Formulation, Optimization, in Vitro and in Vivo Investigation of Ketoprofen - Fumaric Acid Co-crystal for the Solubility Enhancement of Ketoprofen

Formulation, Optimization, in Vitro and in Vivo Investigation of Ketoprofen - Fumaric Acid Co-crystal for the Solubility Enhancement of Ketoprofen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Formulation, Optimization, in Vitro and in Vivo Investigation of Ketoprofen - Fumaric Acid Co-crystal for the Solubility Enhancement of Ketoprofen Meenakshi Bhatia, Ashwani Kumar, Vikas Verma, Snehlata Yadav, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-905168/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present piece of research work is framed as improving the solubility of ketoprofen by forming co-crystal using fumaric acid as a coformer. Co-crystal of ketoprofen and fumaric acid were prepared by simple solvent assisted grinding. The independent variables i.e. drug and coformer were mixed in 1:1 molar ratio and dependent variables were assumed to be solubility and % drug release. Differential scanning calorimetry, fourier transform infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance and scanning electron microscopy techniques were used to characterize the preparation of optimized batch of co-crystal and further, evaluated for in-vitro and in-vivo anti-inflammatory and analgesic activities. Based on results of solubility and dissolution rate studies the drug showed 4-5 fold improvement in both the properties on co-crystallisation. The values of Gibbs free energy are negative at all levels of carrier demonstrating spontaneity of drug solubilization process. The IC 50 value of optimized batch of co-crystal formulation and pure drug was observed as 327.33 µg/ml and 556.11 µg/ml, respectively, demonstrating that co-crystal formulation possesses more percentage protection against protein denaturation than the drug ketoprofen. In-vivo (anti-inflammatory and analgesic) activities revealed that optimized batch of co-crystal formulation delivered a rapid pharmacological response in wistar rats and albino mice when compared with standard drug. Drug Delivery ketoprofen fumaric acid Co-crystal non-covalent interactions solubility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Numerous strategies to improve bioavailability of drugs with poor/low solubility include drug micronization in to amorphous form [ 1 ], complexation with hydrophilic carrier [ 2 – 4 ], solid dispersion [ 5 , 6 ], micellar solubilization [ 7 ], nanoparticle technology [ 8 – 11 ], self-emulsifying drug delivery systems [ 12 – 14 ], salt formation [ 15 ], liposomes [ 16 ], nanostructured lipid carriers (NLC) [ 17 , 18 ], prodrug [ 19 ] and formation of co-crystal [ 20 – 25 ] etc. However, there are disadvantages associated with these techniques like agglomeration, instability during storage, requirement of advanced/or sophisticated instruments, tacky product etc. Therefore, co-crystallization appears to be a potential method for improving the solubility, dissolution and thus bioavailability of crystalline materials being a direct, viable, economical and green method. Co-crystal is defined as a multicomponent crystalline material possessing two or more molecules (i.e. drug and coformers) that are held together by noncovalent interactions in the same crystal lattice [ 26 ]. Co-crystallization can appreciably reorganize the physiochemical properties of active pharmaceutical ingredient (API) by introducing a coformer that interconnected with the target API in a defined stoichiometric ratio through intermolecular interactions [ 27 ]. The enhancement in solubility is explained by the two step mechanism as first the solute molecules are released from the crystal lattice followed by the solvation of released molecules. Also the Gibbs free energy (∆G) associated with this system (∆G solution ) involves free energy allied with release of solute molecules from the lattice i.e. ∆G lat and solvation barrier (∆G solv ) that may be attributed as: ∆G solution =∆G lat +∆G solv (1) At the moment, the free energy equated with lattice interaction and solvation barrier because trivial, the dissolution of co-crystal is improved due to drop free energy change for solubilization [ 28 , 29 ]. Here, in this study ketoprofen, a nonsteroidal anti-inflammatory drug, belonging to the BCS class II was selected as a model drug that pertain low solubility and high permeability. Ketoprofen works by inhibiting the enzyme cyclooxygenase-I and II, resulting in decreased production of precursors of prostaglandins and thromboxanes, thereby, displaying antipyretic, anti-inflammatory and analgesic activities. Nanosuspensions with Phosphol-ipon 80 [ 30 ], soild dispersion [ 31 ], microemulsion-based gel [ 32 ], emulgels [ 33 ], nanoparticles [ 34 – 36 ], solid lipid nanoparticles [ 37 ], nanostructured lipid carriers [ 38 ], prodrugs [ 39 ], micro and nanocomposites with PLGA [ 40 ] and liquisolid [ 41 ] etc. have already been used to increase the solubility of ketoprofen. However, co-crystal of ketoprofen with nicotinamide by grinding method are reported in literature that depicted higher anti-inflammatory activity [ 42 ]. The dicarboxylic acid viz. fumaric acid was selected as co-crystal coformer in the present study. Fumaric acid is a popular coformer that has been widely explored for the production of co-crystal of different active pharmaceutical ingredients like glycine [ 43 ], meloxicam [ 44 ], (DL)-phenylalanine [ 45 ], adenine [ 46 ], 5-Fluorocytosine [ 47 ], arginine [ 48 ] berberine chloride [ 49 ], Ketoconazole [ 50 ] etc. The ketoprofen and fumaric acid were selected on the basis of the pK a rule. Fumaric acid exhibits an aqueous solubility of ∼0.6 g/l at 24°C and has pK a value of 3.03 whereas pK a value for ketoprofen is 3.88 and the value of ΔpK a (pK aacid − pK abase ) is − 0.85. According to Bhogala et al., at negative values of ΔpK a co-crystal formation is expected [ 51 ]. There is no study reported on co-crystal formation of ketoprofen with fumaric acid. In the present piece of research-work formation of ketoprofen co-crystal with fumaric acid is described with the objective to enhance aqueous solubility of drug utilizing simple and reproducible technique of solvent-assisted grinding. The preparation of co-crystal was achieved as per experimental design protocol as recommended by the 2-factor, 3 level CCD (central composite experimental design, Design Expert software version 11.0). The solubility, Gibbs free energy, entrapment efficiency and in-vitro drug release for each batch was determined and numerically optimized. The optimized batch as suggested by design expert was characterized by FT-IR, DSC, XRD, SEM and NMR studies. The evaluation of optimized batch was carried out by in-vitro / in-vivo anti-inflammatory and analgesic activity employing suitable animal models like rat paw edema and tail flick methods. Further, the mechanism of drug release was determined by fitting the release data in various release kinetics models. Experimental Materials Ketoprofen (ket) was received as a gift sample from Infinity Laboratories Pvt. Ltd (Behra, India). Fumaric acid (FA) was supplied by Central Drug House (P) Ltd., New Delhi. Ethanol, potassium chloride, di-sodium hydrogen orthophosphate, potassium di-hydrogen orthophosphate, sodium chloride and carrageenan were obtained from Hi-Media lab. Pvt. Ltd. All other chemicals & reagents were of analytical grade and used as received. The chemical structures of ketoprofen and fumaric acid were obtained from pubchem database [https://pubchem.ncbi.nlm.nih.gov]. Method Preparation of ketoprofen-fumaric acid co-crystal Ketoprofen-fumaric acid co-crystal (Ket-FACo) were prepared by the simple solvent-assisted grinding technique as reported earlier [52, 53]. Ketoprofen and fumaric acid were used in stoichiometrically equal ratio and after carefully weighing were ground using a mortar and pestle for 30 minutes with the dropwise addition of ethanol. The powder was dried, preserved in airtight vials and stored in a desiccator till further use. Experimental Design The preparation of co-crystals using ketoprofen and fumaric acid was optimized using 2-factor, 3 level central composite experimental design. The concentration of ketoprofen (254.29-508.58 mg) (X 1 ) and concentration of FA (116.07-232.14 mg) (X 2 ) were designated as formulation variables whereas the % drug release and solubility (µg/ml) were selected as response variables (Table I) and each of the independent variable was considered at 3 levels (-1, 0, and 1). Experimental design and statistical analysis of the data was realized by Design Expert software (version 11.0). Solubility studies To determine the solubility of ketoprofen and each batch of Ket-FACo formulations carrying drug equivalent to 5 mg and pure drug (5 mg) was dispersed in 20 ml of distilled water and phosphate buffer solution pH-7.4, separately and were kept on continuous shaking at room temperature for 48 h. The obtained solution was then filtered by 0.45µm millipore filter paper and the drug content was measured by taking absorbance at 260 nm using uv-vis spectrophotometer. The amount of drug was measured using the calibration curve in water [5]. The Gibbs free energy of transfer (ΔG) of ketoprofen present in different batches of co-crystal is determined using equation 2. ΔG = -2.303RT log S o /S s (2) S o is the solubility of the co-crystal in water and Ss is the solubility of pure drug in water, R = 8.31 J k -1 mol -1 and T = 298.15°C. Percentage Drug content To ascertain the drug content of every batch of Ket-FACo formulation, co-crystal equivalent to 5 mg were weighed and dissolved separately in 25 ml of phosphate buffer (pH 7.4) in volumetric flask with continuous stirring for 24 h on a magnetic stirrer [ 54 ], after proper dilutions, drug content was determined using uv-vis spectrophotometrically at 260 nm. The following equation was used to calculated Total Drug Content (TDC) \(\text{T}\text{o}\text{t}\text{a}\text{l} \text{d}\text{r}\text{u}\text{g} \text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t} \left(\text{\%}\right)=\frac{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{i}\text{n} \text{c}\text{o}-\text{c}\text{r}\text{y}\text{s}\text{t}\text{a}\text{l}}{ \text{W}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{c}\text{o}-\text{c}\text{r}\text{y}\text{s}\text{t}\text{a}\text{l} }\) ×100 (3) In vitro drug release profile In vitro dissolution studies of pure drug (Ketoprofen) and each batch of Ket-FACo formulation containing Ketoprofen equal to 20mg were conducted in 900ml PBS (phosphate buffer solution, pH-7.4) at 37 ± 0.5 o C with constant stirring speed of 50 rpm. The powder was dispersed over the dissolution medium. Aliquots of sample (5ml) were withdrawn at different time intervals for 1 h and restored with an equal volume of the dissolution medium to keep sink conditions in the course of the experiment. The 0.45µm milipore filters was used for the sample filtration and the drug concentration in the samples was determined by measuring the absorbance of the samples at a wavelength of 260 nm using the uv–vis spectrophotometer followed by determination of mechanism of release by fitting the release rate data in various release kinetic models [ 6 ]. In vitro drug release profile In vitro dissolution studies of pure drug (Ketoprofen) and each batch of Ket-FACo formulation containing Ketoprofen equal to 20mg were conducted in 900ml PBS (phosphate buffer solution, pH-7.4) at 37±0.5 o C with constant stirring speed of 50 rpm. The powder was dispersed over the dissolution medium. Aliquots of sample (5ml) were withdrawn at different time intervals for 1 h and restored with an equal volume of the dissolution medium to keep sink conditions in the course of the experiment. The 0.45µm milipore filters was used for the sample filtration and the drug concentration in the samples was determined by measuring the absorbance of the samples at a wavelength of 260 nm using the u v – vis spectrophotometer followed by determination of mechanism of release by fitting the release rate data in various release kinetic models [6]. Characterization Fourier Transform Infrared Spectroscopy (FT-IR) analysis FT-IR analysis was used for the interaction between drug and carrier. FTIR spectral analysis of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation was done by FT-IR Perkin-Elmer, Spectrum, US spectrophotometer and the spectrum was documented in the wavelength region of 4000cm -1 to 400 cm -1 using KBr pellet method. X-ray diffraction analysis (XRD) analysis The XRD spectra of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation were obtained using an X-ray diffractometer (Miniflex 2, Rigaku, Japan) at room temperature and at 30kV. The scanning diffraction angle (2θ) ranging from 0 o to 80 o . The Miller index (d hkl ) is used to establish direction and plane in the crystal and is determined using Bragg’s equation (Eq. 4 ). $$\text{nλ=2d}\text{hkl}Sin\theta$$ 4 Here λ and n denote the wavelength (1.5418Å) and order (n = 1, first order), respectively; θ is the Bragg’s angle. Differential scanning calorimetry (DSC) DSC thermograms of ketoprofen, Fumaric acid and optimized batch of Ket-FACo formulation were recorded using DSC (Mettler Toledo, Switzerland), the samples were heated within the temperatures range of 20-400°C with a scanning rate of 10°C/min in aluminum pans under nitrogen flow at a rate of 50 ml/min. Scanning electron microscopy (SEM) The surface morphology and shape of optimized batch of Ket-FACo formulation was observed using scanning electron microscope (JSM-6100 scanning microscopy, Japan). Nuclear Magnetic Resonance (NMR) Spectroscopy The NMR spectra of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation after dissolving in DMSO-d 6 were examined using Bruker Avance AV 400 NMR spectrometer (Bruker, Karlsruhe, Germany) to get solution 13 C NMR data at a temperature of 293 K using Tetramethylsilane (TMS) as an internal standard [55]. Data was interpreted using Mnova program (Mestrelab Research, Santiago de Compostela, Spain). Stability studies The optimized batch of co-crystal was kept for the accelerated stability studies according to ICH guidelines (40 ± 2 o C and 75 ± 5% RH) for a period of 6 months in a stability chamber. The samples were placed in hermetically sealed vials containing rubber plugs and aluminum bung. The stored co-crystal were taken out after 6 months and evaluated for the drug content (according to the method described in earlier section of drug content, n=3 ) and for any physical changes [47]. Biological Evaluation of Ket-FACo In-vitro anti-inflammatory activity Egg albumin denaturation method was used to determine in-vitro anti-inflammatory activity of drug ketoprofen and optimized batch of Ket-FACo [ 56 – 58 ]. The mixtures containing of fresh hen’s egg albumin (0.2ml), Phosphate buffered saline 7.4 (2.8ml) and different concentrations of Ket-FACo formulation (2 ml, containing ketoprofen equivalent to 10 mg) (125, 250, 500, 750 & 1000 µg/ml in Dimethyl sulfoxide as solvent). The mixture was placed in an incubator at a temperature of 37 ± 2 o C for 20 min incubation followed by heating at 70 o C. After cooling at room temperature, the absorbance measured at λ max of 660nm to determine % inhibition. Similar procedure was done for the drug (ketoprofen) with the similiar concentrations as engaged for the formulation as a reference or control. The percentage protection from protein denaturation was calculated as per Eq. 5 % protection from denaturation= \(\frac{Abs. of control-Abs. of sample}{Abs. of control}\) X 100 (5) The half maximal inhibitory concentration (IC 50 ) values of ketoprofen and optimized formulation was calculated by nonlinear regression analysis. In-vivo carrageenan-induced anti-inflammatory activity The protocol with registration no. CPCSEA Reg. no-IAEC/2021/10–19 was approved for animal study by the Animal Ethical Committee, Guru Jambheshwar University of Science and Technology, Hisar, India. Wistar rats (150–180 g) were distributed into three groups comprising of six animals each. Group I (control treated) with carrageenan was kept as control, Group II (standard drug) was treated with drug-ketoprofen (10 mg/kg) and Group III (test compound) was treated with Ket-FACo (equivalent to ketoprofen 10 mg/kg body weight) that is administrated orally. 1% suspension of carrageenan (0.1 ml) in normal saline, was administered as subplantar injection in the left hind paw of albino wistar rats, after 1 h of oral administration of the test materials. The paw volume was measured using vernier caliper at 1, 2, 3, 4 and 5 h after the carrageenan injection. The % inhibition in paw volume was calculated using Eq. 6 , $$\% inibition=\frac{Vc-Vt}{Vc}\times 100$$ 6 Where V c and V t is the inflammatory increase in paw volume control group and test group respectively [ 59 ]. Analgesic activity (Tail Flick Method) Tail flick method was used to measure analgesic activity using a radiant type analgesiometer. Three different groups (control, test and standard) of Swiss albino mice (25-30g) containing six mice each group were taken for study. The tail flick reaction time for each animal was recorded six times before administering the drug and the mean was used as predrug reaction time. A dose of the standard drug (Ketoprofen) and test compound (optimized batch of Ket-FACo) containing ketoprofen equivalent to 5 mg/kg of body weight in 0.9% w/v sterile saline was orally administered to mice. After administration of the drug, the tail flick reaction time was measured at 0, 1, 2, 3, 4 and 5 hrs. % Analgesic activity (PAA) was calculated as per Eq. 7. PAA= (T 2 -T 1 )/T 1 ˣ 100 (7) Where T 1 are reaction time in second before treatment of drug(s) and T 2 are reaction time in second after treatment of drug(s) [ 60 , 61 ]. Data was analyzed by one-way ANOVA followed by Tukey’s post-hoc test and statistically was denoted as P value. Results And Discussion The preparation of co-crystal using ketoprofen and fumaric acid was optimized using 2-factor, 3 level CCD. The concentration of ketoprofen (X 1 ) and concentration of fumaric acid (X 2 ) were designated as formulation variables whereas the % drug release and solubility (µg/ml) were picked as response variables. The TDC of different batches of ket-FACo was found to be between 94.48 to 97.81 %, thus depicting that handsome amount of drug has been loaded and also no physical changes were observed during stability studies and even after six months. In different batches of ket-FACo solubility in phosphate buffer (PBS pH-7.4) values range from 29.08 to 10.63 µg/ml whereas ketoprofen solubility was found to be 3.64 µg/ml. As presented in Table 1, solubility of ket-FACo varied in the range of 30.68-57.44µg/ml. The pure ketoprofen dispensed a solubility of 11.24 µg/ml in water at room temperature. Table 1. Formulation parameters and responses for central composite experimental design. Batch Conc. Of Ketoprofen (mg) Conc. Of Fumaric acid (mg) Solubility (µg/ml) Solubility in PBS pH-7.4 (µg/ml) ∆G (KJ/MOL) % Drug release in 60 min. Drug content (% ) F1 254.29 116.07 30.68±0.01 10.63±0.32 -3.49 62.21±0.03 95.89±0.06 F2 508.58 116.07 42.04±0.03 18.36±0.12 -1.92 70.01±0.05 95.66±0.01 F3 254.29 232.14 48.3±0.01 23.39±0.02 -3.04 71.94±0.02 95.33±0.04 F4 508.58 232.14 57.44±0.05 29.08±08 -2.68 83.68±0.01 97.81±0.01 F5 254.29 174.10 45.97±0.2 21.59±0.22 -2.54 70.52±0.06 96.39±0.06 F6 508.58 174.10 51.08±0.3 28.46±0.13 -3.19 80.59±0.07 95.60±001 F7 381.43 116.07 36.47±0.1 15.16±0.41 -3.32 66.52±0.02 95.04±0.02 F8 381.43 232.14 53.66±0.09 28.52±0.17 -2.93 79.71±0.04 97.47±0.07 F9 381.43 174.10 49.08±0.08 27.46±0.27 -3.36 75.54±0.05 97.25±0.03 F10 381.43 174.10 48.14±0.4 26.94±0.31 -3.08 75.04±0.2 95.53±0.05 F11 381.43 174.10 49.51±0.04 27.67±0.29 -3.04 75.59±0.3 94.88±0.01 F12 381.43 174.10 50.23±0.06 26.54±1.1 -3.15 73.52±0.1 94.48±0.06 F13 381.43 174.10 50.16±0.03 26.83±0.11 -3.12 73.02±0.03 96.35±0.08 Ketoprofen 11.24±0.03 3.64±0.14 23.05±0.01 All values are expressed as mean ± S.D., n=3. Table 1 shows the results of solubility of different batches of Ket-FACo organized according to design protocol. The responses produced were fitted into several polynomial models using CCD. The response solubility was fitted greatest into quadratic model with none transformation of the data. The polynomial models for the responses solubility (Y 1 ) can also be expressed by the equation (8) with determination correlation (R 2 ) of 0.9659. Y 1 = 49.36+4.27X 1 +8.37X 2 -0.5550X 1 X 2 -0.6822X 1 2 -4.14X 2 2 (8) Table 2 summarizes the results of ANOVA on the solubility and % drug release response surface model, demonstrated that model was found significant with lack of fit as non-significant. Adequate precision of solubility is found to be 28.33 indicates an adequate signal. The adequate precision measuring signal to noise ratio (greater than 4) is desirable. Fig. 1 (a) show the collective effect of concentration of ketoprofen and fumaric acid on solubility. It may be reckoned from the plots that a curvilinear relationship exists between independent and dependent variables. It is also inferred from the plot that higher level of ketoprofen and fumaric acid results in increase in solubility. However, the effect of the concentration of fumaric acid (X 2 ) seems to be more pronounced as compared to the concentration of ketoprofen (X 1 ). This increase in solubility may be due to formation of soluble complex between ketoprofen and fumaric acid. Fumaric acid presents higher solubility than the drug that comes out of the crystal lattice. The drug in co-crystal get supersaturated in aqueous medium and possesses more energy as compared to crystalline phase, thereby, exhibit marked increase in solubility than the pure drug. In vitro drug release Results of in-vitro drug release (Table 1) revealed that 62.21 to 83.68% and 23.50% of ketoprofen got released from different batches of Ket-FACo and pure drug solution respectively, in 1 h study. This rise in percentage drug release from Ket-FACo as compared to pure drug may be associated to the solubility data. As previously mentioned that the fumaric acid form the soluble complex with the drug and thereby increase the drug wettability that leads to a better solubility and further heads towards better rate of drug release. The adjusted polynomial equation obtained for the in-vitro drug release (Y 2 ) is shown in equation 9 with determination correlation of adjusted R² of 0.958 and predicted R 2 of 0.905. The predicted is in reasonable agreement with the adjusted R² with a difference less than 0.2. Y 2 ­=74.84+4.93X 1 +6.10X 2 +0.985X 1 X 2 -0.0336X 1 2 -2.47X 2 2 (9) Table 2 recapitulating the results of ANOVA of in-vitro release data on response surface model fitted best in quadratic model (after none transformation of the data). The responses observed were fitted into different polynomials models using the experimental design. Adequate precision of in-vitro drug release is found to be 25.57 indicates an adequate signal. Table 2. Model summary statistics. Model Lack of Fit Response factor(Y) F-value Prob.>F R 2 Adeq.Prec. C.V (%) F-value Prob.>F Y 1 69.00 <0.0001 0.9659 28.33 2.79 4.09 0.1035 Y 2 56.65 <0.0001 0.975 25.57 1.60 0.937 0.501 To attain stability a natural tendency to acquire minimum Gibbs energy is always there. All the values of ΔG are negative (Table 1) at all levels of carrier demonstrating spontaneity of drug solubilization process. Optimization The optimization equations 8 and 9, involving the response and independent factors were assembled based on a quadratic model. To the responses i.e. solubility and in-vitro drug release the desirability function was applied with constraints to obtain the higher level of both, the batch F4 comes out to be optimized batch. In this fashion, the formulation containing fumaric acid (228.82mg) as coformer and drug content (508.58 mg) with addition of ethanol, established the maximum desirability, was organized and evaluated. The mathematical optimization tool with desirability method was employed to prepare co-crystal. The constraints of maximum solubility and maximum % release was imposed on independent variables for optimization. The parameters recommended by the design were concentration of ketoprofen (508.58 mg) & concentration of fumaric acid (228.82 mg) that provide co-crystal with solubility of 56.06 µg/ml (predicted value 56.63 µg/ml) and % drug release 83.35% (predicted value 84.22%). The closer agreement between predicted and observed values indicated the high prognostic ability of the model. Fig. 2 shows the in vitro release profile of ketoprofen as pure drug and optimized batch (F4) of co-crystal formulation. The release rate data of ketoprofen from co-crystal and from drug solution was fitted into several kinetic models to estimate release kinetics and mechanism of drug release. The release rate data was found to be put best into Higuchi model (with R 2 =0.985) of release kinetics. Further, the value of n=0.475 (0.43<n<0.85), release exponent of Korsemeyer and Peppas equation, indicated that the release of ketoprofen from co-crystal occurs by diffusion and erosion of the matrix. Fourier Transform Infrared Spectroscopy (FT-IR) analysis FTIR is an excellent analytical technique to study the deviations in the position caused by the vibration modes of the functional groups. This technique reveals the shift in characteristic peaks of drug and coformer due to co-crystal formation involving H bonding between the corresponding functional groups. The spectra of ketoprofen fig. 3 (a) showed characteristic absorption band at 2979.27 cm -1 due to –CH stretching. The peak appearing at 1697.76 cm -1 can be ascribed to -C=O stretching of acid while peak appearing at 1655.77 cm -1 is due to -C=O stretching of ketone. The absorption bands at 1598.67 cm -1 (-C=C= stretching), 1442.21 cm -1 (-C=O stretching of aromatic ring), 1420.59 cm -1 (-C-H deformation of -CH 3 asymmetrical) and 1370.04 cm -1 (-C-H deformation of -CH 3 symmetrical) also appeared. The FTIR spectra of fumaric acid exhibit peaks at 3084.26 cm -1 , 1684.31 cm -1 , 1422 cm -1 ascribed to the -O-H stretching, -C=O stretching vibration and –C-C aromatic stretching, respectively. The FTIR spectra of ket-FACo displayed that -C=O stretching of –COOH group of ketoprofen get shifted from 1697.76 cm -1 to 1667.30 cm -1 and the peak due to -C=O stretching of fumaric acid at 1655cm -1 got disappeared. Therefore, FT-IR analysis confirms the interactions occurring between ketoprofen and fumaric acid. These interactions are essentially hydrogen bonds between the carboxylic group of the fumaric acid and the main functional groups of ketoprofen (-C=O and -O-H) which are able to generate supramolecular heterosynthons. Powder X-ray diffraction analysis (PXRD) The powder X-ray diffraction (PXRD) pattern of ketoprofen, fumaric acid and optimized batch of ket-FACo illustrated in fig. 3 (b). The diffraction peaks (and Miller indices) at 2θ of 12.74 (100), 18.52 (200), 22.85 (211), 24.00 (221), 26.32 (222), 28.920 (300), 36.71 (322) and 22.86 (211), 28.92 (300), 29.49 (310), and 30.03 (311) showed crystalline structure of ketoprofen and fumaric acid respectively. The major diffraction peaks at 2θ, 18.57 (200), 23.27 (211), 28.01 (222) and 29.28 (300) were also observed in PXRD spectra of co-crystal that portrayed crystalline nature of resultant product. The distinctive PXRD pattern of the ket-FACo was distinguishable from ketoprofen and fumaric acid, this outcome specifies the formation of a new crystal phase [62]. Differential scanning calorimetry (DSC) DSC thermograms [fig. 4 (a)], reported that pure ketoprofen showed a sharp endotermic peak at 94.5º that corresponds to its melting point. The peak at 280.4ºC attributed to melting point of fumaric acid. In the thermogram of the prepared co-crystal, peaks were found to be displaced and difference in intensity is also observed from that of its constitutional components indicating the occurrence of weak cohesive forces that bonded together by reversible hydrogen bonding, suggesting the development of co-crystal formation. The thermal behavior of the ket-FACo was prominent, with a different melting transition from that seen with either of the constitutional components; this recommends the formation of a new phase. Scanning electron microscopy (SEM) The SEM image of the optimized batch of ket-FACo [fig. 4(c)] depicted good crystalline characteristics. This crystalline character was reinforced by the XRD data, as discussed earlier. The voids over the surface of the co-crystal may brace the imbibition of the solvent and biological fluids and thereby proliferating the solubility and bioavaibility of ketoprofen as estimated. Nuclear magnetic resonance (NMR) Spectroscopy NMR spectroscopy is used to characterize the co-crystal by studying the chemical environment of their nuclei and hydrogen bonding and it also offers valuable information regarding interactions. In the NMR pattern of ket-FACo, the carbonyl carbon of ketoprofen corresponding to 196.46 ppm and140.13 ppm has shifted to 196.10 ppm and 142.22 ppm, respectively. A deviation in the carbonyl carbon of carboxylic group in fumaric acid shifted from 166.41 ppm and 140.51 ppm to 175.52 ppm and 137.45 ppm, respectively (Fig. 5). This suggests an interaction between alcoholic group of fumaric acid and –COOH group of ketoprofen in ket-FACo. Computational studies The constituents of the co-crystal interact through weak non covalent interactions (NCI) [63] and in order to determine the points of contacts between the ketoprofen and coformer fumaric acid, molecular electrostatic surface potential (MESP) analysis was performed and the extreme positive and negative values from MESP are displayed in fig. 6. The magnitude of these values signifies that both H-bond donor and acceptor are present in the crystals of both compounds. MESP of ketoprofen exhibits both positive (+53.53 kcal/mol) and negative (-37.40 and -32.94 kcal/mol) extreme values thus proving that it can form intermolecular as well as intramolecular hydrogen bonds during process of co-crystalization. Similarly, fumaric acid displays positive (+69.87 and +69.85 kcal/mol) and negative (-33.5 and -33.46 kcal/mol) extreme values which confirms its hydrogen bonding capability. It has stronger hydrogen bond donor ability due to higher positive extreme value and on the other hand ketoprofen has higher hydrogen bond acceptor ability due to higher negative extreme value. According to Etter's rule [64], there is more probability of interactions between most polar parts of the molecules in a co-crystal. Therefore, these two compounds will pair in the co-crystal through hydrogen bonding. This pairing of these molecules was accomplished by combining two molecules and energy minimization of combined form. The interaction diagram of the molecules in combination is shown in fig. 7 (a, b). Reduced density gradient (RDG) analysis [fig. 7 (a)] exhibits formation of two hydrogen bonds between two fumaric acid and ketoprofen. Two hydroxyl groups made these two hydrogen bonds (shown as blue colored discs) with two carbonyl oxygen atoms of ketoprofen. Further, van der Waals interactions can be observed between double bond region of fumaric acid and phenyl ring of ketoprofen (shown as green and brown color). The results of RDG calculations are also in line with the MESP predictions. The hydrogen bonding interactions between these two molecules were further confirmed by Hirshfeld surface mapped by electron density with promolecular approximation analysis [fig. 7 (b)]. This calculation shows three regions of high electron density; two are the same as found in RDG analysis while the third is between carbonyl oxygen of fumaric acid and phenyl hydrogen of ketoprofen. These observations are also in agreement with the findings of MESP analysis. The positive parts of one molecule are interacting with negative parts of another molecule. The geometry of the both molecules individually as well as in combined form was minimized using MOPAC [65] with latest PM7 method choosing value of gnorm as 0.01 after optimization with molecular mechanics method. Molecular energy and other various properties of the minimized discrete molecules and co-crystal were calculated with Firefly [66] by density functional theory (DFT) taking 6-31G* basis set in B3LYP method. The MESP calculation, RDG analysis and Hirshfeld surface mapped by electron density with promolecular approximation [67] calculations were performed with Multiwfn 3.8 [68] and visualization was done with the help of VMD [69]. Biological evaluation of the co-crystal In-vitro anti-inflammatory activity The % protection from denaturation of protein is comparably plotted at different concentration of optimized batch of formulation and pure drug ketoprofen (fig. 8). Egg albumin protein denaturation method displayed concentration dependent anti-inflammatory activity by protecting the protein. Half maximal inhibitory concentration (IC 50 ) values of ketoprofen and optimized formulation was calculated by nonlinear regression analysis. The IC 50 values for pure drug ketoprofen and optimized formulation was observed to be 556.11 µg/ml and 327.33 µg/ml respectively. Thus it can be inferred that optimized co-crystal formulation is additionally effective as compared to pure drug in generating anti-inflammatory response. In-vivo anti-inflammatory activity The improvement in activity of ketoprofen and co-crystal formulation was comparatively assesed by the increase in paw volume of control groups. The paw edema volume (before and after drug administration) and % inhibition of edema at different time interval was convinced and displayed in Table 3. The ketoprofen and co-crystal showed inhibition of paw edema as 49.34±0.18% and 60.39±0.15 at the end of 5 h, respectively thus demonstrating quick onset of action by co-crystal in contrast with the pure drug ketoprofen. Statical Analysis:- Data was compared by ANOVA followed by Tukey’s test. The p value is <0.0005 is considered as significant. Table 3. Effect of ketoprofen and optimized batch of ket-FACo formulation on the paw edema induced by carrageenan in Wistar rats. Time (min) Paw volume (mm) Inibition (%) Control Pure drug Co-crystal Pure drug Co-crystal 60 4.25±0.07 4.12±0.03 * 4.02±0.09 * 1.05±0.03 5.17±0.08 # 120 4.54±0.10 3.86±0.06 * 3.53±0.04 * 10.97±0.01 16.65±0.09 # 180 4.96±0.014 3.48±0.04 * 3.22±0.06 * 29.83±0.03 35.08±0.27 # 240 5.49±0.06 3.31±0.05 * 3.04±0.04 * 39.70±0.10 44.62±0.03 # 300 6.11±0.022 3.09±0.03 * 2.42±0.035 * 49.34±0.18 60.39±0.15 # All values are expressed as mean ± S.D., n=6. * Significant (p<0.05) compared to control. # Significant (p<0.05) compared to pure drug (ketoprofen). Analgesic activity The results of the % analgesic activity (PAA) of test, reference and control group are shown in Table 4. The PAA (equation 8) was comparatively evaluated for Ket-FACo and pure drug based on its potential to suppress pain. Ket-FACo showed significant effect in enhancing the pain thershold to a certain extent when compared to that of drug (ketoprofen), thus, stipulating that an improvement in solubility further tweaked the pharmacological response. Table 4. % Analgesic effect of ketoprofen and optimized batch of Ket-FACo by tail flick method in mice. Treatment PAA 0h 1h 2h 3h 4h 5h Standard (ketoprofen) 0.06±0.020 31.30±0.71 * 35.18±0.12 * 42.36±0.17 * 50.93±0.14 * 71.96±0.17 * Test (Ket-FACo) 0.09±0.021 32.14±0.24 * 50.44±0.14 *# 60.23±0.18 *# 65.41±0.12 *# 75.68±0.22 *# Control (vehicle) 0.01±0.012 0.91±0.19 1.32±0.02 0.83±0.10 1.1±0.20 0.021±0.03 All values are expressed as mean ± S.D., n=6. * Significant (p<0.05) compared to control. # Significant (p<0.05) compared to pure drug (ketoprofen). Conclusion The present study demonstrated the effectiveness of ketoprofen co-crystal towards improved solubility and anti-inflammatory activity. Co-crystal of ketoprofen with fumaric acid prepared via simple solvent-assisted grinding technique were systematically characterized through DSC, PXRD, FTIR and NMR studies was further evaluated for in-vitro and in-vivo anti-inflammatory and analgesic activities. The solubility and % drug release of different batches of co-crystal was found to be between 30.68–57.44 µg/ml and 62.21–83.68%, respectively. The IC 50 values for pure drug ketoprofen and optimized formulation was observed to be 556.11 µg/ml and 327.33 µg/ml respectively. Thus it can be inferred that optimized co-crystal formulation is additionally effective as compared to pure drug in generating anti-inflammatory response. Thus, the reported co-crystal have important implications for the use of co-crystallization approach to improve drugs solubility and efficacy of BCS- II drugs. Declarations Consent for Publication Not applicable. Availability of Data and Materials All the data is available in the manuscript. Author Contributions Sunita Devi- Conceptualization, Writing – Original Draft Preparation; Meenakshi Bhatia- Conceptualization, Supervision; Ashwini Kumar- Review & Editing, Software; Vikas Verma- Writing – Review & Editing; Snehlata Yadav– Review & Editing, Data Curation. Ethical Approval and Consent to Participate This investigation is approved by IAEC, Guru Jambheshwar University of Science and Technology, Hisar, India under CPCSEA reg. no-IAEC/2021/10-19. Funding None. Acknowledgement None. Conflict of Interest The authors declare that there is no conflict of interest. References Rasenack N, Müller BW. Micron-size drug particles: common and novel micronization techniques. Pharmaceutical development technology. 2004;9(1):1–13. https://doi.org/10.1081/PDT-120027417 . Jain NK, Gupta U. Application of dendrimer–drug complexation in the enhancement of drug solubility and bioavailability. Expert Opin Drug Metab Toxicol. 2008;4(8):1035–52. https://doi.org/10.1517/17425255.4.8.1035 . 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Stewart, Stewart Computational Chemistry, web: HTTP://OpenMOPAC.net. Days left: 237. Alex A, Granovsky. Firefly version 8, www http://classic.chem.msu.su/gran/firefly/index.html . Tian Lu F, Chen. Quantitative analysis of molecular surface based on improved Marching Tetrahedra algorithm. J Mol Graph Model. 2012;38:314–23. DOI: 10.1016/j.jmgm.2012.07.004 . Tian Lu F, Chen. J Comput Chem. 2012;33:580–92. Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics. J Molec Graphics. 1996;14(1):33–8. Supplementary Files graphicalabstractKETFACOCopy.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-905168","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":55980791,"identity":"568fe663-6afa-4a6e-bcbf-6f9daa15ac69","order_by":0,"name":"Meenakshi Bhatia","email":"","orcid":"","institution":"Guru Jambheshwar University of Science \u0026 Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meenakshi","middleName":"","lastName":"Bhatia","suffix":""},{"id":55980792,"identity":"61bca2f8-d7d3-4749-93e0-ee4b1e20ff82","order_by":1,"name":"Ashwani Kumar","email":"","orcid":"","institution":"Guru Jambheshwar University of Science \u0026 Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ashwani","middleName":"","lastName":"Kumar","suffix":""},{"id":55980793,"identity":"d503c342-31c6-4229-8a01-af6afd01252b","order_by":2,"name":"Vikas Verma","email":"","orcid":"","institution":"Guru Jambheshwar University of Science \u0026 Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Vikas","middleName":"","lastName":"Verma","suffix":""},{"id":55980794,"identity":"b902315a-7ab4-41c4-8cd5-4af591b5a5ca","order_by":3,"name":"Snehlata Yadav","email":"","orcid":"","institution":"Indira Gandhi University Meerpur","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Snehlata","middleName":"","lastName":"Yadav","suffix":""},{"id":55980795,"identity":"cac2b893-4183-4e9e-91a7-ec72952e94f4","order_by":4,"name":"SUNITA DEVI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2PMUvDQBTHT4RMatdA4e4TCC8EOvZjOF8oJEsidcsgNFNcpPNJBr9Ci9D5hYNOcQ+cg+4ON4mTmjsdm1i3gvcbjneP/48/jxCH40BB+/qEyHfwqN08/6bgt3IkSX4W2g3fq8Ynx5I0NCrMZ0hhIqulvt7QUXUL8qqcJPcX8qVrmdLzYrcC7SVH3KrQf2pA3pVxtlYxdMosnGCP4qeA6KloZYbTcputK24UjDY9ChNG+VCLH+UzCapEDyqk7ZJ1qbipkyeNx9k4HW6B5hXwcakC0cbzWuResBqnc+TQfwu7SUOdvyk2ErMHrcFjrEq6IZ/SPmVHr03CvnHbW/wl7XA4HP+BL+KVbtO1BmEYAAAAAElFTkSuQmCC","orcid":"","institution":"Guru Jambheshwar University of Science \u0026 Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"SUNITA","middleName":"","lastName":"DEVI","suffix":""}],"badges":[],"createdAt":"2021-09-14 17:05:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-905168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-905168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14448322,"identity":"8fc0f8e8-80a9-4537-b17f-c77dbb6aa335","added_by":"auto","created_at":"2021-10-12 14:37:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":705062,"visible":true,"origin":"","legend":"Contour plots (a, c) and response surface plots (b, d) showing the effect of concentration of ketoprofen \u0026 fumaric acid on solubility (Y1) and drug release (Y2) respectively.","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/46e2eb23924252e97c3aa861.png"},{"id":14448320,"identity":"27f1ebe2-5ce5-4139-9451-fc22be2a2057","added_by":"auto","created_at":"2021-10-12 14:37:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":142386,"visible":true,"origin":"","legend":"In-vitro release profile of ketoprofen and optimized batch of ket-FACo.","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/16fb9736f732bd887189dc1b.png"},{"id":14447871,"identity":"3701b4df-91d3-4d67-b3fe-cd2d19d15079","added_by":"auto","created_at":"2021-10-12 14:34:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42685,"visible":true,"origin":"","legend":"FTIR spectra (a) and (b) XRD patterns of Ketoprofen, Fumaric acid and optimized batch of ket-FACo.","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/41cb55c030c59d0876254a61.png"},{"id":14447869,"identity":"0a890529-4fdf-4835-8726-fb42ebe6ee63","added_by":"auto","created_at":"2021-10-12 14:34:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286970,"visible":true,"origin":"","legend":"DSC curves of ketoprofen, optimized batch of Ket-FACo (a) and fumaric acid (b) and SEM image of optimized batch of ket-FACo (c). ","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/bbc24f814e90d2860b0ab65c.png"},{"id":14448756,"identity":"bce06e32-d877-4754-be97-5ff1bab851ac","added_by":"auto","created_at":"2021-10-12 14:40:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83286,"visible":true,"origin":"","legend":"NMR spectra of ketoprofen (a), fumaric acid (b) and optimized batch of ket-FACo (c).","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/8f5a7fc2d1aa09a6939476e2.png"},{"id":14448757,"identity":"a7ce368d-018b-4932-9b80-535b29c822d7","added_by":"auto","created_at":"2021-10-12 14:40:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90617,"visible":true,"origin":"","legend":"Extreme values (positive and negative values in kcal/mol) from MESP of individual ketoprofen and fumaric acid.","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/a822855d1d3db657162b4878.png"},{"id":14447863,"identity":"befc9663-d0e7-40e5-a6ae-e9ca9e6ecd18","added_by":"auto","created_at":"2021-10-12 14:34:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":79624,"visible":true,"origin":"","legend":"Non-covalent interactions between ketoprofen and fumaric acid (a) and Hirshfeld surface mapped by electron density with promolecular approximation showing hydrogen bonding between ketoprofen and fumaric acid (b). (Red: high electron density, White: low electron density, Blue: zero electron density).","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/cefb2292307f1d3c593085bd.png"},{"id":14447870,"identity":"a3386208-b073-44c5-a1b3-02a2598f05bc","added_by":"auto","created_at":"2021-10-12 14:34:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":349615,"visible":true,"origin":"","legend":"% protection from protein denaturation of ketoprofen and optimized formulation.","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/9ae3992825093df3166610cd.png"},{"id":15024326,"identity":"bbbb4f2d-653c-4465-afdf-a5d9a1fa10f9","added_by":"auto","created_at":"2021-10-29 13:30:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2162650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/9e0ce83c-16da-48d0-a068-9aed0f680e81.pdf"},{"id":14447866,"identity":"7ec476bc-937f-44e9-bfed-0525445bad61","added_by":"auto","created_at":"2021-10-12 14:34:57","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":4158244,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstractKETFACOCopy.tif","url":"https://assets-eu.researchsquare.com/files/rs-905168/v1/b058d18e0a3f4110e36b4abf.tif"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFormulation, Optimization, in Vitro and in Vivo Investigation of Ketoprofen - Fumaric Acid Co-crystal for the Solubility Enhancement of Ketoprofen\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNumerous strategies to improve bioavailability of drugs with poor/low solubility include drug micronization in to amorphous form [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], complexation with hydrophilic carrier [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], solid dispersion [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], micellar solubilization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], nanoparticle technology [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], self-emulsifying drug delivery systems [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], salt formation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], liposomes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], nanostructured lipid carriers (NLC) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], prodrug [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and formation of co-crystal [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] etc. However, there are disadvantages associated with these techniques like agglomeration, instability during storage, requirement of advanced/or sophisticated instruments, tacky product etc. Therefore, co-crystallization appears to be a potential method for improving the solubility, dissolution and thus bioavailability of crystalline materials being a direct, viable, economical and green method. Co-crystal is defined as a multicomponent crystalline material possessing two or more molecules (i.e. drug and coformers) that are held together by noncovalent interactions in the same crystal lattice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Co-crystallization can appreciably reorganize the physiochemical properties of active pharmaceutical ingredient (API) by introducing a coformer that interconnected with the target API in a defined stoichiometric ratio through intermolecular interactions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The enhancement in solubility is explained by the two step mechanism as first the solute molecules are released from the crystal lattice followed by the solvation of released molecules. Also the Gibbs free energy (∆G) associated with this system (∆G\u003csub\u003esolution\u003c/sub\u003e) involves free energy allied with release of solute molecules from the lattice i.e. ∆G\u003csub\u003elat\u003c/sub\u003e and solvation barrier (∆G\u003csub\u003esolv\u003c/sub\u003e) that may be attributed as:\u003c/p\u003e \u003cp\u003e∆G\u003csub\u003esolution\u003c/sub\u003e=∆G\u003csub\u003elat\u003c/sub\u003e+∆G\u003csub\u003esolv\u003c/sub\u003e (1)\u003c/p\u003e \u003cp\u003eAt the moment, the free energy equated with lattice interaction and solvation barrier because trivial, the dissolution of co-crystal is improved due to drop free energy change for solubilization [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, in this study ketoprofen, a nonsteroidal anti-inflammatory drug, belonging to the BCS class II was selected as a model drug that pertain low solubility and high permeability. Ketoprofen works by inhibiting the enzyme cyclooxygenase-I and II, resulting in decreased production of precursors of prostaglandins and thromboxanes, thereby, displaying antipyretic, anti-inflammatory and analgesic activities. Nanosuspensions with Phosphol-ipon 80 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], soild dispersion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], microemulsion-based gel [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], emulgels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], nanoparticles [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], solid lipid nanoparticles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], nanostructured lipid carriers [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], prodrugs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], micro and nanocomposites with PLGA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and liquisolid [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] etc. have already been used to increase the solubility of ketoprofen. However, co-crystal of ketoprofen with nicotinamide by grinding method are reported in literature that depicted higher anti-inflammatory activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe dicarboxylic acid \u003cem\u003eviz.\u003c/em\u003e fumaric acid was selected as co-crystal coformer in the present study. Fumaric acid is a popular coformer that has been widely explored for the production of co-crystal of different active pharmaceutical ingredients like glycine [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], meloxicam [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], (DL)-phenylalanine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], adenine [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], 5-Fluorocytosine [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], arginine [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] berberine chloride [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], Ketoconazole [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] etc. The ketoprofen and fumaric acid were selected on the basis of the pK\u003csub\u003ea\u003c/sub\u003e rule. Fumaric acid exhibits an aqueous solubility of \u0026sim;0.6 g/l at 24\u0026deg;C and has pK\u003csub\u003ea\u003c/sub\u003e value of 3.03 whereas pK\u003csub\u003ea\u003c/sub\u003e value for ketoprofen is 3.88 and the value of ΔpK\u003csub\u003ea\u003c/sub\u003e (pK\u003csub\u003eaacid\u003c/sub\u003e \u0026minus; pK\u003csub\u003eabase\u003c/sub\u003e) is \u0026minus;\u0026thinsp;0.85. According to Bhogala et al., at negative values of ΔpK\u003csub\u003ea\u003c/sub\u003e co-crystal formation is expected [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is no study reported on co-crystal formation of ketoprofen with fumaric acid. In the present piece of research-work formation of ketoprofen co-crystal with fumaric acid is described with the objective to enhance aqueous solubility of drug utilizing simple and reproducible technique of solvent-assisted grinding. The preparation of co-crystal was achieved as per experimental design protocol as recommended by the 2-factor, 3 level CCD (central composite experimental design, Design Expert software version 11.0). The solubility, Gibbs free energy, entrapment efficiency and \u003cem\u003ein-vitro\u003c/em\u003e drug release for each batch was determined and numerically optimized. The optimized batch as suggested by design expert was characterized by FT-IR, DSC, XRD, SEM and NMR studies. The evaluation of optimized batch was carried out by \u003cem\u003ein-vitro\u003c/em\u003e/\u003cem\u003ein-vivo\u003c/em\u003e anti-inflammatory and analgesic activity employing suitable animal models like rat paw edema and tail flick methods. Further, the mechanism of drug release was determined by fitting the release data in various release kinetics models.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKetoprofen (ket) was received as a gift sample from Infinity Laboratories Pvt. Ltd (Behra, India). Fumaric acid (FA) was supplied by Central Drug House (P) Ltd., New Delhi. Ethanol, potassium chloride, di-sodium hydrogen orthophosphate, potassium di-hydrogen orthophosphate, sodium chloride and carrageenan were obtained from Hi-Media lab. Pvt. Ltd. All other chemicals \u0026amp; reagents were of analytical grade and used as received. The chemical structures of ketoprofen and fumaric acid were obtained from pubchem database [https://pubchem.ncbi.nlm.nih.gov].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of ketoprofen-fumaric acid co-crystal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKetoprofen-fumaric acid co-crystal (Ket-FACo) were prepared by the simple solvent-assisted grinding technique as reported earlier\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[52, 53]. Ketoprofen and fumaric acid were used in stoichiometrically equal ratio and after carefully weighing were ground using a mortar and pestle for 30 minutes with the dropwise addition of ethanol. The powder was dried, preserved in airtight vials and stored in a desiccator till further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of co-crystals using ketoprofen and fumaric acid was optimized using 2-factor, 3 level central composite experimental design. The concentration of ketoprofen (254.29-508.58 mg) (X\u003csub\u003e1\u003c/sub\u003e) and concentration of FA (116.07-232.14 mg) (X\u003csub\u003e2\u003c/sub\u003e) were designated as formulation variables whereas the % drug release and solubility (\u0026micro;g/ml) were selected as response variables (Table I) and each of the independent variable was considered at 3 levels (-1, 0, and 1). Experimental design and statistical analysis of the data was realized by Design Expert software (version 11.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolubility studies\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the solubility of ketoprofen and each batch of Ket-FACo formulations carrying drug equivalent to 5 mg and pure drug (5 mg) was dispersed in 20 ml of distilled water and phosphate buffer solution pH-7.4, separately and were kept on continuous shaking at room temperature for 48 h. The obtained solution was then filtered by 0.45\u0026micro;m millipore filter paper and the drug content was measured by taking absorbance at 260 nm using \u003cem\u003euv-vis\u003c/em\u003e spectrophotometer. The amount of drug was measured using the calibration curve in water\u003csup\u003e\u0026nbsp;\u003c/sup\u003e[5].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Gibbs free energy of transfer (\u0026Delta;G) of ketoprofen present in different batches of co-crystal is determined using equation 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026Delta;G = -2.303RT log S\u003csub\u003eo\u003c/sub\u003e/S\u003csub\u003es\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (2)\u003c/p\u003e\n\u003cp\u003eS\u003csub\u003eo\u003c/sub\u003e is the solubility of the co-crystal in water and Ss is the solubility of pure drug in water,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eR = 8.31 J k\u003csup\u003e-1\u003c/sup\u003emol\u003csup\u003e-1\u003c/sup\u003e and T = 298.15\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePercentage Drug content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ascertain the drug content of every batch of Ket-FACo formulation, co-crystal equivalent to 5 mg were weighed and dissolved separately in 25 ml of phosphate buffer (pH 7.4) in volumetric flask with continuous stirring for 24 h on a magnetic stirrer [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e], after proper dilutions, drug content was determined using \u003cem\u003euv-vis\u003c/em\u003e spectrophotometrically at 260 nm.\u003c/p\u003e\n\u003cp\u003eThe following equation was used to calculated Total Drug Content (TDC)\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\text{T}\\text{o}\\text{t}\\text{a}\\text{l} \\text{d}\\text{r}\\text{u}\\text{g} \\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t} \\left(\\text{\\%}\\right)=\\frac{\\text{W}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\text{o}\\text{f} \\text{d}\\text{r}\\text{u}\\text{g} \\text{i}\\text{n} \\text{c}\\text{o}-\\text{c}\\text{r}\\text{y}\\text{s}\\text{t}\\text{a}\\text{l}}{ \\text{W}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\text{o}\\text{f} \\text{c}\\text{o}-\\text{c}\\text{r}\\text{y}\\text{s}\\text{t}\\text{a}\\text{l} }\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e\u0026times;100 (3)\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eIn vitro\u003c/span\u003e \u003cstrong\u003edrug release profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e dissolution studies of pure drug (Ketoprofen) and each batch of Ket-FACo formulation containing Ketoprofen equal to 20mg were conducted in 900ml PBS (phosphate buffer solution, pH-7.4) at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003eo\u003c/sup\u003eC with constant stirring speed of 50 rpm. The powder was dispersed over the dissolution medium. Aliquots of sample (5ml) were withdrawn at different time intervals for 1 h and restored with an equal volume of the dissolution medium to keep sink conditions in the course of the experiment. The 0.45\u0026micro;m milipore filters was used for the sample filtration and the drug concentration in the samples was determined by measuring the absorbance of the samples at a wavelength of 260 nm using the \u003cem\u003euv\u0026ndash;vis\u003c/em\u003e spectrophotometer followed by determination of mechanism of release by fitting the release rate data in various release kinetic models [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003edrug release profile\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;In vitro\u003c/em\u003e dissolution studies of pure drug (Ketoprofen) and each batch of Ket-FACo formulation containing Ketoprofen equal to 20mg were conducted in 900ml PBS (phosphate buffer solution, pH-7.4) at 37\u0026plusmn;0.5\u003csup\u003eo\u003c/sup\u003eC with constant stirring speed of 50 rpm. The powder was dispersed over the dissolution medium. Aliquots of sample (5ml) were withdrawn at different time intervals for 1 h and restored with an equal volume of the dissolution medium to keep sink conditions in the course of the experiment. The 0.45\u0026micro;m milipore filters was used for the sample filtration and the drug concentration in the samples was determined by measuring the absorbance of the samples at a wavelength of 260 nm using the\u0026nbsp;\u003cem\u003eu\u003c/em\u003e\u003cem\u003ev\u003c/em\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003cem\u003evis\u003c/em\u003e spectrophotometer followed by determination of mechanism of release by fitting the release rate data in various release kinetic models [6].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier Transform Infrared Spectroscopy (FT-IR) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFT-IR analysis was used for the interaction between drug and carrier. FTIR spectral analysis of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation was done by FT-IR Perkin-Elmer, Spectrum, US spectrophotometer and the spectrum was documented in the wavelength region of 4000cm\u003csup\u003e-1\u003c/sup\u003e to 400 cm\u003csup\u003e-1\u003c/sup\u003e using KBr pellet method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX-ray diffraction analysis (XRD) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe XRD spectra of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation were obtained using an X-ray diffractometer (Miniflex 2, Rigaku, Japan) at room temperature and at 30kV. The scanning diffraction angle (2\u0026theta;) ranging from 0\u003csup\u003eo\u003c/sup\u003e to 80\u003csup\u003eo\u003c/sup\u003e. The Miller index (d\u003csub\u003ehkl\u003c/sub\u003e) is used to establish direction and plane in the crystal and is determined using Bragg\u0026rsquo;s equation (Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ1\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\text{n\u0026lambda;=2d}\\text{hkl}Sin\\theta$$ 4\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eHere \u0026lambda; and n denote the wavelength (1.5418\u0026Aring;) and order (n\u0026thinsp;=\u0026thinsp;1, first order), respectively; \u0026theta; is the Bragg\u0026rsquo;s angle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential scanning calorimetry (DSC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDSC thermograms of ketoprofen, Fumaric acid and optimized batch of Ket-FACo formulation were recorded using DSC (Mettler Toledo, Switzerland), the samples were heated within the temperatures range of 20-400\u0026deg;C with a scanning rate of 10\u0026deg;C/min in aluminum pans under nitrogen flow at a rate of 50 ml/min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface morphology and shape of optimized batch of Ket-FACo formulation was observed using scanning electron microscope (JSM-6100 scanning microscopy, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNuclear Magnetic Resonance (NMR) Spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe NMR spectra of ketoprofen, fumaric acid and optimized batch of Ket-FACo formulation after dissolving in DMSO-d\u003csub\u003e6\u003c/sub\u003e were examined using Bruker Avance AV 400 NMR spectrometer (Bruker, Karlsruhe, Germany) to get solution \u003csup\u003e13\u003c/sup\u003eC NMR data at a temperature of 293 K using Tetramethylsilane (TMS) as an internal standard [55].\u003csub\u003e\u0026nbsp;\u003c/sub\u003eData was interpreted using Mnova program (Mestrelab Research, Santiago de Compostela, Spain).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimized batch of co-crystal was kept for the accelerated stability studies according to ICH guidelines (40 \u0026plusmn; 2 \u003csup\u003eo\u003c/sup\u003eC and 75 \u0026plusmn; 5% RH) for a period of 6 months in a stability chamber. The samples were placed in hermetically sealed vials containing rubber plugs and aluminum bung. \u0026nbsp;The stored co-crystal were taken out after 6 months and evaluated for the drug content (according to the method described in earlier section of drug content, \u003cem\u003en=3\u003c/em\u003e) and for any physical changes [47].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiological Evaluation of Ket-FACo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e anti-inflammatory activity\u003c/p\u003e\n\u003cp\u003eEgg albumin denaturation method was used to determine \u003cem\u003ein-vitro\u003c/em\u003e anti-inflammatory activity of drug ketoprofen and optimized batch of Ket-FACo [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. The mixtures containing of fresh hen\u0026rsquo;s egg albumin (0.2ml), Phosphate buffered saline 7.4 (2.8ml) and different concentrations of Ket-FACo formulation (2 ml, containing ketoprofen equivalent to 10 mg) (125, 250, 500, 750 \u0026amp; 1000 \u0026micro;g/ml in Dimethyl sulfoxide as solvent). The mixture was placed in an incubator at a temperature of 37\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003eo\u003c/sup\u003eC for 20 min incubation followed by heating at 70\u003csup\u003eo\u003c/sup\u003eC. After cooling at room temperature, the absorbance measured at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e of 660nm to determine % inhibition. Similar procedure was done for the drug (ketoprofen) with the similiar concentrations as engaged for the formulation as a reference or control. The percentage protection from protein denaturation was calculated as per Eq.\u0026nbsp;5\u003c/p\u003e\n\u003cp\u003e% protection from denaturation= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{Abs. of control-Abs. of sample}{Abs. of control}\\)\u003c/span\u003e\u003c/span\u003e X 100 (5)\u003c/p\u003e\n\u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of ketoprofen and optimized formulation was calculated by nonlinear regression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003e carrageenan-induced anti-inflammatory activity\u003c/p\u003e\n\u003cp\u003eThe protocol with registration no. CPCSEA Reg. no-IAEC/2021/10\u0026ndash;19 was approved for animal study by the Animal Ethical Committee, Guru Jambheshwar University of Science and Technology, Hisar, India. Wistar rats (150\u0026ndash;180 g) were distributed into three groups comprising of six animals each. Group I (control treated) with carrageenan was kept as control, Group II (standard drug) was treated with drug-ketoprofen (10 mg/kg) and Group III (test compound) was treated with Ket-FACo (equivalent to ketoprofen 10 mg/kg body weight) that is administrated orally. 1% suspension of carrageenan (0.1 ml) in normal saline, was administered as subplantar injection in the left hind paw of albino wistar rats, after 1 h of oral administration of the test materials. The paw volume was measured using vernier caliper at 1, 2, 3, 4 and 5 h after the carrageenan injection. The % inhibition in paw volume was calculated using Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e,\u003c/p\u003e\n\u003cdiv class=\"Equation\" id=\"Equ2\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$\\% inibition=\\frac{Vc-Vt}{Vc}\\times 100$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere V\u003csub\u003ec\u003c/sub\u003e and V\u003csub\u003et\u003c/sub\u003e is the inflammatory increase in paw volume control group and test group respectively [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAnalgesic activity (Tail Flick Method)\u003c/p\u003e\n\u003cp\u003eTail flick method was used to measure analgesic activity using a radiant type analgesiometer. Three different groups (control, test and standard) of Swiss albino mice (25-30g) containing six mice each group were taken for study. The tail flick reaction time for each animal was recorded six times before administering the drug and the mean was used as predrug reaction time. A dose of the standard drug (Ketoprofen) and test compound (optimized batch of Ket-FACo) containing ketoprofen equivalent to 5 mg/kg of body weight in 0.9% w/v sterile saline was orally administered to mice. After administration of the drug, the tail flick reaction time was measured at 0, 1, 2, 3, 4 and 5 hrs.\u003c/p\u003e\n\u003cp\u003e% Analgesic activity (PAA) was calculated as per Eq.\u0026nbsp;7.\u003c/p\u003e\n\u003cp\u003ePAA= (T\u003csub\u003e2\u003c/sub\u003e-T\u003csub\u003e1\u003c/sub\u003e)/T\u003csub\u003e1\u003c/sub\u003e ˣ 100 (7)\u003c/p\u003e\n\u003cp\u003eWhere T\u003csub\u003e1\u003c/sub\u003e are reaction time in second before treatment of drug(s) and T\u003csub\u003e2\u003c/sub\u003e are reaction time in second after treatment of drug(s) [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e]. Data was analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test and statistically was denoted as P value.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eThe preparation of co-crystal using ketoprofen and fumaric acid was optimized using 2-factor, 3 level CCD. The concentration of ketoprofen (X\u003csub\u003e1\u003c/sub\u003e) and concentration of fumaric acid (X\u003csub\u003e2\u003c/sub\u003e) were designated as formulation variables whereas the % drug release and solubility (\u0026micro;g/ml) were picked as response variables. The TDC of different batches of ket-FACo was found to be between 94.48 to 97.81 %, thus depicting that handsome amount of drug has been loaded and also no physical changes were observed during stability studies and even after six months. In different batches of ket-FACo solubility in phosphate buffer (PBS pH-7.4) values range from 29.08 to 10.63 \u0026micro;g/ml whereas ketoprofen solubility was found to be 3.64 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003eAs presented in Table 1, solubility of ket-FACo varied in the range of 30.68-57.44\u0026micro;g/ml. The pure ketoprofen dispensed a solubility of 11.24 \u0026micro;g/ml in water at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eFormulation parameters and responses for central composite experimental design.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eBatch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eConc. Of Ketoprofen (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.191582002902758%\"\u003e\n \u003cp\u003eConc. Of Fumaric acid (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eSolubility (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eSolubility in PBS pH-7.4 (\u0026micro;g/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e∆G (KJ/MOL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e% Drug release in 60 min.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eDrug content (% )\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e254.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e116.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e30.68\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e10.63\u0026plusmn;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e62.21\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.89\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e508.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e116.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e42.04\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e18.36\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e70.01\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.66\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e254.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e232.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e48.3\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e23.39\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e71.94\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.33\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e508.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e232.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e57.44\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e29.08\u0026plusmn;08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e83.68\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e97.81\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e254.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e45.97\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e21.59\u0026plusmn;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e70.52\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e96.39\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e508.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e51.08\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e28.46\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e80.59\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.60\u0026plusmn;001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e116.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e36.47\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e15.16\u0026plusmn;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e66.52\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.04\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e232.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e53.66\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e28.52\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-2.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e79.71\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e97.47\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e49.08\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e27.46\u0026plusmn;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e75.54\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e97.25\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e48.14\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e26.94\u0026plusmn;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e75.04\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e95.53\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e49.51\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e27.67\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e75.59\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e94.88\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e50.23\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e26.54\u0026plusmn;1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e73.52\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e94.48\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eF13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e381.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.191582002902758%\"\u003e\n \u003cp\u003e174.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e50.16\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e26.83\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e-3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e73.02\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.62699564586357%\"\u003e\n \u003cp\u003e96.35\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003eKetoprofen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.191582002902758%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e11.24\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e3.64\u0026plusmn;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.04644412191582%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e\u0026nbsp;23.05\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.62699564586357%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAll values are expressed as mean \u0026plusmn; S.D., n=3.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 shows the results of solubility of different batches of Ket-FACo organized according to design protocol. The responses produced were fitted into several polynomial models using CCD. The response solubility was fitted greatest into\u0026nbsp;quadratic\u0026nbsp;model with none transformation of the data. The polynomial models for the responses solubility (Y\u003csub\u003e1\u003c/sub\u003e) can also be expressed by the equation (8) with determination correlation (R\u003csup\u003e2\u003c/sup\u003e) of 0.9659.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e = 49.36+4.27X\u003csub\u003e1\u003c/sub\u003e+8.37X\u003csub\u003e2\u003c/sub\u003e-0.5550X\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e-0.6822X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e-4.14X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (8) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 summarizes the results of ANOVA on the solubility and % drug release response surface model, demonstrated that model was found significant with lack of fit as non-significant. Adequate precision of solubility is found to be\u0026nbsp;28.33 indicates an adequate signal. The adequate precision measuring signal to noise ratio (greater than 4) is desirable. Fig. 1 (a) show the collective effect of concentration of ketoprofen and fumaric acid on solubility. It may be reckoned from the plots that a curvilinear relationship exists between independent and dependent variables. It is also inferred from the plot that higher level of ketoprofen and fumaric acid results in increase in solubility. However, the effect of the concentration of fumaric acid (X\u003csub\u003e2\u003c/sub\u003e) seems to be more pronounced as compared to the concentration of ketoprofen (X\u003csub\u003e1\u003c/sub\u003e). This increase in solubility may be due to formation of soluble complex between ketoprofen and fumaric acid. Fumaric acid presents higher solubility than the drug that comes out of the crystal lattice. The drug in co-crystal get supersaturated in aqueous medium and possesses more energy as compared to crystalline phase, thereby, exhibit marked increase in solubility than the pure drug.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003edrug release\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults of \u003cem\u003ein-vitro\u003c/em\u003e drug release (Table 1) revealed that 62.21 to 83.68% and 23.50% of ketoprofen got released from different batches of Ket-FACo and pure drug solution respectively, in 1 h study. This rise in percentage drug release from Ket-FACo as compared to pure drug may be associated to the solubility data. As previously mentioned that the fumaric acid form the soluble complex with the drug and thereby increase the drug wettability that leads to a better solubility and further heads towards better rate of drug release. The adjusted polynomial equation obtained for the \u003cem\u003ein-vitro\u003c/em\u003e drug release (Y\u003csub\u003e2\u003c/sub\u003e) is shown in equation 9 with determination correlation of adjusted R\u0026sup2; of 0.958 and predicted R\u003csup\u003e2\u003c/sup\u003e of 0.905. The predicted is in reasonable agreement with the adjusted R\u0026sup2; with a difference less than 0.2.\u003c/p\u003e\n\u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e\u0026shy;=74.84+4.93X\u003csub\u003e1\u003c/sub\u003e+6.10X\u003csub\u003e2\u003c/sub\u003e+0.985X\u003csub\u003e1\u003c/sub\u003e X\u003csub\u003e2\u003c/sub\u003e -0.0336X\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e -2.47X\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(9) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 recapitulating the results of ANOVA of \u003cem\u003ein-vitro\u003c/em\u003e release data on response surface model fitted best in quadratic model (after none transformation of the data). The responses observed were fitted into different polynomials models using the experimental design. Adequate precision of \u003cem\u003ein-vitro\u0026nbsp;\u003c/em\u003edrug release is found to be\u0026nbsp;25.57 indicates an adequate signal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eModel summary statistics.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"top\" width=\"76.26459143968872%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Model\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"23.735408560311285%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLack of Fit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.234375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponse factor(Y)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProb.\u0026gt;F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.328125%\"\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.578125%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdeq.Prec.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.203125%\"\u003e\n \u003cp\u003e\u003cstrong\u003eC.V (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProb.\u0026gt;F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.234375%\"\u003e\n \u003cp\u003eY\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e69.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.328125%\"\u003e\n \u003cp\u003e0.9659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.578125%\"\u003e\n \u003cp\u003e28.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.203125%\"\u003e\n \u003cp\u003e2.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e4.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e0.1035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.234375%\"\u003e\n \u003cp\u003eY\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e56.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.328125%\"\u003e\n \u003cp\u003e0.975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.578125%\"\u003e\n \u003cp\u003e25.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.203125%\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.9609375%\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.8671875%\"\u003e\n \u003cp\u003e0.501\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTo attain stability a natural tendency to acquire minimum Gibbs energy is always there. All the values of \u0026Delta;G are negative (Table 1) at all levels of carrier demonstrating spontaneity of drug solubilization process.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOptimization\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe optimization equations 8 and 9, involving the response and independent factors were assembled based on a quadratic model. To the responses \u003cem\u003ei.e.\u003c/em\u003e solubility and \u003cem\u003ein-vitro\u003c/em\u003e drug release the desirability function was applied with constraints to obtain the higher level of both, the batch F4 comes out to be optimized batch. In this fashion, the formulation containing fumaric acid (228.82mg) as coformer and drug content (508.58 mg) with addition of ethanol, established the maximum desirability, was organized and evaluated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mathematical optimization tool with desirability method was employed to prepare co-crystal. The constraints of maximum solubility and maximum % release was imposed on independent variables for optimization. The parameters recommended by the design were concentration of ketoprofen (508.58 mg) \u0026amp; concentration of fumaric acid (228.82 mg) that provide co-crystal with solubility of 56.06 \u0026micro;g/ml (predicted value 56.63 \u0026micro;g/ml) and % drug release 83.35% (predicted value 84.22%). The closer agreement between predicted and observed values indicated the high prognostic ability of the model. Fig. 2 shows the \u003cem\u003ein vitro\u003c/em\u003e release profile of ketoprofen as pure drug and optimized batch (F4) of co-crystal formulation.\u003c/p\u003e\n\u003cp\u003eThe release rate data of ketoprofen from co-crystal and from drug solution was fitted into several kinetic models to estimate release kinetics and mechanism of drug release. The release rate data was found to be put best into Higuchi model (with R\u003csup\u003e2\u003c/sup\u003e =0.985) of release kinetics. Further, the value of n=0.475 (0.43\u0026lt;n\u0026lt;0.85), release exponent of Korsemeyer and Peppas equation, indicated that the release of ketoprofen from co-crystal occurs by diffusion and erosion of the matrix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier Transform Infrared Spectroscopy (FT-IR) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR is an excellent analytical technique to study the deviations in the position caused by the vibration modes of the functional groups. This technique reveals the shift in characteristic peaks of drug and coformer due to co-crystal formation involving H bonding between the corresponding functional groups. The spectra of ketoprofen fig. 3 (a) showed characteristic absorption band at 2979.27 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003edue to \u0026ndash;CH stretching. The peak appearing at 1697.76 cm\u003csup\u003e-1\u003c/sup\u003e can be ascribed to -C=O stretching of acid while peak appearing at 1655.77 cm\u003csup\u003e-1\u003c/sup\u003e is due to -C=O stretching of ketone. The absorption bands at 1598.67 cm\u003csup\u003e-1\u003c/sup\u003e (-C=C= stretching), 1442.21 cm\u003csup\u003e-1\u003c/sup\u003e (-C=O stretching of aromatic ring), 1420.59 cm\u003csup\u003e-1\u003c/sup\u003e (-C-H deformation of -CH\u003csub\u003e3\u003c/sub\u003e asymmetrical) and 1370.04 cm\u003csup\u003e-1\u003c/sup\u003e (-C-H deformation of -CH\u003csub\u003e3\u003c/sub\u003e symmetrical) also appeared. The FTIR spectra of fumaric acid exhibit peaks at 3084.26 cm\u003csup\u003e-1\u003c/sup\u003e, 1684.31 cm\u003csup\u003e-1\u003c/sup\u003e, 1422 cm\u003csup\u003e-1 \u0026nbsp;\u003c/sup\u003eascribed to the \u0026nbsp;-O-H stretching, -C=O stretching vibration and \u0026ndash;C-C aromatic stretching, respectively. The FTIR spectra of ket-FACo displayed that -C=O stretching of \u0026ndash;COOH group of ketoprofen get shifted from 1697.76 cm\u003csup\u003e-1\u003c/sup\u003e to 1667.30 cm\u003csup\u003e-1\u003c/sup\u003e and the peak due to -C=O stretching of fumaric acid at 1655cm\u003csup\u003e-1\u003c/sup\u003e got disappeared. Therefore, FT-IR analysis confirms the interactions occurring between ketoprofen and fumaric acid. These interactions are essentially hydrogen bonds between the carboxylic group of the fumaric acid and the main functional groups of ketoprofen (-C=O and -O-H) which are able to generate supramolecular heterosynthons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePowder X-ray diffraction analysis (PXRD)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe powder X-ray diffraction (PXRD) pattern of ketoprofen, fumaric acid and optimized batch of ket-FACo illustrated in fig. 3 (b). The diffraction peaks (and Miller indices) at 2\u0026theta; of 12.74 (100), 18.52 (200), 22.85 (211), 24.00 (221), 26.32 (222), 28.920 (300), 36.71 (322) and 22.86 (211), 28.92 (300), 29.49 (310), and 30.03 (311) showed crystalline structure of ketoprofen and fumaric acid respectively. The major diffraction peaks at 2\u0026theta;, 18.57 (200), 23.27 (211), 28.01 (222) and 29.28 (300) were also observed in PXRD spectra of co-crystal that portrayed crystalline nature of resultant product. The distinctive PXRD pattern of the ket-FACo was distinguishable from ketoprofen and fumaric acid, this outcome specifies the formation of a new crystal phase [62].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential scanning calorimetry (DSC)\u003c/strong\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDSC thermograms [fig. 4 (a)], reported that pure ketoprofen showed a sharp endotermic peak at 94.5\u0026ordm; that corresponds to its melting point. The peak at 280.4\u0026ordm;C attributed to melting point of fumaric acid. In the thermogram of the prepared co-crystal, peaks were found to be displaced and difference in intensity is also observed from that of its constitutional components indicating the occurrence of weak cohesive forces that bonded together by reversible hydrogen bonding, suggesting the development of co-crystal formation. The thermal behavior of the ket-FACo was prominent, with a different melting transition from that seen with either of the constitutional components; this recommends the formation of a new phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SEM image of the optimized batch of ket-FACo [fig. 4(c)] depicted good crystalline characteristics. This crystalline character was reinforced by the XRD data, as discussed earlier. The voids over the surface of the co-crystal may brace the imbibition of the solvent and biological fluids and thereby proliferating the solubility and bioavaibility of ketoprofen as estimated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNuclear magnetic resonance (NMR) Spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR spectroscopy is used to characterize the co-crystal by studying the chemical environment of their nuclei and hydrogen bonding and it also offers valuable information regarding interactions. In the NMR pattern of ket-FACo, the carbonyl carbon of ketoprofen corresponding to 196.46 ppm and140.13 ppm has shifted to 196.10 ppm and 142.22 ppm, respectively. A deviation in the carbonyl carbon of carboxylic group in fumaric acid shifted from 166.41 ppm and 140.51 ppm to 175.52 ppm and 137.45 ppm, respectively (Fig. 5). This suggests an interaction between alcoholic group of fumaric acid and \u0026ndash;COOH group of ketoprofen in ket-FACo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComputational studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe constituents of the co-crystal interact through weak non covalent interactions (NCI) [63] and in order to determine the points of contacts between the ketoprofen and coformer fumaric acid, molecular electrostatic surface potential (MESP) analysis was performed and the extreme positive and negative values from MESP are displayed in fig. 6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe magnitude of these values signifies that both H-bond donor and acceptor are present in the crystals of both compounds. MESP of ketoprofen exhibits both positive (+53.53 kcal/mol) and negative (-37.40 and -32.94 kcal/mol) extreme values thus proving that it can form intermolecular as well as intramolecular hydrogen bonds during process of co-crystalization. Similarly, fumaric acid displays positive (+69.87 and +69.85 kcal/mol) and negative (-33.5 and -33.46 kcal/mol) extreme values which confirms its hydrogen bonding capability. It has stronger hydrogen bond donor ability due to higher positive extreme value and on the other hand ketoprofen has higher hydrogen bond acceptor ability due to higher negative extreme value. According to Etter\u0026apos;s rule [64], there is more probability of interactions between most polar parts of the molecules in a co-crystal. Therefore, these two compounds will pair in the co-crystal through hydrogen bonding. This pairing of these molecules was accomplished by combining two molecules and energy minimization of combined form. The interaction diagram of the molecules in combination is shown in fig. 7 (a, b).\u003c/p\u003e\n\u003cp\u003eReduced density gradient (RDG) analysis [fig. 7 (a)] exhibits formation of two hydrogen bonds between two fumaric acid and ketoprofen. Two hydroxyl groups made these two hydrogen bonds (shown as blue colored discs) with two carbonyl oxygen atoms of ketoprofen. Further, van der Waals interactions can be observed between double bond region of fumaric acid and phenyl ring of ketoprofen (shown as green and brown color). The results of RDG calculations are also in line with the MESP predictions. The hydrogen bonding interactions between these two molecules were further confirmed by Hirshfeld surface mapped by electron density with promolecular approximation analysis [fig. 7 (b)]. This calculation shows three regions of high electron density; two are the same as found in RDG analysis while the third is between carbonyl oxygen of fumaric acid and phenyl hydrogen of ketoprofen. These observations are also in agreement with the findings of MESP analysis. The positive parts of one molecule are interacting with negative parts of another molecule.\u003c/p\u003e\n\u003cp\u003eThe geometry of the both molecules individually as well as in combined form was minimized using MOPAC [65] with latest PM7 method choosing value of gnorm as 0.01 after optimization with molecular mechanics method. Molecular energy and other various properties of the minimized discrete molecules and co-crystal were calculated with Firefly [66] by density functional theory (DFT) taking 6-31G* basis set in B3LYP method. The MESP calculation, RDG analysis and Hirshfeld surface mapped by electron density with promolecular approximation [67] calculations were performed with Multiwfn 3.8 [68] and visualization was done with the help of VMD [69].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiological evaluation of the co-crystal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e anti-inflammatory activity\u003c/p\u003e\n\u003cp\u003eThe % protection from denaturation of protein is comparably plotted at different concentration of optimized batch of formulation and pure drug ketoprofen (fig. 8). Egg albumin protein denaturation method displayed concentration dependent anti-inflammatory activity by protecting the protein. Half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of ketoprofen and optimized formulation was calculated by nonlinear regression analysis. The IC\u003csub\u003e50\u003c/sub\u003e values for pure drug ketoprofen and optimized formulation was observed to be 556.11 \u0026micro;g/ml and 327.33 \u0026micro;g/ml respectively. Thus it can be inferred that optimized co-crystal formulation is additionally effective as compared to pure drug in generating anti-inflammatory response.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vivo\u003c/em\u003e anti-inflammatory activity\u003c/p\u003e\n\u003cp\u003eThe improvement in activity of ketoprofen and co-crystal formulation was comparatively assesed by the increase in paw volume of control groups. The paw edema volume (before and after drug administration) \u0026nbsp;and % inhibition of edema at different time interval was convinced and displayed in Table 3. The ketoprofen and co-crystal showed inhibition of paw edema as 49.34\u0026plusmn;0.18% \u0026nbsp; and 60.39\u0026plusmn;0.15 at the end of 5 h, respectively thus demonstrating quick onset of action by co-crystal in contrast with the \u0026nbsp;pure drug ketoprofen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatical Analysis:- Data was compared by ANOVA followed by Tukey\u0026rsquo;s test. The p value is \u0026lt;0.0005 is considered as significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Effect of ketoprofen and optimized batch of ket-FACo formulation on the paw edema induced by carrageenan in Wistar rats.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003eTime (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003ePaw volume\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003eInibition (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003ePure drug \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003eCo-crystal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003ePure drug\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003eCo-crystal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e4.25\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e4.12\u0026plusmn;0.03\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e4.02\u0026plusmn;0.09\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e1.05\u0026plusmn;0.03 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e5.17\u0026plusmn;0.08\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e4.54\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.86\u0026plusmn;0.06\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.53\u0026plusmn;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e10.97\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e16.65\u0026plusmn;0.09\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e4.96\u0026plusmn;0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.48\u0026plusmn;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.22\u0026plusmn;0.06\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e29.83\u0026plusmn;0.03 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e35.08\u0026plusmn;0.27\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e240\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e5.49\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.31\u0026plusmn;0.05\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.04\u0026plusmn;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e39.70\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e44.62\u0026plusmn;0.03\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e6.11\u0026plusmn;0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e3.09\u0026plusmn;0.03\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e2.42\u0026plusmn;0.035\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e49.34\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.666666666666668%\"\u003e\n \u003cp\u003e60.39\u0026plusmn;0.15\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAll values are expressed as mean \u0026plusmn; S.D., n=6. \u003csup\u003e*\u003c/sup\u003eSignificant (p\u0026lt;0.05) compared to control. \u003csup\u003e#\u003c/sup\u003eSignificant (p\u0026lt;0.05) compared to pure drug (ketoprofen).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalgesic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the % analgesic activity (PAA) of test, reference and control group are shown in Table\u0026nbsp;4.\u0026nbsp;The PAA (equation 8) was comparatively evaluated for Ket-FACo\u0026nbsp;and pure drug based on its potential to suppress pain. Ket-FACo\u0026nbsp;showed significant effect in enhancing the pain thershold to a certain extent when compared to that of drug (ketoprofen), thus, stipulating that an improvement in solubility further tweaked the pharmacological response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e % Analgesic effect of ketoprofen and optimized batch of Ket-FACo by tail flick method in mice.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.071856287425149%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" width=\"85.92814371257485%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;PAA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.156794425087108%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.550522648083625%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.073170731707318%\"\u003e\n \u003cp\u003e\u003cstrong\u003e2h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.073170731707318%\"\u003e\n \u003cp\u003e\u003cstrong\u003e3h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.073170731707318%\"\u003e\n \u003cp\u003e\u003cstrong\u003e4h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.073170731707318%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.071856287425149%\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ketoprofen)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.023952095808383%\"\u003e\n \u003cp\u003e0.06\u0026plusmn;0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.221556886227544%\"\u003e\n \u003cp\u003e31.30\u0026plusmn;0.71\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e35.18\u0026plusmn;0.12\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e42.36\u0026plusmn;0.17\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e50.93\u0026plusmn;0.14\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e71.96\u0026plusmn;0.17\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.071856287425149%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(Ket-FACo)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.023952095808383%\"\u003e\n \u003cp\u003e0.09\u0026plusmn;0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.221556886227544%\"\u003e\n \u003cp\u003e32.14\u0026plusmn;0.24\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e50.44\u0026plusmn;0.14\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e60.23\u0026plusmn;0.18\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e65.41\u0026plusmn;0.12\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e75.68\u0026plusmn;0.22\u003csup\u003e*#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.071856287425149%\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e (vehicle)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.023952095808383%\"\u003e\n \u003cp\u003e0.01\u0026plusmn;0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.221556886227544%\"\u003e\n \u003cp\u003e0.91\u0026plusmn;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e1.32\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e0.83\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e1.1\u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.67065868263473%\"\u003e\n \u003cp\u003e0.021\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAll values are expressed as mean \u0026plusmn; S.D., n=6.\u003csup\u003e*\u003c/sup\u003eSignificant (p\u0026lt;0.05) compared to control.\u003csup\u003e#\u003c/sup\u003eSignificant (p\u0026lt;0.05) compared to pure drug (ketoprofen).\u003c/em\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study demonstrated the effectiveness of ketoprofen co-crystal towards improved solubility and anti-inflammatory activity. Co-crystal of ketoprofen with fumaric acid prepared via simple solvent-assisted grinding technique were systematically characterized through DSC, PXRD, FTIR and NMR studies was further evaluated for \u003cem\u003ein-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e anti-inflammatory and analgesic activities. The solubility and % drug release of different batches of co-crystal was found to be between 30.68\u0026ndash;57.44 \u0026micro;g/ml and 62.21\u0026ndash;83.68%, respectively. The IC\u003csub\u003e50\u003c/sub\u003e values for pure drug ketoprofen and optimized formulation was observed to be 556.11 \u0026micro;g/ml and 327.33 \u0026micro;g/ml respectively. Thus it can be inferred that optimized co-crystal formulation is additionally effective as compared to pure drug in generating anti-inflammatory response. Thus, the reported co-crystal have important implications for the use of co-crystallization approach to improve drugs solubility and efficacy of BCS- II drugs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data is available in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSunita Devi- Conceptualization, Writing \u0026ndash; Original Draft Preparation; Meenakshi Bhatia- Conceptualization, Supervision;\u0026nbsp;Ashwini Kumar-\u0026nbsp;Review \u0026amp; Editing, Software; Vikas Verma- Writing\u0026nbsp;\u0026ndash; Review \u0026amp; Editing;\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003eSnehlata Yadav\u0026ndash; Review \u0026amp; Editing, Data Curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis investigation is approved by IAEC, Guru Jambheshwar University of Science and Technology, Hisar, India under CPCSEA reg. no-IAEC/2021/10-19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRasenack N, M\u0026uuml;ller BW. 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J Molec Graphics. 1996;14(1):33\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ketoprofen, fumaric acid, Co-crystal, non-covalent interactions, solubility","lastPublishedDoi":"10.21203/rs.3.rs-905168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-905168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present piece of research work is framed as improving the solubility of ketoprofen by forming co-crystal using fumaric acid as a coformer. Co-crystal of ketoprofen and fumaric acid were prepared by simple solvent assisted grinding. The independent variables i.e. drug and coformer were mixed in 1:1 molar ratio and dependent variables were assumed to be solubility and % drug release. Differential scanning calorimetry, fourier transform infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance and scanning electron microscopy techniques were used to characterize the preparation of optimized batch of co-crystal and further, evaluated for \u003cem\u003ein-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e anti-inflammatory and analgesic activities. Based on results of solubility and dissolution rate studies the drug showed 4-5 fold improvement in both the properties on co-crystallisation. The values of Gibbs free energy are negative at all levels of carrier demonstrating spontaneity of drug solubilization process. The IC\u003csub\u003e50\u003c/sub\u003e value of optimized batch of co-crystal formulation and pure drug was observed as 327.33 µg/ml and 556.11 µg/ml, respectively, demonstrating that co-crystal formulation possesses more percentage protection against protein denaturation than the drug ketoprofen. \u003cem\u003eIn-vivo\u003c/em\u003e (anti-inflammatory and analgesic) activities revealed that optimized batch of co-crystal formulation delivered a rapid pharmacological response in wistar rats and albino mice when compared with standard drug.\u003c/p\u003e","manuscriptTitle":"Formulation, Optimization, in Vitro and in Vivo Investigation of Ketoprofen - Fumaric Acid Co-crystal for the Solubility Enhancement of Ketoprofen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-12 14:34:55","doi":"10.21203/rs.3.rs-905168/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9d28ba7-ebf4-4b6b-a042-8d183ec45503","owner":[],"postedDate":"October 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":7792037,"name":"Drug Delivery"}],"tags":[],"updatedAt":"2021-10-29T13:30:04+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-12 14:34:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-905168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-905168","identity":"rs-905168","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}