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Colonic absorption of salmon calcitonin using tetradecyl
maltoside (TDM) as a permeation enhancer
Signe Beck Petersena,b, Lisette Gammelgaard Nielsenb, Ulrik Lytt Rahbekb,
Mette Guldbrandtb, David J Braydena,*
a
UCD School of Veterinary Science and UCD Conway Institute, University College Dublin,
Belfield, Dublin 4, Ireland; bNovo Nordisk, ADME department, Novo Nordisk park, 2860
Måløv, Denmark
*Corresponding author. Tel:. +353 1 7166013; fax: +353 1 7166104
E-mail address: [email protected]
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Abstract
Calcitonin is used as a second line treatment of postmenopausal osteoporosis, but widespread
acceptance is somewhat limited by subcutaneous and intranasal routes of delivery. This study
attempted to enable intestinal sCT absorption in rats using the mild surfactant, tetradecyl
maltoside (TDM) as an intestinal permeation enhancer. Human Caco-2 and HT29-MTX-E12
mucus-covered intestinal epithelial monolayers were used for permeation studies. Rat in situ
intestinal instillation studies were conducted to evaluate the absorption of sCT with and
without 0.1% w/v TDM in jejunum, ileum and colon. TDM significantly enhanced sCT
permeation across intestinal epithelial monolayers, most likely due to combined paracellular
and transcellular actions. In situ, TDM caused an increased absolute bioavailability of sCT in
rat colon from 1.0% to 4.6%, whereas no enhancement increase was observed in ileal and
jejunal instillations. Histological analysis suggested mild perturbation of colonic epithelia in
segments instilled with sCT and TDM. These data suggest that the membrane composition of
the colon is different to the small intestine and that it is more amenable to permeation
enhancement. Thus, formulations designed to release payload in the colon could be
advantageous for systemic delivery of poorly permeable molecules.
Keywords: salmon calcitonin, oral bioavailability, permeation enhancers, epithelial drug
transport, oral peptide delivery, Caco-2 and HT29-MTX-E12 monolayers
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1. Introduction
The 32 amino acid peptide, salmon calcitonin (sCT), is widely used to treat postmenopausal
osteoporosis, Paget’s disease, bone associated pain conditions and hypercalcemia (Chesnut,
III et al., 2008). Calcitonin is secreted by the parafollicular (C-cells) of the thyroid gland, and
in tandem with parathormone (PTH) maintains bone mass by regulating calcium metabolism,
through opposing excessive resorption by osteoclasts (Karsdal et al., 2008;de Paula and
Rosen, 2010). sCT is currently limited to either parenteral or intranasal administration with
interchangeable protection against fracture outcome as a second line treatment in
osteoporosis, albeit with 10 fold lower doses required for injection. Intranasal doses of 200IU
per day for 12-24 months reduce the risk of new vertebral fractures in postmenopausal woman
with osteoporosis (Karsdal et al., 2008; Chesnut, III et al., 2000). Aside from the use in
osteoporosis, there is potential for wider use of sCT due to its anti-inflammatory actions and it
is being researched as a potential adjunct treatment for osteoarthritis (Maricic, 2012; Nielsen
et al., 2011; Manicourt et al., 2006). The oral route would however, be preferred over nasal
sCT considering the long-term dosing requirement and potential for nasal irritation, but
enzymatic degradation and low intestinal membrane permeability lead to insufficient oral
bioavailability in the absence of formulation to address these issues (Lee and Sinko, 2000).
Studies examining potential oral delivery of sCT can be found in the literature. An example is
the oral formulation with sCT and the Eligen® (Emisphere Corp., USA) carrier, 5-CNAC,
which showed greater efficacy in suppression of bone resorption compared to a marketed
nasal formulation, but it still failed the primary endpoint in Phase III trial for osteoporosis
(Karsdal et al., 2011; Maricic, 2012). An alternative oral formulation is OstoraTM (Unigene/
Tarsa Therapeutics (USA), comprising vesicles of sCT and the peptide-stabilising excipient,
citric acid in a pH-dependent Eudragit® L30 D-55 -coated tablet, which completed a Phase III
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trial (ORACAL) and was superior to nasal sCT and placebo in increasing bone mineral
density and reducing bone turnover over 48 weeks; it also appeared to be generally safe and
well tolerated despite some intestinal side effects in 50% of subjects (Binkley et al., 2012).
Acyl carnitines have been used in Unigene’s oral PTH formulations as permeation enhancers
since much higher oral bioavailability will be needed for such peptides compared to sCT
(Stern and Gilligan, 1999), while the medium chain fatty acids, sodium caprate and caprylate
have been used as enhancers in recent oral peptides delivery clinical trials by others (Maher
and Brayden, 2012).
Aegis Therapeutics (USA) has developed a delivery system (Intravail®) containing the alkyl
maltosides, tetradecyl maltoside (TDM) and/or dodecylmaltoside (DDM) as permeation
enhancers that has reached Phase I nasal delivery trials for sCT, parathyroid hormone 1-34,
and diazepam (Maggio and Pillion, 2012). In preclinical studies, TDM improved nasal,
pulmonary and ocular delivery of insulin and calcitonin (Ahsan et al., 2001; Ahsan et al.,
2003).
In initial assessments of its potential for use in oral delivery, it enhanced low
molecular weight heparin bioavailability following oral gavage in rats (Yang et al., 2005),
while studies in mice also showed increased oral delivery of a leptin analogue (Lee et al.,
2010) and octreotide (Maggio and Grasso, 2011). Recent mechanistic evaluation of TDM as
an intestinal permeation enhancer in rat intestinal mucosae mounted in Ussing chambers by
our group found that TDM increased colonic mucosae permeation to flux markers, mannitol
and FITC-dextran 4kDa, in the absence of histological damage, whereas remarkably, there
was no increase across jejunal mucosae (Petersen et al., 2012). Paracellular and transcellular
paths across colonic epithelia were exploited by TDM, as evidenced from in vitro high
content analysis and tissue histopathology, in keeping with TDM’s designation as a mild nonionic surfactant. The aim of the current study was, therefore, was to replace flux markers with
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a peptide, sCT, and to examine regional intestinal absorption in the presence of TDM using
rat in situ instillations of jejunal, ileal and colonic regions as well as in two different types of
human intestinal epithelial monolayer systems. In doing so, we attempted to study in vitro-in
vivo relationships of the effects of TDM and to assess its mechanism of action in detail in
separate bioassay systems.
Human Caco-2 and mucus-covered HT29-MTX-E12 (E12) monolayers were used to assess
whether TDM could increase the permeability of sCT in vitro. Furthermore, the impact of the
mucus layer on permeation of sCT and 3H-mannitol were tested in E12 monolayers
incubated with the mucolytic, n-Acetyl Cysteine (NAC). In situ instillation experiments
elucidated whether TDM was an effective intestinal absorption enhancer for sCT absorption
in different segments of the rat intestine; jejunum, ileum and colon. To assess whether TDM
causes damage to intestinal epithelia in vitro, TDM was evaluated by lactate dehydrogenase
(LDH) assay in Caco-2 and E12 monolayers. Furthermore, tissues from the in situ
experiments were assessed histologically in order to seek any relationship between
permeability increases and potential cytotoxicity. The hypothesis therefore was that TDM
may have potential as a non-toxic colonic permeation enhancer for poorly permeable peptides
and that it can eventually be converted from nasal formulations in the clinic to controlled
release oral ones.
2. Materials and methods
2.1 Cell culture
Human intestinal epithelial Caco-2 cells (p31-48) and E12 cells (p51-55) were grown in
DMEM with L-glutamine (2mM) (Lonza, Switzerland), 1%v/v nonessential amino acids
(Gibco, Denmark), penicillin (100U)/streptomycin (100g/mL) (Lonza) and 10%v/v fetal
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bovine serum (Gibco) on 75 cm2 tissue culture flasks at 95% O2/ 5% CO2 at 37C in a
humidified environment and split when 80-90% confluence was reached. Caco-2 cells were
seeded at a density of 1x105 cells/well on 12 mm Transwells® (polycarbonate, pore size 0.4m)
(Corning Costar Corp., USA) and grown for 21 days in DMEM with media change every
second day (Hubatsch et al., 2007). E12 cells were seeded at a density of 1 x105 cells/well and
cultivated for 14-16 days (Behrens et al., 2001). TEER was measured before experiment start
and at the conclusion of the 60 min sCT transport studies using a Millicell®ERS-2 Epithelial
Volt-Ohm Meter (Millipore, USA).
2.2 Permeability of 3H-mannitol and sCT across monolayers
Permeability of sCT (ChemPrep Inc., FL, USA) and 3H-mannitol (Perkin Elmer, USA) were
measured on Caco-2 and E12 monolayers. The donor-side apical concentration of sCT used
was 25µM and contained TDM concentrations; 0.01, 0.05 and 0.1% w/v. The apical
concentration of 3H-mannitol was 0.8 Ci/mL. Transport experiments on E12 cells were
carried out in the presence and absence of 10mM NAC (Sigma-Aldrich, Denmark) added 15
min prior to sCT (Keely et al., 2005) in order to evaluate the influence of the mucous layer on
TDM’s enhancing abilities. Transport buffer consisted of HBSS (Gibco), 25mM HEPES
(Gibco) and 0.1% w/v BSA (Sigma-Aldrich). BSA was added to prevent sCT adhesion to
plastic (Torres-Lugo et al., 2002). Before transport experiments, DMEM media was changed
to HBSS (0.4 mL apical side; 1 mL basolateral) and left to equilibrate for 60 min before test
solution was added. Basolateral samples of 200L were taken every 15 min over 60 min,
while an apical sample of 10L was taken at time zero. 50L of the basolateral sample was
used for sCT EIA analysis and 50L was used for 3H-mannitol analysis. The apical sample
was diluted 20000 and 50 times before sCT and 3H-mannitol analysis, respectively. Apical
and basolateral samples containing 3H-mannitol was mixed with 100µL scintillation fluid
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and read in scintillation counter (Packard TopCount NTX). Papp values for sCT and 3Hmannitol were calculated according to the following equation:
Papp= (dQ/dt) (1/A*C0)
where dQ/dt is the flux, A is the surface area exposed (1.12 cm2) and C0 is the starting
concentration of sCT or 3H-mannitol. Papp was expressed as diffusion of solutes per second
(cm/s). N numbers in Transwell® experiments refer to independent replicates from separate
studies. For in vitro sCT permeation, apical and basolateral samples were analysed using an
sCT EIA (Phoenix Pharmaceuticals, Inc., CA, USA). Standards were made from the sCT test
solutions in the used HBSS transport buffer with the following concentrations; 25000, 10000,
4000, 1600, 640, 256, 102 and 0 pM. The standard curve was used to calculate the sCT
concentration in the apical and basolateral compartments.
2.3 Monolayer LDH release
Apical samples (50µL) from sCT transport studies on Caco-2 and E12 monolayers were
withdrawn after 60 min to test for LDH release. LDH assay was performed in accordance
with manufacturer’s guide, where positive controls were created by adding lysis buffer from
the kit to a set of control wells and incubating for 20 min (CytoTox-ONETM Homogeneous
Membrane Integrity Assay, Promega, USA).
2.4 In vivo rat intestinal instillation
2.4.1 Animals
Experimental protocols using the in situ instillation rat model were approved by the
University College Dublin (UCD) Animal Research Ethics Committee and carried out at
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UCD Biomedical Facility under a license obtained from the Irish Department of Health and
Children. Additional studies were carried out at Novo Nordisk animal facility under a
protocol approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.
Adult male Wistar rats (approx. 300-400g) were purchased from either Charles River Labs,
UK or Taconic, Europe, and housed under controlled environmental conditions regarding
humidity and temperature with a 12:12 h light/dark cycle. The rats received tap water and
standard laboratory chow ad lib. All studies were carried out in adherence with the
“Principles of Laboratory Animal Care,” (NIH Publication #85-23, revised in 1985).
2.4.2 Rat intravenous injection of sCT
Rats were anaesthetised by the sub-cutaneous route (s.c) with a Hypnorm®-Dormicum® 1:1
mixture (2ml/kg). Anaesthesia was maintained at a dose of 1ml/kg s.c every 30-40 min during
catheter surgery and the 4 h experiment. Separate catheters were inserted 2-3cm into right
jugular vein and left carotid artery and rats were placed on a heating pad to maintain body
temperature. Following i.v. dosing of sCT (20µg/kg, ChemPrep Inc., FL, USA), 200-400µL
aliquots of blood were retrieved via the arterial catheter at 0, 5, 15, 30, 60, 120, 180 and 240
min. Blood samples were centrifuged at 6000 rpm for 5 min at 4C, and serum was aliquoted
for sCT analysis and stored at -20C until assayed. Rats were subsequently euthanized with
CO2.
2.4.3 Rat intestinal instillation
Intestinal instillations with sCT and TDM (0.1% w/v) were modified from an in situ closeloop instillation protocol (Sugita et al., 2007). Rats were given 5% glucose water for 2 days
before experiments start and fasted for 18 hours. They were anaesthetised by intraperitoneal
(i.p.) injection of ketamine (75mg/kg) and xylazine (10mg/kg) and placed on a heating pad to
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maintain body temperature. Anaesthesia was maintained by inhalation of isoflurane (1-2%)
vaporised in oxygen and administered at a flow rate of 1L/min. sCT was dissolved in PBS
with or without 0.1% w/v of TDM. Following a midline laparotomy, sCT (400g/kg)containing solutions were injected using a 30G micro-syringe into the surgically-exposed
proximal jejunum, ileum (the 2-3 cm before cecum) or distal colon. Following instillation,
blood 200-400L was sampled from the orbital vein at 0, 5, 15, 30, 60, 120, 180 and 240 min.
Serum samples were prepared for analysis as described above. Rats were euthanized by
injecting 0.5mL Euthatal® into either the heart or the tail vein. Following euthanasia, the
intestinal segments were excised and placed in 10% (v/v) buffered formalin for histology.
2.5 sCT and calcium analysis
Serum samples were analysed for sCT using EIA kits from Bachem Corp. and Phoenix
Phamarceuticals (USA). Both EIA’s were performed as recommended in the manufacturer’s
guides, but with minor modifications for the latter: 10µL sample was mixed with 40µL EIA
buffer and the EIA plate was incubated at 4ºC overnight to increase sensitivity. Standards
were made from pooled male Wistar rat serum (SeraLab, UK) in the presence of sCT
(ChemPrep Inc., FL., USA). The rationale for use of two different EIA kits for serum samples
was that 10-fold lower serum volumes could be used for the Phoenix assay and it also had a
wider linear concentration range. The Bachem EIA had greater sensitivity at low pM
concentrations, which the Phoenix assay could not detect. Samples from instillations was
analysed using the Bachem EIA in order to cater for very low serum sCT concentrations,
while samples following i.v. administration were analysed with the Phoenix EIA, where interrat variation was less at higher serum sCT concentrations. Pharmacokinetic (PK) data was
plotted as meanSEM after subtraction of the individual sCT concentration at time zero. The
peak sCT concentration (Cmax), the time to reach max (tmax), area under the curve (AUC) and
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half-life (t½) were calculated using WinNonLin® 5.3, noncompartmental analysis (PharSight
Corporation, USA). The absolute bioavailability (F) of sCT was calculated: F (%) = (AUC04h*Di.v./AUCi.v.*Dinst)
*100, where AUC0-4h was the area under the sCT serum concentration
curve between 0 and 4 h and AUVi.v was the area under the sCT serum concentration curve
after i.v. administration (20g/mL). I.V. data was fitted to a two-compartment model using
WinNonLin®, from which initial serum () - and terminal () t½ were determined, and these
values were used to deconvolute the colonic instillation data. Serum calcium was analysed
with a Randox Laboratory clinical chemistry analyser (Ryan et al., 2009).
2.6 Histology
Following instillation studies, intestinal tissues were placed in 10% (v/v) buffered formalin
for at least 48 hours. Tissues were processed and stained with hematoxylin & eosin (H&E),
alcian blue and neutral red. The slides were scanned in 20X scanning mode on a NanoZoomer
2.0-HT (Hamamatsu) and viewed at 20X.
2.7 Data analysis
Statistical analysis was carried out using Prism-5® (GraphPad®, San Diego, USA). Two-tailed
unpaired Students t-tests were used for comparison. Results are presented as the mean ±
standard error of the mean (SEM). A significant difference was considered if p<0.05.
3. Results
3.1 Effects of TDM on TEER and permeability across Caco-2 and E12 monolayers
Caco-2 and E12 monolayers were exposed to three different concentrations of TDM (0.01,
0.05 and 0.1% w/v) to assess effects on apical-to-basolateral side fluxes of 3H-mannitol and
sCT over 60 min. Mean basal TEER values were 971 Ω.cm2 for Caco-2 monolayers (n=3,
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each in triplicate), 1009 Ω.cm2 for E12 monolayers (n=4), and 1026 Ω.cm2 for E12 after preincubation with 10mM NAC (n=2). TEER values were significantly decreased for each TDM
concentration in Caco-2, E12 and in E12 monolayers exposed to NAC at 60 min compared to
controls (p<0.01 for 0.01% TDM, p<0.0001 for 0.05 and 0.1% w/v TDM). TEER decreased
across both Caco-2 monolayers and E12 monolayers by 32%, 78% and 81% and by 51%,
74% and 74%, respectively in the presence of 0.01, 0.05 and 0.1% w/v TDM compared to
initial values. For E12 monolayers exposed to 10mM NAC for 15 min, TEER decreased by
53%, 72% and 73% for 0.01, 0.05 and 0.1% w/v TDM.
Papp values for both sCT and 3H-mannitol on Caco-2 monolayers and E12 monolayers
revealed concentration-dependent increase in the presence of TDM (Fig. 1). This was
reflected in increased enhancement ratios (Fig. 1e,f), noting slightly higher ratios for the E12
monolayers than the Caco2 monolayers. 0.05% w/v TDM statistically increased the Papp for
sCT and 3H-mannitol by ~50% and by 10-20%, across E12 and Caco-2 monolayers
respectively. The mean basal Papp for sCT on Caco-2 monolayers was 7.8 x10-8 cm/s, within
the range of previous reports (Song et al., 2005;Youn et al., 2006), while it was 6.5 x10-8 cm/s
for E12 monolayers. These values are consistent with very low paracellular permeation. The
mean basal Papp for 3H-mannitol was 1.1 x10-6 cm/s for Caco-2 monolayers while it was 6.2
x10-7 cm/s for E12. The Papp values for sCT and 3H-mannitol across E12 monolayers preincubated with 10mM NAC for 15 min before addition of TDM concentrations were
comparable to those in E12 monolayers in the absence of NAC; the mucolytic had no effect
on flux (Fig. 2a,b). While, enhancement ratios with TDM application were higher for sCT in
NAC-treated versus untreated E12 monolayers, this reflects a slightly lower basal Papp in the
former and absolute values were not different. Similarly, [3H]-mannitol Papp enhancement
ratios were unaffected by NAC in E12 monolayers (Fig. 2c) and basal values were similar in
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the presence and absence of NAC. TDM was therefore an effective permeation enhancer for
mannitol and sCT in both in Caco-2 monolayers as well as in mucous covered E12
monolayers.
3.2 Assessment of toxicity on Caco-2 and E12 monolayers
Cytotoxicity induced by TDM following 60 min flux studies in Caco-2, E12 and E12
monolayers exposed to NAC was assessed by LDH release. LDH release from the apical side
of the Caco-2 monolayers showed a significant increase in the presence of TDM (Fig. 3),
which correlated with data from the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay in Caco-2 cells (Petersen et al., 2012). LDH release from
the apical side of the E12 monolayers also revealed a significant increase for all TDM
concentrations (Fig. 3), whereas application of NAC did not change the LDH release profile.
LDH release was significantly higher in the Caco-2 cell monolayers than the E12 monolayers
at most TDM concentrations, suggesting that the mucus layer provides some protection (Fig.
3). The key data was that 75% cell death was seen with 0.05% w/v TDM at 60 min in Caco-2
and E12 monolayers, the same concentration that induced large Papp changes for sCT and
[3H]-mannitol. This would infer that the two processes are difficult to separate at an in vitro
level.
3.3 In situ instillation of jejunum, ileum and colon
Fig. 4 shows the serum calcium changes when 400g/kg sCT was administered in the
presence and absence of 0.1% w/v TDM to the jejunum, ileum and colon. The decrease in
calcium levels following sCT administration to the three segments were not statistically
significant different between sCT and sCT in the presence of TDM, however there were clear
reductions in levels in both formats in all three tissues compared to PBS controls. Serum
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calcium levels were however, statistically reduced in the presence of TDM at time 60 min
time point compared to sCT alone in colonic instillations (p 0.05). Overall, with this
exception, the pharmacodynamic effects on serum calcium in the three regions were
equivalent for TDM on sCT action, confirming the limitations of this assay as a
discriminating tool for sCT formulation screening.
PK parameters (Cmax, Tmax and AUC) derived by non-compartmental analysis (NCA) for
serum [sCT] following instillations are shown (Table 1). The mean AUC for rats administered
sCT by the i.v. route was 667743±45275 min.pM, and this was used to calculate absolute
bioavailability (F) for instillations. No statistical PK differences were seen however when
sCT was instilled to jejunal and ileal segments in the presence and absence of 0.1% TDM
(Fig. 5b, c). The absolute bioavailability (F) for sCT in the absence and presence of TDM for
jejunal and ileal instillations was also very low and not different (Table 2), hence there was no
statistical difference in AUC of jejunal and ileal sCT instillation in the presence and absence
of TDM and the relative F was therefore not calculated. Importantly, there was a statistical
increase in AUC upon colonic instillation of sCT with 0.1% TDM (Table 1, Fig. 5d) and the
relative F for colon administration of sCT in the presence of TDM was 22.5% compared to
sCT in its absence. The mean absolute colonic F for sCT was 1.0% (control) and 4.6% in the
presence of 0.1% TDM, calculated from the AUC derived by NCA in WinNonLin® (Table 2).
Performing model fitting in WinNonLin® of i.v. data; the i.v. data fitted a two-compartment
model with the macro-constants  and  determined to 0.13 min-1 and 0.019 min-1 equivalent
to  and  half-life (t½) of 5.3 min and 36.1 min, respectively. A similar t½ () was previous
reported in rats (Ryan et al., 2009; Sinko et al., 1995) and dogs (Lee and Sinko, 2000). The
derived macro-constants was used to perform deconvolution of colon data in WinNonLin®,
which gave a mean absolute F of 1.4% and 5.1% in the absence and presence of TDM,
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respectively, correlating well to the absolute F derived from the colonic AUC’s and calculated
absolute F by NCA. Deconvolution revealed that the maximum absorption rate was at 5 min.
The harmonic mean of the apparent t½ for administration in colon was 22.5 min (range 13.751.5) in the absence of TDM and 20.9 min (range 12.2-109.0) in the presence of 0.1% w/v
TDM, as calculated by NCA. This was not statistically different from the apparent t½ of 29.2
min detected following i.v. administration, also calculated byNCA. Furthermore, it was not
statistically significant from the terminal t½ () derived by fitting i.v. data to a twocompartment (by NCA). Unlike the calcium serum assay, PK data therefore confirmed that
both basal sCT absorption and the permeation enhancing effects of TDM on sCT absorption
were substantially increased in the colon compared to small intestinal regions.
3.4 Histology data
Colonic mucosae were assessed for gross histology in the presence and absence of 0.1% w/v
TDM after 4 h instillation. Colonic tissue exposed to sCT without TDM generally appeared as
healthy intestinal mucosa, whereas TDM-exposed tissue showed mild oedema and evidence
of cell sloughing to a varying degree (Fig. 6). Some mucous secretion within the colonic
segments was also observed, corresponding well with results from rat isolated colonic
mucosae used in Ussing chambers (Petersen et al., 2012). These data suggest that the
cytotoxicity data seen for TDM in intestinal epithelial cell lines is much exaggerated over that
detected in the intestine in vivo and that histology data in tissue is more relevant and reliable.
4. Discussion
Attempts to formulate an oral sCT as a second line treatment for osteoporosis have been made
without success for over 20 years. Typically, very low oral bioavailability has been reported,
but even 1% would be of the same order as current nasal sCT products (Illum, 2012; Grant
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and Leone-Bay, 2012), and this seems to be the case in the recent Phase III ORACAL trial for
Ostora™ where primary end-points were met (Binkley et al., 2012). Our data suggests that
incorporation of alkyl maltoside permeation enhancers that work selectively in the colon may
increase oral bioavailability of sCT over approaches aimed at the small intestine. This has not
been shown before. The Unigene/Tarsa oral sCT formulation that completed Phase III
releases the protected peptide in the duodenum as the pH rises. Even though basal sCT
permeability is lower there than in colon and despite relatively low sCT bioavailability, it may
still compete with marketed nasal counterparts. Unigene/Tarsa’s oral sCT formulation
contains citric acid incorporated in vesicles with the peptide primarily in order to protect it
from small intestinal peptidases, which have sub-optimal activity at acidic pH values. These
vesicles are then entrapped in a capsule with a pH-dependent coating designed to release in
the duodenum. The formulation does not contain an enhancer unlike their PTH formulations,
which contain lauroyl carnitine (LeCluyse et al., 1991; Mehta, 2010). This is because a much
higher oral bioavailability is required for PTH than for sCT.
The low nasal bioavailability seen with the highly potent licenced peptides means that there is
substantial waste that would not be acceptable for other peptides. Therefore, there is a search
on for effective epithelial enhancers with potential for both nasal and oral peptide delivery as
these would increase the range of less potent peptides that could be formulated. Our approach
was therefore to examine whether one of the main alkyl maltosides present in the Intravail®
technology may be leveraged from nasal delivery to a colonic delivery application. We were
also influenced by recent studies showing that Intravail® could increase the oral
bioavailability of octreotide and a leptin-like peptide in mice (Lee et al., 2010; Maggio and
Grasso, 2011). In addition, our in vitro data revealed that TDM increased fluxes of [14C]mannitol and FD-4 across isolated rat colonic but not small intestinal mucosae and moreover,
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the enhancement caused by the non-ionic detergent was at concentrations well above its
critical micelle concentration (Petersen et al., 2012). This led to the current study where we
placed the emphasis on rat in vivo instillations in different intestinal regions with sCT.
Firstly, we showed that TDM increased the Papp value of sCT across Caco-2 and mucuscovered E12 monolayers in a concentration-dependent fashion and these data matched the
pattern seen for [3H]-mannitol. Basal sCT Papp values across Caco-2 monolayers corresponded
well with previous studies (Song et al., 2005; Torres-Lugo et al., 2002; Youn et al., 2006),
and the TDM-induced concentration-dependent increase in the [3H]-mannitol Papp value
across Caco-2 cell monolayers matches previous findings (Petersen et al., 2012; Yang et al.,
2005). Since TEER reductions were seen in parallel, this supported an initial conclusion that
the effect was paracellular. Such a conclusion was somewhat invalidated by LDH release
assay cytotoxicity results showing substantial cell death during 60 min incubation at the same
concentrations of TDM that increased Papp values. These findings are consistent with the nonionic surfactant properties of TDM, which lead to reductions in plasma membrane potential
and mitochondrial membrane potential in Caco-2 cells following short-term exposure
(Petersen et al., 2012). Papp values for sCT and [3H]-mannitol across E12 monolayers in which
mucus was removed by NAC (Keely et al., 2005) were very similar to values obtained in E12
and Caco-2. While this was in keeping with reports that mucus produced by other HT29MTX sub-clones is not a major hindrance to either hydrophilic or hydrophobic drug diffusion
across the epithelium (Pontier et al., 2001), data from other mucus-covered HT29-H subclones suggested that the mucus elaborated by that line prevented flux of the highly lipophilic
molecule testosterone (Karlsson et al., 1993). In sum, the in vitro data indicated that mucous
lying above the E12 monolayers did not prevent the permeation-enhancement effects of TDM
and that in vivo effects in the intestine should similarly not be influenced.
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In vitro Caco-2 cell LDH data raised a concern that toxicity and permeation enhancement
might be intimately related, however LDH release from isolated colonic mucosae exposed to
the same concentrations of TDM for 60 min was not as high (Petersen et al., 2012), compared
to observations in Caco-2 monolayers. This was further supported by gross histology of
TDM-exposed tissue from the in situ instillation experiments where, although some cell
sloughing and mild oedema was seen, the epithelial barrier was still intact. That Caco-2 cell
cytotoxicity studies overestimate actual intestinal tissue damage by enhancers is well
established (Aungst, 2012). The likely reason is the lack of capacity for in vitro models to
rapidly repair in the face of damage compared to in vivo (Cano-Cebrian et al., 2005;Maher et
al., 2009). In sum, despite the caveat on cytotoxicity, the in vitro data demonstrating sCT
permeation enhancement in the presence of TDM supported the carrying out of in vivo
studies.
In situ instillations to different rat intestinal regions proved that TDM acts selectively in the
colon to increase sCT absorption and this confirmed the flux data for the presence of TDM in
rat intestinal tissue mucosae mounted in Ussing chambers (Petersen et al., 2012). Basal
absorption of sCT was significantly higher across the in situ colon compared to other regions.
Others demonstrated an increased hypocalcemic effect of sCT in rats after intra-colonic
administration in a PEGylated format compared intra-jejunal instillation of the same
formulation (Mansoor et al., 2005). A very low absolute F of 0.02% was also reported
following rat intra-duodenal administration of sCT (Sinko et al., 1995). This suggests that
permeability to small hydrophilic molecules is inherently increased across colon compared to
the small intestine. Others report that the effectiveness of the related dodecyl maltoside
(DDM) as an enhancer was in the order: rectum > colon > ileum > jejunum, where little
17
absorption was detected (Murakami et al., 1992;Fetih et al., 2005), so it would seem that there
is a class effect for alkyl maltosides relating to large intestine permeability enhancement.
Even though the colonic mucosa seems to be generally more responsive to permeation
enhancers than ileal and jejunal regions (Maroni et al., 2012), some agents are effective in the
small intestine. An example is sodium caprate, which is a component of several peptide
formulations designed to release payload in the small intestine (Maher et al., 2009).
The small intestine must withstand regular exposure to 30mM bile salts and mixed micelles in
the fed state, so it requires a high level of structural robustness compared to colon. There are
also lower fluid volume and longer residence times in colon, along with potential solvent drag
effects, which may favourably impact permeability (Maher and Brayden, 2012). In general,
the level of proteolysis in the colon of humans are lower than that in the small intestine
(Gibson et al., 1989) and this may in part explain why sCT has a shorter t½ in jejunal fluid
than in colonic (Mansoor et al., 2005). On the other hand, proteases originating from bacterial
fauna are found selectively in the colon (Gibson et al., 1989) and human CT was still
susceptible to degradation in rat caecal contents (Tozaki et al., 1997). In the Ussing chamber
studies demonstrating colon-specific effects of TDM and DDM (Petersen et al., 2012),
stability was not an issue for [3H]-mannitol or FD-4 and this would suggest that a difference
in membrane composition between jejunum and colon relating to fluidity is the most
important differentiating factor.
Placing the in situ colonic sCT bioavailability in context, the absolute F value of 4.6%
achieved with 0.1% TDM appears impressive, although it must be emphasised that regional
intestinal instillations do not factor in potential losses during GI transit or the impact of
dilution for an oral formulation. Therefore, this is the best data that can be detected in rats if
18
sCT and TDM could be reliably protected, delivered and released together in the colon in a
controlled release formulation. Still, enteric-coated sCT capsules for small intestinal delivery
have achieved an absolute F of up to 1.8% after oral administration to rats (Wu et al., 2010),
so the current data would suggest that this can be improved upon by targeting the colon and
using an appropriate enhancer. A colonic F for sCT approaching 5% would be significantly
higher than current nasal formulations and other oral formulations in clinical trials targeted at
the jejunum and ileum.
Finally, there is the question of how to compare serum calcium data in response to sCT.
The calcium decrease seen with sCT and TDM did not show any significant difference
compared to native sCT at a dose of 400 µg/kg in any region, except at 60 min in the colon.
The calcium decrease of 20-30% seen in all intestinal segments was in the range of previous
reports following s.c., intrajejunal and intracolonic administration of sCT in rats (Cheng and
Lim, 2009;Cetin et al., 2008;Wu et al., 2010;Mansoor et al., 2005). A maximum
hypocalcemic threshold level exists due to calcium homeostasis and moreover, even i.v.
dosing of sCT does not induce serum calcium decreases in a strict dose-dependent fashion
(Ryan et al., 2009;Mansoor et al., 2005;Cheng and Lim, 2009). Profound elevations in serum
sCT are not accompanied by major hypocalcaemia, since low levels of calcium will lead to a
rebound in PTH secretion, which acts on kidney and bone to mobilise plasma calcium levels
(Friedman, 2011). A stabile circulating calcium level is vital for the electrophysiological
performance of most cells and consequently, a self-regulating system with several interacting
hormones exists (de Paula and Rosen, 2010). As a read-out, serum calcium reductions in
response to oral sCT can therefore provide useful information as to whether the peptide is
bioactive in the intestine, however they provide no insight into rank ordering effective
formulations and consequently PK data is what should be relied upon.
19
5. Conclusion
In summary, for the first time TDM proved to be effective as an intestinal permeation
enhancer for sCT across Caco-2 and E12 monolayers. In rat in situ instillations, TDM
preferentially increased absorption of sCT in the colonic region of the intestine compared to
proximal jejunum or ileum. Importantly, in contrast to in vitro cytotoxicity studies, in vivo
toxicity assessment confirmed a wide margin between doses of TDM that induce permeation
and those which damage tissue. These novel data indicate that oral peptide formulations
utilizing TDM as an absorption enhancer may potentially be developed to target the colonic
region for improved systemic bioavailability.
Acknowledgements
Novo Nordisk A/S supported for the post-doctoral fellowship of SBP. Additional support was
from Science Foundation Ireland grant Number 07/SRC/B1154 (The Irish Drug Delivery
Network). Thanks to Ms. Margaret Coady for assistance with tissue histology and to Tanira
Aguirre for assistance with instillations, both at UCD. Also thanks to Sebastian Beck
Jørgensen, Kent Emil Pedersen and Helle Andersen for assistance with i.v. rat studies, and to
Christian Rosenquist and Susanne Halkier for discussion on the sCT EIA, and to Flemming
Seier Nielsen for discussion and interpretation of WinNonLin® data (all at Novo-Nordisk).
20
Figure legends
Fig 1. Papp of [3H]-mannitol and sCT across Caco-2 and E12 monolayers with TDM (0.01,
0.05 and 0.1% w/v). (a) Caco-2: Papp [3H]-mannitol, (b) E12: Papp [3H]-mannitol, (c) Caco-2:
Papp sCT, (e) E12: Papp sCT, (e) Caco-2 enhancement ratios versus controls in the absence of
TDM, (f) E12 enhancement ratios versus controls in the absence of TDM. (n=3 for Caco-2
and n=4 for E12).
Fig 2. Papp of [3H]-mannitol and sCT across E12 monolayers incubated with NAC (10 mM)
and TDM (0.01, 0.05 and 0.1% w/v). (a) Papp [3H]-mannitol, (b) Papp sCT, (c) enhancement
ratios versus controls in the absence of TDM. (n=2).
Fig 3. LDH release after 60 min exposure of Caco-2 and E12 monolayers with TDM.
Caco-2 monolayers (n=3),
E12 monolayers (n=3),
E12 monolayers with 10mM
NAC (n=2). LDH release by lysis buffer was set to 100%. P < 0.001 for 0.01% TDM; P <
0.0001 for 0.05 and 0.1% TDM versus untreated controls; P < 0.05 between Caco-2 and E12
monolayers for 0.01% and 0.05% w/v.
Fig 4. Serum calcium decrease in % of initial value for rat sCT instillation studies. (a)
Jejunum, (b) Ileum and (c) Colon. N number as for Fig.5. (○) sCT control, () sCT with 0.1%
w/v TDM, (□) PBS control (n=2 for each intestinal segment).
Fig 5. Serum sCT in rats. (a) i.v. (n=4), (b) Jejunal instillations (n=7 sCT control, n=5 with
0.1% w/v TDM), (c) Ileal instillations (n=5 control, n=6 with TDM), (d) Colonic instillations
(n=6 control, n=5 with TDM). (○) sCT solution, () sCT with 0.1% w/v TDM. The i.v. dose
of sCT: (20µg/kg); instillation dose: (400µg/kg).
21
Fig 6. Histology of rat colonic tissue after 4 h colonic instillations with sCT (400µg/kg). (a)
sCT solution, (b) 0.1% w/v TDM with sCT. Upper panel: H&E; lower panel: alcian blue and
neutral red (Bar=200µm).
22
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Caco-2
E12
25
(a)
***
20
Papp (cm/sec) (x10-6)
***
15
10
5
0
***
15
***
10
5
***
0.
1%
0.
05
%
0.
01
%
C
tl
0.
1%
0.
05
%
0.
01
%
C
tl
0
60
60
(c)
Papp (cm/sec) (x10-7)
***
***
40
20
0
(d)
***
40
***
20
**
(e)
0.
1%
C
tl
0.
1%
0.
05
%
0.
01
%
C
tl
0
0.
05
%
sCT
Papp (cm/sec) (x10-7)
(b)
20
0.
01
%
[3H]-mannitol
Papp (cm/sec) (x10-6)
25
(f)
0.01%
0.05%
0.1%
[3H]-mannitol
1.1
14.1
17.0
sCT
2.1
49.7
55.2
27
0.01%
0.05%
0.1%
[3H]-mannitol
2.1
17.3
23.1
sCT
4.6
46.8
68.3
Fig. 1
28
Papp (cm/sec) (x10-6)
25
(a)
20
***
15
***
10
5
***
0.
1%
0.
05
%
0.
01
%
C
tl
0
Papp (cm/sec) (x10-7)
60
(b)
***
***
40
20
**
0.
1%
0.
05
%
0.
01
%
C
tl
0
0.01%
0.05%
0.1%
[3H]-mannitol
2.3
19.3
24.8
sCT
7.7
92.3
98.1
(c)
Fig. 2
150
***
100
***
***
*** ***
***
*
*
50
***
*** ***
0.
1%
0.
05
%
0.
01
%
on
tr
ol
0
C
%LDH
*
Fig. 3
Jejunum
(a)
(b)
110
% drop in [Ca]
110
100
90
80
70
100
90
80
70
0
50
100
150
200
0
50
Time, min.
100
150
200
Time, min.
Colon
(c)
110
% drop in [Ca]
% drop in [Ca]
Ileum
100
90
80
*
70
0
50
100
150
200
Time, min.
Fig. 4
I.V.
Jejunum
80
(a)
15000
(b)
sCT [pM]
sCT [pM]
60
10000
5000
40
20
0
0
50
100
150
200
0
250
0
50
Time, min.
200
250
200
250
Colon
20000
(c)
(d)
15000
sCT [pM]
300
sCT [pM]
150
Time, min.
Ileum
400
100
200
10000
5000
100
0
0
0
50
100
150
Time, min.
200
250
0
50
100
150
Time, min.
Fig. 5
(a)
(b)
Fig. 6
33