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.

Investigations of Piperazine Derivatives as Intestinal Permeation Enhancers in Isolated Rat Intestinal Tissue Mucosae.

A limiting factor for oral delivery of macromolecules is low intestinal epithelial permeability. 1-Phenylpiperazine (PPZ), 1-(4-methylphenyl) piperazine (1-4-MPPZ) and 1-methyl-4-phenylpiperazine (1-M-4-PPZ) have emerged as potential permeation enhancers (PEs) from a screen carried out by others in Caco-2 monolayers. Here, their efficacy, mechanism of action and potential for epithelial toxicity were further examined in Caco-2 cells and isolated rat intestinal mucosae. Using high-content analysis, PPZ and 1-4-MPPZ decreased mitochondrial membrane potential and increased plasma membrane potential in Caco-2 cells to a greater extent than 1-M-4-PPZ. The Papp of the paracellular marker, [14C]-mannitol, and of the peptide, [3H]-octreotide, was measured across rat colonic mucosae following apical addition of the three piperazines. PPZ and 1-4-MPPZ induced a concentration-dependent decrease in transepithelial electrical resistance (TEER) and an increase in the Papp of [14C]-mannitol without causing histological damage. 1-M-4-PPZ was without effect. The piperazines caused the Krebs-Henseleit buffer pH to become alkaline, which partially attenuated the increase in Papp of [14C]-mannitol caused by PPZ and 1-4-MPPZ. Only addition of 1-4-MPPZ increased the Papp of [3H]-octreotide. Pre-incubation of mucosae with two 5-HT4 receptor antagonists, a loop diuretic and a myosin light chain kinase inhibitor, reduced the permeation enhancement capacity of PPZ and 1-4-MPP for [14C]-mannitol. 1-4-MPPZ holds most promise as a PE, but intestinal physiology may also be impacted due to multiple mechanisms of action.

Local delivery of macromolecules to treat diseases associated with the colon.

Current treatments for intestinal diseases including inflammatory bowel diseases, irritable bowel syndrome, and colonic bacterial infections are typically small molecule oral dosage forms designed for systemic delivery. The intestinal permeability hurdle to achieve systemic delivery from oral formulations of macromolecules is challenging, but this drawback can be advantageous if an intestinal region is associated with the disease. There are some promising formulation approaches to release peptides, proteins, antibodies, antisense oligonucleotides, RNA, and probiotics in the colon to enable local delivery and efficacy. We briefly review colonic physiology in relation to the main colon-associated diseases (inflammatory bowel disease, irritable bowel syndrome, infection, and colorectal cancer), along with the impact of colon physiology on dosage form design of macromolecules. We then assess formulation strategies designed to achieve colonic delivery of small molecules and concluded that they can also be applied some extent to macromolecules. We describe examples of formulation strategies in preclinical research aimed at colonic delivery of macromolecules to achieve high local concentration in the lumen, epithelial-, or sub-epithelial tissue, depending on the target, but with the benefit of reduced systemic exposure and toxicity. Finally, the industrial challenges in developing macromolecule formulations for colon-associated diseases are presented, along with a framework for selecting appropriate delivery technologies.

An Assessment of the Permeation Enhancer, 1-phenyl-piperazine (PPZ), on Paracellular Flux Across Rat Intestinal Mucosae in Ussing Chambers.

PURPOSE: 1-phenyl piperazine (PPZ) emerged from a Caco-2 monolayer screen as having high enhancement potential due to a capacity to increase permeation without significant toxicity. Our aim was to further explore the efficacy and toxicity of PPZ in rat ileal and colonic mucosae in order to assess its true translation potential. METHODS: Intestinal mucosae were mounted in Ussing chambers and apparent permeability coefficient (Papp) values of [(14)C]-mannitol and FITC-dextran 4 kDa (FD-4) and transepithelial electrical resistance (TEER) values were obtained following apical addition of PPZ (0.6-60 mM). Exposed issues were assessed for toxicity by histopathology and lactate dehydrogenase (LDH) release. Mucosal recovery after exposure was also assessed using TEER readings. RESULTS: PPZ reversibly increased the Papp of both agents across rat ileal and distal colonic mucosae in concentration-dependent fashion, accompanied by TEER reduction, with acceptable levels of tissue damage. The complex mechanism of tight junction opening was part mediated by myosin light chain kinase, stimulation of transepithelial electrogenic chloride secretion, and involved activation of 5-HT4 receptors. CONCLUSIONS: PPZ is an efficacious and benign intestinal permeation enhancer in tissue mucosae. However, its active pharmacology suggest that potential for further development in an oral formulation for poorly permeable molecules will be difficult.

Current status of selected oral peptide technologies in advanced preclinical development and in clinical trials.

The development of oral dosage forms that allows absorption of therapeutic peptides to the systemic circulation is one of the greatest challenges for the pharmaceutical industry. Currently, a number of technologies including either mixtures of penetration enhancers or protease inhibitors and/or nanotechnology-based products are under clinical development. Typically, these formulations are presented in the form of enteric-coated tablets or capsules. Systems undergoing preclinical investigation include further advances in nanotechnology, including intestinal microneedle patches, as well as their combination with regional delivery to the colon. This review critically examines four selected promising oral peptide technologies at preclinical stage and the twelve that have progressed to clinical trials, as indicated in www.clinicaltrials.gov. We examined these technologies under the criteria of peptide selection, formulation design, system components and excipients, intestinal mechanism of action, efficacy in man, and safety issues. The conclusion is that most of the technologies in clinical trials are incremental rather than paradigm-shifting and that even the more clinically advanced oral peptide drugs examples of oral bioavailability appear to yield oral bioavailability values of only 1-2% and are, therefore, only currently suitable for a limited range of peptides.

The mycotoxin patulin increases colonic epithelial permeability in vitro.

The gastrointestinal lumen is directly exposed to dietary contaminants, including patulin, a mycotoxin produced by moulds. Patulin is known to increase permeability across intestinal Caco-2 monolayers. This study aimed to determine the effect of patulin on permeability, ion transport and morphology in isolated rat colonic mucosae. Mucosal sheets were mounted in Ussing chambers and voltage clamped. Apical addition of patulin (100-500 µM) rapidly reduced transepithelial electrical resistance (TEER) and increased permeability to [(14)C] mannitol (2.9-fold). Patulin also inhibited carbachol-induced electrogenic chloride secretion and histological evidence of mucosal damage was observed. To examine potential mechanisms of action of patulin on colonic epithelial cells, high-content analysis of Caco-2 cells was performed and this novel, quantitative fluorescence-based approach confirmed its cytotoxic effects. With regard to time course, the cytotoxicity determined by high content analysis took longer than the almost immediate reduction of electrical resistance in isolated mucosal sheets. These data indicate patulin is not only cytotoxic to enterocytes but also has the capacity to directly alter permeability and ion transport in intact intestinal mucosae. These data corroborate and extend findings in intestinal cell culture monolayers, and further suggest that safety limits on consumption of patulin may be warranted.

CriticalSorb™ promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers.

PURPOSE: CriticalSorb™ is a novel absorption enhancer based on Solutol(®) HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth hormone. The hypothesis was that permeating enhancement effects of the Solutol(®)HS15 component would translate to the intestine. METHODS: Rat colonic mucosae were mounted in Ussing chambers and Papp values of [(14)C]-mannitol, [(14)C]-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calculated in the presence of CriticalSorb™. Tissues were fixed for H & E staining. Caco-2 monolayers were grown on Transwells™ for similar experiments. RESULTS: CriticalSorb™(0.01% v/v) significantly increased the Papp of [(14)C]-mannitol, FD-4 [(14)C]-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of [(14)C]-mannitol FD-4 and [(14)C]-antipyrine over 120 min. In both monolayers and tissues, it acted as a moderately effective P-glycoprotein inhibitor. There was no evidence of cytotoxicity in Caco-2 at concentrations of 0.01% for up to 24 h and histology of tissues showed intact epithelia at 120 min. CONCLUSIONS: Solutol(®) HS15 is the key component in CriticalSorb™ that enables non-cytotoxic in vitro intestinal permeation and its mechanism of action is a combination of increased paracellular and transcellular flux.

Lymphocyte migration through the blood-brain barrier (BBB) in feline immunodeficiency virus infection is significantly influenced by the pre-existence of virus and tumour necrosis factor (TNF)-alpha within the central nervous system (CNS): studies using an in vitro feline BBB model.

AIMS: In human immunodeficiency virus infection, macrophage-tropic and lymphotropic viruses exist in the host. Central nervous system (CNS) infection is an early and ongoing event, important to understand when developing strategies to treat infection. Some knowledge exists on macrophage-tropic virus interactions with the blood-brain barrier (BBB), and the aim of this study was to investigate lymphotropic lentivirus interactions with the BBB. METHODS: Interactions of the lymphotropic feline immunodeficiency virus (FIV) with an in vitro model of the feline BBB were evaluated in scenarios to mimic in vivo infections. RESULTS: Cell-free FIV crossed the BBB in very low quantities, and in the presence of tumour necrosis factor (TNF)-alpha, BBB integrity was unaffected. However, cell-associated FIV readily crossed the BBB, but BBB integrity was not significantly altered. Transmigration of uninfected and infected lymphocytes increased in response to TNF-alpha, accompanied by a moderate disruption of barrier integrity and an upregulation of vascular cell adhesion molecule-1 rather than intercellular adhesion molecule-1. Significant enhancement of migration and disruption of BBB tight junctions occurred when infected cells and TNF-alpha were added to the brain side of the BBB and this enhancement was not mediated through additional TNF-alpha production. CONCLUSIONS: Small quantities of virus in the brain together with TNF-alpha have the potential to stimulate greater cell and viral entry into the CNS and this is likely to involve important factors other than further TNF-alpha production. Lymphotropic lentivirus entry to the CNS is governed by many factors similar to macrophage-tropic strains.

Selamectin is a potent substrate and inhibitor of human and canine P-glycoprotein.

The transport of the antiparasitic agents, ivermectin, selamectin and moxidectin was studied in human intestinal epithelial cell monolayers (Caco-2) and canine peripheral blood lymphocytes (PBL). Both models expressed the mdr1-coded 170 kDa ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp). Fluxes of the P-gp substrate rhodamine-123 (Rh-123) across Caco-2 monolayers showed that ivermectin and selamectin acted as potent P-gp inhibitors with IC50 values of 0.1 microm. In contrast, moxidectin was a weaker P-gp inhibitor with an IC50 of 10 microm. The transport of radiolabelled ivermectin, selamectin and moxidectin through Caco-2 monolayers showed that ivermectin, selamectin and moxidectin were P-gp substrates with secretory/absorptive ratios of 7.5, 4.7 and 2.6 respectively. Secretory transport of [3H]-ivermectin and [3H]-selamectin was blocked by the P-gp inhibitor, verapamil. Ivermectin and selamectin inhibited the efflux of Rh-123 from PBL and the concentration of inhibition was similar to that of verapamil. In contrast, moxidectin did not have a significant effect on Rh-123 efflux from PBL. The data suggest that ivermectin and selamectin are potent P-gp substrates, while moxidectin is a weak one.

Microparticle vaccine approaches to stimulate mucosal immunisation.

Entrapment of antigens in biodegradable particles for mucosal immunisation has given successful outcomes in animals, but not as yet in man. Formulations using genuinely stable biocompatible nanoparticles with co-entrapped mucosal adjuvants and/or with surface-conjugated human M-cell-targeting ligands may lead to better uptake of intact antigen by Peyer's patch M cells and delivery to antigen-presenting cells.

Oral vaccination in man using antigens in particles: current status.

Over 30 publications suggest that antigens given orally to mice in biocompatible microspheres stimulate an immune response and, in some cases, can give rise to protective immunity. Of those responses in mice that have been reproduced, confirmation in large animal models and in Phase 1 studies has not resulted. Particles containing antigens given orally and mixed with soluble adjuvants like cholera toxin have generally not produced any better data in mice than that seen with mixed solutions of unprotected antigens and adjuvants. Peyer's patch M cell targeting of antigens in particles remains however a relatively untested hypothesis. While binding and uptake of M cell-targeted latex particles and stable liposomes by mouse M cells has been clearly shown using the mouse M cell-specific lectin, Ulex europaeus 1 (UEA-1), a direct relationship between M cell particle uptake and immune outcome remains illusive. Some studies have produced increased serum antibodies from UEA-1- and cholera toxin B (CTB)-coated liposomes containing antigens. Other groups are currently working on developing novel human M cell ligands for attachment to stable particles for oral delivery. Use of untargeted antigen-containing particles with adjuvants administered by the nasal route remains an alternative option.

Encapsulation in biodegradable microparticles enhances serum antibody response to parenterally-delivered beta-amyloid in mice.

Poly(lactide-co-glycolide) (PLG) microspheres were tested as a parenteral delivery system for human beta-amyloid (1-42) (Abeta), a potential immunotherapeutic undergoing assessment in Phase 1 studies for Alzheimer's disease (AD). Abeta was successfully encapsulated in PLG microspheres of average sizes of 3 or 15 microm diameter. Swiss Webster (SW) mice were injected by the sub-cutaneous (s.c.) or intra-peritoneal (i.p.) routes with 3-33 microg Abeta. Abeta-PLG microparticles (3 microm) induced dose-dependent antibody responses, which were maximal at 33 microg Abeta, while Abeta in phosphate-buffered saline (PBS) produced weak antibody responses at the same doses by both routes. Significantly increased antibody responses were seen for both small and large particle formulations given by the i.p. route in comparison to the s.c route. It was previously reported that passive immunisation with Abeta-specific antibodies cleared amyloid plaques in a mouse model of AD (Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the nervous system and reduce pathology in a mouse model of Alzheimer disease. Nature Med 2000;6:916-19), an indication that induction of serum antibody is a prerequisite for efficacy.

Protection against Bordetella pertussis infection following parenteral or oral immunization with antigens entrapped in biodegradable particles: effect of formulation and route of immunization on induction of Th1 and Th2 cells.

The immunogenicity and protective efficacy of systemically and orally delivered pertussis antigens entrapped in either microparticle poly-lactide-co-glycolide (PLG) or nanoparticle PLG formulations were evaluated in a murine respiratory challenge model for infection with Bordetella pertussis. The results demonstrate that immunization with two parenteral doses of 1 microg or three oral doses of 100 microg of pertussis toxoid (PTd) and filamentous haemagglutinin (FHA) encapsulated in PLG conferred a high level of protection against B. pertussis challenge. Furthermore protection could be generated with a single parenteral immunization with a combined microparticle and nanoparticle formulation. However, the route of immunization and the size of the particles affected the type of T cell response induced. Parenteral immunization with PTd and FHA entrapped in PLG microparticles elicits a potent type 1 T cell response and potent antibody response when given by the intraperitoneal (i.p.) or intramuscular (i.m.) route. In contrast, nanoparticle formulations favoured the induction of Th2 cells.

A distinctive electrophysiological signature from the Peyer's patches of rabbit intestine.

1. Rabbit small intestinal segments containing Peyer's patches (PP) were examined in Ussing chambers using short-circuit current (Isc) recording. By comparison with control small intestinal mucosal segments, rabbit PP-containing epithelia exhibited decreased basal Isc, increased transepithelial resistance (TER) and unchanged potential difference (PD). 2. Carbachol caused a decrease in Isc in rabbit PP epithelia. Forskolin, dibutyryl cyclic GMP, histamine and the calcium ionophore, A23187, were without effect. In contrast, control epithelial segments of rabbit intestine responded to carbachol and forskolin with an increased Isc, indicative of electrogenic chloride secretion. The EC50 for carbachol was approximately 2 microM in both types of epithelia. Methacholine also caused an outward current in rabbit PP epithelia which had similar properties to that of carbachol. The effect of the cholinomimetics on rabbit PP was basolateral-sided, reversible, and sensitive to low concentrations of the general muscarinic cholinoceptor blockers, atropine, scopolamine and also to the M1 cholinoceptor blocker, pirenzepine. 3. The Isc response to cholinomimetics in rabbit PP was insensitive to bumetanide, amiloride, TEA, barium, acetazolamide, piroxicam and omeprazole, but was attenuated in the presence of ouabain. Using bilaterally-substituted solutions, the carbachol effect on rabbit PP Isc was abolished in chloride/bicarbonate-free, but not in chloride-free solutions, suggestive of stimulation of electrogenic bicarbonate absorption by the agent. Substitution for sodium abolished both the basal current and the Isc response to carbachol. Part of the effect of carbachol on PP Isc appeared to be mediated by submucosal neurones because addition of tetrodotoxin reduced the effect by 60%. 4 As microfold (M) epithelial cells predominate in the PP of the rabbit, the unusual phenotype of cholinomimetic-induced outward current may be used as an electrophysiological marker for these potential sites of oral vaccine delivery, and in particular it may also be of use as a marker for rabbit M cells.

Stilbenes stimulate T84 Cl- secretion by elevating Ca2+.

Basolateral but not apical application of 10-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to T84 monolayers produced a transient increase in short-circuit current (Isc), followed by a sustained inhibition. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS) had no effect. The increase in Isc produced by DIDS represents Cl- secretion and appears to result from Ca2+ elevation, because in all respects except time course the response to DIDS mimicked the response to the Ca(2+)-elevating agent thapsigargin. Fura-2 measurements established that thapsigargin elevates Ca2+ in T84 cells, but Ca2+ responses to DIDS could not be established directly because DIDS absorbs strongly at the critical wavelengths. Responses to DIDS and thapsigargin were 1) blocked by bumetanide; 2) not blocked by basolateral Ba2+; 3) completely nonadditive; 4) strongly synergistic with basal levels of Isc or with Isc increases produced by elevating adenosine 3',5'-cyclic monophosphate (cAMP; with forskolin) or guanosine 3',5'-cyclic monophosphate (with heat-stable enterotoxin); and 5) reversibly abolished by removal of basolateral Ca2+. Interactions between Ca2+ and cAMP-elevating agents strongly support a model of Cl- secretion in which apical Cl- conductance is activated by cyclic nucleotides but not by Ca2+ while basolateral K+ channels are activated by Ca2+. In contrast with this mechanism, whole cell patch-clamp recordings of nonconfluent T84 cells indicated that DIDS and other Ca(2+)-elevating agents stimulated an increase in Cl- conductance. Thus increases in cytosolic free Ca2+ in nonconfluent T84 cells activate conductances that differ from those in confluent monolayers.

Synchronous transporting activity in epithelial cells in relation to intracellular calcium concentration.

Cultured monolayers of human sweat-gland epithelia have been used to measure electrogenic sodium transport, as short-circuit current, and intracellular Ca2+ concentration ([Ca]i) from Fura-2 fluorescence. The sodium currents in response to the agonists lysylbradykinin, histamine and carbachol show oscillatory behaviour in the 1-2 per minute frequency range. The oscillations can be terminated either by using specific antagonists or with amiloride, which prevents sodium entry into the epithelium. Oscillatory behaviour is also seen when [Ca]i is measured and occurs in the same frequency range. Sodium transport in these cultured epithelia is thought to result from an increase in [Ca]i, which in turn activates calcium-sensitive potassium channels, so increasing the electrochemical gradient for sodium entry. The oscillatory behaviour implies that the epithelial cells behave in synchrony to increase [Ca]i, so inducing synchronous changes in sodium current. It is shown that the behaviour is not unique to sodium-absorbing epithelia, and the possible utility of synchronous behaviour in epithelial tissues is discussed.

Cystic fibrosis, the CFTR, and rectifying Cl- channels.

The human genetic disease cystic fibrosis is caused by a single defective gene on chromosome 7 that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The defect causes a profound reduction of Cl- permeability in several tissues, which in turn impairs salt absorption and fluid secretion. A 25-80 pS, rectifying Cl- channel has been targeted as the exclusive or primary channel affected in CF. However, we have found no evidence for significant activation or spontaneous activity of this channel in cell-attached patches of normal lymphoblasts or dog tracheal cells. However, in dog tracheal cells, we find lower conductance, linear Cl- channels that are spontaneously active in unstimulated cells and may show increased activity in stimulated cells. Attempts to correlate the expression of mRNA for the CFTR protein in various types of cells with the presence of the rectifying Cl- channel show a lack of correlation: i.e., depolarization-activated rectifying Cl- channesl have been found in excised, inside-out patches from all cell types that we have examined to date, but the CFTR mRNA has so far only been detected in a subset of epithelial cells.

Ion transport in cultured epithelia from human sweat glands: comparison of normal and cystic fibrosis tissues.

1. Cultured epithelia derived from whole human sweat glands, isolated secretory coils, isolated reabsorptive ducts and whole glands from cystic fibrosis (CF) subjects have been used to examine drug sensitivity by use of short circuit current recording. 2. Short circuit current increases were observed with lysylbradykinin, carbachol and histamine in epithelia of different origins. All responses were due to stimulation of electrogenic sodium absorption, evidenced by the inhibition of these responses by amiloride. The latter also abolished the basal current. The terpenes, thapsigargin and forskolin had no effect on transport. 3. The stimulation of a sodium current by agonists was dependent upon calcium, responses being inhibited by lanthanum ions and EGTA. Further A23187 induced a sodium current. 4. Pronounced oscillations in the sodium currents were a feature of the responses, implying synchronous, regulated cell activity. 5. Forskolin produced a ten fold increase in adenylate cyclase activity. All agonists listed in 2 except forskolin caused an increase in intracellular calcium [Ca]i, [Ca]i responses in CF cells were not different from those of normal cells, except with thapsigargin where the responses were smaller. 6. It is concluded that in culture, cells develop ductal characteristics, whether derived from normal or CF glands, coils or ducts. An increase in [Ca]i followed by activation of calcium-sensitive potassium channels and apical membrane hyperpolarization may be the major mechanism for increasing sodium influx.

Altered sensitivity to amiloride in cystic fibrosis. Observations using cultured sweat glands.

1. Using cultured epithelia from sweat glands, derived from both cystic fibrosis and normal subjects, the relationship between amiloride concentration and the inhibition of electrogenic sodium transport was measured, under short circuit conditions. 2. The Kd for amiloride in cultures from normal subjects was 0.64 microM (n = 6) while in cultures derived from CF patients the value was 1.07 microM (n = 4). The values were significantly different (P less than 0.02, ANOVA). 3. In cultures from normal sweat glands, bathed in solutions free of permeable anions (chloride/bicarbonate), the Kd for amiloride rose to a value greater than found in CF tissues (2.3 microM). In CF epithelia subject to the same conditions the abnormally high value increased further, so that in solutions without permeable anions normal and CF cultures behaved similarly. 4. In cultures derived from normal and CF tissues lowering the sodium concentration to 10 mM also lowered the Kd for amiloride, however the shift was greater for CF cultures. 5. Several possible explanations for the results are discussed. The most probable is that the relatively more positive apical membrane potential in CF epithelia opposes the interaction of amiloride with the sodium channel, implying that complex formation is potential sensitive.