THE DEVELOPMENT OF GLIBENCLAMIDE-SACCHARIN COCRYSTAL TABLET FORMULATIONS TO INCREASE THE DISSOLUTION RATE OF THE DRUG

Objective: Cocrystallisation is a promising method in order to increase the solubility and dissolution of poorly water-soluble drugs. The aim of this study was to prepare, formulate and evaluate glibenclamide (GCM) cocrystal in direct compress tablet dosage form using saccharin (SAC) as the coformer. 
Methods: GCM cocrystal with various stoichiometric ratios were prepared by the solvent drop grinding method. The co-crystal was characterized by a saturated solubility test and dissolution rate test, Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), and Powder X-Ray Diffraction (PXRD). The tablet dosage form of GCM was formulated and evaluated compare with the conventional dosage form. 
Results: The solubility and disso­lution rate of GCM-SAC cocrystals increased significantly compared with pure GCM, especially for 1:2 of ratio. The dissolution rate of cocrystal with ratio 1:2 increased by almost 91.9% compared with pure GCM. Based on the FTIR analysis, it showed the shifting of characteristic bands of GCM in the spectrum and there was no chemical reaction in GCM cocrystal. In PXRD measurement, the new crystalline peak was detected in the crystal habit of cocrystal compared with pure GCM and coformer. The new single melting of GCM-SAC cocrystal also was detected in DSC measurement. The tablets of GCM-SAC cocrystal were successfully prepared by direct compression method which rapidly disintegrated (1 min) and has higher dissolution compared with its pure form (32.36% greater than glibenclamide after 45 min). 
Conclusion: The tablet dosage form of GCM cocrystal with SAC as coformer was successfully prepared, formulated and improved its solubility and dissolution rate.


INTRODUCTION
Solubility and dissolution are important factors in the pharmacological effect of the drug [1]. Although the level of drug solubility is needed for oral absorption, almost 40% of the drug in the market shows low solubility in water. Due to the low solubility, the drug is absorbed slowly and the levels of the drug in the blood are lower than the required [2,3]. In the biopharmaceutical classification system (BCS), glibenclamide (GCM) is included in class II, which means it has high permeability and low solubility. GCM is a type 2 antidiabetic drug used for controlling glycemia [4]. Glibenclamide has a low solubility of about 4 mg/l [5] and its bioavailability is only 40-45%.
Several studies have been reported about methods improving glibenclamide solubility such as fast dissolving tablet (FDT) [6], lipid nanocrystal [7], nanoparticles [8,9], solid dispersion [10,11], selfemulsifying drug delivery systems (SEEDS) [12] and liquisolid technology [13]. Cocrystal is one method that can increase the solubility of the API [14]. Cocrystal can be defined as complex crystal formations with stoichiometric multi-component system connected by a synthon that in pharmaceutical crystal engineering is called "a non-covalent interaction involving hydrogen bonds, in which two distinct components are solid under ambient conditions". The previous study showed that cocrystallization method can increase the solubility of GCM compared with pure GCM with saccharin and aspartame as the coformer [14,15].
In this study, glibenclamide cocrystal was prepared by solvent drop grinding using saccharin as the coformer. Saccharin (SAC) is an artificial sweetener that commonly used in cocrystallization as the coformer. Some several studies reported that using SAC as coformer can increase the solubility of carbamazepine, ketoprofen and GCM compared with pure drugs [14,16,17]. In the last decades, the research of cocrystal has grown rapidly, but only a few research conduct about the preparation and evaluation of cocrystal in the tablet dosage form. Thus, the aim of this study was to prepare, formulate and evaluate GCM cocrystal in tablet dosage form using SAC as the coformer. The tablet formulation was designed as direct compress tablet to minimize manufacturing process impact to GCM cocrystal performance.

Materials
GCM was obtained from Indofarma (Indonesia) with a purity>99% and SAC and ethanol pro analysis were obtained from Merck (Germany).

Preparation of cocrystal
The cocrystal of GCM-SAC was employed with solvent drop grinding method. GCM and SAC were weighted and mixed with different molar ratio (1:1, 1:2 and 2:1). The mixture was ground by mortar and pestle, during grinding add ethanol as a solvent for 15 min. The mixture was dried overnight at ambient temperature.

Solubility study
The sample equivalent to 20 mg GCM was placed in vials containing water. The vials were agitated using a mechanical agitator at room temperature and allowed to stand for equilibrations for 24 and 48 h. The samples were filtered through a 0.45 μm membrane filter, diluted with distilled water and analyzed spectrophotometrically at 266 nm [15].

Particulate dissolution study
Dissolution studies of cocrystals were performed using type 2 apparatus (paddle). A sample of cocrystals weighed in equal to 50 mg GCM then put into 900 ml buffer phosphate pH 8 and stirred at 75 rpm, the temperature was maintained at 37 ± 0.2 °C. The 5 ml of samples were filtered through a syringe filter of 0.45 μm pore size periodically (at 0, 10, 15, 30, 45, and 60 min) and analyzed by UV spectrophotometer at 266 nm [15].

FTIR analysis
FTIR analysis was employed to evaluate the interaction between drug and coformer. The cocrystal was dispersed homogeneously in KBr pellet. The samples were analyzed using an infrared spectrophotometer (Shimadzu®) at room temperature in a range of wavenumbers from 400-4000 cm -1 [18]. The X-ray generator was operated using Phillip PW 1835 at 40 kV and 40 mA using CuKα radiation. The scans were performed between 3 ᵒ and 40 ᵒ with a scanning rate of 4 ᵒ/min at room temperature [14].

Differential scanning calorimetry (DSC)
DSC analysis was performed using Linseis DSC PT1000. Approximately 5 mg of the powder sample was placed in an aluminum pan and heated at a rate of 10 °C/min, from 0 to 300 °C temperature range, under nitrogen stream [19,20].

Formulation of cocrystal tablet
An accurately weighed quantity of cocrystal equivalent to drug dose (5 mg) and all other ingredients was mixed. The mixture was directly compressed into tablets. Round concave tablets of 100 mg mass were obtained [21]. Table 1 outlines the composition of tablet formulations.

Evaluation of cocrystal powder (pre-compression parameters)
The mixture of drug and excipient were evaluated for tapped density, bulk density, and flow properties and compressibility parameters. Flow properties of powder were determined by the angle of repose and compressibility by Carr's index [22,23].

The thickness and weight variation
Twenty tablets were randomly taken from each formulation and thickness was measured using Vernier caliper. The results are expressed as mean±standard deviation (SD). The average weight of twenty tablets was determined using an electronic balance and tablet were weighed individually and compared to average weight.

Hardness test
Ten tablets were randomly selected from each batch and hardness of tablets was determined by using hardness tester. The mean values and standard deviation for each batch were calculated [24].

Friability test
The friability of tablets was measured using friability tester. Twenty tablets were placed in plastic friability tester attached to motor revolving at a speed of 25 rpm for 4 min. The tablets were then deducted, reweighed, and percent weight loss was calculated using the formula, % friability = ((initial weight-final weight)/initial weight)×100 [23].

In vitro disintegration time
Determination in vitro disintegration time (DT) using distilled water at 37±2 °C. The time when the tablets were disintegrated completely without any residue remaining in the apparatus was recorded as mean±SD [24].

In vitro dissolution study
Dissolution test of tablet glibenclamide cocrystal was performed using type 2 apparatus (paddle type). A sample tablet put into 900 ml buffer phosphate pH 8 and stirred at 100 rpm, temperature 37 ± 0.2 °C. Sampling (5 ml) until 60 min at predetermined interval times (5, 10, 15, 30, 45, 60 min) and 5 ml of fresh medium was added after each sampling. The sample was analyzed using a UV Spectrophotometer to quantify the amount of dissolved GCM concentration [25].

Preparation of cocrystal
The preparation of the GCM and SAC cocrystal was carried out by solvent drop grinding (SDG). The SDG method is reliable in the discovery of new cocrystals because the presence of a solvent can improve the rate of cocrystal formation and is suitable for cocrystal constituents with the equimolar ratio [26]. By grinding with minimal addition the solvent, the SDG can control over the polymorphic outcome of cocrystal [27]. In this experiment, saccharin was chosen as the coformer because of its stability, polarity, and ability to form a heterosynthon in cocrystal [2]. The purpose of using this method is to demonstrate an 'environmentally friendly' method of generating GCM-SAC cocrystals [28].

Solubility test
Based on fig. 1, the solubility of cocrystal (1:2) was higher than the solubility of its pure form and other cocrystals (1:1 and 2:1) for 24 and 48 h. This indicated that the molecular interaction between GCM with SAC has successfully occured and can form a cocrystals state [25]. The interaction between hydrogen atoms of the amide groups from GCM and oxygen atoms of sulfonyl from SAC might have formed the hydrogen bonds [15]. Saccharin has a strong hydrogen bond acceptor (C=O) and strong donors (N-H) to form a robust homodimer synthon in the crystal structure with GCM [28]. In addition, the higher hydrophilicity of coformer tend to ease the solubilization process of compound [29].
It is assumed that the GCM cocrystal has a weaker crystalline structure that can increase the dissolution rate of GCM. Moreover, the changed crystallinity pattern, crystal habit, size and shape of cocrystal can be attributed to increasing dissolution of GCM [25]. Increasing the dissolution rate is related to the diffusion constant, the function of the surface area, boundary-layer thickness, and solubility [26].

Fig. 2: In vitro dissolution study of the cocrystal (All the values were calculated as mean±SD)
Characterization of co-crystal FTIR analysis

Fig. 3: Overlay Infrared spectrum of pure glibenclamide (green line), saccharin (black line), and glibenclamide-saccharin cocrystal (red line)
The weakened C=O peak could confirm hydrogen bond formation between GCM and SAC (table 2). Interaction of GCM and SAC was predicted between amide groups of glibenclamide and sulfonyl group of saccharin [14]. In FTIR analysis, there were no new peaks observed, indicating that no chemical reaction occurred during cocrystal preparation.

Powder X-ray diffraction
PXRD was performed to verify the formation of the cocrystal of GCM. All crystal forms of a compound show the characteristic on diffractogram pattern of drug [26]. The overlay of the diffractogram showed peaks and intensity at an angle of 2θ of pure GCM is 10.7 °, 12.3 °, 19.5 °, 19.8 °, 20.9 °, 22.1 ° and intensity at an angle of 2θ of SAC is 9.01 °, 15.2 °, 19 °, dan 24 °. A different powder X-ray pattern for the GCM-SAC cocrystals from those of the constituent GCM and saccharin confirms the formation of a new cocrystal phase [28].

Differential scanning calorimetry (DSC)
DSC studies are performed to observe the solid-state interaction of two compounds or more by giving heat energy to the co-crystals to evaluate thermodynamic changes (endothermic or exothermic peaks) [26]. The thermogram fig. 5 showed that the melting point of cocrystal (144.5-166.9 °C) was lower than pure GCM (169.6-185.1 °C). The change in thermal properties (melting point) indicated as evidence for the formation of cocrystal. A decrease in the melting point of the cocrystal directly correlates with increased solubility of GCM in the cocrystal system [26].

Evaluation pre-compression and post-compression
The bulk density of all the formulations was found to 0.56 g/ml, tapped density was 0.64 g/ml, angle of repose 15.94 °, and index carr was 11.27 % indicating good flow property of tablet granules and tablet of cocrystal could be compressed directly. Results of postcompression such as weight variation, diameter, thickness, hardness, friability, and disintegration time are reported in table 4. These evaluations indicated that the stoichiometry ratio of cocrystal was not affected mechanical properties of GCM.

In vitro dissolution study of tablet cocrystal
The drug release of all cocrystal formulations were more than 80% at 60 min. The tablets of 1:2 cocrystal was the highest drug release percentage among other tablets. However, cocrystallization glibenclamide was still potential because the tablet was made by direct compression method. Drug release percentage of pure glibenclamide tablet after 45 min, cocrystal 1:1, 1:2, 2:1 were 65.32 %; 94.06 %; 97.68 %; and 88.65 % respectively.

CONCLUSION
The solubility and dissolution rate of GCM-SAC cocrystals increased significantly compared with pure GCM, especially for ratio of 1:2.
The dissolution rate of cocrystal with ratio 1:2 increased by almost 91.9% compared with pure GCM. Based on the FTIR analysis, it showed the shifting of characteristic bands of GCM in the spectrum and there was no chemical reaction in GCM cocrystal. In PXRD measurement, the new crystalline peak was detected in the crystal habit of cocrystal compared with pure GCM and coformer. The new single melting of GCM-SAC cocrystal also was detected in DSC measurement. The tablets of GCM-SAC cocrystal were successfully prepared by direct compression method which rapidly disintegrated (1 min) and has higher dissolution compared with its pure form (32.36% greater than glibenclamide after 45 min).