In Vitro and In Vivo Evaluation of Oxatomide β-Cyclodextrin Inclusion Complex

The objective of this study was to evaluate the influence of oxatomide β-cyclodextrin inclusion complex on the physicochemical properties and bioavailability of the drug. Oxatomide β-cyclodextrin solid complex was prepared with equimolar ratio of both oxatomide and β-cyclodextrin in presence or absence of water soluble polymers using different techniques. The coevaporated complex prepared in presence of PVP-K15 showed a prompt drug release and significantly increased % dissolution efficiency (P < 0.05) compared to the pure oxatomide. Moreover, the results of bioavailability evaluation of this complex in rabbits compared to commercial drug product indicated a 73.15% increase in the oral bioavailability of oxatomide. In conclusion, inclusion complex of oxatomide with β-cyclodextrin prepared by coevaporation in presence of PVP-K15 not only results in an enhancement of the oxatomide dissolution rate but also improves the bioavailability of oxatomide.


Introduction
Oxatomide is an antihistaminic drug that has been reported to have applications in the treatment of a number of different types of allergic and other hypersensitivity reactions. ese include but are not restricted to the symptomatic treatment of allergic rhinitis and chronic urticaria, the classical nasal and ocular symptoms associated with hay fever, the reduction of the severity of the erythema, and pruritus in cases of chronic urticaria [1,2]. Although oxatomide has been extensively investigated and approved for the use in the treatment of a broad range of diseases, it still possesses bioavailability limitations and poor dissolution properties that restrict its full clinical use [1].
One of the major current challenges of the pharmaceutical industry is related to strategies that improve the water solubility of drugs [3,4]. Low solubility can cause low bioavailability or give rise to large �uctuations in the fraction absorbed in humans that can oen not be compensated by a high permeability. Furthermore, low solubility may be associated with stability problems and difficulties in developing an acceptable formulation [5]. By improving the drug release pro�le of these drugs, it is possible to enhance their bioavailability and reduce side effects [6][7][8][9].
Cyclodextrins have been utilized extensively in pharmaceutical formulations to enhance oral bioavailability [10]. Cyclodextrins are a family of cyclic oligosaccharides that are composed of -1, 4-linked glucopyranose subunits. ese macrocyclic carbohydrates with lipophilic central cavities and hydrophilic outer surfaces can form complexes with and solubilize many normally water-insoluble compounds. e lipophilic cavity of cyclodextrin molecules provides a microenvironment into which appropriately sized nonpolar moieties can enter to form inclusion complexes [11]. Cyclodextrins are of three types: -cyclodextrin,cyclodextrin, and -cyclodextrin. Currently, -cyclodextrin is the most common, the most accessible, the lowest-priced, and generally the most useful cyclodextrin in pharmaceutical formulations [12,13]. In addition, a drug--cyclodextrin solid inclusion complex is convenient for oral administration [14].
e purpose of study was to improve oxatomide water solubility by preparation of inclusion complex withcyclodextrin and evaluate the in�uence of complex formation on the physicochemical properties and bioavailability of the drug.

Materials and Methods
2.1. Materials. Oxatomide was kindly donated by Janssen Pharmaceutica, Germany. -cyclodextrin hydrate was purchased from Acros Organics, New Jersey, USA. Hydroxy propyl methyl cellulose (HPMC) and polyvinyl pyrrolidone K15 (PVP-K15) Fluka, Chemie, USA. Oxatomide tablets (Tinset 30 mg) were purchased from local pharmacy. All other reagents used in this study were of analytical grade. All chemicals were used as received without any further puri�cation.

Construction of the Oxatomide-Cyclodextrin Phase Solubility
Diagrams. An excess of oxatomide was added to screw-capped vials containing aqueous solutions of various concentrations of -cyclodextrin. e vials were mechanically shaken for 48 hrs at room temperature. Aer 2 days, aliquots were withdrawn and �ltered using 0.45 m pore size �lter. e �ltrate was suitably diluted and analyzed spectrophotometrically at the wavelength of 226 nm. e same procedure was repeated with cyclodextrin aqueous solution containing either 0.25% w/v PVP-K15 or 0.1% w/v HPMC [15].

Preparation of Oxatomide-Cyclodextrin Solid Complexes.
Oxatomide -cyclodextrin solid complex was prepared with equimolar ratio of both oxatomide andcyclodextrin by using �ve different techniques as follows.
Physical Mixing Technique. e technique simply relies on the idea that both oxatomide and -cyclodextrin were properly blended together in a ceramic mortar using pestle.
Kneading Technique. Into a ceramic mortar, -cyclodextrin was wetted with a few drops of distilled water and properly kneaded. en, oxatomide was added slowly, and the combined mixture was kneaded with the addition of few drops of distilled water. e dough mass was pressed and stretched with the hand �ngers, folded over, and rotated through 90 ∘ , repeatedly. is process was continued for additional 15 min until the dough is elastic and smooth and then the obtained dough mass was le to dry under fume hood at room temperature for 24 hrs.
Coevaporation Technique. Coevaporation is another technique for formation of solid oxatomide -cyclodextrin complex. Simply it consists of the simultaneous deposition of the oxatomide and -cyclodextrin under vacuum conditions. is is simply done by dissolving weighed amount of oxatomide in 10 mL of 60% methanol solution in a glass beaker. In another glass beaker, -cyclodextrin was dissolved in 50 mL distilled water. e contents of the two beakers are then mixed together, and the obtained solution is put in sonicator for 25 min at room temperature to obtain a clear solution. e resultant solution is dried by being subjected to evaporation using Büchi Rotavapor (R200V800, Büchi Labortechnik, Switzerland) at 70 ∘ C under vacuum (55 mbar).
Spray-Drying Technique. Spray drying was performed in a Büchi mini spray drier (B 290, Büchi Labortechnik, Switzerland). is is simply done by dissolving weighed amount of oxatomide in 10 mL 60% methanol solution, in 100 mL glass beaker. In another 100 mL glass beaker -cyclodextrin was dissolved in 50 mL distilled water. e contents of the two beakers were then mixed together and sonicated at room temperature for 25 min to obtain and ensure a clear solution. e obtained solution is dried by spray drying under puri�ed nitrogen gas. e inlet adjusted to a �ow rate of 880 mL/h and at temperature 120 ∘ C. e nitrogen gas adapted to a �ow rate of 357 NL/h u, and outlet temperature was 85 ∘ C.
Freeze-Drying Technique. Freeze-drying process was typically carried out in Heto Power Dry LL 3000 freeze dryer (ermo Electron Corporation, Czech Republic) at −50 ∘ C, under vacuum (0.128 mbar). Brie�y, in a 100 mL glass beaker, oxatomide was dissolved in 10 mL 60% methanol solution. In another 100 mL glass beaker, -cyclodextrin was dissolved in 50 mL distilled water. e contents of the two beakers are then mixed together, and the obtained solution is put in sonicator at room temperature for 25 min to ensure that a clear solution is obtained. e resultant solution is then frozen by placing at −30 ∘ C freezer for at least 24 hrs to ensure that the solution was completely frozen. en, frozen solution was kept for 24 hrs into the vacuum chamber at −50 ∘ C where the freeze-drying process takes place.

Preparation of Oxatomide-Cyclodextrin Solid Complex in Presence of Water Soluble Polymer.
Oxatomidecyclodextrin solid complexes were prepared with a molar ratio of 1 : 1 in addition to 21% of water soluble polymer such as PVP-K15, HPMC, or PEG 6000 using coevaporation, spray-drying, and freeze-drying techniques.

Characterization of Oxatomide-Cyclodextrin Complex.
e oxatomide--cyclodextrin complex obtained from each technique is subjected to different chemical and thermal characterizations as follows.
Infrared (IR) Spectroscopy. IR spectra of oxatomide,cyclodextrin, and the prepared oxatomide -cyclodextrin solid complexes were obtained at room temperature using an infrared spectrometer (IR100/IR200, ermo Nicolet Corp., USA). Samples were prepared in KBr disks by means of a hydrostatic press. e scanning range was 400 to 4000 cm −1 , and the resolution was 4 cm −1 .
Differential Scanning Calorimetry (DSC). e thermal properties of oxatomide, -cyclodextrin, and the prepared oxatomide -cyclodextrin solid complexes were characterized using Shimadzu Differential Scanning Calorimeter, DSC60, Japan. e measurements were carried out at a heating rate of Journal of Pharmaceutics 3 10 ∘ C/min. In order to provide the same thermal history, each sample (1.2 to 1.8 mg) was heated from room temperature to 200 ∘ C and rapidly cooled down to room temperature, then the DSC scan was recorded by heating from 30 to 200 ∘ C.

X-Ray Diffraction Studies (XRD)
. e diffractograms of the different samples were obtained by Philips PW 3040 equipment. e measurements were carried out at conditions: Ni-�ltered CuK radiation, voltage 50 kV, current 30 mA, scanning speed 1 ∘ (2 )/min, and investigating the samples in the 2 range 0-30 ∘ .

In Vitro Dissolution Properties of Oxatomide-Cyclodextrin
Complexes. e dissolution studies of oxatomide powder and the prepared oxatomidecyclodextrin solid complexes were performed according to the USP XXIII rotating basket method. e samples corresponding to 30 mg of oxatomide were placed into rotating basket. Dissolution medium was 900 mL of distilled water (pH 6.8). e stirring speed was 50 rpm, and the temperature was maintained at 37 ± 0.5 ∘ C. At predetermined times intervals samples of 5 mL were withdrawn by using syringe �lter (0.45 m Millipore �lter) and analyzed spectrophotometrically for oxatomide concentration at max 277 nm. e withdrawn samples were replaced by equal volume of fresh dissolution medium. Each test was performed in triplicate.

Bioavailability Study.
e study was performed to assess the bioavailability of the suggested formula (oxatomide--cyclodextrin-PVPK15) selected on the basis of the dissolution studies in comparison with the reference oxatomide commercial product (Tinset 30 mg tablets).
(1) Study Design. e study was approved by the Animal Ethics Committee of Faculty of Pharmacy, Helwan University. Guidelines of the ethics committee were followed for the study. Sixteen New Zealand white male rabbits (15 weeks old and weight 2.5-3 kg) were used in the study. All rabbits were housed and received similar diet. e rabbits were divided randomly into two groups; each was of eight rabbits and all rabbits were fasted overnight for 12 hrs with free access to water. On the day of experiment, each group received a single oral dose equivalent to 5 mg/kg oxatomide from each formulation by intragastric feeding tube. e doses of both products were suspended in a solution of 4.6% glycerin, 87.6% polyethylene glycol 400, and 7.8% distilled water.
e blood samples were withdrawn from central ear artery 1 hr before the drug administration and aer administration at speci�c time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hrs). Blood samples were collected in heparinized tubes. e blood samples were centrifuged at 3,000 rpm for 10 min, and the plasma was transferred to separate glass tubes to be kept frozen until analysis.

(2) Analysis of Plasma Levels of Oxatomide
Oxatomide Sample Preparation. Prior to analysis of plasma samples, aliquots of plasma (0.5 mL) spiked with 75 ng/mL �unarizine hydrochloride (internal standard) were vortexed for two min with (100 L × 1 M) sodium hydroxide solution (100 L × 1 M) and 5.0 mL ethyl ether. e samples were centrifuged at 2000 rpm for 10 min at room temperature. e upper organic layer was transferred to another centrifuge tube and evaporated to dryness under a gentle stream of nitrogen gas in a water bath at 40 ∘ C. e residues were then redissolved in 200 L mobile phase under vortex and centrifuged at 18000 rpm for 8 min. Aliquots of 5 L of the supernatant were injected into the liquid chromatography-mass spectrometry (LC-MS system).
LC-MS System. Plasma samples were analyzed for oxatomide concentration by using a validated LC-MS assay [16] with some modi�cations. Brie�y, LC-MS analysis was performed on an Agilent 1100 series LC/MSD chromatographic system (Agilent, USA) consisting of a water HPLC system equipped with an on-line solvent degasser, binary solvent delivery system, autosampler, and an Agilent technologies single quadrupole mass spectrometer with an electrospray ionization (ESI) interface. e MS detector has electrospray capability with a mass range of 50-3000 and a mass accuracy of 0.1 amu. A liquid chromatographic separation was achieved on a Phenomenex C18 (250 × 2.0 mm i.d.) column, which was maintained at 40 ∘ C. e mobile phase consisting of 85% methanol and 15% (v/v) aqueous ammonium acetate solution (10 mM, pH 4.0) was pumped at an isocratic �ow rate of 0.2 mL/min. e total run time was 7.5 min for each injection.
e ESI source was set at positive ionization mode. e [M + H] + , 427.10, for oxatomide and [M + H] + , 405.05, for �unarizine hydrochloride were selected as detecting ions, respectively. e MS operating conditions were optimized as follows: nebulizer gas rate, 1.5 L/min; CDL temperature, 250 ∘ C; block temperature, 200 ∘ C; probe voltage, �4.5 kV. e quanti�cation was performed via peakarea ratio. e lower limit of quanti�cation was 5 ng/mL. e standard calibration curve for oxatomide was linear (correlation coefficients were >0.9975) over the studied concentration range (5-200 ng/mL). Data acquisition and processing were accomplished using Envirolab version 5 for the LC-MS system.
(3) Pharmacokinetic Analysis. Oxatomide pharmacokinetics parameters were determined by noncompartmental kinetics [17]. e elimination rate constant ( el ) was estimated by least square regression of plasma concentration-time data points in the terminal log-linear region of the curves. Half life ( 1 2 ) was calculated as 0.693 divided by el . e area under the plasma concentration-time curve from zero to the last measurable plasma concentration at time (AUC 0− ) was calculated using linear trapezoidal rule. e area under the curve from zero to in�nity, AUC 0−∞ , was calculated as AUC 0−∞ = (AUC 0− ) + el , where is the last measured concentration at the time t. Peak plasma concentration ( max ) and the time to peak concentration ( max ) were obtained directly from the individual plasma concentration versus time curve.

Statistical Analysis.
In order to compare the results Student's t-test (SPSS program; version 12.0) was used. Data reported as mean ± standard deviation (SD). A statistically signi�cant difference was considered at value < 0.05.

Results and Discussion
3.1. Oxatomide-Cyclodextrin Phase Solubility Diagrams. e phase solubility studies for oxatomide with -cyclodextrin were carried out according to the method described by Higuchi and Connors [18]. e phase solubility diagrams of oxatomide in different concentration of -cyclodextrin in distilled water, 0.1% HPMC solution, and 0.25% PVP solution are presented in Figure 1. e phase solubility pro�le of oxatomide -cyclodextrin complex showed a typical Bstype solubility curve, where the initial ascending portion is followed by a plateau region and then a decrease in total oxatomide solubility accompanied by precipitation of a microcrystalline complex [18].
e extent of complexation in aqueous media is characterized by the stability constant . e values of were calculated according to the equation of Higuchi and Connors and from the initial straight line portion of the solubility diagrams by assuming that a 1 : 1 complex was initially formed according to the following equation: where is the stability constant for the complex and 0 is the solubility of oxatomide in absence of -cyclodextrin.

Characterization of Oxatomide-Cyclodextrin Complex
3.2.1. IR Spectroscopy. IR spectra of oxatomide and its inclusion complexes with -cyclodextrin prepared by different methods are presented in Figure 2. Oxatomide is characterized by the absorption of the carbonyl (C=O) group at 1702 cm −1 ; in the spectra of the inclusion complex, this band was shied towards higher frequencies at 1727 cm −1 in case of coevaporated solid complex and to lower frequencies at 1682 cm −1 in spray-dried solid complex and decreased in intensity in freeze-dried complex. e absorption of N-H group in oxatomide at 3172 cm −1 was shied toward lower frequencies at 2936-2924 cm −1 in all types of solid complexes. As spectral changes always concern C-OH, -CH 2 , and CH groups of the -CD, it should be suggested that the hostguest interactions are dominated by hydrogen bonds among the above-mentioned groups.

DSC Studies.
e thermal behavior of oxatomidecyclodextrin inclusion complexes was studied using DSC to con�rm the formation of the solid complexes. �hen guest molecules are incorporated in the cyclodextrin cavity or in the crystal lattice, their melting, boiling, and sublimation points usually shi to a different temperature or disappear within the temperature range at which the cyclodextrin  lattice is decomposed [19]. e DSC curves for all the complexes are represented in Figure 3. e DSC thermogram of oxatomide exhibited one endothermic peak at 159.95 ∘ C. e DSC thermogram of -cyclodextrin shows a very broad endothermic peak around 95 ∘ C corresponding to the release of water molecules. e thermogram of the physical mixture showed the melting endothermic peak characteristic of pure  (2), physical mixture (3), kneaded mixture (4), coevaporated (5), freeze-dried (6), and spray-dried (7) inclusion complexes of equimolar ratio of oxatomide and -cyclodextrin.
oxatomide and the broad peak corresponding to the dehydration of -CD as if this thermogram was the superposition of those components, indicating the absence of interaction between oxatomide and -cyclodextrin in such mixture. ermograms of oxatomide -cyclodextrin complexes prepared by kneading displayed a broad endothermic at 71.80 ∘ C due to the dehydration of the complex, and the peak at 160 ∘ C can still re�ect the presence of a few drug crystals in the preparation. However, this thermal effect appeared more broadened and reduced in intensity, which suggests some drug-cyclodextrin interaction [20]. In addition to changes in the broad peak of -cyclodextrin dehydration provided a further indication of the existence of inclusion complexes in these systems. However, the kneading and physical methods do not provide complete inclusion, and oxatomide was dispersed in the free state between inclusion complexes. e complete disappearance of the endothermic peak of pure oxatomide was observed in thermograms of freeze-dried, spray-dried, and coevaporated complexes and is attributed to the formation of an amorphous solid product or inclusion of the drug inside the -cyclodextrin cavity or both [21].

X-Ray
Diffraction. e X-ray diffraction patterns of pure oxatomide, -cyclodextrin, and their physical and kneaded mixture, coevaporated, freeze-dried, and spraydried inclusion complexes are represented in Figure 4. e diffractograms of oxatomide and -cyclodextrin exhibited a series of intense peaks which are indicative of their crystallinity.
e differences in the diffraction patterns of physical mixture, coevaporated, kneaded, freeze-dried, and spraydried complexes from each isolated constituent seem to  indicate the formation of a new solid phase as a result of formation of inclusion complexes which is in good agreement with DSC studies. Furthermore, a reduction in peak height in diffraction pattern of the binary systems that was observed in all complexes indicated also a reduction in crystallinity. Figure 5, it is clear that all inclusion complexes prepared by coevaporation, spray drying, and freeze drying exhibited higher dissolution characteristics than kneaded mixture, physical mixture, and pure oxatomide in distilled water. e percentages of drug release aer 5 min ( 5 %), 90 min ( 90 %), % dissolution efficiency (%DE) at 5 min (%DE 5 min ), and 60 min (%DE 60 min ) were determined and presented in Table 1. e %DE was calculated as the percent ratio of area under the dissolution curve up to time (5 and 60 min), to that of the area of the rectangle described by 100% dissolution at the same time [22].

Dissolution Properties of Oxatomide-Cyclodextrin Complex. As illustrated in
Regarding 5 , the inclusion complexes prepared by spray drying, freeze drying, and coevaporation ( Figure 5) showed a prompt drug release compared to pure oxatomide ( 0.05). e complex prepared by coevaporation in presence of PVP-K15 showed the highest release with 5 % and 90 % values of 98.33 ± 1.02% and 100%, respectively (Table 1 and Figure 6(b)). All inclusion complexes signi�cantly increased the %DE 5 min and %DE 60 min ( 0.05) compared to the pure drug (Table 1). Also, the complex prepared by coevaporation in presence of PVP-K15 showed the highest %DE. e increase in dissolution of drug in presence of both -cyclodextrin and water soluble polymers may be attributed to both improvement of drug wettability and formation of T 1: e percentage of drug release aer 5 min ( 5 %), 90 min ( 90 %), and % dissolution efficiency (%DE) at 5 min (%DE 5 min ) and 60 min (%DE 60 min ) of oxatomide from different inclusion complexes in distilled water (pH 6.8 ) at 37 ∘ C ± 0.5 ∘ C. readily soluble complexes in dissolution medium. When oxatomide -cyclodextrin inclusion complexes come in contact with an aqueous dissolution medium, the hydrophilic carrier dissolves and results in precipitation of the embedded drug into �ne particles, which increase the dissolution surface available [23].

Bioavailability
Study. e mean plasma oxatomide concentration-time pro�le following administration of single oral dose of oxatomide--cyclodextrin-PVPK15 complex and the commercial drug product (Tinset) are shown in Figure 7. Pharmacokinetic parameters ( max , max , AUC 0-4 and AUC 0-∞ ) are calculated individually on the basis of concentration-time data. From individual pharmacokinetic parameters, their mean values ± S.D, were obtained and are shown in Table 2 for both formulations.
e individual AUC 0-∞ values for oxatomide-cyclodextrin-PVPK15 complex were compared to those for the commercial product to determine the relative bioavailability. e mean relative bioavailability of oxatomide--cyclodextrin-PVPK15 complex to the commercial product was 173 15 ± 16 53%. is result indicated that 73.15% increase in the oral bioavailability of oxatomide was achieved by the complex formulation. Although oxatomide -cyclodextrin complex improves the bioavailability of oxatomide in terms of extent of absorption from the GI tract, it did not change the rate of drug absorption in terms of max and achieved signi�cantly lower max values in comparison with the commercial drug product. Comparing the values of elimination rat constants ( el ) for oxatomide--cyclodextrin-PVPK15 complex with those for commercial product reveals a marked effect of complexation on increasing the duration of action of oxatomide.

Conclusion
e results reported here suggest that inclusion complex of oxatomide with -cyclodextrin prepared by coevaporation in presence of PVP-K15 results not only is an enhancement of the oxatomide dissolution rate but also improves the bioavailability of oxatomide in terms of extent of absorption from the GI tract and increases the duration of action of oxatomide.