It is highly desirable that these therapeutic proteins and peptides possess an “active targeting” capability to reach intended target cells and leave the normal cells unharmed.

Table1 List of Protein-based Drugs ^1,2

Drug	Indication
Insulin	Diabetes
Rituximab	Non-Hodgkin's disease
Erythropoeitin	Anaemia
Glatiramer acetate	Multiple sclerosis
Interferons	Hepatitis C

Routes of Administration:

1. Parenteral Drug Delivery Systems:

· Biodegradable polymer based drug delivery systems

· Liposome based drug delivery systems

· Hydro gel based drug delivery systems

· Emulsions

2. Non-Invasive Drug Delivery Systems:

· Oral delivery systems

· Buccal delivery systems

· Nasal delivery systems

· Rectal delivery systems

· Transdermal delivery systems

· Pulmonary delivery systems

Parenteral systemic drug delivery:

Biodegradable polymer based drug delivery systems:

Microspheres are produced using naturally occurring or synthetic polymers to produce particulate systems in the size range of 0.1 to 500 μm³.They are generally biocompatible, can provide high bioavailability, and are capable of sustained release for long periods of time. Disadvantages of microspheres include difficulty of large-scale manufacturing, inactivation of drug during fabrication, and poor control of drug release rates. The fate of microspheres is dependent on the polymeric material chosen, the preparation technique. Microspheres are physically and chemically more stable. So they are useful carrier system for proteins and peptides. Natural polymers like collagen, gelatin, chitosan, dextrin and starch. These are used as drug carriers for site specific targeted drug delivery. They are readily available, inexpensive and capable of a magnitude of chemical modification. Most widely used synthetic biodegradable polymers include poly lactic acid (PLA), poly glycolic acid (PGA) and PLGA, Random copolymer of d,l-lactide and glycolide. Biodegradation occurs through homogenous hydrolytic chain cleavage mechanism. In the mechanism of biodegradation, first random chain scission occurs, then soluble oligomeric products are formed which then gets converted to soluble monomers. Poly peptides like calcitonin, LHRH and somatostatin have been formulated in lactide or glycolide copolymers as injectable, controlled release formulation. Insulin has been formulated in controlled release micro beads and pellets. PEGylation improves macromolecule solubility and stability by shielding it from carbohydrate and peptide receptor clearance mechanisms and immune system cells. PEG-Intron™ has reduced the dosing required for interferon beta for treatment of hepatitis from rapid hydrolysis or degradation within the body, thus prolonging its action.

Table 2 List of PEGYlated Proteins for clinical use ^{4,5,6,7,8,9,10}

Company	Product	API	Indication
Enzon	Adagen	PEG-Aminodeaminase	Severe combined immuno deficiency(SCID)
Roche	PEGasys	PEG-interferon-alfa-2a	Hepatitis C
Pfizer	Somavert	PEG-hGH antagonist	Acromegaly
Amigen	Neulasta	PEG-GCSF	NeuTtropenia
UCB	Cimzia	Citrolizumab Pegol	Chron's disease

Liposome based drug delivery system:

Liposomes are microscopic vehicles composed of one or more lipid layers that enclose aqueous compartments. Lipid-based vesicles are a very promising approach to treat diseases such as cancer, chronic infections and auto-immunity. Modern drug encapsulation methods allow efficient packing of therapeutic substances inside liposomes, thereby reducing the systemic toxicity of the drugs. Specific targeting can enhance the therapeutic effect of the drugs through their accumulation at the diseased site. They have high degree of biocompatibility and used as carrier systems to facilitate the parentral administration of therapeutic agents. Phosphatidylcholines (lecithin), phosphatidylethanolamines (PE), sphingomyelins, phosphatidylserines, phosphatidylglycerols (PG), and phosphatidylinositols (PI) are important components for preparation of liposomes. Liposomes behavior is governed by physical size, membrane permeability, and percentage of entrapped solutes, chemical composition, quantity and purity of materials. Liposome membranes are semi permeable and can be used as controlled delivery systems. Bleomycin a peptide with anti tumour activity encapsulated in thermo sensitive liposomes which potentially enhance the activity and reduce the toxicity. Liposomes have recently used as vehicles for vaccines. In the vaccine field, the integration of functional viral envelope proteins into liposomes has led to an antigen carrier and delivery system termed a virosome, a clinically proven vaccine platform for subunit vaccines with an excellent immunogenicity and tolerability profile. Liposome-encapsulated horseradish peroxidase acquires the ability to cross the hemato-encephalic barrier, whereas the native enzyme cannot¹¹. Moufti et al.¹²were able to produce a 50% reduction in blood glucose level in normal rats by insulin-containing liposomes. Dobre et al. illustrated a lowering of blood glucose level in normal rats following oral administration of insulin entrapment in phosphatidylcholine (PC) and cholesterol (CH) liposomes¹³.

Hydrogel based drug delivery system:

Hydrogels are very attractive delivery systems for hydrophilic macromolecules such as peptides and proteins. Hydrogels produced by rapid swelling of polymers upon exposure to an aqueous environment. The cross linking nature and the thermodynamic nature of the components of these hydrogels play a key role in their diffusion behaviour, molecular mesh size changes and the associated molecular stability of the incorporated bioactive agents. These are biocompatible and highly responsible for specific degradation. Hydrogels are suitable for delivery of proteins and peptide due to their ability to respond P^H changes in GI tract and provide protection from harsh environment of GI tract.

Emulsions:

Emulsions can be used for parentral delivery of proteins and peptides. Multiple emulsions prolong the release of drug. Micro emulsions are clear, stable, isotropic mixtures of oil, water and surfactant, frequently in combination with a co surfactant. These systems are currently of interest to the pharmaceutical scientist because of their considerable potential to act as drug delivery vehicles by incorporating a wide range of drug molecules.

Subcutaneous administration of muramyldipeptide w/o emulsion significantly potentiated the immune effect.

Non-invasive routes for delivery of Proteins and Peptides:

Oral route of drug delivery:

Oral administration of different dosage forms is the most commonly used method due to greater flexibility in design of dosage form and high patient acceptance, but the gastrointestinal tract presents several formidable barriers to drug delivery. The major obstacles for oral delivery of proteins are potential degradation by the strongly acid environment in the stomach and by the proteolytic enzymes in the intestinal tract, as well as presystemic elimination in the liver. Various oral delivery systems have been attempted to deliver the proteins and peptides. Enzyme (Protease) inhibitors which are present the delivery systems prevent the degradation of drug by proteolytic enzymes. Eudragit L₁₀₀ microspheres containing various protease inhibitors were able to protect insulin from proteolytic degradation. Paracellular permeability enhancers (PPEs) reversibly open the tight junctions which allow the water soluble proteins to pass¹⁴. Modifying the chemical structure of a peptide or protein is another approach to enhance bioavailability by increasing stability against possible enzymatic degradation or its membrane permeation. Replacement of residue with another retains the receptor-binding activity of the peptide, which yields enhanced resistance to peptidase activity. Replacement of an L-amino acid with L-praline or N-methylation of amide nitrogen leads to increase in resistance to enzymatic activity. Successful drug delivery is achieved by using liposomes as drug carriers. Insulin entrapped liposomes cause dose dependent hypoglycaemia.

Nasal route of drug delivery:

Nasal cavity is potential cavity for drug delivery. Nasal cavity is selective, so efficiency of transport is highly dependent on peptide structure and size. Nasal drug delivery offers rapid absorption, ease of administration as well as bypassing of first pass effect by liver and gut enzymes. There is a possibility of achieving drug delivery transsynaptically directly into the brain by using nasal drug delivery¹⁵. Peptidase enzyme barrier exists in nasal cavity. In order to overcome the barrier to nasal absorption of proteins and peptides , two main approaches have been utilized, modification of permeability of nasal membrane by employment of absorption enhancer, such as surfactants, bile salts, phospholipids, and fatty acids, and use of the mucoadhesive system such as bio adhesive, liquid formulation microsphere powder and liquid gelling, formulation that decreases the mucociliary clearance of the drug formulation and thereby increase contact time between the drug and site of the absorption. Optimisation of bioavailability after nasal instillation involves consideration of following:

a) Deposition of drug in the nasal cavity

b) Clearance from the nasal cavity.

c) Rate of movement of drug in to nasal blood supply.

The way in which the drug is deposited in to nasal cavity has a dramatic influence on the invivo fate of the administered dose.

Pharmaceutical drugs as well as endogenous hormones such as luteinizing-hormone-releasing hormone (LHRH), thyrotrophin-releasing hormone (TRH) ¹⁶, vasopressin¹⁷, calcitonin, oxytocin, ACTH¹⁸, glucagon, insulin¹⁹ , interferons ²⁰, and enkephalins,have been shown to be absorbed nasally in animal and human. The studies of the nasal delivery of a number of peptide-based drugs demonstrated that systemic bioavailability can be improved by nasal route.

Rectal route of delivery:

Rectal route of drug delivery offers an advantage of reduction in first pass metabolism and rapid systemic absorption. Rectum also consists of large number of lymphatic vessels that may target drug delivery to the lymphatic circulation through rectal absorption. Absorption of proteins and peptides from the rectum is less and has low systemic bioavailability. Co administration of absorption promoting agents like sodiumglycholate, salicylates and EDTA enhance rectal absorption of insulin.

Buccal route of delivery:

The buccal mucoadhesive formulations are to be an alternative to the conventional oral small amount of medicaments as they can be readily attached to the buccal cavity retained for a longer period of time and removed at any time. The epithelium of the mouth is accessible with small surface area approximately 100 cm ². Peptide absorption occurs across the mucosa by passive diffusion and it is unlikely that there is a carrier-mediated transport mechanism²¹.The principal pathway is probably via the intercellular route where the major permeability barrier is represented by organized array of neutral lipids in the superficial layers of the epithelium. An absorption enhancer such as salicylates is needed to deliver the proteins across the mucosal tissues. These promoters provide little specificity with respect to substance whose absorption they facilitate. Parameters such as molecular weight, charge, conformation and molecular size play an important role in absorption of peptide and protein drugs. Bioadhesive formulations designed for buccal application should exhibit suitable rheological and mechanical properties, including pseudoplastic or plastic flow with thixotrophy, ease of application, good spreadability, appropriate hardness, and prolonged residence time in the oral cavity. Dosage forms for buccal delivery include adhesive tablets, gels and adhesive patches. Proteins such as oxytocin, vasopressin, calcitonin, insulin and interferon are absorbed through buccal mucosa.

Occular route of delivery:

The ocular route may also be used for the systemic delivery of peptides and protein based pharmaceutical drugs. The ocular route is the site of choice for the localized delivery of ophthalmologically active peptide and protein for the treatment of ocular disease in the pharmaceutical dosage form of solutions, suspensions and ointment. Several peptide and protein pharmaceuticals, such as enkephalins, thyrotropin releasing hormone, LHRH, glucagon, and insulin were reportedly absorbed. Drugs are absorbed by two ways. Those are

a) Drugs instilled in to precorneal cavity can reach the systemic circulation via blood vessels

underlying the conjuctival mucosa.

b) Overflow of drug solution in to the nasolacrimal drainage system followed by absorption through nasal mucosa.

By incorporating absorption promoters such as bilesalts (sodium salt of glycolic acid) and polyoxyethylene-9-lauryl ether, bioavailability of insulin was improved. Factors which affect ocular absorption are hydrophilicity, large molecular size, susceptibility to enzymatic barriers formulation. Use of nanoparticles, liposomes, gels and ocular inserts may improve ocular delivery of peptide based pharmaceuticals.

Transdermal route of delivery:

This is a topical medication in which drug was absorbed through the skin. The advantages of transdermal drug delivery (TDD) have therapeutic benefits such as sustained delivery of drugs to provide a steady plasma profile, particularly for drugs with short half-lives, and hence reduced systemic side effects; reducing the typical dosing schedule to once daily or even once weekly, hence generating the potential for improved patient compliance; and avoidance of the first-pass metabolism effect for drugs with poor oral bioavailability. The non-invasive delivery of TDD makes it accessible to a wide range of patient populations a highly acceptable option for drug dosing. Approaches for TDDS of peptide drugs are iontophoresis, phonophoresis, electroporation and application of penetration enhancers. Iontophoresis represents novel non invasive method for local systemic delivery where an electric current is used to drive the molecules across the skin surface²². Phonophoresis is enhanced absorption via thermal effect of ultrasonic waves and subsequent alterations in physical structure of skin. Electroporation is enhanced membrane permeability using short duration of electrical impulses which increases the drug deliver^23,24. Permeation enhancers like dimethyl sulfoxide, oleic acid and terpenes are used to improve the peptide and protein drug delivery.

Pulmonary route of delivery:

Pulmonary route of delivery is an attractive route of proteins and peptides than other alternative routes of administration. The lungs offer a large surface area for drug absorption, of approximately 80-140 m ². The alveolar epithelium is very thin (approximately 0.1-0.5 mm thick), thereby permitting rapid drug absorption. The alveoli can be effectively targeted for drug absorption by delivering the drug as an aerosol, with a mass median aerodynamic diameter of less than 5 μm. Furthermore, the first-pass metabolism of the GIT is avoided. Although metabolic enzymes are found in the lungs, the metabolic activities and pathways may differ from those observed in the GIT, and this makes the pulmonary administration of many peptides and proteins very promising. The pulmonary route, through aerosol delivery systems is for the administration of drugs molecules to treat pulmonary diseases, such as asthma. Three devices available for pulmonary delivery of drugs are metered dose inhalers (MDI), jet or ultrasonic nebulizer and dry powder inhaler ^{25, 26}. When penetration enhancers were incorporated into aerosol system complete absorption of insulin occurred.

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Received on 17.11.2012

Modified on 29.11.2012

Accepted on 25.12.2012

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(1): January- February, 2013, 7-11