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Challenges in delivery of proteomics

Ambikanandan MisraThursday, December 16, 2010, 08:00 Hrs  [IST]

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.

 
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