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Permeapad™ for investigation of passive drug permeability: The effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF)
Accepted Manuscript Title: PermeapadTM for investigation of passive drug permeability: the effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF) Author: Hanady Ajine Bibi Massimiliano di Cagno Rene Holm Annette Bauer-Brandl PII: DOI: Reference:
S0378-5173(15)30047-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.028 IJP 15032
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
13-5-2015 8-7-2015 9-7-2015
Please cite this article as: Bibi, Hanady Ajine, di Cagno, Massimiliano, Holm, Rene, Bauer-Brandl, Annette, PermeapadTM for investigation of passive drug permeability: the effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF).International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PermeapadTM for investigation of passive drug permeability: the effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF) Hanady Ajine Bibia, Massimiliano di Cagnob, Rene Holmc, Annette Bauer-Brandla,* a
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark b
Drug Transport and Delivery Research Group, Department of Pharmacy, UiT - The Arctic University of Norway, Universitetsveien 57, 9037 Tromsø, Norway c
Biologics and Pharmaceutical Science, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
*
Corresponding author: e-mail: [email protected], Tel: +45 65504497, Fax: +45 66158760
Abstract
The aim of the present work was to investigate the potential of the new and innovative artificial barrier, PermeapadTM, when exposed to surfactants and co-solvents, often employed for poorly water soluble compounds. The barrier was in addition also exposed to Fasted and Fed state simulated intestinal fluids versions 1 and 2 (FaSSIF and FeSSIF), all of which the PermeapadTM barrier was compatible with based upon relative comparison of the permeability of the hydrophilic marker calcein in phosphate buffer. The new barrier therefore holds a huge potential due to its functional stability and robustness. It can be used as a standard tool to investigate permeability of drugs in the presence of different surfactants and co-solvents, from DMSO stock solutions at even high concentrations and for the evaluation of permeability in the presence of Biomimetic Media (BMM)..
Keywords: apparent permeability; PermeapadTM; FaSSIF, FeSSIF; surfactants, co-solvents
Chemical compounds studied in this article
Calcein (PubChem CID: 65079); Caffeine (PubChem CID: 2519); Triton-X (PubChem CID: 5590); Polysorbate 80 (PubChem CID: 5284448); MacrogolG R (PubChem CID: 81307); Dimethylsulphoxide (DMSO) (PubChem CID: 679); sodiumdocecylsulphate (SDS) (PubChem CID: 8778).
1. Introduction In drug discovery and the early stages of drug development, physicochemical characterisation of new chemical entities is of fundamental significance. In particular, two parameters are of importance at this stage, namely the solubility and the permeability properties, because of their direct connection to bioavailability. Permeability, generally expressed by the apparent permeability coefficient (Papp), describes the ability of a drug molecule to permeate through a biological barrier, as a function of its chemical properties. The most common in vitro assays for permeability investigations are cell-based models like the Caco-2 assay (Uchida et al., 2009), and non-cell-based assay like the Parallel Artificial Membrane Permeability Assay (PAMPA) (Kansy et al., 1998) and the Phospholipid Vesicle Based Permeation Assay (PVPA) (Flaten et al., 2006). A common feature of both cell-based and non-cell-based permeability assays is that their permeation properties can be compromised in the presence of the various excipients used in drug formulations such as surfactants and co-solvents (Fischer et al., 2011a). Drugs classified by the Biopharmaceutics Classification System (BCS), (Amidon et al., 1995; Yu et al., 2002) as class II, III and IV, associated to poor water solubility or/and poor permeability, are in many cases presented in formulation systems designed to overcome these issues, thereby seeking a sufficient bioavailability of the compounds to ensure the desired therapeutical
effect. Therefore, such formulations may contain surfactants or solvents in high concentrations. These formulations can, hence, not be evaluated by the classical in vitro permeability methods, and evaluation and ranking of such formulations are often conducted in non-clinical models. For permeability studies carried out on cell-based assays, buffered salt solutions are commonly used as a medium on both the donor and the acceptor side. Examples of commonly used media are the Hanks’ balanced salt solution (HBSS), standard phosphate buffer saline (PBS) adjusted to isotonic conditions, and the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) at pH 7.4. Also in non-cell-based assays, PBS solutions adjusted to isotonic conditions are commonly employed. While these buffers simulate the physiological pH and osmolality of the intestine, they lack to reflect the presence of bile salts and phospholipids available within the gastrointestinal (GI) tract, elements crucial for the solubilisation of BCS II and IV compounds under physiological conditions. The importance of physiological relevant surfactants for dissolution studies have been reflected by the introduction of Biomimetic media (BMM) in order to mimic in vivo conditions when drugs are dissolved in the GI tract (Dressman et al., 1998; Dressman and Reppas, 2000; Galia et al., 1998). This initial work has led to standardized compositions of Fasted and Fed state simulated intestinal fluid, FaSSIF and FeSSIF, which have also been introduced in both the European Pharmacopeia and Untied States Pharmacopeia. Over the years FaSSIF and FeSSIF version 2 media have been developed based on new learning on the physiology within the GI tract (Jantratid et al., 2008) however, these have not yet been incorporated into the pharmacopeias. A large number of publications have used BMMs for in vitro dissolution to obtain a better correlation to the in vivo situation. The same media have also been investigated in in vitro permeability models. (Ingels et al., 2004; Ingels et al., 2002) demonstrated that FaSSIF could be used in Caco-2 cell studies, whereas FeSSIF could not. Studies investigating the possibility of combining both dissolution and permeability using BMM have also been published, describing
excellent in vitro in vivo correlations (IVIVC) (Buch et al., 2010), however, generally it is difficult to get these models to function as the cell layer is very sensitive to the BMM. Therefore, there clearly is a need for a different cell system or another artificial barrier capable of withstanding the harsh BMMs.
The innovative permeability barrier PermeapadTM, recently introduced (di Cagno; Bauer-Brandl, 2014), has proven to be a powerful tool for a fast and reliable determination of drugs passive permeation properties (di Cagno et al., 2015). Limited information is available on PermepadTM’s functional resistance towards both pharmaceutical excipients and BMMs. Based upon these considerations the purpose of the present study was to evaluate how surfactants, co-solvents and BMM’s would affect the integrity and functional stability of the PermeapadTM barrier using a hydrophilic marker calcein.
2. Materials and Methods
2.1 Chemicals Caffeine, calcein, dimethylsulphoxide (DMSO), sodiumdocecylsulphate (SDS), metoprolol and Triton-X-100 were obtained from Sigma-Aldrich (St. Louise, MO, USA). Soy phosphatidylcholine (PC) S-100 was a generous gift from Lipoid GmbH (Ludwigshafen, Germany). Glacial acetic acid (CH3COOH), maleic acid, sodium dihydrogen phosphate monohydrate ([NaH2PO4] H2O), sodium dihydrogen phosphate dihydrate ([NaH2PO4]·2H2O), di-sodium hydrogen phosphate dodecahydrate ([Na2HPO4]·12H2O), sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium chloride (NaCl) were used for the preparation of the different buffers and were all obtained from Sigma-Aldrich. Polysorbate 60 (Tween 60) and polysorbate 80 (Tween 80) were obtained from Fluka Chemie
(Steinheim, Germany). Macrogolglycerol Ricinoleate (macrogolG R) (formerly known as Cremophor®) was purchased from Caelo GmbH (Caesar & Lorenz, Hilden, Germany). FaSSIF, FeSSIF, FaSSIF-V2 and FeSSIF-V2 powders were all purchased from Biorelevant (Surry, United Kingdom). Water used in the experiments was obtained from a Millipore purification system (Odense, Denmark)
2.2 Methods 2.2.1 Preparation of biomimetic barrier PermeapadTM barrier was prepared as previously described by (di Cagno; Bauer-Brandl, 2014). Soy phosphatidylcholine S-100 was used as the lipid layer. In brief, a thin layer of lipid was applied on a support sheet (Pütz GmbH, Taunusstein, Germany). The final barrier was composed by the support layer and the lipid layer. All PermeapadTM barriers employed in this work were stored at room temperature protected against sunlight.
2.2.2 Preparation of sample solutions. A 74 mM phosphate buffer saline (PBS) at pH 7.40 (± 0.05) was used for all the permeability and stability studies. PBS was prepared by mixing 2.5 % (mass/volume, M/V) a sodium dihydrogen phosphate dihydrate solution with a 1.8 % (M/V) di-sodium hydrogen phosphate dodecahydrate solution in a 1:4 ratio. The pH of the solution was adjusted to 7.4 by addition of NaOH, and osmolality was adjusted to 285 (± 5) mOsm, by adding NaCl (osmolality measured by Semi-Micro Osmometer K7400, Herbert Knauer GmbH, Berlin, Germany). All the sample and surfactant solutions were prepared in PBS.
BMM solutions; FaSSIF, FaSSIF-V2, FeSSIF and FeSSIF-V2, were prepared according to the manufacturer’s instructions. FaSSIF solutions were stored for 2 hours prior to use (equilibrium time) whereas FaSSIF-V2 and FeSSIF-V2 were stored for 1 hour prior to use. Table 1 shows the composition of the four different BMMs (Biorelevant, 2014a; Biorelevant, 2014b)
2.2.3 Permeability studies of hydrophilic marker calcein in the presence of surfactants For all permeability studies, Franz diffusion cells were employed (SES GmbH-Analysesysteme, Bechenheim, Germany) with a surface area of 1 cm2, and an upper chamber nominal volume of 2 mL, lower chamber volume 8 mL. The donor (upper) chamber was filled with 1.5 mL of a solution containing the hydrophilic marker, calcein (5 mM), in different surfactant and solvent solutions see Table 2. The acceptor compartment (lower chamber) was filled with 8 mL PBS (pH value 7.40 ± 0.05). PermeapadTM was placed between the donor and acceptor compartment and the flux (J) of calcein was measured over time. The permeability studies were conducted over a 5 hours period and samples of 200 µL were withdrawn from the acceptor chamber every 30 min for the first 2 hours and every 60 min thereafter. The volume was replaced with an equal amount of fresh PBS at each sample time point. The samples were analysed by fluorescence spectroscopy using a BMG Fluostar Omega 96 plate reader with excitation at 485-512 nm and emission at 520 nm (BMG Labtech GmbH, Ortenberg, Germany).
2.2.4 Permeability studies of hydrophilic marker calcein in presence of BMM The permeability experiments of calcein through PermeapadTM employing BMM as acceptor medium were also carried out. The BMMs were employed as acceptor media to avoid interactions between the hydrophilic marker and the medium, since this might affect the availability of calcein for permeation,
and thus leave unclear if a change in permeability is due to such interaction or to the BMM’s effect on PermeapadTM. Permeability studies with FaSSIF and FaSSIF-V2 were performed in Franz diffusion cells as described above. The donor phase consisted of 1.5 mL calcein solution (5 mM) in PBS and the acceptor phase consisted of 8 mL FaSSIF or FaSSIF-V2. All the experiments were performed in triplicates, and the permeation followed over a period of 5 hours. Samples of 200 µL were withdrawn every 30 min for the first 2 hours and then every 60 minutes. The withdrawn volumes were replaced with fresh FaSSIF or FaSSIF-V2 solutions, respectively.
For FeSSIF and FeSSIF-V2, the permeation experiments of calcein were carried out using a side-by-side diffusion chamber (Ussing chambers, SES GmbH-Analysesysteme, Bechenheim, Germany) due to their more efficient stirring, which was necessary for these media. The donor compartment was filled with 5 mL of calcein solution (5 mM in PBS) and acceptor chamber was filled with 5 mL FeSSIF or FeSSIF-V2 solution, respectively, and both chambers were stirred (surface area 1.76 cm2). Samples of 0.5 mL were withdrawn every 30 min for the first 2 hours and every 60 minutes thereafter up to 5 hours; sample volumes were replaced by fresh FeSSIF or FeSSIF-V2 solutions as described above.
2.2.5 Explorative barrier functionality stability studies Explorative barrier functionality stability experiments were carried out in order to determine whether 5 % macrogolG R or FeSSIF would alter the permeation properties for PermeapadTM. In each case, a permeability experiment with calcein solution (5 mM) containing 5 % macrogolG R or FeSSIF as the donor and PBS as acceptor phase was carried out over 5 hours with the Ussing chambers. After 5 hours, the Ussing chambers were emptied and washed 3 times with PBS. Immediately
thereafter, a new permeability experiment was carried out on the very same barrier with a fresh calcein solution (5 mM) as donor and PBS as acceptor (5 h). In both studies samples were withdrawn as described above.
2.2.6 Permeability studies with caffeine and metoprolol with FeSSIF as acceptor media In order to determine FeSSIF’s effect on the PermeapadTM barrier, a new set of permeability studies with caffeine and metoprolol were carried out in Franz diffusion cells. In these investigations, the lower compartment (8 mL) was used as the donor and the upper compartment (1.5 mL) as acceptor. The study was carried out using PermeapadTM or the empty barrier support. Caffeine (50 mM) or metoprolol (25 mM) was prepared in FeSSIF blank buffer and added into the donor chamber. FeSSIF was used as acceptor medium. Samples were withdrawn every 30 min for 5 hours and the removed volume was replaced with FeSSIF. The samples were analysed by UV-vis spectroscopy on a Genesis 10 UV/VIS (Thermo Electron Corporation, Cambridge, UK).
2.2.7. Data analysis / permeability calculations For each permeation experiment, the amount of permeated drug over the surface area (dQ/A) was calculated over the respective time interval (dt). The flux (J), represented by the slope of the linear regression in the plot containing the cumulative amount of permeated drug as a function of time according to:
The obtained flux values were used to calculate the apparent permeability coefficient (Papp) by dividing the flux with the initial concentration of permeated drug (C0), which was supposed to be constant
during the experiment as sink conditions, was kept.
2.2.8. Statistical analysis Significant changes in permeability were evaluated by a two-sided student’s t-test and ANOVA Tukey Post-hoc test. P ≤ 0.05 was considered as significantly different. Moreover, a Thomson Tau test was used to identify possible outlier values.
3. Results and discussion The aim of this work was to evaluate the functional resistance of the recently introduced PermeapadTM barrier to excipients, additives and Biomimetic media (BMM). For this purpose, the apparent permeability coefficient (Papp) of the hydrophilic marker calcein was measured in the presence of different surfactants and co-solvents commonly used in enabling formulations. Calcein is a very hydrophilic and highly water soluble substance (MW: 622.53 g/mol; log P: -3.1)(Pubchem, 2015). Further, it is a widely used marker of barrier integrity in in vitro studies ((di Cagno et al., 2015; Flaten et al., 2006). In addition, the permeability of calcein through PermeapadTM was investigated using four BMMs; FaSSIF versions 1 and 2 and FeSSIF versions 1 and 2 as acceptor media.
3.1 Permeability studies in the presence of surfactants and co-solvents through PermeapadTM The Papp value for calcein in PBS through PermeapadTM barrier is presented in Table 3 and used as the reference value. The permeation of calcein through the empty barrier support was significantly faster than the permeation through the PermeapadTM (see Figure 1). This result was expected, as it has been proven that the lipid layer contained in PermeapadTM has a significant retention on the permeability of
hydrophilic compounds (di Cagno et al., 2015). The standard deviations (SD) for the experiments were low, 8 % and 6 % for the PermeapadTM and support layer respectively. Therefore an impact of the surfactants on the integrity of the PermeapadTM barrier would be observable through an increased Papp value for calcein.
The surfactants tested on the PermeapadTM barrier are reported in Table 2. The studied concentrations of Polysorbate 80, MacrogolG R, DMSO, SDS and EtOH were chosen in order to compare with published in vitro studies using other models (Flaten et al., 2008). SDS has previously been used as a positive control in Caco-2 cells studies in a concentration of 0.1 % (W/V) and upwards for the lysis of the cells (Sakai et al., 1998). Also Triton-X is known to be harsh towards in vitro models and is used as positive control in the concentration range of 0.1-1 % for both caco-2 cells and PVPA (Fischer et al., 2011a; Fischer et al., 2011b). Figure 2 illustrates the permeability of calcein through PermeapadTM barrier in the presence and absence of surfactants and co-solvents in the form of Papp values. Although the permeability of calcein in some cases was increased by up to 30 %, and for most of the excipients a statistical difference was noted, the permeability was still considered very low compared to the “barrier support” (1.65 ± 0.1 · 105 cm/s) and hence the barrier intact. Permeability experiments with surfactants and co-solvents were carried out as consecutive rows of single experiments with increasing concentrations for each surfactant and checked for plausibility. Explorative barrier stability studies were carried out on the surfactants that showed a very high increase in permeability.
PVPA has been reported to be compatible with 6 % EtOH and 4 % DMSO (Flaten et al., 2008). However, the reported standard deviation of calcein permeability generally was much higher in the PVPA models than the deviations found in the present study, indicating a better tolerance of the PermeapadTM barrier towards these two solvents - with the appropriate reservations when comparing standard deviations across studies. The permeability of calcein through PermpeapadTM with 4 % EtOH and 10 % DMSO was 0.12 · 10-5 cm/s and 0.15 · 105 cm/s, respectively, which was not statistical significant from the results obtained in PBS, and these co-solvents were considered compatible with PermeapadTM. PAMPA has been reported to use 5 % DMSO in the stock solutions of drugs and 5 % EtOH, and is thus also compatible with these co-solvents at the given conentrations (Liu et al., 2003). Both PVPA and Caco-2 cells have been investigated with respect to MacrogolG R compatibility: PAMPA and PVPA have been reported to be incompatible with respectivly 0.5 and 4 % MacrogolG R (Liu et al., 2003), (Flaten et al., 2008), and Caco-2 cells have been reported to be compatible with MagrogolG R up to 10 % (Nerurkar et al., 1996). The permeability of calcein through PermpeapadTM in the presence of 5 % MacrogolG R was 0.23 · 10-5 cm/s. That is approximately a 2 fold the value of the permeability in PBS and statistically significantly different (p < 0.05). Therefore more explorative barrier functionality stability studies were carried out. The explorative studies were carried out in a different setup (Ussing chambers instead of Franz cells) so consecutive experiments could be carried out on the very same PermeapadTM barrier: the acceptor and donor compartments were emptied, washed and refilled with new media, without destroying or affecting the barrier. Results obtained for the permeability of calcein through PermeapadTM with 5 % macrogolG R in the Ussing chambers was 0.12 ± 0.01 · 105 cm/s, which was much lower and similar to the permeability observed in PBS, showing the PermeapadTM barrier to be compatible with 5 % MacrogolG R. The data
obtained in Franz cells probably lead to differences in the observed permeability due to insufficient stirring of the macrogolG R solution next to the barrier surface.
Interestingly, one of the tested surfactants resulted in a lower permeability value than the value obtained in PBS: The permeability of calcein in the presence of 5 % Polysorbate 80 was 0.06 · 10-5 cm/s, which was half the permeability obtained in PBS. A decrease in permeation does not indicate any destruction of the barrier, but rather the opposite. The decrease in permeability might be explained by a number of different hypotheses: The first could be that an interaction between Polysorbate 80 micelles (with or without calcein) and the surface of the barrier had occurred, resulting in micelle deposition on the surface of the barrier and thereby an increased resistance of the diffusion layers that calcein has to pass. A second possibility could be that a limited efficiency of stirring in the donor compartment next to the barrier surface might have resulted in a lower diffusion coefficient and thereby a lower permeation rate. Furthermore, for the 5 % Polysorbate 80 solution, a lag time of 1 hour was observed, which was only observed for the higher polysorbate concentrations. This supports both of the hypotheses above. Other in vitro models have previously been reported to be largely affected by the presence of surfactants such as polysorbate 80. For example, PVPA were not compatible with Polysorbate 80 in concentrations as low as 0.5 %, where the permeability of calcein was significantly increased, probably due to Polysorbate 80’s destruction of the lipid barriers (Flaten et al., 2008; Naderkhani et al., 2014). Caco-2 cells have been reported to be compatible with Polysorbate 80 with concentrations up to 1 % (Nerurkar et al., 1996), and PAMPA is compatible up to 5 % (Liu et al., 2003) however experiments are often carried out with only 0.2 % Polysorbate 80 (Liu et al., 2003). For
PermeapadTM the permeability decreased, which indicates that Polysorbate 80 did not disrupt the barrier, but instead interacts with calcein or with the barrier.
A lag time of 1 hour was also observed for 4 % Polysorbate 60. This indicates that both surfactants above their CMC interact with calcein and / or the barrier in a way that slows the diffusion of calcein through PermeapadTM. However, the permeability of calcein in the presence of 4 % Polysorbate 60 was different in comparison to Polysorbate 80. This was probably due to different molecular sizes of the two Polysorbate types. Polysorbate 80 is a larger molecule than Polysorbate 60, which results in a slightly lower diffusion coefficient and a slightly higher hydrophilic/lipophilic balance (HLB) 15 and 14.9 respectively.
The most interesting observation made was with Triton-X, a substance that in many cell-based in vitro models is used as a positive control in a concentration range of 0.1-1 % (Fischer et al., 2011b), i.e. destroying the barrier for recovery measurement. Studies with PVPA (Naderkhani et al., 2014) have used Triton-X in a concentration of 0.05 %, as a positive control, showing that PVPA loses its integrity when in contact with this surfactant. Results obtained for PermeapadTM show that Triton-X (up to a much higher concentration of 1 % M/V) did not affect the integrity of the barrier at all. The permeability of calcein through PermeapadTM was unchanged when comparing to the PBS reference, both resulted in Papp values of 0.12 · 10-5 cm/s. In summary all these results clearly show that PermeapadTM can withstand the different surfactants, co-solvents and Triton-X listed in Table 2 without affecting the integrity of the barrier and thereby the permeability of calcein.
3.2 Permeability studies with PermeapadTM, using simulated intestinal fluids FaSSIF and FeSSIF are interesting to use as media in both dissolution and permeation studies of oral drugs and oral dosage forms, since they can be used to mimic the gastrointestinal fluids in both the fasted and fed state. Using the BMM in in vitro studies may, hence give a more representative indication of how a drug might behave in vivo upon oral administration. The compositions of the four BMMs used, are shown in Table 1. Permeability studies with the different BMM’s were carried out on two different permeability setups; FaSSIF and FaSSIF-V2 were conducted in the Franz diffusion cells whereas FeSSIF and FeSSIF-V2 studies were carried out on the Ussing chamber, due to difficulties when employed in the Franz cell. When FeSSIF and FeSSIF-V2 were used in the Franz cells calcein was not homogeneously distributed: a layer of calcein accumulated on the top next to the barrier, not mixing properly with the rest of the acceptor phase. Increased viscosity was assumed to be the problem; however, viscosity measurements did not show increased viscosity for FeSSIF nor FeSSIF-V2 (data not shown). It is therefore assumed that the stirring in the Franz cell was not sufficient to distribute calcein properly when large amounts of sodium taurocholate NaTC were present in the media. Both media contain substantial amounts of NaTC and lecithin, when compared to the FaSSIF media. FeSSIF and FeSSIF-V2 were therefore investigated in the Ussing chamber where the magnetic stirring was more vigorous close to the barrier resulting in an even distribution of calcein in the respective cells. The results obtained from the permeability studies with the BMMs are presented in Table 3. The results show that the permeability values for calcein in FaSSIF, FaSSIF-V2 and FeSSIF-V2 were slightly
increased in comparison to PBS. In contrast to this, FeSSIF increased the permeability of calcein significantly (Papp = 0.53 ± 0.09 · 105 cm/s), thus more explorative barrier stability experiments were carried out with this medium. The barrier was first exposed to FeSSIF for 5 hours, then washed and re-tested for permeability using calcein solution in PBS as donor and PBS in the acceptor compartment. With this procedure a Papp for calcein in PBS of 0.27 ± 0.05 · 10-5 cm/s was found, after a 5 hour exposure to FeSSIF, which clearly indicates that the integrity of the barrier was not compromised, but that the effect is rather associated with the FeSSIF. A possible explanation could be that the high osmolality of FeSSIF (635 ± 10 mOsm) creates an osmotic pressure difference, which may induce an increase in permeability. To further investigate the effect of FeSSIF additional experiments were conducted with FeSSIF as acceptor media using caffeine and metorpolol as permeability markers. Caffeine was chosen due to its high permeability and high UV absorbance, while Metoprolol is a moderate permeability drug. The results for caffeine and metoprolol are presented in Figure 3. The permeability of caffeine through PermeapadTM was not significantly different when replacing PBS with FeSSIF as the acceptor medium (P > 0.05). The results for metoprolol also show that the permeability of metoprolol was not affected by the presence of the FeSSIF (P > 0.05). The barrier is clearly not disrupted by the medium, but other parameters influence the data. Hence, studies should be planned carefully when using FeSSIF together with the PermeapadTM barrier.
The effect of FaSSIF on Caco-2 cells has earlier been investigated by (Fossati et al., 2008; Ingels et al., 2004; Ingels et al., 2002) and was found compatible with Caco-2 cells, whereas FeSSIF was not (Ingels et al., 2002). However, data was also presented showing that sodium taurocholate (NaTC) has an inhibitory effect on P-glycoprotein (P-gp), which affects the active efflux of drugs (Ingels et al., 2004).
Studies with PermeapadTM in the different BMMs have shown the barrier to be stable over time and in the presence of the different BMMs, however, PermeapadTM is designed as a tool for predicting passive diffusion of drugs and therefore the effect of NaTC on P-gp will not be measurable.
Other artificial barriers have also been investigated together with BMMs. The PVPA model has been shown to be compatible with FaSSIF, although an increase in SD was observed (Buckley et al., 2012). The PAMPA model has been used to determine passive permeability of lipophilic molecules, using FaSSIF-V2 and FeSSIF-V2 as donor solutions (Markopoulos et al., 2013). The publication demonstrated no data to ensure the compatibility, but it must be assumed.
PAMPA is a non-cell based artificial membrane for the screening of drugs in relation to passive diffusion. It has over the recent years become more abundant, and different PAMPA models have been developed (Avdeef, 2005). PAMPA has the advantage of screening large amounts of new drug entities, due to the 96-well microtiter plate format – making it very ideal for High Throughput Screening (HTS) (Kansy et al., 1998). However, comparing PermeapadTM with PAMPA some advantages are noted in favour of PermeapadTM. Especially highly lipophilic drugs (Log P > 4) tend to accumulate in the lipidic phase of PAMPA, instead of the acceptor phase (Avdeef, 2005). This issue has not been a problem for PermeapadTM: all the permeability experiments have yielded high drug recovery values (90-100 % - data not shown). The clear advantages of PAMPA are the reproducibility and its ability to HTS, where PermeapadTM is still carried out on laboratory scale.
The present study has looked into the potential use of the new artificial barrier, PermeapadTM, in a number of media normally not compatible with other in vitro models. The PermeapadTM generally
showed good barrier function even when exposed to the tested excipients or media, suggesting a barrier with huge potential within particular in vitro models with combined dissolution and permeability, which future studies will investigate.
4. Conclusion The functionality of the new biomimetic barrier PermeapadTM was evaluated in the presence of different surfactants, co-solvents and simulated intestinal fluids. The obtained results showed that PermeapadTM was compatible with a wide range of surfactants/co-solvents including Polysorbate 80, DMSO and Triton-X in relevant and high concentrations. Moreover, four different Biomimetic Media (namely FaSSIF, FaSSIF-V2, FeSSIF, and FeSSIF-V2), were also found compatible with PermeapadTM. These results indicate that PermeapadTM is well suited for fast and reliable prediction of passive drug permeability even in the presence of typical components used in enabling formulations. References Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharmaceutical Research 12, 413-420. Avdeef, A., 2005. The rise of PAMPA. Expert Opinion on Drug Metabolism and Toxicology 1, 325342. Bauer-Brandl, A.d.C., Massimiliano 2014. Assembly for assessing drug permeability with adjustable biomumetic properties, Danish Patent Application PA 2014 70708, Denmark Biorelevant, C.o.F.a.F.-V., 2014a. Available from: http://biorelevant.com/fassif-v2-powder/containssodium-taurocholate-lecithin/ (Accessed on 8 May 2015). Biorelevant, C.o.F.a.F.-V., 2014b. Available from: http://biorelevant.com/fessif-v2-powder/containstaurocholic-acid-sodium-salt-lecithin/ (Accessed on 8 May 2015). Buch, P., Holm, P., Thomassen, J.Q., Scherer, D., Branscheid, R., Kolb, U., Langguth, P., 2010. IVIVC for fenofibrate immediate release tablets using solubility and permeability as in vitro predictors for pharmacokinetics. Journal of Pharmaceutical Sciences 99, 4427-4436.
Buckley, S.T., Fischer, S.M., Fricker, G., Brandl, M., 2012. In vitro models to evaluate the permeability of poorly soluble drug entities: Challenges and perspectives. European Journal of Pharmaceutical Sciences 45, 235-250. di Cagno, M., Bibi, H.A., Bauer-Brandl, A., 2015. New biomimetic barrier Permeapad™ for efficient investigation of passive permeability of drugs. European Journal of Pharmaceutical Sciences 73, 29-34. Dressman, J.B., Amidon, G.L., Reppas, C., Shah, V.P., 1998. Dissolution testing as a prognostic tool for oral drug absorption: Immediate release dosage forms. Pharmaceutical Research 15, 11-22. Dressman, J.B., Reppas, C., 2000. In vitro-in vivo correlations for lipophilic, poorly water-soluble drugs. European Journal of Pharmaceutical Sciences 11, S73-S80. Fischer, S.M., Brandl, M., Fricker, G., 2011a. Effect of the non-ionic surfactant Poloxamer 188 on passive permeability of poorly soluble drugs across Caco-2 cell monolayers. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 79, 416-422. Fischer, S.M., Flaten, G.E., Hagesæther, E., Fricker, G., Brandl, M., 2011b. In-vitro permeability of poorly water soluble drugs in the phospholipid vesicle-based permeation assay: The influence of nonionic surfactants. Journal of Pharmacy and Pharmacology 63, 1022-1030. Flaten, G.E., Dhanikula, A.B., Luthman, K., Brandl, M., 2006. Drug permeability across a phospholipid vesicle based barrier: A novel approach for studying passive diffusion. European Journal of Pharmaceutical Sciences 27, 80-90. Flaten, G.E., Luthman, K., Vasskog, T., Brandl, M., 2008. Drug permeability across a phospholipid vesicle-based barrier. 4. The effect of tensides, co-solvents and pH changes on barrier integrity and on drug permeability. European Journal of Pharmaceutical Sciences 34, 173-180. Fossati, L., Dechaume, R., Hardillier, E., Chevillon, D., Prevost, C., Bolze, S., Maubon, N., 2008. Use of simulated intestinal fluid for Caco-2 permeability assay of lipophilic drugs. International Journal of Pharmaceutics 360, 148-155. Galia, E., Nicolaides, E., Hörter, D., Löbenberg, R., Reppas, C., Dressman, J.B., 1998. Evaluation of various dissolution media for predicting In vivo performance of class I and II drugs. Pharmaceutical Research 15, 698-705. Ingels, F., Beck, B., Oth, M., Augustijns, P., 2004. Effect of simulated intestinal fluid on drug permeability estimation across Caco-2 monolayers. International Journal of Pharmaceutics 274, 221232. Ingels, F., Deferme, S., Destexhe, E., Oth, M., Van Den Mooter, G., Augustijns, P., 2002. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. International Journal of Pharmaceutics 232, 183-192.
Jantratid, E., Janssen, N., Reppas, C., Dressman, J.B., 2008. Dissolution media simulating conditions in the proximal human gastrointestinal tract: An update. Pharmaceutical Research 25, 1663-1676. Kansy, M., Senner, F., Gubernator, K., 1998. Physicochemical high throughput screening: Parallel artificial membrane permeation assay in the description of passive absorption processes. Journal of Medicinal Chemistry 41, 1007-1010. Liu, H., Sabus, C., Carter, G., Du, C., Avdeef, A., Tischler, M., 2003. In Vitro Permeability of Poorly Aqueous Soluble Compounds Using Different Solubilizers in the PAMPA Assay with Liquid Chromatography/Mass Spectrometry Detection. Pharmaceutical Research 20, 1820-1826 Markopoulos, C., Imanidis, G., Vertzoni, M., Symillides, M., Parrott, N., Reppas, C., 2013. In vitro and Ex vivo investigation of the impact of luminal lipid phases on passive permeability of lipophilic small molecules using PAMPA. Pharmaceutical Research 30, 3145-3153. Naderkhani, E., Isaksson, J., Ryzhakov, A., Flaten, G.E., 2014. Development of a Biomimetic Phospholipid Vesicle-based Permeation Assay for the Estimation of Intestinal Drug Permeability. Journal of Pharmaceutical Sciences 103, 1882-1890. Nerurkar, M.M., Burton, P.S., Borchardt, R.T., 1996. The use of surfactants to enhance the permeability of peptides through caco-2 cells by inhibition of an apically polarized efflux system. Pharmaceutical Research 13, 528-534. Pubchem, P.c.p.o.c., 2015. Available from http://pubchem.ncbi.nlm.nih.gov/compound/calcein (Accessed 2 July 2015). Sakai, M., Imai, T., Ohtake, H., Otagiri, M., 1998. Cytotoxicity of absorption enhancers in caco-2 cell monolayers. Journal of Pharmacy and Pharmacology 50, 1101-1108. Uchida, M., Fukazawa, T., Yamazaki, Y., Hashimoto, H., Miyamoto, Y., 2009. A modified fast (4 day) 96-well plate Caco-2 permeability assay. Journal of Pharmacological and Toxicological Methods 59, 39-43. Yu, L.X., Amidon, G.L., Polli, J.E., Zhao, H., Mehta, M.U., Conner, D.P., Shah, V.P., Lesko, L.J., Chen, M.L., Lee, V.H.L., Hussain, A.S., 2002. Biopharmaceutics classification system: The scientific basis for biowaiver extensions. Pharmaceutical Research 19, 921-925.
S0378-5173(15)30047-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.028 IJP 15032
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
13-5-2015 8-7-2015 9-7-2015
Please cite this article as: Bibi, Hanady Ajine, di Cagno, Massimiliano, Holm, Rene, Bauer-Brandl, Annette, PermeapadTM for investigation of passive drug permeability: the effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF).International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PermeapadTM for investigation of passive drug permeability: the effect of surfactants, co-solvents and simulated intestinal fluids (FaSSIF and FeSSIF) Hanady Ajine Bibia, Massimiliano di Cagnob, Rene Holmc, Annette Bauer-Brandla,* a
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark b
Drug Transport and Delivery Research Group, Department of Pharmacy, UiT - The Arctic University of Norway, Universitetsveien 57, 9037 Tromsø, Norway c
Biologics and Pharmaceutical Science, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
*
Corresponding author: e-mail: [email protected], Tel: +45 65504497, Fax: +45 66158760
Abstract
The aim of the present work was to investigate the potential of the new and innovative artificial barrier, PermeapadTM, when exposed to surfactants and co-solvents, often employed for poorly water soluble compounds. The barrier was in addition also exposed to Fasted and Fed state simulated intestinal fluids versions 1 and 2 (FaSSIF and FeSSIF), all of which the PermeapadTM barrier was compatible with based upon relative comparison of the permeability of the hydrophilic marker calcein in phosphate buffer. The new barrier therefore holds a huge potential due to its functional stability and robustness. It can be used as a standard tool to investigate permeability of drugs in the presence of different surfactants and co-solvents, from DMSO stock solutions at even high concentrations and for the evaluation of permeability in the presence of Biomimetic Media (BMM)..
Keywords: apparent permeability; PermeapadTM; FaSSIF, FeSSIF; surfactants, co-solvents
Chemical compounds studied in this article
Calcein (PubChem CID: 65079); Caffeine (PubChem CID: 2519); Triton-X (PubChem CID: 5590); Polysorbate 80 (PubChem CID: 5284448); MacrogolG R (PubChem CID: 81307); Dimethylsulphoxide (DMSO) (PubChem CID: 679); sodiumdocecylsulphate (SDS) (PubChem CID: 8778).
1. Introduction In drug discovery and the early stages of drug development, physicochemical characterisation of new chemical entities is of fundamental significance. In particular, two parameters are of importance at this stage, namely the solubility and the permeability properties, because of their direct connection to bioavailability. Permeability, generally expressed by the apparent permeability coefficient (Papp), describes the ability of a drug molecule to permeate through a biological barrier, as a function of its chemical properties. The most common in vitro assays for permeability investigations are cell-based models like the Caco-2 assay (Uchida et al., 2009), and non-cell-based assay like the Parallel Artificial Membrane Permeability Assay (PAMPA) (Kansy et al., 1998) and the Phospholipid Vesicle Based Permeation Assay (PVPA) (Flaten et al., 2006). A common feature of both cell-based and non-cell-based permeability assays is that their permeation properties can be compromised in the presence of the various excipients used in drug formulations such as surfactants and co-solvents (Fischer et al., 2011a). Drugs classified by the Biopharmaceutics Classification System (BCS), (Amidon et al., 1995; Yu et al., 2002) as class II, III and IV, associated to poor water solubility or/and poor permeability, are in many cases presented in formulation systems designed to overcome these issues, thereby seeking a sufficient bioavailability of the compounds to ensure the desired therapeutical
effect. Therefore, such formulations may contain surfactants or solvents in high concentrations. These formulations can, hence, not be evaluated by the classical in vitro permeability methods, and evaluation and ranking of such formulations are often conducted in non-clinical models. For permeability studies carried out on cell-based assays, buffered salt solutions are commonly used as a medium on both the donor and the acceptor side. Examples of commonly used media are the Hanks’ balanced salt solution (HBSS), standard phosphate buffer saline (PBS) adjusted to isotonic conditions, and the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) at pH 7.4. Also in non-cell-based assays, PBS solutions adjusted to isotonic conditions are commonly employed. While these buffers simulate the physiological pH and osmolality of the intestine, they lack to reflect the presence of bile salts and phospholipids available within the gastrointestinal (GI) tract, elements crucial for the solubilisation of BCS II and IV compounds under physiological conditions. The importance of physiological relevant surfactants for dissolution studies have been reflected by the introduction of Biomimetic media (BMM) in order to mimic in vivo conditions when drugs are dissolved in the GI tract (Dressman et al., 1998; Dressman and Reppas, 2000; Galia et al., 1998). This initial work has led to standardized compositions of Fasted and Fed state simulated intestinal fluid, FaSSIF and FeSSIF, which have also been introduced in both the European Pharmacopeia and Untied States Pharmacopeia. Over the years FaSSIF and FeSSIF version 2 media have been developed based on new learning on the physiology within the GI tract (Jantratid et al., 2008) however, these have not yet been incorporated into the pharmacopeias. A large number of publications have used BMMs for in vitro dissolution to obtain a better correlation to the in vivo situation. The same media have also been investigated in in vitro permeability models. (Ingels et al., 2004; Ingels et al., 2002) demonstrated that FaSSIF could be used in Caco-2 cell studies, whereas FeSSIF could not. Studies investigating the possibility of combining both dissolution and permeability using BMM have also been published, describing
excellent in vitro in vivo correlations (IVIVC) (Buch et al., 2010), however, generally it is difficult to get these models to function as the cell layer is very sensitive to the BMM. Therefore, there clearly is a need for a different cell system or another artificial barrier capable of withstanding the harsh BMMs.
The innovative permeability barrier PermeapadTM, recently introduced (di Cagno; Bauer-Brandl, 2014), has proven to be a powerful tool for a fast and reliable determination of drugs passive permeation properties (di Cagno et al., 2015). Limited information is available on PermepadTM’s functional resistance towards both pharmaceutical excipients and BMMs. Based upon these considerations the purpose of the present study was to evaluate how surfactants, co-solvents and BMM’s would affect the integrity and functional stability of the PermeapadTM barrier using a hydrophilic marker calcein.
2. Materials and Methods
2.1 Chemicals Caffeine, calcein, dimethylsulphoxide (DMSO), sodiumdocecylsulphate (SDS), metoprolol and Triton-X-100 were obtained from Sigma-Aldrich (St. Louise, MO, USA). Soy phosphatidylcholine (PC) S-100 was a generous gift from Lipoid GmbH (Ludwigshafen, Germany). Glacial acetic acid (CH3COOH), maleic acid, sodium dihydrogen phosphate monohydrate ([NaH2PO4] H2O), sodium dihydrogen phosphate dihydrate ([NaH2PO4]·2H2O), di-sodium hydrogen phosphate dodecahydrate ([Na2HPO4]·12H2O), sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium chloride (NaCl) were used for the preparation of the different buffers and were all obtained from Sigma-Aldrich. Polysorbate 60 (Tween 60) and polysorbate 80 (Tween 80) were obtained from Fluka Chemie
(Steinheim, Germany). Macrogolglycerol Ricinoleate (macrogolG R) (formerly known as Cremophor®) was purchased from Caelo GmbH (Caesar & Lorenz, Hilden, Germany). FaSSIF, FeSSIF, FaSSIF-V2 and FeSSIF-V2 powders were all purchased from Biorelevant (Surry, United Kingdom). Water used in the experiments was obtained from a Millipore purification system (Odense, Denmark)
2.2 Methods 2.2.1 Preparation of biomimetic barrier PermeapadTM barrier was prepared as previously described by (di Cagno; Bauer-Brandl, 2014). Soy phosphatidylcholine S-100 was used as the lipid layer. In brief, a thin layer of lipid was applied on a support sheet (Pütz GmbH, Taunusstein, Germany). The final barrier was composed by the support layer and the lipid layer. All PermeapadTM barriers employed in this work were stored at room temperature protected against sunlight.
2.2.2 Preparation of sample solutions. A 74 mM phosphate buffer saline (PBS) at pH 7.40 (± 0.05) was used for all the permeability and stability studies. PBS was prepared by mixing 2.5 % (mass/volume, M/V) a sodium dihydrogen phosphate dihydrate solution with a 1.8 % (M/V) di-sodium hydrogen phosphate dodecahydrate solution in a 1:4 ratio. The pH of the solution was adjusted to 7.4 by addition of NaOH, and osmolality was adjusted to 285 (± 5) mOsm, by adding NaCl (osmolality measured by Semi-Micro Osmometer K7400, Herbert Knauer GmbH, Berlin, Germany). All the sample and surfactant solutions were prepared in PBS.
BMM solutions; FaSSIF, FaSSIF-V2, FeSSIF and FeSSIF-V2, were prepared according to the manufacturer’s instructions. FaSSIF solutions were stored for 2 hours prior to use (equilibrium time) whereas FaSSIF-V2 and FeSSIF-V2 were stored for 1 hour prior to use. Table 1 shows the composition of the four different BMMs (Biorelevant, 2014a; Biorelevant, 2014b)
2.2.3 Permeability studies of hydrophilic marker calcein in the presence of surfactants For all permeability studies, Franz diffusion cells were employed (SES GmbH-Analysesysteme, Bechenheim, Germany) with a surface area of 1 cm2, and an upper chamber nominal volume of 2 mL, lower chamber volume 8 mL. The donor (upper) chamber was filled with 1.5 mL of a solution containing the hydrophilic marker, calcein (5 mM), in different surfactant and solvent solutions see Table 2. The acceptor compartment (lower chamber) was filled with 8 mL PBS (pH value 7.40 ± 0.05). PermeapadTM was placed between the donor and acceptor compartment and the flux (J) of calcein was measured over time. The permeability studies were conducted over a 5 hours period and samples of 200 µL were withdrawn from the acceptor chamber every 30 min for the first 2 hours and every 60 min thereafter. The volume was replaced with an equal amount of fresh PBS at each sample time point. The samples were analysed by fluorescence spectroscopy using a BMG Fluostar Omega 96 plate reader with excitation at 485-512 nm and emission at 520 nm (BMG Labtech GmbH, Ortenberg, Germany).
2.2.4 Permeability studies of hydrophilic marker calcein in presence of BMM The permeability experiments of calcein through PermeapadTM employing BMM as acceptor medium were also carried out. The BMMs were employed as acceptor media to avoid interactions between the hydrophilic marker and the medium, since this might affect the availability of calcein for permeation,
and thus leave unclear if a change in permeability is due to such interaction or to the BMM’s effect on PermeapadTM. Permeability studies with FaSSIF and FaSSIF-V2 were performed in Franz diffusion cells as described above. The donor phase consisted of 1.5 mL calcein solution (5 mM) in PBS and the acceptor phase consisted of 8 mL FaSSIF or FaSSIF-V2. All the experiments were performed in triplicates, and the permeation followed over a period of 5 hours. Samples of 200 µL were withdrawn every 30 min for the first 2 hours and then every 60 minutes. The withdrawn volumes were replaced with fresh FaSSIF or FaSSIF-V2 solutions, respectively.
For FeSSIF and FeSSIF-V2, the permeation experiments of calcein were carried out using a side-by-side diffusion chamber (Ussing chambers, SES GmbH-Analysesysteme, Bechenheim, Germany) due to their more efficient stirring, which was necessary for these media. The donor compartment was filled with 5 mL of calcein solution (5 mM in PBS) and acceptor chamber was filled with 5 mL FeSSIF or FeSSIF-V2 solution, respectively, and both chambers were stirred (surface area 1.76 cm2). Samples of 0.5 mL were withdrawn every 30 min for the first 2 hours and every 60 minutes thereafter up to 5 hours; sample volumes were replaced by fresh FeSSIF or FeSSIF-V2 solutions as described above.
2.2.5 Explorative barrier functionality stability studies Explorative barrier functionality stability experiments were carried out in order to determine whether 5 % macrogolG R or FeSSIF would alter the permeation properties for PermeapadTM. In each case, a permeability experiment with calcein solution (5 mM) containing 5 % macrogolG R or FeSSIF as the donor and PBS as acceptor phase was carried out over 5 hours with the Ussing chambers. After 5 hours, the Ussing chambers were emptied and washed 3 times with PBS. Immediately
thereafter, a new permeability experiment was carried out on the very same barrier with a fresh calcein solution (5 mM) as donor and PBS as acceptor (5 h). In both studies samples were withdrawn as described above.
2.2.6 Permeability studies with caffeine and metoprolol with FeSSIF as acceptor media In order to determine FeSSIF’s effect on the PermeapadTM barrier, a new set of permeability studies with caffeine and metoprolol were carried out in Franz diffusion cells. In these investigations, the lower compartment (8 mL) was used as the donor and the upper compartment (1.5 mL) as acceptor. The study was carried out using PermeapadTM or the empty barrier support. Caffeine (50 mM) or metoprolol (25 mM) was prepared in FeSSIF blank buffer and added into the donor chamber. FeSSIF was used as acceptor medium. Samples were withdrawn every 30 min for 5 hours and the removed volume was replaced with FeSSIF. The samples were analysed by UV-vis spectroscopy on a Genesis 10 UV/VIS (Thermo Electron Corporation, Cambridge, UK).
2.2.7. Data analysis / permeability calculations For each permeation experiment, the amount of permeated drug over the surface area (dQ/A) was calculated over the respective time interval (dt). The flux (J), represented by the slope of the linear regression in the plot containing the cumulative amount of permeated drug as a function of time according to:
The obtained flux values were used to calculate the apparent permeability coefficient (Papp) by dividing the flux with the initial concentration of permeated drug (C0), which was supposed to be constant
during the experiment as sink conditions, was kept.
2.2.8. Statistical analysis Significant changes in permeability were evaluated by a two-sided student’s t-test and ANOVA Tukey Post-hoc test. P ≤ 0.05 was considered as significantly different. Moreover, a Thomson Tau test was used to identify possible outlier values.
3. Results and discussion The aim of this work was to evaluate the functional resistance of the recently introduced PermeapadTM barrier to excipients, additives and Biomimetic media (BMM). For this purpose, the apparent permeability coefficient (Papp) of the hydrophilic marker calcein was measured in the presence of different surfactants and co-solvents commonly used in enabling formulations. Calcein is a very hydrophilic and highly water soluble substance (MW: 622.53 g/mol; log P: -3.1)(Pubchem, 2015). Further, it is a widely used marker of barrier integrity in in vitro studies ((di Cagno et al., 2015; Flaten et al., 2006). In addition, the permeability of calcein through PermeapadTM was investigated using four BMMs; FaSSIF versions 1 and 2 and FeSSIF versions 1 and 2 as acceptor media.
3.1 Permeability studies in the presence of surfactants and co-solvents through PermeapadTM The Papp value for calcein in PBS through PermeapadTM barrier is presented in Table 3 and used as the reference value. The permeation of calcein through the empty barrier support was significantly faster than the permeation through the PermeapadTM (see Figure 1). This result was expected, as it has been proven that the lipid layer contained in PermeapadTM has a significant retention on the permeability of
hydrophilic compounds (di Cagno et al., 2015). The standard deviations (SD) for the experiments were low, 8 % and 6 % for the PermeapadTM and support layer respectively. Therefore an impact of the surfactants on the integrity of the PermeapadTM barrier would be observable through an increased Papp value for calcein.
The surfactants tested on the PermeapadTM barrier are reported in Table 2. The studied concentrations of Polysorbate 80, MacrogolG R, DMSO, SDS and EtOH were chosen in order to compare with published in vitro studies using other models (Flaten et al., 2008). SDS has previously been used as a positive control in Caco-2 cells studies in a concentration of 0.1 % (W/V) and upwards for the lysis of the cells (Sakai et al., 1998). Also Triton-X is known to be harsh towards in vitro models and is used as positive control in the concentration range of 0.1-1 % for both caco-2 cells and PVPA (Fischer et al., 2011a; Fischer et al., 2011b). Figure 2 illustrates the permeability of calcein through PermeapadTM barrier in the presence and absence of surfactants and co-solvents in the form of Papp values. Although the permeability of calcein in some cases was increased by up to 30 %, and for most of the excipients a statistical difference was noted, the permeability was still considered very low compared to the “barrier support” (1.65 ± 0.1 · 105 cm/s) and hence the barrier intact. Permeability experiments with surfactants and co-solvents were carried out as consecutive rows of single experiments with increasing concentrations for each surfactant and checked for plausibility. Explorative barrier stability studies were carried out on the surfactants that showed a very high increase in permeability.
PVPA has been reported to be compatible with 6 % EtOH and 4 % DMSO (Flaten et al., 2008). However, the reported standard deviation of calcein permeability generally was much higher in the PVPA models than the deviations found in the present study, indicating a better tolerance of the PermeapadTM barrier towards these two solvents - with the appropriate reservations when comparing standard deviations across studies. The permeability of calcein through PermpeapadTM with 4 % EtOH and 10 % DMSO was 0.12 · 10-5 cm/s and 0.15 · 105 cm/s, respectively, which was not statistical significant from the results obtained in PBS, and these co-solvents were considered compatible with PermeapadTM. PAMPA has been reported to use 5 % DMSO in the stock solutions of drugs and 5 % EtOH, and is thus also compatible with these co-solvents at the given conentrations (Liu et al., 2003). Both PVPA and Caco-2 cells have been investigated with respect to MacrogolG R compatibility: PAMPA and PVPA have been reported to be incompatible with respectivly 0.5 and 4 % MacrogolG R (Liu et al., 2003), (Flaten et al., 2008), and Caco-2 cells have been reported to be compatible with MagrogolG R up to 10 % (Nerurkar et al., 1996). The permeability of calcein through PermpeapadTM in the presence of 5 % MacrogolG R was 0.23 · 10-5 cm/s. That is approximately a 2 fold the value of the permeability in PBS and statistically significantly different (p < 0.05). Therefore more explorative barrier functionality stability studies were carried out. The explorative studies were carried out in a different setup (Ussing chambers instead of Franz cells) so consecutive experiments could be carried out on the very same PermeapadTM barrier: the acceptor and donor compartments were emptied, washed and refilled with new media, without destroying or affecting the barrier. Results obtained for the permeability of calcein through PermeapadTM with 5 % macrogolG R in the Ussing chambers was 0.12 ± 0.01 · 105 cm/s, which was much lower and similar to the permeability observed in PBS, showing the PermeapadTM barrier to be compatible with 5 % MacrogolG R. The data
obtained in Franz cells probably lead to differences in the observed permeability due to insufficient stirring of the macrogolG R solution next to the barrier surface.
Interestingly, one of the tested surfactants resulted in a lower permeability value than the value obtained in PBS: The permeability of calcein in the presence of 5 % Polysorbate 80 was 0.06 · 10-5 cm/s, which was half the permeability obtained in PBS. A decrease in permeation does not indicate any destruction of the barrier, but rather the opposite. The decrease in permeability might be explained by a number of different hypotheses: The first could be that an interaction between Polysorbate 80 micelles (with or without calcein) and the surface of the barrier had occurred, resulting in micelle deposition on the surface of the barrier and thereby an increased resistance of the diffusion layers that calcein has to pass. A second possibility could be that a limited efficiency of stirring in the donor compartment next to the barrier surface might have resulted in a lower diffusion coefficient and thereby a lower permeation rate. Furthermore, for the 5 % Polysorbate 80 solution, a lag time of 1 hour was observed, which was only observed for the higher polysorbate concentrations. This supports both of the hypotheses above. Other in vitro models have previously been reported to be largely affected by the presence of surfactants such as polysorbate 80. For example, PVPA were not compatible with Polysorbate 80 in concentrations as low as 0.5 %, where the permeability of calcein was significantly increased, probably due to Polysorbate 80’s destruction of the lipid barriers (Flaten et al., 2008; Naderkhani et al., 2014). Caco-2 cells have been reported to be compatible with Polysorbate 80 with concentrations up to 1 % (Nerurkar et al., 1996), and PAMPA is compatible up to 5 % (Liu et al., 2003) however experiments are often carried out with only 0.2 % Polysorbate 80 (Liu et al., 2003). For
PermeapadTM the permeability decreased, which indicates that Polysorbate 80 did not disrupt the barrier, but instead interacts with calcein or with the barrier.
A lag time of 1 hour was also observed for 4 % Polysorbate 60. This indicates that both surfactants above their CMC interact with calcein and / or the barrier in a way that slows the diffusion of calcein through PermeapadTM. However, the permeability of calcein in the presence of 4 % Polysorbate 60 was different in comparison to Polysorbate 80. This was probably due to different molecular sizes of the two Polysorbate types. Polysorbate 80 is a larger molecule than Polysorbate 60, which results in a slightly lower diffusion coefficient and a slightly higher hydrophilic/lipophilic balance (HLB) 15 and 14.9 respectively.
The most interesting observation made was with Triton-X, a substance that in many cell-based in vitro models is used as a positive control in a concentration range of 0.1-1 % (Fischer et al., 2011b), i.e. destroying the barrier for recovery measurement. Studies with PVPA (Naderkhani et al., 2014) have used Triton-X in a concentration of 0.05 %, as a positive control, showing that PVPA loses its integrity when in contact with this surfactant. Results obtained for PermeapadTM show that Triton-X (up to a much higher concentration of 1 % M/V) did not affect the integrity of the barrier at all. The permeability of calcein through PermeapadTM was unchanged when comparing to the PBS reference, both resulted in Papp values of 0.12 · 10-5 cm/s. In summary all these results clearly show that PermeapadTM can withstand the different surfactants, co-solvents and Triton-X listed in Table 2 without affecting the integrity of the barrier and thereby the permeability of calcein.
3.2 Permeability studies with PermeapadTM, using simulated intestinal fluids FaSSIF and FeSSIF are interesting to use as media in both dissolution and permeation studies of oral drugs and oral dosage forms, since they can be used to mimic the gastrointestinal fluids in both the fasted and fed state. Using the BMM in in vitro studies may, hence give a more representative indication of how a drug might behave in vivo upon oral administration. The compositions of the four BMMs used, are shown in Table 1. Permeability studies with the different BMM’s were carried out on two different permeability setups; FaSSIF and FaSSIF-V2 were conducted in the Franz diffusion cells whereas FeSSIF and FeSSIF-V2 studies were carried out on the Ussing chamber, due to difficulties when employed in the Franz cell. When FeSSIF and FeSSIF-V2 were used in the Franz cells calcein was not homogeneously distributed: a layer of calcein accumulated on the top next to the barrier, not mixing properly with the rest of the acceptor phase. Increased viscosity was assumed to be the problem; however, viscosity measurements did not show increased viscosity for FeSSIF nor FeSSIF-V2 (data not shown). It is therefore assumed that the stirring in the Franz cell was not sufficient to distribute calcein properly when large amounts of sodium taurocholate NaTC were present in the media. Both media contain substantial amounts of NaTC and lecithin, when compared to the FaSSIF media. FeSSIF and FeSSIF-V2 were therefore investigated in the Ussing chamber where the magnetic stirring was more vigorous close to the barrier resulting in an even distribution of calcein in the respective cells. The results obtained from the permeability studies with the BMMs are presented in Table 3. The results show that the permeability values for calcein in FaSSIF, FaSSIF-V2 and FeSSIF-V2 were slightly
increased in comparison to PBS. In contrast to this, FeSSIF increased the permeability of calcein significantly (Papp = 0.53 ± 0.09 · 105 cm/s), thus more explorative barrier stability experiments were carried out with this medium. The barrier was first exposed to FeSSIF for 5 hours, then washed and re-tested for permeability using calcein solution in PBS as donor and PBS in the acceptor compartment. With this procedure a Papp for calcein in PBS of 0.27 ± 0.05 · 10-5 cm/s was found, after a 5 hour exposure to FeSSIF, which clearly indicates that the integrity of the barrier was not compromised, but that the effect is rather associated with the FeSSIF. A possible explanation could be that the high osmolality of FeSSIF (635 ± 10 mOsm) creates an osmotic pressure difference, which may induce an increase in permeability. To further investigate the effect of FeSSIF additional experiments were conducted with FeSSIF as acceptor media using caffeine and metorpolol as permeability markers. Caffeine was chosen due to its high permeability and high UV absorbance, while Metoprolol is a moderate permeability drug. The results for caffeine and metoprolol are presented in Figure 3. The permeability of caffeine through PermeapadTM was not significantly different when replacing PBS with FeSSIF as the acceptor medium (P > 0.05). The results for metoprolol also show that the permeability of metoprolol was not affected by the presence of the FeSSIF (P > 0.05). The barrier is clearly not disrupted by the medium, but other parameters influence the data. Hence, studies should be planned carefully when using FeSSIF together with the PermeapadTM barrier.
The effect of FaSSIF on Caco-2 cells has earlier been investigated by (Fossati et al., 2008; Ingels et al., 2004; Ingels et al., 2002) and was found compatible with Caco-2 cells, whereas FeSSIF was not (Ingels et al., 2002). However, data was also presented showing that sodium taurocholate (NaTC) has an inhibitory effect on P-glycoprotein (P-gp), which affects the active efflux of drugs (Ingels et al., 2004).
Studies with PermeapadTM in the different BMMs have shown the barrier to be stable over time and in the presence of the different BMMs, however, PermeapadTM is designed as a tool for predicting passive diffusion of drugs and therefore the effect of NaTC on P-gp will not be measurable.
Other artificial barriers have also been investigated together with BMMs. The PVPA model has been shown to be compatible with FaSSIF, although an increase in SD was observed (Buckley et al., 2012). The PAMPA model has been used to determine passive permeability of lipophilic molecules, using FaSSIF-V2 and FeSSIF-V2 as donor solutions (Markopoulos et al., 2013). The publication demonstrated no data to ensure the compatibility, but it must be assumed.
PAMPA is a non-cell based artificial membrane for the screening of drugs in relation to passive diffusion. It has over the recent years become more abundant, and different PAMPA models have been developed (Avdeef, 2005). PAMPA has the advantage of screening large amounts of new drug entities, due to the 96-well microtiter plate format – making it very ideal for High Throughput Screening (HTS) (Kansy et al., 1998). However, comparing PermeapadTM with PAMPA some advantages are noted in favour of PermeapadTM. Especially highly lipophilic drugs (Log P > 4) tend to accumulate in the lipidic phase of PAMPA, instead of the acceptor phase (Avdeef, 2005). This issue has not been a problem for PermeapadTM: all the permeability experiments have yielded high drug recovery values (90-100 % - data not shown). The clear advantages of PAMPA are the reproducibility and its ability to HTS, where PermeapadTM is still carried out on laboratory scale.
The present study has looked into the potential use of the new artificial barrier, PermeapadTM, in a number of media normally not compatible with other in vitro models. The PermeapadTM generally
showed good barrier function even when exposed to the tested excipients or media, suggesting a barrier with huge potential within particular in vitro models with combined dissolution and permeability, which future studies will investigate.
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