Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-015-0280-7

ORIGINAL PAPER

Combining Chemical Permeation Enhancers for Synergistic Effects Trizel du Toit1 • Maides M. Malan1 • Hendrik J. R. Lemmer1 • Chrisna Gouws1 Marique E. Aucamp1 • Wilma J. Breytenbach2 • Josias H. Hamman1

•

 Springer International Publishing Switzerland 2015

Abstract Currently, macromolecular drugs such as proteins are mainly administered by means of injections due to their low intestinal epithelial permeability and poor stability in the gastrointestinal tract. This study investigated binary combinations of chemical drug absorption enhancers to determine if synergistic drug absorption enhancement effects exist. Aloe vera, Aloe ferox and Aloe marlothii leaf gel materials, as well as with N-trimethyl chitosan chloride (TMC), were combined in different ratios and their effects on the transepithelial electrical resistance (TEER), as well as the transport of FITC-dextran across Caco-2 cell monolayers, were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers, such as complex formation. In terms of FITCdextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption

& Josias H. Hamman [email protected] 1

Faculty of Health Sciences, Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa

2

Statistical Consultation Services, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa

enhancers at lower concentrations compared to the single components.

Key Points In vitro evaluation of binary combinations of drug absorption enhancers. Determination of synergism by means of an adapted isobole method. Synergism occurred in some of the combinations in terms of TEER reduction and transport of model compound. Due to synergism, absorption enhancers can produce increased effects at lower concentrations.

1 Introduction Due to ease of administration and patient acceptability, the oral route of administration remains the preferred means of drug delivery [1, 2]. The term ‘‘drug absorption’’, with respect to oral administration, refers to the transport of drug molecules from the site of administration across the intestinal epithelium and into the blood surrounding the gastrointestinal tract [3]. Despite advances in biotechnology and the emergence of protein- and peptide-based drugs as therapeutics for the treatment of diseases such as diabetes mellitus [4], these therapeutic agents are mainly administered by means of the parenteral route due to their low intestinal epithelial permeability and poor stability in the gastrointestinal tract [5, 6]. The parenteral route of

T. du Toit et al.

administration (e.g. subcutaneous injection) is associated with discomfort, a risk of infection, hypertrophy of subcutaneous fatty tissue and immune response of the skin [7]. One of the major challenges to achieve effective oral delivery of protein and peptide drugs is the poor oral bioavailability due to poor penetration of the intestinal mucosa. Inclusion of safe and effective absorptions enhancers in oral dosage forms is one approach to ensure therapeutic levels after oral administration [8–10]. Absorption enhancers are compounds that temporarily disrupt or reversibly remove the intestinal barrier with minimum tissue damage, thus allowing a drug to penetrate the epithelial cells and enter the blood or lymph circulation [11]. Several compounds have shown the ability to enhance the absorption of drugs across the intestinal epithelium. Chitosan and its derivative, N-trimethyl chitosan chloride (TMC), have shown the ability to influence the integrity of epithelial tight junctions to increase paracellular transport of large hydrophilic compounds [12, 13]. Aloe vera gel enhanced the bioavailability of co-administered vitamins when taken orally in humans [14]. The gel and whole leaf materials from different aloe species as well as precipitated polysaccharides from these materials improved insulin transport across in vitro models such as Caco-2 cell monolayers and excised animal tissues [15, 16]. Synergism is a concept which refers to a situation where the effect of a mixture of compounds exceeds that expected from the effects of the individual components [17]. The use of binary combinations of permeation enhancers to create synergistic drug absorption enhancing effects has been investigated within the Caco-2 cell model. Some of the enhancer formulations (i.e. a combination of hexylamine and chembetaine) have increased mannitol transport 15-fold and FITC-dextran transport 8-fold, indicating the potential of achieving synergistic effects with combinations of absorption enhancers [18]. One of the most effective and practical methods, in terms of experimental design to demonstrate synergism, is the isobole method. This method is based on the concept of dose equivalence, which leads to the observation that if a combination (da, db) is represented by a point in a graph, the axes of which represent doses of A and B respectively, the point lies on the straight line joining Da and Db, thus satisfying the equation Ddaa þ Ddbb ¼ 1, if and only if there are no drug interactions [19]. The aim of this study is to determine if a synergistic drug absorption enhancement effect can be obtained when combinations in different ratios of leaf gel materials of three aloe species, namely A. vera, Aloe ferox and Aloe marlothii, as well as combinations with N-trimethyl chitosan chloride (TMC), are applied to Caco-2 cell monolayers. Isobolograms were constructed from the transport data of a model compound (FITC-dextran) to determine

which combinations of absorption-enhancing agents produced synergistic effects.

2 Materials and Methods 2.1 Materials Aloe vera gel powder was sourced from Warren Chem (Johannesburg, South Africa), A. ferox gel was obtained by freeze drying leaf pulp collected in the Western Cape Province of South Africa by Organic Aloe Pty Ltd. (Albertinia, South Africa) and A. marlothii gel was obtained by freeze-drying leaf pulp collected in the Northwest Province of South Africa. Caco-2 cells were purchased from the European Collection of Cell Cultures (ECACC by Sigma-Aldrich, South Africa), Transwell plates (6.5 mm inserts, 24-well plates with a 0.33 cm2 membrane surface area) and Transwell plates (24 mm inserts, 6-well plates with a 4.67 cm2 membrane surface area) were purchased from Corning Costar Corporation (Manassas, United States of America). Other materials for the cell culture experiments were sourced from the Scientific Group (Randburg, South Africa), including HEPES [n(2-hydroxyethyl), piperazine-N-(2-ethanesulfonic acid)] buffer solution, amphotericin B, foetal bovine serum (FBS) and Hank’s Balanced Salt Solution (HBSS) without phenol red. Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, 4.0 mM L-glutamine, sodium pyruvate and penicillin/streptomycin solution were purchased from Separations (Randburg, South Africa). L-glutamine (200 mM), nonessential amino acids (NEAA, 1009) and trypsin-versene (EDTA) mixture (19) were purchased from Whitehead Scientific (Cape Town, South Africa). The following materials were purchased from Sigma-Aldrich (Johannesburg, South Africa): Fluorescein isothiocyanate (FITC) dextran (MW = 4000), trypan blue solution (0.4 %) and phosphate buffered saline (PBS). ChitoClear (Chitosan) was purchased from Primex (Siglufjordur, Iceland). 2.2 Absorption Enhancer Combinations The binary combinations of the selected absorption enhancers are shown in Table 1, which were each tested in five different ratios namely 10:0, 8:2, 5:5, 2:8, 0:10 and at two concentrations of 0.1 and 0.5 % w/v for the TEER reduction studies. 2.3 Chemical Fingerprinting of Aloe Leaf Gel Materials All the aloe gel materials investigated in this study were chemically fingerprinted by means of proton nuclear

Combining Chemical Permeation Enhancers Table 1 Composition of binary combinations of absorption enhancers investigated in five different ratios

2.4.3 Prolongation of Reaction Step 2

Combination

Composition

1

A. vera gel and A. marlothii gel

At the end of reaction step 2, prior to precipitation of the product, an additional 5 ml of iodomethane and 10 ml of a 15 % (w/v) aqueous sodium hydroxide (NaOH) were added. The reaction was then allowed to continue for another hour at 60 C. The product was precipitated with absolute ethanol, washed with diethyl ether and dried under vacuum.

2

A. vera gel and A. ferox gel

3

A. marlothii gel and A. ferox gel

4

A. vera gel and TMC

5

A. ferox gel and TMC

6

A. marlothii gel and TMC

2.4.4 Ion-Exchange Step 1

magnetic resonance ( H-NMR) spectroscopy to determine the presence of marker molecules, which are commonly used to identify fresh aloe leaf gel material and to certify aloe-containing products [20]. An amount of 35 mg of each gel material was dissolved separately in 2 ml of deuterium oxide (D2O) with 5 mg 3-(trimethylsilyl)-propionic acid-D4 sodium salt (TPS) in an NMR tube and filtered through cotton wool. The 1HNMR spectra were recorded with an Avance III 600 Hz NMR spectrometer (Bruker BioSpin Corporation, Rheinstetlen, Germany) [21]. 2.4 Synthesis of N-Trimethyl Chitosan Chloride The N-trimethyl chitosan chloride (TMC) was synthesised based on the modified reductive methylation method previously described [22, 23]. 2.4.1 Reaction Step 1 For the first reaction step, 4 g of chitosan was dissolved in 160 ml of 1-methyl-2-pyrrolidinone. This solution was heated in a water bath to 60 C and 9.6 g of sodium iodide, 22 ml of a 15 % (w/v) aqueous sodium hydroxide (NaOH) solution and 23.5 ml of iodomethane were added. A Liebig’s condenser was used to keep the iodomethane in reaction. After reaching 60 C, the mixture was stirred for an hour and then removed from the water bath. An excess of absolute ethanol was added to the mixture and it was left to precipitate overnight. 2.4.2 Reaction Step 2 The product obtained from reaction step 1 was washed several times with diethyl ether on a glass filter and dried under vacuum. The polymer obtained was dissolved in 160 ml 1-methyl-2-pyrrolidinone and 9.6 g of sodium iodide, 22 ml of a 15 % (w/v) aqueous sodium hydroxide (NaOH) solution and 23.5 ml of iodomethane were added. The reaction was carried out in the presence of a Liebig’s condenser at 60 C, where it was stabilised for an hour.

To exchange the iodide ions on the product with chloride ions, the product obtained in the aforementioned step was dissolved in 100 ml of 10 % (w/v) sodium chloride solution and consequently precipitated using ethanol and diethyl ether. To remove the residual sodium chloride, the products were repeatedly dissolved in water and precipitated with ethanol and diethyl ether. The final product was thoroughly dried under vacuum. 2.5 Determination of the Degree of Quaternisation The TMC polymer obtained from the synthesis reaction was chemically characterised by means of proton nuclear magnetic resonance (1H-NMR) spectroscopy with an Avance III 600 Hz NMR spectrometer (Bruker BioSpin Corporation, Rheinstetlen, Germany). A sample of the polymer (35 mg) was dissolved in 2 ml D2O and a spectrum obtained from the NMR spectrometer at 80 C with suppression of the water peak. The degree of quaternisation was calculated from the 1H-NMR spectrum using the combined integrals of the H-3, H-4, H-5, H-6 and H-60 (6H) peaks at d 3.6–4.5 and H-2 peak at 3.10 ppm. The following equation was used to calculate the degree of quaternisation [24]:   ½NðCH3Þ3 6 % N-Trimethylation =  ½H-2, H-3, H-4, H-5, H-6, H-60  9 ð1Þ where [N(CH3)3] is the integral of the N-trimethyl singlet peak (3.30 ppm) and the integral H-3, H-4, H-5, H-6 and H-60 (6H) at d 3.6–4.5 ppm and H-2 peak at d 3.10 ppm represent six protons. The quaternisation degree is expressed as the percentage trimethylation (Ru´narsson et al. [24]:2662). 2.6 High-Performance Liquid Chromatography Analysis of FITC–Dextran Quantification of fluorescein isothiocyanate (FITC)–dextran in the transport samples was carried out using high-

T. du Toit et al.

performance liquid chromatography (HPLC) with size exclusion separation and fluorescence detection. The chromatographic system and conditions were as follows: liquid chromatographic system equipped (Spectra-Physics, CA, USA) with a pump (model P1000); autosampler (model AS3000); fluorescence detector (model FL2000), excitation wavelength 494 nm and emission wavelength 518 nm; PolySep-GFC-P Linear size exclusion column, 300 9 7.80 mm; and PolySep-GFC-P guard column, 35 9 7.80 mm (Phenomenex, United States of America distributed by Separations, Johannesburg, South Africa). The mobile phase consisted of acetonitrile: 0.05 M phosphate buffer (12:88) delivered at a flow rate of 1 ml/min. The buffer component of the mobile phase was prepared with deionised water and the pH was adjusted to 6.5. 2.7 Seeding and Culturing of Caco-2 Cell Monolayers Caco-2 cells (passages 52–60) were used for the TEER and in vitro transport studies. The cells were seeded and grown into monolayers on tissue culture treated polycarbonate permeable supports with an area of 0.33 cm2 in Costar Transwell 24-well plates for the TEER studies and on permeable supports (area 4.67 cm2) in Costar Transwell 6-well plates at a concentration of 2 9 104 cells/ml for both studies. Growth medium, consisting of Dulbecco’s Modified Eagle’s Medium (DMEM, pH 7.4) supplemented with 10 % v/v foetal bovine serum (FBS), 2 mM L-glutamine, 1 % v/v amphotericin B, 1 % v/v non-essential amino acids (NEAA) and 1 % v/v penicillin/streptomycin solution, was added to both the donor and acceptor chambers. The growth medium was changed every second day and the cell monolayers were used 21–23 days after seeding. Caco-2 cells were cultured at 37 C in a humidified atmosphere of 95 % air and 5 % CO2. 2.8 Transepithelial Electrical Resistance Studies A TEER value of the Caco-2 cell monolayers on the 24-well Transwell plates of at least 750 X (247.5 X cm2) was required prior to the commencement of the TEER experiments. The growth medium was removed from the basolateral chambers using an aspirator (Integra Vacusafe, Zizers, Switzerland) and replaced with 1 ml pre-warmed Hank’s Balanced Salt Solution (HBSS) and incubated at 37 C for 30 min. The TEER of the Caco-2 cell monolayers was measured using a Millicell ERS-2 meter (Millipore, Bedford, MA, USA) connected to chopstick electrodes. The TEER was measured at 20 min intervals starting 1 h prior to the addition of the test solutions on the apical chamber of the cells and continued for 2 h after the addition of the

test solutions (i.e. combinations of absorption enhancers as shown in Table 1 at concentrations of 0.1 and 0.5 % w/v). TEER measurements for the control groups were recorded under the same conditions. The normal control group consisted of the Caco-2 cells alone without addition of any chemical permeation enhancer. The positive control group consisted of a solution of TMC at a concentration of 0.1 and 0.5 % w/v, respectively, for the different experiments, all of which were done in triplicate with the Transwell plates kept in a CO2 incubator at 37 C in a humidified atmosphere of 95 % air and 5 % CO2 [16]. 2.9 In Vitro Transport Studies A TEER value of the Caco-2 cell monolayers on the 6-well Transwell plates of at least 250 X (1167.5 X cm2) was required prior to the commencement of the transport experiments. Although the TEER experiments, as an initial screening for the effects of the chemical permeation enhancer combinations, were conducted at two concentrations (i.e. 0.1 and 0.5 % w/v), the transport studies were conducted at the lowest concentration only (0.1 % w/v). The growth medium was removed from the basolateral chambers using an aspirator and each basolateral chamber was filled with 2.5 ml pre-warmed DMEM buffered with 25 mM HEPES (a mixture of 39 ml DMEM and 1 ml HEPES) and incubated at 37 C for 30 min. The medium in the apical chambers was then removed and 2.5 ml of each of the test solutions (i.e. combinations of absorption enhancers as shown in Table 1 at a concentration of 0.1 % w/v) were applied. Samples of 400 ll were taken at 0, 20, 40, 60, 80, 100 and 120 min from the basolateral chamber. The samples withdrawn were immediately replaced with an equal volume of buffered DMEM. The normal control group contained a solution of FITC-dextran without any permeation enhancer and the positive control group contained TMC (0.1 % w/v) together with FITC-dextran. Samples withdrawn were stored in HPLC vials until quantification by HPLC. 2.10 Isothermal Microcalorimetry To determine whether physical and/or chemical interactions occurred between different permeation enhancer combinations for each ratio, the method of isothermal microcalorimetry was used. The usefulness of this method lies within its ability to detect small, low energy interactions between compounds. A Thermal Activity Monitor (TAMIII) apparatus (TA Instruments, New Castle, DE, USA) equipped with an oil bath with a stability of ±100 lK over 24 h was used during this study. The temperature of the samples (absorption enhancer combinations as shown in Table 1 at 0.1 % w/v) was maintained at 60 C

Combining Chemical Permeation Enhancers

throughout the monitoring of the heat flow. To determine interactions between the different materials used in the combinations studies, the heat flow was measured for the single components as well as the combinations. The samples were run against an inert reference (an empty sealed ampoule). The calorimetric outputs observed for the individual samples were summed to give an additive hypothetical response. This calculated hypothetical response represents an expected calorimetric output if the two materials do not interact with each other. If the materials interact, the measured calorimetric response will differ from the calculated hypothetical response. A heat flow difference of more than 100 lW/g was considered a significant difference indicative of a physical and/or chemical interaction between two compounds. Correlation of the interaction data obtained by microcalorimetry with the transport data enabled us to relate such interactions with either synergistic or antagonistic effects. The physical and/or chemical interactions between absorption enhancers in the different combinations, at each ratio, were therefore used to help interpret or explain the effects obtained on the transport of FITC-dextran. 2.11 Data and Statistics Analysis 2.11.1 Percentage TEER Reduction The percentage TEER reduction was obtained by subtracting the percentage TEER values at times 60 and 120 min from the TEER value at time 0 (i.e. 100 %), which quantitatively expresses the extent to which each experimental group opened the tight junctions between Caco-2 cells in the monolayers. 2.11.2 Apparent Permeability (Papp) Coefficient Values Apparent permeability is defined as the initial flux of a compound across the membrane normalised by membrane surface area and donor concentration. This index is widely used as part of a general screening process to study drug absorption with in vitro and ex vivo experiments and is calculated by means of the following equation [25]:   dQ 1 Papp ¼ ð2Þ dt ðA  60  C0 Þ where Papp is the apparent permeability coefficient (cm s-1), dQ/dt is the permeability rate (amount permeated per minute), A is the diffusion area of the monolayer (cm2) and C0 is the initial concentration of the model drug. 2.11.3 Isobole Method According to Berenbaum [19], the zero interaction or additive effect relies on the mechanism that the combined

effect of two components is a pure summation effect (Eq. 3). This means the components do not interact and the line connecting the point is representative of the single doses with the same effect as the combinations, will be a straight line [19, 26]. If synergism occurs, the total effect of two components that are applied together as a mixture must be greater than it would be expected by the summation of the component’s separate effects [27, 28]. This will result in a concave curve and is defined by Eq. 4. The opposite applies for antagonism, in which case an overall effect of two components is less than expected from the summation of the effects obtained from the individual components [19, 26, 28]. Antagonistic interactions will result in a convex curve and can be defined by Eq. 5. Eðda ; db Þ ¼ Eðda Þ þ Eðdb Þ

ð3Þ

Eðda ; db Þ [ Eðda Þ þ Eðdb Þ

ð4Þ

Eðda ; db Þ \Eðda Þ þ Eðdb Þ

ð5Þ

where E is the observed effect, and da and db are the doses of components a and b. Since the isobole method was originally designed to use the doses of two or more drugs, with constant potency ratios needed to achieve a specific therapeutic effect, it had to be modified to accommodate components of unknown molecular weight. The need for this modification arises from the difficulty in isolating the individual components of a complex mixture such as the aloe gel and whole leaf materials used in this study. To achieve this, the isobole method was extended to a higher dimensional multivariable problem in which the isobologram is seen as the n-dimensional reflection from an (n ? 1)-dimensional hyperspace containing the drug ratios and observed effects, where n is the number of drugs being tested. This (n ? 1)dimensional isobologram depends explicitly on the observed effects and relates the ratios of the therapeutic agents to its corresponding effects in such a way that all the information usually found in the classic n-dimensional isobologram is maintained. This enables the researcher to obtain the desired drug interaction information directly from the ratios and its corresponding effects. Although mathematical proof is not presented in this paper, it can be shown mathematically that the drug ratio-effect data can be expressed as vectors in Rn?1 which all extend from the origin to an n-dimensional plane that is normal to the ratio axes. This (n ? 1)-dimensional isobologram can be related to the classic n-dimensional isobologram by the matrix T, in that T: V ? W is a linear transformation, where V is the basis drug ratio-effect vectors and W is the basis drug dose vectors of the isobologram. If a polynomial is fitted to the points on the isobologram, a similar polynomial can be fitted to the drug ratio-effect points, containing the same maxima, minima and inflection points. The method used to

T. du Toit et al.

draw the (n ? 1)-dimensional isobolograms and the procedure can easily be adapted to any computer software package. The procedure (presented here for n = 2): •

Express the ratios and corresponding effects as vectors in matrix form, e.g. 2 3 1 0 Eda 6 0:8 0:2 Eðda ; db Þ 7 6 7 A ¼ 6 0:5 0:5 Eðda ; db Þ 7 4 0:2 0:8 Eðd ; d Þ 5 a b 0 1 Edb •

Find the equation of a plane that extends from the origin through the points (1, 0, Eda) and (0, 1, Edb) to the point (1, 1, Eda þ Edb ). Let p be a point on the plane and let n be a vector orthogonal to the plane, which can be found by:   i j k   det 1 0 Eda  0 1 Edb  The Cartesian equation of the plane through the origin can therefore be found from the point products ðx; y; zÞ  ðnÞ ¼ ð pÞ  ðnÞ •

Calculate the values of z (the effect axis) which correspond to the different ratios. These z-values represent the expected additive effect values. Express the ratios and corresponding additive effects as vectors in matrix form, e.g.: 2

6 6 B¼6 4 •

ð6Þ

1 0:8 0:5 0:2 0

0 0:2 0:5 0:8 1

Eda Eda þ Edb Eda þ Edb Eda þ Edb Edb

3 7 7 7 5

The matrices A and B were plotted to obtain a 3D graph containing the experimental and expected additive values associated with each drug ratio.

2.11.4 Statistical Analysis of Results The following statistical tests were done using Statistica software (StatSoft, Inc. 2012, Tulsa, Oklahoma, United States of America) to determine if the mean effects obtained from the combinations of permeation enhancers differed from those of the control group by a statistically significant amount. All tests were performed at the 0.05 level of significance.

One-way analyses of variance (ANOVA) were done to determine if statistical significant differences exist between the mean percentage TEER reduction values of the experimental groups and each of the control groups in general. These procedures were also done when analysing the mean Papp values to determine significant differences between the experimental groups and each of the control groups. These were done for TEER data on concentrations 0.1 and 0.5 % w/v and for transport data on concentration 0.1 % w/v. Levenes’ tests were performed in each ANOVA’s case to assure equality of variances. In cases of inequality of variances, Welch tests were performed. Normal probability plots on the residuals were done in each analysis to ensure that the data was fairly normally distributed [29]. Dunnett’s post hoc tests were finally done in each ANOVA’s case to determine which of the test compounds’ means differed statistically significant from the means of each of the control compounds.

3 Results and Discussion 3.1 Chemical Fingerprinting of Aloe Leaf Gel Materials The 1H-NMR spectra obtained for A. vera, A. marlothii and A. ferox are respectively illustrated in Fig. 1. It is evident, from the 1H-NMR spectrum of A. vera leaf gel material, that the marker molecules for fresh A. vera gel material namely aloverose (partly acetylated polymannan or acemannan), glucose and malic acid are present together with low levels of lactic acid and formic acid. In general, high amounts of lactic acid can indicate bacterial degradation due to Lactobacillus, while acetic acid and formic acid are present due to hydrolysis of aloverose and thermal degradation of glucose during storage. According to the 1H-NMR spectra of the A. marlothii and A. ferox leaf gel materials, glucose and small amounts of lactic acid are present. Other phytochemicals such as malic acid, acetic acid, formic acid, citric acid and benzoic acid are also identifiable on the spectra, but aloverose is absent. These findings are in accordance with previously published data, which showed aloe species indigenous to South Africa (e.g. A. ferox) do not contain aloverose [30]. 3.2 Degree of Quaternisation of N-Trimethyl Chitosan Chloride The 1H-NMR spectrum obtained for the synthesised TMC is shown in Fig. 2, followed by the calculation of the degree of quaternisation.

Combining Chemical Permeation Enhancers Fig. 1 1H-NMR spectra of the a Aloe vera leaf gel material, b Aloe marlothii leaf gel material and c Aloe ferox leaf gel material investigated in this study

T. du Toit et al.

% N-Trimethylation   ½N ðCH3Þ3 6  100 ¼  ½H-2, H-3, H-4, H-5, H-6, H-60  9   ½22:21 6   100 ¼ 50:81 % ¼ ½28:78þ0:36 9 where [N(CH3)3], [N(CH3)2], [N(CH3)] are the integrals of the N-trimethylamino (3.30 ppm), N-dimethylamino (d 2.87 or 3.00 ppm) and N-monomethylamino (d 2.77 or 2.80 ppm) singlet peaks, respectively. 3.3 Transepithelial Electrical Resistance (TEER) Studies The percentage TEER reduction values after 120 min exposure to the different absorption enhancer combinations at concentrations of 0.1 and 0.5 % w/v, respectively, are shown in Fig. 3a, b. The TEER of the negative control group (i.e. Caco-2 cell monolayers without chemical enhancer addition) remained constant at, or slightly above, the initial TEER value, which indicated the cell monolayers stayed intact and therefore a 0 % reduction was recorded for this group. It is clear from Fig. 3a that some of the single absorption enhancers as well as some of the combinations between the different aloe species gel materials had a statistically significant (p B 0.05) reduction effect on the TEER of the Caco-2 cell monolayers when compared to the negative control group. Some of the aloe material combinations with TMC showed a higher TEER reduction effect compared to Fig. 2 1H-NMR spectrum of Ntrimethyl chitosan chloride (TMC)

those of the positive control group (i.e. TMC alone which is a well-known tight junction modulator and absorption enhancer). In general, the TEER reduction effect of all combination ratios at concentration 0.5 % w/v was higher than at concentration 0.1 % w/v. A reduction in TEER is associated with opening of tight junctions between epithelial cells that enhances paracellular transport of macromolecules. However, a risk of toxicity exists with absorption enhancers that modulate opening of tight junctions. This toxicity may include cell membrane disruption and/or influx of unwanted solutes other than drugs [31]. The cytotoxicity of aloe gel and whole leaf materials has been investigated [32] as well as for TMC [33]. These studies indicated that the absorption enhancers investigated in this study did not exhibit cytotoxic effects on epithelial cells at the concentrations utilised in this study. Some of the ratio combinations between A. vera and A. marlothii at 0.1 % w/v, as well as between A. vera and A. ferox at 0.5 % w/v showed enhanced TEER reduction effects when compared to the single components. Almost all ratios of TMC and aloe gel combinations (i.e. combination 4, 5 and 6) had a statistically significant higher reduction effect (p B 0.05) on the TEER compared to those of the negative control group. The following ratio combinations between aloe gel material and TMC resulted in increased TEER reduction effects compared to that of at least one of the single components: A. vera and TMC at ratios 8:2; 5:5 and 2:8; A. ferox and TMC at 5:5 and 2:8; A. marlothii and TMC at ratios 5:5 and 2:8.

Combining Chemical Permeation Enhancers

100

a

90

70

*

*

*

8:2 5:5 2:8 0:10

10:0

* 0:10

* * 10:0 8:2 5:5 2:8 0:10

5:5 2:8

8:2

10:0

5:5 2:8 0:10

10:0 8:2

*

2:8

*

*

10

*

*

*

20

0

*

8:2 5:5 2:8

*

30

*

*

*

10:0

* *

0:10

40

10:0 8:2 5:5

50

*

*

*

60

0:10

% TEER Reduction

80

AM / TMC AV / AF AV / AM AM / AF AV / TMC AF / TMC Combination 1 Combination 2 Combination 3 Combination 4 Combination 5 Combination 6

100

b

*

90

*

% TEER reduction

80

*

*

*

*

*

70

* *

60

*

*

50

*

*

40

*

30 20

0:10

5:5 2:8

8:2

10:0

0:10

5:5 2:8

8:2

10:0

5:5 2:8 0:10

10:0 8:2

0:10

5:5 2:8

10:0 8:2

0:10

8:2 5:5 2:8

10:0

0:10

5:5 2:8

0

10:0 8:2

10

AM / TMC AF / TMC AV / TMC AM / AF AV / AF AV / AM Combination 1 Combination 2 Combination 3 Combination 4 Combination 5 Combination 6 Fig. 3 Percentage TEER reduction of Caco-2 cell monolayers at 120 min for all combinations at a concentration 0.1 % w/v and b concentration 0.5 % w/v. Bars on the graph marked with asterisk

indicates statistically significant differences with the negative control group (p B 0.05). AVAloe vera, AM Aloe marlothii, AF Aloe ferox, TMC N-trimethyl chitosan chloride

The results from the TEER studies therefore indicate potential interactions between the components of some of the combinations investigated, which may result in improved drug absorption enhancement effects. To determine whether these combinations of absorption enhancers produce additive, synergistic or antagonistic effects in terms of drug absorption, their effects on FITC-dextran transport across Caco-2 cell monolayers were measured.

3.4 In Vitro Transport Studies The FITC-dextran transport results (i.e. % transport plotted as a function of time) were processed to calculate the apparent permeability coefficient (Papp) values, which are shown in Table 2. From Table 2, it can be concluded that most of the combinations of absorption enhancers had higher effects on

T. du Toit et al. Table 2 The apparent permeability coefficient values (Papp) for FITC-dextran in the presence of different combinations of absorption enhancers (0.1 % w/v) Absorption enhancers

Papp 9 10-8 (cm/s) Ratio 10:0

Ratio 8:2

Ratio 5:5

Ratio 2:8

Ratio 0:10

Combination 1

1.5 ± 0.09

2.6 ± 0.08

3.3 ± 0.18

13.6 ± 0.27*

2.2 ± 0.04

Combination 2

1.5 ± 0.09*

2.6 ± 0.002*

2.6 ± 0.02*

4.2 ± 0.04*

7.6 ± 0.04*

Combination 3 Combination 4

2.2 ± 0.04 1.5 ± 0.09

5.2 ± 0.05 3.7 ± 0.03

7.0 ± 0.05 61.1 ± 0.73*

24.4 ± 0.82* 18.9 ± 0.22*

7.6 ± 0.04 5.2 ± 0.01

Combination 5

7.6 ± 0.04*

11.0 ± 0.04*

14.1 ± 0.34*

9.3 ± 0.02*

5.2 ± 0.01*

29.4 ± 1.81*

17.0 ± 0.47

2.8 ± 0.02

5.2 ± 0.01

Combination 6

2.2 ± 0.04

Negative control (FITC-dextran alone)

0.7 ± 0.002

Positive control (FITC-dextran and TMC)

5.2 ± 0.01

Values marked with * are statistically significantly different from the negative control group (p B 0.05) (n = 3, mean ± SD)

FITC-dextran transport than each of the components on their own. Although all the ratios (10:0, 8:2, 5:5, 2:8, 0:10) of combination 1 and combination 3 produced higher Papp values for FITC-dextran transport than the negative control group (FITC-dextran alone), only ratio 2:8 of each of these combinations exhibited a statistically significant (p B 0.05) higher transport of FITC-dextran. All the ratios of combinations 2 and 5 had a statistically significant effect (p B 0.05) on FITC-dextran transport when compared to the negative control group. The isobolograms for all the combinations investigated in this study are shown in Fig. 4. Figure 4a suggests that synergism, in terms of FITCdextran transport enhancement across Caco-2 cell monolayers, was obtained at all ratios of combination 1 (i.e. A. vera gel combined with A. marlothii gel). This is in-line with the TEER reduction results obtained for combination 1 at a concentration of 0.1 % w/v, which indicated improved TEER reduction effects at most of the ratios compared to those of the single components. Microcalorimetric data did not indicate any interactions occurring between the A. vera and A marlothii gel materials. Therefore, it can be deduced that the two compounds contribute individually to the synergistic effect observed with the enhanced transport of FITC-dextran across the Caco-2 cell monolayers. Conversely, combination 2 (i.e. A. vera gel combined with A. ferox gel as shown in Fig. 4b) resulted in an additive effect (or zero interaction) at ratio 8:2, whilst the other two ratios (i.e. 5:5 and 2:8) resulted in antagonism. This is in-line with the TEER reduction results obtained for combination 2 at a concentration of 0.1 % w/v. A possible explanation for this negative interaction between A. vera gel and A. ferox gel, in terms of FITC-dextran transport, may be a physical or chemical interaction between the phytochemicals of these two gel materials. The microcalorimetric results indicated that interactions did occur at ratios 8:2, 5:5 and 2:8 of combination 2.

Combining A. marlothii gel with A. ferox gel (combination 3), as well as combining A. ferox and TMC (combination 5), resulted in synergistic effects on FITC-dextran transport as evident from Fig. 4c, e. A combination of A. vera with TMC (combination 4 as shown in Fig. 4d), resulted in synergism at ratios 5:5 and 2:8 in terms of FITCdextran transport enhancement, whilst an additive effect was obtained at ratio 8:2. The isothermal microcalorimetry results indicated no interaction between A. vera and TMC in ratios 5:5 and 2:8, therefore showing the synergistic effect on the FITC-dextran transport is not effected through an interaction, but rather through the combined effect of each separate compound results in enhanced FITC-dextran transport. However, microcalorimetric evaluation of the 8:2 ratio of combination 4 showed an interaction between A. vera and TMC. This interaction influenced the FITCdextran transport detrimentally. For combination 6 (i.e. TMC and A. marlothii), synergism was observed at ratios 8:2 and 5:5, whilst antagonism was observed at ratio 2:8, where TMC was in majority. From the results of the isothermal heat-conduction calorimetry, it was evident that an interaction between A. marlothii and TMC occurred at ratio 2:8 which can explain the antagonistic effect at this specific combination ratio.

4 Conclusion The results from this study indicated that combinations of certain drug absorption enhancers can produce synergetic effects in terms of tight junction modulation of epithelial cell monolayers, whilst others cause additive or antagonistic effects. Furthermore, the type of effect is dependent on the concentration and ratio of the binary mixture. Contradictory effects between ratios of the same combination could be explained by physical or chemical interactions between the components of the materials at

Combining Chemical Permeation Enhancers

Fig. 4 Isobolograms of the apparent permeability coefficient (Papp) values of FITC-dextran in the presence of different ratios of a combination 1, b combination 2, c combination 3, d combination 4, e combination 5 and f combination 6

that specific ratio microcalorimetry.

combination

as

indicated

by

Acknowledgments This work was carried out with the financial support of the National Research Foundation of South Africa. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and therefore the NRF do not accept any liability with regard thereto.

Conflict of interest

The authors report no conflict of interest.

References 1. Daugherty AL, Mrsny RJ. Transcellular uptake mechanisms of the intestinal epithelial barrier: part one. Pharm Sci Tech Today. 1999;2:144–51.

T. du Toit et al. 2. Renukuntla J, Vadlapudi AD, Patel A, Boddu SHS, Mitra AK. Approaches for enhancing oral bioavailability of peptides and proteins. In J Pharm. 2013;447:75–93. 3. Hamman JH. Oral drug delivery, biopharmaceutical principles, evaluation and optimization. Pretoria: Content Solutions; 2007. p. 86. 4. Antosova Z, Mackova M, Kral V, Macek T. Therapeutic application of peptides and proteins: parenteral forever? Trends Biotechnol. 2009;27:628–35. 5. Crommelin D, Van Winden E, Mekking A. Delivery of pharmaceutical proteins. In: Aulton ME, editor. Aulton’s pharmaceutics: the design and manufacture of medicines. New York: Churchill Livingstone; 2002. p. 616–25. 6. Al-Hilal TA, Park J, Alamb F, Woo Chung S, Park JW, Kim K, Kwon IC, Kim IS, Kim SY, Byun Y. Oligomeric bile acid-mediated oral delivery of low molecular weight heparin. J Control Release. 2014;175:17–24. 7. Nolte MS, Karam MD, Karam JH. Pancreatic hormones and antidiabetic drugs. In: Weitz M, Lebowitz H, editors. Basic and clinical pharmacology. San Francisco: McGraw-Hill; 2003. p. 693–714. 8. Legen I, Salobir M, Kercˇ J. Comparison of different intestinal epithelia as models for absorption enhancement studies. Int J Pharm. 2005;291:183–8. 9. Hamman JH, Enslin GM, Kotze´ AF. Oral delivery of peptide drugs. BioDrugs. 2005;19:165–77. 10. Brayden DJ, Maher S. Oral absorption enhancement: taking the next steps in therapeutic delivery. Ther Deliv. 2010;1:5–9. 11. Muranishi S. Absorption enhancers. Crit Rev Ther Drug Carrier Syst. 1990;7:1–33. 12. Kotze´ AF, Lueßen HL, De Leeuw BJ, De Boer AG, Verhoef JC, Junginger HE. Effect of the degree of quaternization of N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2). Eur J Pharm Biopharm. 1999;47:269–74. 13. Rosenthal R, Gu¨nzel D, Finger C, Krug SM, Richter JF, Schulzke J-D, Fromma M, Amasheh S. The effect of chitosan on transcellular and paracellular mechanisms in the intestinal epithelial barrier. Biomaterials. 2012;33:2791–800. 14. Vinson JA, Al Kharrat H, Andreoli L. Effect of Aloe vera preparations on the human bioavailability of vitamins C and E. J Phytomed. 2005;12:760–5. 15. Beneke C, Viljoen A, Hamman J. In vitro absorption enhancement effects of Aloe vera and Aloe ferox. Sci Pharm. 2012;80:475–86. 16. Lebitsa T, Viljoen A, Lu Z, Hamman JH. In vitro drug permeation enhancement potential of aloe gel materials. Curr Drug Deliv. 2012;9:297–304. 17. Howard GJ, Webster TF. Generalized concentration addition: a method for examining mixtures containing partial agonists. J Theor Biol. 2009;259:469–77.

18. Whitehead K, Karr N, Mitragotri S. Discovery of synergistic permeation enhancers for oral drug delivery. J Control Release. 2008;128:128–33. 19. Berenbaum MC. What is synergy? Pharmacol Rev. 1989;41:93–129. 20. Chen W, Lu Z, Viljoen A, Hamman J. Intestinal drug transport enhancement by Aloe vera. Planta Med. 2009;76:587–95. 21. Campestrini LH, Silveira JLM, Duarte MER, Koop HS, Noseda MD. NMR and rheological study of Aloe barbadensis partially acetylated glucomannan. Carbohydr Polym. 2013;94:511–9. 22. Polnok A, Borchard G, Verhoef JC, Sarisuta N, Junginger HE. Influence of methylation process on the degree of quaternization of N-trimethyl chitosan chloride. Eur J Pharm Biopharm. 2004;57:77–83. 23. Sieval AB, Thanou M, Kotze´ AF, Verhoef JC, Brussee J, Junginger HE. Preparation and NMR characterization of highly substituted N-trimethyl chitosan chloride. Carbohydr Polym. 1998;36:157–65. 24. Ru´narsson OV, Holappa J, Nevalainen T, Hja´lmarsdo´ttir M, Ja¨rvinen T, Loftsson T, Einarsson JM, Jo´nsdo´ttir S, Valdimarsdo´ttir M, Ma´sson M. Antibacterial activity of methylated chitosan and chitooligomer derivatives: synthesis and structure activity relationships. Eur Polym J. 2007;43:2660–71. 25. Palumbo P, Picchini U, Beck B, Van Gelder J, Delbar N, Degaetano A. A general approach to the apparent permeability index. J Pharmacokinet Phar. 2008;35:235–48. 26. Williamson EM. Synergy and other interactions in phytomedicines. J Phytomed. 2001;8:401–9. 27. Wagner H, Ulrich-Mezenich G. Synergy research: approaching a new generation of phytopharmaceuticals. J Phytomed. 2009;16:97–110. 28. Breitinger HG. Drug synergy–mechanisms and methods of analysis. 2012. http://www.intechopen.com/books/toxicity-and-drugtest. Accessed 2 July 2014. 29. Tabachnick BG, Fidell LS. Using multivariate statistics. Boston: Allyn and Bacon; 2001. 30. O’Brien C, Van Wyk B-E, Van Heerden FR. Physical and chemical characteristics of Aloe ferox leaf gel. S Afr J Bot. 2011;77:988–95. 31. Kondoh M, Takahashi A, Yagi K. Spiral progression in the development of absorption enhancers based on the biology of the tight junctions. Adv Drug Del Rev. 2012;64:515–22. 32. Du Plessis L, Hamman J. In vitro evaluation of the cytotoxic and apoptogenic properties of aloe whole leaf and gel materials. Drug Chem Toxicol. 2014;37:169–77. 33. Thanou MM, Kotze´ AF, Scharringhausen T, Lueßen HL, de Boer AG, Verhoef JC, Junginger HE. Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal Caco-2 cell monolayers. J Control Release. 2000;64:15–25.