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

Sam Maher1†*, Joanne Heade2*, Fiona McCartney2, Seona Waters1, Sinéad B. Bleiel3, David J. Brayden2

1School of Pharmacy, Royal College of Surgeons in Ireland, Dublin 2, Ireland, 2UCD School of Veterinary Medicine and UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland, 3AnaBio Technologies Ltd, IDA Business Park, Carrigtwohill, Co. Cork, T45 RW24. *Joint first authors.

†Corresponding author: Tel.: +3534022362, Email: sammaher@rcsi.ie

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ABSTRACT

Surfactant-based intestinal permeation enhancers (PEs) are constituents of several

oral macromolecule formulations in clinical trials. This study examined the interaction

of a test panel of surfactant-based-PEs with isolated rat colonic mucosae mounted in

Ussing chambers in an attempt to determine if increases in transepithelial

permeability can be separated from induction of mucosal perturbation. The aim was

to establish assess if increases in permeability (i) intestinal permeability (the

apparent permeability coefficient (Papp) of [14C]-mannitol), (ii) epithelial histology, and

(iii) short-circuit current (ΔIsc) responses to a cholinomimetic (carbachol, CCh).

Enhancement ratio increases for Papp values followed the order: C10 > C9 = C11:1 > a

bile salt blend > sodium choleate > sucrose laurate > Labrasol® >C12E8 > C12 >

Cremophor® A25 > C7 > sucrose stearate > Kolliphor® HS15 > Kolliphor® TPGS.

Exposures that increased the Papp by ≥2-fold over 120 min were accompanied by

histological damage in 94% of tissues, and by a decreased ΔIsc response to CCh of

83%. A degree of separation between the increased Papp of [14C]-mannitol,

histological damage, and diminution of the ΔIsc response to CCh was observed at

selected PE concentrations (e.g. Labrasol® at 2 mg/mL). Overall, this surfactant-

based PE selection caused transcellular perturbation at similar concentrations to

those that enhanced permeability.

KEYWORDS: Ussing chamber; intestinal permeation enhancers; surfactant toxicity;

intestinal epithelial damage

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GLOSSARY

BA: oral bioavailability

C7 sodium enanthate

C9 sodium pelargonate

C10: sodium caprate

C11:1: sodium undecylenate

C12: sodium laurate

C12E8: polyoxyethylene-8 lauryl ether

CCh: Carbachol

ER: Enhancement ratio

ΔISC: change in short circuit current

LFCS: Lipid Formulation Classification System

MCFA: medium chain fatty acids

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

Papp: apparent permeability coefficient

PE: Permeation enhancer

TEER: transepithelial electrical resistance

TPGS: D-α-Tocopherol polyethylene glycol 1000 succinate.

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1. INTRODUCTION

Biomedical research has played a significant role in identification and elucidation of

new drug targets as well as discovery of drugs with improved efficacy, safety and

tolerability. Chemical entities that exhibit these desirable characteristics are generally

larger and of greater molecular complexity (Leeson, 2016). Drugs have therefore

become progressively larger and more complex over the last 75 years, making oral

formulation more difficult. Sub-optimal oral bioavailability can be distilled into three

key challenges low solubility (Williams et al., 2013) and/or poor permeability (Aungst,

2012). Strategies to improve drug solubility have enabled oral formulation of more

lipophilic drugs, which has in turn enriched the pharmaceutical pipeline with poorly

soluble leads that were inconceivable only 50 years ago. Technologies to improve

intestinal permeability of macromolecules have not had the same success.

Several technologies have been demonstrated to increase oral absorption of poorly

permeable drugs, but few have progressed beyond rodent models (Maher et al.,

2014). Technologies that have progressed to clinical testing frequently tend to

include intestinal permeation enhancers (PEs) as core constituents. PEs increase

permeation across the paracellular route by either opening tight junctions (TJs) or via

transcellular perturbation, or a combination of both (Aungst, 2012). Over 250

molecules have been shown to reduce the intestinal barrier in preclinical in vitro and

in vivo models. These are typically grouped as: calcium chelators, bile salts,

surfactants, microbial toxin derivatives, and synthetic TJ-modulator peptides (Maher

et al., 2016).

The most effective PEs should ideally exhibit a fast onset and efficacious intestinal

epithelial permeation enhancement that is reversible. These target properties were

designated as “Class I” in our attempt to create a “Permeation Enhancer

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Developability Classification System”(Maher et al., 2016). When these metrics are

taken into account, the number of intestinal PEs with true development potential in

oral permeability enhancement is quite low. Selective, targeted and reversible

opening of TJs is however, considered safer than either non-specific TJ opening or

transcellular perturbation (Kondoh et al., 2012). Nonetheless, surfactant-based PEs

with a multiplicity of mechanisms to increase permeability are in clinical trials for a

number of poorly permeable drugs (e.g. C8 (Melmed et al., 2015), salcaprozate

sodium (Davies et al., 2017), sodium caprate (C10) (Walsh et al., 2011)), whereas

selective TJ-openers have not yet reached clinical testing (Aguirre et al., 2016).

Even so, the potential for surfactant PE-induced toxicity may ultimately be an

impediment for clinical application (Kondoh et al., 2006; Lemmer and Hamman,

2013; Okuda et al., 2006). The narrow window between concentrations that induce

efficacy and cytotoxicity in cell culture models has raised concerns that surfactant-

based PEs might lead to GI damage (Ward et al., 2000). Yet, safety concerns have

not been to the fore in publications to date of clinical trials where surfactant-based

PE formulations have been tested in hundreds of subjects without major toxicity

problems (Leonard et al., 2006; Maher et al., 2014; McCartney et al., 2016; Walsh et

al., 2011). Perhaps the better-than-expected toxicity profiles seen for PEs in clinical

trials is due to their relatively short duration, in keeping with the highly effective and

fast epithelial repair mechanisms present in the human small intestine in vivo

(McCartney et al., 2016).

In many cases, surfactant-based PEs can alter paracellular permeability at low

concentrations and transcellular perturbation at higher concentrations (Maher et al.,

2016). A recent evaluation by high content image analysis showed that permeation

enhancement by surfactant-based PEs (medium chain fatty acids, MCFAs) can be

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uncoupled from transcellular perturbation and cytotoxicity in Caco-2 monolayers

(Brayden et al., 2015). However, the relatively low mM concentrations of the MCFAs

associated with a specific TJ-opening effect in Caco-2 monolayers are often lower

than the effective concentrations required for less reductionist delivery models

including rat intestinal tissue mucosae in Ussing chamber and in rat in situ intestinal

loop instillations (Maher et al., 2016; Maher et al., 2009c).

The focus of this study was to evaluate the potential relationship between intestinal

permeability, histological damage, and tissue function for a panel of surfactants with

established use in pharmaceutical formulation and food science. The aim was to

determine if increases in permeability can be separated from mucosal perturbation

across a range of concentrations in order to identify new PEs with potential

application in oral delivery of poorly permeable molecules.

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2. MATERIALS AND METHODS

2.1 Materials

Unless otherwise stated, analytical grade chemicals were obtained from Sigma-

Aldrich (Ireland). Other sources included: sodium undecylenate (C11:1n-1, abbreviated

to C11:1) (Chemos, Germany), C9 (Tokyo Chemical Industry, UK), CCh (Merck

Biosciences, UK), sodium heptanoate (C7) (Fisher Scientific, USA), sodium

taurochenodeoxycholate (Chemos, Germany), sucrose laurate (monoester)

(Millipore, UK), and [14C]-mannitol (Perkin Elmer, UK) were obtained from their

respective suppliers. A synthetic sodium choleate mixture without impurities (bilirubin

and sulphated ash) was prepared by admixing bile salts in the following proportions

(in % w/w) sodium taurocholate (37.4%); sodium cholate (30.1%) sodium

taurochenodeoxycholate (15.8%) taurodeoxycholate (13%) and taurolithocholate

(3.7%) (Story and Barnwell, 1990). Labrasol® was a gift from Gattefosse (France)

and sucrose stearate (mixed ester blend, Pharma Grade, hydrophilic lipophilic

balance (HLB): 16) was a gift from Mitsubishi Chemicals (Japan). Surfactants were

selected for screening based on one or more of the following criteria: current use in

food or pharmaceutical products, a HLB (>10) and/or ability to form micelles (soluble

surfactants). Hydrophobic moieties were either linear aliphatic or aromatic, and

hydrophilic moieties were comprised of sodium carboxylate, ethoxylate, or sucrose

esters. The test selection included an antifungal agent (C11:1), a digestive aid (sodium

choleate), solubilizing oral excipients (Kolliphor® TPGS, Kolliphor® HS15,

Labrasol®, sucrose laurate, sucrose stearate), food additives (medium chain fatty

acids, sucrose laurate, sucrose stearate) and excipients in topical formulations (e.g.

Cremophor® A25)) and the positive control C10 – a PE that has been successfully in

rectal delivery of ampicillin (DoktacillinTM, Meda, Sweden(Lindmark et al., 1997)) and

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a core constituent of the GIPETTM (Merrion Pharma) formulation that reached Phase

II for oral insulin (Brayden et al., 2015).

2.2 Tissue dissection

Experiments involving the use of rat tissues were carried out in accordance with the

current University College Dublin Animal Research Ethics Committee Protocol

[AREC-14-28-Brayden]. All experiments complied with the EU directive on the

protection of animals used for scientific purposes (2010/63/EU). Male Wistar rats

(200-220g; Charles River, UK) were euthanized by stunning and cervical dislocation.

Following a midline laparotomy, the colon was removed and rinsed in pre-warmed

Krebs Henseleit (KH) buffer (37°C). Dissection was performed as previously

described (Maher et al., 2009a). In brief, mucosa/sub-mucosa preparations were

prepared by opening the colon along the mesenteric border, pinning the tissue

mucosal-side down on a dissection board and the underlying circular/longitudinal

muscles were removed by blunt dissection using a size 5 watchmaker’s forceps.

2.3 Electrophysiology

Muscle-stripped colonic tissue mucosae were mounted in Ussing chambers with a

circular window area of 0.63 cm2 (WPI, UK), bathed bilaterally with KH buffer and

continuously bubbled with an O2/CO2 mixture (95/5%) (Maher et al., 2009a). The

temperature of the chambers was maintained at 37°C by an outer glass water jacket

connected to a heated recirculating water bath. In experiments with MCFA salts,

calcium was omitted from KH buffer bathing the mucosal surface in order to avoid

precipitation (Anderberg et al., 1993). Transepithelial potential difference (PD, mV)

was recorded in an open circuit for a period of 20 min after which the tissue was

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voltage-clamped using an epithelial voltage clamp (WPI, UK). Isc (µA.cm-2) and PD

were monitored by switching from clamp to open circuit respectively for 3 s every 30

s using a Pro 4 timer (WPI, UK). Analogue signals were then digitised using a

Powerlab® data acquisition module and analysed with Chart® software (AD

Instruments). The tissue was equilibrated for a further 30 min before commencement

of transport studies. Transepithelial electrical resistance (TEER) was calculated

using Ohm’s Law (V = IR). Tissues with a baseline TEER of < 70 Ω.cm2 were

excluded (Ungell et al., 1998).

2.4 Transepithelial fluxes across rat colonic mucosae

The apparent permeability coefficient (Papp) of [14C]-mannitol was used as a marker of

epithelial barrier integrity (Lazorova et al., 1998). After the tissue equilibration period,

[14C]-mannitol (0.1 µCi/mL) was added to the apical chamber and T0 samples (100

µL) were taken from the apical and basolateral immediately prior to addition of test

PE. Basolateral samples were subsequently taken at T20 min, T40, T60, T80, T100 and

T120. The sampled basolateral volumes were replaced with pre-warmed KH buffer in

order to maintain both sink conditions and equal volumes on each side. Samples

were mixed with scintillation fluid (Ecoscint A; National Diagnostics, USA) and

analysed in a scintillation counter (Tricarb 2900 TR, Packard, USA). The Papp of [14C]-

mannitol was calculated using the equation: Papp (cm/s) = (dQ/dt) ∙ (1/A.C0) where

dQ/dt is the transport rate (DPM (disintegrations per minute)/s); A is the surface area

of intestinal mucosa (0.63 cm2); C0 is the initial concentration in the donor

compartment (DPM/mL) (Maher et al., 2009a). The Enhancement Ratio (ER) was

defined as the ratio of the Papp in the presence of a specified concentration of PE

treatment to the Papp of the control group (ER = Test Treatment Papp/control Papp). At

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the end of each transport experiment, the capacity of tissue to produce a transient

inward short circuit current (ΔIsc) in responsive to stimulation by the cholinomimetic,

carbachol (CCh) (0.1-10 µM) was used as a measure of induced secretory function

(Baldassano et al., 2009).

Test agent preparation and addition

PEs were tested at three concentrations (2, 5 and 10 mg/mL). The lowest test

concentration (2 mg/mL) was selected because it corresponds to the quantity of

sodium caprate used as an experimental control in Ussing chamber experiments.

The two higher test concentrations (20, 50 mg/5mL) were selected to gain

information for formulation of mini-tablets (20, 50 mg) for animal studies. Surfactant

dispersions were prepared in dH2O at a concentration of 10, 20 or 50x and added to

the apical chamber in a volume of 0.5, 0.25 or 0.05 mL – removing a sufficient

volume of KH buffer prior to addition. Mixing in the chamber was achieved by

bubbling at a rate of 1 bubble /second. In cases where mixing led to foaming, the

mixing rate was lowered to 1 bubble / 2 second. Following addition of each

surfactant, visual inspection was performed to ensure that addition of surfactant did

not result in precipitation in the apical chamber.

2.5 Histology

Tissues were removed from Ussing chambers, fixed in neutral buffered formalin

(10% v/v), processed for embedding in paraffin wax, and sectioned (5 µm) on a

microtome (Leica Leitz 1512; GMI, USA). Sections were mounted on adhesive

coated slides, stained with haemotoylin and eosin (H&E) and visualised by light

microscopy (Lobophot-2; Nikon®, Japan). Electronic images were captured with an

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interfaced camera (MicroPublisher® 3.3; QImaging, Canada) and processing

software (Image-Pro® Plus 6; Media Cybernetics, USA). Histology scoring was

carried out on a selection of eight PEs at three concentrations (2, 4, 10 mg/mL). To

enable an objective assessment, we used a blinded score card (McCartney et al.,

2015) adapted from previous studies (Daig et al., 1996; Fix et al., 1986). The effect

of each PE on tissue on morphology of the intestinal mucosae was characterised as

(I) absence of histological damage, (II) mild mucosal perturbation, (III)

moderate/superficial mucosal injury and (IV) severe mucosal injury with extensive

inflammation. Score categories were composed of a number of observed histology

parameters of increasing severity score (see Appendix 1). Here, category I

observations were assigned a score of 0, category II: 5, category III: 10 and category

IV: 20) [20]. The sum of observations for each specimen was used to generate an

overall score (I (< 15), II (15 to < 35), III (35 to < 95) and IV (≥ 95)).

2.6 Data analysis

Unless otherwise stated, each experiment was carried out on three independent

occasions (one colonic tissue mucosa from each of three rats). All values are

expressed as the mean ± SEM. Statistical analysis of Papp values was performed by

Student’s unpaired t-test with Welsh correction when variance was unequal (F >

FCRITICAL)). GraphPad Prism® Version 5 was used for data analysis. The threshold for

significance was set at P ≤ 0.05. For histology scoring, samples were blinded to the

observer and scoring was assigned to two independent observers.

3. RESULTS

3.1 Effect of PEs on TEER and Papp of [14C]-mannitol in isolated rat colonic mucosae

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Baseline TEER values from rat colonic mucosae were 126 ± 47 Ω.cm2 (n = 152).

These values were consistent with previously published data (Maher et al., 2009a).

Apical addition of TPGS (Fig. 1A), HS15 (Fig. 1B) and sucrose stearate (Fig 1C)

caused a partial reduction in TEER (>45% of control) in rat colonic mucosae over 60

min. At incubation times of 60-120 min these three surfactants reduced TEER to

between 20-30% of initial control values.

Other PEs induced a rapid concentration-dependent decrease in TEER to 20-30% of

control values in less than 30 min (Fig. 1D-N). The % TEER remaining after

incubation with PE at 10 mg/mL for 120 min followed the order: TPGS (46%), HS15

(50%), sucrose stearate (34%), C7 (12%), C10 (11%), C12 (11%), sucrose laurate

(10%), Labrasol® (10%), C12E8 (9%) C9 (7%), sodium choleate (6%), C11:1 (5%),

and the bile salt blend (3%).

Colonic mucosae had an average Papp of 0.31 ± 0.03 × 10-6 cm/s for [14C]-mannitol

across rat colonic mucosae (n = 36). These values were consistent with previously

published data (Maher et al., 2007). The three PEs that induced only a partial

reduction in TEER (TPGS, HS15, Sucrose stearate) did not increase the Papp of [14C]-

mannitol (Table I). Low concentrations of PEs that partially reduced TEER also had

no effect on Papp (e.g. C7, bile salt blend) (Table I). Apart from these treatments,

higher PE concentrations had ER values ranging from 2 (e.g. C9 at a concentration

of 2 mg/mL) to 13 (sucrose laurate at a concentration of 5 mg/mL). ER values

obtained at the highest PE concentration (10 mg/mL in each case) were in the order:

C10 > C9 = C11:1 > bile salt blend > sodium choleate > sucrose laurate > Labrasol®

>C12E8 > C12 > Cremophor® A25 > C7 > sucrose stearate > HS15 > TPGS (Table I).

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3.2 Effect of PEs on active ion transport in isolated rat colonic mucosae

Exposure of mucosae to TPGS (Fig 2A), HS15 (Fig 2B), Labrasol® (Fig 2C), C7 (Fig

2D), bile salt blend (Fig 2K), sucrose laurate (Fig 2L) and sucrose stearate (Fig 2I)

led to a partial attenuation of the CCh-stimulated Isc in a PE-concentration-

dependent manner. In contrast, exposure to C9 (Fig 2E), C10 (Fig 2F), C11:1 (Fig 2G),

C12 (Fig 2H), Cremophor® A25 (Fig 2I), sodium choleate (Fig 2J) and C12E8 (Fig 2M)

led to complete loss of the CCh-stimulated Isc (μA).

3.3 Histology scores for selected PEs in isolated colonic mucosae

Histological assessment was performed on 75 tissue specimens treated with control

or PE for 120 min. In order to determine if there was a direct relationship between

Papp and tissue damage, a sample of PEs representing low (0-1), medium (2-5) and

high (6+) ER values were assigned for morphological testing. The low ER value

group included Kolliphor® TPGS (2-10 mg/mL), C7 (2-4 mg/mL), and the bile salt

blend (2 mg/mL). The PEs in the medium ER group were C7 (10 mg/mL), C9 (2

mg/mL), bile salt blend (4 mg/mL), and sodium choleate (2-4 mg/mL). The high ER

group comprised C9 (4-10 mg/mL), C10 (2-10 mg/mL), C11:1 (2-10 mg/mL), bile salt

blend (10mg/mL), and sodium choleate (10 mg/mL). Fig. 3 shows the histology

scoring for eight PEs at all three concentrations over 120 min, while Fig. 4 shows

representative H&E images of eight PE treatments at concentrations of 10 mg/mL.

The highest concentration of Kolliphor® TPGS tested (10 mg/ml) caused no

histological damage (Fig. 3A, 4A). All other PEs led to a concentration-dependent

increase in histology scores (Fig. 3, 4). At the lowest concentration tested (2 mg/ml),

Labrasol® (Fig. 3B), C7 (Fig. 3C) and bile salt blend (Fig. 3H) had no effect on

histology score. The highest degree of mucosal damage was observed at the highest

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PE concentration tested (10 mg/mL) in the rank order: C10 > C11:1 > sodium choleate

> Labrasol® > C9 = bile salt blend.

3.4 Association between permeability of [14C]-mannitol and histology score

A plot of the average Papp of all tissue segments that scored 1, 2, 3 or 4 shows a

relationship between tissue histology score and permeability (Fig. 5A). Three out of

four PE treatments that did not increase Papp had a histology score of 1 (normal

mucosa): bile salt blend (2 mg/mL) and Kolliphor® TPGS (10 mg/mL). Labrasol® (2

mg/mL) was the only PE treatment that increased Papp to over 1 ×10-5 (1.6 × 10-5

cm/s; ER 5) without causing mucosal damage (Table I, Fig. 3B). PEs that had ER

values of ≥ 2 were associated with a histology damage score ranging between

scores of 2 (mild mucosal perturbation) and 4 (severe mucosal injury) (Fig. 3). PEs

exhibiting this score range included the lowest tested concentration (2mg/mL) of

each of the following: sodium choleate (Papp 0.7 × 10-5 cm/s; ER 2; score 2), C9 (Papp

0.7 × 10-5 cm/s; ER 2; score 2), C10 (Papp 1.9 × 10-5 cm/s; ER 6; score 3) and C11:1

(Papp 1.3 × 10-5 cm/s; ER 4; score 3). Histology scores of 2-4 were associated with

significant increases in Papp in 92% (i.e.12/13) of treatments. Similarly, 94% of

treatments with ER values of ≥ 2 were associated with histology scores of 2-4. A plot

of the average Papp of tissues in each histology score category revealed a linear

increase in permeability of mannitol with increasing histology score (Fig 5B). Here

tissue segments that had a histology score of 1 had an average Papp of 0.6 cm/s

which increased to 1.6 cm/s for a histology score of 2, 1.8 cm/s for a histology score

of 3, and 2.2 cm/s for a score 4 (Fig. 5B).

3.5 Association between permeability and CCh-induced ΔIsc

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A plot of the mean cumulative ΔIsc induced by CCh (0.1-10 µM) (denoted: ΔIsc (Σ CCh 10-

7-10

-4M)) versus mean Papp of 14 PEs revealed an l overall reduction in electrogenic ion

transport function with increasing Papp values, although there were a number of

outliers (Fig. 6). The majority of treatments (80%) increased Papp to a value > 1 x 10-5

cm/s reduced ΔIsc to < 15% of control. This was not the case with Labrasol® (2, 4, 10

mg/mL) or sucrose laurate (2, 4 mg/mL). Over 80% (25/30) of PE treatments that

had an ER value of ≥ 2 reduced the ΔIsc by over 75%. However, this was not the

case with the two lowest test concentrations of sucrose laurate (2, 4 mg/mL) and all

concentrations of Labrasol® (2, 4, 10 mg/mL). Interestingly, 40% of PE treatments

with an ER value of 1 were still found to reduce ΔIsc by ≥ 50% (e.g. C7 (2-4 mg/mL),

Kolliphor® HS15 (4 mg/mL) and sucrose stearate (4 mg/mL)).

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4. DISCUSSION

Surfactants are one of the most widely tested PE categories. As they are known to

cause mucosal perturbation, there are concerns about their safety in oral

formulations designed to increase intestinal permeability (Lemmer and Hamman,

2013; Ward et al., 2000). The study aimed to determine if increases in rat colonic

mucosal permeability of mannitol could be uncoupled from both morphological

damage and impaired ion transport for a panel of surfactants with a track record of

use in man. Evidence of histological damage and/or alteration to induced

electrogenic ion transport in response to CCh was reviewed at two thresholds of

increased permeability: i) a two fold increase in the Papp and ii) an increase in the

Papp to values > 1 × 10-5 cm/s. The latter is a value that, when observed in human

colonic mucosae mounted in Ussing chambers, correlated with an intra-colonic

fraction of drug absorbed of >70% in man (Sjöberg et al., 2013). The permeability

and toxicology data from the current study was therefore intended to assist

identification of PEs that have potential application in oral delivery of poorly

permeable drugs.

This study shows that the selected surfactants have potential to alter intestinal

permeability, which may be associated with transmucosal perturbation. Seven

surfactants were shown to increase the Papp > 1 × 10-5 cm/s at all test

concentrations, namely C10, C11:1, C12, Labrasol®, sucrose laureate, C12E8, and

Cremophor® A25. Although the data does not test potencies relative to a current

16

gold standard PE, C10, the efficacy of PEs at concentrations of the same order as

this positive control suggest they also warrant further evaluation. At both of the Papp

thresholds, increased permeability was typically accompanied by histological

damage and/or reduced ion transport functional capacity. These data indirectly

suggest that the selected surfactants act in part via transcellular perturbation. A

proposed mechanism by which surfactants alter membrane integrity is illustrated in

Fig. 7. Here, the monomeric form is thought to insert into the plasma membrane

leading to destabilisation and removal of phospholipids and membrane fragments

into micellar structures, which then facilitates vectorial flux (Maher et al, 2016).

The lowest concentration of sucrose laurate increased the Papp > 1 x 10-5 cm/s and

retained the capacity for electrogenic ion transport in response to CCh. The lowest

concentration of Labrasol® (2 mg/mL) did not cause histological damage despite

inducing an average Papp of > 1 x 10-5 cm/s, therefore the data suggests some of

separation between an increase in Papp and mucosal perturbation. This must be

treated with caution however, as in neither case did the increase in Papp reach

significance. The mechanism by which low concentrations of Labrasol® can increase

intestinal permeability without mucosal perturbation is unclear. Labrasol® is a Class

III agent according to the Lipid Formulation Classification System (LFCS) and is

composed of 90% caprylocaproyl macrogol-8 glycerides (mono- and di- esters), free

PEG, and 10% medium chain glyceride esters (mono-, di- and tri- esters of C8 and

C10). The blend has previously been shown to increase mannitol permeability without

altering TEER across Caco-2 monolayers (Sha et al., 2005). Therefore, a possible

paracellular increase in flux of the hydrophilic molecule is likely using low

concentrations of Labrasol®, especially as the same study showed an alteration to

17

localisation of the TJ associated proteins, ZO-1 and F-actin without loss of

monolayer viability. One interpretation is the destabilisation of TJ by extraction of

loosely-held TJ proteins from the low fluidity regions of lipid raft micro-domains, as

was previously suggested for other surfactants (Maher et al., 2009b).

Performance of PEs in the wider group varied considerably: Kolliphor® HS15,

Kolliphor® TPGS and sucrose stearate did not increase permeability; neither did

they alter histology scores, nor the Isc response to CCh. The data for Kolliphor®

HS15 was consistent with studies in Caco-2 cells in which exposure to 5 times the

CMC did not lead to loss of membrane integrity (Alani et al., 2010). Sucrose stearate

has previously been shown to increase intestinal absorption of alendronate (Alama

et al., 2016) and carboxyfluorescein (Yamamoto et al., 2014) in rat intestinal in situ

loop instillations; this is in contrast to its lack of efficacy in the current study. These

differences are possibly due to different grades of sucrose ester, where different

levels of esterification give rise to differences in the HLB.

Sodium choleate (Ox bile extract) is a food grade bile salt mixture and GRAS food

additive. It is composed of bile salts (50-60%), bilirubin (5-10%) and sulphated ash

(10-20%). The major bile salts in choleate are taurocholate [17.5 %], cholate [14%],

taurochenodeoxycholate [7.4%], taurodeoxycholate [6.1%] and taurolithocholate

[1.7%] (Story and Barnwell, 1990) Each of these bile salts increased intestinal

permeability in intestinal models (Maher et al., 2016). Sodium choleate increased

permeability at the highest concentration tested (10 mg/mL) however, and therefore

would appear to have lower enhancement potential compared to other candidate

PEs. In order to determine if non-surfactant constituents contributed to the reduced

18

effiacy and to establish if a more purified ox bile extract could be used in delivery of

poorly permeable molecules, a synthetic choleate mixture (a bile salt blend) was

prepared without bilirubin and the sulphated ash impurities. The composition of the

bile salt blend was (% w/w): sodium taurocholate (37.4); sodium cholate (30.1)

sodium taurochenodeoxycholate (15.8) taurodeoxycholate (13) and taurolithocholate

(3.7). Despite enrichment, the bile salt blend did not increase permeation

enhancement at low concentrations (2-4 mg/mL), and therefore choleate may have

less potential in oral formulations of poorly permeable molecules relative to other

PEs.

All test concentrations of the positive control, C10, increased the Papp > 1 × 10-5 cm/s,

consistent with previous studies (Maher et al., 2009a). C11:1 and C12 also increased

the Papp above both effect thresholds and caused near complete attenuation of

secretory capacity. C11:1 had induced comparable colonic histology scores as C10 at

low concentrations (both scoring 3, at 2-4 mg/mL), but a lower score of damage at

the highest concentration (3 versus 4. at 10 mg/mL). In a previous head-to-head

comparison, C11:1 was considered as effective as C10 (Brayden and Walsh, 2014). To

our knowledge, C11:1 has not been tested clinically as an oral PE, however, the acid

form (undecylenic acid) is an OTC antifungal therapy and a nutritional supplement.

In general, longer chain length MCFAs (C12, C11:1 and C10) had greater increases on

the Papp and histology scores at concentrations of 2 mg/mL than those with shorter

chain lengths (C7 and C9). The highest concentration of C7 (10 mg/mL) increased Papp

by 2-fold, but did not reach the designated cut-off of 1 x 10-5 cm/s, as was the case

with all other fatty acids. The lowest concentration of C9 did not increase Papp value to

> 1 × 10-5 cm/s, unlike C10, C11:1 and C12. We previously reviewed the

19

physicochemical properties of surfactants that give rise to variation in permeation

enhancement action (Maher et al., 2016). In general, the capacity of surfactants to

perturb biological membranes is a delicate balance between the concentration of

monomeric surfactant available to act on the membrane (the form responsible for

detergent-like perturbation) and its capacity to efficiently insert. Fatty acids with

shorter acyl chains (C7 and C9) have higher CMCs (a direct measure of the solubility

of a surfactant in the monomeric form) than fatty acids with additional carbo chaiin

length (C10, C11:1, C12), but their shorter hydrocarbon tails are likely to make them less

efficient than the latter group at penetration into cell membranes to elicit transcellular

perturbation.

The data does not preclude the possibility that permeation enhancement and toxicity

can be uncoupled for other PEs (e.g. C10). In a high content analysis Caco-2 study

using fluorophores to detect sub-lethal events, 2.5mM C10 increased intracellular

Ca2+, but typical evidence of cytotoxicity was only observed at higher concentrations

≥ 8.5 mM over 60 min (Brayden et al., 2014). However, concentrations less than

8.5mM did not stimulate an increase in the Papp of [14C]-mannitol in rat or human

colonic mucosae (Shimazaki et al., 1998). Similarly, the concentration of C10 (2.5

mM) that increased intracellular Ca2+ in Caco-2 monolayers only partially reduced

TEER to 40% after 1 h, suggesting that a Ca2+-dependent paracellular effect may not

appreciably increase intestinal permeability (Brayden et al., 2014). It is therefore

likely that transcellular perturbation plays a prominent role in the permeability

enhancement induced by PEs reported to act on both paracellular and transcellular

pathways.

20

The current study assisted in identifying surfactant PEs that increase permeability to

the same extent as C10. As with most in vitro intestinal transport studies performed in

monolayers and tissues, the exposure period of 2 h in the Ussing chambers may not

accurately model the relatively short exposure time to high concentrations moving

along the upper GI tract in vivo. Both permeability and toxicity effects of PEs in the

dynamic conditions of the small intestine are likely to be less pronounced than in

static systems used for in vitro study due to the shorter residence time, arising from

the processes of intestinal transit, dilution in the lumen, and spreading across the

surface area of intestinal regions. It is difficult to establish whether the test

concentrations of PE used here are actually achievable in the lumen of the small

intestine in vivo. These in vitro concentrations (2, 5, 10 mg/mL) were approximated

from an oral dosage form containing 500 mg of C10 (Maher et al., 2009b; Maher et

al., 2016). When dissolved in the mean fasted state (54 mL) or fed state (105 mL)

intestinal fluid volume, they were estimated to yield concentrations of 5-9 mg/mL

(Schiller et al., 2005). Nonetheless, PE still exhibit intestinal permeation

enhancement action in man when dosed at high levels, as is the case with C10

(Walsh or Leonard), acyl carnitines (Enteris REF) and SNAC (2017 Phase II already

cited)

There is has been little evidence that surfactant-based PEs induced adverse events

in clinical trials for oral macromolecules carried out in hundreds of volunteers over

the last decade. It is almost certain that they cause temporary and rapidly reversible

perturbation in the period that they are exposed to the small intestinal epithelium

(McCartney et al, 2016). It is not practical to directly evaluate such effect of oral

formulations in man because histological assessment requires an invasive

21

procedure. Yet, a relationship between permeation enhancement and histological

damage was observed in human subjects when C10 was administered to human

subjects as a rectal suppository comprising ampicillin with C10 and hard fat. The

improved BA of ampicillin from 13 to 23% in man was accompanied by mild and

reversible histological damage (Lindmark et al., 1997), although this appeared to be

partly due to a hyperosmolar effect in addition to the presence of C10. Such in vivo

data, albeit from a rectal administration study, was in keeping with a link between

increased permeability and histological changes reported here for rat colonic

mucosae exposed to surfactant-based PEs in vitro. PEs inducing a histology score of

3 in tissue mucosae might cause moderate/superficial mucosal injury in relatively

static compartments like the rectum. Assessing the likelihood of mucosal damage in

the upper GI tract is difficult because dilution and fast transit may reduce the local

concentration of PEs and limit interaction with the epithelium, although this is usually

compensated for by dosing PEs at high concentrations (Brayden et al., 2015; Narkar

et al., 2008). Thus, the histology and functional data presented here for surfactant-

based PEs in vitro represents potential for intestinal interaction that is dependent on

sufficient exposure. It is noteworthy that co-presenting the PE and payload in the

small intestine can significantly improve intestinal permeability in vivo. For example,

the bioavailability of cefoxitin when co-instilled in rat jejunum with a PE, palmitoyl

carnitine, was 77% in ligated tissue conditions but only 18% in un-ligated (Sutton et

al., 1993). While the colon is known to be more sensitive than the small intestine to

surfactant-based PEs (Baluom et al., 1998; Fetih et al., 2005), its use here was

because the isolated colonic mucosa is more amenable to such an in vitro PE screen

due to its capacity to better withstand the dissection and to retain viability in the

Ussing chamber compared to small intestinal mucosae.

22

Colonic epithelial responsiveness to CCh, as indicated by an increase in electrogenic

ion transport, is a measure of maintenance of mucosal function in the presence of

PEs. However, this assay is sensitive to false positives arising from pharmacological

inhibition of electrogenic chloride secretion that may be unrelated to transcellular

perturbation or toxicity. It is well known that low concentrations of MCFAs can inhibit

Ca2+ - and cAMP -dependent anion secretion in rat intestinal mucosa via inhibition of

anion transporters and/or modulation of intracellular signalling (Schultheiss et al.,

2001). In the current study, surfactants that caused histological damage were

consistently associated with near complete inhibition of the Isc response to CCh. At

the same time, all test samples with a histology score of 1 retained capacity to

respond to CCh. Function-associated parameters including CCh-stimulated Isc can

provide initial in vitro toxicology information on tissue viability, but only when

pharmacological interaction with ion channels or transporters can be ruled out. In

order to interpret, such data needs to be assessed in the context of structural and

additional functional assessments, including measurement of TEER. The evidence

from the TEER data suggests that many of the surfactants increased permeability

within 30 min. However, as the histology and secretory responsiveness experiments

were performed at 2 h incubation, it was not possible to conclude that surfactants

cause transmucosal perturbation at initial time points. Previous studies have shown

that incubation with C10 for 60 min causes detergent like perturbation of Caco-2 cells

as indicated by electron microscopy (Brayden et al., 2015) and high content image

analysis (Brayden et al., 2014), but this is a different in vitro model from the current

study.

23

5. CONCLUSIONS

This study identified a group of surfactants that can increase intestinal permeability

across isolated rat colonic mucosae to the same degree as one of the leading PEs in

clinical development, C10. Surfactant-induced intestinal permeation enhancement

was typically accompanied by histological damage and a reduced capacity to

undergo active anion transport. Since most of these surfactants have not yet

presented safety concerns in oral formulations tested in man, the present work

suggests that PEs should not be prematurely discarded because of toxicity concerns

in an in vitro isolated intestinal tissue model with known limitations concerning low

viability compared to in vivo. The study revealed a rank order of PE efficacy that may

be useful to compare with in vivo preclinical models.

ACKNOWLEDGEMENTS

The study was financially supported by the Irish Research Council Employment-

based postgraduate programme (EBPPG/2015/133), AnaBio Technologies Ltd

(Ireland), and also by Science Foundation Ireland (SFI) and European Regional

Development Fund (Grant Number 13/RC/2073).

24

Table I: Effect of PEs on Papp of [14C]-mannitol across isolated rat colonic mucosae. Each value represents the mean ± SEM of treatment versus control: NS, not significant, * P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001

PE (mg/mL) ~Mw (Da) Type pKa HLB Papp (× 10-5)(cm/s)

ER Value

P Value

Control ― ― ― ― 0.31 ± 0.03 1 ―Kolliphor® TPGS (2)

1513 Non ionic ― 13

― ― ―Kolliphor® TPGS (4) 0.24 ± 0.01 1 nsKolliphor® TPGS (10) 0.38 ± 0.06 1 *Kolliphor HS 15 (2)

963 Non-ionic ― 13-15

― ― ―Kolliphor HS 15 (4) 0.42 ± 0.09 1 nsKolliphor HS 15 (10) 0.33 ± 0.05 1 *C7 (2)

152 Anionic 4.9 230.34 ± 0.06 1 ns

C7 (4) 0.36 ± 0.03 1 nsC7 (10) 0.48 ± 0.02 2 nsC9 (2)

180 Anionic 5 220.66 ± 0.23 2 ns

C9 (4) 1.57 ± 0.09 5 ***C9 (10) 2.12 ± 0.21 7 ***C10 (2)

194 Anionic 4.9 211.89 ± 0.35 6 *

C10 (4) 2.56 ± 0.88 8 nsC10 (10) 2.24 ± 0.19 7 **C11:1 (2)

206 Anionic 4.9 211.32 ± 0.18 4 **

C11:1 (4) 1.90 ± 0.26 6 **C11:1 (10) 2.10 ± 0.30 7 **C12 (2)

222 Anionic 5.3 201.78 ± 0.46 6 *

C12 (4) 1.47 ± 0.18 5 *C12 (10) 1.32 ± 0.13 4 **Cremophor® A25 (2)

1360 Non-ionic ― 15-17

1.61 ± 0.51 5 nsCremophor® A25 (4) 1.62 ± 0.26 5 *Cremophor® A25 (10) 1.06 ± 0.10 3 **Labrasol® (2)

― Non-ionic ― 12-14

1.56 ± 0.52 5 nsLabrasol® (4) 1.61 ± 0.51 3 *Labrasol® (10) 1.63 ± 0.16 5 **bile salt blend (2)

― Anionic ― ―0.28 ± 0.03 1 ns

bile salt blend (4) 0.56 ± 0.06 2 nsbile salt blend (10) 1.95 ± 0.25 6 **Na Choleate (2)

― Anionic ― ―0.65 ± 0.07 2 ns

Na Choleate (4) 0.66 ± 0.17 2 nsNa Choleate (10) 1.86 ± 0.47 6 nssucrose laurate (2)

525 Non-ionic ― 16

1.36 ± 0.79 4 nssucrose laurate (5) 3.95 ± 2.08 13 *sucrose laurate (10) 1.72 ± 0.14 6 **C12E8 (2)

539 Non-ionic ― 13

1.62 ± 0.49 5 *C12E8 (5) 1.36 ± 0.56 4 nsC12E8 (10) 1.62 ± 0.41 5 *Sucrose stearate (2)

609 Non-ionic ― 16

0.32 (n = 1) 1 ― Sucrose stearate (4) 0.35 (n = 1) 1 ―Sucrose stearate (10) 0.36 (n = 1) 1 ―

25

FIGURE LEGENDS

Fig. 1: Effect of PEs on TEER across isolated rat colonic mucosae. Each value

represents the mean ± SEM. Symbols denote: ● Control, □ 2 mg/ml, ▲4 mg/ml, ◊ 10

mg/ml. (A) Kolliphor® TPGS, (B) Kolliphor® HS15, (C) sucrose stearate (D)

Labrasol® (E) C7 (F) C9 (G) C10 (H) C11:1 (I) C12 (J) Cremophor® A25 (K) sodium

choleate (L) sodium choleate admixture, (M) sucrose laurate (N) C12E8.

Fig. 2: Effect of PEs on secretory response to CCh (ΔISC) in isolated rat colonic

mucosae. The capacity of intestinal tissue to undergo active ion transport in

response to CCh was used as an initial measure of the ability of surfactants to

abrogate essential secretory processes in the intestinal epithelium. Each value

represents the mean ± SEM. Symbols denote: ● Control, □ 2 mg/ml, ▲4 mg/ml, ◊ 10

mg/ml. (A) Kolliphor® TPGS, (B) Kolliphor® HS15, (C) Labrasol® (D) C7 (E) C9 (F)

C10 (G) C11:1 (H) C12 (I) Cremophor® A25 (J) sodium choleate (K) sodium choleate

admixture, (L) sucrose laurate (M) C12E8 (N) sucrose stearate.

Fig. 3: Histology scores for selected PEs in isolated rat colonic mucosae. Symbols

denote: ● Control, □ 2 mg/ml, ▲4 mg/ml, ◊ 10 mg/ml. (A) Kolliphor® TPGS, (B)

Labrasol®, (C) C7, (D) C9, (E) C10, (F) C11:1, (G) sodium choleate (H) sodium choleate

admixture.

Fig. 4: Representative histology images for a selected group of eight PEs (10

mg/mL) exposed to the apical side of isolated rat colonic mucosae for 120 min. (A)

Kolliphor® TPGS, (B) Labrasol®, (C) C7, (D) C9, (E) C10, (F) C11:1, (G) sodium

choleate (H) sodium choleate admixture.

26

Fig. 5: Relationships between permeability and histological damage for a selection of

PE treatments in isolated rat colonic mucosae. (A) histology score for individual PE

treatments versus their corresponding Papp of [14C]-mannitol, (B) Mean Papp observed

for a pool of all PE treatments in each histology category.

Fig. 6: Relationships between permeability (Papp of [14C]-mannitol) and active ion

transport (CCh induced ΔIsc) for a selection of PE treatments in isolated rat colonic

mucosae.

Fig. 7: Proposed mechanism by which surfactant-based PEs may alter membrane

integrity resulting in histological damage. Surfactant PE monomers insert into the

phospholipid bilayer resulting in altered phospholipid packing and removal of

phospholipids and/or membrane fragments into PE micelles leading to the formation

of PE phospholipid mixed micelles.

27

28

29

30

31

32

33

34

Appendices

Appendix 1: Histology score card to determine the effect of PEs on isolated rat colonic mucosae.

SEVERE MUCOSAL INJURY WITH EXTENSIVE INFLAMMATION

Absence of enterocytes

(extensive)

Surface erosion

(extensive)Exposure of basal lamina

sloughing (Extensive)

Immune cell

infiltration (extensive)

Detachment &

discontinuation

(extensive)

Score Per Row

SCORE 4 95 - 190

YES □ NO □

YES □ NO □

YES □ NO □

YES □ NO □

YES □ NO □

YES □ NO □    

MODERATE/SUPERFICIAL MUCOSAL INJURY

Flattened epithelium

(focal)

Moderate immune

cell infiltration

Necrosis & cell

perturbation (focal)

sloughing (focal)

Surface erosion

(extensive) ―

Score Per Row

SCORE 3 35-95

YES □ NO □ YES □ NO

□ YES □ NO

□ YES □ NO

□ YES □ NO

□ ―    

MILD MUCOSAL PERTURBATION

Cuboidal epithelium Oedema

Neutrophils in

the lamina

Changes to secretory

cells

Necrosis & cell

abnormalitiesDetachment

(focal)

Score Per Row

SCORE 2 15-35

YES □ NO □ YES □ NO □ YES □ NO

□ YES □ NO

□ YES □ NO

□ YES □ NO

□    

NORMAL MUCOSAColumnar epithelium (proximal)

Intact epithelium ― ― ― ―

Score Per Row

SCORE 1 0-15

YES □ NO □ YES □ NO □ ― ― ― ―    NOTES:

35

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