Cystic ovary disease impairs transport speed, smooth muscle contraction, and epithelial ion transport in the bovine oviduct.

Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment.

LabrafacTM MC60 is an efficacious intestinal permeation enhancer for macromolecules: Comparisons with Labrasol® ALF in ex vivo and in vivo rat studies.

Labrafac™ MC60 (glycerol monocaprylocaprate) is a lipid-based excipient used in oral formulations as a solubiliser. Due to the high proportions of established permeability enhancers, caprylate (C8) and caprate (C10), in Labrafac™ MC60, we hypothesised that it might behave as an intestinal permeation enhancer. We therefore evaluated this using two paracellular markers (ex vivo) and insulin (in vivo) as model molecules. Ex vivo studies were conducted in isolated muscle-stripped rat colonic mucosae mounted in Ussing chambers. Apical addition of Labrafac™ MC60 (8, 12, and 16 mg/ml) enhanced the apparent permeability coefficients (Papp) of [14C] mannitol and FITC-dextran 4 kDa (FD4) across colonic mucosae. Similar effects were observed in isolated jejunal mucosae, but at higher concentrations (40 mg/ml). The enhancing capacity of Labrafac™ MC60 was transient due to reversibility of reductions in transepithelial electrical resistance (TEER) upon wash-out and effects on fluxes were molecular weight-dependent (MW) as suggested by fluxes of a set of high MW FITC-dextrans. The permeability enhancing effects of Labrafac™ MC60 ex vivo were maintained in the presence of simulated intestinal fluids, FaSSIF and FaSSCoF, in both jejunal and colonic mucosae, respectively. Following intra-intestinal regional instillations to rats, the relative bioavailability of 50 IU/kg insulin ad-mixed with Labrafac™ MC60 was 5 % in jejunum (40 mg/ml) and 6 % in colon (8 mg/ml). When Labrafac™ MC60 was combined with PEG-60 hydrogenated castor oil (1 % v/v), this further increased the bioavailability of insulin to 8 % in jejunum. Absorption enhancement was also maintained in the presence of FaSSIF in jejunal instillations. Histology after 120 min exposure to Labrafac™ MC60 in vivo for both jejunum and colon was similar to untreated control. Labrafac™ MC60 therefore acts as a non-damaging intestinal permeation enhancer for macromolecules and can be considered as another excipient in screening programmes to develop orally administered macromolecules.

Challenges in Permeability Assessment for Oral Drug Product Development.

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed.

Impact of PEGylation on an antibody-loaded nanoparticle-based drug delivery system for the treatment of inflammatory bowel disease.

Nanoparticle-based oral drug delivery systems have the potential to target inflamed regions in the gastrointestinal tract by specifically accumulating at disrupted colonic epithelium. But, delivery of intact protein drugs at the targeted site is a major challenge due to the harsh gastrointestinal environment and the protective mucus layer. Biocompatible nanoparticles engineered to target the inflamed colonic tissue and efficiently penetrate the mucosal layer can provide a promising approach for orally delivering monoclonal antibodies to treat inflammatory bowel disease. The study aims to develop mucus-penetrating nanoparticles composed of poly(lactic-co-glycolic acid, PLGA) polymers with two different polyethylene glycol (PEG) chain lengths (2 kDa and 5kDa) to encapsulate monoclonal antibody against tumor necrosis factor-α (TNF-α). The impact of different PEG chain lengths on the efficacy of the nanosystems was evaluated in vitro, ex vivo, and in vivo. Both PLGA-PEG2k and PLGA-PEG5k nanoparticles successfully encapsulated the antibody and significantly reduced TNF-α secretion from activated macrophages and intestinal epithelial cells. However, only antibody-loaded PLGA-PEG2k nanoparticles were able to alleviate the experimental acute colitis in mice demonstrated by improved colon weight/length ratio, histological score, and reduced tissue-associated myeloperoxidase activity and expression of proinflammatory cytokine TNF-α levels compared with the control group. The results suggest that despite having no significant differences in the in vitro cell-based assays, PEG chain length has a significant impact on the in vivo performance of the mucus penetrating nanoparticles. Overall, PLGA-PEG2k nanoparticles were presented as a promising oral delivery system for targeted antibody delivery to treat inflammatory bowel disease. STATEMENT OF SIGNIFICANCE: There is an unmet therapeutic need for oral drug delivery systems for safe and effective antibody therapy of inflammatory bowel disease. Therefore, we have developed PEGylated PLGA-based nanoparticulate drug delivery systems for oral targeted delivery of anti-TNF-α antibody as a potential alternative treatment strategy. The PEG chain length did not affect encapsulation efficiency or interaction with mucin in vitro but resulted in differences in in vitro release profile and in vivo efficacy study. We demonstrated the superiority of anti-TNF-α mAb-PLGA-PEG2k over mAb-PLGA-PEG5k nanoparticles to effectively exhibit anti-inflammatory responses in an acute murine colitis model. These nanoparticle-based formulations may be adjusted to encapsulate other drugs that could be applied to a number of disorders at different mucosal surfaces.

Barriers to the Intestinal Absorption of Four Insulin-Loaded Arginine-Rich Nanoparticles in Human and Rat.

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 ± 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa.

Comparison of the effects of the intestinal permeation enhancers, SNAC and sodium caprate (C10): Isolated rat intestinal mucosae and sacs.

SNAC and C10 are intestinal permeation enhancers (PEs) used in formulations of peptides for oral delivery in clinical trials. Our aims were to compare their: (i) mechanism of action in isolated rat intestinal mucosae mounted in Ussing chambers and in non-everted gut sacs, (ii) effects on mucosa integrity in those models and also in in situ intra-jejunal instillations and (iii) interactions with intestinal mucus. SNAC increased the apparent permeability coefficient (Papp) of the paracellular marker, FITC-dextran 4000 (FD4), across isolated rat gastric mucosae in concentration-dependent fashion, whereas C10 did not, while both reduced the transepithelial electrical resistance (TEER). In isolated jejunal and colonic mucosae, both agents increased the Papp of [14C]-mannitol and FD4 whereas C10 but not SNAC reduced TEER. 20 mM SNAC was required to achieve the efficacy of 10 mM C10 in jejunal and colonic mucosae. In isolated non-everted jejunal and colonics sacs, FD4 flux increases were observed in the presence of both PEs. Histology of mucosae revealed that both PEs induced minor epithelial damage to the mucosa at concentrations that increased fluxes. Jejunal tissue withstood epithelial damage in the following order: intra jejunal in situ instillations > jejunal sacs > isolated jejunal mucosae. Both PEs modulated viscoelastic properties of porcine jejunal mucus without altering rheological properties. In conclusion, SNAC and C10 are reasonably efficacious PEs in rat intestinal tissue with common overall mechanistic features. Their potency and toxic potential are low, in agreement with clinical trial data.

Permeability-enhancing effects of three laurate-disaccharide monoesters across isolated rat intestinal mucosae.

Laurate (C12)-sucrose esters are established intestinal epithelial permeation enhancers (PEs) with potential for use in oral delivery. Most studies have examined blends of ester rather than specific monoesters, with little variation on the sugar moiety. To investigate the influence of varying the sugar moiety on monoester performance, we compared three monoesters: C12-sucrose, C12-lactose, and C12-trehalose. The assays were: critical micellar concentration (CMC) in Krebs-Henseleit buffer, MTS and lactate dehydrogenase assays in Caco-2 cells, transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [14C] mannitol across isolated rat intestinal mucosae, and tissue histology. For CMC, the rank order was C12-trehalose (0.21 mM) < C12-sucrose (0.34 mM) < C12-lactose (0.43 mM). Exposure to Caco-2 cells for 120 min produced TC50 values in the MTS assay from 0.1 to 0.4 mM. Each ester produced a concentration-dependent decrease in TEER across rat mucosae with 80% reduction seen with 8 mM in 5 min, but C12-trehalose was less potent. C12-sucrose and C12-lactose increased the Papp of [14C] mannitol across mucosae with similar potency and efficacy, whereas C12-trehalose was not as potent or efficacious, even though it still increased flux. In the presence of the three esters, gross intestinal histology was unaffected except at 8 mM for C12-sucrose and C12-lactose. In conclusion, the three esters enhanced permeability likely via tight junction modulation in rat intestinal tissue. C12-trehalose was not quite as efficacious, but neither did it damage tissue to the same extent. All three can be considered as potential PEs to be included in oral formulations.

Synthesis and In Vivo Evaluation of Insulin-Loaded Whey Beads as an Oral Peptide Delivery System.

For many diabetics, daily, lifelong insulin injections are required to effectively manage blood glucose levels and the complications associated with the disease. This can be a burden and reduces patient quality of life. Our goal was to develop a more convenient oral delivery system that may be suitable for insulin and other peptides. Insulin was entrapped in 1.5-mm beads made from denatured whey protein isolate (dWPI) using gelation. Beads were then air-dried with fumed silica, Aerosil®. The encapsulation efficiency was ~61% and the insulin loading was ~25 µg/mg. Dissolution in simulated gastric-, and simulated intestinal fluids (SGF, SIF) showed that ~50% of the insulin was released from beads in SGF, followed by an additional ~10% release in SIF. The omission of Aerosil® allowed greater insulin release, suggesting that it formed a barrier on the bead surface. Circular dichroism analysis of bead-released insulin revealed an unaltered secondary structure, and insulin bioactivity was retained in HepG2 cells transfected to assess activation of the endogenous insulin receptors. Insulin-entrapped beads were found to provide partial protection against pancreatin for at least 60 min. A prototype bead construct was then synthesised using an encapsulator system and tested in vivo using a rat intestinal instillation bioassay. It was found that 50 IU/kg of entrapped insulin reduced plasma glucose levels by 55% in 60 min, similar to that induced by subcutaneously (s.c.)-administered insulin (1 IU/kg). The instilled insulin-entrapped beads produced a relative bioavailability of 2.2%. In conclusion, when optimised, dWPI-based beads may have potential as an oral peptide delivery system.

Silica-Coated Nanoparticles with a Core of Zinc, l-Arginine, and a Peptide Designed for Oral Delivery.

Nanoparticle constructs for oral peptide delivery at a minimum must protect and present the peptide at the small intestinal epithelium in order to achieve oral bioavailability. In a reproducible, scalable, surfactant-free process, a core was formed with insulin in ratios with two established excipients and stabilizers, zinc chloride and l-arginine. Cross-linking was achieved with silica, which formed an outer shell. The process was reproducible across several batches, and physicochemical characterization of a single batch was confirmed in two independent laboratories. The silica-coated nanoparticles (SiNPs) entrapped insulin with high entrapment efficiency, preserved its structure, and released it at a pH value present in the small intestine. The SiNP delivered insulin to the circulation and reduced plasma glucose in a rat jejunal instillation model. The delivery mechanism required residual l-arginine in the particle to act as a permeation enhancer for SiNP-released insulin in the jejunum. The synthetic process was varied in terms of ratios of zinc chloride and l-arginine in the core to entrap the glucagon-like peptide 1 analogue, exenatide, and bovine serum albumin. SiNP-delivered exenatide was also bioactive in mice to some extent following oral gavage. The process is the basis for a platform for oral peptide and protein delivery.

An Enteric-Coated Polyelectrolyte Nanocomplex Delivers Insulin in Rat Intestinal Instillations when Combined with a Permeation Enhancer.

The use of nanocarriers is being researched to achieve oral peptide delivery. Insulin-associated anionic polyelectrolyte nanoparticle complexes (PECs) were formed that comprised hyaluronic acid and chitosan in an optimum mass mixing ratio of 5:1 (MR 5), followed by coating with a pH-dependent polymer. Free insulin was separated from PECs by size exclusion chromatography and then measured by HPLC. The association efficiency of insulin in PECs was >95% and the loading was ~83 µg/mg particles. Dynamic light scattering and nanoparticle tracking analysis of PECs revealed low polydispersity, a negative zeta potential range of -40 to -50 mV, and a diameter range of 95-200 nm. Dissolution studies in simulated small intestinal fluid (FaSSIF-V2) revealed that the PECs were colloidally stable. PECs that were coated with Eudragit® L-100 delayed insulin release in FaSSIF-V2 and protected insulin against pancreatin attack more than uncoated PECs. Uncoated anionic PECs interacted weakly with mucin in vitro and were non-cytotoxic to Caco-2 cells. The coated and uncoated PECs, both concentrated further by ultrafiltration, permitted dosing of 50 IU/kg in rat jejunal instillations, but they failed to reduce plasma glucose or deliver insulin to the blood. When ad-mixed with the permeation enhancer (PE), sucrose laurate (100 mM), the physicochemical parameters of coated PECs were relatively unchanged, however blood glucose was reduced by 70%. In conclusion, the use of a PE allowed for the PEC-released bioactive insulin to permeate the jejunum. This has implications for the design of orally delivered particles that can release the payload when formulated with enhancers.

Striving Towards the Perfect In Vitro Oral Drug Absorption Model.

Oral drug delivery systems have multiple goals, assessing and enabling intestinal absorption at efficacious doses being one of them. Here we highlight the in vitro advances in modeling drug absorption, which more faithfully reflect human intestinal physiology and reduce the reliance on animal models.

Evaluation of Sucrose Laurate as an Intestinal Permeation Enhancer for Macromolecules: Ex Vivo and In Vivo Studies.

Oral delivery of macromolecules requires permeation enhancers (PEs) adaptable to formulation. Sucrose laurate (SL) (D1216), a food grade surfactant, was assessed in Caco-2 monolayers, isolated rat intestinal tissue mucosae, and rat intestinal instillations. Accordingly, 1 mM SL increased the apparent permeability coefficient (Papp) of [14C]-mannitol and reduced transepithelial electrical resistance (TEER) across monolayers. It altered expression of the tight junction protein, ZO-1, increased plasma membrane potential, and decreased mitochondrial membrane potential in Caco-2 cells. The concentrations that increased flux were of the same order as those that induced cytotoxicity. In rat colonic tissue mucosae, the same patterns emerged in respect to the concentration-dependent increases in paracellular marker fluxes and TEER reductions with 5 mM being the key concentration. While the histology revealed some perturbation, ion transport capacity was retained. In rat jejunal and colonic instillations, 50 and 100 mM SL co-administered with insulin induced blood glucose reductions and achieved relative bioavailability values of 2.4% and 8.9%, respectively, on a par with the gold standard PE, sodium caprate (C10). The histology of the intestinal loops revealed little damage. In conclusion, SL is a candidate PE with high potential for emulsion-based systems. The primary action is plasma membrane perturbation, leading to tight junction openings and a predominant paracellular flux.

Labrasol® is an efficacious intestinal permeation enhancer across rat intestine: Ex vivo and in vivo rat studies.

Labrasol® ALF (Labrasol®), is a non-ionic surfactant excipient primarily used as a solubilising agent. It was investigated here as an intestinal permeation enhancer in isolated rat colonic mucosae in Ussing chamber and in rat in situ intestinal instillations. Labrasol® comprises mono-, di- and triglycerides and mono- and di- fatty acid esters of polyethylene glycol (PEG)-8 and free PEG-8, with caprylic (C8)- and capric acid (C10) as the main fatty acids. Source components of Labrasol® as well as Labrasol® modified with either C8 or C10 as the sole fatty acid components were also tested to determine which element of Labrasol® was responsible for its permeability-enhancing properties. Labrasol® (4, 8 mg/mL) enhanced the transport of the paracellular markers, [14C] mannitol, FITC-dextran 4000, and FITC-insulin across colonic mucosae. The enhancement was non-damaging, transient, and molecular weight-dependent. The PEG ester fraction of Labrasol® also had enhancing properties. When insulin was administered with Labrasol® in instillations, it had a relative bioavailability of 7% in jejunum and 12% in colon. C8- and C10 versions of Labrasol® and the PEG ester fraction also induced similar bioavailability values in jejunal instillations: 6, 5 and 7% respectively. Inhibition of lipases in instillations did not reduce the efficacy of Labrasol®, suggesting that its mechanism as a PE is not simply due to liberated medium chain fatty acids. Labrasol® acts as an efficacious intestinal permeation enhancer and has potential for use in oral formulations of macromolecules and BCS Class III molecules.

The UCD nanosafety workshop (03 December 2018): towards developing a consensus on safe handling of nanomaterials within the Irish university labs and beyond - a report.

Careful handling of the nanomaterials (NMs) in research labs is crucial to ensure a safe working environment. As the largest university in Ireland, University College Dublin (UCD) has invested significant resources to update researchers working with NMs. Due to sizes often <100 nm, the NMs including nanoparticles, harbor unprecedented materialistic properties, for example, enhanced reactivity, conductivity, fluorescence, etc. which albeit conferring the NMs an edge over bulk materials regarding the applied aspects; depending on the dose, also render them to be toxic. Thus, a set of regulatory guidelines have emerged regarding safe handling of the NMs within occupational set-ups. Unfortunately, the current regulations based on the toxic chemicals and carcinogens are often confusing, lack clarity, and difficult to apply for the NMs. As a research-intensive university, a diverse range of research activities occur within the UCD labs, and it is difficult, at times impossible, for the UCD Safety, Insurance, Operational Risk & Compliance (SIRC) office to develop a set of common guidelines and cater throughout all its labs conducting research with the NMs. Hence, a necessity for dialog and exchange of ideas was felt across the UCD which encouraged the researchers including early stage researchers (e.g. PhDs, Postdocs) from multiple schools to participate in a workshop held on the 03 December 2018. The workshop tried to follow a pragmatic approach, where apart from discussing both the in vitro and in vivo scenarios, practical cases simulating situations faced frequently in the labs were discussed. This report summarizes the findings made during the workshop by this emerging critical mass in UCD.

Effects of surfactant-based permeation enhancers on mannitol permeability, histology, and electrogenic ion transport responses in excised rat colonic mucosae.

Surfactant-based intestinal permeation enhancers (PEs) are constituents of several oral macromolecule formulations in clinical trials. This study examined the interaction of a test panel of surfactant-based PEs with isolated rat colonic mucosae mounted in Ussing chambers in an attempt to determine if increases in transepithelial permeability can be separated from induction of mucosal perturbation. The aim was to assess the effects of PEs on (i) apparent permeability coefficient (Papp) of [14C]-mannitol (ii) histology score and (iii) short-circuit current (ΔIsc) responses to a cholinomimetic (carbachol, CCh). Enhancement ratio increases for Papp values followed the order: C10 > C9 = C11:1 > a bile salt blend > sodium choleate > sucrose laurate > Labrasol® >C12E8 > C12 > Cremophor® A25 > C7 > sucrose stearate > Kolliphor® HS15 > Kolliphor® TPGS. Exposures that increased the Papp by ≥2-fold over 120 min were accompanied by histological damage in 94% of tissues, and by a decreased ΔIsc response to CCh in 83%. A degree of separation between the increased Papp of [14C]-mannitol and histological damage and diminution of the ΔIsc response to CCh was observed at selected concentrations of Labrasol®. Overall, this surfactant-based PE selection caused transcellular perturbation at similar concentrations to those that enhanced permeability.

Physicochemical, pharmacokinetic and pharmacodynamic analyses of amphiphilic cyclodextrin-based nanoparticles designed to enhance intestinal delivery of insulin.

Due to excellent efficacy, low toxicity, and well-defined selectivity, development of new injectable peptides is increasing. However, the translation of these drugs into products for effective oral delivery has been restricted due to poor oral bioavailability. Nanoparticle (NP) formulations have potential to overcome the barriers to oral peptide delivery through protecting the payload and increasing bioavailability. This study describes the rational design, optimization and evaluation of a cyclodextrin-based NP entrapping insulin glulisine for intestinal administration. A cationic amphiphilic cyclodextrin (click propyl-amine cyclodextrin (CD)) was selected as the primary complexing agent for NP development. Following NP synthesis, in vitro characterization was performed. The insulin glulisine NPs exhibited an average size of 109 ±â€¯9 nm, low polydispersity index (0.272) negative zeta potential (-25 ±â€¯3 mV), high association efficiency (71.4 ±â€¯3.37%) and an insulin loading of 10.2%. In addition, the NPs exhibited colloidal stability in intestinal-biorelevant media (SIF, supplemented-SIF 1% (w/v) and FaSSIF-V2) for up to 4 h. Proteolysis studies indicated that the NPs conferred protection to the entrapped insulin relative to free insulin. In vivo rat jejunal instillation studies demonstrated that the NPs mediated systemic insulin absorption, accompanied by a decrease in blood glucose levels. The relative bioavailability of the instilled insulin (50 IU/kg) from the NP was 5.5% compared to subcutaneous administration of insulin solution (1 IU/kg). The pharmacodynamic and pharmacokinetic data indicate that this cyclodextrin-based formulation may have potential for further research as an oral insulin dosage form.

Safety concerns over the use of intestinal permeation enhancers: A mini-review.

Intestinal permeation enhancers (PEs) are key components in ∼12 oral peptide formulations in clinical trials for a range of molecules, primarily insulin and glucagon-like-peptide 1 (GLP-1) analogs. The main PEs comprise medium chain fatty acid-based systems (sodium caprate, sodium caprylate, and N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC)), bile salts, acyl carnitines, and EDTA. Their mechanism of action is complex with subtle differences between the different molecules. With the exception of SNAC and EDTA, most PEs fluidize the plasma membrane causing plasma membrane perturbation, as well as enzymatic and intracellular mediator changes that lead to alteration of intestinal epithelial tight junction protein expression. The question arises as to whether PEs can cause irreversible epithelial damage and tight junction openings sufficient to permit co-absorption of payloads with bystander pathogens, lipopolysaccharides and its fragment, or exo- and endotoxins that may be associated with sepsis, inflammation and autoimmune conditions. Most PEs seem to cause membrane perturbation to varying extents that is rapidly reversible, and overall evidence of pathogen co-absorption is generally lacking. It is unknown however, whether the intestinal epithelial damage-repair cycle is sustained during repeat-dosing regimens for chronic therapy.