Pulmonary delivery offers direct drug targeting for local diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Inhalation therapy may also be applied to treating systemic diseases, as it has some advantages over other routes of administration. The lungs provide a large surface area and high blood supply for drug absorption into the systemic circulation. It also avoids the first pass hepatic metabolism that orally delivered drugs undergo. Furthermore, inhalation is less invasive than injection, which is the conventional delivery method for many proteins. Thus, interest in developing inhaled proteins and peptides for systemic treatments has been increasing in recent decades. The respiratory route has favorable properties for the absorption of proteins and peptides. On the other hand, the lungs also have pulmonary clearance and metabolic pathways to guard against foreign macromolecules. These natural defense mechanisms can inadvertently oppose therapeutic protein delivery and need to be overcome if absorption is to increase. Active research is being carried out in pulmonary delivery of proteins, but so far very few respirable proteins have been marketed and practically no absorption enhancers have been approved. Most of the in vivo efficacy and toxicity studies have been conducted on animals, and not yet on humans. As proteins and peptides are complex and fragile molecules, there are formulation challenges that need to be overcome. Knowledge in this field is continually progressing and inhalation will become a convenient administration method for proteins and peptides, particularly for local delivery, in the future.

The most commonly used cell-penetrating peptides (CPPs): Tat, oligoarginine, and transportan, have all been demonstrated to facilitate the entry of protein/peptide cargoes into cells both in vitro and in vivo. In cellular systems, transportan has displayed greater internalization properties than the aforementioned arginine-rich CPPs and their uptake efficiencies can be depicted as follows: transportan > oligoarginine > Tat peptide. When picking the “right” CPP sequence for cargo delivery, both of these aspects need to be considered, and when lower concentrations are used, the transportan or oligoarginine should be preferred. In vivo, however, the uptake efficiency, specificity, and toxicity have not been extensively studied for the different CPPs. Nevertheless, it is evident that for targeted delivery, some extra motifs need to be added to the CPP sequence. A rather promising aspect for CPP-mediated delivery of peptides/proteins is that transcytosis in endothelial cells requires caveolin. Several CPPs, but especially transportan, exploit the caveolin pathway for cell entry, possibly giving transportan a beneficial “edge” in vivo. Despite the number of obstacles and pending challenges still faced today, the growing number of examples of in vivo delivery, Tat-HSP70 for neuronal rescue and Tat-Bcl-x(L) for improved neuronal precursor cell survival, confirm that the problems can be overcome in one way or another.

This chapter discusses the hazards of peptide and protein delivery. If the product quality is lacking and foreign species are present—such as protein aggregates of active ingredients or other dangerous contaminants—endogenous antibodies may be produced. These have the potential to reduce or eliminate the efficacy of the drug, or to cross-react and neutralize other endogenous proteins. The drug's route of administration, product quality, dosing frequency and duration, as well as the individual patient's immune system, can all impact such an immune response. This can have devastating results, compounding patient suffering and potentially leading to mortality. Progress is continuously being made in retaining product quality during manufacture and supply. There are many examples of how to stabilize proteins in aqueous solutions and dried solids based on previous experience and well designed experiments. This selection of appropriate conditions allows such products to remain stable for supply as effective medicines. As more research is carried out in these areas, the selection and design of appropriate molecules and conditions to maintain stability during manufacture and supply based on fundamental understanding become possible. Combining this information with quality by design (QbD) approaches will better enable robust manufacturing and supply of biopharmaceutical products.

This chapter highlights the significant advances in brain delivery of peptide and protein drugs made over the past three decades. While many limitations still exist regarding the cerebral delivery of peptide and protein drugs, several strategies show considerable promise for improvement of brain uptake. The chapter reviews the various strategies that are applicable for enhancing the delivery of protein/peptide delivery to the CNS. A wide variety of peptides and proteins have been identified as promising therapeutic agents for the treatment of various brain pathologies. Peptides and proteins are present in the whole nervous system, with unique distribution patterns. They can exert numerous biological actions in the brain, such as regulation of the internal environment of the brain and of the cerebral blood flow; modulation of the permeability of the blood–brain barrier (BBB) to nutrients, neurotransmission, and neuromodulation; various roles in the immune system; and hormonal regulation in the endocrine system. They are also involved in temperature control; food and water intake; cardiovascular, gastrointestinal, and respiratory control; memory and affective states; and potently modulate nerve development and regeneration. The multiplicity of biological actions of peptides suggests that these agents may be used as pharmaceuticals in the treatment of a variety of disorders of the brain and spinal cord, as indeed is borne out in the examples provided in this chapter.

The purpose of this chapter is to review the different nanoparticulate matrices; the stability of nanoparticles and the encapsulated peptides/proteins; the issues related to the bioavailability, biocompatibility, and toxicity of the nanoparticles; and other related topics such as techniques used to characterize the delivery systems and clinical applications. The major proteolytic enzyme in the stomach is pepsin and those in the small intestine include trypsin, chymotrypsin, carboxypeptidase, and elastase. In the gastrointestinal tract, enzymatic degradation can occur at the lumen, brush border, mucosal mesh, etc. Metabolism of proteinaceous drugs inevitably reduces their bioavailability. The small intestine is generally the major site for absorption of drugs, but enzymatic activity of proteases is also higher in this region than any other part of the gastrointestinal (GI) tract. The efficiency and mechanism of drug absorption change between the different segments of the small intestine. Towards the distal sections, the villi become smaller and fewer, thereby reducing the surface area of absorption. For instance, it is reported that in the upper jejunum the mucosal area per cm of serosal length is 98 cm2, whereas it is only 20 cm2 at the ileal side. The development of a successful oral peptide/protein delivery system has been actively investigated for the past few decades and is still a challenging area of research. Recent advances in biochemistry and biotechnology have led to the discovery and mass production of therapeutic peptides and proteins. However, their bioavailablity via oral delivery is still almost nil because of the many barriers in the GI tract, such as proteolytic degradation and the inability of these macromolecules to penetrate the intestinal cell wall.

Several case studies are described in this chapter that present the application of peptide and protein drugs in various surgical techniques and medicines. The formulation of these drugs is relatively complex and draws on existing polymeric carriers for their delivery to a local site. This is interesting, since it is a move away from more familiar protein delivery systems which are generally based on subcutaneous or intravenous injection or polymer encapsulation technologies for controlled delivery (e.g., Zoladex Implant). It is therefore important to be aware of this class of medical devices, since it offers great potential for emerging biomaterials research to be translated into medical practice. The design of the formulation is clearly central to the method of application and will continue to draw on multidisciplinary research themes such as protein engineering, surface adsorption, polymer synthesis, protein–polymer interactions, etc. The outcomes of the human clinical trials have been vital to determining the scope and utility of these medical devices and play a clear role in directing the field. The clinical need for this class of medical devices will continue to grow as the field of regenerative medicine becomes ever more integrated into healthcare.

Nasal delivery is needle-free, non-invasive, and painless; it does not require sterile preparation, can be self-administered, and facilitates the rapid onset of both local and systemic drug action. In order to maximize patient compliance and expand/extend any existing patent position, the nasal route of administration is the focus of much attention as a non-invasive alternative to the peroral route and to invasive parenteral application. Moreover, the nose to brain pathway opens the door for targeting drugs to the central nervous system. The physical barrier of the nasal epithelium is the main limitation with respect to drug absorption, especially for large molecules such as proteins and peptides. Another important obstacle is the rapid mucociliary clearance which limits the time available for drug absorption. Enzymes (particularly proteases) that are present in the nasal mucosa and the mucus layer are of less importance (certainly when compared to oral delivery), but not negligible. Another possible drawback is the relatively small amount of drug that can be nasally administered; this is related to the method and technique of administration which, if poor, may lead to drug absorption problems depending on the actual site of drug deposition. To achieve a stable, safe, and effective nasal delivery of peptides and proteins, these limitations have to be overcome.

This chapter discusses why bioconjugation of peptides and proteins is a useful tool to improve the biological performance of biotech drugs. The covalent conjugation of poly(ethylene glycol) (PEG) chains to proteins, known as PEGylation, has been successfully developed as a means of varying the in vivo properties of protein drugs. This technique offers less frequent administration to patients, greater convenience, and improved efficacy. PEG is a hydrophilic, biocompatible polymer approved by the main regulatory agencies, namely EMEA and FDA, for parenteral administration. The PEGylation of a protein can be achieved by random or selective conjugation protocols that yield the covalent attachment of one or more polymer chains to specific anchoring functions on the protein surface. Polymer conjugation induces size enlargement, charge and surface modifications, and protein shielding, which ultimately result in improved solubility, stability, enhanced immunological profile, prolonged permanence in the body and altered tissue localization, and cellular uptake. The beneficial effects of PEGylation usually compensate for the reduced biological activities of proteins modified by polymer conjugation. PEGylation has a strong effect on the pharmacokinetic profiles of proteins because it prolongs their presence in the bloodstream, increases their bioavailability, and modifies their biodistribution profiles. Renal clearance represents one of the main routes of protein elimination from the bloodstream. The glomerular capillary walls in the kidneys are organized into highly structured architectures with specialized barrier properties that control the ultrafiltration of hydrophilic macromolecules, namely proteins and polymers, and their reabsorption at the level of the proximal tubule. Both ultrafiltration and reabsorption depend on the composition, sizes, and charges of the circulating molecules.

This chapter provides an outline to some of the key events that have led to the current level of interest in peptide and protein drugs and defines the terms “peptide” and “protein.” A consensus has not been properly reached with respect to the use of these latter two terms, with the 51 amino acid, mature human insulin being a good example of the ambiguity, since it is generally described as a peptide but also as a protein by some. As a guide, peptides can be considered to be up to 50 amino acids in length, with proteins being larger than this. This boundary corresponds approximately to the upper limit of routine peptide synthesis in the solid phase. The rise of molecular biology as a tool by which to generate biopharmaceutical drugs—those that include proteins, DNA, conjugates, viruses, etc., initially far outpaced the development of delivery technologies. These biomacromolecules almost inevitably do not survive the stomach and intestinal environment due to pH and the presence of proteases. Nor do they readily transit the epithelial barrier due to the presence of cell-cell tight junctions, the semipermeable cell membrane, and, for intestinal epithelia, efflux proteins such as P-glycoprotein and the cell glycocalyx. Furthermore, biomacromolecules are very much larger than organic drugs, have short plasma half-lives, are involved in active transport processes, are susceptible to chemical and physical degradation, and are very potent. The need to overcome these challenges to their delivery has resulted in huge volumes of research, drawn from many disciplines including pharmaceutical materials, chemical engineering, biophysics, analytical methodology, cell and molecular biology, and in vivo studies.

This chapter discusses the modulation of the intestinal tight junctions using bacterial enterotoxins. Epithelial cell sheets limit the movement of solutes through the intercellular space by forming tight junctions (tjs) between adjacent epithelial cells, and therefore act as the major barrier between the internal and external environment of the body. There are three transepithelial pathways for molecules to pass from the intestinal lumen to the bloodstream: the passive transcellular pathway, the carrier-mediated transcellular pathway, and the paracellular pathway. Physicochemical properties, such as hydrophobicity, allow a molecule to passively partition from the intestinal lumen through the lipid bilayer into the cell. Some hydrophilic molecules, such as sugars and amino acids, are absorbed by specifically interacting with active transport systems on the cell membrane. Drug delivery via the paracellular pathway is less dependent on the physicochemical properties of the drug, and does not require a specific interaction with a transport system, and so is suitable for a large variety of molecules including peptides and proteins. Since opening of the tjs can cause the influx of other foreign substances, absorption enhancers acting via the paracellular pathway are required to modify the tight junctional structure reversibly. Early development of paracellular absorption enhancers was limited due to the lack of knowledge regarding the composition of the tjs and their regulatory role, resulting in unacceptable side-effects. Study of canonical enterotoxins (a class of exotoxin that acts on the intestinal epithelium) such as those released by Clostridium perfringens and Vibrio cholerae has increased our understanding to the point where one can exploit their mechanisms to facilitate peptide and protein delivery via the paracellular pathway.