Journal of Controlled Release 69 (2000) 421–433 www.elsevier.com / locate / jconrel
NMR characterisation and transdermal drug delivery potential of microemulsion systems a, b c Mads Kreilgaard *, Erik J. Pedersen , Jerzy W. Jaroszewski a Department of Pharmaceutics, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Institute of Chemistry, University of Copenhagen, The H.C. Ørsted Institute, Universitetsparken 5, DK-2100 Copenhagen, Denmark c Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark b
Received 26 June 2000; accepted 30 August 2000
Abstract The purpose of this study was to investigate the influence of structure and composition of microemulsions (Labrasol / Plurol Isostearique / isostearylic isostearate / water) on their transdermal delivery potential of a lipophilic (lidocaine) and a hydrophilic model drug (prilocaine hydrochloride), and to compare the drug delivery potential of microemulsions to conventional vehicles. Self-diffusion coefficients determined by pulsed-gradient spin-echo NMR spectroscopy and T 1 relaxation times were used to characterise the microemulsions. Transdermal flux of lidocaine and prilocaine hydrochloride through rat skin was determined in vitro using Franz-type diffusion cells. The formulation constituents enabled a broad variety of microemulsion compositions, which ranged from water-continuous to oil-continuous aggregates over possible bicontinuous structures, with excellent solubility properties for both lipophilic and hydrophilic compounds. The microemulsions increased transdermal flux of lidocaine up to four times compared to a conventional oil-in-water emulsion, and that of prilocaine hydrochloride almost 10 times compared to a hydrogel. A correlation between self-diffusion of the drugs in the vehicles and transdermal flux was indicated. The increased transdermal drug delivery from microemulsion formulations was found to be due mainly to the increased solubility of drugs and appeared to be dependent on the drug mobility in the individual vehicle. The microemulsions did not perturb the skin barrier, indicating a low skin irritancy. 2000 Elsevier Science B.V. All rights reserved. Keywords: Microemulsion; Transdermal drug delivery; PGSE NMR; T 1 relaxation; Local anaesthetics
Abbreviations: Cv , drug concentration in the formulation; D, self-diffusion coefficient; d, duration of the z gradient pulse (PGSE NMR); HLB, hydrophilic / lipophilic balance; HPLC, high performance liquid chromatography; Ig , intensities of NMR signal in the presence of field gradient pulses; Io , intensities of NMR signal in the absence of field gradient pulses; II, isostearyle isostearate; J, flux; LAB, Labrasol; NMR, nuclear magnetic resonance; o / w, oil-in-water; PEG, polyethylene glycol; PGSE, pulsed-gradient spin-echo; PI, Plurol Isostearique; S, drug solubility; w / o, water-in oil; kp , permeability coefficient; D, time interval between the gradient pulses (PGSE NMR); g, gyromagnetic constant for 1 H (PGSE NMR) *Corresponding author. H. Lundbeck A / S, Neurochemistry & Discovery ADME (845), Ottiliavej 9, DK-2500 Valby, Denmark. Tel.: 145-3644-2425, ext. 3057; fax: 145-3630-3482. E-mail address:
[email protected] (M. Kreilgaard). 0168-3659 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 00 )00325-4
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1. Introduction The transdermal route of drug administration offers several advantages compared to the oral and parental route in terms of decreased drug degradation before entrance into the systemic circulation and higher patient comfort and compliance, respectively. However, the poor permeability of the epidermal stratum corneum often limits the possibilities for choosing the topical administration route for novel drug formulations. Several reports have indicated that microemulsion vehicles may increase transdermal delivery of both lipophilic and hydrophilic drugs, compared to conventional vehicles, depending on the constituents used for the microemulsion vehicle [1–7]. It has been suggested that microemulsion formulations may increase cutaneous drug delivery by means of the high solubility potential for both lipophilic and hydrophilic drugs, which creates an increased concentration gradient towards the skin [2,6,7], and / or by using constituents with penetration enhancer activity [5,6,8]. Microemulsion systems, which increase permeation rate by means of the latter mechanism, are often of limited clinical relevance, due to the possibility of inducing skin irritancy [9]. Besides the individual characteristics of the applied constituents for pharmaceutical microemulsion formulations, transdermal drug delivery potential of the systems has been demonstrated to be highly dependent on the incorporated ratio of the respective constituents [2,7,10,11]. However, the mechanism behind the drug delivery potential of a given microemulsion vehicle, and relationship to the fractional composition / internal structure of the phases, has not yet been elucidated. Microemulsion structures may vary from emulsion-like normal or reverse swollen micelles, to aggregates with typical sizes of 10–100 nm, over bicontinuous structures, depending on the ratio of the constituents [12]. They are dynamic systems in which the interface is continuously and spontaneously fluctuating [13]. Determination of self-diffusion coefficients of the components by pulsed-gradient spin-echo (PGSE) NMR has proven to be a valuable, general tool for characterisation of microemulsion structures [10,12,14–17]. The purpose of the present study was to investi-
gate the transdermal drug delivery potential of microemulsions, formulated with a novel low-irritant non-ionic surfactant system, and to characterise these microemulsions. The specific aims of the investigations were 3-fold: (A) To investigate the influence of drug disposition in microemulsions, solubility potential and the self-diffusion rate of the constituents in the vehicles on the transdermal delivery rate of a lipophilic and a hydrophilic model drug. (B) To optimise the presented microemulsion formulations for high transdermal drug delivery rate, and compare the microemulsion formulations to marketed conventional vehicles. (C) Finally, to assess potential cutaneous irritancy of the microemulsions in terms of disrupted barrier function of the skin.
2. Materials and methods
2.1. Chemicals Labrasol (a mixture consisting of 30% mono-, diand triglycerides of C 8 and C 10 fatty acids, 50% of mono- and di-esters of poly(ethylene glycol) (PEG 400) and 20% of free PEG 400), Plurol isostearique (isostearic acid ester of polyglycerol, containing 30– 35% of diglycerol, 20–25% of triglycerol, 15–20% of tetraglycerol, and 10% of pentaglycerol and higher oligomers) and isostearylic isostearate (92% purity) were products of Gattefosse´ (Lyon, France) and were obtained from Bionord (Hellerup, Denmark). The same batch of the microemulsion components was used in all experiments. Lidocaine and lidocaine hydrochloride were purchased from Unikem (Copenhagen, Denmark) and prilocaine hydrochloride from Sigma (St Louis, USA). Prilocaine was ¨ ¨ a gift from Astra (Sodertalje, Sweden). All chemicals were used as received. EMLA (2.5% lidocaine, 2.5% prilocaine), Xylocain 5% cream (lidocaine) and Xylocain 2% gel (lidocaine hydrochloride) (Astra) are commercial formulations. Distilled water was filtered through a Milli-Q filter (Millipore, Bedford, MA, USA) prior to use. Solvents were of HPLC grade, and all other chemicals were of analytical grade.
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2.2. Microemulsions A microemulsion is defined as a system of water, oil and surfactants, which is a transparent, single optically isotropic and thermodynamic stable liquid solution [18,19]. Pseudo-ternary phase diagrams of microemulsion regions were constructed by slow titration of a given blend of surfactant / co-surfactant (Labrasol / Plurol Isostearique) and oil (isostearylic isostearate) with water. The titrated mixture was continually stirred at room temperature and microemulsion regions were identified as transparent, low viscous and isotropic mixtures. No attempt was made to identify any other structural regions in the phase diagrams. After the microemulsions region borders were established by the first titration, they were confirmed by a second titration using the same procedure but titrating a blend of the surfactants and water with the oil. Microemulsion formulations were formed spontaneously by admixing appropriate quantities of the components and drug with gentle mixing at room temperature. All systems were examined for lack of birefringence by polarised light microscopy. Furthermore, pH was determined and viscosity measured with a Bohlin V88 viscometer (Bohlin Instruments, Gloucestershire, UK) at 248C. Unless stated otherwise, all microemulsion compositions and drug concentrations are presented as (%, w / w). Solubility of lidocaine and prilocaine hydrochloride in the seven microemulsion systems and their components was determined by adding excess amounts of the drugs and continuously mixing for at least 72 h at 248C. The mixtures were centrifuged (35003g, 20 min), dissolved in isobutanol, and concentrations of the drugs were determined by HPLC.
2.3. NMR spectroscopy NMR measurements were performed at 258C on a Bruker AMX 400 or a Varian Unity 400 system equipped with a Peforma 1 pulsed-field gradient unit (9 Gauss / cm), using 5-mm inverse probes. Chemical shifts of all microemulsion components were determined relative to internal tetramethylsilane (neat Labrasol, Plurol Isostearique and isostearylic iso-
423
stearate, lidocaine in isostearylic isostearate) or sodium 4,4-dimethyl-4-silapentanesulfonate (aqueous lidocaine and prilocaine hydrochloride), and the same values were adopted for the NMR spectra of the microemulsion formulations, which were determined without internal standards. Longitudinal (T 1 ) relaxation times were determined using the inversion recovery method with 18 relaxation delay increments. Microemulsion formulations containing D 2 O / H 2 O in a ratio of 1:9 were used. Oxygen was not excluded from the microemulsions and their components. Self-diffusion coefficients were measured using the gradient spin-echo pulse sequence, modulating the amplitude of fixed-length gradient pulses; 21 amplitude values were used for each experiment. Microemulsion formulations containing D 2 O / H 2 O in a ratio of 1:1 were used in the diffusion measurements. Self-diffusion coefficients (D) were derived from a slope of line defined by ln(Ig /Io ) 5 2 [g 2 d 2 G 2 (D 2 d / 3)]D, where Ig and Io are intensities of NMR signal in the presence and absence of field gradient pulses, g is the gyromagnetic constant for 1 H, d is the duration of the z gradient pulse, and D the time interval between the gradient pulses. With neat water, a value of 1.71310 210 m 2 s 21 was determined, which corresponds well to accepted value. Two or more experiments were carried out with each microemulsion using several values of d (typically between 10 and 80 ms) to cover the span of diffusion coefficient values characterising the system. In some cases (see text) the data showed biexponential behaviour with different diffusion coefficients, the limiting values of which were determined from the plot.
2.4. HPLC assay Lidocaine and prilocaine were quantified using a high-performance liquid chromatography (HPLC) system (Merck–Hitachi, Darmstadt, Germany), consisting of a D-7200 autosampler (20 ml loop), a 655A-11 pump (1.5 ml / min), a 655A UV-detector (205 nm), a D-7500 integrator and a Merck LiChrospher 100 RP-18 column (5 mm, 12534.6 mm i.d.). The mobile phase consisted of acetonitrile– 0.05 M aqueous Na 2 HPO 4 –triethylamine (40:60:0.01, v / v) adjusted to pH 7.0. The peak area correlated linearly with lidocaine and prilocaine
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concentrations (r 2 51.000) in the range 2–100 mg / l. Limit of quantitation was 4.9 and 0.36 mg / l; coefficient of variation was 1.7 and 1.7% at 2 mg / l and 2.2 and 1.9% at 100 mg / l for lidocaine and prilocaine, respectively.
dependent experiments, and data are expressed as mean6standard deviation (S.D.), unless stated otherwise.
3. Results and discussion
2.5. Transdermal permeation 3.1. Microemulsion formulation The permeation studies were carried out in Franztype [20] glass diffusion cells (7 ml receptor volume, 1.8 cm 2 area of diffusion) from Crown Glass (Somerville, NJ, USA). Excised dorsal skin (subcutaneous fatty tissue removed) from male Wistar rats (300– 400 g), shaved and sacrificed immediately prior to the experiments, was used as a barrier membrane. The receptor compartment was filled with an isotonic buffer (0.09 M NaCl, 0.05 M KH 2 PO 4 , pH 7.4), magnetically stirred and maintained at 328C during the entire experiment. Test sample (1 ml) was applied in the donor compartment. At predetermined times, 1-ml samples were withdrawn from the receptor compartment and were immediately replaced by fresh buffer solution. Sink conditions were maintained at all times. All samples were centrifuged (35003g, 10 min) and the supernatant assayed for lidocaine or prilocaine content by HPLC. Accumulated amount of drug (Q, mg) in the receptor compartment was plotted as function of time (t, h), and steady-state flux (J, mg h 21 cm 22 ) was calculated as the slope from the linear part of the curve (20–28 h) divided by the application area. Permeability coefficients (kp , mg h 21 cm 22 ) were calculated as kp 5 J /Cv , where Cv (%, w / w) is drug concentration in the formulation. To examine potential cutaneous irritancy, 1 ml of water or neat microemulsion A was initially applied in the donor compartment for a 20-h period. Subsequently, the compartment was emptied and refilled with 1 ml of microemulsion A 2.4% prilocaine hydrochloride, and the permeation measurements carried out as described above. Flux and permeation coefficients were calculated from the 3–9-h sampling period after application of the active microemulsion (23–29 h after mounting the rat skin). Two-tailed t-test was used for statistic analysis of kp and J values of the formulations, P,0.05 being considered statistically significant. All transdermal permeation values were calculated from three in-
Pseudo-ternary phase diagrams of microemulsion regions of existence with Labrasol, Plurol Isostearique, isostearylic isostearate and water mixtures and the final compositions are presented in Fig. 1. The large microemulsion regions of existence found with the present components, enabled a broad variety of possible formulation compositions with different internal structure and solubility properties for lipoand hydrophilic drugs. Based on the phase diagrams, seven microemulsion compositions (referred to as system A–G, Table 1) were selected from the regions of existence. All microemulsion compositions studied maintained microemulsion characteristics after addition of lidocaine or prilocaine hydrochloride in the concentrations of interest in the temperature range of 24–328C.
3.2. Solubility The solubility of lidocaine and prilocaine hydrochloride in the microemulsion systems and neat components is shown in Table 2. Comparison of the measured solubility of the drugs to that calculated from the solubilities in neat microemulsion components and the respective weight fraction of the components in the vehicles, showed a 28–62% increase in lidocaine solubility and a 24–40% increase in prilocaine hydrochloride solubility in the microemulsion structures. The large increase in drug solubility is most likely related to the formation of an interfacial surfactant-film between the oil and water phase, which may lead to additional solubilisation sites for the drugs, compared to the molecular organisation of the bulk surfactants. This agrees well with results of Malcolmson and co-workers [21–23], who suggested that the major solubilisation advantage of microemulsions could be ascribed to the surfactant interfacial film of micellar structures, and the solubility of (lipophilic) drugs would be further
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Fig. 1. Pseudo-ternary phase diagrams of microemulsion regions of existence (within the connected lines) with different surfactant / cosurfactant ratios ((a) 1:1; (b) 2:1; (c) 3:1) composed of Labrasol, Plurol Isostearique, isostearyle isostearate and water (%, w / w). Composition of microemulsion system A–G is indicated on the diagrams and collected in Table 1.
increased over that of micellar solutions by increasing oil content of the microemulsion. For further studies, the seven microemulsion systems selected (Table 1) were formulated with 4.8% (w / w) of lidocaine and 2.4% (w / w) of prilocaine hydrochloride (except for system D, due to the low solubility). Additionally, each microemulsion system was formulated with a drug concentration approximately 10% lower than the assessed saturation value, in order to determine transdermal drug flux for each
of the microemulsion formulations with optimised thermodynamic activity.
3.3. NMR characterisation of microemulsions 3.3.1. T1 relaxation investigations 1 H NMR spectra of microemulsions could be readily assigned by comparison with the spectra of pure microemulsion components. However, since several of the components were derived from fatty
M. Kreilgaard et al. / Journal of Controlled Release 69 (2000) 421 – 433
426 Table 1 Microemulsion compositions System
% Water
% Isostearylic isostearate
% Labrasol
% Plurol Isostearique
A B C D E F G
20 20 20 7 11 55 65
10 10 10 70 26 8 3
35 a 47 b 53 c 11.5 a 42 b 25 b 24 c
35 a 23 b 17 c 11.5 a 21 b 12 b 8c
a
Labrasol–Plurol Isostearique, 1:1 (w / w). Labrasol–Plurol Isostearique, 2:1 (w / w). c Labrasol–Plurol Isostearique, 3:1 (w / w). b
acids (i.e., isostearylic isostearate, Labrasol and Plurol Isostearique), many of the resonances present in the spectra of microemulsions corresponded to multiple species. Unique signals included the ethylenedioxy group (OCH 2 CH 2 O) of the PEG moiety and the resonances of the drugs. The microemulsions, their components and microemulsion formulations were characterised by longitudinal (spin-lattice, T 1 ) relaxation times and self-diffusion coefficients determined by PGSE NMR. Relaxation times are related via correlation times to the mobility of a molecule, and may thus be used to assess the site of incorporation in the microemulsion phases. Examples of the determined T 1 values are shown in Table 3. While relaxation times of
individual groups may vary according to their internal mobility, the trends observed were identical for all groups. Thus, the relaxation times of lidocaine in all microemulsions were closely similar to that found in neat isostearylic isostearate, and were significantly shorter than in water due to the increased viscosity of the medium. This indicates that lidocaine is primarily located in the oil phase, and that a large proportion of the drug is free, i.e., not immobilised in the surfactant interface. This is in agreement with the better solubility of lidocaine in the lipophilic components than in water (Table 2). The relaxation times of prilocaine hydrochloride in the microemulsions were also significantly shortened compared to aqueous solution, indicating a less dynamic behaviour.
Table 2 Viscosity, measured (Smea ) and calculated (Scalc ) solubility of lidocaine and prilocaine hydrochloride in microemulsion systems and components at 248C, and pH of saturated solutions System
A B C D E F G Labrasol Isostearylic isostearate Plurol Isostearique Water
Viscosity
Lidocaine
(Pa)
Smea (%, w / w)
Scalc (%, w / w)
pH
0.39 0.21 0.20 0.11 0.19 0.10 0.02 – –
26 30 27 20 28 14 11 26 12
18 19 19 14 19 10 9 – –
8.5 8.6 8.3 7.8 8.0 8.3 7.7 – –
–
22
–
–
–
8.0
–
0.4
Prilocaine hydrochloride Smea (%, w / w)
Scalc (%, w / w)
6 6 6
5 5 5
pH
–
–
4 15 16 ,1 ,1
3 11 12 – –
4.7 4.2 4.2 – 4.5 4.2 4.8 – –
2
–
–
18.5
–
4.3
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Table 3 Longitudinal (T 1 ) relaxation times (s) of prilocaine hydrochloride (2.4%) and lidocaine (4.8%) in neat microemulsion components and microemulsion systems, respectively, at 258C System
Water Isostearylic isostearate A B C D E F G
Compound Prilocaine hydrochloride d
Lidocaine d
1.74 0.80 a
1.96 0.77
0.68 / 0.76 b 0.81 0.78 0.81 0.80 1.04 1.03 / 0.91 c
0.83 0.81 0.82 – 0.83 0.88 0.77
a
Prilocaine-free base. 4.8% prilocaine hydrochloride. c 14% prilocaine hydrochloride. d Determined with the aromatic signals. b
From the solubility data, it would appear that prilocaine hydrochloride is dissolved in the aqueous phase. However, the T 1 results point to a partial adhesion or integration of the drug into the surfactant system, which becomes more pronounced as water / surfactants ratio decreases. This cohesion is presumably attributable to the weak surface active properties of prilocaine hydrochloride.
3.3.2. PGSE assessment of self-diffusion coefficients Self-diffusion coefficient of a compound is inversely related to the macromolecule / aggregate radius and viscosity of the medium, and reflects the degree of structural encapsulation and association of phases [12]. PGSE NMR-determined self-diffusion coefficients are presented in Fig. 2. Deviations of replicate assessments were generally very small (CV,5%), and self-diffusion coefficients were, therefore, generally determined by single assessments. Since the amplitude of resonances in PGSE NMR experiments are modulated by apparent transverse relaxation times (T 2* ) and the PGSE spectra have to be displayed in the magnitude mode, selfdiffusion coefficients could not be determined for components giving weak resonances, especially those on the foot of strong signals. Although the
Fig. 2. Self-diffusion coefficients (D, m 2 s 21 310 11 ) of, respectively, (a) unloaded and (b) lidocaine (4.8%) or (c) prilocaine hydrochloride (2.4%) loaded microemulsion systems and single constituents (Labrasol [Lab], Plurol Isostearique [PI], water and isostearylic isostearate [II]), determined by PGSE NMR at 258C. The systems are arranged according to increasing water content. The self-diffusion coefficients for the two common signals for Lab, PI and II were determined from resonance of CH 3 and CH 2 , respectively. The self-diffusion coefficients for the unique signal of Lab were determined from the resonance of the OCH 2 CH 2 group of the polyethylene glycol moiety. Symbols with dashed lines indicate biphasic behaviour. Self-diffusion coefficient of prilocaine in neat isostearylic isostearate was determined with the free base form of the drug, and that of lidocaine in neat water with 0.4% lidocaine.
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signals of terminal methyl groups and of the methylene groups of the fatty acid residues correspond to multiple species including Labrasol, isostearylic isostearate and Plurol Isostearique, PGSE experiments yielded linear plots for these signals in most systems, indicating that these molecules either had identical self-diffusion coefficients or the behaviour of the resonances was completely dominated by one component. Generally, self-diffusion of Labrasol, Plurol Isostearique and isostearylic isostearate in all microemulsion systems was very low (10 212 m 2 s 21 range), indicating that they are included in conjugated frame-like structures, i.e., ‘swollen micelles’ or aggregates in bicontinuous structures. Only the selfdiffusion determined from the methyl and methylene groups of the system D remained similar to those of the neat components (10 211 m 2 s 21 range). Due to the high oil content in D, these values are presumed to reflect isostearylic isostearate diffusion, indicating the presence of the oil in a continuous phase. Even though water diffusion was not measurable for system D due to its low content, it is thus assumed that the microemulsion D structure consists of waterin-oil (w / o) droplet-like aggregates. The behaviour of the unique OCH 2 CH 2 O resonance of PEG-containing components of Labrasol in microemulsions F and G indicated the presence of two distinct self-diffusion coefficients. One of them was in the 10 212 m 2 s 21 range, being similar to those determined with the fatty acids residues, and another one order of magnitude larger. This is most likely due to the heterogeneous nature of Labrasol, which contains PEG esters of different molecular size. Thus, the diffusion data demonstrate the presence of PEG in the continuous phase outside the surfactant frame structures in the systems F and G. Self-diffusion coefficients of water in microemulsions A–C were about 10 times lower than that of neat water, indicating that water is partially encapsulated in the surfactant framework in these systems, possibly in bicontinuous structures. In microemulsion E, the water self-diffusion coefficient was almost of the same order as for the surfactants, indicating a w / o droplet-like structure. Water diffusion in microemulsion F and G was relatively fast, pointing towards water constituting a continuous free phase in these systems, forming o / w droplet-like aggregates. The slow diffusion of lidocaine in all systems
confirmed the association with the lipophilic phase, in agreement with the T 1 relaxation results. The diffusion was fastest in the microemulsions containing the smallest amounts of surfactants (D, F and G). The microemulsion structures were not substantially affected by the addition of lidocaine, neither at 4.8% drug load (Fig. 2) or at near saturated levels (examined for systems A and G; data not shown). The low diffusion coefficients of prilocaine hydrochloride confirmed the presumed association to the surfactant structures, which, however, appears to be inversely related to the water content and prilocaine hydrochloride concentration in the microemulsions. Although no major structural changes were observed upon the addition of prilocaine hydrochloride, selfdiffusion of the surfactants in systems A, F, and G was somewhat altered compared to those of unloaded microemulsions, which became more pronounced at higher drug concentrations (systems A and G; data not shown). Self-diffusion coefficient of prilocaine hydrochloride was substantially increased at near saturated systems (from 0.6 to 1.4310 211 m 2 s 21 for system A and from 1.3 to 7.6310 211 m 2 s 21 for system G). This provides corroborative evidence for the integration of the drug with the surfactant structure at low concentrations.
3.4. Transdermal drug delivery potential of microemulsions Transdermal flux of a drug in a simple solution is theoretically proportional to the partition coefficient between the skin and the vehicle and to the concentration of the drug in the vehicle, which is also referred to as activity [24]. In colloidal systems, the relationship is more complex due to the multiple phases and surfactant film, and the activity of a drug in the vehicle is not readily assessable [3,25–27]. The present investigations have therefore assessed transdermal drug delivery potential of the microemulsions at both equal drug concentrations and at theoretically maximal thermodynamic activity (at saturated level).
3.4.1. Correlation between transdermal permeation rate and structure characterisation of microemulsion formulations at equal drug load Transdermal permeation rate of lidocaine and prilocaine hydrochloride from microemulsion sys-
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429
Table 4 Steady-state flux (J; 20–28 h) of lidocaine (4.8%, w / w) and prilocaine hydrochloride (2.4%, w / w), through rat skin from microemulsion systems A–G with equal drug load System
Lidocaine 4.8% J (mg h 21 cm 22 )
A
7.160.3
B C D E F G
10.361.1 8.262.3 14.667.8 8.360.8 25.162.0 26.366.5
Prilocaine hydrochloride 2.4% J (mg h 21 cm 22 ) 6.261.7 1.060.1 a,c 3.261.2 b,c 2.560.8 c 10.762.3 10.163.5 – 6.260.7 16.364.9 6.161.2
a
Skin pre-treated for 20 h with water. Skin pre-treated for 20 h with neat microemulsion A. c Sample interval: 3–9 h after application of active microemulsion. b
tems containing equal drug concentrations is presented in Table 4. A considerable variation in mean transdermal flux was observed, depending on the microemulsion composition. This confirms that the drug delivery potential of microemulsions is greatly dependent on the internal structure / fractional composition of the phases [2,7,10,11]. Correlation of transdermal flux with drug selfdiffusion values determined by PGSE NMR (Fig. 3) indicated a linear relationship for both lidocaine (r 2 50.79, P50.01) and prilocaine hydrochloride (r 2 50.78, P50.02). This suggests that the transdermal drug delivery potential of microemulsion vehicles, with given constituents, may be hampered by diffusion hindrances due to the internal structure of the microemulsion. It should be noted, however, that the high correlation coefficient for the prilocaine hydrochloride formulations is primarily attributable to microemulsion F (without system F: r 2 50.30, P50.34), and more studies would be desirable to confirm the relationship indicated by this study. The current findings compare well with earlier studies by Osborne et al., where a correlation has been indicated between water self-diffusion coefficients in three microemulsion vehicles and transdermal flux for both glucose [11] and water [10] (assuming that glucose is primarily dissolved in the aqueous phase and the self-diffusion coefficient of glucose can be related to that of water). The significant influence of
Fig. 3. Correlation between self-diffusion coefficients (D) and transdermal flux (J) of (a) lidocaine (n57) and (b) prilocaine hydrochloride (n56) in microemulsion systems. Error bars represent S.D. (n53). Solid line represents best linear fit.
the drug diffusion in the vehicle on the transdermal delivery rate is also supported by in vitro flux investigations of diclofenac diethylamine from five phospholipid formulations with various colloidal structures, ranging from liposomal dispersions via microemulsions to lamellar liquid crystals [2]. The diffusion rate of the drug in these vehicles was assessed indirectly by estimating the release rate of the drug from the formulations through a non-ratelimiting silicone membrane and compared well with permeation rate of the drug through excised human stratum corneum.
3.4.2. Comparison of optimised microemulsion vehicles to conventional vehicles for transdermal drug delivery Transdermal drug delivery results for microemulsion systems containing near saturated drug concentrations are collected in Table 5. There was a
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Table 5 Steady-state flux (J; 20–28 h) and permeation coefficient (kp ) of lidocaine and prilocaine hydrochloride (HCl) through rat skin from microemulsion systems A–G with near maximum drug load and comparison with EMLA 5%, Xylocain 5% o / w-cream and Xylocain 2% hydrogel Formulation
A B C D E F G EMLAa EMLAa,c Xylocain 5% Xylocain 2%
Lipophilic model drug
Hydrophilic model drug
[Lidocaine] (%, w / w)
J (mg h 21 cm 22 )
kp (mg h 21 cm 22 )
23 27 24 17 25 12 9.1 2.5 2.5 c 5 –
36.264.2 41.168.9 44.864.3 56.564.6 50.868.6 44.664.3 78.363.9 52.3619 57.8618.8 c 22.266 –
1.660.2 1.560.3 1.960.2 3.360.3 2.060.3 3.760.4 8.760.4 20.967.6 23.167.5 c 4.461.2 –
[Prilocaine HCl] (%, w / w)
J (mg h 21 cm 22 )
5 5 5
8.162.6 6.361.3 6.462.1
–
–
2.4 b 13 14 – – – 2d
6.260.7 24.764.3 29.769.9 – – – 3.161.0
kp (mg h 21 cm 22 ) 1.660.5 1.360.3 1.360.4 – 2.660.3 1.960.3 2.160.7 – – – 1.660.5
a
n55. Near maximum concentration equals 2.4%. c Prilocaine-free base. d Lidocaine hydrochloride. b
large increase in lidocaine flux from all systems upon the increased drug load, and, apart from system F, the individual flux ranks of the systems did not diverge significantly from those obtained with the low drug load systems. This indicates that the differences in relative thermodynamic activity were not the main determinant for the flux ranks observed with the systems with equal drug load. All microemulsion systems provided a significantly larger transdermal flux for lidocaine, with microemulsion G 9.1% L increasing average lidocaine flux almost 4-fold (P50.0002), compared to the conventional o / w-emulsion (Xylocain 5%). However, apart from system G, permeation coefficients of lidocaine from the microemulsions were not larger than that from the conventional o / w-emulsion, indicating that the unique microemulsion structures do not generally increase drug delivery compared to regular emulsions. Thus, at theoretically equivalent thermodynamic activity, overall transdermal flux is apparently increased due to the higher concentration gradients enabled by the excellent solubilisation properties of the microemulsions, without a concurrent decrease in partition coefficient for the drug. Lidocaine and prilocaine form a neat oil phase (eutectic mixture) in EMLA, and the drugs, therefore, have a unique thermodynamic activity in this formu-
lation. Accordingly, a superior transdermal permeation coefficient of lidocaine was found, compared to the microemulsions (Table 5). Nevertheless, average transdermal flux of lidocaine from microemulsion G 9.1% L was approximately 50% larger (P50.03), compared to that from EMLA, which can be explained by the almost 4-fold larger concentration gradient from the microemulsion. The similar permeation coefficients of lidocaine and prilocaine from EMLA, indicate that the transdermal permeation of the drugs in their free base form are not discernible, and it is therefore assumed that the permeation coefficient of prilocaine hydrochloride is comparable with that of lidocaine hydrochloride. Only microemulsion systems F and G displayed a significantly increased transdermal flux of the hydrophilic model drug by increasing the vehicle load, with system G 14% P increasing flux almost 10-fold compared to Xylocain 2% hydrogel (P50.04). The corresponding flux increase for system G with the approximately 6-fold increase in drug load, was substantially larger than for system F. This may be explained by the low diffusivity of the drug in system G at low drug load, indicating a strong adhesion of prilocaine hydrochloride to the surfactant film, which hampers partitioning of the drug to the skin. The self-diffusion coefficient was substantially
M. Kreilgaard et al. / Journal of Controlled Release 69 (2000) 421 – 433
increased at high drug load (approaching that of system F at low drug load), suggesting unhampered diffusivity. Presumably, the low mobility of the hydrophilic solute in systems A–C impedes transdermal flux in spite of the increased concentration gradient. The permeation coefficients of the model drugs from the vehicles with high drug load did not diverge significantly from those with low drug load. Hence it appears that the increased transdermal flux is mainly due to higher concentration gradients of the drugs in the individual vehicles. These findings correlate well with results from a lecithin / isopropyl palmitate / water-based microemulsion in vitro study by Dreher et al. [[6], who found higher drug permeability coefficients from neat oil compared to microemulsions, but a higher overall transdermal flux of indomethacin and diclofenac from the microemulsion, due to a higher solubility in the latter. However, in the present study the absolute concentration gradient (i.e., maximum solubility potential) was not the main determinant for the transdermal delivery potential of lidocaine for the microemulsions, as indicated by the highest flux obtained from systems F and G, with the lowest absolute lidocaine solubility. As suggested by Osborne et al. [10,11], the present investigations indicate that free diffusion of the drug in the vehicle is a more important factor, which determines the drug delivery potential of a microemulsion, emphasising the importance of characterising the internal structure of microemulsion systems to optimise cutaneous drug delivery.
3.5. Cutaneous irritancy To investigate potential skin irritancy of the microemulsions, control experiments with pre-treatment of the skin barrier with microemulsion A were carried out (Table 4). No statistically significantly difference in the flux of prilocaine hydrochloride through rat skin from microemulsion system A 2.4% P with or without 20 h pre-treatment of the skin with, respectively, neat microemulsion A (P50.47) or water (P50.09) was observed. A significant higher flux of prilocaine hydrochloride from microemulsion A 2.4% P without pre-treatment in the time interval from 20 to 28 h was found, compared to 3–9 h (P50.03). However, this was a general
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trend for all tested formulations, and can be related to a well-known effect of initial increase of skin hydration caused by the infinite dose technique of the experimental in vitro model, increasing skin permeability, or an incomplete drug distribution in the skin. Hence, while these results do not exclude a general interaction of the microemulsions with the stratum corneum, they do not point towards a continuous disruption of the stratum corneum barrier function. The low permeation coefficients from the microemulsion systems with large surfactant content, A–C and E (Tables 4 and 5), provide further evidence that the transdermal drug delivery potential is not due to a general enhancing effect caused by the surfactant interaction with the stratum corneum. The negligible stratum corneum perturbation suggests a low skin irritancy and thus a high clinical relevance of these microemulsions, even though the surfactant content is high. The low skin irritancy of the surfactant system used in this work has also been indicated by unchanged transepidermal water loss from the skin of human volunteers before and after a 3-h treatment with topically applied microemulsions (Labrasol / Plurol Isostearique / ethyl oleate / water) [5].
4. Conclusions The components used in this study were shown to enable a broad variety of microemulsion compositions, with a high solubility potential for both a lipophilic and a hydrophilic model drug. PGSE NMR combined with T 1 relaxation time determinations provided valuable information about microemulsion structures and drug incorporation. The microemulsions were shown to increase transdermal delivery of a lipophilic model drug up to four times compared to a conventional o / w-emulsion vehicle, and delivery of a hydrophilic model drug almost 10 times compared to a hydrogel, depending on the microemulsion structure and drug load. The superior transdermal flux appears to be mainly due to the large solubility capacity of the microemulsions, which leads to larger concentration gradients towards the skin. This study indicates that the increased drug delivery potential of the individual microemulsion vehicles is dependent on the drug mobility in the vehicle, and that mea-
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surement of self-diffusion coefficients is valuable to optimise fractional composition of a given microemulsion vehicle, in order to maximise drug delivery. The negligible skin barrier perturbation indicates that the microemulsions are promising vehicles for future topical applications with low skin irritancy. The in vivo relevance of the increased in vitro permeation rate obtained with the optimised microemulsion vehicles is currently being investigated in both rats and humans, and will be presented in forthcoming papers.
Acknowledgements This study was supported by LEO Pharmaceutical Products Ltd., Bionord A / S and Astra AB. Lona L. Christrup, Erik Didriksen and Aksel Jørgensen are thanked for valuable discussions and comments. Mads B. Larsen and Kirsten Dayan are thanked for technical assistance.
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