Evaluation of alkylmaltosides as intestinal permeation enhancers: Comparison between rat intestinal mucosal sheets and Caco-2 monolayers

European Journal of Pharmaceutical Sciences 47 (2012) 701–712

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Evaluation of alkylmaltosides as intestinal permeation enhancers: Comparison between rat intestinal mucosal sheets and Caco-2 monolayers Signe Beck Petersen a,b, Gavin Nolan a, Sam Maher a, Ulrik Lytt Rahbek b, Mette Guldbrandt b, David J. Brayden a,⇑ a b

UCD School of Veterinary Science and UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Novo Nordisk, ADME Department, Novo Nordisk Park, 2860 Måløv, Denmark

a r t i c l e

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Article history: Received 17 June 2012 Received in revised form 8 August 2012 Accepted 8 August 2012 Available online 23 August 2012 Keywords: Tetradecyl maltoside Dodecyl maltoside Intestinal permeation enhancers Caco-2 monolayers Intestinal epithelia Oral peptide delivery

a b s t r a c t Alkylmaltosides are a class of non-ionic surfactant currently in clinical trials to improve nasal permeation of peptide drugs, however few studies have detailed their potential effects on intestinal permeation enhancement. Tetradecyl maltoside (TDM, C14), was examined in Caco-2 monolayers and in isolated rat jejunal and colonic mucosae mounted in Ussing chambers. Dodecyl maltoside (DDM, C12) was examined in mucosae. Parameters measured included critical micelle concentration (CMC), transepithelial electrical resistance (TEER), and apparent permeability coefficients (Papp) of paracellular and transcellular flux markers. TDM and DDM decreased TEER and increased the Papp of [14C]-mannitol and FD-4 across Caco-2 monolayers and colonic mucosae in the concentration range of 0.01–0.1% w/v, concentrations much higher than the CMC. Remarkably, neither agent had any effect on the TEER or fluxes of jejunal mucosae. Histopathology, cell death assays (MTT and LDH) and sub-lethal high content cytotoxicity analyses (HCA) were carried out with TDM. Exposure of colonic mucosae to high concentrations of TDM had no major effects on gross histology and ion transport function was retained. In Caco-2, HCA data at sublethal concentrations of TDM was consistent with the action of a mild non-ionic surfactant. In conclusion, alkylmaltosides are effective non-toxic permeation enhancers in isolated colonic tissue and their inclusion in oral peptide formulations directed to that intestinal region warrants further study. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Delivery of protein and peptides via the oral route is very attractive due to the likely improvement in patient compliance compared to injectable delivery. In addition, patenting for a new chemical entity may be extended using enabling oral formulations and the high costs associated with sterile manufacturing may be offset. The pharmacodynamic argument for oral peptide delivery is that the delivery via the intestine may be more physiological than a bolus injection and there are benefits in targeting the liver in the case of molecules aimed at treating type II diabetes including insulin and glucagon-like Peptide-1 agonists. Oral delivery of most protein and peptide drugs is however, limited by poor bioavailability due to susceptibility to peptidases and low intestinal epithelial permeation (Aungst, 2012). One approach to overcome the latter is the use of absorption enhancers co-administered with the protein or peptides in coated matrix tablets (Walsh et al., 2011a). Another is to construct nanoparticle formats to ensure co-release of a protected peptide along with an enhancer in high concentrations close to the intestinal wall (Makhlof et al., 2011). Oral enhancers to ⇑ Corresponding author. Tel.: +353 1 7166013; fax: +353 1 7166104. E-mail address: [email protected] (D.J. Brayden). 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.08.010

enable an increase in peptide and macromolecule bioavailability include sodium caprate (C10), acyl carnitines, bile salts and acylated amino acids; incorporating them in formulations has led to some encouraging clinical trial data for calcitonin, insulin and octreotide (Aungst, 2012; Maher and Brayden, 2012). A recurring issue for current enhancers however, is the large intra-subject variability at the relatively low extent of oral bioavailability detected, which limits current technologies to payloads that are cheap, potent and have wide safety margins. A considerable interest therefore exists in leveraging materials with a good human safety record that have already displayed clinical potential by other routes in order to examine their potential for intestinal permeation enhancement. Tetradecyl maltoside (TDM) and dodecyl maltoside (DDM) are non-ionic surfactants comprising 14 and 12 carbon alkyl chains respectively, which are being investigated as nasal peptide enhancers. Aegis Therapeutics (USA) has recently advanced its IntravailÒ enhancement system comprising TDM and DDM into nasal clinical trials for a parathyroid hormone analogue (Illum, 2012). Their mild non-denaturing nature, rapid effectiveness at low concentrations and low toxicity has led to their acceptance as ‘generally regarded as safe’ (GRAS) in a range of product applications: these are key initial properties sought in promising epithelial permeation candidate enhancers (Maggio, 2006; Arnold et al., 2010). In preclinical

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studies, ad-mixed solutions of TDM significantly improved nasal, ocular and pulmonary delivery of calcitonin and insulin (Ahsan et al., 2001, 2003), and efficacy was related to alkyl chain length in the case of nasal and ocular peptide delivery (Ahsan et al., 2001; Pillion et al., 2002). A rat nasal study showed that FITC-labelled insulin was internalised in epithelia exposed to 0.125% w/ v TDM, and peptide was present in endocytotic vesicles in response to perturbation of the cell membrane (Arnold et al., 2004). Another nasal study revealed altered morphology of the nasal tissue 15 min after exposure to 0.125% TDM, but no cell death; the tissue displayed restored morphology after 2–4 h recovery (Arnold et al., 2010). An increase of [14C]-mannitol by TDM on 16HBE14o human bronchial epithelial cells and C2BBe1 human colonic monolayers reflected tight junction opening and increased paracellular permeability (Ahsan et al., 2003; Yang et al., 2005). From these studies, the mechanism through which alkylmaltosides enhance transepithelial transport appears multi-modal: disruption of tight junctions along with fluidization of the epithelial membrane leading to increased paracellular and transcellular transport. Alkylmaltosides have been less studied as potential oral permeation enhancers given the focus on their development for advanced nasal peptide formulations. Permeation of insulin across rat buccal tissue in the presence of DDM seemed to be more efficacious than nasal or rectal tissue (Aungst, 1994), but a relationship between chain length, critical micellar concentration (CMC) and permeation enhancement potential was not apparent. Apical addition of TDM (0.06, 0.12, 0.25% w/v) to C2BBe1 monolayers rapidly increased enoxaparin (low molecular weight heparin) permeability, which translated to a 2.5-fold increase in oral bioavailability in rats following oral gavage at the highest concentration (Yang et al., 2005), one that proved effective in nasal delivery (Arnold et al., 2004; Pillion et al., 2002). IntravailÒ also increased the oral bioavailability of octreotide and the leptin analogue, ([D-Leu-4]OB3) following oral gavage in mice, with a relative oral bioavailability of 4% in each case (Maggio and Grasso, 2011; Lee et al., 2010). Since TDM-induced permeation enhancement involves perturbation of the epithelial cell membrane, potential toxicity of TDM by the oral route requires further investigation. Ideally, permeation enhancers should cause a significant, rapid, temporary reversible enhancement of epithelial permeation without causing significant damage (Maher et al., 2008). Importantly, if cytotoxicity is observed, the tissue should have the capacity to repair and regenerate (Aungst, 2012). The aim of this study was therefore to further elucidate the capacity of alkylmaltosides for regional intestinal permeation enhancement, their mechanism of action, as well as cytotoxic potential. Using TDM as a prototype, we compared fluxes and histology from isolated rat jejunal and colonic mucosae mounted in Ussing chambers with Caco-2 monolayers. Additional data was obtained with DDM in tissue mucoase for comparison with TDM. To assess detailed sub-lethal damage to intestinal epithelia, TDM was evaluated in Caco-2 cells by the highly sensitive quantitative method of high content analysis (HCA), which measures contemporaneous changes in multiple intracellular parameters in live cells over time (Rawlinson et al., 2010). 2. Materials and methods All materials were from Sigma–Aldrich unless otherwise stated.

platinium plate method was used to measure CMC (Benincasa et al., 2004) and the temperatures of the buffers were set to 37 °C in the 50 ml bath. The surface tension was measured for 11 different concentrations of TDM over a range of 2  106– 1  103% w/v (i.e. 0.04–21 lM). The CMC was determined as no further reduction in surface tension was observed (n = 3). 2.2. Caco-2 cell monolayers Caco-2 cells (p56-63) were grown in Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (2 mM), 1% nonessential amino acids, penicillin (100U)/streptomycin (100 lg/ml) and 10% foetal bovine serum (Gibco, Biosciences Ireland) on 75 cm2 tissue culture flasks at 95% O2/5% CO2 at 37 °C in a humidified environment. Cells were split at 80–90% confluence. Caco-2 cells (p56-61) were seeded at a density of 3  105 cells/well on 12 mm TranswellÒ filters (polycarbonate, pore size 0.4 lm) (Corning Costar Corp., USA) and grown for 21 days in DMEM for transport experiments (Hubatsch et al., 2007). Transepithelial electrical resistance (TEER, X cm2) was measured across the monolayers using an EVOMÒ voltohmmeter with a chopstick-type electrode (EVOMÒ, WPI, UK). TEER measurements were made prior to transport study and then every 20 min thereafter. 2.3. Rat intestinal tissue mucosae: dissection and electrophysiology Studies were carried out in accordance with the UCD Animal Research Ethics Committee policy, ‘Regarding the use of post mortem animal tissue in research and research (2007) (www.ucd.i.e./ researchethics/pdf/arec_post_mortem_tissue_policy.pdf) as well as in adherence with the ‘‘Principles of Laboratory Animal Care,’’ (NIH Publication #85-23, revised in 1985). Male Wister rats (250–300 g) (Charles River, UK) were euthanized by stunning and cervical dislocation. Colon and jejunum were removed, opened along the mesenteric border and rinsed in warm oxygenated Krebs–Henseleit buffer (KH) according to previous methods (Cuthbert and Margolius, 1982). Ten to fifteen cm of jejunum was excised at a point 2–3 cm distal to the caecum and directly above the ileum. Tissue was pinned with the mucosal side down on a dissection board to expose the external muscularis, which was carefully removed with a size 5 forceps. The tissue was then mounted in Ussing chambers with a circular window area of 0.63 cm2, bathed bilaterally with 5 ml KH and continuously gassed with 95% CO2/5% O2 at pH 7.4 maintained at 37 °C. The transepithelial potential difference (PD, mV) and short circuit current (Isc, lA) were measured across the colonic and small intestinal tissue. The tissue was voltage clamped to zero for 30 s and switched to open circuit configuration for 3 s by an automatic voltage clamp (EVC4000 amplifier) and Pro-4 timer (both WPI, UK). Analogue data was digitized with a Mac PowerlabÒ data acquisition unit and analysed with ChartÒ software (AD instruments, UK). Following an equilibration period of 45 min, PD and Isc were measured and TEER was calculated at regular time points from 0 to 120 min using Ohm’s law. The acceptable cut-off TEER values for rat colonic tissue and small intestinal tissue were 70 O cm2 and 30 O cm2 respectively, tissues with lower values were not used (Ungell et al., 1998). The capacity of the tissue to generate an inward Isc response to basolateral addition of the cholinomimetic, carbachol (CCh, 0.1– 10 lM), was used as a measure of retained epithelial ion transport secretory function at the experimental endpoint.

2.1. Critical micelle concentration (CMC) 2.4. Permeability of [14C]-mannitol, FD-4 and [3H]-propranolol The CMC of TDM was measured in the three different buffers; Krebs–Henseleit buffer (KH), Hank’s Balanced Salt Solution (HBSS, Gibco, Biosciences Ireland) with 25 mM HEPES (Gibco) and 11 mM glucose, as well as PBS. A Kruss K12 tensiometer (Bristol, UK),

Transport of the paracellular flux markers, [14C]-mannitol (Perkin Elmer, USA) and FITC-Dextran 4000 (FD-4), was examined across mucosae and Caco-2 cell monolayers. The transport buffer

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for the Caco-2 cell monolayers consisted of HBSS, 25 mM HEPES and 11 mM glucose. DMEM media was replaced with HBSS transport buffer and equilibrated for at least 30 min on both sides of the monolayers. At time zero, the apical-side HBSS (0.55 ml) was replaced by test solution containing [14C]-mannitol and FD-4 in HBSS transport buffer. [14C]-mannitol (0.1 lCi/ml), FD-4 (Caco-2; 250 lg/ml, Ussing; 2.5 mg/ml) together with TDM (0.01, 0.03, 0.05, 0.08 and 0.1% w/v, equivalent to 0.186, 0.557, 0.928, 1.49 and 1.86 mM) were then added to the apical chamber. Basolateral samples were taken every 20 min for 120 min, while an apical sample was taken at time zero in order to calculate the apparent permeability coefficient (Papp). Basolateral samples were replaced with fresh HBSS buffer (Caco-2) or KH (mucosae). [14C]-mannitol samples were mixed with scintillation fluid and assayed in a scintillation analyzer (Packard Tricarb 2900 TR). Fluorescence of FD-4 samples were measured in a spectrofluorimeter (MD Spectramax Gemini) with kex/kem of 490/525 nm. The Papp for each marker was calculated according to the following equation:

Papp ¼ ðdQ =dtÞð1=A  C 0 Þ where dQ/dt is the flux, A is the surface area exposed (Caco-2; 1.12 cm2, Ussing; 0.63 cm2) and C0 is the starting concentration of flux marker on the apical side. The Ussing chamber studies with DDM used the following concentrations; 0.01, 0.05 and 0.1% w/v (equivalent to 0.198, 0.979 and 1.96 mM, respectively) and the Papp values of [14C]-mannitol and FD4 across rat colon and distal jejunum were assessed. Transport of the transcellular probe [3H]propranolol (Perkin Elmer, USA) was examined in rat colonic tissue only, in both apical-to-basolateral and reverse direction. For the former, [3H]-propranolol was added apically (0.1 lCi/ml) with TDM (0.1% w/v); unlabelled propranolol (100 lM) was added both apically and basolaterally in order to reduce adsorption of the radiolabelled compounds to chamber components (Dube et al., 2010). For the reverse direction, [3H]-propranolol was added basolaterally to colonic tissue at time zero and sampling was carried out from the apical side. Test samples were mixed with scintillation fluid and read in a scintillation analyzer (Packard Tricarb 2900 TR). 2.5. MTT assay Cytotoxic potential following incubation of exponentially grown Caco-2 cells was elucidated by an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as previously described (Mosmann, 1983). Briefly, Caco-2 cells (p57-63) were seeded at a density of 2  104 cells/well on 96 well plates and incubated overnight. Fresh DMEM was applied and TDM (0.005–1% w/ v) or TritonÒ-X-100 (0.1% v/v) were added and incubated for 1, 2, 12 and 24 h. Fresh DMEM and MTT (0.5 mg/ml in PBS) were then added to the wells and incubated at 37 °C at 95% O2/5% CO2 in a humidified atmosphere for 4 h. The resulting media was removed and DMSO was added to the wells and the plates were shaken for 5 min at 500–900 rpm to solubilise the formazan crystals after which the absorbance was read in a multi-plate reader at 550 nm with a reference wavelength set to 690 nm. Percent viable cells were measured relative to untreated media control. Experiments were repeated on three different occasions using multiple comparisons on each plate. 2.6. High content analysis (HCA) Cytotoxicity of TDM on Caco-2 cells was examined using a HCA protocol that has been validated as a sensitive and selective in vitro assessment of hepatotoxicity (O’Brien et al., 2006) and recently applied to Caco-2 cells (Rawlinson et al., 2010). Caco-2 cells (p53-63) were seeded at a density of 6  103 cells/well, on F96 MicroWell™

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plates (Nunclon™ D Surface, Collagen I and Poly-D-Lysine, Nunc) overnight to adhere. Spent DMEM media was replaced with media containing increasing concentrations of TDM (0.005–1% w/v; 0.09– 18.6 mM) in triplicate wells for 30, 60 min and 24 h and incubated at 37 °C and 95% O2/5% CO2. Positive controls including carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP) (a mitochondrial membrane uncoupler, 100 lM), ionomycin (a calcium ionophore, 10 lM), and TritonÒ-X-100 (non-ionic detergent, 0.05%) were included. Before data acquisition, each well was incubated with dye cocktail mixture containing a final concentration of Hoechst 33342 (0.8 lM) (measures cell number (CN), nuclear intensity (NI) and nuclear area (NA)), Fluo-4 AM (1 lM) (measures intracellular calcium (IC)), TOTOÒ-3 iodide (1 lM) (measures plasma membrane potential (PMP)) and tetramethyl rhodamine methyl ester (TMRM; 20nM) (measures mitochondrial membrane potential (MMP)) for 37 °C, protected from light. The positive controls were added after 50 min incubation with the dye cocktail. Data was captured for each plate at 10  (cell number) and 20  (other parameters) objective magnification in the selected excitation and emission wavelengths of Hoechst 33342 (Ex/Em 360/460 nm), Fluo-4 AM (Ex/Em 480/535 nm), TMRM (Ex/Em 535/600 nm) and TOTOÒ-3 iodide (Ex/Em 620/700 nm). The exposure times were optimised for the Caco-2 cell set-up, Hoechst (700 ms), Fluo-4 AM (250 ms), TMRM (550 ms) and TOTOÒ-3 iodide (400 ms). For each well six random field-of-view images were acquired to examine each parameter except for cell number, where ten fields were used. Data for CN, NA, NI, MMP, PMP and IC were acquired on an InCellÒ 1000 High Content Analyzer (GE Healthcare, UK) which analyses epifluorescence of individual cell events. All experiments were carried out in triplicate on three independent occasions. 2.7. Tissue lactate dehydrogenase release (LDH) Test aliquots (200 ll) for analysis of LDH release from rat colonic tissue exposed to different concentrations of TDM were taken from the apical chamber at 0, 60 and 120 min. LDH assay was performed according to the manufacturer’s instructions. (TOX-7, Sigma) and quantification was carried out as previously described (Uchiyama et al., 1999). Percentage LDH release was measured relative to Triton-X-100 (10% v/v). 2.8. Histology For experiments with colonic mucosae exposed to TDM and DDM (0.01–0.1% w/v), tissues were removed after 120 exposures in Ussing chambers and fixed in 10% (v/v) buffered formalin for at least 48 h. Tissues were processed and stained with haematoxylin and eosin (H&E), alcian blue and neutral red. The slides were scanned in 20  scanning mode on a NanoZoomer 2.0-HT light microscopy (Hamamatsu) and viewed at 10. For transmission electron microscopy (TEM), TDM-exposed colonic mucosae were removed from the chambers after 40 min and fixed overnight in glutaraldehyde (2.5% (v/v)), then post-fixed in osmium tetroxide (1% w/v), dehydrated in ethanol (70–100% (v/v)) and embedded in epoxy resin. Light and ultra-sections were cut with a histology (500 nm) and ultra (70 nm) diamond knife, respectively (Diatome, Switzerland). Ultra-thin sections were mounted on 200 mesh copper grids, contrasted with uranyl acetate and lead citrate and examined using an FEI Tecnai 12 transmission electron microscope. 2.9. Data analysis Statistical analysis was carried out using Prism-5Ò software (GraphPad, San Diego, USA) using two-tailed unpaired Students t-test for all experiments except for MTT and HCA data where

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one-way ANOVA was used. Results are presented as the mean ± standard error of the mean (SEM). A significant difference was considered if P < 0.05.

2.0  107 cm/s for [14C]-mannitol and 2.3  108 cm/s for FD4 in agreement with previous data (Hubatsch et al., 2007; Kamm et al., 2000).

3. Results

3.3. TDM reduces TEER and increases permeability across rat colonic tissue mucosae

3.1. CMC measurements of TDM

The average basal TEER value 116 ± 4 O cm2 for all colonic tissue (n = 77), which was above the acceptable cut-off value (Tanaka et al., 1995; Ungell et al., 1998). Similar to Caco-2 monolayers, apical addition of TDM caused a significant concentration-dependent TEER decrease across colonic mucosae, with the maximum decrease observed at 40 min (Fig. 2a). TEER values decreased to 27% of the basal value in the presence of 0.08 and 0.1% w/v TDM. Control tissue exhibited a gradual decrease over 120 min to 69%, whereas the TDM concentrations 0.03–0.1% w/v induced a further and significant decrease from 5 to 120 min compared to control. The lowest TDM concentration of 0.01% w/v also resulted in a significant TEER decrease between 15 and 100 min compared to controls. In parallel, there was a significant increase in the Papp values for [14C]-mannitol and FD4 in the presence of TDM (0.03–0.1% w/v) (Fig. 2b). The mean Papp value of [14C]-mannitol and FD4 for untreated colonic tissue was 1.8  106 and 4.7  107 cm/s, respectively, within the normal range described previously (Maher et al., 2009a; Nakamura et al., 2003). Control jejunum exhibited a gradual decrease over 120 min to 76.2% (Fig. 2c). Basal TEER values were 54 ± 2 O cm2 for jejunal tissue used (n = 78), in correspondence with previous data (Tanaka et al., 1995; Tsutsumi et al., 2003). Surprisingly, decreases in TEER were not observed for jejunal tissue exposed TDM even at concentrations as high as 0.1% w/v. Likewise, no significant increase in Papp values was seen of [14C]-mannitol and FD4 on distal jejunal tissue in the presence of TDM (Fig. 2d). The mean Papp values of [14C]-mannitol and FD4 on jejunal tissue were 4.8  106 and 7.2  107 cm/s, respectively, within the range of previous reports (Nakamura et al., 2003; Tsutsumi et al., 2003). The enhancement ratios across colon increased in a concentration-dependent manner (Fig. 2e), although the enhancement effect of TDM was not as high as that observed across Caco-2 monolayers.

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Basal TEER for Caco-2 monolayers was 2407 ± 43 O cm2 monolayers (n = 30), within the range of TEER values typically reported by this lab (Brayden et al., 2012). Monolayers were exposed to apical addition of TDM for 120 min in a concentration range 0.01–0.1% w/v and TEER values assessed (Fig. 1a). All concentrations of TDM decreased the TEER values to less than 10% of the initial value. Following incubations, TDM was removed and fresh DMEM introduced; monolayers were then incubated for 24 h to assess TEER recovery. Monolayers regained the initial TEER value at the lowest concentration of 0.01% w/v , while monolayers exposed to 0.03% w/ v yielded 50% partial restoration of initial TEER values. Exposure to 0.05–0.01% w/v concentrations of TDM for 120 min did not result in TEER recovery, reflecting an irreversible deterioration of the monolayer barrier. The Papp values for [14C]-mannitol and across monolayers statistically increased in a concentrationdependent manner in the presence of TDM (0.01–0.1% w/v) (Fig. 1b) and these were reflected by large increases in enhancement ratios (Fig. 1c). The mean Papp for controls monolayers were

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3.2. TDM reduces TEER and increases permeability across Caco-2 cells monolayers

%

CMC values were obtained for TDM in the three buffers at 37 °C that were used for the transport studies: PBS (13.0 lM), HBSS (13.2 lM) and KH (12.6 lM) (equivalent to 0.0007 w/v), which correspond well with values previously determined in water (Ericsson et al., 2005; Mustafa et al., 2004). There was therefore no evidence that different physiological buffers altered the CMC values of TDM. Importantly, the CMC values were more than 10fold lower than the concentrations required to influence TEER and permeability in the intestinal models.

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Fig. 1. TDM decreases TEER and increases fluxes [14C]-mannitol and FD-4 across Caco-2 monolayers. (a) % Change in TEER expressed as % of TEER at t = 0, followed by recovery in fresh buffer. n = 12 (Control) and n = 6 (test groups). (b) Papp for [14C]-mannitol and FD4 . Symbols: Control (s), 0.01% TDM (j), 0.03% (N), 0.05% (.), 0.08% () and 0.1% (d). n = 6 (Control) and n = 3 (test). (C): Enhancement ratios across Caco-2 monolayers in the presence of TDM, P < 0.05, P < 0.01 and P < 0.001 versus untreated controls.

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S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712

Fig. 2. TDM decreases TEER and increases fluxes of [14C]-mannitol and FD-4 across rat colon but not jejunum. (a) % Change in colonic TEER, (n = 29 control, n = 10–11 test). (b) Papp for [14C]-mannitol and FD4 in colon (n = 14–15 control, n = 5 test). (c) % TEER change in jejunum (n = 26 control, n = 10–11 test groups). (d) Papp for [14C]-mannitol and FD4 in jejunum, (n = 10–16 control, n = 5–6 test group). Symbols: control (s), 0.01% TDM (j), 0.03% (N), 0.05% (.), 0.08% () and 0.1% (d). (e): Enhancement ratios in colon in the presence of TDM, P < 0.05, P < 0.01 and P < 0.001 versus untreated controls.

Addition of DDM to rat colonic tissue in the same concentration range also induced a concentration-dependent TEER decrease similar to TDM (Fig. 3a). Likewise, DDM increased colonic Papp values of [14C]-mannitol and FD4 in a concentration-dependent fashion (Fig. 3b). Similar to TDM, DDM caused no alteration in jejunal TEER and Papp values (Fig. 3c and d). Consistent with a minimal effect on Papp value in jejunal mucosae, only the highest concentration of 0.1% w/v DDM gave an increased enhancement ratio of 1.8 in jejunum (data not shown), however while such supra-maximal concentrations may be marginally effective, they are likely to be toxic. No further jejunal studies were carried out for alkylmaltosides on jejunal tissue as they were clearly not effective permeation enhancers in that region. Importantly, while the enhancement ratios of DDM on colonic tissue (Fig. 3e) were somewhat increased compared to TDM at the higher end of the concentration range (Fig. 2e), overall increases were interchangeable and there was no difference in the Papp values of TDM and DDM at equivalent concentrations (% w/v), so the focus of the remaining studies was on TDM in colonic mucosae since the mechanism of action of both agents is likely to be similar.

in the presence of 0.1% w/v TDM. Both apical (A)-to-basolateral (B) and (B)-to-(A) Papp values were obtained. The basal Papp for (A)-to-(B) transport was 2.9  105 cm/s (n = 7) whereas the basal Papp for (B)-to-(A) transport was 2.2  105 cm/s (n = 4), similar to previous reports (Polentarutti et al., 1999), with no statistical significance between them. Apical addition of TritonÒ-X-100 (0.1% v/v) was used as a positive control for facilitating transcellular transport. The Papp of [3H]-propranolol in the presence of TritonÒ-X-100 was increased by 1.7-fold to 4.9  105 cm/s (n = 7) in the (A)-to-(B) direction (P < 0.01). Adding 0.1% w/v TDM apically, however, resulted in an (A)-to-(B) Papp of 2.5  105 cm/s for [3H]propranolol, not different from basal Papp values in either direction. These data suggest that TDM does not facilitate transport of a readily permeable agent via the transcellular route even at high concentrations and therefore acts differently to the strong non-ionic detergent, TritonÒ-X-100. The potential relevance of the transcellular route for TDM cannot however, be discounted from this study alone.

3.4. TDM does not increase flux of a transcellular marker across rat colon

Cytotoxicity of TDM on Caco-2 cells was assessed by MTT assay following incubation with TDM concentrations (0.005–1%) for 1, 2, 12 and 24 h. The number of viable cells decreased as a function of concentration and incubation time (Table 1) and the lowest concentration of 0.005% reduced viability by 15% after only1 h.

Next, we tested whether TDM also facilitated transcellular transport in rat colonic tissue by measuring Papp of [3H]-propranolol

3.5. Assessment of TDM cytotoxicity on Caco-2 cells

50

** 0

0 -50

0

50

100

(e)

DDM (%w/v)

100

10

50

5

0

0

0.01

0.05

0.1

[ C]-mannitol

1.3

6.0

8.5

FD4

2.0

16.4

26.4

14

%

15

C

Time, min

150

1%

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20

(d)

0.

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05 %

(c)

on tr ol

%Initial TEER (drop)

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on

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%

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%

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tr ol

%Initial TEER (drop)

(a)

Papp (cm/sec) (x10-7), FD-4

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Papp (cm/sec) (x10 -7), mannitol

S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712

Papp (cm/sec) (x10-7), mannitol

706

Fig. 3. DDM decreases TEER and increases fluxes of [14C]-mannitol and FD-4 across rat colon but not jejunum. (a) % Change in colonic TEER. (b) Papp for [14C]-mannitol and FD4 across colonic mucosae, (n = 4 control, n = 3–5 test). (c) % Change in jejunal TEER. (d) Papp for [14C]-mannitol and FD4 across jejunal mucosae, (n = 6 control, n = 3–4 test). Symbols: control (s), 0.01% TDM (j), 0.05% (N) and 0.1% (d). (e). Enhancement ratios in colon in the presence of DDM, P < 0.05, P < 0.01 and P < 0.001 versus untreated control.

Exposure to 0.08% reduced viability by >90% at 1 h, similar to 0.1% Triton-X-100. IC50 values for TDM were 0.022% and 0.017% at 1 h and 2 h exposures respectively. Previously, Shah et al. (2004) demonstrated that it took a 10-fold higher concentration of TDM to kill >80% of Caco-2 cells during exposures from 2 to 24 h, perhaps reflecting methodological differences. 3.6. High content analysis for TDM on Caco-2 cells HCA was used to follow both the short-term and long-term sublethal effects of TDM using six toxicity markers in Caco-2 cells. TritonÒ-X-100 and ionomycin increased plasma membrane potential

(PMP) and intracellular calcium (IC) by 2.5-fold and 6.5-fold, respectively, compared with untreated controls following 10 min incubation (P < 0.0001), while FCCP decreased mitochondrial membrane potential (MMP) by 5-fold, also following 10 min of incubation (P < 0.0001), reference data for HCA in Caco-2 cells that corresponds to previous findings (Walsh et al., 2011b). Incubation with TDM had no effect on cell number (CN) at 30 min and 1 h at any concentration, however, it decreased CN at all concentrations after 24 h (Table 2, Fig. 4). Low concentrations significantly influenced nuclear intensity (NI), PMP and MMP. NI was significantly increased at 0.01% w/v, whereas PMP increased significantly with 0.02% w/v (at 30 min), at 0.01% (at 24 h) indicating that TDM

Table 1 MTT assay for TDM on Caco-2 cells. Control

0.005%

0.01%

0.02%

0.03%

0.05%

1h 2h 12 h 24 h

100 ± 2.3 100 ± 2.3 100 ± 2.2 100 ± 2.4

84.3 ± 6.0 85.3 ± 3.2 88.3 ± 6.1 61.3 ± 6.1

77.5 ± 4.6 67.9 ± 4.2 56.1v4.5 20.6 ± 2.4

58.1 ± 3.3 40.5 ± 2.6 7.7 ± 0.8 2.9 ± 0.3

36.3 ± 3.3 20.1 ± 2.3 3.9 ± 0.5 3.8 ± 0.5

22.4 ± 1.6 10.4 ± 1.6 2.9 ± 0.4 3.7 ± 0.3

1h 2h 12 h 24 h

0.08% 5.4 ± 1.1 4.8 ± 0.8 2.7 ± 0.3 2.8 ± 0.4

0.1% 5.7 ± 0.8 4.6 ± 0.4 3.8 ± 0.4 NS 3.4 ± 0.4

0.15% 6.3 ± 0.7 6.5 ± 0.9 2.9 ± 0.5 4.1 ± 0.6

0.5% 6.0 ± 0.6 4.4 ± 0.6 3.7 ± 0.3 3.9 ± 0.5

1% 4.8 ± 1.1 5.2 ± 0.9 3.2 ± 0.5 3.5 ± 0.6

TritonÒ (0.1%) 4.8 ± 0.6 4.1 ± 0.5 4.0 ± 0.2 4.0 ± 0.4

Percentage viable cells relative to media control given as mean ± SEM. TDM concentrations (% w/v) show statistical significance versus media control at each respective time point. P < 0.001 for all groups (n = 3), except for 0.005% TDM at 12 h (NS: not-significant). TritonÒ-X-100 (0.1% v/v) was the positive control.

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S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712 Table 2 Sub-lethal effects of TDM on Caco-2 cells as measured by HCA. CN

NA

NI **

***

0.5 h

NS

0.03 –1%

1h

NS (0.03% (;)*) (0.08% (;)*)

0.05**–1%*** (;) (0.02% (")***)

24 h

0.005–1% (;)***

0.005–1% (;)***

(;)

IC **

0.005 –0.5%

***

(")

MMP *

PMP ***

(;)

0.02–1% (")***

NS (0.01% (")**) (0.5% (")*) (1% (")**)

0.01 –1%

0.005–1% (")***

NS (0.03% (;)**) (0.05% (;)*) (1% (")***)

0.02–1% (;)*** (0.005% (")**)

0.02–1% (")***

0.005–1% (")***

NS (0.005% (")***) (1% (")**)

0.005–1% (;)***

0.01–1% (")***

Concentration range (% w/v) is given where a significant change in the metric are observed. Concentration in brackets gives significance level at that particular concentration. (", ;) = Significant increase or decrease. NS = not significant. Mean of three independent determinations at each concentration and time point. * P < 0.05. ** P < 0.001. *** P < 0.0001.

perturbs the cell membrane at concentrations in the same order as those that reduced viability in Caco-2 cells by measured by MTT assay. A concentration- and time-dependent decrease in MMP was observed and following 24 h incubation of TDM, all tested concentrations showed significance. The MMP curves (Fig. 4), corresponding closely with concentrations that were cytotoxic by MTT assay. The IC curves showed an initial low decrease followed by a gradual increase as function of concentration for the short incubation times, and were only significant for the high concentration. The 24 h incubation with TDM gave significant increase in IC compared to control for the lowest concentration. Hereafter the curve decreased and a significant increase was detected for the highest TDM concentration (Table 2, Fig. 4). In summary, these data suggest that sub-lethal actions of TDM on Caco-2 cells are due to multiple mechanisms ranging from membrane perturbation to intracellular calcium concentration changes, consistent with mild surfactant actions likely to dominate at concentrations above the CMC. 3.7. Assessment of toxicity on rat colonic mucosae: LDH release and CCh-stimulated Isc LDH leakage from the apical side of colonic mucosa was dependent on concentration of TDM after 1 h and 2 h of incubation (Fig. 5a and b). However, only 25% LDH release was seen with 0.1% w/v TDM after 2 h exposure compared to TritonÒ-X-100, which suggests that tissue mucosae are far more robust and can resist the perturbation actions of high concentrations of surfactants without displaying the same degree of cell damage observed in Caco-2 cells. At the termination of colonic TDM exposures at 120 min, an inward Isc induced by carbachol (CCh, 0.1–10 lM), was used to estimate remaining functional capacity of the tissue. Overall, while colonic tissue exposed to TDM (0.01–0.1% w/v) showed a trend in reductions in the CCh-stimulated DIsc compared to untreated tissue, only the 0.03% w/v concentration statistically reduced Isc of 10 lM CCh to D 30 lA cm2, a value still very acceptable. All CCh-stimulated Isc responses were substantial in the presence of TDM and any reductions were not concentration-related (Fig. 5c). This data supports the thesis that TDM does not inhibit colonic tissue secretory capacity at concentrations that enhance permeability and the data is in keeping with the relatively low production of tissue LDH induced. 3.8. Histology and TEM of rat colon exposed to TDM Rat colonic mucosae were assessed by gross histology and by TEM, in the presence and absence of TDM. Tissue was assessed

with TDM (0.01–0.1% w/v) following Ussing chamber transport experiments at 2 h, whereas tissue for TEM was collected after 40 min when the maximum TEER decrease was observed with 0.1% TDM in order to optimise the chances of detecting mucosal alterations. Colonic tissue revealed generally healthy intact intestinal mucosa, with some occasional minor oedema (Fig. 6a). In the presence of TDM, some cell sloughing into the intestinal lumen was observed with concentrations higher than 0.03%. Mild oedema was observed for the highest TDM concentrations compared to the untreated tissue. Some mucous secretion from the goblet cells was also observed in the presence of increasing concentrations of TDM (Fig. 6b–f). Histology of DDM-exposed colonic tissue gave the same pattern as TDM: slight oedema in untreated colonic tissue whereas some cell sloughing and cell detachment was seen in the presence of the highest concentration of DDM. Some mucous secretion from the goblet cells could be seen when 0.1% w/v DDM was added (data not shown). TEM revealed that colonic mucosae exhibited well-defined striated borders consisting of intact microvilli, intact junctional complexes and intracellular content (Fig. 7a and b). Colonic exposure to 0.1% TDM, however, induced some morphological changes at the epithelial cell surface, including detachment of cells and cell sloughing, a shortening of microvilli and less pronounced tight junctions than that of control tissue, (Fig. 7c and d). Overall, the colon histology and TEM data yielded complementary data to the relatively low level of tissue LDH release along with the maintained capacity for ion secretion in the presence of TDM. Taken together, TDM induced some membrane perturbation, but tissue barrier and secretory capacity were still at an acceptable level. 4. Discussion The aim of this study was to elucidate the intestinal permeation-enhancing capacity and mechanism of action of TDM and to detail its effects on cytotoxicity using in vitro intestinal tissue models. For an oral enhancer to show promise, a concentration window needs to be established in which enhancement action is maximal and safety issues are minimal (Aungst, 2012). Both DDM and TDM increase permeability in Caco-2 monolayers and rat colonic mucosae, but not jejunum. The decrease in TEER caused by alkylmaltosides confirms previous findings in a range of epithelial cell lines (Eley and Triumalashetty, 2001; Shah et al., 2004; Yang et al., 2005). This data also supports that of Yang et al. (2005), where TEER was reduced by TDM in C2BBe1 monolayers and oral gavage of enoxaparin led to a small increase in relative oral bioavailability in rats. It is likely that the significant enhancement observed in Caco-2 monolayers is due to transport via tight

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0.5 h

1h

24 h

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CN 0

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Mean Fluorescence Intensity

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0 0.01

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PMP

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0

Log conc. [%w/v] Fig. 4. Effects of TDM on HCA parameters in Caco-2 cells. Rows are cell number (CN), nuclear area (NA), nuclear intensity (NI), intracellular calcium (IC), plasma membrane permeability (PMP) and mitochondrial membrane potential (MMP). Columns are incubation times with TDM: 30 min, 60 min and 24 h. Each value represents the mean fluorescence intensity ± SEM, where the negative control is set to 100%.

junctions as well as across the cell membrane. The membrane is perturbated by 0.02% w/v TDM according to the HCA data for PMP. Increased [14C]-mannitol and FD-4 fluxes are widely accepted as markers for exclusively paracellular transport, but this conclusion is only valid if there is confidence that the epithelial barrier remains fully intact as determined by reversibility, adequate histology matched with low cytotoxicity, and if non-specific surfactant effects on membranes can be discounted. Few of these

criteria can be applied to alkylmaltosides in either intestinal tissue transport model. Moreover, micelle and vesicle formation at low concentrations above the CMC are consistent with both reversible and irreversible alteration to membrane structure. [3H]-propranolol is commonly used as a passive lipophilic transcellular marker for Caco-2 monolayers (Artursson, 1990), although it is also a P-glycoprotein (P-gp) substrate in these cells (D’Emanuele et al., 2004). We did not detect a polarised

S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712

150

(a)

%LDH

*** 100

* 50

* *

150

Tr ito nX

0. 1%

C

on tr ol 0. 01 % 0. 03 % 0. 05 % 0. 08 %

0

(b)

%LDH

*** 100

**

50

*

*

*

C

on tr ol 0. 01 % 0. 03 % 0. 05 % 0. 08 %

0. 1% Tr ito nX

0

ΔISC (μA/cm2)

125

(c)

100 75 50 25 0 1.0×10 -07

1.0×10 -06

1.0×10 -05

Log [CCh] M Fig. 5. (a) % LDH release upon colonic exposure to TDM at 60 min compared to TritonÒ-X-100 (Triton X, 10% v/v). (b) %LDH release at 120 min. (n = 15 control, n = 3–5 test). P < 0.05 and P < 0.01 versus untreated controls. (c) TDM effects on CCh-stimulated rat colonic DIsc. (n = 27 control, n = 9–10 test). P < 0.05 for 10 lM CCh in presence 0.03% TDM versus absence. Symbols: Control (s), 0.01% TDM (j), 0.03% (N), 0.05% (.), 0.08% () and 0.1% ().

[3H]-propranolol flux in rat colonic mucosae, even though P-gp gene expression increases distally from rat jejunum to colon (Cao et al., 2005). These authors have argued that P-gp plays a minimal role in reducing intestinal permeation of highly permeable molecules, and this is consistent with the very high absorptive and secretory Papp values found for [3H]-propranolol. Furthermore, in rat in situ instillations, the potent P-gp substrate rhodamine-123 was not subject to rat colonic P-gp mediated efflux when corrected for water transport in that region as its absorptive flux was not increased by the P-gp inhibitor, verapamil (Iida et al., 2005). In sum, high P-gp expression in rat colon does not necessarily translate to polarised flux for a well-absorbed soluble P-gp substrate and thus [3H]-propranolol would appear to be a valid marker for passive transcellular permeation. Why did TritonÒ-X-100 increase the Papp of [3H]-propranolol whereas TDM did not? The physicochemical properties of TritonÒ-X-100 are significantly different from TDM: both are non-ionic surfactants, but their actions vary depending on the hydrophilic–lipophilic balance (HLB) and the CMC. Therefore, Triton-Ò-X-100 solubilises plasma membranes at relatively

709

low concentrations, thereby allowing additional transcellular flux associated with gross toxicological perturbation. In contrast, TDM is a mild surfactant; even though it causes a reduction in the PMP of Caco-2 cells, formation of endocytotic vesicles in nasal epithelia (Arnold et al., 2004), and causes some histological changes to colonic tissue, our interpretation is that while these effects are not enough to further impact on the flux of a highly permeable molecule like propranolol, they may still contribute to the transcellular flux of poorly permeable substances. Neither DDM nor TDM induced permeation enhancement in jejunal mucosae compared to colon. In colonic mucosae, we have examined the capacity of other permeation enhancers to decrease TEER and to increase the Papp of [14C]-mannitol and FD-4 and find, for example, that 10 mM C10 gives similar TEER decreases and flux increases as 0.08–0.1% TDM (Maher et al., 2009a). This would suggest that alkylmaltosides are quite efficacious in colon since C10 and other medium chain fatty acids are regarded as amongst the most effective enhancers in that region. The increased sensitivity of the colonic mucosa over small intestinal epithelia to many permeation enhancers has been documented (Maroni et al., 2012). For example, a rat in situ instillation study with DDM also showed large permeation enhancement effects in the order: rectum > colon > ileum and jejunum, and none in jejunum (Murakami et al., 1992). In addition, chitosan capsules containing DDM and tested in a rat in situ intestinal loop study confirmed the greatest effect in colon, a moderate effect in ileum and only a small effect in jejunum (Fetih et al., 2005). The regional differences apparent in Ussing chamber studies is likely to be due to increased susceptibility of the apical membrane to surfactants since the small intestine must be able to withstand regular exposure to bile salts and mixed micelles. The difference may be due a more rigid small intestinal apical membrane with less fluidity compared to colon (reviewed in Maroni et al. (2012). This region specific finding shows the importance of evaluating intestinal absorption enhancers in whole tissue models as well as in cell models, since regional differences cannot be detected in Caco-2 monolayers (Cano-Cebrian et al., 2005) or using in oral PK/PD gavage studies. The colonic specificity of the alkylglycosides therefore needs to be taken into account when attempting to formulate alkylmaltosides for oral peptide absorption. Lower fluid volumes, longer residence time and reduced quantities of peptidases are some of the potentially beneficial differences between colon and small intestine. On the contrary, intra-subject variation in intestinal transit times as well as unpredictable dissolution patterns are major challenges in reproducibly achieving colonic peptide delivery with alkylmaltosides. In addition, while temporary reversible permeation enhancement in small intestinal regions has not led to inadvertent pathogen absorption to date in clinical trials with agents that are known to work there (e.g. sodium caprate, acyl carnitines), permeation enhancement in the colon raises separate toxicity issues due to the presence of high levels of bacteria, some of which may be pathogenic. Indeed, increased human colonic epithelial permeability is present in mucosae from ulcerative colitis patients and may contribute to the aetiology (Nejdfors et al., 1998). Exposure to low concentrations of TDM (0.01%) allowed complete recovery of TEER after 24 h in Caco-2 cells whereas higher concentrations did not, which suggests that the line is particularly prone to damage compared to intact tissue. There was considerable overlap in the concentrations of TDM that increased permeability and caused damage, so if we were to rely solely on Caco-2 cell evidence, there would be little encouragement that the damage and permeability effects can be dissociated (Cano-Cebrian et al., 2005). On the other hand, better dissociation was seen in colonic mucosae even if the enhancement observed was not as high as in Caco-2 monolayers, perhaps reflecting the robustness of intestinal tissue found in vivo. The toxicity assays (cell MTT, tissue LDH and

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S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 6. Histology of isolated rat colonic tissue mounted in Ussing chambers and exposed to TDM for 120 min. (a) Control, (b) 0.01%, (c) 0.03%, (d) 0.05%, (e) 0.08%, (f) 0.1%. Upper panels: H&E, lower panels: Alcian blue and neutral red (bar = 250 lm).

Fig. 7. TEM of isolated rat colonic tissue mounted in Ussing chambers exposed to 0.1% TDM for 40 min. (a and b) Control, (c and d) TDM 0.1%. Upper panels; bar = 1 lm. Lower panels; bar = 500 nm.

carbachol-stimulated Isc) showed that high concentrations of TDM resulted in varying degrees of local toxicity and such effects were model-dependent. The cell death assays clearly show that Caco-2

monolayers are more sensitive to the cytotoxic effects of permeation enhancers than isolated tissue. Indeed, some enhancers that are quite cytotoxic in Caco-2 cell studies cause relatively low

S.B. Petersen et al. / European Journal of Pharmaceutical Sciences 47 (2012) 701–712

toxicity when dosed to rats at doses required for absorption enhancement (Aungst, 2000). Histology of TDM-treated colonic mucosae revealed some cell sloughing, detachment of cells and shortening of villi at high concentrations, but the barrier was still intact and functional. DDM also induced mild oedema and goblet cell vacuolization in rectal tissue (Murakami et al., 1992), consistent with its surfactant properties at concentrations above the CMC. HCA using Caco-2 cells has previously been reported by us for mellitin, structural mellitin analogues and the methacrylate-based polymer, pDMAEMA (Rawlinson et al., 2010; Walsh et al., 2011b). Mellitin induced toxicity in Caco-2 cells after long-term incubations by dissipating MMP and elevating IC and PMP, whereas pDMAEMA induced apoptosis in Caco-2 cells. In the current study, short incubation times were more likely to mimic the exposure times likely to be relevant for formulations moving along the intestine in vivo and the concentrations used in HCA reflected those used in the flux studies. The decrease in NA after 24 h incubation with TDM is suggestive of apoptosis (Van and Van Den Broeck, 2002), but it did not occur during short incubation times. Cell death after 24 h of incubation was clearly observed, and may be related to apoptosis. The increase of PMP by short exposures to 0.02% TDM and above confirms that TDM induces membrane perturbation at concentrations similar to those that reduce TEER and increase fluxes across monolayers. Furthermore, the decreases seen in MMP at 0.01% w/v in 30 min correlate with the MTT data; MMP changes will precede the mitochondrial disruption measured by MTT. The increase in NI seen at low concentrations could be due to an excess of dye permeating the cell at low concentrations; at higher concentrations the plasma membrane and nuclear membranes leak and the intensity therefore decreases. The plasma membrane damage to Caco-2 cells caused by TDM has been detected by others using cell death assays, but damage to the nuclear membrane was not seen using propidium iodide staining even after 24 h incubation (Shah et al., 2004). An increase in IC may also be a predictor of apoptosis (Yu et al., 2009). However, an increase of IC was only seen at 1% TDM, and it could be related to either lysis of intracellular stores releasing calcium or influx of extracellular calcium. Overall, TDM does not appear to elevate of intracellular calcium at permeating-enhancing concentrations in contrast to other enhancers including C10 (Maher et al., 2009a,b). To be able to fully evaluate the potential toxicity of TDM, HCA evaluation on human HepG2 liver cells would be informative as TDM could be compared to a massive dataset including more than 200 drugs where the read-outs were highly correlated with in vivo systemic toxicity (O’Brien et al., 2006). Overall, the MTT and HCA data showed that TDM induced cytotoxicity and potential apoptosis of Caco-2 cells when using moderate-to-high concentrations, however both the death and sub-lethal assays used non-polarised cells grown on plastic wells and the histology and secretory function of colonic mucosae did not reflect equivalent toxicity at similar concentrations and exposure times. Since cells are more sensitive to cytotoxic effects of permeation enhancers than whole tissue, this difference will be exacerbated due to the capacity of the intestinal tissue epithelium to repair within hours (Cano-Cebrian et al., 2005; Maher et al., 2009b). 5. Conclusion TDM and DDM are effective in vitro colonic epithelial permeation enhancers at low concentrations working via paracellular and transcellular mechanisms. There was little effect on jejunal permeability, providing further evidence of the increased sensitivity of the colonic plasma membrane to the actions of surfactants. Cytotoxicity was more an issue for exposed Caco-2 monolayers than isolated intestinal mucosae, which display the robustness

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expected in in vivo comparisons. Cytotoxicity was more an issue for exposed Caco-2 monolayers than mucosae, which display robustness expected in in vivo comparisons. Caco-2 cell death was preceded in a concentration- and time-dependent manner by membrane perturbation, indicative of mild detergent-like actions on the plasma membrane. These non-ionic surfactants with an established safety record may have potential to help deliver impermeable peptides across the colon if the formulation can be designed with accuracy to reproducibly reach the region and this can be tested initially with rat in situ intestinal instillations. Acknowledgements The authors would like to thank Novo Nordisk A/S, Denmark, for financial support for the post-doctoral fellowship of S.B.P. Additional support was from SFI Grant Number 07/SRC/B1154 (‘Irish Drug Delivery Network’). Thanks also to Ms. Margaret Coady (UCD) for technical assistance with tissue histology and to Drs. Abina Crean and Emma Louise Hogan at University College Cork for assistance measuring CMCs. References Ahsan, F., Arnold, J., Meezan, E., Pillion, D.J., 2001. Enhanced bioavailability of calcitonin formulated with alkylglycosides following nasal and ocular administration in rats. Pharm. Res. 18, 1742–1746. Ahsan, F., Arnold, J.J., Yang, T., Meezan, E., Schwiebert, E.M., Pillion, D.J., 2003. Effects of the permeability enhancers, tetradecylmaltoside and dimethyl-betacyclodextrin, on insulin movement across human bronchial epithelial cells (16HBE14o-). Eur. J. Pharm. Sci. 20, 27–34. Arnold, J.J., Ahsan, F., Meezan, E., Pillion, D.J., 2004. Correlation of tetradecylmaltoside induced increases in nasal peptide drug delivery with morphological changes in nasal epithelial cells. J. Pharm. Sci. 93, 2205–2213. Arnold, J.J., Fyrberg, M.D., Meezan, E., Pillion, D.J., 2010. Reestablishment of the nasal permeability barrier to several peptides following exposure to the absorption enhancer tetradecyl-beta-D-maltoside. J. Pharm. Sci. 99, 1912–1920. Artursson, P., 1990. Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. J. Pharm. Sci. 79, 476–482. Aungst, B.J., 1994. Site-dependence and structure-effect relationships for alkylglycosides as transmucosal absorption promoters for insulin. Int. J. Pharm. 105, 219–225. Aungst, B.J., 2000. Intestinal permeation enhancers. J. Pharm. Sci. 89, 429–442. Aungst, B.J., 2012. Absorption enhancers: applications and advances. AAPS J. 14, 10– 18. Benincasa, M., Abalos, A., Oliveira, I., Manresa, A., 2004. Chemical structure, surface properties and biological activities of the biosurfactant produced by Pseudomonas aeruginosa LBI from soapstock. Biomed. Life Sci. 85, 1–8. Brayden, D.J., Bzik, V.A., Lewis, A.L., Illum, L., 2012. CriticalSorbÒ promotes permeation of flux markers across isolated rat intestinal mucosae and caco-2 monolayers. Pharm. Res. 64, 644–653. Cano-Cebrian, M.J., Zornoza, T., Granero, L., Polache, A., 2005. Intestinal absorption enhancement via the paracellular route by fatty acids, chitosans and others: a target for drug delivery. Curr. Drug Deliv. 2, 9–22. Cao, X., Yu, L.X., Barbaciru, C., Landowski, C.P., Shin, H.C., Gibbs, S., Miller, H.A., Amidon, G.L., Sun, D., 2005. Permeability dominates in vivo intestinal absorption of P-gp substrate with high solubility and high permeability. Mol. Pharm. 2, 329–340. Cuthbert, A.W., Margolius, H.S., 1982. Kinins stimulate net chloride secretion by the rat colon. Br. J. Pharmacol. 75, 587–598. D’Emanuele, A., Jevprasesphant, R., Penny, J., Attwood, D., 2004. The use of a dendrimer-propranolol prodrug to bypass efflux transporters and enhance oral bioavailability. J. Control. Rel., 447–453. Dube, A., Nicolazzo, J.A., Larson, I., 2010. Chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (+)-catechin and (-)epigallocatechin gallate. Eur. J. Pharm. Sci. 41, 219–225. Eley, J.G., Triumalashetty, P., 2001. In vitro assessment of alkylglycosides as permeability enhancers. AAPS PharmSciTech 2, E19. Ericsson, C.A., Soderman, O., Garamus, V.M., Bergstrom, M., Ulvenlund, S., 2005. Effects of temperature, salt, and deuterium oxide on the self-aggregation of alkylglycosides in dilute solution. 2. n- Tetradecyl-beta-D-maltoside. Langmuir 21, 1507–1515. Fetih, G., Lindberg, S., Itoh, K., Okada, N., Fujita, T., Habib, F., Artersson, P., Attia, M., Yamamoto, A., 2005. Improvement of absorption enhancing effects of ndodecyl–beta-D-maltopyranoside by its colon-specific delivery using chitosan capsules. Int. J. Pharm. 293, 127–135. Hubatsch, I., Ragnarsson, E.G.E., Arthursson, P., 2007. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2, 2111–2119.

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