilicity and aggregation of
proteins also affect their absorption.

Approaches to improve stability of proteins to protease and their delivery
The
stability of proteins to protease can be improved by different
approaches like (a) Substituting unnatural amino acids, including
d-amino acids, for l-amino acids in the primary structure (b)
introducing conformational modification, (c) changing the direction of
the peptide backbone and reversing the chirality of each amino acid, and
(d) acylation or alkylating the N-terminus or altering the carboxy
terminus by reduction or amide formation. (e) Co-administration with
Protease Inhibitors.

Apart from these approaches the attempt have
been made to develop pharmaceutical formulations and New Drug Delivery
Systems that can avoid enzymatic degradation of proteins and improve
their stability and target ability for example protein entrapped in
vesicular carriers, like liposomes, nanoparticles, microparticles.

Oral delivery
Significant
efforts have been made to deliver proteins and peptides through
non-invasive routes, like oral, buccal, nasal, pulmonary, vaginal,
rectal, ocular, and transdermal, because of the limitations of the
parenteral route, that is, frequent dosing, the short half-life of
protein and peptide in blood, pain on administration, poor patient
compliance, and sterility requirement.

Parenteral delivery
Oral
delivery of protein and peptide is unsuitable due to factors like high
molecular weight, poor permeability across gastric mucosa, size, acid
susceptibility, and susceptibility to proteolytic enzyme action in the
gut. Hence, parenteral routes like intravenous (i.v.), subcutaneous
(s.c.), and so on, are the principal preferred routes of drug delivery.
Several controlled release injectable particulate delivery systems have
been investigated for effective delivery of proteins and peptides with
minimal systemic exposure so as to protect the delivered proteins from
degradation by proteolytic enzymes.

The particulate systems
suffer from the disadvantage of low protein loading levels and
difficulty in achieving the desired bio-distribution of the nanocarriers
encapsulating the protein.

Other delivery approaches
The
delivery of peptides or proteins is extremely limited due to
degradation in the gastrointestinal (GI) tract. Attempts have been made
to develop formulation which achieve the effective concentration of
proteins in the blood and to prevents their degradation, avoid hepatic
first-pass metabolism. These formulations can be delivered by various
routes like transdermal, intrauterine, rectal, intranasal, pulmonary
etc.

Stability and evaluation of protein and peptide formulation
A
proteins and peptides are subjected to a number of physicochemical
changes viz. deamination, oxidation, reduction, proteolysis and
conformational changes with time, depending on storage conditions. It
becomes highly complex in multicomponent systems like proteins, where
the presence of excipients changes the properties of proteins. Even the
co-solvents, buffers, and so forth, must be screened. For example, the
addition of co-solvents such as glycerol or polyethylene glycol may
stabilize protein structure by preferential hydration and by decreasing
the contact area of the protein surface with solvent. However, these
changes affect the safety and efficacy of a protein should be
extensively studied.

Currently, proteins and peptides are the
very emerging therapeutics molecules though their whole potential in
therapeutics is yet to be explored completely, they have been widely
acclaimed for cardiovascular, central nervous disorders. The peptide
sequences have microscopic differences among themselves, just differing
by sequential arrangement of amino acids. Hence, it is very difficult to
develop a selective and analyte specific analytical method for
quantitative determination of the same. The biological potency
characteristic of these molecules, degradation products, impurities, and
matrix components places tremendous demands for selectivity on
analytical methodology. The methods used for peptides analysis are
divided into four major categories: (1) measurement of biological
activity, (2) evaluation of purity and stability, (3) quantitative
determination, and (4) structural characterization.

Conclusion
Proteomic
studies have facilitated elucidation of the physiological and
pharmacological role of proteins in a biological system and it has led
to the opening up of several horizons in the field of drug discovery and
drug delivery. However, some basic issues are associated with proteins:
size, structure, high molecular weight, biological barriers to
efficacious protein absorption in vivo, and ability as well as their
vulnerability to various exogenous and endogenous physiochemical factors
have rendered proteins challenging candidates for formulation.

Knowledge
of anatomical and physiological barriers, complexities of body
organization of higher animals, basic chemistry of proteins,
hydrophobicity, conformational and solubility aspects, structure
activity relationship studies, and immunological and various parameters
pertinent to proteins have provided deep insight into the mechanisms of
action of proteins as pharmaceuticals.

This knowledge has been
instrumental in changing the direction of protein related research. Much
work has been undertaken to improve the formulation aspects of
proteins, ranging from designing and incorporating suitable
enhancers/carriers/cell fugogenic ligands/targetters in protein
formulations to development of oral, parenteral, inhalation, transdermal
and rectal delivery systems for proteins. However, in spite of
unprecedented progress achieved in the field of protein delivery, the
dream of achieving targeted, site-specific, controlled drug delivery of
protein remains largely elusive and unfulfilled.

The author is
faculty of technology & engineering, Pharmacy Department, The
Maharaja Sayajirao University of Baroda, Gujarat.