INTRODUCTION:

Solubility properties of a drug are closely related to the bioavailability especially for oral dossage form. The low bioavailability property is the biggest challenge in designing oral dosage form. Several factors such as water solubility, drug permeability, dissolution rate, first-pass metabolism, and pre-systemic metabolism can influence the bioavaibility of drug. The most common causes of low bioavailability are low solubility and permeability¹. However, for some compound with low solubility in water often have low bioavailability and dissolution rate, thus affecting the process of the absorption².

Glibenclamide (GCM) is a drug which has a low solubility in water. GCM is a sulfonylurea oral antihyperglycemic agent used in the treatment of type 2 antidiabetic drug used for controlling glycemia³.

According to the Biopharmaceutical Classification System (BCS), GCM is classified to class II (high permeability but low solubility). The solubility of GCM is practically insoluble in water. However, only 45% GCM from oral doses can be absorbed in the gastrointestinal tract⁴.

Based on the research, the solubility of GCM has been improved by using the Fast Dissolving Tablets (FDT) method⁴, nanoparticle method⁶, solid dispersion method⁷. surface solid dispersion, nanoparticles⁸, and nanoemulsions⁹ and liquisolid technology and co-grinding technique (liquiground technique)¹⁰.

Another simple method for enhancing the solubility of GCM in water is cocrystallization method. Cocrystallization is a method of cocrystal formation with the addition of coformers to Active Pharmaceutical Ingredients (API) however increasing the solubility of GCM without changing the pharmacological activity¹¹.

The previous article reported that the cocrystallization method can improve the solubility of simvastatin (using mallic acid as a coformer) two fold compare with pure simvastatin¹². Furthermore, another study has proved cocrystallization method can increase the solubility of atorvastatin (with benzoic acid as a coformer) from 22.08 ppm to 28.91 ppm, and with aspartame to 30.20 ppm¹³.

In this study, GCM-ascorbic acid (AA) cocrystal were prepared in 1:1 and 1:2 equimolar ratios. AA is an accessible excipient with affordable price. Based on virtual screening, AA can form hydrogen bonds with glibenclamide.

MATERIAL AND METHODS:

Chemicals and reagents:

GCM was obtained from Indofarma Tbk with a purity of > 99%, methanol pro analysis, AA pro analysis, and potassium dihydrogen phosphate pro analysis were obtained from Merck (Darmstadt, Germany).

In silico molecular docking:

2D structures of GCM (Chem Spider ID: 54809) and its coformers in. mol format were downloaded from www.chemspider.com. All of the .mol files of the molecules were converted into. pdb files by employing Open Babel GUI 2.2.3 then it opened in Auto Dock 4.2.3 and converted into. pdbq files by adding polar hydrogen and Kollman charges. The. pdbq files were converted into. pdbqt files by calculating their torsion angles, and were ready to be used for docking. Docking was repeated five times. Parameters observed were both the type and energy (Ei) of interactions¹⁴.

Preparation of glibenclamide cocrystal with solvent evaporation method:

GCM and AA were weighed according to 1:1 and 1:2 equimolar ratios. Each mixed substance was dissolved with methanol in an evaporating dish (the mixture was stirred until completely dissolved). Each solution evaporated on water bath (temperature 45^oC) for 24 hours until all the the solvents completely removed or evaporated. The cocrystal obtained was analyzed for characterization and evaluation¹⁴.

Solubility study:

Cocrystal (equivalent to 20mg of GCM) was weighed and dissolved in 20ml of distilled water. Cocrystal was agitated for 12 and 24 hours respectively using a mechanical agitator (speed 120 rpm) at room temperature. Then the solution was filtered and 1ml sample was diluted to 100ppm with methanol (final volume 10ml). Each sample was measured using UV-Vis spectrophotometer at 266nm (analytical Jena, specord 200®)¹⁵.

Optimization of pH medium dissolution for glibenclamide

Buffer Phosphate solution was made 900ml with pH variation of 6.4, 7.6 and 8.0. The dissolution test of GCM was performed using the type 2 (paddle) dissolution tester (75 rpm) for 60 minutes at 37 + 0, 2^oC. The sample was taken 5ml in interval 5, 10, 15, 30, 45 dan 60 minutes and then replaced with the same amount of phosphate buffer media. The sample was measured by UV spectrophotometry (analytical Jena, specord 200®) at 266nm to determine the concentration of GCM and the optimal pH for dissolution medium¹⁶.

Dissolution test:

The dissolution test was performed by using type 2 dissolution tester (paddle) with the 75rpm speed of rotation for 60 minutes with 900ml phosphate buffer pH 8.0 as a medium on 37 + 0.2^oC. The sample was taken 5 ml in interval time (interval times (5, 10, 15, 30, 45, 60 minutes) and then replaced with the same amount of phosphate buffer media. The aliquot sample was analyzed using a UV Spectrophotometer at 266 nm (analytical Jena, specord 200®) to quantify the amount of dissolved GCM concentration¹⁶.

Characterization of cocrystals:

Fourier Transform infrared spectroscopy (FT-IR) analysis:

One mg of cocrystal of GCM-AA was carefully weighed and homogenously mixed with 250mg of KBr. The sample and Potassium Bromide were mixed homogeneously and compressed to form a disk at 80 Psi of pressure. The disk was measured using infrared spectrophotometer at room temperature on 400- 4000 cm ^-1wave number¹⁷.

Powder X-ray diffraction (XRPD):

Cocrystal of GCM-AA and the pure GCM were recorded by X-ray (Phillips PW1835®) using Cu anode, 40kV voltage, and 40mA stream. Samples were scanned with a detector step size of 0.02^o over an angular range of 2q = 2-50^oC¹⁷.

Differential scanning calorimetry (DSC):

Thermal analyses of the drug, coformer, and solvent evaporation products were performed on a DSC. The thermograph was recorded under a gas flow of 50 mL/min. Samples were analyzed from 30 to 200^oC with a heating rate of 10^oC/min^16,17

Solubility:

The result of the solubility test showed that cocrystallization can increase the solubility of GCM in water. The result of solubility study can be seen in fig. 1

Fig. 1: Solubility Result of GCM and cocrystals.

Dissolution Test:

The dissolution test was performed on the cocrystal with the best equimolar ratio based on the solubility test (glibenclamide- ascorbic acid 1: 2) compared with pure glibenclamide. Optimization of pH of dissolution medium was performed at pH 6.4, 7.6, and 8.0. In Figure 2, the concentration of GCM standard in 60 minutes was 9.91, 16.43, and 45.31 ppm for pH 6.4, 7.6, and 8.0. Based on the results, 8.0 was the optimum pH for the dissolution test of GCM.

Fig. 2: Optimization of pH of dissolution medium

In Fig. 3, the dissolution rate of cocrystal of GCM-AA was higher than its pure form. As stated in the literature, GCM only dissolved 45% in 60 minutes^[21]. In this study, pure GCM dissolved 46.83% in 60 minutes. The result of dissolution rate can be seen in fig. 3.

Fig. 3: The result of dissolution rate of GCM and cocrystal of GCM-AA.

Characterization of Cocrystal:

The objective of cocrystal characterization is to determine the characteristic of the formed cocrystal compared to pure GCM. The characterization was carried out for GCM-AA cocrystal with the best equimolar ratio (1:2). The characterization includes FTIR Spectrophotometry, Differential Scanning Calorimetry (DSC) dan X-Ray Diffraction (XRD).

Fourier Transform Infrared Spectroscopy (FTIR):

FTIR characterization principle is comparing the functional group of sample and pure GCM. The result can be seen in fig. 4.

Fig. 4: Infra Red Spectra of GCM, AA and GCM-AA cocrystal

The FTIR result showed major peaks from GCM including in the area of 3367.71 cm-1 and 3313.71 cm-1 (NH Stretch), then in the area of 3116.97 cm-1 (OH Stretch), 2931 , 80 cm-1 and 2854.65 cm-1 (C - H Stretch), 1716.65 cm-1 (C = O Stretch) and 1616.35 cm-1 (C = C Stretch) (Filho et al., 2018) . For AA, there were major peaks at 3525.88 cm-1, 3410.15 cm-1, 3217.27 cm-1 and 3028.24 cm-1 (OH Stretch), at 2916.37 cm-1 (CH Stretch), 1755,22 cm-1 (C = O Stretch), 1674,21 cm-1 (C = C Stretch) and 1026,13 cm-1 (C=C Stretch).

Based on the result same functional group has found in GCM-AA cocrystal as the constituent material, it showed in the area of 3525.88 cm-1 and 3410.15 cm-1 (OH Stretch), in the area of 3367.71 cm -1 and 3313.71 cm-1 (NH Stretch), then in the area of 2931.80 cm-1 and 2854.65 cm-1 (CH Stretch), the area of 1755.22 cm-1 and 1716.65 cm-1 ( C = O Stretch) and in the area 1674.21 cm-1 and 1616.35 cm-1 (C = C Stretch).

Differential Scanning Calorimetry (DSC):

Thermogram of GCM, AA and cocrystal of GCM:AA (1: 2) can be seen in Fig. 5 and table 2.

Table 2. DSC Analysis Result

Sample	Melting point (⁰C)
Sample	*Onset*	*Peak*	*Endset*
GCM	171.96	174.66	178.33
AA	193.05	202.09	205.44
GCM-AA cocrystal	151.09	158.51	169.47

Fig. 5. DSC Thermogram Result (a) GCM (b) AA (c) GCM-AA cocrystal (1:2).

Powder X-ray diffraction (XRPD)

Fig. 6. GCM, AA and GCM-AA (1:2) Diffractogram

DISCUSSION:

Virtual screening was performed using molecular modeling and the molecular docking program to predict the bonds formed when combining GCM and the coformer structure. Virtual screening was used to determine which coformer should be used¹⁸. Molecular docking was used to determine a parameter of the coformer had a high number of hydrogen bonds and a small value of energy of interaction (Ei), the smaller the Ei, the stronger the bond¹³.

The virtual screening result of GCM and AA showed that three interaction of hydrogen bonds. Hydrogen bond formed from atom O carbonyl of GCM and hydroxy of AA, oxygens atoms of sulfonyl of GCM and hydrogens atoms of AA, and the hydrogen atom of the amide group of GCM and oxygens atom of AA (Table 1).

In addition, π-π and van der Waals bonds could also be used as a selection parameter of the coformer but π-π bonds have lower electronegativity than hydrogen bonds and van der Waals bonds are weaker than hydrogen bonds. Covalent bonds are undesirable because will bind irreversibly to the body and may cause toxicity¹⁵.

Solvent evaporation and grinding method is the most widely used method crystallization process, so the most principle commonly used in cocrystallization is solvent-based and grinding. The preparation of cocrystal with each equimolar mass comparison is related to stoichiometric ratio that can improve the possibility of forming bonds between a molecule¹⁹.

The cocrystal solubility of GCM-AA has better solubility compared to pure GCM with 1:2 equimolar as the most optimal ratio. The greater solubility is due to the addition of coformer (the coformer correlate to barriers, it can reduce the barrier so the solubility process is easier). In this study, AA can enhance GCM solubility in water due to the reduction of barrier (saccharin has good solubility in water) so it can facilitate the solubilization process by ionization²⁰.

Characterization with FTIR spectrophotometry instrument was carried to determine the interaction between GCM with each coformer¹⁶^,17. The absence of new functional groups in GCM-AA cocrystal indicates that no chemical reaction occurred. However, the intensity increased in the area 3525.88 cm^-1from 26.08 to44.29. This increase in intensity occurs due to the formation of hydrogen bonds in GCM-AA cocrystal in the spectra area of O-H group which is widening²².

Differential Scanning Calorimetry (DSC) is an analytic instrument used in characterization to determine the interaction of a solid state interaction between two or more types of drugs, by giving heat energy to the cocrystal to evaluate thermodynamic changes characterized by the formation of exothermic and endothermic peaks on DSC diagrams. If there is a change in the thermodynamic aspects of a solid, there is also a change in the shape of a crystal due to the physical interaction formed from thermal analysis between two or more substances^22,23.

The results of the DSC thermogram analysis showed that the pure GCM contained endothermic peaks at 174.66^oC and 259.89^oC. Then in AA, there was also an endothermic peak at 202.09 ^oC. When compared with pure GCM, cocrystal of GCM-AA has a lower melting point of 158.51^oC. This is in accordance with the literature which states that the cocrystallization process will cause a change in melting point in a compound when compared to the material/constituent substance, that is usually the melting point of the cocrystal will be lower when compared to the active substance and coformer¹⁴.

Characterization using X-Ray Diffraction (XRD) aims to see the differences in the form of crystals compared to pure GCM. X-Ray Diffraction (XRD) is a method used in analyzing the form of polymorphism and to find out whether there is a change in the nature of a crystal and or whether a new phase of crystals is formed from the cocrystallization process that has previously been carried out. Where, when a new crystalline phase is formed on the diffractogram, the formation of a crystallite is indicated²⁴.

Based on the results of the intensity diffraction pattern on cocrystal of GCM-AA (1: 2) shows high intensity compared to the intensity of pure GCM and AA. The highest intensity of GCM-AA cocrystal (1: 2) is at an angle of 10.44 with an intensity of 100%. In addition, the number of peaks formed between pure GCM and cocrystal is different. In pure GCM, there are only 40 peaks whereas in cocrystal there are 47 peaks. From this peak, difference indicates that there is a difference in structure²⁵. Because the number of peaks in each cocrystal increases, it can be predicted that new peaks are formed, where the formation of these new peaks can be predicted for the formation of crystallites²².

CONCLUSION:

The solubility of GCM-AA cocrystal with equimolar ratio of 1:2 was higher 26-fold than pure GCM. GCM-AA cocrystal also can increase the dissolution rate of GCM from 46.83% to 63.248 %. Based on the IR spectrum, a chemical reaction did not occur during the cocrystallization. Characterization of the GCM-AA cocrystal (1:2) including FT-IR, DSC, and PXRD showed the formation of a new solid crystal phase that is different from GCM and AA.

ACKNOWLEDGEMENT:

The authors thanks to Universitas Padjadjaran for financial support in this research.

CONFLICT OF INTERESTS:

All authors have none to declare.

AUTHORS’ CONTRIBUTIONS:

All the authors have contributed equally.

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Received on 11.05.2019 Modified on 12.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5805-5810.

DOI: 10.5958/0974-360X.2019.01005.9