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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
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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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