2.2. Methods
2.2.1. 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 β-cyclodextrin. The vials were mechanically shaken for 48 hrs at room temperature. After 2 days, aliquots were withdrawn and filtered using 0.45 μm pore size filter. The filtrate was suitably diluted and analyzed spectrophotometrically at the wavelength of 226 nm. The same procedure was repeated with β cyclodextrin aqueous solution containing either 0.25% w/v PVP-K15 or 0.1% w/v HPMC [15].
2.2.2. Preparation of Oxatomide-Cyclodextrin Solid Complexes
Oxatomide β-cyclodextrin solid complex was prepared with equimolar ratio of both oxatomide and β-cyclodextrin by using five different techniques as follows.
Physical Mixing Technique. The technique simply relies on the idea that both oxatomide and β-cyclodextrin were properly blended together in a ceramic mortar using pestle.
Kneading Technique. Into a ceramic mortar, β-cyclodextrin was wetted with a few drops of distilled water and properly kneaded. Then, oxatomide was added slowly, and the combined mixture was kneaded with the addition of few drops of distilled water. The dough mass was pressed and stretched with the hand fingers, folded over, and rotated through 90°, repeatedly. This process was continued for additional 15 min until the dough is elastic and smooth and then the obtained dough mass was left to dry under fume hood at room temperature for 24 hrs.
Coevaporation Technique. Coevaporation is another technique for formation of solid oxatomide β-cyclodextrin complex. Simply it consists of the simultaneous deposition of the oxatomide and β-cyclodextrin under vacuum conditions. This is simply done by dissolving weighed amount of oxatomide in 10 mL of 60% methanol solution in a glass beaker. In another glass beaker, β-cyclodextrin was dissolved in 50 mL distilled water. The 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. The 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). This 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 β-cyclodextrin was dissolved in 50 mL distilled water. The contents of the two beakers were then mixed together and sonicated at room temperature for 25 min to obtain and ensure a clear solution. The obtained solution is dried by spray drying under purified nitrogen gas. The inlet adjusted to a flow rate of 880 mL/h and at temperature 120°C. The nitrogen gas adapted to a flow 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 (Thermo Electron Corporation, Czech Republic) at −50°C, under vacuum (0.128 mbar). Briefly, in a 100 mL glass beaker, oxatomide was dissolved in 10 mL 60% methanol solution. In another 100 mL glass beaker, β-cyclodextrin was dissolved in 50 mL distilled water. The 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. The resultant solution is then frozen by placing at −30°C freezer for at least 24 hrs to ensure that the solution was completely frozen. Then, frozen solution was kept for 24 hrs into the vacuum chamber at −50°C where the freeze-drying process takes place.
2.2.3. Preparation of Oxatomide-Cyclodextrin Solid Complex in Presence of Water Soluble Polymer
Oxatomide β-cyclodextrin 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.
2.2.4. Characterization of Oxatomide-Cyclodextrin Complex
The oxatomide-β-cyclodextrin complex obtained from each technique is subjected to different chemical and thermal characterizations as follows.
Infrared (IR) Spectroscopy. IR spectra of oxatomide, β-cyclodextrin, and the prepared oxatomide β-cyclodextrin solid complexes were obtained at room temperature using an infrared spectrometer (IR100/IR200, Thermo Nicolet Corp., USA). Samples were prepared in KBr disks by means of a hydrostatic press. The scanning range was 400 to 4000 cm−1, and the resolution was 4 cm−1.
Differential Scanning Calorimetry (DSC). The thermal properties of oxatomide, β-cyclodextrin, and the prepared oxatomide β-cyclodextrin solid complexes were characterized using Shimadzu Differential Scanning Calorimeter, DSC60, Japan. The measurements were carried out at a heating rate of 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). The diffractograms of the different samples were obtained by Philips PW 3040 equipment. The measurements were carried out at conditions: Ni-filtered CuKα radiation, voltage 50 kV, current 30 mA, scanning speed 1° (2θ)/min, and investigating the samples in the 2θ range 0–30°.
2.2.5. In Vitro Dissolution Properties of Oxatomide-Cyclodextrin Complexes
The dissolution studies of oxatomide powder and the prepared oxatomide β-cyclodextrin solid complexes were performed according to the USP XXIII rotating basket method. The samples corresponding to 30 mg of oxatomide were placed into rotating basket. Dissolution medium was 900 mL of distilled water (pH 6.8). The 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 filter (0.45 μm Millipore filter) and analyzed spectrophotometrically for oxatomide concentration at λmax 277 nm. The withdrawn samples were replaced by equal volume of fresh dissolution medium. Each test was performed in triplicate.
2.2.6. Bioavailability Study
The study was performed to assess the bioavailability of the suggested formula (oxatomide-β-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. The 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. The 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. The doses of both products were suspended in a solution of 4.6% glycerin, 87.6% polyethylene glycol 400, and 7.8% distilled water.
The blood samples were withdrawn from central ear artery 1 hr before the drug administration and after administration at specific time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hrs). Blood samples were collected in heparinized tubes. The 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 flunarizine hydrochloride (internal standard) were vortexed for two min with (100 μL×1 M) sodium hydroxide solution (100 μL×1 M) and 5.0 mL ethyl ether. The samples were centrifuged at 2000 rpm for 10 min at room temperature. The 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. The residues were then redissolved in 200 μL mobile phase under vortex and centrifuged at 18000 rpm for 8 min. Aliquots of 5 μL 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 modifications. Briefly, 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. The MS detector has electrospray capability with a mass range of m/z 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. The mobile phase consisting of 85% methanol and 15% (v/v) aqueous ammonium acetate solution (10 mM, pH 4.0) was pumped at an isocratic flow rate of 0.2 mL/min. The total run time was 7.5 min for each injection.
The ESI source was set at positive ionization mode. The [M+H]+, m/z 427.10, for oxatomide and [M+H]+, m/z 405.05, for flunarizine hydrochloride were selected as detecting ions, respectively. The 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. The quantification was performed via peak-area ratio. The lower limit of quantification was 5 ng/mL. The 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]. The elimination rate constant (Kel) was estimated by least square regression of plasma concentration-time data points in the terminal log-linear region of the curves. Half life (t1/2) was calculated as 0.693 divided by Kel. The area under the plasma concentration-time curve from zero to the last measurable plasma concentration at time t (AUC0-t) was calculated using linear trapezoidal rule. The area under the curve from zero to infinity, AUC0–∞, was calculated as AUC0–∞=(AUC0-t)+Ct/Kel, where Ct is the last measured concentration at the time t. Peak plasma concentration (Cmax) and the time to peak concentration (Tmax) were obtained directly from the individual plasma concentration versus time curve.
2.2.7. 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 significant difference was considered at P value < 0.05.