INTRODUCTION:

Since the 1980s, the advent of recombinant biotechnology has generated a wide range of therapeutic peptides and proteins. The capability to produce novel peptides for the clinic is, however, not matched by corresponding advances in the mode of peptide and protein delivery. Most of the biopharmaceuticals in clinical use are delivered by invasive methods, such as intravenous or subcutaneous injection. Peroral administration of peptide drugs, though intensively sought and much better understood than ever before, remains an elusive goal. The impasse is closely related to the failure of pharmaceutical scientists to breach the twin barriers of enzymatic degradation and low epithelial permeability encountered in the oral delivery of peptide drugs^1-3.

Proteins may have thousands of amino acid residues. Although the terms protein and peptide are sometimes used interchangeably, molecules referred to as polypeptides generally have molecular weights below 10,000 and those called proteins have higher molecular weights. Peptides are molecules formed by linking amino acids in a specific order via peptide (amide) bonds. Naturally occurring sequences are largely restricted to 20 á -L-amino acids and posttranslational modifications thereof.

Analogs of bioactive peptides containing unnatural amino acid residues incorporated into the peptide backbone or linkers, or terminal groups can be developed in order to increase potency or improve stability towards proteases. Peptides play an important role in modulating many physiological processes in our body^4-6.

The field of peptide therapeutics has been rapidly expanding, fuelled by recent advances in target screening using phage display, combinatorial synthesis and high throughput methods. Peptides have a number of therapeutic advantages over both small molecules and proteins. These include low toxicity, low immunogenicity, excellent specificity, high potency and a low probability of drug-drug interaction problems. Synthetic capabilities have increased the diversity of peptide-like molecules, which has allowed for the optimization of potency and metabolic stability and, in some cases, improvement of formulation properties^7-8.

Peptide and polypeptides or proteins are an important class of biological substances which are not only the essential nutrients of human body, but some of the polypeptide hormones like insulin are used in treating various diseases resulting from hormonal deficiency. As this use of peptides and polypeptides for systemic treatment of certain diseases is well accepted in medical practice, research activities are being directed towards the synthesis of large quantities by rDNA technology⁹.

The most common route of administration for protein and peptide drug delivery has been parenteral, although many other routes have been tried with varying degree of success. Routes such as intranasal, transdermal, buccal, intraocular, rectal, vaginal and pulmonary route will deliver the drug to the systemic circulation while avoiding transit through the digestive system. A major factor that limits the usefulness of these substances for their intended therapeutic application is that they are easily metabolized by plasma proteases when they reach the peripheral circulation. In addition, adverse effects associated with applying these drugs to the pulmonary or the other mucosal surfaces, may be limiting⁹.

Protein and peptides when delivered orally would not achieve therapeutically acceptable bioavailability because of the enzymatic barriers, the intestinal epithelial and vascular endothelial barriers, which typically digest them with the help of the GI system. Therapeutic quantities of most macromolecules are able to pass through the skin and mucous membranes with the help of penetration enhancers or penetration enhancing techniques, such as detergents or electric impulses, increasing the likelihood of irritation or other side effects. Penetration enhancers are compounds that are added to increase the absorption of the solute across the biological membranes. Use of surfactants decrease the self association and absorption of the protein on the hydrophobic interface of the delivery matrix. They increase the penetration and stability of protein and peptide formulations⁵.

Designing oral peptide and proteindelivery systems has been a persistent challenge to pharmaceutical scientists because of their several unfavorable physicochemical properties including large molecular size, susceptibility to enzymatic degradation, short plasma half life, ion permeability, immunogenicity, and the tendency to undergo aggregation, adsorption, and denaturation. Consequently, the absolute oral bioavailability levels of most peptides and proteins are less than 1%. The challenge here is to improve the oral bioavailability from less than 1% to atleast 30-50%.¹⁰

Till recently, injections (i.e. intravenous, intramuscular or subcutaneous route) remain the most common means for administering these protein and peptide drugs. Patient compliance with drug administration regimens by any of these parenteral routes is generally poor and severely restricts the therapeutic value of the drug, particularly for disease such as diabetes. Oral administration presents a series of attractive advantages towards other drug delivery. These advantages are particularly relevant for the treatment of pediatric patients and include the avoidance of pain and discomfort associated with injections and the elimination of possible infections caused by inappropriate use or reuse of needles. Moreover, oral formulations are less expensive to produce, because they do not need to be manufactured under sterile conditions. In addition, a growing body of data suggests that for certain polypeptides such as insulin; the oral delivery route is more physiological. The increasing importance of proteins and peptides can be attributed to three main developments:

Ø Improved analytical methods have promoted the discovery of numerous hormones and peptides that have found applications as biopharmaceuticals.

Ø Molecular biology and genetic engineering have enabled the large-scale production of polypeptides previously available only in small quantities.

Ø There is a better understanding of the role of regulatory proteins and peptides in the pathophysiology of human diseases.

Examples of some protein and peptide drugs are Peginterferon α-2a, Peginterferon α-2b, Pegfilgrastim, Doxorubicin, Insulin and Exenatide etc.¹⁰

Successful development of protein and peptide therapeutics requires overcoming challenges in their formulation and delivery. The ideals of room temperature stability and non-invasive delivery have been largely unattainable. Two peptide products available as oral formulations are noteworthy. Desmopressin, a cyclic Nona peptide with high antidiuretic activity, has extremely low oral bioavailability (<1 percent), but cost-effective oral-dosing is enabled by a wide therapeutic index and low cost of goods. Cyclosporine, a cyclic 11-mer and potent immunosuppressant, is extremely hydrophobic, requiring specialized micro emulsion or micro dispersion formulation approaches^11-12.

APPROACHES TO THE ORAL DELIVERY OF PROTEIN AND PEPTIDE DRUGS:

To be absorbed, a peptide drug following oral administration will have to transit along the gastro-intestinal tract (GIT), pass through the mucous/glycocalyx layer to cross the intestinal epithelium into the portal vein and finally drain into the general blood circulation. Most peptide drugs are susceptible to degradation by digestive enzymes present in the gastrointestinal fluid, such as trypsin, chymotrypsin, elastase, carboxypeptidases and amino peptidases, as well as by dipeptidases and tri-peptidases located in the mucous/glycocalyx layers. Further digestion by amino peptidases present in the brush border membrane can occur in the passage across the intestinal enterocytes^13,14.

In addition, some peptides are degraded by specific enzymes, such as the insulin-degrading enzyme present in the cytosol. Even if a substantial amount of the peptide drug dose is successfully transported across the epithelium, extensive first pass metabolism by liver microsomes will further decimate the dose fraction that enters the general circulation. Consequently, very few peptide drugs are able to resist the enzymatic onslaught during the absorption process in the GIT. The biochemical and physical barriers presented in the GIT combine to make the oral delivery of a peptide drug a daunting task. It has not, however, deterred pharmaceutical scientists from adopting a host of approaches to explore the viability of administering a peptide drug via the oral route¹⁵.

Enzyme Inhibitors:

To overcome the enzymatic barrier in the GIT, peroral peptide drugs have been co-administered with protease inhibitors, such as the Bowman-Birk inhibitor from soybeans, ovomucoid and aprotinin bacitracin. Mechanisms employed included:

(1) Competitive inhibition of enzymes;

(2) Non-competitive inhibition of enzymes, and

(3) Sequestration of metal ions essential for maintaining enzyme structure.

Although many of the enzyme inhibitors are associated with minimum cytotoxicity in the short term, long-term administration has been shown to interfere with the digestion of nutritive proteins, and to cause stimulated protease secretion, hypertrophy and hyperplasia of the pancreas. Of greater concern is the development of neoplastic foci following the long-term administration of enzyme inhibitors, such as the trypsin inhibitors derived from raw soybean flour, as these can progress to invasive carcinomas^10,16,18.

Permeation enhancers:

To promote their intestinal permeability, peptide drugs have been co-administered with chemical enhancers, mostly surfactants (e.g. sodium dodecyl sulfate (SDS), polysorbate 80, nonylphenoxy(polyoxyethylene) and polyoxyethylene ethers, and pt- octyl phenol polyoxyethylene-9.5, bile salts (e.g. sodium taurocholate (TC), sodium taurodeoxycholate (TDC), and specific tight junction modifiers, (e.g. chitosan), its analogue N-trimethyl chitosan chloride and zonula occludens toxin. The surfactants work by partitioning into the phospholipids bilayers to disrupt cell membrane integrity. Tight junction modifiers may modulate the function and expression of the Para cellular junction proteins, as in the case of chitosan and its analogues, or they may trigger a cascade of events through protein kinase C-dependent actin reorganization, e.g. the zonula occludens toxin. Unfortunately, many permeation enhancers are associated with unacceptable toxicity, such as reduced cell viability, epithelial damage and histological changes to intestinal tissues. There is also concern that the indiscriminate disruption of protective epithelia may expose the host to exotic toxins and opportunistic microorganisms^8,17-19.

PEGYLATION:

PEG modification is an established method to improve the aqueous solubility and decrease the immunogenicity of a peptide drug. It has been widely applied to prolong the half-life of peptide drugs administered by injections. The increased circulation time was achieved through enhanced drug stability in vivo, reduced renal clearance and prolonged local residence at the injection site. The improved pharmacokinetic properties are often related to the size of the PEG molecules, the larger PEG molecules having a greater affinity for water and therefore providing a more efficient shield to hinder enzyme degradation and renal filtration. As each PEG subunit is capable of binding 2-3 water molecules, a peptide drug conjugated with a PEG could be 5-10 fold larger in size compared to its unmodified counterpart¹⁷.

This increase in size, which might also be attained by multiple conjugation of PEG molecules, could decrease both the epithelial permeability and activity of the peptide drug. In this respect, conjugation with smaller PEG molecules may be more helpful for the noninvasive delivery of peptide drugs. For example, the conjugation of insulin with a PEG of 750 Da was found to increase peptide stability against elastase and pepsin without affecting its biological half-life^20,21.

Poly Ethylene Glycol (PEG) is non-toxic and has been approved by the FDA for use in foods, cosmetics, and pharmaceuticals. PEG polymers can be linear or branched in shape, and can be engineered in a variety of molecular weights. Studies on PEG solution show that each ethylene glycol subunit is tightly associated with two or three water molecules.

PEG modification (PEGylation) increases the plasma half life of protein and reduces the immunogenicity of proteins and in addition, the proteins remain biologically active. Advanced PEGylation, which involves modification of protein, peptide or non-peptide (drug or therapeutic protein) by attaching with specific PEG polymer chains, is a proven method for enhancing the potentials of peptides and proteins as therapeutic agents. The advantage of advanced PEGylation for therapeutic molecules can include enhanced bioavailability, decreased dosing frequency, due to prolonged residence in the body, a decreased degradation by metabolic enzymes, optimized pharmacokinetics, increased efficacy, improved safety profile, a reduction or elimination of protein immunogenicity, improved drug solubility and stability to hydrophobic drugs and proteins. Prodrugs are also prepared by this advance PEGylation technique. The advance PEGylation also offered new opportunities for creating viable peptides and protein drugs by site-specific Pegylation⁵.

Glycosylation:

Glycosylation can also enhance peptide conformational stability and impact distribution. An alternative approach to prolonging circulating half-life is the use of fusion constructs, incorporating macromolecular components such as serum albumin antibody domains. Fusions may be amenable to expression in recombinant systems, although this approach precludes the chemical options outlined above for maximizing metabolic stability. Further, fusions are complex macromolecular structures with their own specific challenges^22,23.

Chemical Modification:

A chemical modification of protein and peptide drugs improves their enzymatic stability and membrane penetration of proteins and peptides. It can also be used for minimizing immunogenicity. Protein modification can be done either by direct modification of exposed side chain amino acid groups of proteins or through the carbohydrate part of glycoprotein’s and glycoenzymes. Despite the promising progress, strategies based on the formulation approach do not resolve the poor intrinsic stability and/or permeability of peptide drugs in the GIT. For this reason, some pharmaceutical scientists sought to chemically modify the peptide drug structures to overcome the enzymatic and permeability barriers encountered in the GIT. Chemical modification strategies involve the conjugation of a peptide drug via a chemical bond with specific functional group(s) that often serve the twin purposes of stabilizing the drug against enzymatic degradation and enhancing its permeability across the intestinal epithelia^10,17,22.

Particulate Delivery System:

Microparticles and, more recently, nanoparticles have been developed as promising platforms for the oral delivery of peptide drugs. Peptide drugs were either encapsulated in the particles or complexed with excipients to form the particles. Particle size was important in governing the efficiency of cellular uptake and extent of tissue distribution of the particles. In one study, polylactic-co-glycolic nanoparticles of 100 nm diameter were shown in an in situ rat intestinal loop model to have higher cellular uptake, particularly in the Peyer's patches, and wider distribution across the basal membrane than larger particles of 500 nm. Although a wide variety of polymers has been employed to formulate the peptide particulate delivery systems, they shared similar mechanisms in that oral peptide bioavailability was generally promoted through increased resistance to enzyme degradation, mucoadhesion, tight junction opening and/or endocytosis. For example, poly-N-isopropyl Acryl Amide (PNIPAAm) nanoparticles have been shown to enhance sCT bioavailability mainly by muco-adhesion, while chitosan-based nanoparticles improved the oral bioavailability of insulin and calcitonin via muco-adhesion and transient tight junction opening. Chitosan nanoparticles also promoted cellular uptake of its cargo via endocytosis, a phenomenon not seen when the polymer was presented as a soluble solution. A peptide drug could also be encapsulated within a polymer matrix to shield the drug from enzymatic degradation until its release at the absorptive site. This strategy has been further improved by incorporating mucoadhesive and/or pH-sensitive block co-polymers, such as the poly(methacrylic-g-ethylene glycol) hydrogels, to prolong the drug residence time of the drug at the site of absorption. However, despite the plethora of promising in vitro and animal data, the majority of peptide particulate delivery systems have not progressed to clinical trials^24,25.

Mucoadhesion:

Several strategies based on mucoadhesion have shown potential for application in the oral delivery of peptide and protein drugs. One of these involved the use of micro tablets as intestinal patches. Coated on one side with ethylcellulose to form an impermeable drug barrier and a mucoadhesive comprising carbopol 934 and pectin, with or without sodium carboxylmethylcellulose on the other end, the micro tablets were designed to adhere to the intestinal mucosa to provide uni-directional drug release at the absorptive site^26,27.

Conjugation with Amphiphilic Molecule:

The amphiphilic group contained one or more alkyl groups, together with one or more ethylene oxide (also called ethylene glycol) oligomer. Conjugation usually occurred via an amide bond between the ε-amine of lysine in the peptide and the carboxyl groupresent in the alkyl or ethylene oxide chains of the amphiphilic molecule. The hexyl-insulin monoconjugate-2 (HIM2) is an example. It consists of insulin conjugated with methyl ethylene glycol heptamer modified hexanoic acid at the lysine moiety of B29²⁸.

Conjugation with Lipids:

Lipids conjugated to a peptide drug do not lose this capacity to interact with membrane phospholipids, yet are able to significantly change the biophysical properties of the peptide drug. Reported lipid conjugation methods include the widely used amide bond lipidization with fatty acid and deoxycholic acid; the reversible, aqueous-soluble lipidization (REAL) with disulfide-bond forming lipids and the aqueous- soluble, non-reversible lipidization with maleimido lipid groups via thio-ether bonds. The lipidized peptides exhibited enhanced stability in vitro and in vivo, enhanced cellular uptake or both. They also showed prolonged half-life and higher efficacy than the corresponding unmodified peptide drugs²⁹.

Conjugation with Transferrin:

Transferrin the natural protein transporter for iron, is relatively stable against digestion by intestinal enzymes, and it promotes cellular iron uptake by binding to the membrane-bound transferrin receptor to initiate receptor-mediated endocytosis. Thus, peptide drugs conjugated with transferrin can hijack the endocytotic transport mechanism for effective translocation into and across epithelial cells. Initially, the peptide drugs were conjugated to transferrin via an intra-disulfide bond. An example was the insulin-transferrin conjugate. More recent methods utilized biological conjugation via a fusion protein expression technology to yield a single well-defined new protein. Granulocyte colony stimulating factor-transferrin fusion protein (G-CSF-Tf) is an example^30-31.

TRANS MUCOSAL DRUG DELIVERY SYSTEM:

The Pulmonary route of protein and peptide drug delivery system has recently received increased attention. Three therapeutic peptides-leuprolide: 9 amino acids; insulin: 51 amino acids; growth hormone: 192 amino acids – were reported to be absorbed in biologically active form from the lungs, with bioavailabilities of 10-25%. These values exceed than nasal delivery of insulin and growth hormone in the absence of permeation enhancers. Current challenges in pulmonary protein and peptide delivery include assessment of the safety of long-term administration, the molecular size limitation of pulmonary absorption and strategies for enhancing permeation and formulation approaches capable of delivering suitable doses of stable proteins to the vast absorptive surface presented by the lung. Pulmonary drug delivery is most commonly used in the case of asthma, but recent advancement technologies by particle engineering and formulation methods to manage particle size, morphology, uniformity, chemical stability and dispersibility are used to manufacture drug powders for inhalation, which have created exciting opportunities to expand the applications for pulmonary delivery to many therapeutic molecules, including proteins and peptides. Pulmonary drug delivery is the key to obtaining effective, non-systemic delivery alternative to injections and is held as a method to directly target disorders of lung, which could not be treated by using oral medications ^32,33.

The Nasal route possesses higher permeability and presents less of an enzymatic barrier than does the oral route. The nasal route has been successful for a number of polypeptide drugs. Nasal formulations for lu-teinizing hormone-releasing hormone (LH-RH) analogs (desmopressin, buserelin, oxytocin and calcitonin) have reached market place. Notably, however, this route of delivery has not been as successful for larger proteins with molecular weights greater than 10 kDa and may be associated with local irritation and toxicity with long-term administration^{32, 34,35}.

TRANSDERMAL DRUG DELIVERY SYSTEM:

Numerous approaches have been suggested for overcoming the skin’s formidable barrier function; although, certain strategies simply act on the drug formulation or transiently increase skin permeability, others are designed to remove the outermost skin layer. Transdermal route for the delivery of proteins and peptides is particularly attractive for a variety of reasons as all other routes (i.e., nasal, buccal, oral, rectal, vaginal and pulmonary) exhibit enzymatic activity and degradation due to gastrointestinal tract and the hepatic first pass metabolism, whereas, skin contains amino peptidases, which exhibit less enzymatic activity. This means that the bioavailability of the peptide drug delivered is increased⁴.

Macroflux^R transdermal patch technology has been developed to deliver biopharmaceutical drugs in a controlled, reproducible manner that optimizes the bioavailability and efficacy without significant discomfort to the patients^36-37.

CONTROLLED DRUG DELIVERY SYSTEM:

The most commercially successful method for achieving controlled release is encapsulation of the peptide within a polymer matrix. Microparticulate systems, consisting of spherical polymeric particles (microspheres) with sizes typically ranging from 10 to 100-ì m, form depots when injected subcutaneously from which the peptide is gradually released over a period of time from one week up to six months. Larger implants (including nonerodible ones) have also been developed for delivery of LHRH superagonists. Although numerous polymer matrices have been used, most of them are biodegradable, such as poly(lactide) (PLA) or poly(lactide-co-glycolide) (PLGA). Due to its excellent biocompatibility and ability to control the drug release profile, PLGA has been successfully applied in numerous products, examples of which include LHRH agonists, antagonists and somatostatin analogs. The release period may be extended by using higher molecular weight polymers or by increasing the ratio of lactide to glycolide (which leads to slower degradation by increasing matrix hydrophobicity) ³².

Microsphere delivery is most suitable for potent compounds, with a maximum dose of about 1 mg/kg per injection, assuming a maximum injectable suspension volume of 2 mL. Another issue to consider is that the release profile is often biphasic. This occurs when a portion of drug is rapidly released initially (“burst”) from the surface, or areas near the surface, of the microparticles. The peptide may be incompletely encapsulated and followed by a more gradual release phase corresponding to hydration and disintegration of the polymer⁴.

To achieve controlled protein and peptide delivery, the use of biodegradable microsphres is being actively pursued, particularly, biodegradable lactic-glycolic acid-coplymer-based microspheres have proved useful for the controlled delivery of several polypeptides and proteins. The first FDA approved system for controlled release of peptide was an injectable poly (lactide-coglyoclide) microsphere formulation of leuprolide acetate. This formulation provides controlled release of the peptide over 30 days for the treatment of prostate cancer. Although, promising, many problems remain, and the more general use of formulations of this type for other biopharmaceuticals is often limited by the instability of the molecule to the stresses of the encapsulation process^38-40.

CONCLUSION:

This paper summarizes current promising approaches and the accompanying mechanisms. It aims to show that, the administration of peptide and protein drugs by the noninvasive routes is sought and much better understood, and can become a reality in near future. But each delivery system has its own pros and cons, these systems can improve patient compliance and bioavailability when compared with the conventional delivery systems. Despite challenges, progress towards the convenient noninvasive delivery of proteins and peptides has been achieved through specific routes of administration.

ACKNOWLEDGEMENTS:

The authors wish to thank Dr. Mohib Khan (Principal), MESCO College of Pharmacy, Hyderabad, for the kind support and encouragement to carry out this work

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Received on 23.12.2009 Modified on 20.02.2010

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 379-384