Development and Characterization of Glimepiride Novel Solid Nanodispersion for Improving Its Oral Bioavailability

: Glimepiride is an antidiabetic drug which is one of the third generation sulfonylureas. It belongs to class II, according to the BCS (Biopharmaceutical Classiﬁcation System), which is characterized by low solubility and high permeability. The aim of this work was to formulate glimepiride as solid dispersion using water-soluble carriers to enhance its aqueous solubility and thus enhance its bioavailability. Nine formulations of glimepiride solid dispersion were prepared by a solvent evaporation technique using three di ﬀ erent carriers (mannitol, polyethylene glycol 6000, and β -cyclodextrin) with three di ﬀ erent drug carrier ratio (1:1, 1:3, and 1:6). Formulation variables were optimized using 3 2 full factorial design. The prepared formulations were evaluated for production yield, drug content, micromeritic properties, thermal analysis, in-vitro release, and in-vivo hypoglycemic e ﬀ ect. All prepared formulations showed high production yield ranged from 98.4 ± 2.8 to 99.8 ± 2.2% and high drug content in the range of 97.2 ± 3.2 to 99.6 ± 2.1%. The micromeritic properties revealed that all prepared glimepiride formulations showed good ﬂowability. The di ﬀ erential scanning calorimetry study revealed the presence of the drug in the more soluble amorphous form. In accordance with the results of in vitro release study, it was found that the solubility of glimepiride was increased by increasing the drug carrier ratio, compared with the pure form of the drug. It was found that F9 showed a high and rapid reduction in blood glucose levels in diabetic rats, which indicated the success of a solid dispersion technique in improving the solubility and hence the bioavailability of glimepiride.


Introduction
Type II diabetes mellitus, previously known as noninsulin-dependent diabetes or adult-onset diabetes, accounts for 90-95% of all diabetes cases [1]. It encompasses individuals who have relative insulin deficiency and have peripheral insulin resistance [2]. At least initially, and often throughout their lifetime, these individuals may not need insulin treatment to survive. Indeed, diabetes requires continuous medical care with multifactorial risk-reduction strategies beyond glycemic control [3]. dependent variable (Y 1 ). The independent variables were the type of carrier (X 1 ), drug carrier ratio (X 2 ), while the dependent variable was the percentage of drug released after 1 hr (Y 1 ). The dependent and independent variables are represented in Table 1. Table 1. Dependent and independent formulation variables and their levels according to 3 2 factorial design. PEG: polyethylene glycol.

The Preparation of GM Solid Dispersion
The solid dispersion of GM was prepared by a solvent evaporation technique [21]. The weighed amounts of GM and the carriers were dissolved in 10 mL of methanol in a round-bottomed flask. The solvent was allowed to evaporate using a Heidolph rotavap (Schwabach, Germany), rotated at 100 rpm at 25 ± 1 • C under 600 mmHg pressure until it is completely dry. Methanol was evaporated, leaving the solid dispersion of GM on the wall of a round-bottomed flask [22]. The prepared GM solid dispersions were collected and further dried in an oven at 40 • C for 24 h, then ground and kept in a desiccator over silica at 60% relative humidity at room temperature for further studies. The composition of the nine formulations of GM solid dispersion is represented in Table 2.
Where +1 is the higher level, −1 is the lower level, and 0 is the medium level for the independent variables.

The Production Yield of GM Solid Dispersion
The production yield of all prepared formulations of GM solid dispersion was estimated by the following equation [23]: The production yield % = weight of the collected solid dispersion Total weight of drug and carrier used × 100 (1)

The Drug Content Uniformity of GM Solid Dispersion
The drug content for all GM solid dispersion was calculated by dissolving an accurate weight of each formulation equivalent to 5 mg of GM in methanol. The methanolic solution of each formulation was subjected to analysis using a UV spectrophotometer (Shimadzu, Kyoto, Japan) at 228 nm after appropriate dilution [17]. The drug content for all formulation was measured through the following equation [24]: The drug content % = the actual drug content the theoretical drug content × 100

The Determination of Micromeritic Properties of GM Solid Dispersion Powders
The flow properties of solid dispersion are important in the pharmaceutical industry, especially in the blending of powders, compression of tablets, and the filling of capsules. Several parameters were used to measure the flow properties of the prepared solid dispersion, such as the bulk density, the tapped density, the angle of repose, Carr's index, and Hausner's ratio.

The Bulk Density
The bulk density of GM solid dispersion formulations was determined by transferring an accurate weight of each formulation (10 g) to a graduated cylindrical measure [25]. The volume of solid dispersion was noted in the cylinder measure without any compacting, and the bulk density was calculated by the following equation [25,26]: The Tapped Density The tapped density of GM solid dispersion was determined by transferring an accurate weight of each formulation (10 g) to a graduated cylindrical measure [27]. The cylinder was tapped until no further change in volume. The tapped density was calculated by the following equation [28]: Tapped density = weight of powder/Tapped volume (4)

Hausner's Ratio
Hausner's ratio of GM solid dispersion was determined to determine the flowability of the formulations [10]. Hausner's ratio was calculated by the following equation [29,30]: Hausner ratio = Tapped density/Bulk density (5) Carr's Index Carr's index is another parameter used to predict the flowability of powder [31]. Carr's index of the prepared GM solid dispersion was calculated by the following equation [32]: The Angle of Repose The angle of repose (θ) of GM solid dispersion powders was estimated using the fixed funnel method. An accurate weight of GM solid dispersion was allowed to pass through a fixed funnel adjusted to 1 cm above the horizontal plane. The passed powder was allowed to form a pile where the apex of the pile was contacted to the end of the funnel [33]. The diameter of the pile was measured, and the angle of repose was calculated by the following equation [34] tan θ = h/r (7) where h is the height of the pile, and r is the radius of the pile base.
2.2.6. The Differential Scanning Calorimetry (DSC) The thermal behavior of the pure GM powder, different carriers used (Mannitol, PEG 6000, and β-cyclodextrin), and the best three formulations: F3, F6, and F9 were determined using a differential scanning calorimeter (Shimadzu, Japan). The selected formulation F3, F6, and F9 were prepared using different carriers; Mannitol, PEG 6000, and β-cyclodextrin, respectively. These formulations were selected based on the highest drug: carrier ratio because it is expected that if there is any interaction between the drug and the carrier, it will be greater in case of a higher ratio. The measurements were done over a temperature range from 0-300 • C under nitrogen purge at 30 mL/min and a scanning rate of 10 • C/min [35,36]. The reference material used in the analysis was pure Indium (In).

The Infrared Spectroscopy Analysis (IR)
The infrared spectroscopy of the pure GM and carriers (Mannitol, PEG 6000, and β-cyclodextrin), F3, F6, and F9 was determined by subjecting the samples to Fourier-Transform Infrared Spectroscopy (FT-IR) using Shimadzu 435 U-O4 IR spectrometer (Tokyo, Japan). The infrared spectroscopy was conducted to ensure the compatibility between the formulation ingredients used in the preparation of GM solid dispersion. Each sample was mixed with potassium bromide and mechanically compressed into a disc [37]. The infrared spectroscopy was measured for the disc of each sample over a wavelength scanning range of 4000 cm −1 to 400 cm −1 [38].

The In-Vitro Release Study of GM from Solid Dispersion
The dissolution study of GM from the prepared solid dispersion was done using an ERWEKA dissolution tester, apparatus II (Erweka, Heusenstamm, Germany). An accurate weight of GM (2 mg) and each solid dispersion formulation equivalent to 2 mg of GM was placed in 900 mL of phosphate buffer (pH 6.8) [39]. The dissolution medium was kept at a temperature of 37 ± 1 • C and a speed of 100 rpm. The samples were withdrawn at different time intervals (5,10,15,20,25,30,45, and 60 min) and replaced with an equivalent volume of fresh medium [40]. The withdrawn samples were filtered and analyzed spectrophotometrically using UV spectrophotometer at 228 nm [41]. The experiment was done in triplicates, and the mean and the standard deviation were measured [40]. The percentage of GM released was plotted against the time, and the data of drug release was kinetically treated to determine the best mechanism of drug release [17].

The Selection of Optimized Formulation of GM Solid Dispersion
The formulation ingredients were optimized to determine the optimum level of X 1 and X 2, which achieve the highest value of Y 1 [35].

The scanning Electron Microscopy of the Optimized Formulation (SEM)
The morphology of the optimized formulation of GM solid dispersion was carried out using a scanning electron microscope (JEOL, Tokyo, Japan) [42]. A dried sample of optimized formulation was spread and adhered to aluminum stub and coated with a thin layer of platinum. The image was taken by SEM at 30 Kv.

The Pharmacological Evaluation of the Antidiabetic Effect of GM Solid Dispersion
Male Wister rats (150-180 g) were obtained from the modern veterinary office for laboratory animals (Cairo, Egypt). Groups of six rats were housed and kept under standard laboratory conditions, with the temperature at 25 ± 1 • C and relative humidity (55 ± 5%). The animals were housed in polypropylene cages, six per cage. All experimental protocols were approved by the Research Ethics Committee at the Faculty of Pharmacy, the British University in Egypt (No. EX-2006, Date 8 July 2020).
All animals were subjected to intraperitoneal injection with 50 mg⁄kg streptozotocin (STZ). The blood glucose level was measured for all rats after 72 h of injection. Rats that completed the in vivo study were selected based on the blood glucose level (>250 mg/dL) [43]. The diabetic rats were divided into three groups, each of six rats, as follows: Group I: control group treated with oral saline. Group II: a diabetic group treated with free GM (0.1 mg/kg; per oral). Group III: a diabetic group treated with F9 (0.1 mg/kg; per oral).
After receiving the previous treatments, blood samples were collected from the tail vein at the end of each hour for 8 h. The collected blood samples were analyzed for the blood glucose level using a commercial glucose kit.

The Statistical Analysis
The results of the in vivo study were subjected to statistical analysis using one-way ANOVA followed by Bonferroni's post-hoc analysis. The difference was considered significant at p values < 0.05.

Results and Discussion
Nine formulations of GM solid dispersion were designed by 3 2 multilevel factorial design using design expert software version 12. The factorial design was used to determine the effect of formulation variables on the dissolution profile of GM.

The Production Yield % (PY%)
Nine formulations of GM solid dispersion were successfully prepared by a solvent evaporation technique with high production yield % ranged from 98.4 ± 2.8% to 99.8 ± 2.2%, as shown in Table 3. The results were in full agreement with Sahoo et al., who have found that the production yield % of GM solid dispersion ranged between 96.5 ± 2.1% and 99.4 ± 1.2% [22].

The Drug Content %(DC%)
As represented in Table 3, the drug content for all formulation of GM solid dispersion was calculated, and it was found in the range of 96.8 ± 2.9-99.6 ± 2.1%. The high value of drug content indicated that the choice of the carriers and the method of preparation were reproducible. These results were in accordance with Sahoo et al., who prepared the GM solid dispersion using PEG 6000, PEG 10000, and Gelucire 44/14 and found that the drug content was in the range of 97.5 ± 1.2-99.5 ± 0.9% [22].

The Bulk and Tapped Density
The flow properties of the solid dispersion were investigated by measuring the bulk density and tapped density. The results of bulk and tapped density were represented in Table 4. It was found that the results of bulk and tapped density were in the range of 0.37 ± 0.05-0.54 ± 0.03 g/cm 3 and 0.43 ± 0.03-0.61 ± 0.02 g/cm 3 , respectively. These results were in agreement with Chavan et al., who prepared nisoldipine solid dispersion using a solvent evaporation technique [44].

Hausner's Ratio
The value of Hausner's ratio was found to give an indication of the flow properties of solid dispersion. The values <1.25 indicate better flowability than values >1.25 [44]. As represented in Table 4, Hausner's ratio of all prepared GM solid dispersion formulations was in the range of 1.11 ± 0.01 to 1.22 ± 0.02, which indicated good flowability [45].

Carr's Index (the Compressibility %)
There is an inverse relationship between the compressibility percentage and the flowability of the solid dispersion [46]. As Carr's index increased, the flowability decreased. The values from 5 to 12 indicate excellent flowability; the values from 12 to 16 exhibit good flowability; the values between 18 and 21 show fair passable flowability; the values ranged from 23 to 35 exhibit poor flowability; while the values between 33 and 38 exhibit very poor flowability [23].
For all prepared GM solid dispersion formulations, the value of Carr's index was in the range of 9.80 ± 0.54-18.18 ± 0.47%, which indicated a good flowability as represented in Table 4. The results were in full agreement with Malviya et al., who prepared paracetamol solid dispersion [47].

Angle of Repose
The flowability of the solid dispersion was found to be affected by the angle of repose [48,49]. Excellent flowability was achieved when the angle of repose was less than 20 • . A value of 20-30 • indicates good flowability, the values between 30 • and 34 • exhibit passable flowability, and the values more than 34 • show very poor flowability [50]. From the results represented in Table 4, it was found that all prepared GM solid dispersion exhibited angle of repose in the range of 14.26-23.44 • which indicated a good flowability.

The Differential Scanning Calorimetry (DSC)
The differential scanning calorimetry (DSC) thermograms of pure GM, Mannitol, PEG 6000, β-cyclodextrin, F3, F6, and F9 were illustrated in Figure 1. It was found that the DSC thermogram of GM showed a single endothermic peak at 212.  The DSC thermogram of F3, which was prepared by mannitol, showed a single endothermic peak at 166.78 °C with an absence of a GM peak. The DSC thermogram of F6, which was prepared by PEG 6000, showed a single endothermic peak at 60.23 °C with an absence of a GM peak. The DSC thermogram of F9 showed an endothermic peak at 97.73 °C with an absence of a GM peak. The disappearance of the peak of GM from the thermograms of F3, F6, and F9 showed that the drug was present in a more soluble amorphous form. These results were in full agreement with Pathak and Kaushik, who prepared a GM solid dispersion for enhancing the solubility using PEG 6000 and observed the disappearance of the endothermic peak of GM in the DSC thermogram of the prepared solid dispersion [54]. The DSC thermogram of F3, which was prepared by mannitol, showed a single endothermic peak at 166.78 • C with an absence of a GM peak. The DSC thermogram of F6, which was prepared by PEG 6000, showed a single endothermic peak at 60.23 • C with an absence of a GM peak. The DSC thermogram of F9 showed an endothermic peak at 97.73 • C with an absence of a GM peak. The disappearance of the peak of GM from the thermograms of F3, F6, and F9 showed that the drug was present in a more soluble amorphous form. These results were in full agreement with Pathak and Kaushik, who prepared a GM solid dispersion for enhancing the solubility using PEG 6000 and observed the disappearance of the endothermic peak of GM in the DSC thermogram of the prepared solid dispersion [54].

The Infrared Spectroscopy (IR)
The infrared spectra of pure GM, Mannitol, PEG 6000, β-cyclodextrin, F3, F6, and F9 were illustrated in Figure 2. The IR spectrum of GM showed characteristic N-H stretch peaks of urea at t 3370 and 3289 cm −1 , peaks at 1707 and 1674 cm −1 corresponding to the carbonyl group, and peaks at 1348 and 1155 cm −1 related to the sulfonamide group. The IR spectrum of Mannitol was characterized by a peak at 3399 cm −1 due to O-H stretching, a peak at 2949 cm −1 due to C-H stretching, and peaks at 1082 cm −1 and 1019 cm −1 due to C-O stretching [55]. The IR spectrum of PEG 6000 is characterized by 3423 cm −1 due to stretching O-H, peak at 2886 cm −1 due to C-H stretching, and peak at 1109 cm −1 due to C-O stretching [56]. The spectra of β-cyclodextrin was characterized by a broad peak due to stretching O-H at 3398 cm −1 , peak at 2925 cm −1 due to stretching C-H., peak at 1644 cm −1 due to H-O-H. while peaks at 1157 and 1030 cm −1 due to vibrations of the asymmetric C-O-C and symmetric C-O-C, respectively [57].

The Infrared Spectroscopy (IR)
The infrared spectra of pure GM, Mannitol, PEG 6000, β-cyclodextrin, F3, F6, and F9 were illustrated in Figure 2. The IR spectrum of GM showed characteristic N-H stretch peaks of urea at t 3370 and 3289 cm −1 , peaks at 1707 and 1674 cm −1 corresponding to the carbonyl group, and peaks at 1348 and 1155 cm −1 related to the sulfonamide group. The IR spectrum of Mannitol was characterized by a peak at 3399 cm −1 due to O-H stretching, a peak at 2949 cm −1 due to C-H stretching, and peaks at 1082 cm −1 and 1019 cm −1 due to C-O stretching [55]. The IR spectrum of PEG 6000 is characterized by 3423 cm −1 due to stretching O-H, peak at 2886 cm −1 due to C-H stretching, and peak at 1109 cm −1 due to C-O stretching [56]. The spectra of β-cyclodextrin was characterized by a broad peak due to stretching O-H at 3398 cm −1 , peak at 2925 cm −1 due to stretching C-H., peak at 1644 cm −1 due to H-O-H. while peaks at 1157 and 1030 cm −1 due to vibrations of the asymmetric C-O-C and symmetric C-O-C, respectively [57]. The IR spectrum of F3, F6, and F9 showed all characteristic peaks of GM, which revealed the absence of any physical or chemical interaction between the drug and the used carriers [21]. These results were in agreement with Kiran et al., who prepared GM solid dispersion [58].

The In-Vitro Release Study of GM From Solid Dispersion
From the results of the in-vitro release, as shown in Figure 3, it was found that the release for all formulations ranged from 65.17 ± 1.64 for F4 to 99.9 ± 2.56% for F9. Also, the release from all formulation was significantly higher than the release of free drug, which indicates that the solid dispersion technique has the ability to enhance the dissolution rate of the GM. This may be attributed to the conversion of the drug into the more soluble amorphous state, and decreasing the interfacial tension between GM and the dissolution medium resulted in enhancing the wettability and the dissolution of the drug [59].
Sci. Pharm. 2020, 88, x FOR PEER REVIEW 10 of 17 The IR spectrum of F3, F6, and F9 showed all characteristic peaks of GM, which revealed the absence of any physical or chemical interaction between the drug and the used carriers [21]. These results were in agreement with Kiran et al., who prepared GM solid dispersion [58].

The In-Vitro Release Study of GM From Solid Dispersion
From the results of the in-vitro release, as shown in Figure 3, it was found that the release for all formulations ranged from 65.17 ± 1.64 for F4 to 99.9 ± 2.56% for F9. Also, the release from all formulation was significantly higher than the release of free drug, which indicates that the solid dispersion technique has the ability to enhance the dissolution rate of the GM. This may be attributed to the conversion of the drug into the more soluble amorphous state, and decreasing the interfacial tension between GM and the dissolution medium resulted in enhancing the wettability and the dissolution of the drug [59].

The Effect of the Formulation Factors in the In-Vitro Release (Y1)
To study the effect of independent formulation variables (X1, and X2) on the in vitro release of prepared GM solid dispersion, a multiple linear regression analysis was done using the Y1 equation: Y1 = 75.31 5.76 X 7.87 X − 0.795 X X 12.42 X 0.0683 X As shown in Figure 4, it was found that the release changed according to the type of carrier (βcyclodextrin > Mannitol > PEG 6000). These results may be attributed to the difference in the hydrophilic effect of the carriers. Also, the release increased for each carrier by increasing the drugcarrier ratio from 1:1 to 1:6, which may be attributed to the increased wettability and surface area as the percentage of water-soluble carriers increased [59,60]. The ANOVA analysis of in-vitro drug release results, as represented in Table 5, revealed that the type of carrier (X1) and the drug: carrier ratio (X2) had a significant effect on (Y1) (p < 0.05).

The Effect of the Formulation Factors in the In-Vitro Release (Y1)
To study the effect of independent formulation variables (X 1 , and X 2 ) on the in vitro release of prepared GM solid dispersion, a multiple linear regression analysis was done using the Y 1 equation: Y1 = +75.31 + 5.76 X 1 + 7.87 X 2 − 0.795 X 1 X 2 + 12.42 X 21 + 0.0683 X 22 (8) As shown in Figure 4, it was found that the release changed according to the type of carrier (β-cyclodextrin > Mannitol > PEG 6000). These results may be attributed to the difference in the hydrophilic effect of the carriers. Also, the release increased for each carrier by increasing the drug-carrier ratio from 1:1 to 1:6, which may be attributed to the increased wettability and surface area as the percentage of water-soluble carriers increased [59,60]. The ANOVA analysis of in-vitro drug release results, as represented in Table 5, revealed that the type of carrier (X1) and the drug: carrier ratio (X2) had a significant effect on (Y1) (p < 0.05).     59 8 In Vitro Drug Release Kinetics The kinetic release study was done for all GM solid dispersions to determine the release behavior. The release data were analyzed with zero-order, first-order, second-order, and Higuchi diffusion models. As represented in Table 6, it was found that all release data fit first-order kinetics. These results were found to be in agreement with those obtained by Mamatha et al., who found that the release of nevirapine from prepared solid dispersion was fitted to first-order kinetics [61]. Also, Pagadala et al. found that the release of GM from prepared solid dispersion was fitted to first-order kinetics [62].

The Selection of Optimized Formulation of GM Solid Dispersion
The independent variables were optimized using Design Expert software (version 12) to determine the optimized formulation. The optimized formula was selected based on the highest drug release (Q 1hr ), which achieved the highest drug release. From the results, F9 was selected as the optimized formulation.

The Scanning Electron Microscopy of the Optimized Formulation (SEM)
The surface morphology of the optimized GM solid dispersion was shown in Figure 5. It was found that the optimized formulation appeared as irregular particles, which may give an indication of the dispersion of GM on the carriers in an amorphous state.

Antidiabetic Effect of GM Solid Dispersion
The antidiabetic effect of F9 was studied in comparison with free GM in STZ (Streptozotocin) diabetic rats. As shown in Figure 6, it was found that F9 showed a high and rapid reduction in blood glucose levels in diabetic rats, which indicated the success of the solid dispersion technique in improving the solubility and hence the bioavailability of GM. It was found that F9 showed a

Antidiabetic Effect of GM Solid Dispersion
The antidiabetic effect of F9 was studied in comparison with free GM in STZ (Streptozotocin) diabetic rats. As shown in Figure 6, it was found that F9 showed a high and rapid reduction in blood glucose levels in diabetic rats, which indicated the success of the solid dispersion technique in improving the solubility and hence the bioavailability of GM. It was found that F9 showed a reduction in blood glucose levels to 45 ± 2.65% of the initial value after 1 h, while the free GM showed a reduction in blood glucose level to 63 ± 3.21% of the initial value after 2 h. These results may be attributed to the enhanced solubility of GM after preparation as solid dispersion using hydrophilic carriers and the presence of GM in a more soluble amorphous from. The results were in full agreement with Li et al., who prepared GM microemulsion for improving its solubility and bioavailability [63].

Antidiabetic Effect of GM Solid Dispersion
The antidiabetic effect of F9 was studied in comparison with free GM in STZ (Streptozotocin) diabetic rats. As shown in Figure 6, it was found that F9 showed a high and rapid reduction in blood glucose levels in diabetic rats, which indicated the success of the solid dispersion technique in improving the solubility and hence the bioavailability of GM. It was found that F9 showed a reduction in blood glucose levels to 45 ± 2.65% of the initial value after 1 h, while the free GM showed a reduction in blood glucose level to 63 ± 3.21% of the initial value after 2 h. These results may be attributed to the enhanced solubility of GM after preparation as solid dispersion using hydrophilic carriers and the presence of GM in a more soluble amorphous from. The results were in full agreement with Li et al., who prepared GM microemulsion for improving its solubility and bioavailability [63]. Figure 6. The antidiabetic activity of F9 in comparison with free glimepiride. Data were analyzed statistically using one-way ANOVA followed by Bonferroni's post-hoc test at p < 0.05. * Versus control and $ versus free glimepiride, n = 6. Figure 6. The antidiabetic activity of F9 in comparison with free glimepiride. Data were analyzed statistically using one-way ANOVA followed by Bonferroni's post-hoc test at p < 0.05. * Versus control and $ versus free glimepiride, n = 6.

Conclusions
The solid dispersion technique was found to be useful in improving the dissolution rate of GM. The type, nature, and the ratio of the carrier used played an important role in the enhancement of the dissolution rate and hence the bioavailability of GM. It was concluded that all prepared formulations have high production yield ranged from 98.4 ± 2.8% to 99.8 ± 2.2% and high drug content in the range of 97.2 ± 3.2% to 99.6 ± 2.1%. The micromeritic properties of all prepared GM formulations showed a high flowability. The authors concluded that the used carriers were arranged according to the enhancement of solubility as follows: β-cyclodextrin > Mannitol > PEG 6000. Also, increasing the drug-carrier ratio from 1:1 to 1:6 caused an increase in the rate of GM release. The formulation of GM as a solid dispersion formulation is a potential way to improve its solubility and enhance its hypoglycemic effect.