1. Introduction
Over the past few decades, diabetes mellitus (DM) has started to appear as one of the major causative factors of deaths globally [
1]. According to the International Diabetes Federation, it is extending at a very alarming rate, with a total estimated increase of 151 million (2000) to 463 million (2019) followed by a probability of increasing up to 700 million in near future [
2]. Though several hypoglycemic drugs are widely used in diabetes management, a complete cure for diabetes still remains distant because of the numerous intrinsic deficiencies and side effects associated with these drugs [
3,
4]. The optimum drug concentration that is required cannot reach focal regions due to proteolytic degradation and chemical instability in a harsh pH environment. In addition, conventional dosage forms cannot be adjusted to tackle extensive fluctuations in glucose that lead to severe hypoglycemia [
5,
6]. Furthermore, other difficulties with drug therapy such as problems in drug absorption, short half-life, low solubility, low bioavailability, and adverse effects on other organs also impairs treatment [
7].
Glimepiride is a third generation sulphonylurea per oral hypoglycemic agent [
8]. It exerts its effect through two mechanisms by increasing the production of intracellular insulin from the (pancreatic) beta cells and by enhancing the sensitivity of intracellular (insulin) receptors to insulin action [
9]. Currently, GMP is one of the most prescribed drugs for the treatment of diabetes, but its (oral) administration presents numerous deleterious side effects, low efficacy, and patient noncompliance [
9,
10]. The major problem associated with GMP is its poor aqueous solubility. To overcome this problem, many studies have been conducted to enhance the solubility of GMP by using a cyclodextrin inclusion complexation [
11].
In our previously published report, we have attempted to devise binary and ternary GMP complexes by employing different hydrophilic polymers (βCD and Gelucire-44/14). The prepared complexes were also characterized using scanning electron microscopy (SEM), infrared (FTIR), powder X-ray diffraction (XRD), and differential scanning calorimeter (DSC). It was concluded that the solubility of GMP was significantly increased by both the GMP/βCD (1:4) and GMP/βCD/GEL-44/16 (1:4, 10%
w/
w) complexes, but the highest solubility was reported with the ternary complex of GMP/βCD/GEL-44/16 [
12]. The GMP/βCD/GEL-44/16 complex (1:4, 10%
w/
w) was selected for the present study [
12].
In the last several years, the topical application of numerous antidiabetic agents has been widely investigated as a promising alternative to an oral approach since it can attain the therapeutic drug concentration through the skin at a controlled and pre-determined rate [
13], bypassing the first pass effect, reducing the frequency of dosing, and the treatment is able to be stopped through simple removal [
14]. However, a safe and efficient permeation not only depends on the physicochemical character of a drug but also on the physical properties and chemical composition of the carrier [
15,
16].
Owing to the dense cellular structure and lipophilic character of stratum corneum, most of the drugs are not eligible to be delivered through the skin using traditional transdermal carriers. The application of nanocarriers for enhancing the transdermal delivery of drugs has emerged as a highly valuable alternative [
17]. Several nanocarriers of GMP that have been investigated in the last ten years include liposomes, nanoethosomes, and self-nanoemulsifying particles, which have been designed for enhancing the transdermal bioavailability by encapsulation or solubilization, enhanced drug permeability, and attaining the controlled release [
18,
19]. Topical nanoemulsion formulations are well known for their ability to enhance skin permeation by temporarily disrupting the highly organized structure of the skin, and the potential of the topical nanoemulsions to deliver GMP was still left unexplored. Previous studies have demonstrated the use of self-nanoemulsifying particles to increase the effectiveness of GMP, but the drawback of these transdermal carriers is that they are only appropriate for smaller molecules that can easily penetrate stratum corneum (SC) [
20].
Clove oil obtained from the flower buds of
syzygiumaromaticum has recently received a lot of attention for its antidiabetic potential, especially in case of type 2 diabetes [
21,
22,
23,
24]. Clove oil contains eugenol and acetyleugenol, which can increase the fluidity of the lipids found in SC and can act as a penetration enhancer [
24]. Advantage of using clove oil in order to deliver GMP is that is expected to improve the permeability and hypoglycemic activity with fewer side effects and higher efficacy. The antidiabetic activity of GMP using clove oil as an oil phase has not been investigated so far. Therefore, a nanoemulsion-based GMP gel using clove oil was formulated.
Hence, in order to enhance the ability of GMP to permeate the skin and to provide a more efficient therapeutic effect in comparison to previously reported studies, a nanoemulsion-based gel of glimepiride was designed. The unique feature of our study is that we introduce a new concept for the incorporation of solubility enhanced GMP (GMP/βCD/GEL-44/16) into the nanoemulsion-based gel (nanoemulgel) and evaluate its permeation and hypoglycemic activity against pure GMP and a marketed brand.
2. Materials and Methods
2.1. Materials
Glimepiride (GMP) was obtained as a gift from Saffron Chemical Industries Ltd. (Pakistan). Tween 20, Tween 40, Tween 80, Span 20, Span 80, propylene glycol, and PEG-400 were purchased from Daejung (Korea), whereas Labrafil-M 2125 CS, Capryol 90, and Gelucire 44/16 were gifted by Gatefosse (France). Ethanol, butanol, propanol, and glycerol were obtained from Merck (Pakistan). Clove oil, eucalyptus oil, peppermint oil, sandalwood oil, cinnamon oil, sesame oil, black seed oil, and rosemary oil were procured from Co Natural and Go Natural (Pakistan). The other chemicals and reagents used in this study were of analytical grade.
2.2. Solubility-Studies
The solubility of GMP was checked in numerous vehicles, which included antidiabetic oils, surfactants, and co-surfactants. For evaluating the solubility, two grams of these vehicles were taken in falcon tubes to which a surplus amount of glimepiride was integrated. These falcon tubes were then vortexed for ten minutes using a vortex mixer and were placed in shaking water bath for 48 h at 37 °C and 75 rpm. After 48 h, the falcon tubes were removed from the shaking water bath and were centrifuged at 3000 rpm for 15 min followed by filtration using a syringe-filter with a pore size of 0.45 µm. The obtained filtrate was then checked on with a UV-Visible spectrophotometer at 228 nm using methanol as blank. The same reagent was also employed for dilution if required [
25].
2.3. Construction of Pseudo-Ternary Phase Diagram
To draw a phase diagram, the water titration method was used In different falcon tubes, oil was thoroughly mixed with surfactant/co-surfactant at different weight ratios of 0.5, 9.5, 1.0:9.0, 1.5:8.5, 2.0:8.0, 2.5:8.5, 3.0:7.0, 3.5:6.5, 4.0:6.0, 4.5:5.5, 5.0:5.0, 6.0:4.0, 7.0:3.0, 8.0:2.0, and 9.0:1.0. Water was then added drop wise into each tube (at 26 ± 2° followed by 2 min of vortex mixing and was allowed to equilibrate after light magnetic stirring for 2 h. After equilibrium establishment, the mixtures were observed for numerous phases, namely transparent, translucent, milky, turbid, gel, and phase separation. The clear emulsion with good flowability was declared as the nanoemulsion. Chemix (cxse 700, ver 4.00) was used to construct the ternary phase diagram [
26].
2.4. Preparation of Nanoemulsion Formulations
Both blank and glimepiride/glimepirde inclusion complex-loaded nanoemulsions were fabricated through the spontaneous emulsification method [
27]. Blank nanoemulsions were formulated by mixing oil and Smix at the optimum formulae that had been chosen from phase diagram on the basis of the nanoemulsion region. After achieving complete mixing, water was added drop-wise to the mixture and was vortexed until a transparent formulation was obtained.
2.5. Preparation of Drug-Loaded Nanoemulsion and Nanoemulsion Gel Formulations
For drug-loaded formulations, an accurately measured quantity of GMP and GMP/βCD/GEL-44/16 were added to the oil phase, which was subsequently followed by the addition of Smix and vortexing to facilitate complete solubilization. Water was then added gradually with proper mixing to attain a clear and homogenous nanoemulsion. The effect of drug loading in terms of the transparency of the NE systems was also studied, and 0.4%
w/
w drug was loaded in the final preparations. For the development of nanoemulsion based gels, the prepared NE formulations of both the GMP and the GMP/βCD/GEL-44/16 (GMP/βCD/GEL-44/16 were gelled using xanthan gum as a gel polymer. For this, an accurately measured amount of xanthan gum (3 g) was dissolved in 100 mL of distilled water in order to formulate a gel base to which subsequent addition of triethanolamine was made to neutralize the pH. The drug and inclusion complex containing nanoemulsions were then added to this gel base in a ratio of 1:1 by magnetic mixing for a period of 15 min at a 250 rpm speed to properly acquire homogenized nanoemulsion based gels [
28].
2.6. Characterization of Nanoemulsion and Nanoemulgel Formulations
For determining the particle size and polydispersity index of the prepared formulations, Malvern Zetasizer (ZS90, UK) with DLS (dynamic scattering technique) was used. All estimations were conducted at 25 °C and at an angle of 90°. Zeta potential (ELS, electrophoretic scattering) was determined using the same instrument at 633 nm by a 1 volt electric field application. All nanoemulsion formulations were diluted 100 times (distilled water) prior to particle size and zeta potential measurement, and separate samples were used for each evaluation. The viscosity of the prepared formulations was tested with a Brookfield viscometer (DVII+ Pro). The spindle used for checking the viscosity was s 63. It was rotated at a speed of 100 rpm for 1 min, and each sample was tested in triplicate. The pH of the both blank and drug/inclusion complex-loaded nanosized emulsion was checked with a pH meter (HI 9811-5 Hanna, United States). Electrical conductivity of both the blank and drug/inclusion complex-loaded formulations were also investigated, and the instrument used for this purpose was a portable EC meter (Hanna
® instruments HI 9811-5 Romania). Nanoemulgel formulations were characterized by tests similar to those performed for NE (pH and viscosity). The spreadability of each nanoemulgel formulation was also assessed using a glass plate according to the method reported by Dantus [
29]. An accurately weighed amount of nanoemulgel was spread on a glass plate, which was pre-marked with a circle measuring 2 cm. The presenting assembly was then topped with another glass plate followed by a weight of 0.5 kg for five minutes. Each formulation was analyzed in triplicate (
n = 3).
2.7. Ex Vivo Permeation Study
Animal study was conducted according to the guidelines of Animal Welfare Act and the those from the Care and Use of Laboratory Animals of National Institutes of Health. All of the protocols were thoroughly studied and consented to by the local Institutional Review Board of GCUF University (GCUF/ERC/2185 Dated: 23 April 2020) after evaluating and ensuring the proper use of animals. In order to conducted permeation studies, rats weighing in the range of 200–300 g were sacrificed, and the hairs on their abdominal area were removed followed by surgical removal of the skin. The excised skin was subsequently rinsed with alcohol and distilled water, and it was stocked at −20 °C until additional application. Before use, it was removed from the freezer and was thawed to room temperature followed by placement in phosphate buffer for at least 60 min in order to properly hydrate the membrane. For the permeation study, a Franz diffusion cell apparatus with two compartments named as donor and receiver compartment was used. As far as the skin is concerned, it was positioned in between the two compartments in such a way that the stratum corneum (SC) side countered the receiver compartment. The GMP dispersed in clove oil and a GMP aqueous suspension with (0.5%) carboxymethyl cellulose were used as controls. The formulations (1 gm, control and sample) to be tested were placed in donor compartment, whereas receiver compartment contained the dissolution medium, i.e., the phosphate buffer (pH 7.4) plus methanol (70:30, %
v/
v), which was constantly stirred with magnetic bars at 300 rpm. The temperature was regulated at 37 °C, and samples were taken at specified time points preceded by replacement with the same amount of dissolution medium. The samples were then scanned using a UV spectrophotometer at a wavelength of 228 nm. Experimentation was done in triplicate, and the obtained data were articulated as the cumulative quantity of GMP permeated across SC against time [
30]. Furthermore, estimations of various skin permeation parameters, namely drug flux (Jss), as well as the permeability co-efficient (K
p) were also determined.
2.8. In Vivo Antidiabetic Study
In order to inspect the anti-diabetic potential of the selected test formulations, male wistar rats weighing 200–250 g were used. All of the animals were accommodated under managed temperature conditions (24 °C) and humidity (70–30%) along with a proper diet consisting of typical rat feed and water. Diabetes mellitus was developed by injecting streptozocin intra-peritoneal at a dose of 50 mg/kg body weight. Prior to the diabetes generation, the rats were fasted for the night; however, only food was restricted, and water was available at all times. After 14 days, the serum blood glucose levels of fasted rats was checked using a glucometer, and the rats with glucose levels between 250–300 mg/100 dL were selected for further studies. The overall experimental design of this study involved six groups with each group consisting of five rats in total. The rats in Group I (normal control) and Group 2 did not receive any treatment at all and served the as normal and diabetic controls, whereas the rats in Group 3 (positive control) were given a per oral glimepiride dose of 10 mg/kg bodyweight [
31]. Rats in Group 4 were given a blank gel formulation, which was prepared by incorporating blank nanoemulsion in xanthan gel. Similarly, the rats in Group 5 and 6 were subjected to the topical application of nanoemulgels, namely GMP loaded nanoemulgels and GMP/βCD/GEL-44/16-loaded nanoemulgels at a dose of 10 mg/kg bodyweight, respectively. For oral administration, the standard oral gavage technique was used. Blood samples were collected from the tip of the tail by nicking the lateral tail vein.
2.9. Skin Irritation Study
The skin irritation study was conducted according to OECD guidelines and for this purpose, the wistar rats weighing in the range of 150–200 g were used. All of the rats were divided completely at random in to four groups: Group 1 (no application), Group 2 (blank nanoemulgel, NEG), Group 3 (GMP-loaded NEG), and Group 4 (GMP/βCD/GEL-44/16-loaded NEG). The hairs on the dorsal surface of each rat were carefully removed by shaving the rats 24 h prior to experimentation, and 0.5 gm of each test formulation was applied onto the shaved skin for 4 h by evenly spreading it over an area of 6 cm
2. The testing area was ascertained for any erythemic or edema reaction after 1, 24, 48, and 72 h of application. The experiential tolerance reaction was scored as 0, 1, 2, and 3 for no, slight, moderate, and severe erythema/edema, in that order. After 72 h, all of the animals were sacrificed through cervical decapitation, and the skin from the testing area was obtained. The obtained skins were stored in 10% neutral buffered formalin solution, fixed in paraffin, cut in 5 µm sections, and stained with H &E dye followed by imaging under light microscopy [
32].
2.10. Statistical Analysis
All of the parameters were taken in triplicate, and the obtained results were presented as mean ± standard deviation. For statistical significance, one-way ANOVA, two-way ANOVA and Tukey’s multiple comparison test were employed in order to assess any significant differences amongst the quantitative variables. Data values having p < 0.05 were marked as statistically significant.