Solubility Enhancement of Raloxifene Using Inclusion Complexes and Cogrinding Method

The objective of the present work was to enhance the solubility and dissolution of practically water-insoluble drug raloxifene HCl (RLX), for the same two approaches that were used. In the first approach, drug was kneaded with hydroxypropyl-β-cyclodextrin (HPβCD), and in the second one drug was cogrinded with modified guar gum (MGG). The drug-cyclodextrin complex and drug-MGG cogrind mixtures were characterized by differential scanning calorimetry, X-ray diffraction studies, scanning electron microscopy, and Fourier transform infrared spectroscopy. The solubility and dissolution study reveals that solubility and dissolution rate of RLX remarkably increased in both methods. It was concluded that the prepared inclusion complex showed a remarkable increase in solubility and dissolution of poorly water-soluble drug raloxifene. In the cogrinding mixture, a natural modified gum is used as a surfactant and enhances the solubility and dissolution of RLX without requiring addition of organic solvent or high temperature for its preparation; thus, process is less cumbersome and cost effective. But when both methods were compared; HPβCD complexation method showed significant enhancement of drug solubility.


Introduction
Solubility of a drug is an important property that mainly in�uences the extent of oral bioavailability. Enhancement of oral bioavailability of poorly water soluble drugs is the most challenging aspect of drug development [1]. Most of the new chemical entities suffer from low bioavailability due to their low aqueous solubility and dissolution. erefore, it is very important to �nd appropriate formulation approaches to improve aqueous solubility and bioavailability of poorly aqueous soluble drugs [2].
Raloxifene (marketed as Evista by Eli Lilly and Company) is an oral second generation selective estrogen receptor modulator (SERM) used to prevent osteoporosis in postmenopausal women. It is 2-(4-hydroxyphenyl)-3-({4-[2-(piperidin-1-yl) ethoxy] phenyl} carbonyl)-1-benzothiophen-6-ol that has estrogenic actions on bone and antiestrogenic actions on the uterus and breast. It belongs to class II drug according to biopharmaceutical classi�cation system (BCS), that is, low solubility and high permeability. But raloxifene has very less bioavailability, only 2%, and it would be advantageous to increase the solubility of such molecule. Raloxifene is available in salt form as raloxifene HCl [3]. e drug is poorly absorbed from the gastrointestinal (GI) tract therefore; it is important to enhance aqueous solubility and dissolution rate which may lead to enhancement of bioavailability from its oral solid dosage forms.
In this study, two strategies were used, which were aimed at improving the aqueous solubility. e �rst one is complexation of drugs with cyclodextrin, and the second one is Cogrinding with natural polymers. Cyclodextrins (CDs) are cyclic macromolecules, obtained by the degradation of starch by -1,4-glucan-glycosyl transferase. ey have also been used to improve drug stability, bioavailability, or toxicity pro-�les. Moreover, chemically modi�ed cyclodextrins have been extensively used. Among the chemically modi�ed cyclodextrins, 2-hydroxypropyl--cyclodextrin (HP--CD) deserves special attention due to its favorable physicochemical and biological properties [4]. Kneading method was employed for the preparation of raloxifene HCl with HP CD, and the effect of complexation on the solubility and dissolution rate of raloxifene was evaluated. In the second approach, Cogrinding of raloxifene HCl with modi�ed guar gum was done. Guar gum (GG) is a gum obtained from the ground endosperms of Cyamopsis tetragonolobus (Leguminosae family). It is composed of galactan and mannan units combined through glycosidic linkages [5]. e natural polymers have surfactant activity [6] that enhances the solubility and dissolution rate of drug, but high viscosity of these polymers is a limitation for them to be used as carriers for dissolution enhancement [7]; this problem is overcome by heating of the polymers at particular time and temperature condition which reduces the viscosity [6].
e present study was carried out to investigate the inclusion complex of raloxifene HCl and HP CD in the solid state and the RLX-MGG Cogrind mixture using Xray diffractometry (XRD) differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). e objective of this study was to enhance solubility and dissolution rate of RLX which may lead to enhancement of bioavailability of this drug [1,8,9].

Materials and Methods
2.1. Materials. Raloxifene (RLX) was obtained as a gi sample from Zydus Cadila Healthcare Ltd., Ahmedabad, India. HP CD was gied by Roquette Pharma, France and, Guar gum (GG) was gied by Lucid Colloids Ltd., Sewari, Mumbai. Methanol and all other reagents used were of analytical grade. [8,10] Development of Inclusion Complex of Raloxifene with HP CD. As raloxifene HCl is practically insoluble in water, an inclusion complex of the antiosteoporotic raloxifene HCl (RLX) in hydroxypropyl--cyclodextrin (HP CD) was prepared and characterized.

Kneading Method
Preparation of RLX-HP CD Inclusion Complex. It was prepared by Kneading method. e mixture of RLX and HP CD in 1 : 1 molar ratio was triturated in a mortar with a small volume of water-methanol (1 : 2 v/v) solution. e thick slurry formed was kneaded for 45 min and then dried at 45 ∘ C. e dried mass was pulverized and sieved through sieve no. 60.

Cogrinding Method
Modi�cation of Polymers [1]. Guar Gum was modi�ed by heating method. Powdered gum was taken in a porcelain bowl and kept in hot air oven at different temperatures and different time intervals. e viscosity and swelling index were studied, which reveals that viscosity decreases as the time and temperature of heating increases but swelling index remains unaffected. It was observed that guar gum produced colour change on heating above 130 ∘ C and 120 ∘ C more than 2 hrs respectively. us, 120 ∘ C and 2 hrs conditions were selected for modi�cation of polymers. Finally, modi�ed gum was sieved through mesh no. 100 and stored in airtight container.

Characterization of Polymers
Swelling Index (SI) [1,9,11]. About 1 gm of GG and MGG were accurately weighed and transferred to 100 mL measuring cylinder. e initial volume of powder in measuring cylinder was noted which is denoted as 0 . Distilled water was added in measuring cylinder up to 100 mL mark, shaken gently, and cylinder was kept aside for 24 hrs. e �nal volume occupied by polymers was noted aer 24 hrs, which is denoted as . Swelling index was calculated according to the following equation: Viscosity Measurement [9]. Viscosity of GG and MGG gums was determined by using �rook�eld DV�E viscometer (�rook�eld engineering laboratory) at 37 ∘ C and 50 rpm. 1% (w/v) solution was prepared in distilled water and used for measuring the viscosity.
Preparation of Cogrind Mixture [1]. Cogrind mixtures of drug and modi�ed gum were prepared in different ratio such as 1 : 1 to 1 : 9. e ratio was optimized by using solubility data. e sample of drug and gum in 1 : 1 w/w ratio was Cogrinded for 25 min, in ceramic mortar and sieved through mesh no. 100. e same method was applied for all ratios of drug with polymer. e Cogrind mixture of RLX with MGG and GG denoted as RLX-MGG and RLX-GG, respectively.
Ratio Optimization (Drug : Polymer). Samples were placed in 10 mL solvent (pH 7 phosphate buffer) in te�on facing screw capped vial and kept at equilibrium for a period of 24 hrs on orbital shaking incubator (Remi Instruments Ltd.) at 37 ± 0.5 ∘ C and 50 rpm. e contents of vials were �ltered through 0.2 micron �lter and analyzed by UV-Visible spectrophotometer (UV 1601, Shimadzu) at 287 nm. As shown in Table 3, the solubility increases as the gum concentration increases, the optimized ratio was found to be 1 : 8 w/w as further increase in ratio to 1 : 9 w/w showed no signi�cant increase in solubility of drug. Cogrinding mixtures of RLX with MGG and GG were prepared in 1 : 8 w/w ratio.

Characterization of HP CD Inclusion Complex and Cogrind
Mixture [1,9] (1) Differential Scanning Calorimetry (DSC). DSC studies of raloxifene HCl, HP CD, MGG, RLX-HP CD inclusion complex, and RLX-MGG Cogrind mixture were performed using differential scanning calorimeter (Mettler Toledo DSC 1 Star System, Zurich, Switzerland) at heating rate of 10 ∘ C/min from 40 to 340 ∘ C in nitrogen atmosphere.
(3) Fourier Transform Infrared Spectroscopy (FT-IR). Raloxifene HCl, HP CD, MGG, RLX-HP CD inclusion complex, and RLX-MGG Cogrind mixture were mixed separately with IR grade KBr in the ratio of 1 : 100, and corresponding pellets were prepared by applying 10 metric ton of pressure in hydraulic press. e pellets were then scanned over a wave range of 4000-400 cm −1 in FTIR instrument (8400 S Shimadzu).
Phase Solubility Studies of Inclusion Complex [10,12]. e phase solubility technique permits the evaluation of the affinity between HP CD and raloxifene in water. Phase solubility studies were performed according to the method reported by Higuchi and Connors [13]. As given in Table  1, raloxifene was taken into vials in an excess amount, and 20 mL of distilled water was added, containing various concentration of HP CD (10-40 mmol). ese vials were sealed and shaken at 20 ∘ C for 4 days. is period was considered sufficient to reach equilibrium. Subsequently, the aliquots were withdrawn, using a syringe, and samples were �ltered through 0.2 micron �lter and appropriately diluted. A portion of the sample was analyzed by UV spectrophotometer (UV 1601, Shimadzu) at 287 nm against blank prepared in the same concentration of HP CD in water so as to cancel any absorbance that may be exhibited by the HP CD. e solubility experiments were conducted in triplicate. e apparent stability constant ( ) of complexes was calculated from the phase solubility diagram using the following equation: e slope obtained from the initial straight line portion of the plot of raloxifene concentration against HP CD concentration, and 0 is the equilibrium solubility of raloxifene in water.

Statistical Evaluation.
All results are expressed as mean ± S.D. Differences between the two related parameters were considered statistically signi�cant for values for less than 0.05. Drug to polymer optimization ratio, solubility determination, and dissolution efficiency results were analyzed by applying one way ANOVA test.

Development of Inclusion Complex of Raloxifene and HP CD.
Cyclodextrin (CD) has a hydrophobic central cavity and hydrophilic outer surface and can encapsulate model substrates to form host-guest complexes or supramolecular species. is usually enhances drug solubility in aqueous solution and affects the chemical characteristics of the encapsulated drug. HP CD is a hydroxyalkylated--cyclodextrin derivative that combines relatively high water solubility with low toxicity and satisfactory inclusion ability. e binding behavior of hydroxypropyl--cyclodextrin with RLX and the solubilization effect of HP CD toward RLX may provide a useful approach to produce a novel RLX formulation with improved bioavailability.

Development of Cogrind Mixture of Raloxifene and
Modi-�ed �uar �um. e natural polymers are mainly evaluated in industry for their new applications. Due to the less toxic effect and low production cost, these polymers mainly used as drug carrier in pharmaceutical industry. Guar gum has surfactant activity [6], which reduces the contact angle and increases wetting of drug particles, thus enhances solubilization and dissolution of drug particles. is gum has limitation as dissolution enhancing carrier due to their high viscosity. ese polymers produce gel layer on the hydrated surfaces which prevents the drug release during drug dissolution and reduced the dissolution [14]. It is reported that the swelling of polymers in�uences improvement of dissolution rate of poorly aqueous soluble drugs [15]. erefore, it is useful to modify the gum in such a way that its swelling ability remains the same and decreases the viscosity.

Viscosity and Swelling Index
Measurement. e results of swelling index and viscosity of polymers are given in Table  2. e result indicates that the viscosity of MGG is lower than that of the GG, and swelling index of MGG was not reduced signi�cantly than the GG. �ecause of swelling nature of the carrier, the extensive surface was increased during the dissolution and thus dissolution rate of drug was enhanced [16].

Differential Scanning Calorimetry (DSC) Study.
As shown in Figure 1, the thermograms of the RLX, HP CD, and MGG showed respective endothermic peaks at 266.44 ∘ C, 97.46 ∘ C and 64.95 ∘ C corresponding to their melting points.
In the thermogram of RLX-HP CD complex, the peak of drug disappeared indicating the complexation of RLX with cyclodextrin, whereas in DSC spectra of RLX-MGG Cogrind mixture, the peak of drug was observed, but the intensity was reduced suggesting the conversion of raloxifene hydrochloride from crystalline form to amorphous form. Figure 2, the X-ray diffraction patterns were recorded for pure RLX, HP CD, MGG, RLX-HP CD inclusion complex, and RLX-MGG Cogrind mixture. PXRD studies were performed in conjunction with DSC to verify the reduction of crystallinity of RLX. Diffraction spectrum of drug sample showed distinct peaks at 2 of 12.812 ∘ , 14.47 ∘ , 15.784 ∘ , 19.153 ∘ , 22.682 ∘ , and 25.876 ∘ . All these peaks, though of relatively lesser intensity, were observed to be in RLX-HP CD complex and RLX-MGG Cogrind mixture. It was thus concluded that the drug was converted from crystalline to amorphous state. Figure 3, FT-IR spectra of RLX exhibited characteristic peaks at 1,642.44 (C=O stretching), 1,596.15 (-C-O-Cstretching), 1,466.91 (-S-benzothiofuran), and 905.61 cm −1 (benzene ring). ey were well preserved in the RLX-HP CD complex and RLX-MGG Cogrind mixture. ese results indicate that no interaction occurred between drug and excipients. Figure 4, the scanning electron microscopy photomicrographs of RLX shown in Figure 4 show the longer crystals with very speci�c morphology, whereas for RLX-HP CD complex and RLX-MGG Cogrind mixture, a decrease in crystallinity due to formation of drug-cyclodextrin complex and molecular dispersion of RLX in the polymer matrix (MGG) was observed, respectively.

Solubility Study
Phase Solubility Study of Inclusion Complex. e phase solubility diagram for the complex formation between RLX and HP CD is shown in Figure 5. e aqueous solubility of RLX increased linearly with a slope 0.3083 ( 2 = 0.9926) as a function of HP CD concentration. e apparent solubility constant , obtained from the slope of the linear phase solubility diagram was found to be 4.5949 mol −1 .

Conclusion
e molecular structure of cyclodextrin creates a bucketlike cavity that can function to complex with drug or functional groups on drug. e investigation suggests from phase solubility study and dissolution rate pro�le of the inclusion complex that the solubility and dissolution rate of raloxifene increases signi�cantly due to HP CD. Whereas, the Cogrinding method enhances the solubility of RLX by converting it to amorphous form, reducing the particle size and increasing wettability. e optimum ratio for Cogrinding mixture was found to be 1 : 8 which shows higher solubility. Moreover, this natural polymer like guar gum has advantage over other synthetic polymers as these polymers are biocompatible, biodegradable, and having low cost.
Hence, from practical point of view, Cogrinding method appeared easier and was considered as the most convenient method. But when both these techniques were compared the    inclusion complex method showed better results as compared to those of the other methods and thus was found to be more effective than Cogrinding method. �e authors report no con�ict of interests.