Development of Ketoprofen-p-Aminobenzoic Acid Co-Crystal: Formulation, Characterization, Optimization and Evaluation

44 Co-crystal is a promising class of solids that may provide options for improved properties . In the 45 present study, ketoprofen- p -aminobenzoic acid (KP-PABA) co-crystal were prepared to sought 46 enhanced solubility and dissolution rate of drug. KP-PABA co-crystal were prepared by solvent 47 evaporation technique employing central composite experimental design, selecting independent 48 variables as concentration of drug and PABA whereas dependent variables were assumed to be 49 solubility and % drug release. The optimized batch as suggested by the experimental design was 50 characterized by FTIR, DSC, XRD, SEM and NMR studies and further, evaluated for in-vitro 51 and in-vivo anti-inflammatory and analgesic activities. The solubility and % drug release of 52 different batches of co-crystal was found to be between 34.20-60.11 µg/ml and 68.11-93.45%, 53 respectively. Co-crystal containing ketoprofen and PABA in molar ratio (1:1) was found to be 54 optimized formulation batch. Physical characterization by X-ray diffraction spectra and 55 differential scanning calorimetric studies confirms the crystallinity of prepared co-crystal. The 56 half maximal inhibitory concentration (IC 50 ) values for in-vitro anti-inflammatory activity comes 57 out to be 34.04 µM for ketoprofen and 4.373µM for optimized formulation, exhibiting almost 8- 58 fold amplification indicating higher anti-inflammatory effect of optimized batch as compared to 59 drug ketoprofen. The results of in-vivo anti-inflammatory activity carried out by rat paw edema 60 method revealed that the optimized batch of co-crystal preparation provided a significant % 61 inhibition in paw volume in contrast to standard drug in wistar rats. In this case, a crystalline 62 molecular complex of drug Ketoprofen, that demonstrate poor aqueous solubility, with p- 63 aminobenzoic acid was recognized that further set out an improvement in solubility and also in 64 anti-inflammatory activity of the drug in wistar rats.


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Solubility studies 140 Ketoprofen pure drug and each batch of KP-PABA co-crystal formulations containing 141 ketoprofen equivalent to 10 mg was dispersed in 10 ml of distilled water, separately and are kept 142 on continuous shaking for 48 h for equilibration at room temperature (25°C) to determine the 143 solubility of ketoprofen. The obtained solution was filtered by 0.45µm millipore filter paper and 144 the drug content was determined by taking absorbance at 260 nm using uv-vis 145 spectrophotometer. The amount of drug was calculated using the calibration curve in water.

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Determination of drug content 147 The different batches of KP-PABA co-crystal formulations containing drug equivalent to 5mg 148 were dissolved separately in 25 ml of phosphate buffer (pH 7.4). The samples were filtered 149 through 0.45µm milipore filters and after appropriate dilution the samples were analyzed by uv- 150 vis spectrophotometer at 260nm [35]. 151 Total Drug Content (TDC) was calculated by the following equation.  under nitrogen flow at a rate of 50 ml/min.

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Scanning electron microscopy (SEM) 185 The surface morphology and shape of optimized batch of KP-PABA co-crystal formulation was 186 examined using SEM (JSM-6100 scanning microscopy, Japan). The sample (optimized batch) 187 after coated with gold was mounted on aluminium stub containing double adhesive carbon tape.

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The photographs were observed at acceleration voltage of 10 kV. The optimized batch of co-crystal was kept for the accelerated stability studies according to ICH 197 guidelines (40 ± 2 o C and 75 ± 5% RH) for a period of 6 months in a stability chamber. The 198 samples were placed in hermetically sealed vials containing rubber plugs and aluminum bung.

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The stored co-crystal were taken out after 6 months and evaluated for the drug content  Where, T 1 and T 2 are reaction time in seconds before and after treatment with drug, respectively.

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Data was analyzed by one-way ANOVA method and further followed by Tukey's post-hoc test, 247 statistically and is denoted as P value.

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The preparation of co-crystal using ketoprofen and PABA was optimized using 2-factor, 3 level  The outcome of solubility (Y 1 ) and in-vitro drug release profile (Y 2 ) of the KP-PABA co-crystal, 258 formulated according to the experimental design expert protocol is exhibited in Table I

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The responses, solubility (Y 1 ) and in-vitro drug release (Y 2 ) were fitted best into the quadratic

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The polynomial models for the responses solubility (Y 1 ) and in-vitro drug release (Y 2 ) can also 275 be expressed by the equation (6) and (7), respectively. The synergistic and antagonist effect is concentration of PABA (X 2 ) seems to be more pronounced as compared with that of 282 concentration of ketoprofen (X 1 ).
The denouement of ANOVA test on the response surface quadratic model are summarized in 286 The optimization equation 6 and 7, relating the response and independent factors, was fabricated

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The release profile of co-crystal is compared with equivalent concentration of pure drug solution 317 to study release rate kinetics. The release of ketoprofen from co-crystal formulation and drug 318 solution was put into various kinetic models to estimate the kinetics and mechanism of release 319 (Table III). The release rate data for the formulation was found to be fitted best into Zero order