Proteins and peptides are becoming increasingly popular to be used as therapeutic molecules. Therapeutic antibodies are the largest class of protein therapeutics today, but this group also includes enzymes, cytokines, hormones, and other proteins. Advances in biotechnology and genomics and, more recently, proteomics, have opened new ways to discover more therapeutic molecules. The progress in biotechnology and bioinformatics over the past 25 years have produced a number of protein- and peptide-based pharmaceutical products approved for the treatment of a broad spectrum of diseases. Also the modern biosynthetic approaches are enabling the availability of increasingly complex proteins including those with multiple specific carbohydrate moieties.
As we are aware that the most commonly used oral route of administering the drugs is not feasible for these molecules as they can be easily degraded in the GI tract. Therefore, so much of research is going on to provide either alternate form of the formulations or alternate route of administration for successful delivery of these molecules. The research was actually revolutionized in 1980s when recombinant insulin came into the market for the diabetic patient. Still people are using injectable form of insulin which is not patient compliant.
Route of administration
Mostly protein/peptide based formulations are lyophilized samples and they are delivered by subcutaneous, intramuscular, or intravenous injection. The mode of delivery limits product potential in the market place because patients do not readily accept injections especially in chronic treatment situations. The other possible routes of administration have been considered including nasal, buccal/sublingual, oral, transdermal, pulmonary, ocular, vaginal, and rectal. The large surface area associated with most of these routes makes them attractive targets for drug delivery.
Recent research areas
Technologies currently under investigation, ranging from those in their early-stage of development, such as laser-assisted delivery to others, where feasibility has already been demonstrated, such as micro needle systems, and finally more mature techniques that have already led to commercialisation for e.g., velocity-based technologies.
Market for protein therapeutics
The protein therapeutics market totaled $63 billion in 2007 ($39 billion in the US alone) and is expected to reach $87 billion by 2010, according to a report by Kalorama Information.
Protein delivery
Protein and peptide-based therapeutic agents have unique physiochemical properties such as high molecular weight, short half life, requirement of a sustained plasma level for the desired therapeutic effect, liable to physical and chemical instability by gastric enzymes and harsh acidic environment as well as first pass metabolism, which makes their delivery a challenge. Despite the efforts on non-invasive delivery methods, invasive delivery remains the main method of administering peptide and protein drugs. Recent developments in injectable, polymeric controlled-release formulations also put an emphasis on hydrogels and particulate systems. What remains, however, is one of the most difficult challenges the development of alternate delivery routes for these macromolecular drugs.
Following are few areas in which the research is going to deliver peptide based drugs at site with less invasiveness and targetability.
Drug delivery system (DDS)
By definition - DDS supplies drug selectively to its site(s) of action(s) in a manner that provides maximum therapeutic activity (though controlled and predetermined drug release kinetics), presents degradation or inactivation during transit to the target sites and protects the body from adverse reactions because of inappropriate disposition. For drugs that have a low therapeutic index, targeted drug delivery proves an effective treatment at a relatively low drug concentration.
Classification of drug targeting: Drug targeting has been classified into three types:
(a) First-order targeting - describes delivery to a discrete organ or tissue;
(b) Second-order targeting - represents targeting to a specific cell type(s) within a tissue or organ (e.g. tumour cells versus normal cells and hepatocytic cells versus Kupffer cells); and
(c) Third-order targeting - implies delivery to a specific intracellular compartment in the target cells (e.g. lyososomes).
Similarly there are three approaches for drug targeting:
● The first approach involves the use of biologically active agents that are both potent and selective to a particular site in the body (magic bullet approach of Ehrlich).
● The second approach involves the preparation of pharmacologically inert form of active drugs that when they reach the active sites become activated by a chemical or enzymatic reaction (pro drug approach).
● The third approach utilizes a biologically inert macromolecular carrier system that directs a drug to a specific site in the body where it is accumulated and affects its response (magic gun or missile approach).
Smart polymers
To fulfill the above criteria the use of control delivery using smart polymers seems promising as they overcome the limitations posed by other routes of delivery. Smart polymers are those polymers which can deliver the drug at site according to the need and condition. Smart polymers increase patient compliance, maintain stability of the drug, and maintain drug level in therapeutic window and are easy to manufacture. Different types of smart polymer-based delivery systems, such as sensitive to temperature, phase, pH, electric charge, light, and biochemicals, and their application in controlling the release of the incorporated drug to obtain a sustained plasma level have come up.
High-velocity particle delivery
High-velocity particle delivery across the skin is the technology of a major drug delivery operation based in UK and USA. Once a drug has been formulated as an appropriate and well-characterized powder, it is then introduced into a compact hand-held device in which a supersonic flow of gas accelerates the particles to a speed high enough that they can collide with the skin having enough energy to penetrate the outer layers and affect drug delivery. The depth and extent of delivery depends on the speed, diameter, and density of the drug particles.
Use of nanocarriers
Various pharmaceutical nanocarriers, such as nanospheres, nanocapsules, liposomes, micelles, cell ghosts, lipoproteins and some others are widely used for experimental (and already clinical) delivery of therapeutic and diagnostic agents including anticancer drugs are being worked upon. Among the most popular and well-investigated drug carriers are liposomes (mainly, for the delivery of water-soluble drugs) and micelles (for the delivery of poorly soluble drugs).
Liposomes
Liposomes are artificial phospholipid vesicles that vary in size from 50 to 1000 nm and can be loaded with a variety of water-soluble drugs (into their inner aqueous compartment) and sometimes even with water-insoluble drugs (into the hydrophobic compartment of the phospholipid bilayer .the component of targetability can be utilized with liposome by including the monoclonal antibodies with them. This increases the efficacy of the liposomal drug and decreases the loss of liposomes and their contents in the reticuloendothelial system (RES). The unique properties of long-circulating and targeted liposomes could be combined in one preparation in which antibodies or other specific binding molecules had been attached to the water-exposed tips of polyethylene glycol (PEG) chains.
Micelles
As these peptide based drugs are unstable to overcome the poor solubility, various micelle-forming surfactants can be used in the formulations of insoluble drug is suggested. This is why micelles, including polymeric micelles, are another promising type of pharmaceutical carrier. Micelles are colloidal dispersions with a particle size between 5 nm and 100 nm. Because of their small size (5-100 nm), micelles demonstrate spontaneous penetration into the interstitium in the body compartments with leaky vasculature (tumours and infarcts) by the EPR effect - a form of selective delivery termed "passive targeting".
Opsonization is the serious limitation with all pharmaceutical nanocarriers. They can easily be recognized as foreign components by the system and removed from the circulation before completion of their function. Pegylation is one of the procedures used to overcome this problem. Nanoparticles with PEG sterically hinder interactions of blood components with their surface and reduce the binding of plasma proteins with PEGylated nanoparticles, as was demonstrated for liposomes. PEG molecules with a molecular weight below 40 kDa are readily excretable from the body via the kidneys. Another approach of tagging nanocarriers with folate, since folate receptor (FR) expression is frequently over expressed in many tumour cells. Liposomal daunorubicin and doxorubicin were delivered into various tumor cells via folate receptor and demonstrated increased cytotoxicity. In vivo studies by Piego et al, showed that the Chitosan-PEG nanocapsules enhanced and prolonged the intestinal absorption of salmon calcitonin. Therefore, by modulating the PEGylation degree of chitosan, it was possible to obtain nanocapsules with a good stability, a low cytotoxicity and with absorption enhancing properties.
Liposomes are also used for targeting of antisense oligonucleotides, in particular for neuroblastoma treatment, exemplified by coated cationic liposomes made of a central core of a cationic phospholipid bound to oligonucleotide, and an outer shell of neutral lipid.
Polysaccharides
Using polysaccharides is a new approach. They are useful as they have lots of functional groups which can be utilized for drug conjugation. Esterfication with carboxylic drugs (used occasionally) and more often by oxidation of the hydroxyl groups to form aldehydes permit bond formation with different drugs.
Cell-penetrating peptides (CPPs)
Big molecules can not pass through membrane but CPPs are smaller peptides. This is another approach to deliver peptides to the system as they have ability to enter cells independent of a membrane receptor, and they show no cell-type specificity. They are small (10-30 residues in length), often positively charged sequences of amino acids. Currently research is going on two CPPs in particular; Tat, originating from the retrovirus human immunodeficiency virus type 1 (HIV-1) and pAntp, derived from the transcription factor Antennapedia. The current view is that each CPP may be internalized via an idiosyncratic. Unusually; CPPs exhibit remarkably low toxicity and have an extensive range of possible cargo types. Unlike viral vectors, CPPs do not have the capacity to integrate the genetic material they deliver, and therefore, there has been no evidence for CPP use resulting in oncogene activation. The immunogenicity raised in response to conjugated CPPs alone is said to be very unlikely. One trial concerns the drug cyclosporine A (CsA) used in ointment for the topical treatment of psoriasis. Previous formulations were ineffective due to the impermeable nature of the skin. However, a CPP-based formulation permits skin penetration. A polyarginine heptamer with a pH-sensitive linker allows the release of CsA at functional pH within the skin. Phase 1 trials were successful. Until the complete picture is revealed however, the therapeutic potential of CPPs cannot be realized, only predicted.
Pulmonary delivery
Since few years pulmonary delivery system has become hot area of research for delivery of drug due its direct accessibility to the site in case of respiratory diseases and availability of a wider area for absorption of the drug in case of other diseases. Inhalers and nebulizers are being used not only for the treatment of respiratory disease but also for the treatment of other non respiratory disease as well. Examples for the product available in the market are given in table 1.
Some more options
Currently, there are a number of alternatives being discovered to the conventional vial and syringe delivery for the drugs like of insulin and other peptide based medicines. This includes cartridge-based pen systems utilizing fine gauge needles, continuous subcutaneous infusion pumps and needle-less injectors for aqueous formulations. Dry powder needle-less system are also under development.
One more system called Transdermal System: (3M's Technologies): Microstructured Transdermal System (MTS) which a moulded plastic array to temporarily disrupt the normal skin barrier and allow intradermal delivery of proteins and peptides. Osmotically driven implantable pumps are also being made by Alza Corp which are being tested with the glucagon-like peptide, GLP-1 and lysozyme.
Use of peptide to deliver other biological molecules
Research is being carried out to deliver peptides to the target site but, peptides themselves are also being used to deliver other biological molecules like DNA/RNA to the target site.
Coralville, IA: Integrated DNA Technologies (IDT), the world leader in oligonucleotide synthesis, has introduced the novel peptide-based double-stranded RNA (dsRNA) transduction delivery system Transducti. Transductin, licensed from Traversa Therapeutics for research applications, is intended for in vitro testing and high-throughput screening projects. Transductin complexes with dsRNAs, such as IDT's Dicer-substrate siRNAs, and delivers them across cell membranes via macropinocytosis. This mechanism minimizes the risk of having the dsRNA trigger an innate immune response and has virtually no toxicity.
There are several products available based on peptide delivery system. Jonas Sal Brian Spencer group at Salk Institute for Biological Studies, has exploited one of the mechanisms that allow the brain to import essential nutrients and molecules such as cholesterol from the bloodstream. Low-density lipoprotein (LDL) receptors, which can be found on the surface of most cells including endothelial cells like shuttle large molecules such as apolipoprotein B across the blood-brain barrier. A fragment of apolipoprotein B was attached to glucocerebroside for the transport of the enzyme into the brain.
Summary
It is expected that in near future we will be getting many more important molecules for the treatment or prevention of many fatal diseases like cancer and AIDS etc. Tremendous research opportunities are still remaining to be explored to find ideal delivery systems for even old and the new molecules in future as well.
(The authors Sujata Basu & Amit Gupta are with JRF, SPTM, NMIMS University, Mumbai and Rachana is with JIITU, Sector 62, Noida)