Discriminating the Molecular Identity and Function of Discrete

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Discriminating the Molecular Identity and Function of Discrete Supramolecular Structures in Topical Pharmaceutical Formulations F. Benaouda,† M. B. Brown,‡,§ S. Ganguly,† S. A. Jones,*,† and G. P. Martin† †

Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom ‡ MedPharm Ltd., Unit 3/Chancellor Court, 50 Occam Road, Surrey Research Park, Guildford, GU2 Guildford, GU2 7AB, United Kingdom § School of Pharmacy, University of Hertfordshire, College Lane Campus, Hatfield, Hertfordshire, AL10 9AB, United Kingdom S Supporting Information *

ABSTRACT: There is a need to understand how solvent structuring influences drug presentation in pharmaceutical preparations, and the aim of this study was to characterize the properties of propylene glycol (PG)/water supramolecular structures such that their functional consequences on drug delivery could be assessed. Shifts to higher wavenumbers in the C−H and C−O infrared stretching vibrations of PG (up to 8.6 and 11 cm−1, respectively) implied that water supramolecular structures were being formed as a consequence of hydrophobic hydration. However, unlike analogous binary solvent systems, water structuring was not enhanced by the presence of the cosolvent. Two discrete populations of supramolecular structures were evident from the infrared spectroscopy: water-rich structures, predominant below a PG volume fraction (f PG) of 0.4 (unmoving water bending vibration at 1211 cm−1) and PG-rich structures, predominant above 0.4 f PG (both C−H and water peaks moved to lower wavenumbers). The un-ionized diclofenac log−linear solubility and transmembrane transport altered dramatically when f PG > 0.55 (a 10-fold increase in transport from 0.28 ± 0.06 μg·cm−2·h−1 at 0.2 f PG to 2.81 ± 0.16 μg·cm−2·h−1 at 0.9 f PG), and this demonstrated the ability of the PG rich supramolecular structures, formed in the PG/water solvent, to specifically modify the behavior of un-ionized diclofenac. KEYWORDS: nonideal mixing, vehicle supramolecular structuring, diclofenac, transmembrane transport, ion-pairs

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INTRODUCTION A number of in silico and experimental studies have demonstrated that the thermodynamic properties displayed by binary solvents are a result of the supramolecular structures formed in these solutions.1−8 For example, Dixit et al. provides evidence, using neutron diffraction techniques, that intermolecular associations of methanol and water in the solution state dictate the properties of the binary solvent mixture.2 Given these findings, it appears incongruent to assume that pharmaceutical preparations, which employ binary solvents, present drug molecules to the patient as a homogeneous arrangement of molecules. However, as the supramolecular structuring of liquid dosage forms is rarely reported when considering their behavior, it is difficult to determine if the influence of this phenomenon has been accounted for during the process of drug administration.9−11 The modification of drug solubility through the manipulation of solvent−solvent interactions, which essentially alters the nature of the solvent supramolecular structuring, has been noted in the pharmaceutical science literature.12−15 For example, Rubino and Yalkowsky investigated solute−solvent interactions in binary mixtures indirectly by modeling the solute’s saturated solubility with a log−linear model.13 Such an © 2012 American Chemical Society

approach allows the molecular interactions in a pharmaceutically relevant vehicle to be interpreted and solubility in analogous systems to be predicted from functional data.16−18 However, there appears to be less information available with regards to the influence of solvent structuring upon other behavioral properties such as cellular uptake, membrane transport, receptor docking, or drug stability, which suggests more work in these areas is warranted. The process of passive transmembrane penetration is most commonly interpreted using the theoretical models proposed by Higuchi19 and Stokes−Einstein as cited in Miller20 and Fick,21 but all of these are based upon the assumption that the agents penetrating a membrane are presented as a homogeneous solution and do not show “specific interactions” with the application vehicle. The data presented by Dixit et al.1,2 and the appearance of numerous other papers detailing the importance of nonideal solvent mixing interactions3,4,6,7,22 suggest that the use of the traditional models to interpret drug transport from Received: Revised: Accepted: Published: 2505

March 6, 2012 June 26, 2012 July 11, 2012 July 11, 2012 dx.doi.org/10.1021/mp300127f | Mol. Pharmaceutics 2012, 9, 2505−2512

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Specac Ltd., UK) fitted with CaF2 windows and a 25 μm mylar spacer (Specac Ltd., UK). Solutions were produced by adding excess of DDEA to PG−water binary mixtures, equilibrating using stirring (at 25 °C, for 48 h) and removing the excess solid via centrifugation (20 min at 13 000 rpm, Biofuge pico, Kendro Laboratory Products plc, UK). The vehicle spectra were compared to that of diclofenac saturated solutions (pH adjusted to 3). Deuterated water (D2O) was employed in the solutions as it dampened the solvent signal in the 1700−1300 cm−1 and 3000−2850 cm−1 ranges. The pH of the vehicles used in this work was tightly controlled (pH = 3 ± 0.1) using phosphate buffer and adjustment of the pH after the addition of the drug with phosphoric acid, to maintain the diclofenac (pKa = 3.830) in an un-ionized state. All spectra were produced using 32 scans collected at a spectral resolution of 4 cm−1 and recorded using a Spectrum One spectrometer (Perkin-Elmer Ltd., UK). Spectral analysis was performed with Spectrum software (version 10, Perkin-Elmer Ltd., UK). Log−Linear Solubility. The diclofenac saturated solubility (X2) in the cosolvent mixtures was determined as described previously for the FTIR analysis. The drug−solvent intermolecular interactions were characterized by comparing the experimentally derived saturated solubility to the log-linear saturated solubility using a log−linear solubility model for binary mixtures (eq 1):13

binary solvents requires re-evaluation. If solvent supramolecular structuring does occur in solvents, the drug−vehicle interactions will be dependent upon the type and number of supramolecular structures present. In this context changing the proportion of a specific component of the vehicle may be more significant than previously suggested, and if this is the case further study in this area may improve the understanding of drug delivery from the solution state. Propylene glycol (PG), a diol commonly used in pharmaceutical formulations, displays nonideal mixing characteristics when combined with water.8,12,15,22 It provides an interesting case study when considering the impact of supramolecular structuring because the published literature detailing the transmembrane penetration characteristics of molecules administered to membranes using PG−water vehicles display a confusing array of drug and cosolvent composition specific effects. For example, the transmembrane transport of benzocaine,23 parabens,24 and hydrocortisone25 has been shown to be insensitive to the composition of the PG− water vehicle, while the transport of oestradiol,26 ibuprofen,27 and piroxicam28 is known to be influenced by the PG fraction in a delivery vehicle. The aim of the present study was to investigate the consequences of vehicle supramolecular structuring on transmembrane penetration of an un-ionized drug molecule using diclofenac as a pharmaceutically relevant model agent. The PG−water system was selected as a consequence of the previously reported nonideal mixing of PG with water.29 PG− water supramolecular structure formation and drug interaction with these structures was measured using Fourier transform infrared spectroscopy (FTIR), and the transmembrane penetration was measured using diffusion cells. It was not the intention of this study to mimic any specific barrier such as that presented by the skin, the airway, or the gastro-intestinal tract; rather it was the intention to determine the passive transmembrane transport in the presence and absence of significant solute partitioning, and hence two model membranes were employed such that the functional consequences of distinct supramolecular structures could be determined.

ln Xi = f ln Xc + (1 − f )ln X w

(1)

where Xi was the log-linear solubility in the cosolvent binary mixture, Xc the solubility in neat cosolvent, Xw the solubility in water, and f the volume fraction of cosolvent PG. The hydrophobicity of the solute, PG, water, and PG−water mixtures was expressed in terms of their predicted solubility parameter calculated using the Fedors method.31 The solubility parameter calculations assumed no significant change in the vehicle volume upon mixing the two solvents. Transmembrane Penetration. Diclofenac Transport. Upright individually calibrated Franz diffusion cells (MedPharm Ltd., UK), with an average of 2.1 ± 0.1 cm2 surface areas and 9.7 ± 0.4 mL receptor compartment volume, were used to study the transmembrane permeation of diclofenac. The cells were fitted with either silicone membrane or RCM. The receiver fluid was 20:80 (v/v) EtOH/PBS (pH 7.2) for silicone membrane transport studies, but PG−water mixtures matched to the application vehicles were used for the RCM transport studies, due to back diffusion of the solvent known to be problematic with RCM. The membrane temperature was thermostatted at 25 °C (under constant stirring in a water bath, Grant Instruments, UK). After a 2 h equilibration, infinite doses (1 mL) of diclofenac-saturated PG−water mixtures (pH 3) were applied to the membrane. At predetermined time intervals 1 mL aliquots were removed from the receiver phase, replaced by thermostatted receiver fluid and analyzed by HPLC. Cumulative amounts of drug penetrating per unit surface of the membrane area (μg·cm−2) were plotted against time (h), and the steady-state transmembrane transport rate (J) was calculated from the slope of the linear portion of the curve (i.e., R2 ≥ 0.97), using at least five points with values above the assay limit of detection (LOD). The enhancement ratio (ER) was determined as per eq 2:

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MATERIALS AND METHODS Materials. Diclofenac, as the diethylamine salt (DDEA) (melting point of 154 °C, BP grade, 99.9%) was provided by Unique Chemicals, India (DDEA was used to generate comparative data to the previously published studies28). Propylene glycol (PG) (ACS reagent grade, ≥99.5%) was supplied from Sigma Aldrich, UK. Acetonitrile and methanol (high-performance liquid chromatography (HPLC) grade) were obtained from Fisher Scientific International, UK, and ethanol (99.7−100% v/v) was provided by BDH Laboratory Supplies, UK. Phosphate-buffered saline (PBS, pH 7.2, 0.172 M) tablets were supplied by Oxoid Ltd., UK. Sheets of silicone membrane (Folioxane), with a thickness of 120 μm, were purchased from Novatech Ltd., France. Regenerated cellulose membrane (RCM) (Visking, cutoff pore size, 12−14000 Da) was purchased from Medicell International Limited, London. Deionized water (electrical conductivity 0.5−1 μS) was used throughout this study. Vehicle−Solute Interactions. Fourier Transform Infrared Spectroscopy (FTIR). Binary PG−water solutions (pH 3, PBS 0.172 M), with volume ratios ranging from 1:0 to 0:1 (v/v) were analyzed using FTIR. The samples were loaded into a demountable universal transmission cell system (Omni-Cell,

ER = 2506

J2 J1

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where J2 was the steady-state transmembrane transport rate of diclofenac from the drug-saturated vehicle and J1 was the diclofenac steady-state rate from the drug-saturated vehicle containing 0.2 f PG. The effective diffusion coefficient of diclofenac was determined using a rearrangement of the Higuchi eq 3: D=

J × γ × h* α

on the basis of the peak area measurements using standard solutions of known diclofenac concentrations dissolved in an identical fluid as the receiver phase for the permeation studies, PBS (pH 7.4) for the solubility studies, and an identical fluid as the extraction fluid for the partitioning studies. The assay was shown to be “fit for purpose” in terms of sensitivity (LOD = 0.68 μg·mL−1, n = 30), suitability, accuracy (100.8 ± 2.2, n = 5), precision ( 0.9998, n = 30) in accordance with the limits described by the International Conference on Harmonisation guidelines.32 Statistical Analysis. All values were expressed as their mean ± standard deviation (SD), and a statistical analysis of data was performed using the statistical package for social sciences SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The normality (Sapiro-Wilk) and homogeneity of variances (Levene’s test) of the data were assessed prior to statistical analysis. Permeation results were analyzed statistically using one way analysis of variance (ANOVA) tests with posthoc Tukey analysis where required. All other data were analyzed using a Student’s t-test. Statistically significant differences were assumed when p ≤ 0.05.

(3)

where D is the diffusion coefficient, J the steady-state transport rate across silicone membrane, h* the diffusional path, γ the effective activity coefficient of diclofenac in the silicone membrane (γ = 1.7, determined experimentally), and α the thermodynamic activity of diclofenac in the vehicle (i.e., in this case α = 1). Since it was not possible to measure h*, the membrane thickness was utilized. The influence of the PG− water vehicle and diclofenac on the membrane swelling was assessed by the measurement of the membrane thickness preand 24 h post-application of vehicles using a Vernier micrometer (Starrett, Jedburgh, UK) (n = 18 for each system). The membrane thickness was found to be unaltered by the application vehicle (variance < ± 5%, n = 18 for drug unloaded cosolvent vehicles and < ± 3%, n = 18 for the drug-saturated vehicles). Membrane Partitioning and Solubility. Diclofenac membrane-vehicle partitioning behavior from two vehicles (0.2 f PG and 0.65 f PG (pH 3)) at multiple drug concentrations (1 and 3.3 μg·mL−1 for 0.2 f PG and 60, 450, and 740 μg·mL−1 for 0.65 f PG; n = 3−4 for each concentration) was determined using silicone membrane. Pieces of membrane (16 cm2 in size, 0.22− 0.33 g in weight) were placed into a vial to which 2 mL diclofenac solutions were added. The vials were incubated in a shaking water bath (120 strokes min−1), at 25 °C for 72 h (the previously determined equilibration,29 data not shown). The drug was extracted from the membranes by immersion in 10 mL of an ethanol/water (2:3 v/v) solution at 50 °C for 30 min with sonication and diclofenac content determined by HPLC (recovery was 98.9 ± 1.6%, n = 3). The membrane-vehicle partition coefficient (K) calculated using eq 4:

K=

Cm Cv

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RESULTS Vehicle−Solute Interactions. The FTIR spectra recorded for the cosolvent mixtures were presented as collected by the transmission method, that is, without deconvolution or solvent subtraction, as they demonstrated significant visible shifts in the regions of interest for the two solvents. The bending vibration of water (D2O) at 1211−1212 cm−133 recorded during the infrared measurement of the PG/water solvent mixtures remained unaffected by the increasing volume fraction of PG from 0 to 0.4 f PG. Above a f PG of 0.4 the water peak shifted to higher wavenumbers; for example, the D2O peak appeared at 1220 cm−1 in a 0.7 f PG cosolvent system (Figures 1a and 2a), but in PG-rich solvents, that is, >0.7 f PG, the bending vibration disappeared. The bands observed at 2972, 2932, and 2878 cm−1 in the pure PG solvent, assigned to the asymmetric C−H stretches of the alkyl groups,34−36 moved to higher wavenumbers as the proportion of water in the cosolvent increased (Figure 1c) as did the C−O stretching motion of PG at 1139 cm−1 34−36 (Figure 1b). Together the peak movements of C−H and C−O suggested a significant hydrophobic hydration between PG and water in the vehicle (Figures 1b,c and 2b). In a similar manner to the bending vibration of water, in the range 0.1−0.4 f PG, the C−O stretching bands remained unaltered, residing at 1150 cm−1, but there appeared to be an inflection point at 0.4 f PG after which the D2O bending and C− O stretching vibration indicated that significant changes in the supramolecular structure occurred (Figure 2). The pattern of changes recorded for the alkyl asymmetric stretching behavior did not accord with the trends observed for the water and C−O PG peak in that it increased progressively with increasing water volume fractions across the whole range of solvent combinations assessed in this work (Figure 2b). No spectral changes in the solvents bands were induced by the addition of the unionized diclofenac at any of the vehicle compositions, which indicated that the vehicle structuring was not significantly affected by the addition of the solute (data not shown graphically). Increasing the volume fraction of PG in the PG/water (pH 3) cosolvent decreased the vehicle solubility parameter in a manner that resulted in it approaching that of the drug (δdiclofenac = 10.86 (cal·cm−3)1/2; Table 1). As a consequence of

(4)

where Cm was the drug concentration in the silicone membrane at equilibrium (mg·g−1) and Cv the drug concentration in the vehicle at equilibrium (mg·g−1). The effect of vehicle composition on the membrane capacity to retain diclofenac was measured by applying an excess amount of the solute in a PG/water (pH 3) vehicles at different cosolvent volume ratios (range of 1:0 to 0:1 (v/v) PG/water) to a 16 cm2 (0.22−0.33 g) piece of silicone membrane (n = 3). Diclofenac was extracted from the membrane and assayed as described previously. Diclofenac Quantification. Quantitative determination of diclofenac was performed using a reverse-phase HPLC system consisting of a Jasco UV detector and pump (Jasco Corporation Ltd., UK). The mobile phase comprised acetonitrile− methanol−formate buffer (25 mM) (50:20:30 (v/v), pH 3.5) set at a flow rate of 1.2 mL·min−1. Diclofenac was separated using a Gemini C18 (250 × 4.6 mm) stationary phase (Phenomenex, UK) at room temperature with a 20 μL injection volume and UV detection at 275 nm. The retention time for diclofenac was 7.5 min. The calibration curves were constructed 2507

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Figure 2. Changes in the Fourier transform infrared wavelength of the bending vibration of deuterated water (D2O) (a) and in the C−O and C−H stretching vibrations of propylene glycol (PG) (b) upon varying the volume fraction of (f PG) in the cosolvent mixture. The inflection point represents the proposed initiation of the shift in the C−O stretching and D2O bending vibrations.

Table 1. Diclofenac Saturated Solubility and Silicone Transmembrane Transport Rate from Drug-Saturated Propylene Glycol (PG)/Water (pH 3), at Different PG Volume Fractions (f PG) (Mean ± SD)a

Figure 1. Fourier transform infrared spectra of propylene glycol (PG)−water (D2O) vehicles at varying PG volume fractions ( f PG): D2O bending region (a); C−O stretching region of PG (b); C−H stretching region of PG (c).

f PG

δ (cal·cm−3)1/2

0 0.2 0.4 0.5 0.6 0.7 0.8 0.9 1

23.4 21.67 19.94 19.08 18.22 17.35 16.49 15.63 14.67

saturated solubilityb (μg·mL−1) 2.7 4.9 25.6 61.1

± ± ± ±

0.3 0.5 0.6 0.4

932.2 2137.9 2436.9 8415.8

± ± ± ±

52.67 100.7 53.2 797.6

transport ratec (μg·cm−2·h−1) 0.28 0.41 1.36 1.88 2.02 2.39 2.81

± ± ± ± ± ± ±

0.06 0.09 0.14 0.08 0.28 0.47 0.16

ER 1 1.5 5.1 6.9 7.5 8.9 8.9

± ± ± ± ± ±

0.4 1.1 1.3 1.7 2.3 1.7

a The enhancement ratio (ER) was the ratio of the flux compared to that when a vehicle containing 0.2 f PG was employed (δ is the vehicle solubility parameters. bn = 3. cn = 5.

the changes in solvent properties, the drug saturated solubility increased exponentially with increasing PG volume fraction in the vehicle (Figure 3a): drug solubility in pure PG (pH 3), at 8415.8 ± 797.6 μg·mL−1, was 3000-fold higher than that in pure water (pH 3) at 2.7 ± 0.3 μg·mL−1. The diclofenac−PG− water system demonstrated a negative deviation from ideal behavior when f PG ranged between 0.1 to 0.6 (with a maximum at 0.2−0.5 f PG, ln X(dev) = −1.0) and a positive deviation in the f PG 0.6−1 range (peak at 0.8 f PG, ln X(dev) = 0.24) (Figure 3b).

Transmembrane Penetration. Diclofenac penetrated silicone rapidly, irrespective of the vehicle, and therefore a penetration lag time could not be measured for any of the cosolvents (Figure 4). The diclofenac saturated vehicles containing a low or moderate volume fraction of PG (0.2, 0.4, and 0.5 f PG) displayed donor phase depletion at 2 h postdose application (36%, 41%, and 7.5% of the applied dose 2508

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having permeated 2 h post dose application, respectively). At 0.6 f PG, donor phase depletion was delayed to 5 h, and using 0.7 f PG the system did not show depletion during the 23 h study. The use of infinite doses allowed steady-state transport rates to be calculated in all of the experiments prior to the occurrence of donor depletion effects (R2 > 0.98 over at least five time points, Figure 4). The silicone membrane penetration rate of diclofenac significantly increased with increasing PG vehicle content up to 0.65 f PG (p < 0.05), above which it remained unchanged (p > 0.05; Table 1). There was a 10-fold enhancement in diclofenac transport when f PG was increased from 0.2 f PG (0.28 ± 0.06 μg·cm−2·h−1) to 0.9 f PG (2.81 ± 0.16 μg·cm−2·h−1; Table 1). The membrane diffusion coefficient of the drug in silicone increased 5-fold when f PG increased from 0.2 f PG to 0.65 f PG (p < 0.05). At 0.2 f PG, the K was calculated as 15.51 ± 4.69 (n = 8), but it was almost 45-fold lower at 0.37 ± 0.11 (n = 12) using 0.65 f PG (Table 2). The PG−water Table 2. Diclofenac Permeation Parameters: Partition Coefficient (K) between Silicone Membrane and Propylene Glycol (PG)−Water Binary Mixtures (pH 3), Transport Rates, and Effective Diffusion Coefficient (D) (Regenerated Cellulose Membrane (RCM) vehicle

Figure 3. (a) Saturated solubility (X2) of diclofenac in propylene glycol (PG)−water mixtures (pH 3) (mean ± SD, n = 3). (b) Deviation (Xdev) of observed diclofenac saturated solubility (X2) from ideal solubility (Xi) in PG−water mixtures (mean ± SD, n = 3) as a function of PG volume fraction (f PG) where the ideal solubility is represented by a line superimposable on the x-axis. The dotted line indicates the inflection point at which the solubility data appears to show a change in the cosolvent supramolecular structure.

parameter

0.2 f PG

Ka Db (× 10−3 cm2·h−1) transport rate (RCM)b (μg·cm−2·h−1) transport rate (silicone)b (μg·cm−2·h−1)

15.51 ± 4.69 5.82 ± 1.15 c 0.28 ± 0.06

0.65 f PG 0.37 38.49 4.01 1.88

± ± ± ±

0.11 2.04 0.31 0.08

a n = 8−9. bn = 5. cValue could not be measured as amounts permeated were below the assay limits of detection.

binary mixtures had no significant influence on the silicone membrane solubility of diclofenac (p > 0.05); it remained unchanged at 0.59 ± 0.18 mg.g−1 (n = 24) irrespective of the PG content in the diclofenac-saturated vehicle (data not shown graphically). Although RCM is a porous hydrophilic membrane, it has been demonstrated previously to provide a barrier for drug transport.37,38 As such, it was deemed suitable to use for the measurement of drug transport, and as in a previous work, it was assumed to not be subject to significant drug partitioning. The RCM transport of diclofenac from the drug-saturated 0.65 f PG did not suffer from dose depletion, and the transport rate was 4.01 ± 0.3 μg·cm−2·h−1 (taken from 1.25 to 5 h as values prior to 1.25 h were below the assay LOD, R2 > 0.97). The drug-saturated 0.2 f PG vehicle did not generate any quantifiable transport.

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DISCUSSION A number of vehicles used to deliver pharmacologically active molecules are now known to display nonideal mixing at a molecular level due to formation of supramolecular structures.1−7,39 However, the influence of supramolecular vehicle structuring on processes relevant to the administration of drug molecules remains unclear. The FTIR data generated in this study suggested that PG−water formed supramolecular structures in a similar manner to the methanol−water1−3,6,39 and ethanol−water vehicles.4,5,7 Like these other binary solvent mixtures, a shift of the alkyl stretching motion to higher wavenumbers was observed when the water volume fraction was increased, and this was assigned to hydrophobic hydration between the alkyl groups and water.40−42 The alkyl group shift

Figure 4. Effect vehicle supramolecular structuring on the transmembrane transport of un-ionized diclofenac across silicone membrane; (a) at propylene glycol volume fractions (f PG) of 0.2 (⧫), 0.4 f PG (◊), and 0.5 f PG (▲) and (b) at 0.6 f PG (⧫), 0.7 f PG (◊), and 0.9 f PG (▲) (mean ± SD, n = 5).

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consequence of water structuring and the consequential inability of PG molecules that support these structures to interact with the solute, while the higher than expected solubility in the PG-rich vehicles can be attributed to PG-rich supramolecular structures having a higher solvency capacity than predicted. These trends in the diclofenac solubility data seemed to broadly support the properties of the solvent described by the FTIR data, but there was a critical “offset” in the appearance of the most significant changes in behavior which were observed in the PG-rich systems, that is in systems >0.55 f PG. This suggested that un-ionized diclofenac solubility was most heavily influenced by the proportion of the PG molecules which were not associated with the water supramolecular structures. Previous work with diclofenac diethylamine ion-pairs showed a mirror image of the data presented in this work, which further supports the presence of a specific interaction between the un-ionized drug and the PG-rich supramolecular structures formed in this binary system.29 The transmembrane transport data recorded for un-ionized diclofenac suggested that PG-rich vehicles facilitated entry into the barrier through the moderation of the drug−membrane interactions in a manner that facilitated the drug transport without perturbing the properties of the barrier. Such a mode of membrane penetration enhancement has been previously reported for PG.47,48 In this work, the unaltered capacity of drug within the membrane and its constant thickness, regardless of cosolvent volume fraction, demonstrated that the physical membrane properties were not influenced by the PG−water system. In this context the inverse relationship between diclofenac partitioning and membrane transport was representative of the ability of the PG to moderate the interactions with the barrier. Combining the data generated for the un-ionized diclofenac with equivalent studies reported in previous work for diclofenac ion-pairs, which demonstrated a slower transport rate in the presence of the PG rich supramolecular structures, provides strong evidence that it is inappropriate to simply suggest that there are appreciable “solvent interactions” between diclofenac and the PG/water vehicle. It may be more accurate to suggest that diclofenac behavior when dissolved in a binary PG/water solvent is a result of the interaction with discrete solvent supramolecular structures, which alter the functional properties of the drug solutions (Figure 5).

is counterintuitive, as conventionally proton donor groups involved in H-bonding interactions move to lower wavenumbers. However, the CH−water interaction is accompanied by C−H bond shortening and an increase in the electron density due to the transfer from the proton acceptor group.40,42 Some of the shifts in band position were relatively small when comparing one single spectrum to a second; however the magnitude of the shifts recorded in the FTIR spectra (in the range of 4−8 cm−1) were in accordance with those reported previously for alcohol−water and surfactant−water interactions.41,43,44 The inflection point in the spectroscopy data, which was at an equivalent point for C−O of PG and O−D of D2O, indicates that the water-rich suprastructures achieve an optimal configuration in solvents with no more than 0.4 f PG. Previous literature supports the conclusion that, up to this critical point, most PG molecules would be involved in supporting water structuring in a manner whereby the hydrogen bonds between PG−water are thought to compensate for the lost hydrogen bonding between water−water as the volume fraction of PG increases.2−4,7 The lack of an inflection point in the CH-stretching shifts suggests the formation a second series of PG−water structures, which are generated through hydrophobic hydration but do not support the water supramolecular structuring identified from the water-rich mixtures. Above the 0.4 f PG composition, it is proposed this second population PG−water arrangements predominate, while the prevalence of the water-rich supramolecular structures recedes, as it was demonstrated previously for ethanol−water and methanol−water vehicles.3,6,7,41 The shifts in the C−O stretching vibration of PG, recorded even when the water peak is lost in the PG-rich vehicles, support this hypothesis. One significant difference for the PG/water solvent compared to the analogous data reported for ethanol/water and methanol/water systems was that the water structuring, most evident in waterrich solvents, was not enhanced by the presence of the PG cosolvent. This demonstrates that the chemical identity of the cosolvent mixture does have a significant influence on the strength and presumably the type of solvent clusters that are formed in vehicles.1,4−7,39 Spectral deconvolution (as described by Singh et al.45) was possible for the presented IR data, but it proved difficult to make accurate assignments for all of the spectral peaks without the support of addition spectroscopic or in silico data, and hence this analysis has not been presented. The exponential increase in un-ionized diclofenac solubility with an increasing volume fraction of PG was comparable with previously reported findings for lipophilic agents.12,13,15,26 This behavior cannot be easily explained by the vehicle solvency capacity according to the solubility parameter and suggests that the nonideal mixing properties of the binary solvent system, not accounted for in Fedors method,31 has a consequential influence on drug solubility. A log−linear solubility model provides a better means of describing drug solubility in cosolvents compared to a linear relationship, but diclofenac solubility also deviates from the log-normal profile, albeit in a manner that has previously been reported for other lipophilic molecules.12,13,15 The range of positive and negative deviations from the ideal log linear solubility for ibuprofen,46 phenytoin,13 and esters of amino- and hydroxyl-benzoates12 are similar to that of diclofenac. This series of agents are characterized by two main regions in the log plots: a negative deviation from ideal solubility in water-rich systems and a positive deviation from ideal solubility in PG-rich systems. The lower than expected solute solubility in the water-rich solvents was probably a

Figure 5. Silicone transmembrane transport rate of diclofenac from drug-saturated binary propylene glycol (PG)/water mixtures as a function of different PG volume fractions (f PG), when diclofenac was presented at the un-ionized form (pH 3) (◊) and as the diclofenac diethylamine ion-pair (pH 7.6, data taken from Benaouda et al.28) (■); mean ± SD, n = 5. 2510

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CONCLUSIONS The concentration-dependent passive transmembrane transport process described by traditional theoretical frameworks cannot be used to explain the manner in which diclofenac molecules pass through a homogeneous model membrane when applied in a PG−water solvent. This is not because of the influence of the permeant thermodynamic activity, which was kept constant in this work, but because the PG−water delivery vehicle is not homogeneous at a molecular level. Importantly, the data presented in this study demonstrate that this effect is not limited to highly hydrophobic barriers and therefore applicable only to formulations applied to external membranes such as the skin, but it is also experienced when barrier transport is not limited by the partitioning process. Therefore it appears pertinent that supramolecular complexes that are present in pharmaceutical dosage forms should be given some consideration when dosage forms are constructed and delivery characteristics are assessed.

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ASSOCIATED CONTENT

* Supporting Information S

FTIR spectra of PG/water at 0.3 and 0.7 PG volume fractions (Figures S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*King’s College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, U.K. Tel.: +44 (0)207 848 4843. Fax: +44 (0)207 848 4800. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Algerian Ministry of Higher Education and Scientific Research is acknowledged for the funding and Yu-Lin Chen for assistance in generating the graphical abstract.

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REFERENCES

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