Systemic delivery of peptides by the oral route: Formulation and medicinal chemistry approaches.

In its 33 years, ADDR has published regularly on the po5tential of oral delivery of biologics especially peptides and proteins. In the intervening period, analysis of the preclinical and clinical trial failures of many purported platform technologies has led to reflection on the true status of the field and reigning in of expectations. Oral formulations of semaglutide, octreotide, and salmon calcitonin have completed Phase III trials, with oral semaglutide being approved by the FDA in 2019. The progress made with oral peptide formulations based on traditional permeation enhancers is against a background of low and variable oral bioavailability values of ~1%, leading to a current perception that only potent peptides with a viable cost of synthesis can be realistically considered. Desirable features of candidates should include a large therapeutic index, some stability in the GI tract, a long elimination half-life, and a relatively low clearance rate. Administration in nanoparticle formats have largely disappointed, with few prototypes reaching clinical trials: insufficient particle loading, lack of controlled release, low epithelial particle uptake, and lack of scalable synthesis being the main reasons for discontinuation. Disruptive technologies based on engineered devices promise improvements, but scale-up and toxicology aspects are issues to address. In parallel, medicinal chemists are synthesizing stable hydrophobic macrocyclic candidate peptides of lower molecular weight and with potential for greater oral bioavailability than linear peptides, but perhaps without the same requirement for elaborate drug delivery systems. In summary, while there have been advances in understanding the limitations of peptides for oral delivery, low membrane permeability, metabolism, and high clearance rates continue to hamper progress.

Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug.

Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.

Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation.

Cell-cell interactions of the mucosal epithelia are important for the maintenance and establishment of epithelial barrier function. During events of inflammation, such cell-cell interactions are often disrupted, resulting in a leaky epithelial barrier, which in turn can lead to various inflammatory and infective dysfunctions. Human junctional adhesion molecule (huJAM), found on the mucosal epithelia and vascular endothelia of many major organ systems, is a membrane glycoprotein which resolves to a doublet band of approximately 40 and approximately 37 kDa under SDS-PAGE analysis, representing differentially glycosylated forms of the same protein. huJAM was localized to the lateral membrane of Caco-2 cells (a human colonic epithelial cell line) monolayers, in an area basolateral of the epithelial tight junctions (TJ). Through functional and biochemical assays, we show huJAM to be able to homotypically associate and to participate in TJ restitution after trypsin-EDTA disruption. Furthermore, we also observed a migration of huJAM expression toward areas of cell-cell contacts during events of cell adhesion and monolayer formation. These qualities makes huJAM a likely player in the regulation of cell-cell contacts and the subsequent formation of TJs.

The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction.

Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were alpha-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-zeta, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.

New approaches to antigen delivery.

An improved understanding of immunologic events associated with immunization, the identification of promising new antigens, and an increased capacity to generate these antigens through chemical and biotechnology methods have led to many new vaccine opportunities. Inappropriate antigen exposure, however, can result in unwanted outcomes, such as incomplete protection, allergic reactions, autoimmunity, infection, or even tolerization. Thus, proper antigen delivery is critical for achieving the desired outcome. A number of vaccination approaches have now been described with varied degrees of success. The relative success of these approaches can be correlated with antigen delivery to specific presentation cells and stimulation of the immune system at sites where protective immunity is most appropriate. In addition, a greater understanding of mechanisms involving cells and effector molecules in the events of immunity may allow for improved possibilities for initiating, augmenting, and maintaining the response to a delivered antigen. This review provides insights into the various strategies currently being explored to optimize antigen delivery and the immune response to that antigen.

Modulation of tight junction function by G protein-coupled events.

Small G proteins or GTPases comprise a growing family of signal transduction molecules with inducible properties dependent upon reversible interactions with guanine nucleotides. Activation status of the proteins is characterized by preferential affinity for triphosphorylated guanine nucleotides, initiating signaling events that control fundamental processes involved in cell migration and contraction. Termination of small G protein signaling activity is in part achieved through intrinsic GTPase activity, which catalyzes the removal of GTP and its replacement with functionally inactive GDP. Recent investigations have implicated various small G proteins as messengers that control cell-cell contact between scaffold proteins and the actin cytoskeleton, suggesting an intrinsic mechanism for the regulation of paracellular permeability in polarized epithelial and endothelial cells. This review will examine current evidence for the control of tight junction permeability by small G proteins, and speculate upon future directions that may be of value in further exploring the biological importance of these key mediators.

Epithelial application of Pseudomonas aeruginosa exotoxin A results in a selective targeting to cells in the liver, spleen and lymph node.

Pseudomonas aeruginosa exotoxin A (PE) is a 67-kDa protein expressed under the selective pressure of a low iron environment. Previous studies using non-toxic PE chimeras containing a viral surface antigen, the V3 loop of MN gp120 from human immunodeficiency virus type 1 (HIV-1), resulted in not only an effective mucosal immunization but also a striking systemic immune response following epithelial application. Presently, we have examined the possibility that such a strong dual immune response was generated by the efficient targeting of critical cells of the immune system. Mice were dosed with 10 microg of toxic PE or a non-toxic mutant of PE (ntPE) by intratracheal instillation. Examination of lung, liver and spleen tissues isolated 4, 8 and 12 h following intratracheal instillation with PE demonstrated specific cell damage in these tissues which was not observed in mice dosed with ntPE. Based upon the location and characteristics of observed responses, the cells targeted by PE appear to be involved in the antigen presentation arm of the immune response. Since ntPE chimeras with inserted peptide antigen epitopes from a wide variety of pathogens are easy to prepare and administer, these results support this approach for mucosal immunization.

Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin.

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.

Noninvasive in vivo analysis of human small intestinal paracellular absorption: regulation by Na+-glucose cotransport.

Activation of intestinal Na+-glucose cotransport increases paracellular movement of inert tracers in cultured monolayers, isolated rodent intestinal mucosae, and in rodents in vivo. However, not all studies have demonstrated comparable effects on human intestinal paracellular absorption. We sought to assess the effects of Na+-glucose cotransport on paracellular absorption in human beings using a simple noninvasive assay. Study subjects drank six 200-ml doses of test solution, composed of 0.8% w/v creatinine (sufficient to overwhelm endogenous creatinine) in 277 mM glucose or mannitol and urine was collected. Intestinal creatinine absorption is paracellular. Once absorbed, creatinine is cleared into the urine. Therefore, urinary creatinine recovery reflects intestinal paracellular creatinine absorption. Total urinary creatinine recovery was 55% +/- 4% of creatinine ingested with glucose and 38% +/- 9% of creatinine ingested with mannitol (p < 0.001). Thus, intestinal paracellular absorption of creatinine is increased by the presence of luminal glucose. Our results are consistent with in vivo human regulation of mucosal permeability by Na+-glucose cotransport.

Hepatocyte growth factor inhibits amiloride-sensitive Na(+) channel function in cystic fibrosis airway epithelium in vitro.

Cystic fibrosis (CF) airway epithelial cells have a reduced cAMP-dependent Cl(-)conductance channel (CFTR) function but an increased level of amiloride-sensitive Na(+)channel (ENaC) activity. Recently, expression of the alpha -subunit of the ENaC protein complex was shown to be down-regulated by activation of the extracellular signal-regulated protein kinase (ERK) pathway. In the present study we have examined the actions of a potent regulator of the ERK pathway, recombinant human hepatocyte growth factor (rhHGF), on the function of ENaC in confluent, polarized monolayers of both primary cultures of CF airway cells and an SV40-transformed CF nasal epithelial cell line (JME CF/15). Treatment of JME/CF 15 cells with rhHGF at concentrations of 100 ng/ml and above was found to dramatically decrease the activity of amiloride-sensitive Na(+)transport. This effect required basolateral exposure of the cytokine. Addition of 100 ng/ml rhHGF to JME/CF 15 cells decreased I(eq)with a t(1/2)of;18 h, with a maximal inhibition of;90% by 36 h. By 48 h, stimulation with rhHGF induced a down-regulation of its receptor, c-met, expressed in these cells. The decrease in I(eq)of JME/CF 15 monolayers was not immediately reversed upon removal of rhHGF. Treatment with rhHGF did not appear to affect monolayer resistances nor Cl(-)currents induced by mediators such as isoproterenol, histamine or bradykinin. Studies with primary cultures of CF airway cell sheets demonstrated comparable sensitivity and time-course properties for the inhibition of amiloride-sensitive currents following rhHGF addition. These observations are consistent with the possible application of an extracellular signalling molecule, such as the cytokine HGF, to reduce the abnormally high activity of amiloride-sensitive Na(+)ion channels observed in CF airway cells.

In vitro evaluation of microparticles and polymer gels for use as nasal platforms for protein delivery.

PURPOSE: Nasal delivery of protein therapeutics can be compromised by the brief residence time at this mucosal surface. Some bioadhesive polymers have been suggested to extend residence time and improve protein uptake across the nasal mucosa. We examined several potential polymer platforms for their in vitro protein release, relative bioadhesive properties and induction of cytokine release from respiratory epithelium. METHODS: Starch, alginate, chitosan or Carbopol microparticles, containing the test protein bovine serum albumin (BSA), were prepared by spray-drying and characterized by laser diffraction and scanning electron microscopy. An open-membrane system was used to determine protein release profiles and confluent, polarized Calu-3 cell sheets were used to evaluate relative bioadhesion, enhancement of protein transport and induction of cytokine release in vitro. RESULTS: All spray-dried microparticles averaged 2-4 microm in diameter. Loaded BSA was not covalently aggregated or degraded. Starch and alginate microparticles released protein more rapidly but were less adhesive to polarized Calu-3 cells than chitosan and Carbopol microparticles. Protein transport across polarized Calu-3 cells was enhanced from Carbopol gels and chitosan microparticles. A mixture of chitosan microparticles with lysophosphatidylcholine increased protein transport further. Microparticles prepared from either chitosan or starch microparticles, applied apically, induced the basolateral release of IL-6 and IL-8 from polarized Calu-3 cells. Release of other cytokines, such as IL-1beta, TNF-alpha, GM-CSF and TGF-beta, were not affected by an apical exposure to polymer formulations. CONCLUSIONS: We have described two systems for the in vitro assessment of potential nasal platforms for protein delivery. Based upon these assessments, Carbopol gels and chitosan microparticles provided the most desirable characteristics for protein therapeutic and protein antigen delivery, respectively, of the formulations examined.

Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses.

We have used a mouse immunization model to evaluate the potential for a chimera protein composed of a nontoxic form of Pseudomonas exotoxin (ntPE) to incite and sustain a mucosal immune response against an integrated antigen. The chimera, termed ntPE-V3MN26, contained 26 amino acids of the gp120 V3 loop region sequence of the MN strain of HIV-1 integrated in place of the Ib region of ntPE. Following either vaginal, rectal, oral or subcutaneous administration and boosting, anti-gp120-specific IgA and IgG levels in serum and saliva samples were assessed by ELISA. All dosing regimens stimulated significant and comparable salivary IgA and serum IgG responses at 1, 2 and 3 months after the initial inoculation. Following a boost at 16 months with ntPE-V3MN26, a strong memory response to the antigen was observed. Isotyping of serum antibodies at this time suggested that both a Thl and a Th2 response had been induced. Responses to ntPE-V3MN26 following subcutaneous injection in the presence or absence of Freund's adjuvant demonstrated that Freund's adjuvant resulted in a three-fold greater enhancement of immune response compared to administration of chimera alone. These results demonstrate that mucosal presentation of a chimera composed of a nontoxic form of Pseudomonas exotoxin can result in a strong mucosal and systemic antigen-specific immune response to an integrated antigen. The profound memory responses induced by this chimera may be particularly useful for practical vaccine applications.

Characterization of V3 loop-Pseudomonas exotoxin chimeras. Candidate vaccines for human immunodeficiency virus-1.

To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.

An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2.

Carcinomas of the exocrine pancreas are poorly understood and have a poor prognosis because of their highly malignant nature. Using two human pancreatic cancer cell lines, AsPc-1 and Capan-2, we have investigated avenues that might be useful in targeting the delivery of antineoplastic agents to such cancers. Qualitative RNA PCRs established the presence of the oligopeptide transporter PEPT 1 in these pancreatic cell lines. Northern analysis confirmed the presence of a 3.3-kb transcript. The transporter is normally expressed primarily in small intestinal epithelial cells for nutrient absorption. It is also expressed in a human intestinal cell line, Caco-2. High levels of PEPT 1 protein expression in AsPc-1 and Capan-2, as multiple glycosylated forms (Mr approximately 90,000-120,000), were confirmed by Western immunoblotting, when compared with Caco-2 cell cultures. Absorption of the model dipeptide glycyl-L-sarcosine by AsPc-1 and Capan-2 cells was similar to glycyl-L-sarcosine absorption by Caco-2 cells and a Chinese hamster ovary cell line expressing human PEPT 1 (CHO-PEPT 1). Uptake was pH dependent and inhibited by several di/tripeptides and bestatin, but it remained unaffected by glycine and tetraglycine. Peptide solute transport by AsPc-1 and Capan-2 cells exhibited binding affinities (Kms) similar to those previously reported for PEPT 1, whereas the transport maximal velocity (Vmax) of the AsPc-1 cells was much greater than those of the Capan-2 and Caco-2 cells. Immunomicroscopy demonstrated PEPT 1 protein localized at the plasma membrane and in intracellular vesicular structures, similar to that observed for Caco-2 and CHO-PEPT 1 cells. These data suggest that the pancreatic cancer cells AsPc-1 and Capan-2 express surprisingly high levels of a solute transporter that was previously thought to be restricted in function to the absorption of nutrients from the small intestine.

Adjuvants and delivery issues related to immunization: a survey of the recent patent literature.

Vaccines have provided generations of individuals with protection against a number of devastating childhood diseases and, in the case of smallpox, have eradicated the disease. Arguably, this preventive approach to health care may have saved more lives than all other therapeutic approaches combined. Being commonly composed of killed-whole or live-attenuated organisms, successful vaccines have been capable of not only presenting crucial epitopes required for protective immunity but also components which stimulate the immune system provide a response of sufficient intensity to provide sustained protection. It is unlikely, however, that these same vaccines would be approved in today's regulatory climate where manufacturers must validate a highly reproducible manufacturing process and demonstrate the exact composition of the final product through stringent quality control (Gupta, 1997). Therefore, companies involved in the identification of new vaccines and their production appear to be moving away from the ill-defined and poorly-controlled immunogens of the past. Instead, they have started employing recombinant biotechnology to produce large quantities of specific antigens with the hope that these antigens will provide protective immunity. Antigen identification can take years of investigation. This process could be speeded up, however, by understanding the mechanisms which pathogens such as viruses and bacteria use to infect the host, their replication cycle and the steps they frequently take to evade the host immune system. Such a rational approach to immunogen design results in what has been termed an "intelligent" vaccine (Plough, 1998). "Intelligent" vaccines will likely be completely composed of artificial or synthetically derived products using recombinant and cell culture technologies and hopefully will allow manufacturers to develop products with the desired efficacy and safety profiles required by regulatory authorities. But unlike the complex and ill-defined immunogens of the past, these specific antigens are frequently poorly antigenic by themselves when compared to whole organisms. Co-delivery with a variety of agents, known as adjuvants, has been shown to increase the antigenicity of these poorly antigenic immunogens. In this editorial, I review the recent patent literature as it pertains to targeting of specific antigens and the role played by various classes of adjuvants to enable or enhance such targeted deliveries.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.