Pharmaceutical co-crystallization is a novel technique to alter the physicochemical properties of API to fine-tune its bioavailability and solid-state properties. In this research work, different co-formers with different functional groups like carboxylic acid, hydroxyl, was tried in different molar or w/w ratio to form co-crystals of ETD. The CA and TA were used as co-crystal formers. Several methods were used in the co-crystal formation i.e. neat grinding method, solvent assisted grinding, and solvent evaporation technique. Different ratios of co-former i.e. CA and TA were used to form co-crystals. Co-crystals were prepared by dry grinding; solvent assisted grinding and solvent evaporation method. Using different ratio of drug-co-former, nine pre-optimized formulations of different compositions were prepared.
4.1 Mechanism of bonding between etodolac, citric acid and tartaric acid
ETD (±, RS-1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl) acetic acid) formed co-crystals with CA (2-hydroxy-1,2,3-propanetricarboxylic acid) and TA (2,3-dihydroxybutanedioic acid) with the help of the intermolecular bonding, Vander Wall bonding and hydrogen bonding. ETD contained acetic acid group which formed hydrogen bond, due to the hydroxyl group which bonded to the hydrogen of CA and TA. The co-crystal formers also produced N–H····N hydrogen bond as well as N–H····O pair-wise hydrogen bonds, and also the weaker aromatic π····π interactions, which jointly took part in the crystal packing. The proposed mechanism of formation of co-crystals was further supported by the pKa values of the compounds. Since, the pKa values had no major difference, the possibility of proton transfer, i.e. salt formation, might not be significant. The non-covalent interactions provided a thermodynamically stable crystal lattice arrangement and imparted a crystalline nature to the co-crystals, with improved solubility and significantly lower melting temperatures in comparison to pure drug crystals as these were weaker bonds than the covalent bonds.
Based on different characterization findings the release of three optimized formulations (SN-3, SN-5 and SN-9) was checked at pH 4.0, 6.8 and 7.4 and it was compared with % cumulative release of ETD. The percentage cumulative release of co-crystals in the acidic pH was found to be minimum due to the acidic nature of the drug. Maximum percentage cumulative release of co-crystals was found to be 83.551% at 12 h. From all in-vitro drug release data of co-crystals with ETD-CA and ETD-TA, It was concluded that the co-crystal with CA (ETD-CA) showed best release profile. For some of the co-crystals formulated with CA, formulations SN-3 and SN-5 showed maximum release for an extended period of time i.e. 24 h. Out of these two, SN-5 displayed 80.5465% release in 6 h and 94.808 % in 24 h at pH (6.8). Therefore, it was selected as the optimized batch. Hence, the release pattern depended on the type of co-former used. From this release profile it could be revealed that the co-crystals formed with CA displayed highest solubility as well as best release profile.
The calculated regression coefficients for zero order, first order, Higuchi’s models and Korsmeyer-peppas were 0.574, 0.804, 0.979 and 0.964 for SN-5 batch of co-crystals. It was found that in vitro % drug release of SN-5 batch of co-crystals was best explained by Higuchi’s models as the plot showed the highest linearity [20; 21].
The solubility studies were performed with the ETD and co-crystals in distilled water and PBS pH 6.8. The solubility of ETD, ETD-CA and ETD-TA was found to be 19.35, 79.99 and 77.98 mg/L in distilled water. The solubility of ETD, ETD-CA and ETD-TA was found to be 18.06, 48.19 and 39.05 mg/L in PBS pH 6.8. The increase in solubility of ETD, ETD-CA and ETD-TA was found to be 1.39, 6.14 and 5.81-fold in distilled water. The increase in solubility of ETD, ETD-CA and ETD-TA was found to be 1.29, 3.38 and 2.91folds in PBS pH 6.8. This denoted that the solubility increase was more in distilled water than PBS pH 6.8. The maximum increase in solubility (61.4%) was seen in SN-5. Based on overlay graph findings dissolution study was performed at three different pH. Batch SN-3, SN-5 and SN-9 showed faster % cumulative release. The SN-5 and SN-9 co-crystals showed good solubility due to the irregular and asymmetrical structure of the co-crystals as seen in SEM images. The irregular shaped co-crystals showed maximum solubility.
These co-crystals were characterized by melting point and change in melting point was observed i.e. melting point was increased due to the different co-formers used and it further denoted increase in solubility of co-crystal due to the ability of a molecule to pack tightly into a crystal lattice. A slight shift in FTIR peaks were found which reflected some interaction between drug and co-formers but no change was seen in the chemical properties of drug. SEM images of co-crystals were observed, they were irregular in shape. This irregularity in shape is probable reason for enhanced solubility [11].
In DSC, the melting point was increased because different co-formers were used with pure drug and it denoted increase in solubility of co-crystals due to ETD-TA and ETD-CA formation, with more hydrogen bonds. Thus, the polarity of the co-crystals increased which confirmed the maximum solubility and maximum melting point of SN-5 (79.99mg/L and 156.59°C).
The PXRD crystallogram pattern of co-crystals showed a variation in peak intensity and formation of new peaks as compared to that of the pure ETD. Hence, it revealed that some chemical bonding occurred between pure ETD and co-formers resulting in the formation of co-crystals. This change in the PXRD pattern could be due to the change in the crystal lattice structure owing to the interaction between the drug and co-formers [11]. Characteristic peaks in crystallogram of pure drug appeared at different position at °2 theta scale- 10.25°, 16.28°, 16.58°, 23.29°, 24.50° with intensities in percentage 100.00, 24.60, 32.42, 45.36, 24.58 respectively. Characteristic peaks in crystallogram of co-crystal with citric acid (ETD-CA, SN-5) as co-former, appeared at different position at °2 theta scale- 10.39°, 16.39°,18.21°, 19.59°, 26.17°, 31.10° with intensities in percentage 86.30, 51.82, 16.02, 15.15, 100.00, 11.87 respectively. The peak at 10.39° was almost same as in the crystallogram of drug but some new peaks were observed at 18.21°, 19.59°, 26.17°, 31.10°. These new peaks confirmed the formation of new crystals lattice pattern in the co-crystal as compared to the pure drug. These studies indicated the formation of new crystalline phase due to physical or chemical interaction between drug and co-former. The main aim behind formation of co-crystals was to increase aqueous solubility of drug and as well as to improve its dissolution profile [22].
Drug content for optimized batch was found to be 85.23%. Capsules of SN-5 were prepared and compared with marketed ETD, % release of SN-5 capsules showed maximum release in 4 h whereas marketed ETD showed maximum release in 12 h. It denoted that the solubility of co-crystals is more as compared to ETD.
The optimized batch SN-5 was used for in vivo study and stability studies. The formulation SN-5 was used for stability studies. Both the formulations were stored at 4 ± 2°C, 30 ± 2°C and 40 ± 2°C, 75 ± 5% RH. The residual drug content values of ETD co-crystal stored at 4 ± 2°C was found to be higher as compared with formulation stored at room temperature and 45 ± 2ºC. So, it can be concluded that the optimized formulation was more stable at 4 ± 2°C and tend to degrade faster at higher temperature. Thus, it is recommended that the formulation should be stored in a refrigerator for a longer shelf life (Table 5).
The enhanced potential of SN5 in decreasing the NO and cytokine levels could be due to enhanced solubility of the co crystals which could have the ability to penetrate the cells.
The pharmacodynamic (PD) and pharmacokinetic (PK) study was performed on albino wistar rat. Oral administration of co-crystal of ETD (20mg/kg) showed enhanced therapeutic efficacy. The statistical result of one-way ANOVA indicated that group treatment differed significantly, the F test value was found to be 5.325 which was greater than tabulated p value (p = 3.10) which showed reduction in inflammation level in comparison with anti-inflammation control group. Paw of rat Group I (treated with 0.9% NaCl), 1) before induction of inflammation, 2) after induction of inflammation at 2 h, revealed that inflammation remained up to 24 h. Percent inhibition of inflammation was greater by co-crystal of ETD than by marketed formulation. Hence, SN-5 was the better formulation than the marketed formulation. In PK, Cmax of ETD and SN-5 was found to be 4.19 µg/ml and 9.12 µg/ml, tmax of ETD and SN-5 was found to be 3.01 h and 4.01 h. Cmax got increased in SN-5 than ETD and tmax decreased in SN-5 than ETD. This indicated enhanced solubility and bioavailability. The t1/2 of formulation increased upto 20.11 h demonstrating prolonged release.