Abstract
The objective of the present investigation was to develop and characterize the self-nanoemulsifying drug delivery system (SNEDDS) of glimepiride, a poorly soluble drug. Solubility of glimepiride in various vehicles was determined, and ternary phase diagrams were constructed using a suitable oil, surfactant, and cosurfactant system to find out the efficient self-emulsification system. A three factor, three level Box-Behnken statistical design was employed to explore the main and interaction effect of independent variables, namely X1 (amount of Capmul MCM), X2 (amount of Acrysol K 140), and X3 (amount of Transcutol P). Percent transmittance value (Y1), droplet diameter (Y2), and percent drug released at 5 min (Y3) were the dependent variables. Formulation optimization was carried out to optimize the droplet diameter and percent drug dissolved at 5 min. The batch prepared according to the optimized formulation showed a close agreement between observed and predicted values. Box-Behnken statistical design allowed us to understand the effect of formulation variables on the rapid dissolution of drug from SNEDDS and to optimize the formulation to obtain a rapid drug dissolution at 5 min.
LAY ABSTRACT: A self-nanoemulsifying drug delivery system of glimepiride has been design, developed, and optimized. A three factor, three level Box-Behnken statistical design was employed to explore the main and interaction effect of independent variables, namely X1 (amount of Capmul MCM), X2 (amount of Acrysol K 140), and X3 (amount of Transcutol P). Percent transmittance value (Y1), droplet diameter (Y2), and percent drug released at 5 min (Y3) were the dependent variables. The Capmul MCM–Akcrysol K 140–Transcutol system was found to be the suitable ternary system that was able to release almost 80% of drug within the first 5 min. The improved dissolution of glimepiride might improve patient compliance.
1. Introduction
Glimepiride (GMP), [1- (p- (2- (3-ethyle-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl) phenyl) sulfonyl)-3-(trans-4-methylcyclohexyl)], Figure 1, is a potent sulfonylurea with established potential benefits such as lower dose, rapid onset, lower insulin levels and less-pronounced glucagonotropic effects, and insulin-sensitizing and insulin-mimetic affects. GMP lowers blood glucose level in patients with type 2 diabetes by directly stimulating the acute release of insulin from functioning beta cells of pancreatic islet tissue by binding to the SUR1 subunits and blocking the ATP-sensitive K+ channel (1). However, GMP is a poorly soluble drug (<0.004 mg/mL in acidic and neutral media) with relatively high permeability through CaCo-2 cell monolayers (2), which warrant it to be classified under Biopharmaceutical Classification System (BCS) Class II classification. It is effectively absorbed from the gastrointestinal tract; however, the presence of food, certain dietary supplements, or hyperglycemia can interfere with its dissolution and in turn its absorption (3), and hence in view of the time required to reach an optimal concentration in plasma, GMP may be more effective if given 30 min before a meal (4). Conversely, this might reduce patient compliance because, if after taking the drug the patient is not able to have the meal it would result in severe hypoglycemia, and if taken with a meal then food sequentially would interfere with its absorption. Hence, improving the dissolution characteristics of GMP might allow concomitant dosing of the drug with food. Various researchers have tried to improve the dissolution characteristics of GMP by various traditional techniques such as addition of surfactants (5), cosolvency (6), solid dispersions and micronization (7), cyclodextrin complexation (8), and others, which could improve its clinical efficacy.
Glimepiride.
Recently, self-nanoemulsifying drug delivery systems (SNEDDS) have been formulated to offer improved dissolution, which could provide a better control over the bioavailability of drug. SNEDDS are isotropic mixtures of oil, surfactants, and cosurfactants that form fine oil-in-water nanoemulsions upon mild agitation, followed by addition into aqueous media, such as gastrointestinal fluids (9). Nanoemulsions (NEs) consist of very small oil droplets in water, exhibiting sizes lower than 300 nm. Like conventional emulsions (with sizes >μm), NEs are, from a thermodynamic point of view, in a non-equilibrium state. However, the kinetics of destabilization of NEs is so slow that they are considered kinetically stable formulations that are easier to manufacture, and they may offer an improvement in dissolution rates and extents of absorption, resulting in more reproducible blood–time profiles due to the nanometer-sized droplets present, which in turn might reduce the fed and fasted variation in the in vivo performance of the drug (10). Surprisingly, no such system has been reported to improve the dissolution characteristics of GMP, which could improve its therapeutic efficacy, lower the dose, and allow concomitant dosing with food. In light of all this, the present investigation was carried out to develop, characterize, and optimize SNEDDS of GMP, to improve the dissolution characteristics of GMP. Preliminarily the ternary phase diagrams were constructed and Box-Behnken design (BBD) was adopted and formulation components were optimized to improve the release of GMP from SNEDDS.
2. Materials and Methods
2.1. Materials
GMP BP was provided as a gift sample by Cadila Pharmaceuticals (Ahmedabad, India); Tween 80 (polyoxyethylene 20 sorbitan monooleate HLB [hydrophyl lipophyl balance] value 15), Tween 60 (polyoxyethylene 20 sorbitan monostearate HLB value 14.9), ethyl oleate, polypropylene glycol (PPG), and polyethylene glycol 400 (PEG 400) were purchased from S. D. Fine Chemicals Limited (Ahmedabad, India). Capryol 90 (propylene glycol monocaprylate, HLB value 6), Labrasol (caprylocaproyl macrogolglycerides, HLB value 14), Labrafac PG (PPG dicaprylocaprate, HLB value 2), Lauroglycol 90 (PPG monolaurate, HLB value 5), and Transcutol P (TP) (diethylene glycol monoethyl ether) were provided by Gattefosse (Mumbai, India) as gift samples. Capmul MCM (CMCM) (glyceryl mono/dicaprylate) was provided as a gift sample from Abitec Corporation (Janesville, WI, USA). Acrysol K 140 (AK 140) (PEG 40 hydrogenated castor oil, HLB value 16) was provided as a gift sample by Corel Pharmachem Ltd. (Ahmedabad, India) and was used after gentle heating at 40–50 °C for 2 min. Size 0 hydroxypropyl methyl cellulose (HPMC) empty capsule shells were received as a gift sample from Amneal Pharmaceuticals Limited (Ahmedabad, India). Double-distilled water was used throughout the study. All the reagents and chemicals used were purchased from S. D. Fine Chemicals Pvt. Ltd.
2.2. Determination of Solubility of GMP
Solubility of GMP was investigated in various buffers, oils, and surfactant solutions (10% w/w) using shake flask method. Briefly, an excess amount of GMP was added to 5 gm of the vehicle. The flasks were then sealed with parafilm and vortexed (Genei, Bangalore, India) for 10 min in order to facilitate the mixing of the drug with the vehicle. Mixtures were then shaken for 3 days in rotary shaker (Remi Instruments, Mumbai, India), maintained at 25 °C and then centrifuged at 4800 rpm (Remi Instruments) for 20 min. The supernatant was then filtered using Whatman filter paper (0.22 μ) and diluted suitably with double-distilled methanol, and the GMP dissolved in various vehicles was measured spectrophotometrically (UV spectrophotometer, Shimadzu 1700) at 228 nm (11).
2.3. Screening of Surfactants for Emulsifying Ability
The emulsification ability of various surfactants was screened to find out the most suitable surfactant system for the selected oily phase. Briefly, 300 mg of selected surfactant was added to 300 mg of selected oil phase. The mixture was then vortexed for 60 s to facilitate the mixing of oil and surfactant. One hundred milligrams of this isotropic system was weighed accurately and diluted to 25 mL of distilled water to yield a fine emulsion. The ease of emulsification was monitored by noting the number of volumetric flask inversions required to give uniform emulsion (12). The formed emulsions were then evaluated for their transmittance at 638.2 nm by UV spectrophotometer, using double-distilled water as blank.
2.4. Screening of Cosurfactants
The cosurfactants have ability to improve the nanoemulsification efficiency. The relative effectiveness of cosurfactants was evaluated by adding 200 mg of surfactant with 100 mg of cosurfactant, and to this 300 mg of Capmul MCM C8 (CMCM) was added and vortexed to homogenize the mixture. The isotropic system was weighed accurately and diluted with double-distilled water to yield a fine emulsion. The ease of emulsification was monitored by noting the number of volumetric flask inversions required to give uniform emulsion. The formed emulsions were then evaluated for their transmittance at 638.2 nm by UV spectrophotometer, using double-distilled water as blank.
2.5. Construction of Ternary Phase Diagrams
The first step towards the formulation development was to identify the self-nanoemulsifying region. To identify the self-nanoemulsifying region, ternary diagrams of oil, surfactant, and cosurfactant were prepared, each representing the apex of the triangle (13). Ternary mixtures with varying composition of oil, surfactant, and cosurfactant were prepared. The oil composition was varied from 25% w/w to 70% w/w, surfactant concentration was varied from 25% w/w to 75% w/w, and the cosurfactant concentration was varied from 0% w/w to 30% w/w. For all mixtures the total of oil, surfactant, and cosurfactant was always 100%. Briefly, the oil, surfactant, and cosurfactant were mixed and vortexed for 60 s to facilitate homogenization, and the efficacy of nanoemulsion formation was assessed by diluting 100 mg of the mixture up to 25 mL of double-distilled water followed by gentle agitation manually. The formulations were assessed for ease of emulsification and visual appearance (12). Only clear or slight bluish dispersions of droplet size less than 200 nm were considered in the nanoemulsion region of the diagram.
3. BBD Experimental Design
BBD was used to optimize and evaluate the main effects, interaction effects, and quadratic effects of the formulation ingredients of GMP-loaded SNEDDS. Fifteen experiments were generated by Minitab 16.0 (Minitab Inc., State College, PA, USA) for the response surface methodology based on the BBD at three factors and three level designs. This design is suitable for exploring quadratic response surfaces and constructing second-order polynomial models. The design consists of replicated center points and the set of points lying at the midpoint of each edge of the multidimensional cube. Three independent variables were selected based on the preliminary screening: the oil phase CMCM (X1), the surfactant AK140 (X2), and cosurfactant TP (X3). The dependent parameters consisted of percent transmittance value (Y1), droplet diameter (Y2), and percent drug released at 5 min (Y3).
4. Preparation of GMP-Loaded SNEDDS
The GMP-loaded SNEDDS were prepared by mixing oil phase CMCM, surfactant AK140, and cosurfactant TP and warming it at 40 °C, then GMP was added to the mixture and vortexed to facilitate the uniform dispersion of GMP. The mixture was then allowed to equilibrate at room temperature. Fifteen such experiments were carried out according to the experimental design with varying concentration of oil, surfactant, and cosurfactant, with the final GMP loading equivalent 10 mg/gm. The prepared GMP-loaded SNEDDS were filled into size 0 HPMC capsule shells.
5. Optimization of Formulation Components
The formulation components were optimized statistically by Minitab 16 subsequent to generating the polynomial equations concerning the dependent and independent variables, and optimization of Y2 (droplet diameter) and Y3 (percent drug released at 5 min) was performed using a desirability function to obtain the levels of X1, X2, and X3, which maximized Y3 and minimized Y2.
6. Characterization of GMP-Loaded SNEDDS
6.1. Droplet Diameter and Zeta Potential Measurement
Droplet diameter and size distribution and zeta potential of SNEDDS were determined using Zetatrac (Microtrac Inc., Montgomeryville, PA, USA). Zetatrac utilizes a high-frequency, alternating current electric field to oscillate the charged particles. The Brownian motion power spectrum is analyzed with the modulated power spectrum technique, a component of power spectrum resulting from oscillating particles. One hundred milligrams of sample was diluted to 25 mL of purified water. Diluted samples were directly placed into a cuvette for measurement of droplet size and zeta potential (14).
6.2. In Vitro Release Studies
Four hundred milligrams of formulation containing 4 mg of GMP were filled in size 0 HPMC capsules. The release of GMP from the capsules filled with SNEDDS formulation was carried out in USP dissolution test apparatus at a rotating speed of 75 rpm in 100 mL pH 7.8 phosphate buffer for a period of 30 min (15). Aliquots of 5 mL each were withdrawn at selected time interval and were filtered through Whatman filter paper (0.22 μ) and replaced with an equivalent amount of fresh dissolution medium. The concentration of GMP was measured spectrophotometrically at 228 nm using the regression equation of standard curve developed in the same range in the linearity range of 2–18 μg/mL.
6.3. Determination of the Emulsification Time
In order to determine the emulsification time (the time needed to reach the emulsified and homogeneous mixture, upon dilution), 100 mg of each formulation was added to 25 mL of pH 7.8 buffer at 37 °C with gentle agitation using magnetic stirrer. The formulations were assessed visually according to the rate of emulsification and the final appearance of the emulsion.
6.4. Statistical Evaluation
The experimental data obtained were validated by analysis of variance (ANOVA) combined with the F-test. The determination coefficient (R2, agreement between the experimental results and predicted values obtained from the model) and the model F-value (Fisher variation ratio, the ratio of mean square for regression to mean square for residual) were applied for statistical evaluation.
7. Results and Discussion
7.1. Solubility Studies
The solubility studies of GMP in various oils, surfactants, and cosurfactants were carried out to identify the suitable oil and surfactant–cosurfactant system to develop an appropriate delivery system that is able to dissolve the total dose of the drug. Various oils with different degrees of saturation were used. As shown in Figure 2, the solubility in oils did not showed any specific pattern, and it was seen that amongs the various assorted oils, GMP showed highest solubility in CMCM, which was then selected as the oily phase, followed by Capryol 90 and Lauroglycol 90. The selection of surfactant and cosurfactant was subsequently done based on their ability to emulsify the oil.
Solubility of GMP is various oils (the data are expressed as average ± standard deviation, n = 6).
7.2. Screening of Surfactants for Emulsifying Ability
Non-ionic surfactants are often considered for pharmaceutical applications and self-nanoemulsifying formulation because these are less toxic and less affected by pH and ionic strength changes. Thus, for the present study, AK 140, Tween 80, and Labrasol were used as surfactants having a higher HLB value. The surfactants were evaluated and compared for their emulsification ability using CMCM and Capryol 90 as the oily phase, as a well formulated self-emulsifying drug delivery system should emulsify within very little time. The transmittance values for different surfactants are shown in Figure 3. The results clearly indicate that AK140 showed the highest emulsification efficiency, requiring only single flask inversions for homogenous emulsion formation with 99.7% transmittance, suggesting the formation of a very fine emulsion. The emulsification efficiency of AK140 was followed by Tween 80, whereas Labrasol was poor emulsifier for GMP. The surfactants showed a wide variation in their emulsification ability even though they had similar HLB values; this could be ascribed to their difference in chain length and structure (12).
Percent transmittance value of various surfactants and cosurfactants.
7.3. Screening of Cosurfactants
As shown in Figure 3 all the cosurfactants—TP, PEG 400, PPG—improved the emulsification ability of AK 140 and Tween 80, increasing the spontaneity of the nanoemulsion formation in varying proportion. Amongs the various assorted cosurfactants, TP showed most promising results and was able to reduce the liquid crystal structures generally observed with AK140, which could be due to the high solving power of TP (13).
7.4. Construction of Ternary Phase Diagrams
Ternary phase diagrams were constructed to identify the self-nanoemulsifying region and to select a suitable concentration of oil, surfactant, and cosurfactant for the development of the SNEDDS. On the basis of preliminary studies, CMCM was selected as the oily phase; Tween 80, Labrasol, and AK 140 were selected as surfactants; and TP was selected as cosurfactant. The phase diagrams of CMCM–Tween 80–TP, CMCM–Labrasol–TP, and CMCM–AK 140–TP are shown in Figures 4a, 4b, and 4c, respectively. The outer parallelogram indicates the area that was explored for locating the nanoemulsification region. The filled circles with a green border indicate the formulation in which nanoemulsions with desired attributes were obtained. From the figure it is clear that CMCM–AK 140–TP showed maximum nanoemulsion region compared to the other systems evaluated, which could be due to the reason that (as seen from the preliminary studies) AK 140 showed maximum emulsification efficiency and was able to emulsify the oil efficiently, which could be attributed to the 18-carbon fatty acid of AK 140, which could help AK 140 molecules insert into the CMCM drop surface uniformly. In addition, the polyethylene glycol structure in both of them could bind together through noncovalent interactions such as hydrogen bonding, which made the combination of oil and surfactant much tighter (16). TP has similar diethylene glycol structure, which could bind with their polyethylene glycol structures to stabilize the nanoemulsion further. Furthermore, co-surfactant increases interfacial fluidity by penetrating into the surfactant film, creating void space among surfactant molecules. As Labrasol was an 8–10 carbon fatty acid, and Tween 80 had no polyethylene glycol structure, they could not emulsify with CMCM and TP as effectively as AK 140. Hence it was decided to load the CMCM–AK 140–TP ternary system with GMP.
The ternary phase diagrams of (a) CMCM–Tween 80–TP, (b) CMCM–Labrasol–TP, and (c) CMCM–AK 140–TP.
7.5. BBD
BBD was used to optimize and evaluate the main effects, interaction effects, and quadratic effects of the formulation ingredients of GMP-loaded SNEDDS. The drug content was kept constant, equivalent to 10 mg/gm of the prepared SNEDDS. Based on the previous results obtained from the ternary phase diagram, the range of each component was selected as follows: X1 (200–400 mg of CMCM), X2 (200–300 mg of AK 140), and X3 (75–125 mg of TP). The percent transmittance of diluted SNEDDS (Y1), mean droplet size (Y2), and cumulative amount of drug released after 5 min (Y3) were selected as the responses and shown in Table I. The observed responses showed a large variation, suggesting that the independent variables had a significant effect (P < 0.05) on the selected dependent variables.
Layout and Observed Responses of Box-Behnken Experimental Design Batches (Average ± Standard Deviation, n = 6)
The coefficients in Table II represent the respective quantitative effect of independent variables (X1, X2, and X3) and their interactions on the response (Y3). It was seen that all the independent variables had a significant effect on the response (P < 0.05). The negative sign of the coefficient indicated that increase in the value of independent variable decreases the value of response, and vice versa. The absolute value of the coefficient indicates the magnitude of effect of the independent variable on the response; the higher the value, the higher the magnitude. The relationship between the dependent and independent variables was further elucidated using response surface plots. All the determination coefficients R2 are larger than 0.9, indicating that over 90% of the variation in the response could be explained by the model and the goodness-of-fit of the model was confirmed. The F- ratio was found to be far greater than the theoretical value with very low probability of less than 0.0001 for each regression model, indicating that the regression model is significant with a confidence level of 95%.
Statistical Analysis of Box-Behnken Experimental Design Batches
The effect of X1 (CMCM) and X2 (AK 140) at the middle level of X3 (TP) is shown in Figure 5a. It is evident from the figure that at lower level (−1) of CMCM, with increase in AK140 from lower level (−1) to higher level (+1), a decrease in the drug release was seen from 81% to 65%. AK 140 at the lowest level was able to emulsify the oil efficiently, and further increase in the concentration of AK 140 lead to decrease in the drug release, which could be attributed to the formation of liquid crystal. Visually also it was seen that at higher levels of AK 140, the SNEDDS preconcentrate did not dispersed rapidly in the dissolution medium; moreover, liquid crystals formed might be responsible for decrement in the drug dissolution by increasing the viscosity of the system, prolonging the time required for phase transition, and finally decreasing drug dissolution. While as CMCM was increased from lower level (−1) to higher level (+1), a moderate decrease in the drug release was seen from 79.26% to 72.58%, which could be attributed to the fact that, as shown in Table III, the solubility of GMP was as follows: AK 140 > TP > CMCM. So when the concentration of CMCM was increased, the total amount of AK 140 decreased, which resulted in increased droplet diameter, and in turn a decrease in total surface area of the emulsifying membrane layer, which might defer drug dissolution (16).
Response surface plot of (a) AK140 and CMCM vs percent drug released at 5 min, (b) AK 140 and TP vs percent drug released at 5 min, and (c) TP and CMCM vs percent drug released at 5 min.
Solubility of GMP in Various Surfactants (Average ± Standard Deviation, n = 6)
The effect of X2 (AK 140) and X3 (TP) at the middle level of X1 (CMCM) is shown in Figure 5b. It was seen that at all levels of TP, there was a decrease in the dissolution of GMP with increase in AK140 from lower level (−1) to higher level (+1), which could be due to the formation of liquid crystals at higher concentration of surfactant (17). It was also seen that when TP was increased from lower level (−1) to higher level (+1), at all level of AK 140, an increase in drug dissolution was seen, which could be ascribed to the fact that higher level of TP would reduce the formation of crystal structure, which would facilitate the dissolution of GMP. Alcohols, when used as cosurfactants, had been found to destroy liquid crystal phases (18), and traditionally Transcutol P had been shown to increase surfactant chain disorder, loosen the packed structure of interface, and increase the mobility of both water and surfactant chains, which resulted in the reduced viscosity of the liquid crystal (19).
The effect of X1 (CMCM) and X3 (TP) at the middle level of X2 (AK 140) is shown in Figure 5c. It was seen that at lower level (−1) of CMCM, an increase in TP from lower level (−1) to higher level (+1) and an increase in the dissolution from 72.25% to 79.26% was seen. It is apparent that only that part of GMP that has been nanoemulsified is detected; moreover, as seen from the preliminary studies, the solubility of GMP was more in the TP as compared to CMCM, and the GMP solubilized in the cosurfactant may dissolve into the medium rapidly even without the help of formation of SNEDDS.
As shown in Figure 6, a high correlation (R2 = 0.9587) was obtained between droplet diameter and percent drug released. As shown in batch F1, droplet diameter 92 nm showed the lowest drug release at 5 min, that is, 59.24%, while formulation F14, having a droplet diameter of 25 nm, showed almost 100% drug release within 5 min. Droplet size, which generally depends upon the formulation excipient, is a significant variable for characterizing SNEDDS. The increased drug released with smaller droplets could be attributed to the reason that a larger interfacial area is available with decreased droplet diameter.
In vitro dissolution profiles of BBD batches (the data are expressed as average ± standard deviation, n = 6).
Based on the equation obtained from BBD, optimization of formulation components was carried out to prepare a SNEDDS that had 25 nm < Y2 < 40 nm and 60% < Y3 < 85%. The desirability function was applied using response optimizer and an optimal D of 0.9943 was obtained at respective levels of X1 (365 mg), X2 (200 mg), and X3 (118 mg), as shown in Figure 7. A final formulation was prepared as per levels obtained in optimization and as shown in Table IV, and as shown in Figure 8 the droplet diameter of the optimized formulation was found out to be 34.10 nm, which was in close agreement with expected value, while as shown in Figure 9 the optimized batch showed significantly (P < 0.001) higher drug release as compared to pure GMP. The zeta potential of the optimized formulation was found to be −42.17 mV, which suggests that no aggregation of droplets took place. The final formulation was stored for 6 months at room temperature and characterized for its stability, and results confirmed that the formulation did not showed any changes during storage.
Optimization plot of GMP-loaded SNEDDS.
Predicted and Observed Response for Optimized GMP-Loaded SNEDDS
Droplet diameter distribution of optimized GMP-loaded SNEDDS.
Comparison of in vitro dissolution profile of pure GMP and optimized GMP-loaded SNEDDS.
8. Conclusions
BBD was effectively employed to optimize the dissolution of GMP from SNEDDS containing CMCM 8–AK 140–TP. All the predetermined independent variables were found to affect the dependent variables from the resultant nanoemulsion. The optimum formulation prepared by response optimizer through desirability function provided a final formulation with D 0.9943, which released 79.85% of GMP within 5 min. The improved formulation could offer an improved drug delivery strategy that might allow concomitant use of GMP with food, which still needs to be correlated with in vivo studies.
Conflict of Interest Declaration
The authors declare that they have no competing interests.
Acknowledgements
The authors are thankful to Cadila Pharmaceuticals Limited, Gattefosse India Limited, Corel Pharmachem limited, and Amneal Pharmaceuticals Limited for providing respective gift samples.
- © PDA, Inc. 2013
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