Identification and Molecular Interpretation of the Effects of Drug

Article pubs.acs.org/molecularpharmaceutics

Identification and Molecular Interpretation of the Effects of Drug Incorporation on the Self-Emulsification Process Using Spectroscopic, Micropolarimetric and Microscopic Measurements A. Mercuri,† P. S. Belton, P. G. Royall,‡ and S. A. Barker* School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, U.K. S Supporting Information *

ABSTRACT: Addition of a drug to a self-emulsifying drug delivery system (SEDDS) can affect the emulsification process after administration, leading to variation in the emulsion droplet size formed and potentially its clinical behavior (Mercuri et al., Pharm. Res., 2011, 28, 1540−1551). However, the mechanisms involved and, in particular, the location of the drug within the system are poorly understood. Here, we have investigated the location of a model drug, ibuprofen, in the emulsions formed from a simple anhydrous SEDDS (soybean oil, Tween 80 and Span 80), using a range of physical characterization techniques. 1H NMR studies showed an interaction between the drug and the polyoxyethylene chains of the surfactant Tween 80. Micropolarity assessment of the emulsion droplet interfacial region, using the chemical probes pyrene and Reichardt’s dye, confirmed this interaction, and suggested that the drug was altering the microenvironment around the surfactants, and hence the behavior of the SEDDS with water during emulsification. Both dielectric spectroscopy and polarized light microscopy highlighted the differential behavior with water of placebo and drug-loaded SEDDS, also seen in the initial visual observational studies on the emulsification performance of the SEDDS. 1H NMR studies with three other NSAIDs indicate that this effect is not confined to ibuprofen alone. The study has therefore indicated that the drug's influence on the emulsification process may be related to interactions within the microenvironment of the surfactant layer. Furthermore, such interactions may be usefully identified and characterized using a combination of micropolarity, spectroscopic and microscopic methods. KEYWORDS: SEDDS, emulsification, ibuprofen, NMR, dielectric, micropolarity

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INTRODUCTION Self-emulsifying drug delivery systems (SEDDS) have been studied extensively as potential oral delivery vehicles for poorly water-soluble drugs, with several recent reviews summarizing the formulation development, therapeutic utility and biological fate of these systems.1−4 SEDDS form a subsection of lipidbased formulations, comprising an anhydrous mixture of drug(s) with oil(s) and surfactant(s). Upon introduction of a SEDDS formulation to an aqueous phase, an emulsion is formed with minimal energy input. Hence, administration of a SEDDS formulation orally should result in the generation of an emulsion in the gastric compartment, the required energy being supplied by normal gastric movement. There are many cases in the literature of SEDDS having been reported as providing increased oral bioavailability of poorly water-soluble drugs, and some commercial products are based on this approach, the first one being ciclosporin A (Sandimmune Neoral). However, given their potential, the number of marketed products is still low. Although the precise mechanisms of bioavailability enhancement are not fully elucidated, proposed mechanisms include presenting the drug to the gastrointestinal tract in a © 2012 American Chemical Society

molecular dispersion, inhibition of efflux transporters such as Pglycoprotein, modification of intestinal membrane permeability, moderation of gastric function such as the stimulation of production of chylomicrons and increase in lymphatic transport.1,5,6 A recent review7 gave a recommendation for the development pattern for a SEDDS product: identification of a suitable solvent system, elucidation of the phase behavior of this system, in vitro assessment of likely in vivo performance, dynamic lipolysis and finally in vivo preclinical assessment. However, the effect that an incorporated drug may have on the physical behavior of a SEDDS, and the predictability of such interactions, may not be explicitly studied, but may be of considerable importance in determining the overall product behavior. Received: Revised: Accepted: Published: 2658

April 18, 2012 August 3, 2012 August 8, 2012 August 22, 2012 dx.doi.org/10.1021/mp300219h | Mol. Pharmaceutics 2012, 9, 2658−2668

Molecular Pharmaceutics

Article

We have previously reported8 that the incorporation of ibuprofen at its clinical dose into a SEDDS formulation altered the emulsification behavior of the system, resulting in a smaller emulsion droplet size, and postulated that this was due to the effect that the drug had on the phase behavior of the system and its tendency to form liquid crystals. Here we use a range of physical characterization techniques to elucidate the mechanism of this interaction, particularly in terms of establishing the location of the drug within the SEDDS formulation excipients and the emulsion formed from the SEDDS. Ibuprofen is a BCS class II drug, showing low aqueous solubility and high permeability, and therefore is representative of a class of drug whereby lipid delivery systems may be recommended to enhance bioavailability. Three other nonsteroidal anti-inflammatory drugs (NSAIDs) were also studied to assess the generalizability of the ibuprofen results: naproxen and flurbiprofen are similar in chemical structure and physicochemical properties to ibuprofen, all three being arylpropionic acid derivatives, whereas indometacin, although a weak carboxylic acid, as are the other drugs, has a much bulkier structure based around an indole moiety. The chemical structures of the model compounds are shown in Figure 1. A

simple system was required to facilitate interpretation of the experimental data. Full details of the chemical composition of the SEDDS excipients are given in the Supporting Information. Briefly, soybean oil is a combination of triacylglycerols of various fatty acids, the major component of Span 80 is 1,4-sorbitan monooleate, and Tween 80 consists of a complex mixture of the partial esters of sorbitol with oleic acid and 20 mol of ethylene oxide per mole of sorbitol. Figure 1 shows the nominal chemical structures of the two surfactants used. For formulation calculations here, nominal molecular weights of 428.6 and 1,310 have been used for Span 80 and Tween 80, respectively. A range of physical analytical techniques were used to characterize the anhydrous SEDDS and the emulsions formed from them, including 1H NMR, dielectric spectroscopy, polarity assessment and optical microscopy. These techniques were chosen because they probed different aspects of the physical structure of the SEDDS emulsions and/or the potential interactions between the excipients and the drugs, thus providing complementary information and allowing more extensive analysis than a single technique would. To our knowledge, this is the first time such a combination of techniques has been used to examine SEDDS formulations. The polarity assessment methods and dielectric spectroscopy are described briefly here and in more detail in the Supporting Information. The interfacial polarity of the emulsion droplet has been assessed using two dissolved chemical probes, pyrene and Reichardt’s dye, which respond in different ways to changes of polarity of the medium. Pyrene (benzo[def ]phenanthrene), shown in Figure 1, exhibits a fluorescence emission spectrum composed of five vibronics bands, and the ratio of the intensities at 373 and 383 nm (the I1/I3 ratio) is commonly used as a polarity index.9−11 The pyrene I1/I3 polarity index can be directly correlated to the dielectric constant of a material, hence it can be used with appropriate calibration to give a measure of the apparent dielectric constant of the system under study. Reichardt’s dye (2,6-diphenyl-4-(2,4,6-triphenyl-1pyridinio)phenolate; betaine 30; RD), shown in Figure 1, shows a differential UV−vis absorbance pattern depending on the nature of the solvent in which it is dissolved. The observed maximum in absorbance can be transformed into the molar electronic transition energy of dissolved RD and used to estimate polarity by reference to a standard scale.12,13 Dielectric spectroscopy is an electrical technique in which an alternating current is induced in a sample by polarization and relaxation of dipoles following the application of a sinusoidal electric field. The measured current will generally vary with the frequency of the applied field, and such variation can aid interpretation of the structure and function of the sample. The overall aim of this study was to use this series of physical techniques to elucidate the location within the SEDDS emulsion of a range of incorporated drugs, and to identify any physicochemical interactions between the drug and the formulation excipients. It is intended that, in this manner, the relationship between drug incorporation and the associated emulsification properties and performance of the corresponding SEDDS formulation may be better understood.

Figure 1. Chemical structures of ibuprofen (1); naproxen (2); flurbiprofen (3); indometacin (4); pyrene (5); the zwitterionic form of Reichardt’s dye (6); 1,4-sorbitan monooleate (Span 80) (7); Tween 80 (8).

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simple SEDDS formulation consisting of soybean oil, Tween 80 and Span 80 was used, the rationale being that these excipients are all pharmaceutically acceptable and their mixtures show a range of self-emulsification behavior depending on the exact ratios used. Additionally, for this exploratory study a relatively

MATERIALS AND METHODS Materials. Span 80 (Sigma, Poole, U.K.), Tween 80 (SigmaAldrich, Gillingham, U.K.), soybean oil (Sigma, Poole, U.K.), flurbiprofen (Sigma, Poole, U.K.), naproxen (Sigma-Aldrich, 2659

dx.doi.org/10.1021/mp300219h | Mol. Pharmaceutics 2012, 9, 2658−2668


Article

in order to perform the blank subtraction. All samples were prepared 24 h prior to the experiment. Steady-state fluorescence measurements were recorded on a FluoroMax-2 spectrofluorimeter (HORIBA Jobin-Yvon Ltd., U.K.) which was calibrated daily. The excitation wavelength used was λex = 332 nm, and the slits were 0.5 nm for both excitation and emission. Emission spectra were recorded between 330 and 450 nm using a quartz Suprasil QS 111 cuvette of 1 cm path length (Hellma GmbH & Co. KG, Germany). Blank subtraction and data analysis were performed in Origin 7.5 (OriginLab, USA). The polarity (n = 3) of pure solvents (water, methanol and ethanol) and water/methanol mixtures (30/70, 50/50 and 85/ 15% w/w) was also measured using a pyrene final concentration of 2 μM. These data were used as calibrants to calculate the apparent dielectric constant of the SEDDS emulsions. Micropolarity Assessment Using Reichardt’s Dye (RD). An ethanolic stock solution of RD was added to 10 mL screw cap glass vials, and the ethanol was evaporated to dryness. To each vial were added 6 μL of SEDDS formulation and 1.5 mL of water, and the mixture was thoroughly mixed for at least 8 h. Immediately prior to collection of the RD spectra, a preconcentrated solution of NaOH/H2O was added to the sample to obtain a final RD concentration of 150 μM and a final pH of 12. This latter dilution step was necessary to avoid hydrolysis of the triacylglyceride ester groups that may be expected after prolonged exposure to such a high pH, which is required to ensure that the RD exists in the zwitterionic state and thus exhibits its solvatochromic properties. Spectra (n = 6) were recorded at 300 ± 1 K between 320 and 900 nm on a Lambda 35 UV−vis spectrophotometer (Perkin-Elmer, USA) using a 1 cm path length quartz cuvette UV 6030 (Hellma GmbH & Co. KG, Germany). Blank SEDDS samples, placebo and drug-loaded, without RD were also run as background comparators. Samples of pure water, pure Tween 80, pure Span 80 and aqueous Tween 80 (6 μL in 5 mL of basic solution) were also tested. Accurate estimates of λmax from the UV−visible spectra were obtained after fitting of each spectrum with a combination of two Gaussian amplitude functions using OriginLab 7.5 (OriginLab, USA). The observed maximum in absorbance can be transformed into the molar electronic transition energy of dissolved RD as defined by the ET(30) value12 and normalized to the dimensionless scale ENT 13 using eqs 1 and 2 respectively.

Poole, U.K.), indometacin (Sigma, Poole, U.K.), ibuprofen (gift from BASF, Ludwigshafen, Germany), Reichardt’s dye (RD) (Fluka, Schnelldorf, Germany), pyrene (Sigma, Gillingham, U.K.), deuterium oxide (Apollo Scientific, Stockport, U.K.), sodium deuteroxide 40% (Aldrich, Gillingham, U.K.) and deuterium chloride 35% (Goss Scientific Instruments, Nantwich, U.K.) were all used as received. Methods. Preparation of SEDDS Formulation Mixtures. Placebo SEDDS formulations (S18, S21 and S24) were prepared by mechanically mixing combinations of Tween 80, Span 80 and soybean oil. Formulation details are given in Table 1 along with the calculated hydrophile−lipophile balance Table 1. Details of Placebo SEDDS Mixtures formulation composition (% w/w) soybean oil Tween 80 Span 80 Tween 80:Span 80 wt ratio molar ratio HLB self-emulsification behavior (CIPAC test) placebo SEDDS drug-loaded SEDDS

S18

S21

S24

65 30 5

65 17.5 17.5

65 5 30

6:1 1:0.5 13.47

1:1 1:3 9.65

1:6 1:18 5.83

bad to poor moderate

good to excellent good

bad to poor moderate

(HLB) values for the placebo mixtures. In all cases, the quantity of the oily phase was kept constant at 65% w/w and the ratio of the two surfactants used varied to provide a range of self-emulsification abilities. Formulation S18 has a high Tween 80:Span 80 ratio (on a weight basis), S21 contains equal amounts of the two surfactants and S24 has a low Tween 80:Span 80 ratio, hence the hydrophilicity and HLB decrease through the range. For the relevant studies, 6% w/w ibuprofen was dissolved into the premixed anhydrous placebo SEDDS formulations. In all cases, the drug dissolved completely within the SEDDS as this concentration is well within the solubility limit of the drug in the anhydrous SEDDS formulations. For the NMR studies with lower concentrations of drug, the drug was added as described below. Visual Assessment of Self-Emulsification Efficiency. The ease of emulsification of SEDDS mixtures was assessed using a modification of the Collaborative International Pesticide Analytical Committee of Europe (CIPAC) test.14 One milliliter of each formulation was introduced into a 100 mL glass measuring cylinder containing deionized water at room temperature (around 293 K); the tendency of the formulation to spontaneously form an emulsion was assessed on a scale of excellent (droplets spread easily throughout the bulk of the measuring cylinder and formed a fine milky emulsion) through to bad (no emulsification observed, even after gentle agitation). Micropolarity Assessment Using Pyrene. A stock solution of pyrene in ethanol was added to 10 mL screw cap glass vials and the solvent evaporated to dryness. To each vial were added 6 μL of SEDDS formulation (placebo and ibuprofen-loaded) and 3 mL of water, so that the final pyrene concentration was 2 μM. This concentration was chosen so as to maintain pyrene in its monomer form. Blank samples without pyrene were also run

E T(30) =

E TN =

hcNA 28591 = (kcal × mol−1) λmax λmax

E T(X) − E T(TMS) E (X) − 30.7 = T E T(H 2O) − E T(TMS) 32.4

(1)

(2)

where h is Plank’s constant, c is the speed of light, NA is Avogadro’s number, λmax is the wavelength of maximum absorption, ET(X) is the ET(30) value of solvent X, and ET(TMS) and ET(H2O) are the ET(30) values for tetramethylsilane (TMS) and water, respectively. ENT will take the values 0 for TMS and 1 for water. 1 H NMR. Data Acquisition and Processing. All spectra were recorded on a 400 MR spectrometer (Varian Inc., CA) at the proton frequency of 400 MHz, with a relaxation delay of 2.256 s, a 45.0 degree pulse and a spectral width of 6000.6 Hz; 64 2660



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Table 2. Pyrene and Reichardt’s Dye Micropolarity Data for SEDDS Emulsions pyrene data sample

ibuprofen content (% w/w)

S18

0 6 0 6 0 6

S21 S24

I1/I3 (mean ± SD) 0.9893 0.9645 0.9766 0.9743 0.9701 0.9664

± ± ± ± ± ±

0.0012 0.0048 0.0021 0.0022 0.0028 0.0052

Reichardt’s dye data

εapp (mean ± SD) 6.117 3.427 4.725 4.475 4.028 3.627

± ± ± ± ± ±

0.1261 0.5269 0.2221 0.2347 0.3046 0.5600

λmax (mean ± SD)

ET(30)

ENT

± ± ± ± ± ±

53.51 55.47 58.80 56.57 56.42 56.99

0.7040 0.7699 0.8674 0.7984 0.7938 0.8115

534.3 513.9 486.3 506.0 506.9 501.7

6.36 15.83 18.27 15.18 19.29 20.30

plots, in that the ideal system shows gradients of 0, −1, −2, and −1 for the low frequency capacitance, low frequency dielectric loss, high frequency capacitance and high frequency dielectric loss, respectively. Equivalent gradients for a real system are denoted (−s), (−p), (−2 + s) and (−1 + n), respectively.16 Polarized Light Microscopy. Microscopy images were collected using an optical microscope (Leica DM LS2 HSM) equipped with magnification lenses (×4 and ×20) and a video camera (Panasonic WV CL310) interfaced to a computer through a capture device (o100vc.dll OSPREY CAPTURE CARD 1); data were recorded and analyzed using the software Studio Capture (version 1.4) and Studio Player (version 2.1) from Studio86Designs. The experiment was performed at 298 ± 1 K. Anhydrous samples were prepared as described above. Samples containing 10 to 90% w/v water were prepared by added the required amount of water to the relevant anhydrous mixture and vortex mixing (n = 4).

scans being co-added. No chemical shift standard was added to the system in order to avoid any undesired interaction with the emulsion droplets. Chemical shifts were referenced to the maximum of the acyl methyl peak, which was set at 0.90 ppm. This reference was chosen rather than water, as the water peak is sensitive to both temperature and pH, and may additionally be affected by the presence of the dissolved drugs. The acyl peak has been well characterized for oils15 and represents a reference from within the emulsion droplet, hence general susceptibility shifts due to compositional changes within the emulsion droplet (e.g., presence of dissolved drugs) are accounted for, allowing better interpretation of any observed shifts. Sample Preparation. Systems matching those to be used for the 1H NMR studies (see below) were prepared with water as the aqueous phase, and the pH was measured using a S20 SevenEasy pH meter (Mettler Toledo, Leicester, U.K.) equipped with a temperature probe and a pH electrode (Inlab 413, ARGENTHAL reference system). For the deuterated samples to be used for the 1H NMR studies, ethanolic stock solutions containing sufficient quantities of naproxen, ibuprofen, flurbiprofen and indometacin were added to 10 mL screw cap glass vials and evaporated to dryness. To each vial were added 2 μL of SEDDS formulation S21 and 1.5 mL of D2O, and the mixture was thoroughly mixed until equilibrium was reached. The final concentration of the model drugs was 300 μM. Corresponding placebo samples were prepared, and the pD of these was adjusted by addition of DCl to match the pH of the aqueous drug-loaded samples previously measured. Samples containing RD were prepared as described above for the micropolarity studies, with the exception that D2O and NaOD were used rather than water and NaOH. Dielectric Spectroscopy. Dielectric spectroscopy was conducted using a BDC-N broad band dielectric converter (Novocontrol Technologies, Germany) linked to a Solartron Frequency Response Analyzer (Solartron, U.K.). The sample (1 mL) was incorporated into a cell with parallel flat-faced stainless steel electrodes of area 254.5 mm2 and separation distance 1.5 mm. A sinusoidal voltage of 0.5 V rms was applied to the sample over a frequency range of 10−2 to 106 Hz. The sample temperature was maintained at 298 ± 1 K throughout the study. Samples were prepared as for the microscopy studies below. Experimentally, the results are expressed as the capacitance, C(ω), and dielectric loss, G(ω)/ω, of the material, where G is the conductance through the sample and ω is the angular frequency (=2πf where f is the applied frequency in Hz), displayed against applied frequency. The data were analyzed using the Dissado−Hill modification of the biphasic Maxwell− Wagner response.16 Deviation from ideal electrical behavior is described by assessment of the gradients of the log10−log10

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RESULTS Visual Assessment. The ease of emulsification of the placebo and ibuprofen-loaded SEDDS as assessed by the modified CIPAC test is summarized in Table 1. S21 placebo showed the best self-emulsification properties, being rated as “good to excellent”. However, in the presence of 6% w/v ibuprofen, the apparent ease of emulsification of the formulation worsened to merely “good”. S18 and S24 placebos both showed “bad to poor” emulsification behavior, which were improved to “moderate” upon addition of the drug. These results indicate a macroscopic, visually observable, effect of the drug on the behavior of the SEDDS formulation. Micropolarity Assessment Using Pyrene. The micropolarity as measured by pyrene emission spectroscopy and the calculated apparent dielectric constant of the three SEDDS emulsions in the presence and absence of ibuprofen (6% w/w in the anhydrous SEDDS) are shown in Table 2. A linear relationship between the apparent dielectric constant and the I1/I3 ratio for the anhydrous pure solvents and solvent mixtures was observed [εapp = (I1/I3 − 1.117) × 108.7 + 20, r2 = 0.9904], similar to literature data.17 Similarly, there was an almost linear correlation between the HLB of the anhydrous surfactant mixture and the I1/I3 ratio for the placebo systems (I1/I3 = 0.0025 HLB + 0.9544, r2 = 0.9664), although with three data points only, there is a danger of overinterpreting the data. A slight, but not statistically significant, lowering of the I1/I3 ratio in the presence of the ibuprofen was observed for the emulsions generated from formulations S24 and S21, with low and medium concentrations of Tween 80, but a statistically significant (t test, p < 0.05) decrease in the micropolarity was observed with formulation S18, which contained the highest level of Tween 80. Micropolarity Assessment Using Reichardt’s Dye (RD). Table 2 summarizes the results of the micropolarity studies 2661



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using Reichardt’s dye for the three SEDDS emulsions in the presence and absence of ibuprofen (6% w/w in the anhydrous SEDDS). Figure 2 shows representative UV absorbance spectra

Figure 3. 1H NMR spectrum of a representative placebo SEDDS emulsion containing Tween 80, Span 80, soybean oil and D2O, showing resonance peaks assignment as described in Table 3.

Figure 2. UV absorbance spectra resulting from the intramolecular charge transfer process for Reichardt’s dye incorporated into the placebo SEDDS emulsions.

Table 3. Assignment of the Main Proton Signals of the 1H NMR Spectrum of the Representative Placebo SEDDS Emulsion Formulation Containing Tween 80, Span 80, Soybean Oil and D2O Shown in Figure 3a

resulting from the intramolecular charge transfer process for Reichardt’s dye incorporated into the placebo SEDDS emulsions. The values of the polarity indices ET(30) and ENT were found to be significantly different for the three placebo SEDDS emulsions (t test, p < 0.05), with formulation S21 showing the highest micropolarity value. Addition of the drug had varying effects: the measured micropolarity of formulation S24, with the lowest HLB value, remained essentially unchanged; that of formulation S21, with the intermediate HLB value, showed a substantial decrease; and formulation S18, with the highest HLB value, exhibited a large increase in micropolarity. However, the final values for the three drugloaded systems were reasonably similar, with the addition of the drug reducing the differences seen for the placebos. 1 H NMR: Peak Assignment. Assignment of soybean oil signals was made following literature data.15,18,19 Assignment of Tween 80 signals was made using Tween 60 as a reference20 and a knowledge of its average fatty acid composition (manufacturer’s literature). The core structure of Span 80 is the same as that of Tween 80 but without the additional polyoxyethylene (POE) chains, as shown in Figure 1, hence the 1 H NMR spectrum of Span 80 was assigned using Tween 80 as a reference along with knowledge of the average fatty acid composition of both surfactants (manufacturer’s literature). Overall, therefore, the peak assignment of the complex spectrum of the SEDDS emulsion formulation was determined on the basis of the contribution of the individual components. Figure 3 shows the 1H NMR spectrum of a representative placebo SEDDS emulsion formulation containing Tween 80, Span 80, soybean oil and D2O, with the signal assignment being shown in Table 3. 1 H NMR: Interaction between Added Drugs and SEDDS Emulsion Components. The presence of ibuprofen in the SEDDS emulsions (6% w/w in the anhydrous SEDDS) resulted in a broadening of the peak centered on 3.71 ppm (labeled as peak no. 7 in Figure 3 and Table 3) with an increase in intensity in the high frequency region of this peak; negligible effects were seen on the other peaks. All three SEDDS formulations showed the same effect. As this peak has been

proton description peak

chemical shift (ppm)

compound

1

5.32

−CHCH−

olefinic

2 3 4 5

5.23 4.83 4.31 4.28

6 7

4.09 3.71

8

2.76

glycerol hydroxyl glycerol methylene group glycerol methylene group diacyl

9 10

2.25 2.04

11

1.59

12

1.36, 1.38

−CH−O−CO−R HOD −CH2−O−CO−R −O−CH2−CH2− O−CO−R −CH2−O−CO−R −O−CH2−CH2− O− −CHCH− CH2−CH CH− −CH2−COOR −CH2−CH CH− −CH2−CH2− COOR −CH2−

13

0.97

−CH3

14

0.90

−CH3

α-carboxyl α-olefinic β-carboxyl methylene group methyl group methyl group

all unsaturated fatty acids triacylglycerols deuterated water triacylglycerols oxyethylene units triacylglycerols oxyethylene units linoleic and linolenic acids all acyl chains all unsaturated fatty acids all acyl chains all acyl chains linolenic acid all acyl chains

a

Peaks are generally broad and are composites of a number of underlying signals. The chemical shift indicated is the maximum of the composite peaks.

assigned to methylene resonances in the polyoxyethylene (POE) region of the Tween 80, the results suggest a positioning effect of the drug in the POE region of the surfactant chain in the formulation. Similar observations were made for the emulsions with and without RD, suggesting that the probe is also located within the POE region of the surfactant in the emulsion. 2662



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indometacin and naproxen. All four drugs showed an effect on the POE peak: the presence of ibuprofen or naproxen in the emulsion resulted in a broadening of the peak in the high frequency region compared to that of the placebo, although the positioning of the main peak was essentially unchanged. The addition of indometacin led to a shift to lower frequencies of the main peak, but had limited effects only on the high frequency region. Flurbiprofen showed the greatest effect of the four drugs, with both peak broadening in the high frequency region and a shift to lower frequencies in the main peak being observed. Dielectric Spectroscopy. Soybean oil showed a very limited dielectric response, essentially behaving as an insulator. The dielectric response of water followed a modified Maxwell− Wagner dispersion, manifest by a diffusive electrode barrier layer seen at sub-Hz frequencies with the higher frequency region showing a bulk conductivity (essentially a direct current (dc) process) and a frequency-independent capacitance. The two surfactants also individually showed a Maxwell−Wagnertype dispersion, but greatly reduced in magnitude and shifted to lower frequencies compared to that of water, Span 80 showing a lower response than Tween 80 as expected from its less polar nature. Anhydrous SEDDS formulations generally exhibited Maxwell−Wagner-type dielectric behavior correlated to their compositions. An additional Debye response, consisting of a peak in the dielectric loss and a step change in the capacitance, was observed at higher frequencies, centered on 5.12 × 103 Hz;

To ascertain whether this effect is generalizable or specific to ibuprofen, a range of other NSAIDs were studied using SEDDS formulation S21. In this case, the concentration of the drug in the sample was standardized at 300 μM to account for molecular weight differences and variability in the solubility of the drugs in the formulation. Figure 4 shows the POE region of

Figure 4. The 1H NMR methylene peak of the POE units for SEDDS emulsions S21 placebo and formulations containing naproxen, ibuprofen, flurbiprofen and indometacin (300 μM).

the 1H NMR spectra (3.8 to 3.6 ppm) for the placebo SEDDS mixture and those loaded with 300 μM ibuprofen, flurbiprofen,

Figure 5. Low-frequency dielectric spectra of anhydrous SEDDS. (A) Spectra over the whole frequency range. (B) Magnification of the spectra in the frequency region 101 to 106 Hz. Bars represent standard deviation and standard error (n = 4). 2663



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20% v/v and above, whereas with the ibuprofen-loaded sample, this comparability of response was reached at 30% v/v water. Subtleties in the dielectric response can be observed by examining the exponents of the power law behavior for the various samples, most easily observed as gradients of the linear portions of the log10−log10 plot.16 The gradients of the high frequency loss slope between 102 and 105 Hz decrease from −0.80 for the placebo and −0.86 for the ibuprofen-loaded system (i.e., values of n = 0.2 and 0.14, see the definition of n given above), indicating an element of conduction by chargehopping, to circa −0.98 for both formulations, representing essentially dc charge transport; these values stabilize at 20% v/v water for the placebo and 30% v/v water for the active samples. A similar result was observed for the gradients of the capacitance slope between 102 and 103 Hz, decreasing from approximately zero, indicating a very limited barrier function, to approximately −1.6 (i.e., s = 0.4, see the definition of s given above), demonstrative of a dispersive barrier function. In this case, however, 30% v/v water and 40% v/v was required for stabilization of the results for the placebo and drug-loaded formulations, respectively. Polarized Light Microscopy. Polarized light microscopy images of the SEDDS formulation S21, both placebo and with 6% w/w ibuprofen, in the presence of increasing amounts of water are shown in Figure 7. At 10% v/v water, both samples showed the formation of liquid crystal mesophases. With 20% v/v water content the structure of the placebo formulation appeared “spongy” and it had lost the birefringence shown at

this was most apparent for S18, less so for S21 and almost negligible for S24, shown in Figure 5. This response indicates some local structuring of the surfactants and oil, and may be a consequence of a small amount of sorbed water in the theoretically anhydrous system. If so, it is most likely to be associated with the level of the more hydrophilic surfactant Tween 80, as the extent of this structuring diminished with decreasing Tween 80 concentration. Addition of drug reduced the dielectric response of the anhydrous systems, but did not change their dielectric behavior. The low frequency dielectric response of placebo and drugloaded SEDDS S21 upon addition of varying amounts of water is shown in Figure 6. For reasons of clarity only samples

Figure 6. Low-frequency dielectric spectra of S21 at different water contents: (A) placebo; (B) formulation containing ibuprofen (6% w/ w). Closed and open symbols represent the capacitance and loss, respectively. Squares (■, □) 0% v/v; circles (●, ○) 10% v/v; triangles up (▲, △) 20% v/v; triangles down (▼, ▽) 30% v/v; diamonds (◆, ◇) 40% v/v. Data obtained at higher water contents are not shown for clarity, but they superimpose onto the 40% v/v water samples.

containing up to 40% v/v water are shown, as samples at higher water content were superimposable to those containing 40% v/ v of water. A shift in the dielectric response from that of the anhydrous system to that of water was observed, i.e., the response shifted to higher frequencies and magnitudes upon the addition of water. Samples containing 10 and 20% v/v water showed the greatest step changes in response compared to the relevant anhydrous system; in fact, the placebo samples showed essentially superimposable responses at water levels of

Figure 7. Microscopy images of placebo (1) and drug loaded (6% w/w ibuprofen) S21 (2) with 10 (a), 20 (b), 30 (c), 40 (d) and 50% v/v (e) water content. 2664



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the lower water content, whereas the birefringence was retained by the drug-loaded sample at this water level. At water contents of 30% v/v and above, an emulsion was formed in the absence of the drug, while the water content had to be increased to be at least 40% v/v before emulsification occurred in the drug-loaded sample.

Applying these data to the current situation, it is likely that as the drug is located in the POE region of the Tween 80, it will alter the molecular conformation of the surfactant molecule and hence affect the hydrogen bonding between water and the POE chains. Such interaction is likely then to have the consequence of changing the behavior of the system on a macroscopic scale and may be expected to be observed by a change in the selfemulsification efficiency. Slight differences in the interaction of the four model compounds with the surfactant were observed. Naproxen, ibuprofen, and flurbiprofen are all arylpropionic acid derivatives sharing a core chemical motif, but varying in the substituent groups and hence slightly in physicochemical properties, whereas indometacin is an indole derivative. All four molecules are weak acids. The differences in the peak positioning and broadening seen between the four drugs may indicate slight variation in the solubilization locus with the POE system, with the naproxen and ibuprofen possibly showing a slightly more interior location than the other two compounds. However, flurbiprofen and, in particular, indometacin are bulkier molecules with calculated molecular radii of 197.6, 193.4, 204.4, and 288.7 Å for naproxen, ibuprofen, flurbiprofen and indometacin, respectively.26 Perhaps more significantly, flurbiprofen and indometacin both contain electronegative halogen atoms (fluorine and chlorine, respectively), which may also play a role in reducing electron density in the neighboring aromatic rings and enhancing interactions with the surfactant. Hence these two drugs may be expected to show more perturbation of the POE moieties with resultant effects on the POE peak broadening and shifting. The 1H NMR results indicate that the RD is also located within the POE region of the surfactant in the SEDDS emulsions, which may help to explain both the RD micropolarity data and the observed variation between the placebo and drug-loaded formulations. Placebo S18, which has the highest concentration of the hydrophilic surfactant Tween 80 and hence the highest HLB value, would be expected to show the highest micropolarity. Similarly, placebo S24 would be expected to show the lowest micropolarity. However, the present results show the opposite trend for these two formulations, and indeed the maximum in micropolarity was observed with placebo formulation S21. This latter result may be partly explained by the reported observation that a 1:1 weight ratio mixture of Tween 80 and Span 80 (as seen in S21) showed the highest water solubilization capacity of any such mixtures studied.27 Consequently, any probe located at the palisade layer of the emulsion droplet formed from this mixture would be exposed to a more hydrophilic environment, and hence a higher micropolarity would be measured. Addition of the ibuprofen had various effects on the measured micropolarity: S18 and S21 showed an increase and a decrease, respectively, with S24 remaining essentially unchanged. Overall, the presence of the ibuprofen appeared to have a “normalizing” effect on the measured micropolarity of the emulsions, with the drug-loaded systems all showing similar results. Ultimately, this may be explained by the RD experiencing the same environment in all drug-loaded samples, irrespective of the overall concentration of the two surfactants. This has most likely resulted from a series of relatively complex interactions involving the drug, the RD, the surfactants and the water. On a molar basis, the ibuprofen concentration is approximately double that of that RD in the final emulsion. It is possible that the two are forming a charge complex, given that the RD exists

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DISCUSSION We have previously reported8 that the addition of a model drug at its therapeutic level to a SEDDS formulation alters its emulsification behavior, and the current investigation attempts to elucidate the physicochemical principles behind the macroscopic observations. The anhydrous SEDDS formulation studies here were selected following an analysis of the phase behavior and self-emulsifying potential of mixtures of soybean oil, Tween 80 and Span 80 reported in our previous paper.8 The overall ratio of 65% oil to 35% surfactants is in line with other published SEDDS formulations.2 Formulation S21 was chosen to provide excellent behavior as a placebo, which was observed to worsen upon the addition of ibuprofen. Formulations S18 and S24 were chosen to keep the overall ratio of oil to surfactants the same, but varying the ratios of the individual surfactants. Both S18 and S24 were poor selfemulsifiers in the placebo state, but their self-emulsification behavior improved upon addition of the drug. The three formulations therefore form a useful homologous series in which to examine the physical interactions between the drug and the SEDDS excipients. The 1H NMR data indicate an interaction between the ibuprofen and the POE chains of the surfactant, Tween 80, reflecting the close physical proximity of these two components of the sample under study and in turn implying the presence of the drug at the oil−water interface. Mechanistically, the 1H NMR data can be ascribed to the ring current effect of the aromatic groups of the drug on the POE protons. The effect of addition of benzene and water on the 1H NMR spectra of the nonionic surfactant system, polyoxyethylene glycol dodecyl ethers with different POE numbers, has previously been studied.21 In that work, the peak due to the POE was a composite, resulting from overlap of the responses of the individual POE units, those toward the terminal POE unit showing a lower chemical shift than those toward the interior of the molecule. Addition of water or benzene resulted in a differential broadening of the POE peak, water having an effect on the next-to-terminal POE units and benzene showing an effect on the more interior POE units, leading the authors to conclude that the additives were predominantly localized at different sites within the surfactant system. By analogy, the current results would tend to suggest that the relatively broad Tween 80 POE peak seen for the placebo SEDDS emulsions is a result of the interaction of the next-to-terminal POE units with water, and that the increased POE peak broadening seen with the drug-loaded systems is a result of further interaction between the more interior POE units and the relatively hydrophobic drugs. Infrared spectroscopic studies22 and molecular dynamics simulations23,24 have been used to elucidate the spatial conformations and water interactions of POE molecules. In particular, water was found to form a hydrogen bond network with the ether oxygens on the oxyethylene groups and was bound inside the helix formed by the POE chains. Conformational changes in the oxyethylene headgroup of nonionic surfactants resulted in dehydration of the headgroups.25 2665



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Figure 8. Representational diagram of the structure of a SEDDS emulsion droplet; in the enlargement box on the left are shown the layers formed by the surfactants and the oil, with the preferential solubilization site for ibuprofen dissolved in a SEDDS droplet.

drug-loaded systems in the presence of varying amounts of water. Both techniques showed that some local structuring, possibly liquid crystal formation, occurred upon addition of small amounts of water, and that this was a stage in the emulsification process. Addition of the drug clearly altered this emulsification process, necessitating an increased amount of water to be present at each stage of the emulsification process, as seen visually in the microscopy images, and resulting in a smaller emulsion droplet size distribution (full details of the effects of incorporated drug and the experimental method used for particle sizing on the results of the emulsion droplet size distribution have been previously presented8). The differences in the dielectric data upon addition of 6% w/w ibuprofen to S21 confirm that the drug is having a substantial effect on the behavior of the system. Both techniques suggested that emulsification is essentially complete after the addition of 30% v/v water to the placebo SEDDS system, but that 40% v/v water is required for the ibuprofen-loaded system, further water acting merely to dilute the emulsion. Taken together, the results from the various experimental investigations are remarkably consistent, particularly given that the measuring techniques arise from a variety of physical stimuli. All indicate that the addition of the model drug, ibuprofen, to a SEDDS formulation will modify its behavior by alteration of the interaction of the surfactants with added water. This is achieved by the drug being physically located at the oil− water−surfactant interface during emulsification, thus altering the micropolarity of the local environment and ultimately affecting the macroscopic emulsification process. In some respects, the emulsification step may be considered to be of prime importance for the oral bioavailability of the drug, and hence it is vital that it is understood and that any effects of added drug on it are examined and quantified. The precise extent of the interaction will depend on the chemical nature of the drug, as indicated by the study here with the four NSAIDs. Figure 8 shows a representational diagram of the proposed structure of a SEDDS emulsion droplet based on the data generated in this study. The Tween 80 POE units form the bulk of the palisade layer between the internal oil core and the external aqueous phase and are shown in an expanded form. The Span 80 occupies the bottom of the palisade layer, as

in a permanent zwitterionic state and that, in an aqueous phase, the drug will exist almost entirely in the negatively ionized state at the pH at which these studies were conducted (a high pH was required to ensure that the RD was in the zwitterionic state and hence would behave as a micropolarity probe). Alternatively, the presence of the drug is forcing the RD away from the interface, toward the interior of the emulsion droplet. Both of these scenarios individually or in combination would explain the micropolarity data. Pyrene, by virtue of its chemical structure, is likely to be located within the oil phase of the emulsion droplet or at the inner ring of the surfactant palisade layer with the surfactants’ hydrophobic tail groups and thereby is able to determine the micropolarity at this location. It is logical therefore to expect, as observed, that the I1/I3 ratio would decrease with decreasing HLB value of the placebo SEDDS system, as this reflects an increase in the relative concentration of the more hydrophobic surfactant Span 80. What is interesting to note is that the only sample that showed a significant change (a decrease) in the measured micropolarity after addition of ibuprofen was S18, which has the lowest concentration of Span 80 and the highest of Tween 80. This again may be a consequence of the interactions of the drug with the POE chains of the Tween 80. A possible explanation is that the conformational changes observed for the POE chains upon addition of drug will result in the “pushing” of the Span 80 away from the palisade layer and deeper into the interior of the emulsion droplet, hence resulting in an increase in hydrophobicity and thereby a decrease of the polarity as measured by the I1/I3 ratio. The effect of the interactions between added drug and the SEDDS components, as determined here by the physical characterization techniques, may be expected to result in a behavioral change of the SEDDS system, especially if the physical interactions involve water. This was in fact observed: formulation S21, which showed “good to excellent” selfemulsification behavior as a placebo, was graded only “good” when 6% w/w ibuprofen was added, whereas S18 and S24, which showed “poor to bad” behavior as placebos, improved to “moderate” in the presence of the drug. Dielectric spectroscopy and polarized light microscopy were used to examine this effect in more detail by assessing the behavior of the S21 placebo and 2666



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reflected by its more hydrophobic nature. The drug ibuprofen is shown interacting with the POE units on the Tween 80. Depending on the precise position of the incorporated drug, which will be dependent on its chemical nature and molecular size, the POE chains will be oriented differently and therefore present a different surface for interaction with the aqueous phase, potentially resulting in variation of emulsification efficiency. Surfactants which are in the more expanded state could be considered to be more mobile and more likely to interact with added water than those in a more collapsed state, which will then be reflected in the ease and speed of emulsification. Given that it is still not completely understood how SEDDS improve the bioavailability of the incorporated drug, with droplet size and lipid digestion both being mooted as possible contributors,28,29 any change in the physical arrangements of the formulation components would seem to be of relevance in determining the clinical effectiveness of the formulation.

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AUTHOR INFORMATION

Corresponding Author

*School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K. E-mail: [email protected]. Tel: +44-(0)1603592843. Fax: +44-(0)1603-592003. Present Addresses †

Bioos Italia S.r.l, Via Salvatore Quasimodo 136, 00144 Roma, Italy. ‡ Institute of Pharmaceutical Science, King’s College London, Fifth Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the University of East Anglia for the financial support of A.M. and Prof. David Russell (School of Chemistry, University of East Anglia) for providing fluorimetric measurement facilities.

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CONCLUSIONS

This study has sought to explain, using a range of physical characterization techniques, the previous observation that the addition of ibuprofen to a relatively simple anhydrous SEDDS formulation modified its emulsification behavior and resultant droplet size.8 The data presented here suggest that the dissolved drug is located at the palisade layer of the droplet during the emulsification process. As such, it will alter the molecular orientation of the surfactants and thus affect their interaction with water during the emulsification process. Macroscopically, this is seen by changes in the ease of emulsification and the droplet size. Variation of the chemical structure of the drug via a series of NSAIDs showed that there was a generic effect of this type of drug, i.e., poorly watersoluble weak acids, but the specific effects of the drug are dependent on its molecular architecture. Overall, therefore, it is evident that the incorporation of a drug into a SEDDS formulation may change its behavior and that this should not be ignored. It is therefore recommended that the effect of any such drug be assessed early in the formulation stage. In this study, a relatively simple SEDDS formulation of one oil and two surfactants was used to facilitate the analysis and interpretation of the data. It would be useful to extend this work to assess other commonly used oils, surfactants and cosurfactants, so as to provide a bedrock of physicochemical and functional information on the excipients to aid formulation selection. Additionally, the effect of incorporation of minor components, such as ethanol or oilsoluble preservatives, into the SEDDS formulations remains to be evaluated. The methods used here provide a template for such experimental investigation.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Further detailed information on the chemical composition of the formulation excipients used and more detailed explanation of the theories and analysis of dielectric spectroscopy and polarity assessment using pyrene and Reichardt’s dye. This material is available free of charge via the Internet at http:// pubs.acs.org. 2667



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Identification and Molecular Interpretation of the Effects of Drug

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