2014 年 62 巻 2 号 p. 135-143
Naproxen (Np) is an example of a non-steroidal anti-inflammatory drug (NSAID) commonly used for the reduction of pain and inflammation. In order to develop an alternative formulation for the topical administration of Np, microemulsions were evaluated as delivery vehicles. Four formulations were prepared using isopropyl myristate (IPM) as oil phase, Span 80, Labrafil M, Labrasol, Cremophor EL as surfactants, ethanol as co-surfactant and distilled water or 0.5 N NaOH solution as aqueous phase. The final concentration of Np in the microemulsion system was 100 mg/g (w/w). The physicochemical properties such as electrical conductivity, droplet size, viscosity, pH and phase inversion temperature of microemulsions were measured. Stability tests of the formulations were also performed at 5±2, 25±2 and 40±2°C. The abilities of various microemulsions and selected commercial (C) formulation to deliver Np through the skin were evaluated in vitro using diffusion cells fitted with rat skins. The in vitro permeation data showed that microemulsions increased the permeation rate of Np between 4.335–9.040 times over the C formulation. Furthermore Np successfully permeated across the skin from the microemulsion with the highest flux rate (1.347±0.005 mg·cm−2·h−1) from a formulation (M4Np) consisting of IPM (2.36 g), Labrosol (0.13 g), Span 80 (0.62 g), ethanol (5.23 g), 0.5 N NaOH solution (0.66 g) and Np (1 g). According to the histological investigations, no obvious skin irritation was observed for the studied microemulsions. These results indicate that the microemulsion formulation may be appropriate vehicles for the topical delivery of Np.
Microemulsions are defined as a dispersion consisting of oil, surfactant, co-surfactant and aqueous phase. Microemulsions are single optically isotropic and thermodynamically stable liquid solution. In principle, microemulsion can be used to deliver drugs to the patients via several routes, but the topical application of microemulsion has gained increasing interest.1,2) The administration of microemulsion offers a lot of advantages in dermal and transdermal drug delivery. Three main mechanisms had been proposed to explain the advantages of microemulsion for the dermal delivery of drugs. First, the high solubility potential for both lipophilic and hydrophilic drugs of microemulsion systems might increase thermodynamic activity towards the skin. Second, ingredients of microemulsion, acting as permeation enhancers, might destroy the structure of the stratum corneum (SC) and increase the flux of drugs via the skin. Third, the permeation rate of the drug from microemulsion may be increased because the affinity of a drug to the internal phase could be modified easily to favor partitioning into the SC.3–5)
Naproxen (Np), (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, is a non-steroidal anti-inflammatory drug (NSAID) compound with analgesic and antipyretic effects, used for the treatment of rheumatoid arthritis, osteoarthritis and traumatic contusions.6) NSAIDs are most commonly used to reduce pain and inflammation.7–9) Oral use of NSAIDs is very effective, but the clinical use is often limited because of their potential to cause adverse effects such as irritation and ulceration of the gastro-intestinal mucosa. Administration of these agents via the dermal route can bypass these disadvantages of the oral route and may maintain relatively consistent plasma levels for long term therapy from a single dose.10,11)
The aim of this study was to formulate an appropriate microemulsion system for the topical application of Np and to compare the in vitro permeation rate of Np from these microemulsion formulations with the commercial (C) formulation. Therefore, the stable microemulsion systems consisting of isopropyl myristate (IPM), Span 80, Labrafil M, Labrasol, Cremophor EL, ethanol and water or 0.5 N sodium hydroxide (NaOH) solution were prepared, and its physicochemical properties, permeation ability of Np in vitro and skin irritation were also evaluated.
Naproxen (Np) (98% purity) was a gift from Deva Holding (Turkey). IPM, Cremophor-EL (Polyoxyethylenglyceroltriricinoleat 35), acetonitrile, ethanol were purchased from Sigma (Germany). Span 80 (Sorbitan monooleate), methanol was purchased from Merck (Germany). Labrafil-M, Labrasol (Glyceryl caprylate/caprate and PEG-8 caprylate/caprate) were gift from Gattefosse (France). All the other chemicals and solvents were analytical reagent grade.
Methods. Microemulsion PreparationConstruction of Pseudo-Ternary Phase Diagrams: Pseudo-ternary phase diagrams were constructed using water titration method at 25±2°C to obtain the appropriate components, and their concentration ranges that resulted in a large existence area of microemulsion were chosen. A combination of Cremophor EL: Labrafil-M and Span 80: Labrasol was used as a mixed surfactant and prepared at weight ratios of 1 : 2 and 1 : 5 respectively. For each phase diagrams, the ratio of surfactant (S) to co-surfactant (CoS) were varied as 1 : 1, 1 : 2, 1 : 3, 1 : 4, 1 : 5, 1 : 6, 1 : 7, 1 : 8, 1 : 9, 2 : 1, 3 : 1, 4 : 1, 5 : 1, 6 : 1, 7 : 1, 8 : 1 and 9 : 1. The oil and the mixture of S/CoS diluted with water phase (0.5 N NaOH solution or distilled water) under magnetic stirring. After being equilibrated, the mixtures were assessed visually and determined to be microemulsions. The concentrations of components were recorded in order to complete the pseudo-ternary phase diagrams, and then the contents of oil, S, CoS and water at appropriate weight ratios were selected based on these results.
Preparation of Naproxen Loaded MicroemulsionsMicroemulsion formulations selected from the constructed phase diagrams were prepared according to the composition presented in Table 1. Np was slowly incorporated into the microemulsion under stirring. After Np was entirely dissolved in the miroemulsion, the clear microemulsion-based formulation was obtained. No phase change was noted after the addition of the drug. The final concentration of Np in microemulsion systems was 10% (w/w) (100 mg/g).
| Formulations | M1 (w/o) | M2 (w/o) | M3 (w/o) | M4 (w/o) | ||||
|---|---|---|---|---|---|---|---|---|
| Formulation components | Formulation components | Formulation components | Formulation components | |||||
| HLB value | 6.833 | 5.916 | 6.833 | 5.916 | ||||
| M1 (%) (w/w) | M1Np (g) | M2 (%) (w/w) | M2Np (g) | M3 (%) (w/w) | M3Np (g) | M4 (%) (w/w) | M4Np (g) | |
| IPM | 29.44 | 2.650 | 27.68 | 2.491 | 31.10 | 2.8 | 26.23 | 2.36 |
| Labrafil-M | 4.43 | 0.398 | — | 5.20 | 0.468 | — | — | |
| Labrasol | — | 1.23 | 0.111 | — | 1.38 | 0.13 | ||
| Span 80 | — | 6.18 | 0.556 | — | 6.91 | 0.62 | ||
| Cremophor EL | 2.21 | 0.199 | — | 2.60 | 0.234 | — | — | |
| Ethanol | 59.81 | 5.383 | 59.39 | 5.345 | 54.67 | 4.920 | 58.10 | 5.23 |
| Distilled water | 4.11 | 0.370 | 5.52 | 0.497 | — | — | — | |
| 0.5 N NaOH solution | — | — | 6.43 | 0.578 | 7.38 | 0.66 | ||
| Naproxen | — | 1 | — | 1 | — | 1 | — | 1 |
The solubility of Np was determined in distilled water, 0.5 N NaOH solution, IPM, ethanol, Labrafil-M, Labrasol, Cremophore EL, Span 80, and M1–M4 microemulsion formulations at 25±2°C. An excess of Np was added into 2 mL solvent and then allowed to equilibrate under continuous mixing for 72 h at room temperature (25±2°C). The suspensions were filtrated through a membrane filter (0.2 µm Nylon, Millipore Millex-GN) and the drug concentration in the filtrate was determined using high pressure liquid chromatography (HPLC) method after the appropriate dilution with mobile phase.12,13) Each experiment was replicated three times.
Determination of n-Octanol–Water Partition CoefficientTo determine the n-octanol–water partition coefficient of Np, n-octanol phases were saturated with distilled water for at least 24 h before the experiment. A solution of Np (10−4 M) was prepared with distilled water. Two milliliters of this solution was transferred to 10 mL assay tubes containing 2 mL of the organic phase. The tubes were stoppered and agitated for 24 h at room temperature. After centrifugation at 3500 rpm for 15 min, the concentration of the drug in water phase was analyzed by HPLC; the drug in n-octonol layer was diluted with acetonitrile before assaying it by HPLC. Six replicates were used for the concentrations of n-octanol–distilled water solutions for partition coefficient calculations.12,14)
Characterization of the MicroemulsionsThe average droplet size and polydispersity index (PDI) of microemulsions in the presence or absence of Np were studied using photon correlation spectroscopy (Nano ZS, Malvern Instruments, U.K.). The viscosity, pH value and refractive index of various microemulsions in the presence or absence of Np were measured at 25±2°C using a viscosimeter (ULA spindle, Brookfield, U.S.A.), a digital pH-meter (HI 221, Mauritius) and a refractometer (Atago RX-7000 CX, Japan) respectively.
The conductivity and phase inversion temperature (PIT) of the microemulsion formulations in the presence or absence of Np were determined at 25±2°C using a conductometer (Jenway 4071, U.K.). Each microemulsion formulation of 20 mL was placed in a beaker. An electrode was totally immersed and fixed in the microemulsion. The beaker was heated in a water bath. The temperature of the bath was increased at 1°C/min steadily. The microemulsion was agitated with a stirrer and the change in the conductivity was recorded.15) The experiments were carried out in triplicate for each sample, and the results are presented as an average±S.D.
Stability of MicroemulsionsThe chemical and physical stabilities of the microemulsions incorporated with Np were studied at 5±2, 25±2 and 40±2°C for 12 months. The clarity, phase separation, droplet size, pH, viscosity, electrical conductivity and concentration of Np were investigated to judge the monthly storage temperature. Centrifuge tests were also carried out to assess the physical stability of the microemulsions.16)
In Vitro Skin Permeation Studies. Preparation of SkinsMale wistar albino rat weighing 250±20 g were purchased from the Experimental Animal Center of Ege University (Izmir, Turkey) for the permeation studies. The experimental protocol was approved by the Local Animal Ethical Committee of Ege University, Faculty of Pharmacy (Approval No. 2008/3–1). The abdominal part of the rat skin was carefully shaved with a razor. Rats were sacrificed by corbondioxide gas 1 d later. The abdominal skin was excised from the abdomen and the subcutaneous fat and connective tissue were trimmed. The obtained skins were washed and examined for integrity, stored at −20±2°C overnight and then used for the permeation experiments.8,17,18)
In Vitro Permeation StudiesThis experiment was performed using vertical diffusion cells with an effective diffusion area of 1.326 cm2. The excised skin samples were clamped between the donor and the receptor chamber of vertical diffusion cells with the SC side facing upwards into the donor compartment and the dermal side facing downwards into the receptor compartment. Five hundred milligrams Np loaded microemulsions (M1Np–M4Np) (containing 50 mg Np) or 500 mg C formulation (containing 50 mg Np) was administrated on the SC side on individual skin samples. The cell was then covered with aluminum foil. The receptor chambers were filled with 10 mL of phosphate buffer (pH 7.4), its temperature was maintained at 32±0.5°C and the solution in the receptor chambers was stirred at 600 rpm throughout the experiment. Approximately 10 mL of the receptor medium was withdrawn at predetermined intervals (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 h) and replaced immediately with an equal volume of receptor solution to maintain a constant volume. All samples were filtered through a membrane filter (0.2 µm Nylon, Millipore Millex-GN) and immediately injected into an HPLC system that consisted of a UV spectrometric detector and C18 column (270 nm). Three replicates of each experiment were performed. All experiments were performed at 25±2°C. Sink conditions were maintained in the receptor compartment during in vitro permeation studies.14,19,20)
Data AnalysisThe cumulative amount of drug permeated was plotted as a function of time, and the flux (Js, mg/cm2·h) was calculated from the steady-state part of the curve. Lag time (TL, h) was defined as the initial detection of the drug. The effect of microemulsions as carrier on dermal administrations, —enhancement ratio (ER)— is determined by the following Eq. 18,9,11,20,21):
 | (1) |
The HPLC analysis method was modified from a previous study.22) The samples were analysed using an HPLC (HP Agilent 1100 series) system that included a separations module, a UV spectrometric pump and detector. The column was a Agilent ZORBAX Eclipse XDB-C18 column (4.6×150 mm, 5 µm). The mobile phase contained methanol–acetonitrile–purified water (20 : 28 : 52 v/v/v %) and 0.4 mL triethylamine (adjusted to pH 3.2 using orthophosphoric acid). The flow rate was adjusted to 1.5 mL/min, and the injection volume was 20 µL. The UV detection wavelength was 270 nm and the retention time of Np was 7.7 min. No interface of the other formulation components was observed. All samples were filtrated through an aqueous 0.2 µm pore size membrane filter (0.2 µm Nylon, Millipore Millex-GN) before injection.
Histopathology StudiesTo confirm the microemulsion formulations safety, a histopathological study was carried out using healthy Albino rats. Rats were divided into three groups of seven animals each, which were treated with the following:
Group I: Microemulsions group (M1Np, M2NP, M3Np and M4Np)
Group II: Negative control group (serum physiologic (SP))
Group III: Positive control group (commercial formulation (C))
Abdominal skin was carefully shaved 12 h before the experiment using razor with no apparent lesions or wounds. Rats were sacrificed by carbon dioxide gas. Skin was excised from the abdomen and the subcutaneous fat and connective tissue were trimmed. Five hundred milligrams of microemulsion formulation (containing drug 50 mg) (M1NP–M4NP), SP (negative control) or C formulation (containing drug 50 mg) (positive control) were applied for 24 h on the excised abdominal skin mounted on a surface area of 1.326 cm2 on the vertical diffusion cell. The formulations were removed; the skins were wiped off with tissue paper and fixed in a 10% (v/v) formaline solution in saline for approximately 24 h; then washed with tap water, dehydrated through a increasing ethanol series, immersed in xylene and were finally embedded in paraffin wax at 56°C. Paraffin blocks were cut serially at 5 µm using a rotary microtome (RM 2145, Leica Co., Nussloch, Germany). Sections were stained with hematoxylene & eosine and examined using light microscope (Olympus BX-51, Japan).23)
Statistical Data AnalysisAll experiments were repeated three times and data were expressed as the mean value±S.D. Statistical data were analyzed by one way ANOVA. A multiple comparison test was used to compare different formulations, and p-value of 0.05 was considered to be significant.
Phase studies were performed to investigate the effect of S to CoS ratio on the extent of stable w/o microemulsion region.24) The pseudo-ternary phase diagrams with various weight ratios of oil, water and S/Cos are shown in Fig. 1. The studied systems were composed of proper constituents including IPM as oil phase, distilled water and a mixture of Cremophor EL: Labrafil M/ethanol as S/Cos for M1 or including IPM as oil phase, distilled water and mixture of Span 80: Labrasol/ethanol as S/Cos for M2 (Figs. 1A, B). Figures 1C and D are the ternary phase diagram, in which the aqueous phase is a 0.5 N NaOH solution, the oil and S/CoS used are the same as those in Figs. 1A and B respectively. In this study, S/Cos 1 : 9 ratio was selected for M1 and M3 formulations and 1 : 8 ratio was selected M2 and M4 formulations, because these ratios are the most stable after the formation of microemulsions. Moreover HLB values of the systems were calculated as 6.833 and 5.916, respectively. The translucent microemulsion regions were presented in the shaded regions of the phase diagrams. No distinct conversion from water-in-oil (w/o) to oil-in-water (o/w) microemulsions was observed. The unshaded region on the phase diagrams represents the turbid and conventional emulsions based on visual observations.
(A) The microemulsion is composed of IPM, Labrafil M, Cremophor EL, ethanol and distilled water. The area of the microemulsion region is 112.77. (B) The microemulsion is composed of IPM, Labrasol, Span 80, ethanol and water. The area of the microemulsion region is 203.53. (C) The microemulsion is composed of IPM, Labrafil M, Cremophor EL, ethanol and 0.5 N NaOH solution. The area of the microemulsion region is 117.21. (D) The microemulsion is composed of IPM, Labrasol, Span 80, ethanol and 0.5 N NaOH solution. The area of the microemulsion region is 215.48.
The influence of weight ratio of distilled water or 0.5 N NaOH solutions on the area of microemulsion region was investigated on the pseudo-ternary phase diagram. As shown in the Fig. 1, the area of microemulsions isotropic region increased slightly in size with 0.5 N NaOH solution. With the increase in the ratio of surfactant and 0.5 N NaOH solution as water phase the areas of the microemulsion region and isotropic regions on the diagram also increased. From a formulation viewpoint, the increased surfactant and the 0.5 N NaOH solution as water phase in microemulsions may provide a greater opportunity for the solubilization of poor water soluble drugs such as Np (Table 2). Therefore the selection of the 0.5 N NaOH solution as water phase in the microemulsion formulations was on the basis of its ability to solubilize the desired amount of Np. A similar result was obtained from Gao and Singh. particularly; the effect of weight ratio of S to Cos on the area of o/w microemulsion regions was studied, as well as the stabilization of microemulsions and the solubilization of the drug.25)
| Component | Solubility (mg/mL) |
|---|---|
| IPM | 4.798±0.509 |
| Labrafil-M | 0.396±0.041 |
| Labrasol | 0.550±0.003 |
| Span 80 | 0.178±0.209 |
| Cremophor EL | 0.547±0.021 |
| Ethanol | 123.427±0.288 |
| Distilled water | 1.141±0.631 |
| 0.5 N NaOH solution | 117.296±2.565 |
| M1 | 102.512±1.203 |
| M2 | 103.338±3.870 |
| M3 | 105.766±2.699 |
| M4 | 110.150±2.149 |
Values are means of three experiments±S.D.
Microemulsion formulations for further studies were selected from the gravity center of these pseudo-ternary diagrams (Figs. 1A–D). The concentrations of microemulsios are provided in Table 1. After the preparation of the microemulsion, Np was dissolved into the microemulsion (M1–M4) under stirring. The clear microemulsion-based formulation was observed. The final concentration of Np (M1Np–M4Np) was 10% (w/w) (100 mg/g) (Table 1).
The Role of Microemulsion Components in the Solubility of NaproxenThe solubility of Np determined in various formulations and components is shown in Table 2. The maximal drug solubility was measured in ethanol (123.427±0.288 mg/mL), 0.5 N NaOH solution (117.296±2.565 mg/mL), M4 (110.150±2.149 mg/mL), M3 (105.766±2.699 mg/mL), M2 (103.338±3.870 mg/mL) and M1 (102.512±1.203 mg/mL). It seems that the microemulsion structure contributes to solubilization. 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 solubilization sites for the drugs, compared to the molecular organization of bulk surfactants. Furthermore, investigations have indicated that the unique structural organization of the phases in microemulsions may contribute to additional solubility regions, increasing the load capacity of microemulsions, compared to non-structured solutions containing the same fraction of the constituents.26,27) This concurs well with results of Malcolmson and co-workers who suggested that the major solubilization advantage of microemulsions could be ascribed to the surfactant interfacial film of miceller structure and the solubility of lipophilic drugs would be further increased over that of miceller solutions.28)
Determination of n-Octanol–Water Partition CoefficientPhysicochemical parameters, such as aqueous solubility and lipophilicity, have been shown to influence membrane flux, therapeutic activity, and pharmacokinetic profiles of medicines. Lipophilicity is very important for dermal permeation because the SC, the major barrier to drug permeation, is lipid in nature and generally favors permeation by lipophilic drugs.29,30) Therefore, in this study, the aqueous solubility and lipophilicity of Np were determined. The log P value for Np was 3.18. This value was sufficiently non-polar for its permeation rate to be controlled primarily by the aqueous strata of the Np layer in skin with disrupted SC.10,12,30)
Characterization of the MicroemulsionThe physicochemical parameters of microemulsions are reported in Table 3. All dermal microemulsions were determined w/o phase systems. The pH values of microemulsions in the presence of Np ranged from 6.0 to 7.2. The pH close to 7.0 indicated that all formulations could result in reduced stimulation to the skin.31) The viscosities of the tested microemulsions are shown in Table 3. These results indicate that the dynamic viscosity of the microemulsion vehicles has a very low range with no significant difference among all formulations. Reduction in the viscosity of microemulsions was reported after the incorporation of short chain alcohol as a co-surfactant.13)
| pH | Conductivity (µs/cm) | Viscosity (cP) | Mean droplet size (nm) | PDI | Refractive index | PIT (°C) | |
|---|---|---|---|---|---|---|---|
| M1 | 6.9±0.01 | 31.6±0.05 | 13.3±0.58 | 1.697±0.050 | 0.412±0.053 | 1.400±0.001 | 52±0.002 |
| M2 | 6.0±0.01 | 22.5±0.1 | 15.0±1 | 1.917±0.109 | 0.376±0.019 | 1.421±0.003 | 48±0.001 |
| M3 | 7.2±0.01 | 155.7±0.01 | 14.3±1.53 | 2.874±0.044 | 0.448±0.032 | 1.379±0.001 | 41±0.005 |
| M4 | 7.1±0.01 | 149.5±0.1 | 15.3±0.58 | 1.700±0.011 | 0.389±0.049 | 1.452±0.002 | 50±0.010 |
| M1Np | 5.8±0.01 | 23.0±0.05 | 13.3±0.58 | 1.590±0.114 | 0.396±0.040 | 1.405±0.002 | 44±0.001 |
| M2Np | 5.0±0.03 | 18.7±0.02 | 15.0±1 | 1.701±0.060 | 0.369±0.029 | 1.429±0.006 | 40±0.020 |
| M3Np | 6.5±0.05 | 147.9±1 | 14.3±1.53 | 2.486±0.064 | 0.479±0.016 | 1.385±0.002 | 38±0.020 |
| M4Np | 6.4±0.03 | 141.5±0.1 | 15.3±0.58 | 1.405±0.040 | 0.399±0.043 | 1.468±0.002 | 41±0.006 |
Values are means of three experiments±S.D.
There is a strong correlation between the specific structures of microemulsion systems and their electrical conductive behavior.15,32) In the microemulsions containing Np, the electrical conductivity slightly increased (Table 3). If a small amount of an aqueous electrolyte is added into nonionic microemulsion, the electrical conductivity of the system will change.33) Thus, in this study, a 0.5 N NaOH aqueous solution was used in the preparation of the M3–M4 microemulsion samples in place of distilled water. It was also observed that when the water phase was changed to 0.5 N NaOH solutions, the conductivity increased by 5 to 7 times. Furthermore, adding small electrolyte amounts to the water phase of the microemulsions leads to a slight increase in the area of the microemulsion region (Fig. 1).
The refractive index of in the presence and absence of Np microemulsion formulations are provided in Table 3. When the refractive index values for the microemulsions with and without Np were compared, it was found that there was no significant difference between the values (p>0.05).
The droplet sizes of the prepared microemulsions were monitored using photon correlation spectroscopy. The average droplet size of all formulations ranged from 1.405±0.040 to 2.874±0.044 nm. PDI value describes the homogeneity of the droplet size. All PDI values were smaller than 0.5. Therefore, these results indicate that droplet sizes have appropriate homogeneity. The mean droplet size of Np loaded microemulsion slightly decreased compared to the mean droplet size of microemulsion in the absence of Np. According to a recent study, mean particle size was decreased noticeably after loading drug. The possible reason for this is that as is the case with Np, there are aldehyde groups and hydroxyl groups in the drug molecule structure. When a drug molecule is dissolved and dispersed into the emulsifying membrane layer (composed of S and CoS) and oil phase, these groups in the drug molecule can react with components of the microemulsion producing a lot of hydrogen bonds. Thus the whole particle shrinks due to the interaction of above hydrogen bonds and consequently, the mean particle size of the resultant microemulsion decreases.35)
Another interesting point is the phase inversion temperature (PIT), where the cloudy dispersions turn into completely transparent microemulsions. The microemulsion, which was prepared with a nonionic surfactant, tended to form a w/o type emulsion at higher temperatures, and an o/w type at lower temperatures. Experimental evidence showed that as the temperature increased, electrical conductivity of the microemulsion changed.36,37) In this study the phase inversion temperatures of microemulsions were detected and these results were reported in Table 3. Moreover, PIT was slightly decreased in the microemulsions loaded with Np (Table 3). It should be noted that the presence of an electrolyte or in some cases a drug, especially if lipophilic in nature as Np, can act to lower the PIT, illustrating the importance of determining microemulsion phase behaviour in the presence of drug.38)
Stability of the MicroemulsionsNon-ionic surfactants have been observed to be sensitive to changes in temperature. In this study only the M3Np formulation was separated to phases during storage at 40±2°C, but it was readily recovered after shaking at room temperature. The microemulsion vehicles were centrifuged for 30 min at 13000 rpm. The microemulsions had isotropic transparent dispersions and after centrifugation no phase separation was observed. This demonstrated the good physical stability of the tested microemulsions. The fact that no changes in droplet size, phase separation, pH, viscosity, electrical conductivity and drug degradation of tested microemulsions were detected for 12 months. These results indicate that the microemulsions prepared were stable and did not change significantly at 5±1, 25±2 and 40±2°C in the presence or the absence of Np for 12 months (p>0.05). The chemical stability of Np in the microemulsion was sufficient at 5±1, 25±2 and 40±2°C. As seen in Fig. 2, 99% of Np remained in the microemulsion after 12 months at all temperatures.
Values are means of three experiments±S.D.
Therefore, it can be concluded that the microemulsion formulations were not only thermodynamically stable but also chemically stable and remained isotropic. Pathan and Setty have shown through the use of the index values of the microemulsions, that the microemulsions were determined to be isotropic, and chemically and thermodynamically stable.34)
In Vitro Skin Permeation StudiesThe permeation ability of the various microemulsions was evaluated using the in vitro permeation experiments. The permeation profiles of Np through rat skin from various formulations are shown in Fig. 3. The permeation parameters of the tested microemulsions and C formulations are presented in Table 4.
Values are means of five experiments±S.D.
| Formulation | Js (mg/cm2/h) | Lag time (h) | r2 | ER | Kc (cm/h) |
|---|---|---|---|---|---|
| M1Np | 0.646±0.013 | 0.768±0.082 | 0.925±0.001 | 4.335 | 1.220±0.018 |
| M2Np | 0.651±0.112 | 0.961±0.131 | 0.994±0.003 | 4.369 | 1.229±0.075 |
| M3Np | 0.984±0.108 | 0.368±0.175 | 0.980±0.009 | 6.604 | 1.856±0.122 |
| M4Np | 1.347±0.005 | 0.311±0.032 | 0.992±0.025 | 9.040 | 2.541±0.020 |
| C | 0.149±0.002 | 0.089±0.010 | 0.998±0.008 | – | 0.374±0.011 |
Values are means of five experiments±S.D. ER=enhancement ratio, C was used as control.
The statistical comparison of the flux throughout rat abdominal skin over 8 h showed that all the microemulsions provided fluxes (p<0.05) higher than the C formulation. In this study, M4Np obtained the highest permeated drug concentration and permeability value for Np. It was observed that when the drug solubility (110.150±2.149 mg/mL) in the microemulsion formulation (M4Np) increased, the drug uptake in the skin, the cumulative amount of drug release and the flux (1.347±0.005 mg/cm2/h) also improved. Moreover, according to particle sizes of microemulsion formulations, the smaller the particles are, the bigger their surface, which leads to an increase solubility pressures of the particles and an increase release (M4Np). A similar result concerning the release of a submicron lipid emulsion formulation was also observed.39–41) Besides, the first time point that Np was detected (the lag time) of formulations ranged from 0.089±0.010 to 0.961±0.131 h, indicating that the drug permeation rates through rat skin were significantly affected by the composition of the formulation (Table 4). Although the composition of vehicles influenced the skin absorption and lag time of drug, the transdermal delivery of drug might have limitation by simple modulation of vehicle compositions. For example; as vehicles, ethanol, polyethylene glycol 400, or propylene glycol was used alone or mixed with a phosphate buffer. Binary vehicles (ethanol : buffer, polyethylene glycol 400 : buffer, propylene glycol : buffer) showed different effects on the skin permeation of Melatonin and its lag time. Compared with the buffer alone, the polyethylene glycol 400 : buffer shortened the lag time of Melatonin but reduced its skin permeation. Ethanol : buffer significantly increased the flux of Melatonin but prolonged the lag time with the content of ethanol. Propylene glycol : buffer did not affect the lag time but slightly increased the skin permeation of Melatonin at the higher content of propylene glycol (≥80%).42)
IPM as a permeation enhancer had a strong permeation enhancing effect and could increase the flux (Jss) in skin, which could result in an increase of the permeation coefficient.41,18) Therefore, the oil with permeation enhancing ability had significant influence on the penetration of Np. Moreover, the close contact with skin might contribute to the direct release of Np from oily droplets into the skin without the transfer of the drug in the continuous phase. Ethanol is widely used as a permeation enchancer for many drugs43) and it has a low irritant and toxicity, therefore ethanol was chosen as the cosurfactant phase in the microemulsion formulations. Moreover, ethanol has been shown to extract SC lipids and to perturb barrier function improving particularly the permeation of more hydrophilic drugs through skin.1) For this reason, these formulations (M1Np, M2Np, M3Np and M4Np) exerted a higher permeation rate for the transdermal release of Np than the C formulation. It has been demonstrated in this study that incorporating Np into the microemulsions enhanced drug penetration through the skin. On the other hand, in M3Np–M4Np formulations, NaOH solution was used as aqueous phase. It was seen that, Np permeation was improved compared to water as aqueous phase. Moreover, M4Np showed a higher permeability coefficient (Kp) (2.541±0.020×10−3 cm·h−1) value than the other formulations.
The enhancement ratio (ER) was significantly increased in the M3Np (6.604 times) and M4Np (9.040 times) formulations compared to C (p<0.05) because these microemulsion formulations contained permeation enhancers like Labrafil M, Labrasol, Span 80, Cremophor EL, IPM and ethanol. The permeability parameters of the M3Np, M4Np and C formulations are shown in Table 4. Shakeel et al. showed that, by using permeation enhancers like Labrafil, Triacetin, Tween 80, and Transcutol P, the ER is increased in nanoemulsions.8)
Histopathology StudiesTo determine the influence of the formulations on skin irritation, the abdominal skin was investigated after the application of the formulations. Microscopic images of rat abdominal skin treated with microemulsion formulations (M1Np, M2Np, M3Np and M4Np) and C formulation are shown in Fig. 4. In Figs. 4A–D, the SC layer became thinner; it was observed that there was no apparent change in skin morphology (other skin layers and dermis) after the application of Group 1 (M1Np, M2Np, M3Np and M4Np). No significant change in epidermal thickness was observed in biopsies from the skin section treated with Group 1, although the section shows a clear disruption of SC organization confirming the reported enhancing capacity of these formulations that accelerate the penetration of the Np. Charoo et al. used the diffusion cells method in their histopathology studies and have shown that no significant change in the skin sections takes place.23)
Visible irritation was not observed after the application of SP (Group 2) (Fig. 4F). In Group 3; the SC layer became thinner and subjacent layers reamined after the application of the C formulation. Furthermore, it was observed that cells rose due to irritation (Fig. 4E). Compared to the positive control (C), the developed microemulsion formulations (M1Np, M2Np, M3Np and M4Np) did not show any statistically significant histological changes on the skin (p>0.05) (Fig. 4). The irritation studies did not show visible irritation after the application of M1Np, M2Np, M3Np and M4Np for 24 h on the dorsal skin of rats. Thus the developed microemulsions are considered to be safe for the use of topical drug delivery.
The images also presented the increased cell gap and a flaky appearance of keratin, which may indicate the denaturation of keratinocytes in the SC layers. SC played an important part in preventing the permeation of drugs, thus it could be inferred that these disruptions of the SC morphology may have contributed to the enhancement effect on drug permeation (Fig. 4). The components of microemulsion played an important role in changing the microstructure of skin. Zhu et al. showed that, when using microemulsions on the skin, the microemulsions influence the SC morphology.31)
(a) 10× (magnification value), (b) 100×, H&E (hematoxylene and eosin). Values are means of seven experiments±S.D. ★, Stratum corneum of epidermis in the rat skin. →, Cells increased in dermis of the skin.
No damage in the epidermal layers and no inflammation in the dermal layers were found in the skins applied with SP (negative control). No inflammation was found in the dermal layers of the skins applied with M4Np formulation and C formulation (positive control), however some damage was observed in the SC (Table 5). Skins applied with the M3Np formulation showed serious damage in the SC, and low damage in the lucidum and granulosum layers, while moderate inflammation was observed in the stratum papillare in the dermal layer. Serious damage was observed in the SC of the skins applied with M1Np and M2Np, while low damage was observed in the stratum lucidum layer. Moderate inflammation was observed in the stratum papillare layer of the skin applied with the M1Np formulation, while low damage was recorded on that of the M2Np formulation (Table 5).
| Groups | Disruption degrees of epidermal layers | Inflammation degrees of dermal layers | ||||||
|---|---|---|---|---|---|---|---|---|
| Stratum corneum | Stratum lucidum | Stratum granulosum | Stratum spinosum | Stratum basale | Stratum papillare | Stratum reticulare | ||
| Group I | M1Np | +++ | + | — | — | — | ++ | — |
| M2Np | +++ | + | — | — | — | + | — | |
| M3Np | +++ | + | + | — | — | ++ | — | |
| M4Np | +++ | — | — | — | — | — | — | |
| Group II | SP | — | — | — | — | — | — | — |
| Group III | C | +++ | — | — | — | — | — | — |
+: Weak; ++: Moderate; +++: Severe.
In this study, new w/o microemulsion systems containing Np were studied for topical application. Different microemulsion formulations were selected using pseudo-ternary phase diagrams. IPM was used as the oily phase of microemulsion due to its permeation enhancing effect for Np. Microemulsions could increase the topical delivery of Np as 4.335–9.040 times compared to the commercial formulation used in this study. The best formulation of M4Np consisted of IPM (2.36 g), Labrosol (0.13 g), Span 80 (0.62 g), ethanol (5.23 g), 0.5 N NaOH solution (0.66 g) and Np (1 g). The addition of 0.5 N NaOH solution to the microemulsion increased the permeation rate of Np via the skin. The results of the physicochemical property and histopathology tests suggest that the microemulsion was a promising vehicle for topical application. Finally, microemulsion formulations containing Np could be promising formulations as an alternative anti-inflammatory dosage form for effective therapy.
This study was supported by University of Ege, Faculty of Pharmacy, and Department of Pharmaceutical Technology (08/ECZ/006).
