6 Routes for the Delivery of Peptides and Proteins | Biotechnology

The following points highlight the six main routes for the delivery of peptides and proteins. The routes are: 1. Oral Route 2. Parental Route 3. Transdermal Route 4. Nasal Route 5. Rectal Route 6. Buccal Route.

1. Oral Route:

Oral route is the most convenient route for administration of drugs. But this route is problematic for protein and peptides drugs Peptides and proteins will be degraded by enzymatic action in the GIT. These molecules cannot cross the epithelial membrane due to their high molecular weight.

If they are absorbed they undergo first pass metabolism in the liver Proteins and polypeptides undergo hydrolysis GIT to form a mixture of smaller peptides and amino acids. But small peptides such as dipeptides and tripeptides remain stable in GIT (example: TRH thyrotropin releasing hormone).

Although these small peptides and a few larger peptides such as cyclosporin (undeca peptide) can remain stable, they are not at all observed to a significant extent. The bioavailability of TRH is only a few percent. The gastric fluids are intestinal fluids and the digestive juices contain many proteolytic enzymes. These enzymes hydrolyte the larger peptides to absorbable product.

A few smaller peptides such As TRH and cyclosporin are absorbed intact. This absorption is done through carrier-mediated transport. Most of the drugs are absorbed by passive transport. Here the rate of absorption is a function of many factors like pKa of the drug, particle size, and partition coefficient, stability in GIT, dose to solubility ratio, and molecular weight of the drug.

High molecular weight substances such as peptides, which have molecular weight above 500, are very poorly absorbed in GIT. Also the mucin layer acts as a physical barrier for the absorption these molecules can occur by the following mechanisms. Water-soluble molecules can diffuse through intercellular spaces (or water pores). Lipid soluble molecules can diffuse through the membrane lipids.

Water-soluble as well as lipid soluble materials can enter into epithelial cells by pinocytosis or phagocytosis. But the absorption by these mechanisms is very less, approximately 1-5 percent. Some peptides, which are absorbed by facilitated transport, are absorbed in greater extent. But this is also insufficient to elicit a pharmacological action.

Oral Bioavailability of Proteins and Peptides:

The oral bioavailability of peptides and protein can be improved by the following methods:

Peptidase Inhibitors:

Protection against the hydrolysis can be given by co-­administration of peptidase inhibitors with proteins and peptides. The enhanced the stability of peptides for example, amistatin acts as a peptidase inhibitor and promotes the absorption of enkephalins

Saturation of peptidases:

The saturation of peptidases can protect the peptides. This can be achieved by either increasing the dose or by administrating another protein or peptide with the drug. Increasing the dose can do saturation of peptidases. But this is possible only if the drug is non-toxic at high concentration. The drugs may be formulated in an enteric coated form to bypass some of the peptidases in GIT.

Polymeric coating cross linked with azoaromatic groups from a tough water-resistant film, which remains intact in stomach and small intestine. The natural microbial flora of colon reduces the azo bonds and hence the drug will be released in large intestine. But the colon is much less permeable to water-soluble drugs then small intestine and this reduces the bioavailability of peptides.

Penetration enhancers:

By the co-administration of penetration enhancers, the absorption of proteins and peptides can be improved. Different penetrations enhancers include surfactants, non- surfactants and chelating agents. Surfactants such as bile salt and detergents increase the membrane fluidity and hence increase the absorption of proteins and peptides.

Micelles formed by the surfactants may interact with the membrane phospholipids of intestine and promote the absorption. Also, pericellular permeation is increased by solubilisation of drugs in micelles formed by bile salts. Bile salt micelles are reported to enhance the absorption of insulin significantly.

Chelating agents such as EDTA and sodium citrate have been proved to enhance the absorption of insulin and heparin. They are supposed to promote the absorption of water soluble drug by chelating the calcium and magnesium ions of epithelial tissue and hence, cause their depletion in the regions of tight junction between the epithelial cells. This will force the water by osmosis and enhances pericellular absorption of peptides and proteins.

Lymphatic Uptake:

A part from enzymatic degradation and poor absorption of peptides from GIT, there is another important factor that is the first pass metabolism in the liver.

The hepatitis first pass metabolism can be avoided by two ways:

i. By blocking specific enzymes

ii. By promoting lymphatic uptake

The lymphatic uptake may be enhanced by surfactants, and bile salts, which facilitate micellar solubilisation. This can also be achieved by using lipid vehicles or by simultaneous feeding of lipid. The lipid enhances absorption via lymph system.

The lipid designation lead to the formation of chylomicrons, in which the drug may be the trapped and selectively absorbed by the lymphatic system Salicylates are also known to enhance lymphatic uptake of peptides. The more hydrophilic is the salicylate, the greater is the activity.

2. Parental Route:

Parental route is the practical method for administration of proteins. Here also the drug can be targeted to a particular tissue in a controlled release manner. Parental targeting is done for the following reasons. Site specific targeting will improve the therapeutic index of the drug by optimising the access, and the nature of interaction with receptors.

Targeting protects the drugs as well the body from unwanted and deleterious disposition. The rate of release can also be controlled and this allows the drugs with short biological half-life (proteins and peptides) and drugs with narrow therapeutic indices to be utilised. A parental controlled release system maintains a constant drug level in the blood and eliminates the problems of patient’s compliance.

Genetically engineered proteins and peptides are ideal candidates for targeting. These have a very short biological half-life in the blood. For example, angiotensin has a half- life of 15 seconds and oxytocin has half-life of 2 minutes.

Similarly the half-life of vasopressin is only 4 minutes and that of insulin is less than 25 minutes. The peptide and protein drugs are chemically similar to the endogenous substances they are intended to replace. Hence their metabolism in the body becomes easy. Both plasma and tissue peptides can breakdown these drugs.

If they are to undergo enzymatic degradation in plasma, they may cause immunological reactions and side effect. Thus by encapsulating the proteins and peptides in a target able drug delivery system; they can be protected from enzymatic degradation until they reach the target tissue or site of action. The biotechnology products are highly potent if they are given in convention dosage forms, whole body will be exposed to them.

Also due to distribution throughout the body, large quantities are required to achieve therapeutic level. The proteins and peptides, especially at high concentration can stimulate immune system. These molecules cannot pass through various membranes of our body due to large molecular weight formulating them in controlled release target able dosage forms will solve all these problems.

Parental Targeting:

Parental drug carrier systems are of two types namely, particulate carriers and soluble carriers. Particle size and surface properties control the fate of colloidal particles administrated intravenously. Particles larger then 7 µm are entrapped in fine capillaries of lungs.

Particles of size range between 100nm and 7µm, which are hydrophobic and carry a negative charge, are taken up by reticuloendothelial system (RES), if they are foreign. Such uptake of particles by the lungs and RES provides opportunity for passive targeting.

Else attaching a site-specific moiety to the carrier can actively target the blood. Passive targeting is the simplest method of parental targeting. Passive targeting to RES can be utilised to treat RES diseases such as viral and bacterial infection and neoplasm’s.

Microphage activating factor for the activation of macrophages to kill the tumors can be formulated into liposome’s, which will be taken up by the macrophages. Muramyl dipeptide is currently under study in a liposome encapsulated form as a macrophage- activating factor.

Inactive targeting, either the drug or the carrier is coupled to a site-specific moiety such as native immunoglubolin, erythrocyte membrane glycoprotein or MAbs. Tumor reactive MAbs are used to target cytotoxic drugs to neoplastic tumors; Synthetic polymers can be chemically conjugated to peptides and proteins to alter pharmacodynamics.

They can increase protein size or reduce unwanted interaction with blood and tissue components. The conjugation with hydrophilic polymers reduces immunogenecity and increase duration of action. Many problems are associated with parental targeting.

Some of them are given below:

1. If targeting is done with particulate carrier, sequestration by RES is the main problem and due to this, the drugs may not reach the site of action.

2. The particles cannot cross the vascular endothelium. Very small particles and some large molecules can pass through. Hence the particulate system should release the drug outside the cell. This leads to metabolism, prior to uptake by the cells.

3. The transport of drugs can occur by either pinocytosis or phagocytosis.

Receptor mediated transcytotic routes exist for only a few macromolecules. Thus, intercellular transport of macromolecules through cell membranes is a major obstacle.

Parental Targeted Drug Delivery Systems:

The parental targeted drug delivery systems for proteins and peptides are either soluble carrier systems or particulate carrier systems.

Soluble carrier systems:

Complexation of peptides and proteins with polymers and lipids enhances the stability and site specificity, while retaining biological activity. Specific properties of target cell are exploited for targeting.

For example, MAbs or other antibodies specific for a variant cell surface antigenic determinant can be complexed with drugs. The drug conjugate may be active at the cell surface, or may become active after endocytosis, or the conjugate may release the drug extracellularly in a controlled manner.

Genetic engineering can be utilised to have site specific sophisticated protein molecule. Hybrid fusion technique has also been used. Such molecule (Hybrid protein) combines future of two to three proteins form drug with target recognition properties as well as pharmacological activity. They have increased half-life, altered immunogenetically, and increased stability.

Particulate carrier systems:

Various particulate systems, which can be used as carriers for targeting of peptides and proteins. These can be targeted passively (according to their particle size) or actively (by conjugating with a tractable moiety like MAb).

Liposomes:

These are tiny spheres formed when phospholipids are combined with water. The structure of liposomes resembles that of cell membranes. They are vesicles made up of either one or more lipid bilayer alternating with aqueous compartments.

Liposome’s are of the following types:

i. Small unilamellar vesicles (SUV) they have a single lipid bilayer of the size of 20.70nm.

ii. Large unilamellar vesicles (LUV) size 100-400nm.

iii. Multilamellar vesicles (MLV) 200nm to several microns size and contain two or more concentric bilayer.

In liposome’s, various molecules can be incorporated to modify their properties. For example, incorporation of cholesterol into the liposome’s result in more stable membrane. Liposome’s can be passively targeted to liver. SUVs can be targeted to in liposomes; various molecules can be incorporated to modify their properties. For example, incorporation of cholesterol into the liposome’s result in more stable membrane.

Liposome’s can be passively targeted to liver. SUVs can be targeted to parenchymal cells of liver due to their small size. They can also be targeted to other organs by attaching immunoglobulins to them.

They are being tested for controlled release of genetically engineered peptides and protein such as macrophage stimulating agent, muramyl dipeptide and enzymes. The use of liposomes for relatively less stable, the entrapped drug may leak and their drug carrying capacity is low.

Microspheres:

They are solid spherical particles with a size range of 1 to 600pm and contain dispersed drug. They are prepared by emulsification; here the dispersed phase consists of droplets of polymer-drug solution. Otherwise, the polymer droplet can be cross-linked chemically, thermally or by gamma radiation.

Entrapment or ionic or covalent binding can be prepared either by natural biodegradable polymers (Gelatin or albumin) or by synthetic polymers (polylactic or polyglycolic acid). Two types of microspheres are possible in first type, the drug is encapsulated in a microcapsule and in the second type, and the drug is dispersed in polymer matrix.

The Microspheres can be passively targeted to lung capillaries or to RES. Active targeting is also possible if they are attached to targeting moieties. Albumin Microspheres mixed with iron oxide can be used as magnetic Microspheres and can be targeted to specific organs by the use of an external magnet.

Nanoparticles:

These are similar to Microspheres but have a particle size ranging between 10 and 1000nm. These can be made from biodegradable material such as albumin such as methyl methacrylate. Targeting the Nanoparticles with targeting the moieties such as monoclonal antibodies increases their specificity of targeting.

Released Erythrocytes:

When red blood cells are suspended in hypotonic solution permeability increases. If the solutions contain dissolved peptides and proteins, they form equilibrium on either side of the membrane. Then changing the ionic concentration of solution to isotonicity and incubating at 37°c restabilises the membrane. The pores will close and make the erythrocytes to reseal.

Using this technique, up to 40 percent of drug can be entrapped in RBCs. The released RBCs can be targeted to liver or spleen because aged, damaged or modified RBCs are taken up by RES and sent to liver. These can be used for enzymes replacement in genetic metabolic disorder. To be targeted by released RBCs. The drug should not be denatured under hypotonic conditions.

Microemulsions:

Colloid sized emulsion droplets can be used for targeting as well as for controlled release of peptides and proteins. The stability and fate of parental emulsion depends upon the emulsifier used.

Most commonly used emulsifying agents in lecithin. RES takes up the emulsion droplets. Vegetable oil emulsions are preferentially deposited in adipose tissue, mammary glands and myocardial muscle and hence can be used to target the drug to these tissues.

3. Transdermal Route:

Till recent times, skin was used only for the treatment of local infections. However, a better understanding of skin structure and functions has led to the utilisation of this tissue for systemic delivery of drugs. The transdermal route has many advantages.

They include:

i. Avoidance of first pass metabolism,

ii. Reduction in frequency of dosing,

iii. Easy termination of dosing

iv. Increased patient compliance

The common disadvantages of this route are:

1. Very low rate of permeation for most of the drugs

2. High variability in intra and inter subject permeation

Peptides and proteins have the difficult of penetration through stratum corneum due to their molecular weight; ionic nature and hydrophilic nature. The penetration can be enhanced by iontophoresis, and utilisation of permeation enhancers.

Iontophoresis:

It is the migration of ions when an electric current is passed through a solution containing ionised species. Drugs in ionic form can be “phoresed” into the body using small current below the pain threshold of the patients. The drug is placed in a gauze pad and is applied on the skin.

An electrode is placed at the distant part of the body and the current is allowed to flow for sufficient time period. To undergo iontophoresis, the proteins and peptides should carry charge. Charge can be included on the protein molecule by controlling the pH and ionic strength of the solution.

Insulin has been successfully studied for iontophoresis through intact rate skin. The rate and extent of transdermal absorption of TRH has been improved by iontophoresis are burning of skin and denaturation of peptides and proteins. They are denatured during charging. Due to current and also due to the heat generated during iontophoresis.

Phonophoresis:

In this method, ultrasonic sound is applied to the coupling contact agent. The ultrasonic waves increase the drug absorption by thermal effects and also by temporary alteration of the physical structure of the skin. In this method also the protein may get denatured due to the heat generation.

Penetration Enhancers:

The substances such as a zone and oleic acid enhance the drug absorption through the skin. They fluidise the intercellular lipid lamellae of stratum corneum. Fatty acids disrupt the packed structure of lipids in the extra cellular spaces. The major problem associated with the use of penetration enhancers is the skin irritation caused by them.

Prodrugs:

Several proteins and peptides including TRH, neurotensin and fibrinopeptides possess a terminal pyroglutamyl residue. This can be easily derived to produce prodrugs with enhanced permeability across the skin. The skin contains enzymes, which hydrolyse the prodrug at physiological pH to generate the active drug.

4. Nasal Route:

The nasal route is another option for delivery of peptides and proteins.

This route of administration has many advantages, which are listed below:

i. It is convenient for administration

ii. Self-medication is possible

iii. The surface area is large and mucosa is highly vascularised

iv. This route bypasses the first pass metabolism

When compared to other routes, this route shows high permeability for peptides and proteins. The bio-availability of peptides through nasal route depends on molecular weight and physicochemical properties. It is of the order of 1-20 percent.

Some of the disadvantages of nasal route include the following:

i. Variation in the mucous secretion affects the extent of absorption

ii. Peptidases present in nasal membrane degrade the peptides.

iii. The absorption will be altered with diseases state such as upper respiratory tract infections, allergic rhinitis and common cold.

iv. Penetration enhancers used may cause damage to mucosal cell membrane and may be ciliotoxic.

The clearance of drugs from nasal cavity is very fast in posterior nasal cavity. Here it is of the order of 10-15 minutes. Thus, nasal sprays are designed to deliver the drugs to the anterior cavity to reduce the clearance.

The absorption of proteins and peptides through nasal mucosa can be enhanced by penetration enhancers. They include surface active agents. These penetration enhancers increase the fluidity of lipid bilayer of the membrane, and also open the aqueous pores as a result of calcium chelation, and thus increase the penetration.

Peptides inhibitors also increase the absorption by decreasing the peptidase activity. But they cause damage to the mucosal membrane and are ciliotoxic in nature. Some penetration enhancers like oleic acid, salicylic acid, linoleic acid and sodium taurodihydrofusidate are safe. Damage to nasal epithelium, which is defence layer against infectious organisms, may lead to severe problems.

Thus the penetration enhancer should leave the nasal defence system intact. The nasal route is successfully used for administration of enkaphalin analogous, insulin and interferon’s. It can also be used for administration of pituitary hormones such as vasopressin and oxytocin. However, this route is suitable for long-term use and is short term delivery of peptides and proteins.

5. Rectal Route:

The large intestine receives the drainage mainly by hepatic portal vein. The lower portion receives drainage by lymphatic. This route is particularly suitable for peptides and proteins because these show greater lymphatic uptake due to their high molecular weight. The first pass metabolism by liver is also avoided. Also the rectal route avoids most of the GIT peptidases.

The bioavailability of peptides and protein from this route is particularly low because of the tight intercellular junctions of the rectal epithelium. Hence, penetration enhancers are required. Surfactants, if used as penetration enhancers cause bleeding and mucosal damage If silicates are used, the above problem is solved. Designing of suitable suppository can be done for peptides and proteins along with a mild penetration enhancer.

6. Buccal Route:

This route has many advantages for the delivery of peptides and proteins:

i. The sensitivity is less when compared to nasal and other routes.

ii. The drug delivery system can be removed at any time. This route has better patient compliance.

But the absorption of proteins and peptides is less when compared to nasal route. The common method for delivery is the tablet form. But there are chances of drug loss due to swallowing of saliva or designing a self-adhesive dosage from, which can retain in the oral cavity in contact with buccal mucosa, can solve accidental swallowing of tablet.

These problems like water-soluble and water insoluble, ionic as well as non-ionic polymers can be used for buccal adhesion. Polyacrylate hydro gels are most commonly used, the adhesive polymer can act as drug carrier. In a second type of device, it acts as adhesive link between disk loaded with drug and the mucosa.

In a third party of device, the disk is fixed to mucosa by self-adhesive polymer shield. This device can be applied for several days. But they interface with eating, drinking as well as talking. So, the maximum time of application should be a few hours. Though peptides and proteins from biotechnology can be delivered by all the above-mentioned routes, the main limitation is the requirement of penetration enhancer.

Milder, less irritating and less damaging penetration enhancer should be developed for the non-conventional routes of administration such as transdermal, nasal, rectal and buccal routes. Among the non-conventional routes, buccal route appears to be more promising because of its least susceptibility to irritation.