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

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-inammatory 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-inammatory 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. (25-30g) containing six each group were taken for study. The tail ick reaction time for each animal was recorded six 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 ick reaction time was measured at 0, 1, 2, 3, 4 and 5 hrs.


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][3][4], solid dispersion [5,6], micellar solubilization [7], nanoparticle technology [8][9][10][11], self-emulsifying drug delivery systems [12][13][14], salt formation [15], liposomes [16], nanostructured lipid carriers (NLC) [17,18], prodrug [19] and formation of co-crystal [20][21][22][23][24][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 de ned 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 de ned stoichiometric ratio through intermolecular interactions [27]. The enhancement in solubility is explained by the two step mechanism as rst 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].
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 e ciency 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-in ammatory and analgesic activity employing suitable animal models like rat paw edema and tail ick methods. Further, the mechanism of drug release was determined by tting the release data in various release kinetics models. Experimental Materials Ketoprofen (ket) was received as a gift sample from In nity 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 ltered by 0.45µm millipore lter 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 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 ask 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)

×100 (3)
In vitro drug release pro le 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 lters was used for the sample ltration 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 tting the release rate data in various release kinetic models [6].
In vitro drug release pro le 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 lters was used for the sample ltration 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 tting 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 (Mini ex 2, Rigaku, Japan) at room temperature and at 30kV. 4 Here λ and n denote the wavelength (1.5418Å) and order (n = 1, rst 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 ow 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 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-in ammatory activity 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= 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-in ammatory 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, 6 Where V c and V t is the in ammatory increase in paw volume control group and test group respectively [59].
Analgesic activity (Tail Flick Method) Tail ick 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 ick 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 ick reaction time was measured at 0, 1, 2, 3, 4 and 5 hrs.
PAA= (T 2 -T 1 )/T 1 100 (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. 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 tted into several polynomial models using CCD. The response solubility was tted 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) Table 2 summarizes the results of ANOVA on the solubility and % drug release response surface model, demonstrated that model was found signi cant with lack of t as non-signi cant. 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 (   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 pro le 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 tted into several kinetic models to estimate release kinetics and mechanism of drug release. The release rate data was found to be put best into Differential scanning calorimetry (DSC) DSC thermograms [ g. 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 [ g. 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 cocrystal may brace the imbibition of the solvent and biological uids 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 g. 6.
The magnitude of these values signi es 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 con rms 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 g. 7 (a, b).
Reduced density gradient (RDG) analysis [ g. 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 con rmed by Hirshfeld surface mapped by electron density with promolecular approximation analysis [ g. 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 ndings of MESP analysis. The positive parts of one molecule are interacting with negative parts of another molecule. Biological evaluation of the co-crystal In-vitro anti-in ammatory activity The % protection from denaturation of protein is comparably plotted at different concentration of optimized batch of formulation and pure drug ketoprofen ( g. 8). Egg albumin protein denaturation method displayed concentration dependent anti-in ammatory 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-in ammatory response.

In-vivo anti-in ammatory 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 signi cant.

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 signi cant 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.

Conclusion
The present study demonstrated the effectiveness of ketoprofen co-crystal towards improved solubility and antiin ammatory 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-in ammatory 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-in ammatory response. Thus, the reported co-crystal have important implications for the use of co-crystallization approach to improve drugs solubility and e cacy of BCS-II drugs.

Consent for Publication
Not applicable.

Availability of Data and Materials
All the data is available in the manuscript.   In-vitro release pro le of ketoprofen and optimized batch of ket-FACo.      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).