The objective of the present study was to examine the influence of cosolvent system and micro-emulsion formulation on in-vitro skin permeation of gabapentin, furthermore, to characterize the physicochemical properties of drug-loaded oil-in-water (o/w) and water-in-oil (w/o) cremophor 40-based microemulsions in comparison to the blank counterparts. The cosolvent system prepared by homogenous mixing is composed of ethanol-water and propylene glycol-water mixture (90:10, 80:20, 70:30 v/v) respectively. The microemulsion consisted of coconut oil, water and mixture of cremophor 40 (surfactant) and ethanol (cosurfactant) and was prepared by aqueous phase titration method. Physicochemical properties of microemulsions were determined using reported procedures. Transdermal flux for gabapentin was studied in-vitro using modified Franz diffusion cells. The physicochemical properties of drug-loaded microemulsions and their blank counterparts were generally alike, however, slight variation in pH and viscosity was observed probably due to the intrinsic properties of the drug. The ethanol-water system (70:30 v/v) gave higher flux for gabapentin when compared to propylene glycol-water system (70:30 v/v). The w/o microemulsion formulations resulted in, higher flux for gabapentin when compared to o/w formulations. FTIR spectra of the untreated stratum corneum, when compared to cosolvent system and microemulsion treated stratum corneum, suggest the mechanism of permeation to be disruption of lipid bilayers and keratin denaturation of the stratum corneum. The results show that incorporation of gabapentin into microemulsions did not change the microemulsion type. The in vitro permeation data obtained from experimental work suggest that the cosolvent system (ethanol-water 70:30 v/v) and w/o microemulsion formulations respectively, can be successfully used as potential vehicles in developing transdermal therapeutic systems for gabapentin.
The in-vitro study on permeation of drug through the skin is very vital in the selection of candidates for the de- velopment of transdermal dosage forms. Investigations of this type are generally done by using a diffusion cell whose donor and receiver compartments are separated by a membrane. Such membranes could come from hu- man skin (cadaver or surgical reduction), mice, pigs, rabbits and rats [
Gabapentin (Pfizer, China), cremophor RH 40 (BASF, Germany), purified coconut oil (Nigeria), ethanol (Sigma and Aldrich, USA). All other chemicals and reagents were of analytical or high-performance liquid chromato- graphy (HPLC) grade, purchased from Sigma and Aldrich (USA).
Ethanol and water or propylene glycol and water were homogenously mixed in different ratios to obtain binary solvent systems of 90:10, 80:20 and 70:30 v/v respectively.
A pseudoternary phase diagram was constructed by using the aqueous phase titration method [
The apparent pH of the formulations was measured in triplicate at 25˚C using a pH meter with combination electrode (Eutech, Japan).
The determination was carried out using photomicrograph (Moticam, Japan). The test was performed on a fil- tered microemulsion sample that was placed on a slide after being diluted with distilled water. The filtration was done through 0.22 mm membrane filter. Polydispersity index was estimated from the ratio of standard deviation to the mean droplet size.
Viscosity of the samples was measured at 25˚C using, a cone and plate viscometer (UTV Gallenkamp, England). A sample volume of 5 ml was used for the viscosity measurement and the test was carried out in triplicate.
Surface tension of the samples was measured at 25˚C using Du Noüy tensiometer. A sample volume of 5 ml was placed on the tensiometer plate and the force required pulling a circular ring out of the sample recorded. The test was performed in triplicate.
Stability of the samples was studied by allowing the microemulsions to stand on the laboratory shelf for 21 days while observing them for phase separation and/or creaming.
The approval to carry out the animal studies was obtained from Faculty of Veterinary Medicine, University of Nigeria, Nsukka animal ethics committee. Male Wister rats were sacrificed with prolonged exposure to chloro- form. Hairs on the skin of animal were removed with electrical clipper. Full thickness skin was surgically re- moved from each rat. The epidermis was prepared from the full thickness skin by heat separation technique [
The studies were done using modified Franz diffusion cells. The diffusional area of the cells was 2.54 cm2. The diffusion cells were connected with a circulating water bath and the temperature was controlled at 37˚C. Phos- phate buffer saline (PBS) used as a receiver fluid was placed in the receiver compartment that has a volume of 25 ml and was stirred by an externally driven Teflon-coated magnetic bar. The prepared epidermis was sand- wiched between the receiver compartment and the donor compartment with the stratum corneum facing upwards. The donor compartment was clamped. One ml of cosolvent system containing 5 mg of gabapentin or drug- loaded microemulsion was applied onto skin surface facing the donor compartment that was covered with a glass lid. The sampling arm of the donor compartment was also covered with aluminum foil. At suitable time interval (0, 1, 2, 4, 6, 8, 10, 12 and 24 h), 1 ml of each sample was withdrawn from the center of the receiver compartment with a syringe connected with a needle. An equal volume of fresh PBS (37˚C) was replenished immediately. The amount of drug in the receiver fluid was determined spectrophotometrically at 210 nm.
The concentrations of gabapentin permeated to the receiver fluids were quantitatively determined by UV/VIS spectrophotometer (Jeenway 6405, England). The samples were analyzed at maximum wavelength of 210 nm. The calibration curve (absorbance versus drug concentration) was constructed by measuring standard solutions of the drug in PBS in the concentration range of 5 - 50 µg/ml. Validation of the method was performed to ensure that the calibration curve was in the linearity range of the assay and the coefficients of variation were less than 2% both intra-day and inter-day.
The skin permeation data were measured as the cumulative drug permeation per unit of skin surface Qt/S (S = 2.54 cm2). The cumulative drug permeation (Qt) was calculated from equation (1):
where, Ct is the drug concentration of the receiver fluid at each sampling time, Ci is the drug concentration of the ith sample, Vr and Vs are the volumes of the receiver fluid and sample respectively. The steady-state fluxes (Jss(6-24 h)) were determined as the slope of the linear portion of the plots obtained by graphing cumulative drug permeation per unit of skin surface versus time. One-way analysis of variance (ANOVA) was used to compare fluxes of different vehicles and a p-value of 0.05 was considered to be statistically significant. The penetration enhancing effect of each vehicle was calculated in terms of enhancement ratio (ER) using Equation (2):
The rat stratum corneum samples were prepared by soaking freshly prepared epidermis membrane in a 0.1 % trypsin solution for 12 h. The SC samples were cleaned by washing with distilled water and blotted dry and used in the FTIR [
The analysis was performed on both untreated and treated rat stratum corneum. The vehicle-treated and un- treated (control) SC samples respectively were vacuum-dried at 25˚C ± 1˚C for 48 h and stored in desiccators to remove traces of solvent. The control samples were treated in phosphate buffer saline (PBS). All samples were treated in the respective vehicles for 24 h prior to been vacuum-dried. The completely dried samples were stu- died by FTIR (Shimadzu 8400S, Japan). Peaks occurring near 2850 cm−1 and 2920 cm−1 that were due to sym- metric and asymmetric C-H stretching vibrations respectively as well as those at 1550 cm−1 and 1650 cm−1 indi- cating amides (C=O) stretching vibrations were characterized.
Transdermal delivery of drugs depends on their permeability through SC that in turn depends on the de- velopment of optimal vehicles. This route of drug delivery is important for drugs of high potency and helps to avoid problems with gastrointestinal intolerance and short half-life, reduces first-pass liver metabolism and eliminates the need for intravenous administration. Our study chose cosolvent system and microemulsion as vehicles to investigate the transdermal delivery of gabapentin. Cosolvent system and microemulsion are amongst the most useful and interesting drug delivery systems [
Pseudoternary phase diagram of coconut oil/water/ cremophor 40 RH:ethanol (1:2) microemulsion formulation
´----´ MEa; ▲-----▲ Ethanol-Water 70:30
■----■ MEc; ·----· Water
. Physicochemical characteristics of the microemulsions
Formulation | pH | Droplet size (nm) | Polydispersity index (PDI) | Surface tension | Viscosity |
---|---|---|---|---|---|
Blank* | 7.54 ± 0.52 | 85.7 ±0.1 | 0.0098 | 83.6 ± 1.2 | 150 ± 17 |
MEa | 7.62 ± 0.14 | 85.8 ± 0.2 | 0.0098 | 84.4 ± 1.2 | 151 ± 20 |
Blank | 7.43 ± 0.21 | 89.8 ± 0.6 | 0.0096 | 85.2 ± 1.1 | 145 ± 14 |
MEb | 7.21 ± 0.33 | 89.9 ± 0.3 | 0.0097 | 86.4 ± 1.1 | 148 ± 12 |
Blank | 7.33 ± 0.62 | 93.7 ± 0.1 | 0.0100 | 84.3 ± 0.9 | 151 ± 20 |
MEc | 7.52 ± 0.41 | 93.7 ± 0.1 | 0.0100 | 85.0 ± 0.9 | 154 ± 15 |
Blank | 7.34 ± 0.22 | 95.2 ± 0.5 | 0.0095 | 84.2 ± 0.4 | 153 ± 17 |
MEd | 7.52 ± 0.73 | 95.2 ± 0.5 | 0.0095 | 84.3 ± 0.6 | 154 ± 17 |
*The apparent similarities in the physicochemical properties of the blank and drug-loaded microemulsions (
permeability coefficients of gabapentin from cosolvent systems and microemulsion formulations are shown in
. Skin permeation parameters of gabapentin through stratum corneum from various vehicles
Vehicles | Jss (µg∙cm−2∙h−1) | Kp × 103 (cm∙h−1) | ER |
---|---|---|---|
Control | 3.569 ± 0.058 | 0.7138 ± 0.007 | - |
10% ethanol | 8.434 ± 0.176 | 1.687 ± 0.080 | 2.4 |
20% ethanol | 14.774 ± 0.409 | 2.955 ± 0.038 | 4.1 |
30% ethanol | 63.290 ± 1.62 | 12.658 ± 0.293 | 17.7 |
10% Pg | 15.984 ± 0.988 | 3.197 ± 0.123 | 4.5 |
20% Pg | 15.761 ± 0.468 | 3.152 ± 0.074 | 4.4 |
30% Pg | 12.263 ± 0.657 | 2.453 ± 0.051 | 3.4 |
MEa | 128.215 ± 1.84 | 25.643 ± 0.325 | 35.9 |
MEb | 5.542 ± 0.587 | 1.108 ± 0.047 | 1.6 |
MEc | 61.137 ± 0.896 | 12.227 ± 0.134 | 17.1 |
MEd | 4.728 ± 0.220 | 0.9456 ± 0.029 | 1.3 |
MEa-d = microemulsion codes, Jss = steady-state flux, Kp = permeability coefficient, ER = enhancement ratio, Pg = propylene glycol
. Peak height of asymmetric, symmetric C-H stretching and amide absorbance of rat stratum corneum
Vehicle | Asymmetric C-H stretching | Symmetric C-H stretching | Amide I stretching vibration | Amide II bending vibration | ||||
---|---|---|---|---|---|---|---|---|
Peak height | % decrease in peak height | Peak height | % decrease in peak height | Peak height | % decrease in peak height | Peak height | % decrease in peak height | |
Untreated | 0.4149 | - | 0.5223 | - | 0.5403 | - | 0.4255 | - |
Ethanol-water 70:30 v/v | 0.2943 | 29.07 | 0.3898 | 25.37 | 0.2546 | 52.88 | 0.2214 | 47.97 |
Mea | 0.1381 | 66.71 | 0.1824 | 65.08 | 0.1245 | 76.96 | 0.1231 | 71.07 |
Both cosolvent system (ethanol-water, 70:30 v/v) and w/o microemulsion formulations (MEa and MEc) very significantly enhanced the skin permeation of gabapentin through the excised rat skin. Results of the study therefore suggest that microemulsion type (w/o) and cosolvent system (ethanol-water, 70:30 v/v) can be suc- cessfully used as vehicles to enhance skin permeation of gabapentin.