Oral biodrug delivery using cell-penetrating peptide

Abstract

During the past few decades, the novel biotherapeutic agents such as peptides and proteins have been contributed to the treatment of several diseases. However, their oral absorption is significantly limited due to their poor delivery through the intestinal mucosa. Therefore, the feasible approaches are needed for improving the oral bioavailability of biodrugs. Recently, cell-penetrating peptides (CPPs) such as HIV-1 Tat, penetratin and oligoarginine are considered as a useful tool for the intracellular delivery of therapeutic macromolecules. Hence, it was expected that the ability of CPPs may be applicable to enhance the absorption of biodrugs through intestinal epithelial membrane. CPPs are likely to become powerful tools for overcoming the low permeability of therapeutic peptides and proteins through the intestinal membrane, the major barrier to their oral delivery. Further advantage of this promising strategy is that this successful intestinal absorption could be achieved by more convenient methodology, coadministration of CPP with drugs via intermolecular interaction among them. Hereafter, the further establishment of delivery system based on CPPs is required to realize the development of the oral forms of therapeutic peptides and proteins. The aim here is to introduce our vision focusing on oral biodrug delivery by the use of CPPs as potential peptide carrier in order to provide new information in the design and development of new oral delivery systems for novel biotherapeutics.

Introduction

A number of new biotechnological therapeutics (biodrugs), such as peptides and proteins, antibodies, siRNAs and other macromolecular drugs, have been developed recently, and there is no doubt that these biodrugs will occupy a significant portion of the drug market in the near future [1]. Biodrugs exhibit high selectivity and an ability to provide effective and potent action with fewer side effects, and these drugs have become available for treating various diseases [2], [3], [4]. Although a wide variety of biodrugs, including peptides and proteins, are now produced on a commercial scale, one challenging task for pharmaceutical researchers is devise ways to deliver these drugs effectively and safely via non-invasive, patient-friendly routes [5]. Problems arise from the unfavorable physicochemical and biological properties of therapeutic peptides and proteins, which affect their absorption. At present, the parenteral route is the most frequently used route of delivery for such biodrugs [6].

Over the past several decades, many approaches have been used to overcome the inherent barriers to the uptake of therapeutic peptides and proteins through the gastrointestinal tract (GIT), transmucosal, and transdermal routes [7], [8]. The oral delivery route remains the most convenient system because it is non-invasive and patient friendly, and can be self-administered; however, it is the most challenging for peptide and protein delivery [9]. The oral administration of biotherapeutic agents is notoriously difficult because of their poor permeability through the intestinal mucosa, which is related to their high molecular weights, hydrophilicity, and susceptibility to enzymatic degradation [10]. To produce therapeutic activity, orally administered biodrugs must be absorbed efficiently from the intestinal lumen into the circulation without being metabolized extensively in the intestine. The low permeability across the epithelial mucosa of the GIT and the lack of stability in the luminal environment remain the two main causes of the poor oral bioavailability of biodrugs [11].

For years, drug delivery scientists have proposed approaches to overcome these limitations on the suitability of peptide substances and macromolecular drugs with poor biopharmaceutical properties. Examples of these approaches include improving the mucosal permeation of peptides and proteins, including the use of absorption enhancers [12], [13], [14], [15], [16] and the use of protease inhibitors [17], [18], [19], mucoadhesive polymeric systems [20], [21], [22], [23], [24], particulate carrier delivery systems [25], [26], [27], and targeted delivery systems [28], [29], [30], [31]. Although these approaches are successful in the laboratory, they have not been accepted widely by clinicians and regulatory bodies. It is clear that strategies directed at overcoming the enzymatic barrier alone will have only limited success in the development of oral forms of peptide and protein drugs. High oral bioavailability is unlikely to be achieved unless one can increase the membrane permeability of macromolecules.

Recently cell-penetrating peptides (CPPs) comprise a growing family of peptides that have opened a new avenue for drug delivery of a vast collection of biomolecules that otherwise do not cross the plasma membrane [32], [33]. Several CPPs, such as human immunodeficiency virus (HIV)-1Tat (48–60) [34], [35], [36], [37], [38] and oligoarginine [34], [35], [36], [37], [39], and amphipathic peptides such as Drosophila Antennapedia homeodomain (penetratin) [40] can overcome the poor permeability of biological agents through the epithelial cell membrane of mammalian cells via adsorption to cell-surface glycosaminoglycans (GAGs) and processing through the endocytic pathway [41], [42], [43], [44], [45], [46], [47], especially macropinocytosis [48], [49]. CPPs are used to deliver a large variety of drugs inside cells, such as proteins [50], DNA [51], oligonucleotides [52], and drug carrier systems including liposomes [53] and nanoparticles [54]. CPPs have promise as potential vectors for the intracellular delivery of various cargo molecules through the membranes of numerous cells.

Dowdy and co-workers were the first to evaluate the in vivo absorption of bioactive macromolecules conjugated to CPPs. The intraperitoneal injection of the 120-kDa β-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, resulted in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for the direct delivery of proteins into patients in the context of protein therapy and for epigenetic experimentation with model organisms. The potential immune responses and toxicity associated with long-term transduction of proteins in vivo are important issues. The injection of 1 mg of a TAT fusion protein per kilogram of body weight daily to a mouse for 14 consecutive days produced no signs of gross neurological problems or systemic distress [55].

Another study examined the absorption of FITC-labeled insulin covalently conjugated with TAT fusion protein across the epithelial cell layer in the gastrointestinal mucosa [56]. The intestinal absorption of FITC-insulin and FITC-insulin/TAT conjugates was compared in vitro in a Caco-2 cell monolayer, an in vitro model used widely for intestinal absorption studies. The transporting efficiency of FITC-insulin/TAT conjugate increased markedly when insulin was linked to the CPP. The transporting efficiency of the insulin/TAT conjugate was 5–8 times higher than that of free insulin, as shown by calculating the effective permeability of insulin and the insulin/TAT conjugate. However, this study was performed in Caco-2 cells and not in a biological membrane. Another study evaluated the usefulness of bioactive drugs covalently conjugated to a CPP in an oral delivery system based on oligoarginine [57]. The effects on the biological ileal permeation of a peptide drug after its coincubation with oligoarginine and the absorption of an oligoarginine-introduced peptide derivative by ileal segments were examined. Leuprolide, a relatively small peptide, was used and was conjugated to oligoarginine. The AUCs did not differ significantly between the administration of leuprolide–d-R6 and that of the original leuprolide. The pharmacological activity of therapeutic peptides and proteins may be lost by CPP conjugation, and the absorption of the leuprolide–d-R6 conjugate was not higher than that of leuprolide alone. These results suggest that the conjugation approach may not be favorable for increasing absorption into circulation.

However, there is only limited information about the effects of CPPs on the permeability of macromolecular drugs through mucosal membranes. The aim here is to introduce our vision for a CPP research strategy for the oral delivery of biodrugs and to focus on some specific aspects of the role of CPPs in the oral delivery of peptides and proteins. We aim to provide an overview that may be used to establish fundamental guidelines for the design and development of non-invasive delivery systems of biotherapeutic drugs using CPPs.