Development of an α-linolenic acid containing a soft nanocarrier for oral delivery-part II: buccoadhesive gel

Abstract

The objective of the present research was to develop and characterize a Carbopol 71G (CP 71G) buccoadhesive gel encompassing an optimized simvastatin-loaded microemulsion (MES4) containing α-linolenic acid as an oil phase for buccal delivery. Crosslinking of the gelling agent was done by adjusting the pH with a neutralizing agent triethanolamine (TEA). The formulations, namely, the drug suspension, the MES4, and the microemulsion based buccal gel containing 4% w/v (MEBG4), 5% w/v (MEBG5) and 6% w/v (MEBG6) CP 71G respectively, were optimized on the basis of the permeation flux of simvastatin (SIM), which was found to be in the range of 0.132–0.482 mg cm⁻² h⁻¹, calculated from an ex vivo permeation study. The optimized buccal gel (MEBG4) showed a significantly higher (P < 0.001) permeation flux (J = 0.443 ± 0.062 mg cm⁻² h⁻¹) compared to the drug suspension (J = 0.132 ± 0.044 mg cm⁻² h⁻¹). The permeation enhancement ratio of MEBG4 was found to be 3.36 fold higher than that of the aqueous suspension. The C_max value (131.208 ± 21.563 ng ml⁻¹) of the buccoadhesive gel (MEBG4) was found to be significantly higher (P < 0.001) when compared to the same dose administered by an oral route (C_max – 68.513 ± 9.821 ng ml⁻¹). The relative bioavailability (Fr) of the optimized MEBG4 buccal gel was about 385.3% higher than that of the oral marketed tablet. The MEBG4 gel followed the Korsmeyer–Peppas equation implying that the gel showed a diffusion type of drug release, due to polymer relaxation or erosion (Case II transport). The texture profile in terms of spreadability (0.8 mJ), adhesiveness (2.9 g), firmness (11.0 g) and extrudability (35.2 mJ) of MEBG4 was evaluated and showed good spreadability and adhesiveness. A rheological study revealed the pseudoplastic behavior of the gel. In conclusion, a consistent and effective buccoadhesive gel with a SIM-loaded microemulsion and improved buccal permeation and pharmacokinetics parameters was developed successfully.

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1. Introduction

Simvastatin (SIM) is a poorly water soluble drug; hence its absorption is dissolution rate-limited.¹ Further, the drug has extensive first pass metabolism and is extensively ionized at intestinal pH, which results in an excretion of about 60% of the unabsorbed drug in feces and results in low oral bioavailability (≤5%).² It belongs to the statins group and is extensively employed in the management of dyslipidemia, hypercholesterolemia, and coronary heart disease. It is a potent inhibitor of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase and catalyzes the conversion of HMG-CoA to mevalonate during the biosynthesis of cholesterol.^2,3

Various formulations such as self-emulsifying drug delivery systems (SMEDDS),⁴ lipid nanocarriers (solid lipid nanocarriers, nanostructured lipid carriers and lipid nanoemulsion),⁵ self-emulsifying granules,⁶ solid dispersion,^7–9 orodispersible tablets,¹⁰ and microemulsions¹¹ have been developed for the delivery of simvastatin. However, no buccal gel formulation containing a simvastatin-loaded microemulsion has been reported to date. The aim of this study was to develop and characterize a novel microemulsion based buccoadhesive gel for the delivery of SIM, in such a manner that the hepatic first pass is bypassed, bioavailability is enhanced and therapeutic action is displayed for a longer duration.

The buccal route of drugs is a pertinent substitute to the oral route to bypass gastrointestinal degradation and the first pass effect.¹² Since the drug directly enters into systemic circulation through the buccal route, via buccal capillaries, it bypasses degradation in the intestine and the first-pass effect in the liver. This enables rapid onset of action, easy administration, and high patient acceptance and compliance.^13,14 The buccal delivery system can be utilized for the local/systemic delivery of drugs.

Properties such as poor aqueous solubility, short half-life (∼3 h), dose size (5 to 80 mg), low molecular weight (418.57), and low bioavailability make SIM an appropriate candidate for administration through the buccal route as a microemulsion based buccoadhesive gel.

Drug delivery through buccal mucosa is a very difficult task, particularly when the drug is poorly soluble in water and has an especially low systemic availability when administered orally.

The rationale behind choosing a microemulsion based bucco/mucoadhesive delivery system is that the majority of lipophilic drugs cannot be incorporated into a gel base directly since solubility acts as a limiting factor and this may affect the release of the drug. Therefore, to surmount this limitation, a microemulsion based approach was used, so that the lipophilic drug candidate can be effectively incorporated and delivered through the gels. Microemulsion facilitates the solubilization of a lipophilic drug, thereby showing quick and proficient penetration through the application site. Microemulsions also have other advantageous properties such as the lowering of interfacial tension between immiscible liquids, thermodynamic stability, spontaneous formation, ease of manufacturing, bioavailability improvement of hydrophobic drugs, and the prospective for permeation enhancement.¹⁵

In our previously reported research work, a novel microemulsion (MES4) containing α-linolenic acid as the oil phase, kolliphor EL40 as a surfactant and Transcutol P as a cosurfactant was developed and optimized for the oral delivery of SIM and the role of α-linolenic acid in the regulation of lipid levels with simvastatin was discussed.¹¹

α-linolenic acid, also known as n-3 fatty acid, has various cardiovascular functions such as reducing triglyceride levels, decreasing the risk of thrombosis by inhibiting platelet aggregation, improving the endothelium-dependent relaxation of hypercholesterolaemia and atherosclerotic, anti-arrhythmic effects, preventing plaque development and also contributing to plaque stabilization through anti-inflammatory effects.^16–18

Linoleic acid (n-6 fatty acid) derived eicosanoids such as prostaglandins and thromboxanes have pro-inflammation, proarrhythmic, vasoconstriction, platelet aggregation and bronchoconstriction effects, while n-3 fatty acid derived eicosanoids have antiarrhythmic, anti-platelet aggregation and anti-inflammatory effects.¹⁹

Linoleic and oleic acid (n-9 fatty acid) derivatives have been developed and evaluated for their anticancer potentials by various researchers.^20–22

Optimized MES4 was utilized for the development of a buccoadhesive delivery system for SIM. The microemulsion was incorporated into a gel base, possibly establishing a better stability and release of the drug than by merely incorporating the drug into the gel base. A gel based system would also increase the time of contact to the buccal mucosa and hence may result in improved patient compliance and efficacy.

Muco/bioadhesive polymers have widely been utilized in the development of buccal drug delivery systems because of their augmented capability to adhere onto biological membranes.²³ Buccal/mucoadhesive formulations may be an alternative to conventional therapy as they can readily adhere to the buccal cavity, be retained for a longer period of time and thus, can improve the effectiveness of treatment.^24–28 In the present study, the bioadhesive gel forming agent Carbopol 71G was used.

Bucco/mucoadhesive formulations intended for buccal application should show appropriate rheological and mechanical properties such as pseudoplastic or plastic flow, ease of application, appropriate hardness, good spreadability, and prolonged residence time at the site of application. These properties can influence the performance of the formulations and their acceptance by patients.

The objective of this study was to develop a novel buccoadhesive gel formulation using optimized SIM-loaded microemulsion bucco/mucoadhesive gels that possess suitable mechanical properties, adhere to the buccal mucosa for a sufficient time, provide sustained action, improve buccal mucosa permeation and enhance the plasma concentration of the drug. To accomplish this objective, microemulsions containing buccoadhesive gel with varied concentrations of Carbopol 71G were prepared. SIM permeation from the prepared gel formulations was examined through ex vivo permeation studies and a mucoadhesive evaluation of the gels was performed via a mucoadhesion test conducted on the buccal mucosa. An in vivo pharmacokinetics study was performed with a final formulation to obtain various pharmacokinetics parameters. The mechanical properties such as hardness, extrudability, adhesiveness and spreadability of the prepared final gel formulation were investigated using texture profile analysis. Rheological studies of the gel formulations were performed with a rheometer and viscosity was measured using a digital viscometer.

2. Experimental

2.1 Materials

SIM and Carbopol 71G were obtained as a gift sample from IPCA Pvt. Ltd. (Mumbai, India). Methylparaben, propylparaben, glycerine, triethanolamine, and propylene glycol were purchased from HiMedia Laboratories (Mumbai, India). All other chemicals used in the study were of analytical grade.

2.1.1 Methods.

2.1.2 Preparation of buccal gel base. The buccal gel base was prepared as follows: propylparaben (0.05% w/v) and methylparaben (0.1% w/v) were used as preservatives and dissolved in a beaker containing a small quantity of double distilled water. These were mixed properly by gentle stirring (100 rpm) and heating (50 °C) on a hot plate digital magnetic stirrer (Tarson, Mumbai, India). A sufficient quantity of water was added in a beaker and Carbopol 71G (7% w/v, polymer) was dispersed slowly into the solution with stirring and allowed to soak for 24 h, followed by the addition of glycerine (10% w/w) and polyethylene glycol 400 (5% w/w) as humectants. Triethanolamine (TEA, pH adjuster) was added drop by drop to the final mixture and stirred thoroughly until a clear, viscous, homogeneous gel was obtained. The pH of the prepared buccoadhesive gel was kept in the range of 6.5–8.0.

2.1.3 Preparation of microemulsion containing buccal gel. As a vehicle for the incorporation of the microemulsion for buccal delivery, Carbopol 71G (CP71G) gels were prepared. A SIM-loaded optimized microemulsion (MES4: preparation and characterization reported elsewhere)¹¹ was utilized for the formulation of the buccal gel. Microemulsion based buccal gels containing 4% w/v (MEBG4), 5% w/v (MEBG5) and 6% (MEBG6) w/v CP 71G were prepared respectively.

To prepare 100 g of the microemulsion based buccoadhesive gel (MEBG), initially 57.14 g, 71.42 g and 85.71 g of gel base were weighed in separate beakers to prepare the 4%, 5%, and 6% gels respectively from the above-prepared gel base (7% w/v). Afterward, 14 g of MES4 was added and the final weight was adjusted with double distilled water to make the concentrations 4%, 5% and 6% w/v of the MEBG gels. The final concentration of the drug in the buccal gel was taken as 1.0% w/w.

The prepared gels were evaluated for homogeneity, color, texture, pH, drug content, buccoadhesive strength, viscosity, and permeation.

2.1.4 Physical examination. The prepared gel formulations were inspected visually for homogeneity, color, and texture.

2.1.5 pH determination. 1.0 gram of the gel was accurately weighed; up to 10 ml of water was added and mixed well and then the pH of the solution was measured using a digital pH meter (Lab India). pH determination was done in triplicate for each formulation.

2.1.6 Drug content analysis. 1.0 g of the gel was accurately weighed and dissolved in 25 ml of methanol in a volumetric flask. The flask was shaken for 10 min and then the mixture was filtered. The volume of filtrate was made up to 50 ml with methanol. One ml of this solution was further diluted to 25 ml with methanol. The total drug content was determined by UV absorbance of the resultant solution at a wavelength of 238 nm. Drug content analysis was performed in triplicate.

2.1.7 Viscosity. The viscosity of the buccoadhesive gels was measured by a Brookfield Viscometer (Digital, Labtronics). The viscosity of the experimental formulations (MBG4–MBG6) was measured at 25 ± 2 °C and at 6 rpm speed using a spindle no. 4. Measurements were executed in triplicate.

2.1.8 In vitro mucoadhesion force. A modified physical balance apparatus was designed and worked out to measure the minimum detachment force. For this experiment, buccal mucosa, acquired from the local slaughterhouse (Lucknow), was carefully removed from the oral cavity of a goat and mounted on two glass bottles as is shown in Fig. 1. This experiment did not encompass any ethical issues since the mucosa was obtained from an animal source slaughtered for food. The prepared gel (500 mg) was placed on one mucosal mounted surface (lower side). Two surfaces (lower and upper) were held in contact with each other for 2.0 minutes to ensure adhesion between them. Afterward, in the second pan, weights were added at a continual increasing rate until the two mucosal membranes were detached from each other. The buccal mucosa was changed for each measurement. The muco/buccoadhesive force (detachment stress, N) was calculated from the minimal weight that separated the buccal mucosal tissue from the surface of each formulation.

Mucoadhesive force (N) = mucoadhesive strength (g) × 9.81/1000 (1)

Fig. 1 Schematic muco/buccoadhesive force measuring device: (A, C) glass bottle, (B) SIM gel, (D) height adjustable platform, (E and F) buccal membranes, (G) modified balance and (H) weights.

2.1.9 Ex vivo drug permeation studies. To check the release behavior and to have an insight on the barrier properties of buccal mucosa, an ex vivo permeation study was carried out for all of the prepared gels using goat buccal mucosa and a modified Franz diffusion assembly, containing donor and receptor compartments.

For the ex vivo permeation studies, excised goat buccal mucosa was collected from the local area abattoir on the same day of the experiment, kept in ringer’s solution, and prepared according to the previously reported method.¹⁴ Prepared buccal mucosa (a diffusional area ∼ 3.14 cm²) was put between the receptor and donor compartments in the Franz diffusion cells to perform ex vivo permeation studies of SIM from the buccoadhesive gel. Approximately 25 ml of PBS (pH 7.4) was filled in the receptor chamber and constantly stirred and thermostated at 37 ± 0.5 °C with the help of the outer water jacket, to hydrate the buccal mucosa. 1.0 ml of PBS was added to the donor chamber, 30 min before the start of the experiment with a formulation to mimic the conditions of the mouth. After 30 min, the donor liquid was removed instantly before the addition of the formulation. The test formulation (1.0 g of buccal gel) was then applied to the buccal mucosa from the donor side compartment; afterward, the donor compartments were covered with Parafilm. A volume of 2.0 ml was withdrawn from each receptor chamber at specific time intervals and replaced with an equal volume of same temperature fresh PBS. The experiments were conducted in triplicate for each formulation. The samples were analyzed spectrophotometrically at 238 nm.

The steady state flux (J_ss) of SIM (mg cm⁻² h⁻¹) was calculated by the previously reported method.²⁸ The cumulative drug release was calculated as the total concentration of the drug in total volume divided by the surface area of the mucosa. The flux (mg cm⁻² h⁻¹) was calculated from the slope of the linear portion of the cumulative amount permeated per unit area versus time plot. The permeability coefficient (K_p) was calculated using the following equation:

K_p = J_ss/C (2)

where J_ss is the steady state flux and C is the initial concentration of simvastatin.

Statistical analysis was performed using one-way ANOVA (Graph prism 6.0 software, trial version) to check the statistical significant difference between data.

In order to study the mechanism of drug release from the prepared buccoadhesive gels, the ex vivo permeation data was evaluated for zero-order release kinetics, first order release kinetics, Hixon–Crowell’s cube root of time equation,²⁹ Higuchi’s square root of time equation,³⁰ and Korsemeyer and Peppas equation.³¹ The goodness of fit was evaluated by comparing the correlation coefficient (r²) values for respective batches.

2.1.10 Texture analysis. Texture analysis of the optimized gel formulation was carried out by an automatic CT3 Texture Analyzer (Brookfield Engineering Laboratories, USA). Extrudability, hardness/firmness, spreadability and adhesiveness were evaluated for the developed buccoadhesive gel.³²

2.1.11 Rheology. The rheology of the sample was measured according to the previously reported method by Shantanu et al.³³ Briefly, a rheology study of the optimized MEBG4 gel was executed by means of a rotational type rheometer (Rheolab QC, M/s Anton Paar GmbH, Vienna, Austria) appended with a water jacket (C-LTD80/QC) for upholding a constant temperature of 25 °C. The analysis was carried out using Rheoplus/32 software version 3.40. For the determination of the rheological behavior of the sample, a spindle DG26 was used.

2.1.12 Stability studies. A stability study of the optimized gel formulation (MEBG4) was performed to determine the stability of the drug and carrier and also to determine the physical stability of the formulation. The optimized gel formulation was stored in well-closed containers for a period of 180 days at ambient room temperature. At predetermined intervals, 0, 30, 60, 120 and 180 days, samples were collected and their physicochemical evaluation parameters such as color, consistency, pH, phase separation and drug content were evaluated. Physical parameters such as color and consistency were observed visually while pH, phase separation and drug content were evaluated after a freeze–thaw cycle.

For one freeze–thaw cycle, the temperature for freezing was −20 °C and for thawing +25 °C. The gel formulation was stored at each temperature for 24 h and evaluated for phase separation (physical instability), pH and drug content.

2.1.13 Histopathological analysis. After the ex vivo permeation experiment, the buccal membranes treated with Carbopol gel formulations were removed from the diffusion cell and examined. An untreated buccal membrane was used as a control, after incubation with phosphate buffer (pH 7.4) on the mucosal and serosal sides. The buccal epithelium samples were fixed in 10% w/v buffered formalin solution, afterwards, were dehydrated with an increasing ethanol concentration i.e. 30%, 50%, 70%, and 95% w/v, and consequently embedded in paraffin wax. The prepared paraffin blocks were cut to 5 μm thickness using a microtome and stained with hematoxylin–eosin dyes. The prepared sample was then analyzed using an optical microscope (RXLr-4) coupled with a digital camera (Canon DSLR 1100).

2.1.14 In vivo pharmacokinetics study. The experiments were carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India under the approval of the Institutional Animal Ethics Committee of Babu Banarasi Das Northern India Institute of Technology, Lucknow (BBDNIIT/IAEC/059/2014). The animals used for the experiments were maintained under standard conditions. The room temperature was maintained between 18 to 29 °C and relative humidity control between 30% to 70% during the experiment and the animals were kept in polyacrylic cages, with a 12 h light/dark cycle, and had free access to standard diet and water. The authors have not performed any experiments on human subjects. Albino Wistar rats weighing around 180–200 g were used for this experiment. An in vivo pharmacokinetics study of gel formulation was performed according to the previously reported method.³⁴ The animals were divided into three groups (n = 6 animals in each group) i.e. group I – kept as a control, group II – given a marketed tablet oral suspension (1 mg dose) and group III – rats were anaesthetized by intraperitoneal injection (i.p.) of phenobarbital sodium (30 mg kg⁻¹), and an extra dose was given periodically to maintain anesthesia. A gel with an amount equivalent to 1 mg of drug was applied on both sides to the inner cheeks of the group III rats. Blood samples were collected from the retro-orbital plexus at 0.25, 0.50, 1, 2, 4, 8, 12 and 18 h after dose administration. Plasma was separated by centrifugation (at 5000 rpm for 10 min) at 4 °C and stored in 2 ml vials at −20 °C until analysis. The assessment of SIM in the blood plasma samples was carried out according to a previously reported HPLC method.¹¹

Briefly, liquid–liquid extraction was done. A gradient HPLC (Waters 2489, with UV-Visible Detector) was used for analysis of the plasma samples. Separation was done with a reverse phase column (Spherosorb C18, 250 × 4.6 mm) with a flow rate of 1.0 ml min⁻¹. The mobile phase contained a mixture of 0.025 M sodium dihydrogen phosphate (HPLC grade, pH 4.5) : acetonitrile (25 : 75 v/v). For analysis of the samples, plasma was mixed with the mobile phase, vortexed and centrifuged (5000 rpm, 10 min) and the supernatant collected in 2 ml centrifuge tubes. The extracted supernatant was dried and reconstituted with the mobile phase, filtered through a 0.22 μm membrane filter before analysis and then analyzed by HPLC.

Pharmacokinetics parameters were determined using WinNonlin software. The relative bioavailability (Fr) of MEBG4 (test) was calculated with respect to the oral drug suspension (standard) using the equation:

Fr (%) = AUC_test/AUC_standard × 100 (3)

Statistical analysis was performed using a t-test with Welch correction (Graph prism 6.0 software, trial version) to check the statistically significant difference between the results.

3. Results and discussion

3.1 Physicochemical analysis

The physicochemical properties of the prepared formulation have been presented in Table 1. It was established that all of the gel formulations were homogenous and smooth with an acceptable consistency, with a pH range of 6.91 ± 0.015 to 7.18 ± 0.024, which was within the pH range (5.6–7.0) of oral mucosa.³⁵ The pH of the developed formulations was near neutral in nature and thus could be considered as a suitable delivery system for avoiding pain, damage or irritation to the oral mucosa tissues. The optimized MEBG4 gel, having a pH of 7.18, could be administered easily through buccal mucosa for systemic delivery of the drug.

Table 1 Physicochemical parameter evaluation of microemulsion gels (n = 3)

Formulation code	Homogeneity	Colour	Texture	pH ± SD^a	Drug content (mg g^−1) ± SD^a
a SD – standard deviation.
MEBG4	Homogenous	Creamy white	Smooth	7.18 ± 0.024	9.94 ± 0.15
MEBG5	Homogenous	Creamy white	Smooth	6.96 ± 0.032	9.88 ± 0.20
MEBG6	Homogenous	Creamy white	Smooth	6.98 ± 0.015	9.90 ± 0.18

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The drug content was determined to check the uniformity of drug distribution in the developed formulation. The drug content of all prepared formulations was found to be in the range of 9.88 ± 0.12 to 9.93 ± 0.20 mg g⁻¹ of gel, the results for which have been recorded in Table 1.

3.2 Viscosity

The viscosity of the semi-solids should be such that they can easily be poured from the container and spread on the desired site. At the same time, formulations should have appropriate retention characteristics to prevent flowing and removal from the application site. Although enhanced viscosity can be more suitable for retention, the ease of application on the desired site as a thin film layer is also an important criterion.

The viscosity of all of the prepared gel formulations was found to be in the range of 30 716.83 ± 125.1 to 50 613.78 ± 218.3 cP as is shown in Table 2. The viscosity was found to increase by increasing the polymer concentration from 4 to 6% w/v. This could be attributed to the augmentation in intermolecular bonds or cross-linking between the polymer chains, hence increasing the gel network complexity^23,36 which happens maximally at about neutral or alkaline pH and was found to increase when the pH of the medium was elevated from acidic to neutral for the CP71G polymer based gel. This behavior is significant for buccal drug delivery since it may lead to adhesive interactions and can enhance polymer retention time over mucosal surfaces where the gel is applied.

Table 2 Results of viscosity, buccoadhesive strength and force (n = 3) of gels

Formulation code	Viscosity (centipoise) ± SD^a	Buccoadhesive force (mN) ± SD^a
a SD – standard deviation.
MEBG4	30 716.83 ± 125.1	185.01 ± 4.84
MEBG5	38 908.42 ± 212.5	261.24 ± 6.57
MEBG6	50 613.78 ± 218.3	374.34 ± 4.18

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3.3 Muco/buccoaadhesive force

Mucoadhesion is the binding of a synthetic or natural polymer-based delivery system to the application site on mucosal tissue, providing prolonged and intimate contact between the delivery system and the absorption site.^35,37,38 Consequently, mucoadhesive delivery systems can immobilize and liberate the drug in specific regions, which contributes to the maintenance of therapeutic concentrations of the drug at the specific site and allows for an adequate and effective therapeutic response.^35,37,39

A mucoadhesion study was performed by using the modified balance method (Fig. 1). It was found that an increased concentration or amount of CP71G in the formulation resulted in an increased mucoadhesive strength. Studies also revealed that the buccoadhesive strength values for the buccal delivery system were between the ranges of 185–374 mN (Table 2) at CP71G concentrations of 4% to 6% w/v. Therefore, it can be concluded that CP71G gels of 4–6% w/v have a good mucoadhesive strength and can be employed to prolong the residence time of the drug at the application site in oral buccal mucosa.

3.4 Permeation study

To study the effect of formulation ingredients on permeation, MEBG4, MEBG5, MEBG6, an aqueous dispersion, and MES4 were investigated for a period of 6 h each and each formulation was analyzed in triplicate (Table 3). The aqueous suspension, MEBG4, MEBG5, MEBG6 and MES4 exhibited 24.75 ± 7.80%, 81.44 ± 4.38%, 63.50 ± 8.25%, 46.84 ± 5.45, and 92.20 ± 6.05% observed drug permeation respectively in 6 h (Fig. 2). The comparison of cumulative permeation as is shown in Fig. 2 between the prepared microemulsion gels and the microemulsion exhibited a significant enhanced drug permeation when compared with the conventional formulation (aqueous suspension). This increment was almost two to four fold when compared with the aqueous suspension.

Table 3 Comparison of permeation parameters of various formulation of SIM (mean ± SD, n = 3)

Formulation code	Mean percent permeation at 6 h	Flux Jss (mg cm^−2 h^−1)	Permeability coefficient Kp (cm^2 h^−1)	Enhancement ratio
MES4	92.20 ± 6.05	0.482 ± 0.033	0.048	3.65
MEBG4	81.44 ± 4.38	0.443 ± 0.062	0.044	3.36
MEBG5	63.50 ± 8.25	0.332 ± 0.048	0.033	2.51
MEBG6	46.84 ± 5.45	0.238 ± 0.094	0.024	1.80
Aqueous suspension of pure drug	24.75 ± 7.80	0.132 ± 0.044	0.013	—

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Fig. 2 Results of permeation studies of different formulations (n = 3, mean ± SD).

Drug permeation from MES4 (92.20 ± 6.05%) was significantly higher (p < 0.001) than that from the aqueous suspension (∼4 folds) of SIM. MEBG4 and MEBG5 based gel showed significantly higher (p < 0.001) drug permeation compared to the aqueous suspension. MEBG4, MEBG5 and MEBG6 showed slower permeation than MES4 which could be attributed to the slower diffusion of the drug through the gel network. Instead of providing the optimum structure and viscosity to the microemulsion for buccal application, Carbopol in the ME gel has the added advantage of excellent adhesive and constant releasing properties.^23,40 The higher polymer concentrations resulted in higher average viscosities of polymer solution which provided higher resistance to drug diffusion, hence slower permeation of the drug as is shown in Fig. 2. The results indicated that the diffusion of SIM from the gel formulation is chiefly dependent on the microviscosity of the water channels of the gel matrix rather than the macroviscosity of the gel formulations and the higher concentration of polymer results in a reduction of the size of the water channels, hence a slower permeation.⁴¹ The difference in permeation can be related to the differences in gel concentrations. An estimation of drug binding in the oral epithelium might imitate the rate of drug disappearance from the oral cavity and appearance in the plasma and accordingly the duration of efficacy of drugs after buccal administration.⁴²

The steady state permeation flux (J_ss) and permeability coefficient (K_p) for all of the formulations are shown in Table 3. MES4 (0.482 mg cm⁻² h⁻¹) and MEBG4 (0.443 mg cm⁻² h⁻¹) showed significantly higher flux (p < 0.001) than the aqueous suspension (0.132 mg cm⁻² h⁻¹) while MEBG5 (0.332 mg cm⁻² h⁻¹) showed a significant higher flux (p < 0.01) than the aqueous suspension. The results demonstrate that MES4 showed a significant enhancement in the permeation of SIM. The permeation enhancement ratio of the MEBG4 gel was 3.36 times higher than that of the aqueous suspension of the drug via buccal mucosa. This could be due to the presence of the microemulsion in the gel formulations that enhanced the permeation of SIM through the buccal mucosa.¹¹ This effect may be due to the presence of the oil and surfactant mixture in the microemulsion which could be responsible for enhanced drug permeation from the buccal membranes by increasing the interaction and fluidity of the membrane. Additionally, the presence of the microemulsion in the buccoadhesive gels changed the permeability of the oral buccal mucosa due to the presence of the surfactant mixture (Kolliphor EL40 and Transcutol HP). The surfactant mixture decreases the interfacial tension between the gel formulation and the lipophilic mucosal layers, resulting in an improved affinity and thereby an enhancement in permeability of the drug across the buccal barrier. Other components of the microemulsion formulations i.e. α-linolenic acid and muco/buccoadhesive polymer may be expected to enhance permeation due to the interaction with mucosal lipids making them suitable ingredients for such formulations.

The MEBG gels demonstrated a slow permeation of SIM compared to MES4. This effect could be due to the release retarding effect of the polymer matrix, mainly because of the enhanced viscosity occurring from polymer gelation.^43,44

Hence, it can be concluded that the polymer concentration and presence of microemulsion in the gel formulations play an important role in the permeation of the drug through buccal mucosa and give a better permeation of the drug when compared with an aqueous suspension of pure drug.

Kinetics modeling of the release profile for various formulations was evaluated to determine the underlying release mechanisms. The correlation coefficient values for various models are listed in Table 4. MEBG4, PDS, and MES4 followed the Korsmeyer–Peppas (KP) model while MEBG5 and MEBG6 followed zero order release kinetics. MEBG4, PDS, and MES4 followed the Korsmeyer–Peppas (KP) model which usually illustrates a drug’s release when the release mechanism is unknown or more than one type of release mechanism is involved. This is an amalgamation of two independent release mechanisms: one process mechanism is related to the transport of drugs in a way that obeys Fick’s law, or Fickian transport, and the second process is a result of the polymer swelling/relaxation phenomenon, which is due to the Case-II transport mechanism.⁴⁴ This model considers both mechanisms, drug diffusion as well as polymer relaxation.^45,46

Table 4 An overview of the different kinetics models followed by various formulations

Formulation code	r^2 values						Best fit model
	Zero-order (ZO)	First-order (FO)	Hixson–Crowell	Higuchi	Korsmeyer–Peppas (KP)
					r^2	Slope (n)
MEBG4	0.980	0.977	0.928	0.969	0.981	0.766	KP
MEBG5	0.991	0.973	0.969	0.954	0.986	0.862	ZO
MEBG6	0.984	0.974	0.965	0.938	0.952	0.957	ZO
Drug suspension	0.980	0.979	0.924	0.974	0.991	1.311	KP
MES4	0.982	0.940	0.916	0.964	0.996	0.642	KP

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The drug release exponent values (n) are utilized to illustrate the different types of release mechanisms. According to literature, an n value of up to 0.50 indicates that drug release from the system occurs by a Fickian diffusion mechanism, and n = 1.00 indicates zero order kinetics, i.e., controlled release by relaxation of the polymer chains or erosion (Case II transport); an ‘n’ value higher than 1 indicates super Case-II transport; and an n value between 0.50 and 1.00 indicates anomalous transport or a combination of both diffusion mechanisms and Case II transport.^45–48

All of the prepared microemulsion based Carbopol gels (MEBG4 to MEBG6) and MES4 as shown in Table 4 showed ‘n’ values between 0.50 and 1.00 except the drug suspension, which indicates anomalous transport or a combination of both diffusion mechanisms and Case II transport. These results were similar to previously reported studies.^49,50

Thus, it can be concluded that the prepared gels promote the release of SIM and the drug release kinetics the from gels are dependent on the Carbopol concentration. Furthermore, the drug release mechanism may be diffusion associated or not associated with gel matrix relaxation. The network complexity, viscosity of the prepared gel, diffusion and dissolution of the drug and the swelling/relaxation of the polymer matrix gel constitute a barrier to the release of the drug. Hence, drug permeation from the gel system (MEBG4 to MEBG6) is sustained when compared with microemulsion (MES4). Based on the different parameters such as the physicochemical analysis, viscosity, in vitro mucoadhesion force, and ex vivo permeation study of the prepared microemulsion based buccal gels, MEBG4 was selected for further evaluations such as texture analysis, rheology, stability studies, histopathology of the buccal membrane and in vivo pharmacokinetics study.

3.5 Texture analysis

Texture analysis was used to imitate the human sensorial interpretation in terms of firmness, spreadability, adhesiveness and extrudability while applying semisolid formulations such as gels, creams etc. on skin.^28,51 These parameters are jointly used to assess the texture profile of the gel formulations for buccal delivery. The texture profile of MEBG4 was analyzed and compared with the gel base (BG contains no microemulsion). The values of the estimated parameters are shown in Table 5. The firmness of MEBG4 and BG were found to be 11.0 g and 16.0 g, respectively. Firmness is the maximum positive force entailed to deform the gel sample by a finger. BG was found to be reasonably harder than MEBG4 implying that BG needed a somewhat higher force to deform its surface. The firmness of MEBG4 was found to be less than that of the BG gel, which could be attributed to the presence of the microemulsion in MEBG4. Spreadability and extrudability are the amounts of work done or energy (mJ) required to penetrate a sample of cream or gel up to a definite depth when applied to the surface and to uniformly extrude out from the packaging tube.⁵² Spreadability is an imitation of human perception of spreading any semi-solid over the skin surface. It depends on the ingredients used in the formulation of cream, ointments or gels. Spreadability and extrudability of semisolid formulations are the imperative texture parameters of an organoleptically aesthetic product.²⁸ The spreadability of MEBG4 (0.8 mJ) was observed to be better than that of BG (1.4 mJ). The work needed to extrude out i.e. extrudability of MEBG4 and BG gels from the tube was found to be 35.2 mJ and 25.5 mJ respectively.

Table 5 Texture profile analysis of blank gel (BG) and optimized MEBG4

Texture profile parameters	Formulations
	BG	MEBG4
Firmness (g)	16.0	11.0
Spreadability (mJ)	1.4	0.8
Extrudability (mJ)	25.5	35.2
Adhesiveness (g)	2.7	2.9

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The adhesiveness of the gel formulation is an important parameter for buccoadhesive character and is defined as the maximum force needed to overcome the attractive force between the surface and the formulation, and it is a measure of the stickiness of the sample.^28,53 The adhesiveness of the MEBG4 and BG gels was found to be 2.9 g and 2.7 g, respectively. Finally, the texture analysis unveiled that the SIM-loaded MEBG4 gel has good gel strength, adequate adhesiveness, and ease of spreading, which are necessary for application and retaining the formulation in the buccal cavity.

3.6 Rheological studies

Pharmaceutical formulations must encompass appropriate rheological properties. These properties are described as gel formulation efficacy, stability, and the consumer’s acceptability.

Sudhakar et al.³⁵ reported that a pseudoplastic or plastic property for a bio/mucoadhesive formulation for buccal application is required for suitable rheological behavior.

The MEBG4 gel showed pseudoplastic behavior as is shown in Fig. 3a and b, in which the increased shear rate resulted in decreased viscosity. The pseudoplastic behavior of a gel system is due to the presence of long molecules and the high molecular weights of the polymer. In solution form, these polymer molecules become entrapped with the immobilized solvent which results in high viscosity and shows flow resistance.

Fig. 3 Rheological behavior of MEBG4 gel (a) pseudoplastic behavior, (b) effect of shear rate on viscosity.

Upon application of shear, the molecules tend to become extricated and align in the direction of the flow and therefore show less flow resistance, with release of the entrapped water, consequently, the viscosity decreases as is shown in Fig. 3b; upon removing the shear stress, a structural reorganization of the system occurs and the viscosity increases hence this process is reversible. For that reason, a pseudoplastic behavior is appropriate for buccal delivery purposes, because as the shear stress increases the viscosity decreases, assisting in the spreading of formulations over tissue.⁵⁴

3.7 Stability study

The color, consistency, pH, phase separation and drug content of the MEBG4 gel were found to be stable and no signs of phase separation or deterioration of drug formulation were observed over a period of 180 days (Table 6). The results indicated the reproducibility of the chemical and physical parameters which ensures a consistent quality of the prepared gel formulation (MEBG4) over a longer time.

Table 6 Stability studies of optimized buccal gel MEBG4

Formulation parameters	Days
	0	30	60	120	180
a Based on sensorial analysis.b Determined after one freeze–thaw cycle.
Color^a	Acceptable	Acceptable	Acceptable	Acceptable	Acceptable
Consistency^a	Acceptable	Acceptable	Acceptable	Acceptable	Acceptable
Phase separation^b	Nil	Nil	Nil	Nil	Nil
pH^b	7.09 ± 0.012	7.08 ± 0.016	7.05 ± 0.022	6.99 ± 0.02	6.98 ± 0.025
Drug content^b (mg g^−1)	9.88 ± 0.035	9.89 ± 0.028	9.80 ± 0.026	9.84 ± 0.04	9.83 ± 0.034

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3.8 Histopathological evaluations

The goat buccal mucosa treated with the MEBG4 gel in comparison with the control established the safety of the prepared formulation with no noticeable histological changes demonstrating the safety of the formulation for buccal use. Fig. 4 shows histological sections of the goat buccal mucosa.

Fig. 4 Histopathological studies (a) control (b) MEBG4 gel treated.

The histological pictures showed that all of the epithelial sections had been successfully detached from the connective tissues with an intact morphology and integrity and no histological changes were observed in formulation treated buccal mucosa when compared with the control.

3.9 In vivo pharmacokinetics study

The plasma concentration and time profile curve to compare the plasma level profiles of SIM after oral administration of the marketed tablet and the microemulsion based buccoadhesive gel (MEBG4) formulations are shown in Fig. 5 and the pharmacokinetics parameters are presented in Table 7. The buccoadhesive gel formulation (MEBG4) was applied to both sides of the cheeks of albino Wistar rats. A dose of marketed tablet (1 mg) as the oral suspension was given to the rats to compare the difference in pharmacokinetics parameters with the buccoadhesive gel (equivalent to 1 mg drug dose).

Fig. 5 Mean plasma concentration (mean ± SD, n = 6) and time curve of simvastatin after buccal administration of the microemulsion based MEBG4 gel and marketed tablet (orally).

Table 7 Pharmacokinetics parameters (mean ± SD, n = 6)

Pharmacokinetics parameters	Marketed tablet	MEBG4
a P < 0.001, statistically significant enhancement of Cmax, AUClast and AUMClast of the optimized MEBG4 as compared to the marketed tablet.
Tmax (h)	2	4
Cmax (ng ml^−1)	68.513 ± 9.821	131.208 ± 21.662^a
AUClast (ng h ml^−1)	279.339 ± 30.416	1076.319 ± 97.648^a
MRTlast (h)	3.899	6.460
AUMClast (ng h^2 ml^−1)	1089.366 ± 119.436	6953.435 ± 308.436^a

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The plasma drug profile indicates that the application of buccoadhesive gel delayed simvastatin delivery and increased the duration of absorption, which in turn increased the absorption of simvastatin into the body when compared to the oral suspension of the drug. The C_max value (131.208 ± 21.563 ng ml⁻¹) of the buccoadhesive gel (MEBG4) was found to be significantly higher (P < 0.001) when compared with the same dose distributed by the oral route C_max (68.513 ± 9.821 ng ml⁻¹).

This could be due to the permeability of the drug increasing through the capillary vessels present in the buccal cavity, thus the drug directly enters into the systemic circulation, and also due to the prevention of drug degradation through the intestine and liver.^12–15 However, the T_max value was found to be significantly increased following buccal gel delivery (4 h) when compared to the oral marketed tablet (2 h). This could be due to the presence of a polymeric gelling agent which sustained the drug release in the buccal cavity.

The area under the curve (AUC) for the optimized MEBG4 buccal gel showed a 2.63-fold increment when compared with the AUC obtained after oral administration of MES4.¹¹ While, at the same time, the AUC_last via the buccal route showed a 3.853-fold increment when compared with the oral marketed tablet (Table 7). The results showed significant differences as indicated by the p values < 0.001, representing enhanced bioavailability through the buccoadhesive gel when compared with MES4 given orally¹¹ and the marketed tablet also given orally.

The enhancement in the AUC value (in the case of buccal gel) signifies the increment in rate and extent (relative bioavailability) from the buccal gel when compared to the orally administered MES4 and marketed tablet.

Similarly, C_max for MEBG4 (131.208 ± 21.662) was also found to be higher when compared to the orally administered tablet formulation (68.513 ± 9.821 ng ml⁻¹) and MES4 (107.84 ± 8.95).¹¹

The relative bioavailability of the MEBG4 buccal gel was 385.30% higher with respect to the orally administered marketed tablet while it was 263% higher when compared to the orally administered MES4.

The increment in relative bioavailability, AUC and C_max could be due to the fact that drug molecules, when absorbed through the buccal route due to the presence of the rich blood vasculature of the buccal oral mucosa, directly enter into the systemic circulation, and also could be due to the avoidance of drug degradation through the intestine and liver. Another reason for the enhancement in AUC and C_max may be that the buccal gel (MEBG4) enhanced the permeability of the buccal mucosa due to the presence of MES4 which contains a surfactant mixture (Kolliphor EL40 and Transcutol HP). The presence of mucoadhesive polymer enabled intimate contact with lipophilic buccal mucosal layers resulting in an increased concentration of the drug in the blood.

The mean residence time (MRT_last) estimates the average time a drug molecule spends in the body. It can be used to interpret the duration of effect for drug molecules. As is observed from Table 7, the MRT value for MEBG4 (MRT-6.460) was 1.6 times higher than the marketed formulation (MRT-3.899). Hence, the sustained effect of the buccal gel is documented when compared with the marketed formulation.

Hence, a buccal gel containing a drug loaded microemulsion could be a good choice for circumventing first pass metabolism and intestinal degradation, and for enhancing the bioavailability of poorly water-soluble drugs.

4. Conclusion

A microemulsion based buccal gel was successfully developed and evaluated for the systemic delivery of SIM through the buccal route. A buccoadhesive gel containing a SIM loaded microemulsion was evaluated for physicochemical parameters, ex vivo and in vivo performances which showed the formation of a consistent, stable and effective delivery system. The results of the permeation study demonstrated the role of the microemulsion for effective buccal permeation of SIM via the buccal route. The pharmacokinetics studies showed the adeptness of the prepared gel toward the proficient absorption of SIM through buccal mucosa as shown by the pharmacokinetics parameters viz. AUC, C_max, T_max, MRT etc. The future perspective includes elaborate clinical and stability studies for developing a microemulsion based bucco/mucoadhesive formulation of SIM for commercial purposes. It was finally concluded that the buccal gel containing the drug loaded microemulsion could be a good choice for circumventing first pass metabolism and intestinal degradation, and also for enhancing the bioavailability of poorly water-soluble drugs.

Conflict of interest

The authors warrant that there is no conflict of interest.

Acknowledgements

The authors gratefully acknowledge the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh (UT), India, for providing their facilities for the evaluation of the formulation. The authors also acknowledge IPCA Pharmaceuticals Ltd., Mumbai, India for gift samples of SIM and Carbopol 71G.

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