Carbamazepine was obtained as gift sample from Medizen Pharmaceutical industries, Alexandria. Dichloromethane was purchased from Al-Nasr Chemical Company, Cairo Egypt. Ethyl cellulose N50 was supplied as a gift sample from Sigma pharmaceutical industries, Quesna, Egypt. Methanol HPLC grade Lobachemie, India. Polyvinyl Alcohol (PVA) was purchased from Nice Chemicals, Kerala, India. All other chemicals used in the present study were of analytical grade.
The concentrations of carbamazepine were in the in vitro samples were determined by UV spectrophotometry using double beam spectrophotometer made by Thermo Fisher Scientific (Madison, USA). Determinations were performed at 284nm and the calibration curve was linear in the concentration range of 2-20 µg/ml and the equation of the calibration curve was Y = 0.0536X + 0.0458). The slope of this standard curve 0.0536 ± 0.0005 and the intercept was 0.0458± 0.0025.
High performance liquid chromatography (HPLC)
The in-vivo study involved determination of carbamazepine concentrations in the plasma. This was achieved using HPLC system (Agilent isocratic controller, Agilent, Germany) which was supported PDA detector and an automatic sampling system with Quat pump. The mobile phase consisted a mixture of methanol and filtered distilled water (50:50, v/v) flowing at a flow rate of 1 ml/minute. The stationary phase was a reversed phase 10 μm particles C18 column, 150 mm in length with and internal diameter of 4.6 mm (Inertsil ODS C18, Waters). Chromatographic separation was conducted at ambient temperature and drug concentration was determined at 285 nm. The whole process including data analysis was controlled by OpenLAB software (Agilent 1260 infinity, Germany) .
The calibration curve was prepared using stock solutions containing 1000μg/ml of CBZ or the internal standard (propyl paraben) which were prepared in methanol. This was used to prepare the working solutions which were prepared by serial dilutions of CBZ in methanol to produced solutions containing 5, 10, 20, 50, 100, 200 and 400μg/ml of CBZ with fixed concentration of the internal standard (100μg/ml). These working solutions were used to construct the calibration curve in the plasma by adding 100μl of each working solution to 500μl of blank plasma followed by vortex mixing. Methanol (400μl) was added to each mixture with mixing to precipitate the proteins. The precipitated protein was separated by centrifugation and 50μl of the supernatant was injected into the HPLC. These procedures produced samples with drug concentration of 0.5, 1, 2, 5, 10, 20 and 40μg/ml with fixed concentration of internal standard (10μg/ml). The calibration curve was constructed by plotting the peak area ration of CBZ to the internal standard as a function of CBZ concentration . The actual plasma samples were treated by adding 100μl of internal standard (100μg/ml) to 500μl of plasma followed by vortex mixing. Methanol (400μl) was added to each mixture with mixing to precipitate the proteins as before.
Preparation of carbamazepine microsponges
Carbamazepine containing microsponges were prepared using the quasi-emulsion solvent diffusion technique. This method is reported to be simple with reproducible results . The composition of microsponges is presented in Table 1. Dichloromethane which solubilize ethyl cellulose and carbamazepine was used as the internal solvent with PVA aqueous solution being employed as external phase. Ethyl cellulose and carbamazepine were dissolved in dichloromethane. This solution was added dropwise to the aqueous solution of PVA using 18G syringe. This process was conducted under continuous stirring at 500 rpm using an overhead stirrer. Stirring continued for 4hours at the end of which the microsponges were harvested by filtration and were left to dry at ambient temperature. The dry microsponges were weighed to calculate the production yield which was calculated using equation 1.
Scanning electron microscopy (SEM)
The morphological characteristics of microsponges were assessed by scanning electron microscopy. The microsponges were coated with gold at room temperature. The morphology of the microsponges was studied using SEM (JEOL JSM-5300, Japan) which is supported with sputter coater device (JFC-1100E ion).
Determination of drug content and encapsulation efficiency
Crushed microsponges (100 mg) were dissolved in 10 ml of methanol and the solution was filtered. The filtrate (1ml) was suitable diluted and the concentration of the drug was quantified spectrophotometrically at 284 nm. These procedures were repeated three times and the drug content was determined using equation and the encapsulation efficiency was calculated using equation 3.
Differential scanning calorimetry (DSC)
DSC was utilized to investigate the effect of encapsulation of carbamazepine into microsponges on the thermal pattern of the drug. This was achieved using differential scanning calorimeter (DSC6 module, Perkin Elmer, Waltham, USA). Carbamazepine (2-4 mg) or equivalent microsponges was encapsulated into aluminum pans. These pans were loaded in the furnace of the equipment with an empty pan serving as a reference. The sample was heated from 30 to 400oC at a heating rate of 10oC/minute. The test was conducted under continuous flow of nitrogen gas. Data acquisition and manipulations were achieved using Pyris® software.
Fourier Transform Infrared spectroscopy (FTIR)
FTIR was performed to monitor possible interactions between carbamazepine and the polymers. The study utilized FTIR spectrophotometer (Bruker Tensor 27, Ettlingen, Germany) which is supported by a DLaTGS detector. Carbamazepine, ethyl cellulose or the corresponding microsponges were mixed with potassium bromide. The mixture was prepared as thin disks by direct compression before loading into the sampling holder of the spectrophotometer. The FTIR spectra were collected in the range of 4000-400cm-1.
X-ray diffraction (XRD)
The crystallinity of carbamazepine was monitored before and after encapsulation into the microsponges. This was achieved using an X-ray powder diffractometer (Bruker MeasSrv- D2-208219/D2-208219) which employed secondary graphite monochromated at 30 kV/10 mA. The data were collected at a scanning steps of 0.03o to cover the two-theta range of 3-60o at a temperature of 25 oC.
Determination of drug release
The release rate of carbamazepine from the prepared microsponges was assessed using USP type II dissolution (paddle method) equipment in which the paddle was rotated at 100 rpm. The release study employed continuous pH variation method in which the formulation was subjected for pH 1.2 for 2 hours at the end of which the pH was adjusted to 7.4 for 6 hours . Briefly, microsponge equivalent to 200mg of carbamazepine was add to dissolution vessels containing 500ml of 0.1N HCl containing 1% sodium lauryl sulphate which were previously equilibrated to 37oC. Samples (5ml) were collected at predetermined time intervals for 2 hours. The dissolution medium was replenished after each sample. The pH was then adjusted to 7.4 by addition of 55 ml of 4N sodium hydroxide and the volume was adjusted to 900 using 20mM sodium dihydrogen phosphate containing 1% sodium lauryl sulphate. Samples were collected and the dissolution medium was replenished as before. The collected samples were filtered and suitably diluted if required before was determination of the drug content spectrophotometrically at 284nm. The cumulative amount of carbamazepine released was expressed as percentage of the dose and was plotted as a function of time to develop the release profile. The release profile was used to compute the release efficiency which was calculated from the area under the release profile relative to the theoretical area of 100% release at all time points .
The release kinetics was computed by fitting the release data for zero, first, Higuchi and Korsmyer and Peppas kinetic models.
In vivo study
The study compared the oral bioavailability of carbamazepine from the microsponges relative to a suspension of the unprocessed carbamazepine. The study utilized 6 male albino rabbits (average weight of 2.18 + 0.1 Kg). The animal study was approved by Faculty of Pharmacy, Tanta University Ethical Committee (approval number (2042016). The study was conducted in a parallel experimental design in which the rabbits were divided into two groups (3 rabbits each). The first group administered carbamazepine-loaded microsponges (group A) and the second group administered carbamazepine aqueous suspension (group B) which served as control. The rabbits were given free access to water with food restriction for overnight before administration. Each rabbit received a dose equivalent to 200 mg carbamazepine suspended in 10 ml water by oral by oral feeding tube. The rabbits were kept in cages and had free access to food and water after 6 h. Serial blood samples (1.0 ml) were withdrawn from the marginal ear vein into a vial containing sodium citrate (3.2%) (50 µl) at predose and post-doses (1, 2, 3, 4, 6, 8, 10,12 and 24 h). The blood samples were mixed and centrifuged at 5000 rpm for 10 min within 1 hour of collection. This separated the plasma which was stored at−20 ◦c until time of analysis. Assay of drug in plasma was conducted using HPLC as described above .
Statistical analysis employed Kruskal Wallis test with tukey`s multiple comparison being used to probe the significance between groups.