Sodium caprate-induced increases in intestinal permeability and

epithelial damage are prevented by misoprostol

David J. Brayden *, Sam Maher, Bojlul Bahar, & Edwin Walsh

School of Veterinary Medicine and Conway Institute, University College

Dublin, Belfield, Dublin 4, Ireland

*Corresponding author: Room 213, School of Veterinary Medicine and The

Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

Tel: +3531 7166013; fax: +3531 7166204. E-mail: david.brayden@ucd.ie

Abstract

1

Epithelial damage caused by intestinal permeation enhancers is a source of debate over

their safety. The medium chain fatty acid, sodium caprate (C10), causes reversible

membrane perturbation at high dose levels required for efficacy in vivo, so the aim was to

model it in vitro. Exposure of Caco-2 monolayers to 8.5mM C10 for 60 min followed by

incubation in fresh buffer led to (i) recovery in epithelial permeability (i.e. transepithelial

electrical resistance (TEER) and apparent permeability coefficient (Papp) of [14C]-

mannitol), (ii) recovery of cell viability parameters (monolayer morphology, plasma

membrane potential, mitochondrial membrane potential, and intracellular calcium) and

(iii) reduction in mRNA expression associated with inflammation (IL-8). Pre-incubation

of monolayers with a mucosal prostaglandin cytoprotectant was attempted in order to

further decipher the mechanism of C10. Misoprostol (100nM), inhibited C10-induced

changes in monolayer parameters, an effect that was partially attenuated by the EP1

receptor antagonist, SC51322. In rat isolated intestinal tissue mucosae and in situ loop

instillations, C10-induced respective increases in the [14C]-mannitol Papp and the AUC of

FITC-dextran 4000 (FD-4) were similarly inhibited by misoprostol, with accompanying

morphological damage spared. These data support a temporary membrane perturbation

effect of C10, which is linked to its capacity to mainly increase paracellular flux, but

which can be prevented by pre-exposure to misoprostol.

Keywords: oral peptide delivery, medium chain fatty acids, sodium caprate, Caco-2

monolayers, intestinal permeation enhancers, cytotoxicity assays

List of Essential Abbreviations

2

HCA: high content analysis

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide

MCFA: medium chain fatty acids from C8-C12

C10: sodium salt of capric acid

uC11: sodium salt of 10-undecylenic acid

PE: permeation enhancer

Papp: apparent permeability coefficient

EC50: concentration required to give a half-maximal effect

mRNA: messenger ribonucleic acid

CN: cell number

NA: nuclear area

NI: nuclear intensity

IC: intracellular calcium

MMP: mitochondrial membrane potential

PMP: plasma membrane permeability (PMP)

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1. Introduction

Increasing oral absorption of peptides via perturbation of the intestinal mucosa has been

achieved using surfactant-based permeation enhancers (PEs) in over 50 clinical studies

[1]. Amongst the leading candidates are amphiphilic agents including medium chain fatty

acids (MCFA), acyl carnitines, alkyl maltosides, and bile salts [2]. It is widely accepted

that these surfactants, as well as several commercial excipients originally designed for

drug solubilisation, can reversibly damage the intestinal mucosa to varying degrees.

There is considerable debate, however, over long term safety of such agents in repeated

oral dosing regimes. The protective response to mucosal challenge in vivo involves rapid

and continuous epithelial cell spreading and renewal, accompanied by secretion of mucus,

prostaglandins, and bicarbonate [3]. One theory is that intestinal tissue damage and

unregulated tight junction opening along the intestine will give rise to bystander

absorption of pathogens leading to local inflammation and sepsis [4, 5]. A contrasting

view supported by a significant body of clinical trial safety data is that, due to the

remarkable capacity of the intestinal mucosa to rapidly repair in response to regular

exposure to dietary agents, this potential toxicity issue is overstated [6, 7].

It is necessary to better understand potential intestinal epithelial toxicity that might be

caused by PEs, since there is often a close association of mucosal perturbation with

increases in drug permeability, along with a correlation in the reversibility of both

processes in vivo. Oral delivery studies in rats with the detergent, sodium dodecyl

sulphate (SDS) and the bile salt, sodium taurocholate, suggested that there was a close

relationship between increased absorption of phenol red with local mucosal damage, but

that this was followed by restoration of the barrier within 1-2 h [8, 9]. Furthermore, the

4

rat jejunal epithelium recovered from mild perturbation within 60 min following

instillation with 100mM of the medium chain fatty acid (MCFA), sodium caprate (C10)

[10], which were similar to the luminal concentrations that are expected to be reached by

Merrion Pharmaceutical’s GIPET® tablets in man [7]. Oral delivery of C10 or sodium

deoxycholate (100 mg.kg-1) caused non-specific histological effects in both ileum and

colon in rats, but the damage caused by C10 was less than the bile salt [11]. The capacity

for epithelial repair in response to challenge was also apparent in rat rectal mucosae

following a 4 h instillation with 100 mM C10 in vivo [12]. Furthermore, a normal mucosa

was present rat intestinal instillations at 180 min following exposure to the related

efficacious MCFA derivative, the sodium salt of undecylenic acid, uC11 [13]. Similar to

C10, this is thought to be due to rapid absorption of the MCFA, followed by epithelial

restitution in the subsequent period. PEs that have progressed to clinical trials (e.g.

Merrion’s GIPET® [7], Chiasma’s Transient Permeation Enhancer system (TPE®) [6]) are

co-released with payload in high concentrations at the small intestinal wall, and this is

when there is likely to be the connection between membrane perturbation and increased

permeation. There is a gap in knowledge, however, about how these processes are related

and this is due in part to lack of reliable in vitro models of epithelial recovery.

Intestinal epithelial cell monolayers on Transwells® as well as isolated muscle-stripped

tissue mucosae in static Ussing chambers with no blood supply are more vulnerable to

membrane damage, but are less equipped to undergo repair than in vivo models [14].

Nevertheless, attempts to demonstrate reversibility of the C10 increase in Caco-2

monolayer paracellular permeability and reversal of the transepithelial electrical

resistance (TEER) decrease in C10-free culture medium has been attempted [15, 16], but

5

methods are not as reproducible as those that provide for the reliable intestinal recovery

data seen in vivo. There has been no systemic study relating monolayer permeability

increases and TEER decreases to C10 concentration, duration, sub-lethal cytotoxic events,

and reversibility. The primary aim of the study was to use measurements of TEER,

apparent permeability coefficient (Papp) of [14C]-mannitol, high content analysis of sub-

lethal cellular parameters, and histological assessment to track the extent of Caco-2

monolayer disruption and capacity to repair. Quantitative expressions of a panel of genes

were evaluated to identify the inflammatory-genes up-regulated during the recovery

period following removal of C10.

As C10-induced permeation enhancement is mediated in part via perturbation of the

mucosal epithelia, then both events might be attenuated by known mucosal protectants

including E-type prostaglandins (PGEs). Endogenous PGEs play a central role in

maintaining mucosal integrity and their protective effect against mucosal perturbants has

been extensively characterised in vivo [17, 18]. PGEs regulate mucosal blood flow,

motility, secretions, immune responses, pro- and anti-inflammatory effects, and gene

expression in intestinal epithelial cells [19]. Pre-treatment of isolated rat and human

gastric glands with dimethyl-PGE2 prior to exposure to indomethacin challenge led to a

concentration-dependent attenuation in lactate dehydrogenase release, ultra-structural

damage, and necrosis compared to indomethacin alone [20]. Misoprostol (Cytotec®,

Pfizer, USA) is a synthetic stable PGE1 methyl ester analogue for the prevention of non-

steroidal anti-inflammatory drug (NSAID)-induced gastric and duodenal ulcers [21]. It

also protects against gastric- and duodenal mucosal perturbation induced by a range of

NSAIDs, including high dose aspirin [22, 23], as well as in alleviating alcohol-induced

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perturbation [24]. Intestinal healing effects are mediated in part by inhibiting production

of tissue damaging, pro-inflammatory Type-1 cytokines and thromboxane [25]. The

second aim of the study therefore was to examine the hypothesis that misoprostol

prevents C10-induced increases in Caco-2 monolayer permeability, as well as in high

content sub-lethal cytotoxicity parameters via an agonist action at the EP1 receptor,

thereby further demonstrating the link between perturbation and increased permeability.

We then sought to confirm that misoprostol could inhibit C10-induced epithelial

permeability increases and histological changes in rat intestinal mucosae mounted in

Ussing chambers and in rat intestinal loop instillations in situ.

2. Materials and Methods

2.1 Materials

C10 was obtained from Fluka Ltd (Dublin, Ireland). PGE2 and misoprostol were purchased

from Sigma-Aldrich (Arklow, Ireland). The EP1-specific receptor antagonist, SC51322,

(8-chloro-2-[3-[(2-furanylmethyl)thio]-1-oxopropyl]hydrazide, dibenz[b,f][1,

4]oxazepine-10(11H)-carboxylic acid), was purchased from Caymen Chemical (Ann

Arbor, USA). Forskolin, and 3-isobutyl-1-methylxanthine (IBMX), were also obtained

from Sigma-Aldrich.

2. 2 [14C]-mannitol flux across Caco-2 monolayers: C10 exposure and recovery

Caco-2 cells (European Collection of Cell Cultures, Passage 52 – 60) were grown on

Transwell® polyester filter inserts (0.4 µm pore size, 12 mm diameter) for 21 – 28 days in

DMEM, supplemented with foetal bovine serum (10%), L-glutamine (1mM), non-

7

essential amino acids (1%) and penicillin-streptomycin (100 IU /100 µ/ml, and

incubated at 37°C with 5% CO2 [26]. In some studies, mucous-covered HT29-MTX-E12

cells, a gift from Professor Per Artursson (University of Uppsala, Sweden), were grown

on filters as previously described [27]. TEER was monitored throughout the culture

period as a measure of monolayer differentiation and integrity using an EVOM™

voltohmmeter with STX2 “chopstick” electrode (World Precision Instruments (WPI),

UK). Acceptable monolayers were required to have a minimum basal TEER of 1200

Ω.cm2. TEER data is presented as the percentage TEER relative to the untreated

monolayer before media replacement, (% T0). To determine [14C]-mannitol apparent

permeability coefficient, (Papp), medium was replaced with 500 l Ca2+free DMEM on the

apical side and 1500 l DMEM on the basolateral side in order to match conditions when

C10 was present on the apical side . The plate was equilibrated for 30 min before 1

µCi/mL [14C]-mannitol was added to the apical chamber, and 50 l was taken from the

apical-side for analysis. C10 was prepared in Ca2+ -free DMEM in order to avoid

precipitation [28] when it was added apically to monolayers. The Papp of [14C]-mannitol

across monolayers were measured by taking basolateral samples (750 l) every 30 min

up to 180 min, followed by replacement with fresh DMEM. In recovery studies to see if

TEER values could be re-established, C10-containing DMEM was replaced with fresh

DMEM, denoting time-zero (T0). DMEM (550 µl) containing [14C]-mannitol (1µCi) was

then added to the apical side. Apical samples (50 µl) were taken at T0 and at every 30 min

up to 8 h after C10 removal and the Papp of mannitol and TEER were determined across

monolayers. All samples were measured using a liquid scintillation analyser (Packard

8

Tricarb 2900 TR) and the apparent permeability coefficient (Papp, cm.s-1) was calculated

according to the equation:

Papp (cm/s) = (dQ/dt) x (1/A x C0)

where dQ/dt is the transport rate (mol x s-1), A is the surface area of the cell monolayer

(cm2), and C0 is the initial concentration in the donor compartment (mol xml-1) [26].

2.3 Transmission Electron Microscopy

Caco-2 monolayers were fixed by adding 2.5% (w/v) glutaraldehyde in 0.1 M Sorenson’s

phosphate buffer to the apical and basolateral sides and incubating at room temperature

for a minimum of 2 h. Monolayers were post-fixed with 1% osmium tetroxide in

Sorensen’s buffer for 1 h, followed by dehydration using ascending concentrations of

ethanol. Following dehydration, ethanol was replaced with propylene oxide. A mixture of

1 part propylene oxide and 1 part epoxy resin, (TAAB-Epon, Agar Scientific, UK), was

then added to the dehydrated monolayers and incubated at room temperature for 1 h. To

ensure complete penetration of the sample, 100% resin was added to the monolayers and

incubated at 37 °C for 2 h. Resin was carefully aspirated from the monolayers and

replaced with fresh 100% resin and plates incubated at 60 °C for 24 h until

polymerisation was complete. Selected areas on each monolayer were identified by

cutting 1 m ‘survey’ sections and staining with 1% toluidine blue for examination by

light microscopy (Leica DMLB, Leica Microsystems, Germany). Ultrathin sections (80 -

100 nm) of the areas were cut using an EM UC6 ultra-microtome (Leica Microsystems,

Germany). Sections were mounted on 200 mesh copper grids and stained with uranyl

9

acetate and lead citrate. The sections were imaged by transmission electron microscopy

(TEM) (Tecnai G2 12 BioTWIN, FEI Company, USA).

2.4 Quantitative real-time PCR and gene expression microarrays

Following C10 exposures, culture medium was removed and monolayers were gently

rinsed with sterile PBS. Total RNA was extracted by collecting the cell population in 1ml

TRIzol® reagent (Life Technologies, USA). The quality and quantity of total RNA was

assayed on an Agilent 2100 Bioanalyser, (Santa Clara, CA, USA), using RNA Nano

LabChips®, (Caliper Technologies Corporation, USA). All RNA samples used for the

gene expression study had an RNA integrity number (RIN) ≥ 8·0. The RIN value

generated by the Agilent 2100 Bioanalyser is an empirical measurement of the integrity

of total RNA that takes into consideration of any sign of degradation of the major RNA

species such as the 28S rRNA and 18S rRNA. RIN value is expressed in the scale of 0

(highly degraded RNA) to 10 (highly intact RNA). For cDNA synthesis, 1 g total RNA

was reverse transcribed using the RT2 First Strand IKit (Qiagen Ltd., Crawley, UK)

according to the manufacturer’s instructions.

The quantitative alteration of Caco-2 cell gene expression following filter-grown

monolayer treatment with 8.5 mM C10 was assessed during the ‘recovery’ period using a

pre-customised TaqMan® 96 well plate array of 48 genes known to be involved in

inflammation and cellular restitution [29]. Prior to the quantitative gene expression

analysis, 50 L cDNA samples from each of the triplicate run for each time point were

pooled. Quantitative real time RT-PCR was performed in a 7300 Real Time PCR system

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(Applied Biosystems, Foster City, CA, USA) and was performed in a 20 µl reaction

mixture per well containing: 1µl pooled cDNA, (after 1:5 dilution), 9 µl water and 10 µl

TaqMan® gene expression master mix, (Applied Biosystems). Thermal cycle conditions

were 94.8 °C for 30 s, followed by 60.8°C for 1 min run over forty cycles. mRNA

quantities were expressed in Ct values, the number of PCR cycles after which the PCR

product crosses a threshold value. Each sample was run in triplicate and Ct values < 35

were used for analysis. The PCR array contained 18S ribosomal RNA gene as an internal

control and 3 reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH),

hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), and beta glucuronidase

(GUSB). The fact that the expression of GAPDH gene was extremely high (Ct value <20)

and highly variable among different treatment groups, the mRNA abundance of only two

reference genes (HPRT1 and GUSB) were chosen for normalization of the mRNA

abundance of the genes included in the PCR array. Normalization of the expression of the

target genes included in the PCR array was performed using the 2–ΔΔCt method [30].

Briefly, average ΔCt was calculated as the difference of Ct values of any target gene

minus average of the Ct value of the two reference genes (HPRT1 and GUSB). Then, fold

change was calculated as 2(-average ΔCt, target gene)/2(-average ΔCt, reference gene). Gene expression was

measured in duplicate per plate for three independent C10 treatment-repair experiments.

2.5 High content analysis of monolayer damage, repair, and cytoprotection

High content analysis (HCA) of filter grown Caco-2 monolayers was performed using a

an established protocol [31]. The fluorescent dye mix in DMEM was added 1 h before

image acquisition. For T0 recovery (i.e. those monolayers analyzed immediately after a

60 min exposure to C10), dyes were added at the same time as C10. For 1 h ‘recovery’, the

11

dyes were added upon removal of C10. For 4, 8 and 24 h ‘recovery’ periods, 50 µl and 150

µl DMEM was removed from the apical and basolateral compartments at 3, 7 and 23 h

post C10 removal respectively and replaced with equivalent volume of dye mix. In

attenuation studies, prostaglandin treatments were removed and 8.5 mM C10 prepared in

Ca2+-free DMEM containing dye mix was added to the apical compartment and 1500 µl

DMEM containing dye mix was added to the basolateral compartment. One media

control and 3 positive control wells containing either ionomycin, carbonyl cyanide-4-

(trifluoromethoxy)phenylhydrazon (FCCP), or Triton® X-100, were included per 12-well

plate.

2.6 Effect of prostaglandins on cAMP levels in monolayers

Apical and basolateral culture medium was removed from Caco-2 monolayers, replaced

with PBS containing 0.2 mM IBMX and incubated at 37 °C / 5% CO2 for 30 min..

Misoprostol, (1-100 nM), PGE2 (1-100 nM), forskolin (10 µM), or IBMX (0.2 mM) were

added bilaterally to for 30 min. Monolayers were trypsinised and transferred to a 1.5 ml

Eppendorf tube and centrifuged at 600g at 4 °C for 10 min. Supernatants were removed

and the pellet washed 3 times with 0.5 ml ice cold PBS containing 0.2 mM IBMX. The

cell pellets were then re-suspended in cell lysis buffer at a concentration of 106 cells/ml,

frozen at -20 °C, thawed to room temperature, vortexed, and the freeze thaw cycle

repeated a second time to ensure lysis, as confirmed by trypan blue exclusion. Eppendorf

tubes were further centrifuged as above, and the lysate removed and stored at -20 °C until

the assay was performed by ELISA [32] using a Parameter™ cAMP ELISA (R&D

Systems®, MN).

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2.7 C10 effects on isolated rat colonic mucosae: PG agonists and antagonists

Rat colonic tissue segments were mounted in Ussing chambers and TEER and [14C]-

mannitol flux measured as previously described [33]. Groups were divided into: 1)

untreated controls, 2) tissue treated with 8.5 mM C10, 3) tissue pre-treated with 1 µM

misoprostol prior to 8.5mM C10 addition and 4) tissue pre-treated with 20 µM SC51322

for 15 min prior to 1 µM misoprostol (15 min), followed by C10. Treatments were added

apically in Ca2+-free Krebs–Henseleit (KH) buffer. The Papp of [14C]-mannitol was

measured from apical to basolateral chambers over 120 min, with basolateral samples

taken every 20 min. Two different C10 treatment designs were investigated. In the first,

C10 and mannitol were added together and incubated for 120 min. In the second, C10

treatment was added for 10 min, removed, and then [14C]-mannitol was added in fresh

KH buffer and incubated for 120 min.

2.9 C10 effects in rat intestinal instillations with PG agonists and antagonists

In vivo colonic instillations were performed in anaesthetized rats as described previously

[34], under a license from the Irish Department of Agriculture following local ethical

review in compliance with EC Directive 86/609/EEC for animal experiments. Intact

loops were pre-treated with 150 µl of 1 µM misoprostol in PBS for 30 min before

instillation of a mixture of 100mM C10 /fluorescein isothiocyanate-dextran 4000 (FD-4)

(40 mg.kg-1) in PBS. Blood samples were taken either by cardiac puncture or retro-orbital

bleed at 0, 5, 15, 30, 45, 60, 90, 120, 150 and 180 min treatment times into 0.5 ml

Eppendorf tubes and stored at 4 °C for a minimum of 60 min prior to centrifugation

13

(6500g, 10 min, 4ºC) and serum collection. Serum FD-4 was quantified by fluorimetry

according to previous methods [33] Gross histology and haemotoxylin and eosin (H & E)

staining was carried out on tissue following Ussing chamber and instillation studies [34].

2.8 Statistical Analysis

Papp, TEER, real-time calcium imaging and instillation data were analysed by one way

ANOVA with Bonferroni’s ad hoc post- test. Gene expression and HCA data was

analyzed by one-way ANOVA with Dunnett’s post-test. All data is presented as the mean

± standard error of the mean (SEM), with significance set at P<0.05.

3. Results

3.1 An experimental design to study TEER and Papp recovery in Caco-2 monolayers

We investigated the relationship between C10 concentration, exposure time, TEER and

Papp of [14C]-mannitol in monolayers. The effects on TEER of an acute 15 min exposure

to C10 concentrations of 8.5, 10 and 12mM were assessed, after which C10 was replaced

with fresh DMEM and TEER re-monitored. C10 (8.5 mM) induced an approximate 90%

reduction in TEER at 15 min, with complete recovery seen in fresh DMEM at 2 h (Fig.

1A). Exposure to 10 and 12 mM C10 irreversibly reduced TEER, as values could not be

recovered in fresh DMEM (Fig. 1A). Incubations were then carried out with the 8.5 mM

concentration for 15, 30 and 60 min, followed by the recovery protocol. TEER was

reduced to the same extent at each time point, but complete recovery in fresh buffer

occurred fastest for the 15 min exposure, with slower recoveries seen with the 30 and 60

14

min exposures respectively (Fig. 1B). In contrast, 8.5 mM concentrations present for

either 15 or 30 min were insufficient to cause [14C]-mannitol Papp increases over 8 h in

fresh buffer following C10 replacement, whereas the 60 min exposure induced a 7-fold

increase in Papp from 1.8 ± 0.4 × 10-7 cm.s-1 to 1.2 ± 0.5 × 10-6 cm.s-1 in the subsequent

period (Fig. 1C). These data suggest that TEER is acutely sensitive to C10 exposure times

in respect of recovery in fresh DMEM, whereas a minimum of 60 min exposure to the

8.5mM concentration was required to trigger an increase in Papp during the subsequent

incubation period in fresh buffer.

Restoration of Caco-2 monolayer basal Papp values of mannitol following the 60 min treat-

ment with 8.5 mM C10 was investigated by assessing Papp values for [14C]-mannitol in set

periods following C10 removal: 0-60, 60-120, 120-240, and 240-420 min, (Fig. 2A). Com-

parison was made with the Papp in monolayers to which C10 was added with [14C]-mannitol

at T0 with continuous incubation for 7 h. The Papp values steadily increased in monolayers

continuously exposed to C10. In monolayers pre-treated with 8.5 mM C10 for 60 min

however, the highest Papp was seen at 2 h post-C10 removal at T0. From 2 h onwards, the

Papp decreased in those monolayers in fresh DMEM and reverted to basal levels within 4

h. This time point correlated with recovery of TEER to levels comparable to those at T0,

(Fig. 2A). Monolayers were examined by TEM after 60 min exposure to 8.5mM C10 and

then at 4 and 24h in fresh DMEM following C10 removal (Fig. 2B). While there was evid-

ence of mild membrane perturbation and loss of microvilli at 60 min exposure, mono-

layer morphology was partially recovered at 4h and fully so at 24h. Taken together, ex-

posure for 60 min of 8.5mM C10 caused TEER reduction accompanied by an increase in

15

the [14C]-mannitol Papp, which was associated with a degree of morphological damage.

When C10 was removed, all three measures were recovered almost completely at 4h. We

then exposed monolayers to C10 using the same experimental design on three successive

days following 24 h recovery periods in fresh DMEM. Upon each repeat exposure, the

capacity of monolayers to recover TEER at the end of the 7 h period post-exposure was

reduced and was associated with corresponding increases in [14C]-mannitol Papp values

(Fig. 3). The data, while demonstrating assay reproducibility, indicated a gradually re-

duced monolayer capacity to recover from perturbation following repeated administra-

tion-recovery cycles. In some studies, HT29-MTX-E12 mucus-covered monolayers were

similarly tested to ascertain a damage-repair protocol for C10, however, results were

highly variable, as 10 mM for 60 min did not reduce TEER below 50% T0, while 8.5 mM

C10 for 90 min did not reduce TEER below 80%, nor was TEER recovery as predictable

in fresh DMEM (data not shown). This was likely due to the significantly lower basal

TEER in E12 compared to Caco-2: 281 ± 14 .cm2 and 2071± 74 .cm2 respectively, as

well as the varying mucous layer thickness between E12 monolayers.

3.2 mRNA expression during Caco-2 monolayer exposure to C10 and recovery

Monolayers on filters were exposed to 8.5 mM C10 for 60 min and mRNA expression was

compared to untreated controls following 1, 4, 8 and 24 h incubation in fresh DMEM

Genes with significant changes at each time point are shown (Table 1), and these are

placed in context of the entire array (Fig. 4). The most significant change was in expres-

sion of the inflammatory signal, IL-8, which was elevated by 11- and 26- fold at 1 h and 4

h recovery respectively, before declining to <3 fold increase at 8 h, and reverting to con-

16

trol levels at 24 h. There was no up-regulation of Ptgs2, which codes for cyclooxy-

genase-2, occasionally induced in cells in response to inflammation and perturbation. Its

expression was however, reduced at the 8- and 24 h restitution time-points. This data sug-

gests that IL-8 expression is induced during monolayer exposure to C10 and that it gradu-

ally reverts to normal, as the monolayer recovers TEER, morphology and normal paracel-

lular flux capacity in fresh DMEM.

3.2 HCA of sub-lethal cellular events during recovery phase

Cellular responses to C10 perturbation and recovery were measured by HCA on filter-

grown monolayers. We confirmed that apical exposure to 8.5 mM C10 for 60 min pro-

duced significant increases in a number parameters: intracellular calcium (IC), mitochon-

drial membrane potential (MMP), plasma membrane permeability (PMP) nuclear intens-

ity (NI), and nuclear area (NA) (Fig. 5). Following C10 removal and incubation in fresh

DMEM, IC and MMP values fully reverted after 1 h; NI at 4 h, and PMP at 8 h. Fluctu-

ations in NA were detected throughout the 24 h recovery period, decreasing significantly

below control levels after 24 h recovery. Importantly, C10 caused no change in cell num-

ber (CN) and, following 24 h in DMEM, it was similar to untreated controls: cellular

replication was not responsible for monolayer recovery. HCA data therefore indicated

that, while monolayer treatment with 8.5 mM C10 for 60 min caused reversible cellular

damage, the cellular changes induced at that time point were sufficient to increase the

Papp of mannitol across monolayers for up to 2h after C10 had been removed (Fig. 2).

HCA recovery data in fresh DMEM was therefore consistent with the corresponding

17

TEM and TEER recoveries over 1-2 h, the Papp recovery from 2-4 h, and the IL-8 gene

expression recovery from 8-24 h.

3.3 Misoprostol attenuates C10-induced monolayer changes: TEER, Papp and TEM

Pre-exposure of monolayers to the stable PGE1 receptor agonist, misoprostol (10, 100

nM) , for 30 min prior to 8.5mM C10 addition for 60 min significantly attenuated C10’s ca-

pacity to reduce TEER and increase the [14C]-mannitol Papp over the subsequent 7 h in

fresh DMEM (Fig. 6A, B). The protective capacity of misoprostol was then tested using a

wider range of pre-treatment concentrations present for 30 min against a subsequent ex-

posure to C10 for 120 min during which the Papp was measured, but without removing the

fatty acid. Significant reductions in the C10-induced Papp increase over this period were

again obtained (Suppl. Fig. 1A), with an EC50 for misoprostol of 6nM (Suppl. Fig. 1B),

and 90% inhibition of the Papp increase seen at 1 µM. There was no evidence that addi-

tion of misoprostol at the same time as C10 or subsequent to it could offset any C10-in-

duced changes in parameters (data not shown). Inhibition of endogenous prostaglandin

synthesis by cell monolayers by pre-incubating with 1 µM piroxicam (30 min) was at-

tempted to see if the cyclooxygenase inhibitor could alter the subsequent Papp response

over 6 h in fresh DMEM, with piroxicam re-added following the 60 min exposure to C10

(Suppl. Fig 2). Piroxicam had no effect, suggesting an insignificant role of endogenous

prostaglandins in monolayer restitution following C10 perturbation. Visual evidence for

the protective effect of pre-incubation with misoprostol against C10 was obtained in mono-

layers by TEM. Compared to the perturbation seen in response to the 60 min exposure to

8.5 mM C10, there was maintenance of microvilli, reduction in the size and frequency of

18

intercellular spaces formed behind the TJs, as well as a reduction in compromised cell-

cell adhesion of monolayers pre-treated with 10 nM misoprostol (Fig. 7).

3.4 HCA deciphers the misoprostol mechanism of action and the receptor basis

Using the same study design as in 3.2, 10 nM misoprostol partially inhibited C10-induced

changes in the IC, PMP and MMP parameters in monolayers (Fig. 8A-C). Moreover, pre-

incubation with the EP1 receptor selective antagonist, SC51322 [35] (20 M) negated the

effects of misoprostol in preventing the C10-induced changes in the three parameters (Fig.

8A-C). Since, misoprostol prevents epithelial ulceration and damage arising from a vari-

ety of causes, we hypothesised that it should also protect against changes induced by

other surfactants, including Triton®-X-100. Similar to the pattern seen with C10, the in-

creases in IC, PMP and MMP induced by Triton®-X-100 were statistically inhibited to a

degree by pre-incubation with 10 nM misoprostol (Fig. 8 D-F). In other studies, both

PGE2 and misoprostol increased monolayer cAMP levels in a concentration-dependent

fashion, (Suppl. Fig. 3), and this may be linked to activation of EP2-4 receptors [36]. Thus,

Misoprostol’s protective mechanism may also involve complex alterations in cellular

ADP/ATP ratio arising from altered membrane ion channel function as a result of C10 –in-

duced membrane fluidity changes and misoprostol-mediated changes in adenylate cyclase

activity.

3.5. Misoprostol attenuates C10-induced permeability enhancement across rat

colonic mucosae in vitro

19

We attempted to replicate some of these findings in rat colonic mucosae mounted in

Ussing chambers. Colon was chosen because is it known to be a region that is especially

sensitive to C10 [37]. Misoprostol (1 µM, 15 min) was added apically in order to mimic

efficacy arising from luminal intestinal instillations in vivo. Misoprostol pre-treatment

attenuated the 8.5 mM C10-induced increase in the Papp of [14C]-mannitol over 120 min

from 1.7 ± 0.3×10-5 cm.s-1 to 1.3 ± 0.4 × 10-5 cm.s-1 (P<0.05, n=6), with control values of

4.5 ± 0.2 × 10-6 cm.s-1 and 3.9 ± 0.2 × 10-6 cm.s-1 obtained for untreated and misoprostol

alone groups respectively. When exposure to C10 was reduced to 10 min to minimise

mucosal perturbation and to model exposure in the small intestine in vivo to a formulation

in transit, inhibition by misoprostol was more efficacious and the resulting Papp was just

slightly above control values. Pre-treatment with SC51322 (20 µM, 15 min) partly

inhibited misoprostol’s attenuating action on C10, but it was not significant (Fig 9A).

Moreover, C10-induced colonic TEER reduction was statistically attenuated by

misoprostol in using this regime, although SC51322 did not prevent misoprostol effect on

this parameter (Suppl. Fig. 4). Histological examination of colonic mucosae upon

exposure to misoprostol revealed an increase in mucous production and/or secretion,

which is likely to be the main basis of the protective effect on tissue (Fig. 9B (i, ii)). This

may explain why misoprostol’s preventative effects on C10-induced Papp and TEER

changes in rat colonic mucosae were not significantly inhibited by SC51322, as mucous

secretion is mediated via EP3 and EP4 receptors in the colon [38]. Importantly,

misoprostol inhibition of C10-induced mannitol Papp increases and TEER reductions in

colonic mucosae were associated with reduced perturbation of the mucosal surface

compared to C10 alone (Fig. 9C, i, ii).

20

3.6 Misoprostol attenuates C10-induced permeability enhancement in rat colonic

loop instillations in vivo

13 µg/kg misoprostol in PBS was instilled into colonic loops 30 min prior to an FD-4 (40

mg.kg-1) / C10 (100 mM) ad-mixture, which was incubated for 180 min. Pre-treatment

reduced the level of significance of the FD-4 serum concentration increase following FD4

/ C10 solution (Fig. 10A, Table 2). In the presence of misoprostol, the FD4 AUC was

reduced by an average of 24%, and there was a 33% reduction in the FD4 Cmax compared

to the C10 group (Fig. 10B, Table 2). Similar to the data in isolated tissue, misoprostol

increased mucous secretion after 30 min (Fig. 10C). Finally, a normal histology profile

for all groups after 180 min according to H & E staining (Fig. 10D). Since restoration of

mucosal surface perturbation in vivo takes place within 60 min of 100 mM C10

instillations [10], this suggests that the protective and inhibitory effects of misoprostol

must take place in the first 15-30 min of co-exposure in vivo.

4. Discussion

The intestinal permeation enhancement effects of C10 are directly related to its

physicochemical properties as an anionic surfactant. Its high hydrophilic-lipophilic

balance (HLB) is indicative of detergent- and solubilization capacity. A concentration of

8.5mM caused a cascade of event in Caco-2 monolayers: increased membrane fluidity

and perturbation, resulting in activation of phospholipase C [39], increased intracellular

calcium [40], depolymerisation of cytoskeletal F-actin [41], and activation of myosin

21

light chain kinase [42]. While these can be viewed as non-specific events, the removal of

the tight junction proteins tricellulin and claudin-5 suggest some specific actions [43, 44].

The result is facilitation of mainly paracellular flux of hydrophilic molecules with

molecular weights up to 10 kDa [43]. Membrane perturbation by many surfactants

follows a model of intercalation into the plasma membrane, equilibration, and part-

dissolution of the bilayer into mixed micelles [45]. Since the C10 concentration required

to maximally increase permeability ( ̴ 10mM) is lower than the critical micellar

concentration of 26mM in aqueous buffers [31], it is possible that the free concentration

at the epithelium in vivo might be of the same order as in vitro, even though in vivo doses

are 10-15-fold higher [7]. This might occur due to dilution and spreading from solid

dosage forms followed by rapid C10 absorption. A further complication is that interaction

of C10 with jejunal bile salts, phospholipids and dietary lipids which form mixed micelles

in vivo may reduce the level of free monomer available at the jejunal interface. C10-

induced mucosal perturbation is therefore difficult to assess in vivo due to the complex

and variable GI environment and very fast repair of mild damage. Recovery studies

highlight the importance of understanding local release profiles to maximize

enhancement action and minimize adverse events.

A reproducible perturbation-repair protocol was established in Caco-2 monolayers and it

enabled characterisation of cellular responses to C10. In the absence of the complete

mechanisms that have evolved in vivo to repair the intestinal barrier [46], concentration

and time- dependent perturbation resulted in reversible damage on a cellular level. This

was sufficient to increase drug permeability both during the exposure phase and for a

22

limited period after the challenge was withdrawn, before reverting to control levels. A

lowering in concentration from 10 mM to 8.5 mM and along with an increase in exposure

from 10 min to 60 min, resulted in a reproducible reversible challenge to the monolayer,

as reflected by measurements of TEER, the [14C]-mannitol Papp, monolayer morphology,

and viability metrics in HCA. C10 concentration was thus matched with duration of

exposure and this enabled the HCA study. In summary, C10 increased IC, PMP, NI, NA,

with a concomitant mitochondrial hyperpolarisation, Within 60 min of removal, IC and

MMP returned to control basal levels, and PMP was decreased significantly. A further

significant drop in PMP was detected 4 h after removal, with NI levels returning to

control values, while PMP finally returned to control levels within 8 h. TEM imaging

revealed that C10 caused focal point cellular damage, with membrane perturbation and

sloughing of microvilli from some cells, but not others. C10 also disrupted TJs of Caco-2

monolayers, and large spaces formed between cells indicating reduced intercellular

adhesion. TEM imaging of monolayers at various stages of recovery over 24 h revealed

restoration of TJ integrity within 1 h, however, cell-cell adhesion remained compromised

with the persistence of large intercellular spaces. Within 4 h of removal however,

monolayers had improved morphological appearance, while at 24 h, recovery resulted in

complete restoration of the membrane barrier, albeit with smaller, truncated microvilli.

All TJs were intact and the intercellular spaces were reduced in number and size at that

time point.

Repeated daily exposure of monolayers to C10 resulted in reduced capacity to fully

recover barrier function, with no recovery of TEER following the third exposure. This

23

was not unexpected for such a delicate in vitro model. The intestinal epithelium is the

most rapidly renewed body tissue and is replaced every 4-5 days [47], which suggests

that recovery from repeat exposure might be less of an issue in vivo. In support of this,

data from monkeys exposed to Chiasma’s TPE ® technology compromising C8 in a liquid

emulsion, did not show an increase in intestinal toxicology following 9 months of daily

exposure [48]. While chronic daily administration of C10 in GIPET® has yet to be tested

in man, over 300 human subjects have been exposed to it in acute clinical trials without

reports of major intestinal issues. On the other hand, C10-induced perturbation may have

some parallels with that induced by aspirin. Examination of human gastric biopsies

excised after oral administration of 600 mg aspirin in saline revealed mucosal focal

damage to 25% of the surface epithelia at 10 min and subsequent recovery within 60 min,

although tight junctions appeared intact at all stages [49]. Importantly, aspirin inhibits

PG-mediated local repair, whereas that mechanism is intact in respect of C10.

Examination of gene regulation throughout the monolayer recovery period revealed that

Caco-2 cells increase IL-8 RNA expression in response to C10. This chemokine has been

shown to promote cell migration over a 24 h period in monolayers of the human colonic

cancer cell line, LIM1215 [50], so it is tempting to associate it with restitution. Yet, IL-8

is a pro-inflammatory cytokine [51], so its production may reflect a protective signal

elaborated in response to perturbation. In support of this, intestinal epithelial cells

increase IL-8 expression in response to bacterial invasion [52], and this was also seen in

intestinal tissue from inflammatory bowel disease patients [53]. In the current study, C10

up-regulated IL-12, which activates dendritic cells to synthesize IFN-γ and additional IL-

24

12; crosstalk between epithelial and dendritic cells has been proposed as a mechanism of

regulating intestinal immune homeostasis [54]. OXSR1 (oxidative stress responsive 1)

was also up-regulated within 1 h of C10 removal, indicative of a stress response to altered

cell volume. It encodes OSR1 protein, a member of the Ser/Thr protein kinase family,

which maintains ion transport and intracellular volume homeostasis [55]. Cytoskeletal

rearrangement via protein kinase C and myosin light chain kinase protein

phosphorylation of F-actin are also involved in cell volume regulation [56]. Thus, C10-

induced changes in membrane fluidity, removal of TJ proteins, and F-actin

depolymerisation contribute to altered cell volume, thereby inducing OXSR1 up-

regulation.

Increased PGE2 production is typically induced in response to mucosal perturbation via

up-regulation of COX-2 (coded by Ptgs2), which converts arachidonic acid to PGH2 [57].

However, no evidence of increased PGE2 synthesis in response to perturbation by C10 was

found in Caco-2 monolayers, as indicated by the lack of effect of piroxicam on any of the

measured parameters. Moreover, addition of exogenous PGE2 or misoprostol to

monolayers following C10 perturbation did not assist restoration of barrier function, in

contrast to pre-treatment. The healing effects of misoprostol on gastric ulcers in vivo was

attributed to the inhibition of pro-inflammatory cytokine synthesis [58], so perhaps the

Caco-2 model cannot replicate that particular mechanism. There is evidence PGE2-

mediated repair of porcine small intestinal tissue in Ussing chambers following

ischaemia-induced injury [59]. They proposed that PGs promote TJ protein

recruitment/reassembly and can stimulate chloride secretion, together leading to closure

25

of the intercellular space via osmotically-driven flow of intercellular fluid to the apical

side of the epithelium. In a similar study, the beneficial role of PGs in ischaemic repair in

isolated porcine ileal mucosae was based on restoring paracellular resistors in the tight

junction and occurred in the absence of histological changes [60]. Given the difference

between the repair demonstrated with PGs in ischaemia-induced injury in previous

studies and the prophylaxis of chemical perturbation of cell culture monolayers and rat

colonic mucosae in the current study, the exact mechanism of action of PG is likely to be

somewhat different.

Misoprostol exerts its in vivo protective effects primarily as an EP2, EP3, and EP4 agonist,

inhibits stomach acid secretion, and stimulates bicarbonate, mucous and chloride

secretion in the duodenum [61]. Despite evidence for the role of the EP1 receptor in the

maintenance and protection of the gastric mucosa [18], misoprostol has a lower affinity

for it compared to the others EP receptors [62], although it still acts via the EP1 receptor

to induce smooth muscle contraction in guinea pig and rat intestine [63]. Misoprostol also

stimulated mucous secretion and MUC5AC-gene mediated mucin production across

human airway epithelial cell monolayers via activation of EP2 and/or EP4 receptors [64].

Similar to PGE2, misoprostol therefore displays affinity for all four EP receptors in

several tissues.

Regarding EP receptor distribution along the GI tract, EP1 and EP2 receptors are found in

rat gastric chief, parietal, and mucous- secreting epithelia, as well as goblet cells in the

small intestine, and in goblet- and other epithelial cells in the colon [17]. EP1 receptors

26

are also located in muscle layers of the rat stomach, small intestine, and colon, while EP3

receptors are expressed by gastric parietal cells, with expression concentrated in the

muscle layers along the remaining length of the GI tract [65]. EP4 receptors are co-

expressed by rat gastric parietal and mucosal epithelial cells [65], with expression also

detected in enterocytes located at villous tips in mouse ileum [66]. In humans, EP2 and

EP3 receptors are present in gastric and colonic epithelial cells, with EP4 receptors

expressed in colonic lateral crypt epithelia [67]. A more recent investigation of EP

receptor expression in human epithelial crypts did not detect any EP receptor mRNA in

ileum, but EP2 and EP4 mRNA were expressed in colon [68]. In relation to the current

study, Caco-2 cells express all four EP receptors [69].

Misoprostol pre-treatment of Caco-2 monolayers on Transwells® attenuated C10-induced

increases in IC, MMP, and PMP, an effect mediated in large part via the EP1 receptor,

since it was inhibited by SC51322. Analysis of a series of MCFAs demonstrated that

increased Ca2+ arising from mitochondria and the endoplasmic reticulum is a consistent

early response to induced cellular perturbation [30] an effect related to lipophilicity and

which reflects capacity to fluidize plasma membranes. Exposure to perturbants is likely to

trigger an early compensatory response, which could involve calcium based signaling

pathways. A study on PGE2-mediated adaptive cytoprotection in Caco-2 cells induced by

low concentrations of ethanol also demonstrated endogenous PGE2-mediated protection

via regulation of intracellular calcium homeostasis [70]. Apart from PGs, several other

agents can offset membrane perturbation effects by assisting cells in dealing with Ca2+

disruption. For example, cytoprotection afforded against C12 by taurine and L-glutamine

27

in Caco-2 cells was mediated in part by activation of plasma membrane calcium-ATPase

(PMAC) and increased mitochondrial Ca2+ buffering [71]. Reductions in cytochrome C

release from mitochondria was also shown to be an additional mechanism of taurine and

L-glutamine protection, indicative of reduced mitochondrial membrane perturbation [72].

PGEs also reinforce the cell membrane via increased phospholipid and glycoprotein

synthesis [73]. It is possible therefore that misoprostol has a dual mechanism of

cytoprotection against C10 in vitro: preparing the cell to compensate for increases in IC,

and reinforcing the plasma membrane against perturbation. Finally, Ussing chamber and

in situ instillation studies revealed mucous secretion to be the major protective

mechanism of misoprostol against C10 in rat intestinal tissue. Increased mucous secretion

is mediated via EP4 receptors in these type of models [17, 18], so EP1 receptor

antagonism might be less significant in them than in Caco-2 monolayers.

5. Conclusions

Low concentrations of C10 over a set period induce reversible cellular damage, direct

membrane fluidization and increased intracellular calcium, an intracellular mediator with

an established role in altered Caco-2 monolayers. C10 also concentration and time

dependent increase in PMP, which is consistent with non-specific surfactant perturbation

effects on the epithelium. In Caco-2 monolayers, this correlated with increased

expression of selected inflammatory and osmotic stress-related kinases. Depending on

exposure level and time, disruption of Ca2+-homeostasis, morphology, membrane

permeability and ultimately intestinal permeability was reversed within hours. C10-

induced increases in cell metrics were significantly attenuated by misoprostol, resulting

28

in reduction in monolayer permeability. In Ussing chamber- and instillation- studies,

misoprostol-stimulated mucous secretion was evident as a protective mechanism against

alteration in intestinal permeability. Overall this study highlights that the concentration

and duration of exposure of the epithelium to surfactant-based permeability enhancers

significantly influences enhancement action and safety.

 

Acknowledgments

This study was funded by Science Foundation Ireland (SFI) Strategic Research Cluster

Grant 07/SRC/B1154 (Irish Drug Delivery Network) and by an Irish Research Council

Ph.D. industry partnership studentship with Merrion Pharmaceuticals Ireland Ltd for

E.W. We thanks Prof. Alan Baird (UCD) for discussion on prostaglandin pharmacology.

Figure Legends

Fig. 1. A. TEER response following 15 min treatment of monolayers with selected C10

concentrations followed by recovery in fresh DMEM. B. Effect of 15, 30 and 60 min

treatment of Caco-2 cell monolayers with 8.5 mM C10 on TEER and its recovery in fresh

DMEM. C. [14C]-mannitol Papp values in fresh DMEM after 8.5 mM C10 was removed

following incubation for 15, 30 and 60 min. **P<0.01 and ***P<0.001 versus control.

Mean ± SEM of 3 independent measures.

Fig. 2. A. [14C]-mannitol Papp was measured across monolayers over a 7 h period

following 60 min apical treatment with 8.5 mM C10. The Papp was calculated separately

over 4 different periods following C10 removal. Statistical change in mannitol Papp for

each time period was calculated by one-way ANOVA with Dunnett’s post-test comparing

29

against untreated controls for the same time period. ***P<0.001 and **P<0.01 and * P<

0.05 versus control. Mean ± SEM of 3 independent measures. B. TEM of monolayers

following exposure to 8.5 mM C10. (i) Control, no treatment: cells have an intact

membrane surface, microvilli (MV), glycogen granules (G), and tight junctions (TJs); (ii)

C10, 60 min: evidence of focal point cellular effects, with MV sloughed from the surface

of some cells. Large intracellular spaces (IS) formed suggesting an effect on cell-cell

adhesion. iii) 240 min incubation following C10 removal: Cells were seen with and

without MV; TJs appeared intact, but large IS remained. (iv) 24 h incubation following

C10 removal: continuous brush border membrane, smaller IS, glycogen granules (G) and

intact TJs were evident. Horizontal bars= 1µm.

Fig. 3. Effect of repeat daily exposure to 8.5 mM C10 for 60 min on monolayer capacity to

recover parameters over 7 h. A. TEER. Code: Control Day 1 (● ); C10 Day 1 (o):

Control Day 2 (■); C10 Day 2 (□): Control Day 3 (▲); C10 Day 3 (∆). B. Papp values over

7 h was measured following the removal of C10 and was found to be significantly higher

for each subsequent day of exposure. Monolayers were further incubated for 14 h in

DMEM before repeat challenge. ***P<0.001 and **P<0.01 versus C10, Day 1. Mean ±

SEM of 3 independent measures.

Fig. 4. A. Changes in Caco-2 gene expression in a panel of 48 genes following

monolayer exposure to 8.5 mM C10 for 60 min followed by recovery in fresh DMEM. (i)

1 h in recovery; (ii) 4 h; (iii). 8 h; (iv). 24 h. Code for genes where significant changes in

expression were detected: 1. IL-8; 2. IL-12A; 3. OXSR-1; 4. IL-1B; 5. GPX-2; 6.

30

TXNRD-1; 7. SELS; 8. TTN; 9. PTGS-2; 10. AOX-1. B. Fold change in IL-8 gene

expression in monolayers over a 24 h recovery period.

Fig. 5. Restoration of normal Caco-2 cell parameters in filter-grown monolayers

following exposure to 8.5 mM C10 for 60 min followed by recovery in fresh DMEM.

Monolayers were examined by HCA at 0, 1, 4, 8, and 24 h incubation in DMEM post

removal. The significant progression of membrane permeability restitution is also

indicated relative to the previous recovery time-point measured. Significance indicated

relative to untreated control, *P<0.05, **P<0.01, and ***P<0.001, with respect to

untreated control unless marked. “----“ represents C10 removal at 60 min followed by

incubation in fresh DMEM. Mean ± SEM of 3 independent determinations.

Fig. 6. Misoprostol (miso) attenuates C10-induced changes in TEER and Papp. Pre-

treatment with Miso was for 30 min; it was removed; C10 (8.5 mM) was then added for 60

min and removed, at which point [14C]-mannitol was added and TEER and Papp were

determined over 7 h in fresh DMEM. A. TEER, B. Papp. One way ANOVA with

Bonferroni ad hoc post-test to compare groups; *P<0.05, **P<0.01, and ***P<0.001.

Mean and SEM of 6 independent determinations.

Fig. 7. TEM images of Caco-2 monolayers fixed within 60 min of incubations. A.

Control; B. 10 nM misoprostol (Miso) for 30 min; C. 8.5 mM C10, 60 min, D. 10 nM

Miso, 30 min, followed by 8.5 mM C10,60 min. IS= intracellular spaces; MV=microvilli;

G=glycogen storage vesicles. Representative images from three monolayers in each case.

31

Fig. 8. HCA assessment of Misoprostol (Miso) pre-treatment of monolayers attenuated

increases induced by C10 and Triton-X-100 (TX-100). A and D: IC; B and E: MMP; C

and F: PMP. Concentrations (bilateral unless stated): Miso (10 nM, 30 min), SC51322

(20 µM, 30 min), C10 (8.5 mM, apical, 60 min), TX-100 (0.05% v/v, apical, 60 min).

Inhibitors were removed at the point when C10 or TX-100 and the dyes were added.

**P<0.01, ***P<0.001, ANOVA with Bonferroni post-test. Mean ± SEM of 3

independent determinations. *P<0.05, **P<0.01, ***P<0.001, ANOVA with Bonferroni

post-test. Miso, SC51322, and diluent controls had no effects on cell parameters, (data

not shown).

Fig. 9. Misoprostol attenuated C10-induced effects in rat colonic mucosae in vitro. A.

[14C]-mannitol Papp values across rat colonic mucosae. SC51322 (20 µM / 15 min),

misoprostol (1 µM, 15 min), C10 (8.5 mM, 10 min). **P<0.01 versus C10, one way

ANOVA with Bonferroni ad hoc post-test. Mean and SEM of 6 – 12 determinations per

group. B. Representative Alcian Blue and Nile Red staining after 15 min incubation in

chambers: (i) untreated control and (ii) misoprostol. Horizontal bar = 100 µm. C. H&E

stained tissue: (i) C10 for 10 min, C10 removal, 2 h in chamber; (ii) misoprostol pre-

treatment followed by 10 min C10, removal, and 2 h in chamber. Horizontal bar = 100

µm.

Fig. 10. Misoprostol pre-treatment reduced C10-induced enhancement of FD4 absorption

across the colon following instillations. 13 µg/kg misoprostol was instilled into colonic

loops 30 min prior to an FD-4 (40 mg.kg-1) / C10 (100 mM) solution. A. Serum FD-4; B.

32

FD4 AUC. Significance determined by one way ANOVA with Bonferroni ad hoc post-

test to compare treatment groups and is indicated relative to control, *P<0.05, **P<0.01,

(n=6). Mean and SEM of 6 – 12 determinations per group. B. Representative Alcian Blue

and Nile Red staining after 30 min instillation period: (i) untreated control and (ii)

misoprostol. Horizontal bar = 50 µm. C. H&E stained tissue: (i) C10 for 180 min; (ii)

misoprostol for 30 min, followed by C10 for 180 min. Horizontal bar = 50 µm. C. H&E

stained tissue: (i) C10 for 10 min, C10 removal, 2 h in chamber; (ii) misoprostol pre-

treatment followed by 10 min C10, removal, and 2 h in chamber. Horizontal bar = 100

µm.

References

[1] S. Maher, B. Ryan, A. Duffy, D. J. Brayden, Formulation strategies to improve oral

peptide delivery. Pharm. Pat. Anal. 3 (2014) 313-336.

[2] B. Aungst, Absorption enhancers: applications and advances. AAPS J. 14 (2012) 10-

18.

[3] L. Laine, K. Takeuchi, A.Tarnawski, Gastric mucosal defense and cytoprotection:

bench to bedside. Gastroenterology, 135 (2008) 41-60.

[4] B. F. Choonara, Y. E. Choonara, P. Kumar, D. Bijukumar, L. C. du Toit, V. Pillay,

A review of advanced oral drug delivery technologies facilitating the protection and absorption of

protein and peptide molecules. Biotechnol. Adv. 32 (2014) 1269-1282.

[5] C. Tscheik, I.E. Blasig, L Winkler, Trends in drug delivery through tissue barriers

containing tight junctions. Tissue Barriers 1 (2013) e24565

33

[6] S. Melmed , V. Popovic, M. Bidlingmaier, M. Mercado, A. J. Van Der Lely, N.

Biermasz, M. Bolanowski, M. Coculescu, J. Schopohl, K. Racz, B. Glaser, M. Goth, Y.

Greenman, P. Trainer, E. Mezosi, I. Shimon, A. Giustina, M. Korbonits, M. D.

Bronstein, D. Kleinberg, S. Teichman, I. Gliko-Kabir, R. Mamluk, A. Haviv, C.

Strasburger, Safety and efficacy of oral octreotide in acromegaly: results of a multicenter

Phase III trial. J. Clin. Endocrinol. Metab. 100, (2015) 1699-1708.

[7] T. W. Leonard, J. Lynch, M. J. McKenna, D. J. Brayden, Promoting absorption of

drugs in humans using medium-chain fatty acid-based solid dosage forms: GIPET™.

Expert Opin. Drug Deliv. 3, (2006) 685-692.

[8] E. S. Swenson, W. Milisen, W. Curatolo, Intestinal permeability enhancement:

efficacy, acute local toxicity, and reversibility. Pharm. Res, 11 (1994) 1132-1142.

[9] Y. Narkar, R. Burnette, R. Bleher, R. Albrecht, A. Kandela, J. Robinson, Evaluation

of mucosal damage and recovery in the gastrointestinal tract of rats by a penetration

enhancer. Pharm. Res. 25 (2008) 25-38.

[10] X. Wang, S. Maher, D. J. Brayden, Restoration of rat colonic epithelium after in

situ intestinal instillation of the absorption promoter, sodium caprate. Ther Del. 1, (2010)

75-82.

[11] D. Fan, X. Wu, W. Dong, W. Sun, J. Li, X. Tang, Enhancement by sodium caprate

and sodium deoxycholate of the gastrointestinal absorption of berberine chloride in rats.

Drug Dev.Ind. Pharm. 39 (2013) 1447-1456.

[12] A. C. Chao, J. V. Nguyen, M. Broughall, A. Griffin, J. A. Fix, P. E. Daddona, In

vitro and in vivo evaluation of effects of sodium caprate on enteral peptide absorption and

on mucosal morphology. Int. J.Pharm. 191, (1999) 15-24.D

34

[13] D. J. Brayden, E. Walsh, Efficacious intestinal permeation enhancement induced by

the sodium salt of 10-undecylenic acid, a medium chain fatty acid derivative. AAPS J.

16 (2014) 1064-1076.

[14] B. J. Aungst, Intestinal permeation enhancers. J. Pharm. Sci. 89, (2000) 429-442.

[15] M. Sakai, T. Imai, H. Ohtake, M. Otagiri, Biopharmaceutics: cytotoxicity of

absorption enhancers in Caco-2 cell monolayers. J. Pharm. Pharmacol. 50, (1988) 1101-

1108.

[16] E. K. Anderberg, T. Lindmark, P. Artursson, P. Sodium caprate elicits dilatations in

human intestinal tight junctions and enhances drug absorption by the paracellular route.

Pharm. Res. 10, (1993) 857-864.

[17] I. Dey, M. Lejeune, K. Chadee, K. Prostaglandin E2 receptor distribution and

function in the gastrointestinal tract. Br. J. Pharmacol. 149, (2006) 611-623.

[18] K. Takeuchi, H. Araki, U. Umeda, Y. Komoike, K. Suzuki, Adaptive gastric

cytoprotection is mediated by prostaglandin EP1 receptors: A study using rats and

knockout mice. J. Pharmacol. Exp. Ther. 297 (2001) 1160-1165.

[19] S. Narumiya, S. Prostanoids and inflammation: a new concept arising from receptor

knockout mice. J. Mol. Med. 87 (2009) 1015-1022.

[20] T. Brzozowski, A. Tarnawski, D. Hollander, S. Sekhon, W. J. Krause, H. Gergely,

Comparison of prostaglandin and cimetidine in protection of isolated gastric glands

against indomethacin injury. J. Physiol. Pharmacol. 56 Suppl 5 (2005) 75-88.

[21] P. W. Collins, Misoprostol: discovery, development, and clinical applications. Med.

Res. Rev. 10 (1990) 149-172.

35

[22] F. Silverstein, M. Kimmey, D. Saunders, D. Levine., Gastric protection by

misoprostol against 1300 mg of aspirin. Digestive Dis. Sci. 31 (1986) 137S-141S.

[23] F. L. Lanza, A guideline for the treatment and prevention of NSAID-induced ulcers.

Am. J. Gastroenterol. 93 (1998) 2037-2046.

[24] R. Liss, R. Letourneau, J. Schepis, Evaluation of cytoprotection against ethanol-

induced injury in gastric mucosa pretreated with misoprostol, cimetidine, or placebo. Dig.

Dis. Sci. 31, (1986) 108-114.

[25] N. M. Davies, J. Longstreth, F. Jamali, Misoprostol therapeutics revisited.

Pharmacotherapy 21 (2001) 60-73.

[26] I. Hubatsch, E. G. E. Ragnarsson, P. Artursson, Determination of drug permeability

and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2 (2007) 2111-

2119.

[27] S. Keely, A. Rullay, C. Wilson, A. Carmichael, S. Carrington, A. Corfield, D. M.

Haddleton, D. J. Brayden, In vitro and ex vivo intestinal tissue models to measure

mucoadhesion of poly(methacrylate) and N-trimethylated chitosan. Pharm. Res. 22

(2005) 38-49.

[28] E. K. Anderberg, T. Lindmark, Artursson P, Sodium caprate elicits dilatations in

human intestinal tight junctions and enhances drug absorption by the paracellular route.

Pharm Res. 10 (1993) 857-864.

[29] E. W. Walsh, The application of medium chain fatty acids as intestinal permeation

enhancers. Appendix III, pp 236-239. Ph.D. thesis, University College Dublin.

[30] K. J. Livak, K. J., T. D. Schmittgen, T. D. Analysis of relative gene expression data

using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25 (2001) 402-408.

36

[31] D. J. Brayden, J. Gleeson, E. Walsh, A head-to-head multi-parametric high content

analysis of a series of medium chain fatty acid intestinal permeation enhancers in Caco-2

cells. Eur. J. Pharm. Biopharm. 88 (2014) 830-839.

[32] S.B. Fowler, S. Poon, R. Muff, F. Chiti, C.M. Dobson, J. Zurdo, Rational design of

aggregation-resistant bioactive peptides: re-engineering human calcitonin, Proc. Natl.

Acad. Sci. U S A. 102 (2005) 10105-10110.

[33] S. B. Petersen, G. Nolan, S. Maher, U. L. Rahbek, M. Guldbrandt, D. J. Brayden,

Evaluation of alkylmaltosides as intestinal permeation enhancers: comparison between rat

intestinal mucosal sheets and Caco-2 monolayers. Eur. J. Pharm. .Sci. 47(2012) 701-712.

[34] T. A. Aguirre, M. Rosa, S. S. Guterres, A. R. Pohlman, I. Coulter, Brayden DJ,

Investigation of coco-glucoside as a novel intestinal permeation enhancer in rat models.

Eur. J. Pharm. Biopharm. 88 (2014) 856-865.

[35] Y. Guan,Y. Zhang Y, J. Wu, Z. Qi, G. Yang, D. Dou, Y. Gao, L. Chen, X. Zhang,

L. S. Davis, M. Wei, X. Fan X, M. Carmosin, C. Hao, J. D. Imig JD, R. M. Breyer, M.

D. Breyer. Antihypertensive effects of selective prostaglandin E2 receptor subtype 1

targeting. J. Clin. Invest. 117 (2007) 2496-2505.

[36] D.F. Legler, M. Bruckner, E. Uetz-Von Allmen, P. Krause, Prostaglandin E2 at new

glance: novel insights in functional diversity offer therapeutic chances. Int. J. Biochem.

and Cell Biol. 42 (2010) 198-201 (2010).

[37] S. Maher, T. W. Leonard, J. Jacobsen, J., D. J. Brayden, Safety and efficacy of

sodium caprate in promoting oral drug absorption: from in vitro to the clinic. Adv. Drug

Deliv. Rev. 61, (2009) 1427-1449.

37

[38] K. Takeuchi, S. Kato, K. Amagase, Prostaglandin EP receptors involved in

modulating gastrointestinal mucosal integrity. J. Pharmacol. Sci. 114 (2010) 248-261.

[39] T. Lindmark, Y. Kimura, P. Artursson, P. Absorption enhancement through

intracellular regulation of tight junction permeability by medium chain fatty acids in

Caco-2 cells. J. Pharm. Exp. Ther., 284 (1998) 362-369.

[40] M. Tomita, M. Shiga, M. Hayashi, S. Awazu, Enhancement of colonic drug

absorption by the paracellular permeation route. Pharm. Res. 5 (1988) 341-346.

[41] Sakai, M., T. Imai., H. Ohtake, H. Azuma, M. Otagiri, M. Effects of absorption

enhancers on cytoskeletal actin filaments in Caco-2 cell monolayers. Life Sci. 63, (1998)

45-54.

[42] L. Feighery, S. Cochrane, T. Quinn, A. Baird, D. O’Toole, D., S. E. Owens, D.

O’Donoghue, R. Mrsny, R., D. Brayden, Myosin light chain kinase inhibition: correction

of increased intestinal epithelial permeability in vitro. Pharm. Res. 25, (2008) 1377-1386.

[43] S. M. Krug, M., Amasheh, I. Dittmann, I. Christoffel, M. Fromm, M., S. Amasheh,,

Sodium caprate as an enhancer of macromolecule permeation across tricellular tight

junctions of intestinal cells. Biomaterials, 34 (2013) 275-282.

[44] G. Del Vecchio, C. Tscheik, K. Tenz, H. C. Helms, L. Winkler, R. Blasig, I. E.

Blasig, Sodium caprate transiently opens claudin-5-containing barriers at tight junctions

of epithelial and endothelial cells. Mol. Pharm. 9 (2012) 2523-2533.

[45] S. Vaidyanathan, B. G. Orr, M. M. Banaszak-Holl, Detergent induction of HEK

293A cell membrane permeability measured under quiescent and superfusion conditions

using whole cell patch clamp. J. Phys. Chem. B. 118 (2014) 2112-2123.

38

[46] A. T. Blikslager, A. J. Moeser, J. L. Gookin, S. L. Jones, J. Odle, Restoration of

barrier function in injured intestinal mucosa. Physiol. Rev. 87 (2007) 545-564.

[47] L. G. van der Flier, H. Clevers, Stem cells, self-renewal, and differentiation in the

intestinal epithelium. Annu. Rev. Physiol. 1 (2009) 241-260.

[48] S. Tuvia, D. Pelled, K. Marom, P. Salama, M. Levin-Arama, I. Karmeli, G. H.

Idelson, I. Landau, R. A. Mamluk, A novel suspension formulation enhances intestinal

absorption of macromolecules via transient and reversible transport mechanisms.

Pharm. Res. 31(2014) 2010-2021.

[49] W. N. Baskin, K. J. Ivey, W.J. Krause, G. E. Jeffrey, R. T. Gemmell, Aspirin-

induced ultrastructural changes in human gastric mucosa: correlation with potential

difference. Annals Internal Med., 85 (1976) 299-303.

[50] A. J. Wilson, K. Byron, P. R. Gibson, Interleukin-8 stimulates the migration of

human colonic epithelial cells in vitro. Clin. Sci., 97 (1999) 385-390.

[51] M. Baggiolini, A. Walz, S. Kunkel, Neutrophil-activating peptide-1/interleukin 8, a

novel cytokine that activates neutrophils. J. Clin. Invest. 84 (1989) 1045-1049.

[52] L. Eckmann, M. F. Kagnoff, J. Fierer, Epithelial cells secrete the chemokine

interleukin-8 in response to bacterial entry. Infect. Immun. 61 (1993) 4569-4574.

[53] R. Daig, T. Andus, E. Aschenbrenner, W. Falk, J. Schölmerich, J., V. Gross,

Increased interleukin 8 expression in the colon mucosa of patients with inflammatory

bowel disease. Gut 38 (1996) 216-222.

[54] M. Rimoldi, M. Chieppa, V. Salucci, F. Avogadri, A. Sonzogni, G. M. Sampietro, A.

Nespoli, G. Viale, P. Allavena, M. Rescigno,Intestinal immune homeostasis is regulated

39

by the crosstalk between epithelial cells and dendritic cells. Nat Immunol, 6 (2005) 507-

514.

[55] E. K. Hoffmann, I. H. Lambert, S. F. Pedersen, Physiology of cell volume regulation

in vertebrates. Physiol. Rev. 89 (2009) 193-277.

[56] E. K. Hoffmann, T. Schettino, T., W. S. Marshall, The role of volume-sensitive ion

transport systems in regulation of epithelial transport. Comp. Biochem. Physiol. Part A:

Molecular and Integrative Physiology, 148 (2007) 9-43.

[57] J. Y. Park, M. H. Pillinger, S. B. Abramson, Prostaglandin E2 synthesis and

secretion: the role of PGE2 synthases. Clin. Immunol., 119 (2006) 229-240.

[58] N. M. Davies, J. Longstreth, F. Jamali, F. Misoprostol therapeutics revisited.

Pharmacotherapy 21 (2001) 60-73.

[59] A. T. Blikslager, M. C. Roberts, R. A. Argenzio, Prostaglandin-induced recovery of

barrier function in porcine ileum is triggered by chloride secretion. American J. Physiol.

276 (199) G28-G36.

[60] A. T. Blikslager, M. C. Roberts, J. M. Rhoads, R. A. Argenzio, Prostaglandins I2 and

E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum. J. Clin.

Invest., 100 (1997) 1928-1933.

[61] Y. Sugimoto, S. Narumiya, Prostaglandin E receptors. J. Biol. Chem. 282 (2007)

11613-11617.

[62] M. Abramovitz, M. Adam, Y. Boie, M.-C. Carrière, D. Denis, C. Godbout, S.

Lamontagne, C. Rochette, N. Sawyer, N. M. Tremblay, M. Belley, M., Gallant, C.

Dufresne, Y. Gareau, R. Ruel, R., H. Juteau, M. Labelle, N. Ouimet, N., K. M. Metters,

The utilization of recombinant prostanoid receptors to determine the affinities and

40

selectivities of prostaglandins and related analogs. Biochim. Biophysica Acta 1483,

(2000) 285-293.

[63] R. M. Eglen, R. L. Whiting, The action of prostanoid receptor agonists and

antagonists on smooth muscle and platelets. Br. J. Pharmacol. 94 (1988) 591-601.

[64] T. Gray, P. Nettesheim, C. Loftin, J-S. Koo, J. Bonner, S. Peddada, R. Langenbach,

Interleukin-1β–induced mucin production in human airway epithelium is mediated by

cyclooxygenase-2, prostaglandin E2 Receptors, and cyclic AMP-protein kinase A

signaling. Mol. Pharmacol. 66 (2004) 337-346.

[65] M. Ding, Y. Kinoshita, K. Kishi, H. Nakata, S. Hassan, C. Kawanami, Y. Sugimoto,

M. Katsuyama, M. Negishi, S. Narumiya, Distribution of prostaglandin E receptors in the

rat gastrointestinal tract. Prostaglandins 53 (1997) 199-216.

[66] K. Morimoto, Y. Sugimoto, M. Katsuyama, H. Oida, K. Tsuboi, K. Kishi, Y.

Kinoshita, M. Negishi, T. Chiba, S. Narumiya, A. Ichikawa, Cellular localization of

mRNAs for prostaglandin E receptor subtypes in mouse gastrointestinal tract. Am. J.

Physiol. 272 (1997) G681-G687.

[67] V. A. Takafuji, A. Evans, K. R. Lynch, J. K. Roche, PGE2 receptors and synthesis in

human gastric mucosa: perturbation in cancer. Prostaglandins, Leukot., Essential Fatty

Acids, 66 (2002) 71-81.

[68] L. T. Olsen Hult, C. R. Kleiveland, K. Fosnes, M. Jacobsen, T. Lea, EP receptor

expression in human intestinal epithelium and localization relative to the stem cell zone

of the crypts. PLoS ONE 6 (2011) e26816.

41

[69] M. J. Rodríguez-Lagunas, R. Martín-Venegas, J. J. Moreno, R. Ferrer, PGE2

promotes Ca2+-mediated epithelial barrier disruption through EP1 and EP4 receptors in

Caco-2 cell monolayers. Am. J. Physiol. 299 (2010) C324-C334.

[70] E. R. Kokoska, G. S. Smith, Y. Deshpande, C. L. Rieckenberg, T. A. Miller,

Adaptive cytoprotection induced by ethanol in human intestinal cells: role of

prostaglandins and calcium homeostasis. Ann. of Surg. 228 (1998) 123-130.

[71] T. Okuda, K. Kadotsuji, C. Takayama, K. Hanada, F. Mukaizawa, F., K.-1 Ogawara,

K. Higaki, T. Kimura, Involvement of intracellular Ca2+ dynamics in cytoprotective

action by amino acids and cytotoxicity by sodium laurate, an absorption enhancer. J.

Pharm. Sci. 95 (2006) 2256-2265.

[72] C. Takayama, F. Mukaizawa, T. Fujita, K.-I Ogawara, K. Higaki, T. Kimura,

Amino acids suppress apoptosis induced by sodium laurate, an absorption enhancer. J.

Pharm. Sci. 98 (2009) 4629-4638.

[73] E. J. Dial, Y-C Kao, L. M. Lichtenberger, Effects of 16,16-dimethyl prostaglandin

E2 on glycoprotein and lipid synthesis of gastric epithelial cells grown in a primary

culture. In Vitro Cell Dev. Biol. 27A (1991) 39-46.

Table 1. Expression of inflammatory markers in the Caco-2 cell in the period following

exposure to 8.5 mM C10 for 60 min.

42

43

Recovery

Time (h)

Gene Fold change

1

Interleukin 8 (IL8) +11.11

Interleukin 12A (IL12A) +2.58

Oxidative stress responsive 1 (OXSR1) +2.45

4

Interleukin 8 (IL8) +26.01

Interleukin 12A (IL12A) +5.39

Thioredoxin reductase 1 (TXNRD1) +2.30

Selenoprotein S (SELS) +2.20

Interleukin 1 beta (IL1B) +2.06

Glutatione peroxidase 2 (GPX2) +1.98

Titin (TTN) -4.33

8

Thioredoxin reductase 1 (TXNRD1) +3.78

Oxidative stress responsive 1 (OXSR1) +3.64

Selenoprotein S (SELS) +2.89

Interleukin 8 (IL8) +2.76

Interleukin 12A (IL12A) +2.20

Interleukin 1 beta (IL1B) -2.00

Titin (TTN) -2.00

Prostaglandin endoperoxidase synthase 2

(PTGS2)-1.78

24

Interleukin 1 beta (IL1B) +3.74

Oxidative stress responsive 1 (OXSR1) +2.51

Prostaglandin endoperoxidase synthase 2

(PTGS2)-4.84

Interleukin 8 (IL8) -2.61

Aldehyde oxidase 1 (AOX1) -1.82

The PCR array was performed on the pooled cDNA samples representing three replicates for each time point (1, 4, 8 and 24 hr) or untreated controls. Fold change in gene expression are expressed relative to the untreated control at each time point. Fold change values ≥ +2.0 or ≤ -2.0 were considered to have biological relevance.

Table 2. FD4 PK data summary following rat colonic instillations (i.c.).

FD4 (i.v.) FD4 (i.c.) Misoprostol-

FD4 (i.c.) C10 (i.c.)

Misoprostol-

C10 (i.c.)

Cmax (µg. ml-

1)

123.9 ±

22.22.7 ± 1.2 1.8 ± 0.7

31.3 ±

13.021.2 ± 5.1

Tmax (min) 5 ± 0 60 ± 53 70 ± 69 24 ± 11 30 ± 15

AUC 0-180

6250.4 ±

462.4

106.6 ±

62.1233.0 ± 70.3

3078.2 ±

1445.4

2329.7 ±

1014.3

ER - 1.0 2.2 28.9 21.9

Fabs (%) - 1.7 3.7 49.2 37.3

Mean and SEM of 6 determinations. Doses and dosing regimes are given in the text. Absolute bioavailability (Fabs) was calculated relative to FD4 (40 mg. kg-1) i.v.0-∞. Enhancement ratio (ER) was calculated relative to FD4 control i.c

44

45

46

47

48

49

50

51