PREPARATION AND CHARACTERIZATION OF METHOCEL K4M, EUDRAGIT RLPO AND PEG 4000 LOADED CARBAMAZEPINE MICROSPHERES

factorial design was made by central composite design where the amount of Methocel K4M and PEG 4000 were selected as independent variables. Thirteen formulations were prepared with MDT, T50%, T80%, and swelling index as dependent variables. Data were analyzed statistically using linear regression model by IBM SPSS version 22. Effect of varying amount of Methocel K4M and PEG 4000 on the drug content, entrapment efficiency, swelling index, surface morphology (SEM), and in-vitro drug release rate were evaluated. Formulations with high amount of Methocel K4M showed good swelling properties. In SEM studies microspheres appeared rough and spherical in shape. Increasing amount of PEG produced pores on the surface of the microspheres due to the diffusion of polymers towards the external phase thus showing better drug release (B1, B3 & B8). Further clarification on release mechanism from spherical particles were studied using the Kopcha kinetics, Weibull model and Baker-Lonsdale model. In Kopcha kinetics all the formulations had ratio of A/B greater than 1 where release was primarily controlled by a Fickian diffusion. Weibull Model has parameters that are more sensitive to release kinetic data, the value of β was <1 and Td value was low showing enhance drug release. In Baker-Lonsdale model most spherical matrix had uniformly distributed drugs with consistent drug release by diffusion mechanism which was seen as linear graphs.

Modulated carbamazepine prolonged release dosage forms, microspheres have been prepared by the emulsion solvent evaporation method using Methocel K4M, PEG 4000 and Eudragit RLPO. A 2 2 factorial design was made by central composite design where the amount of Methocel K4M and PEG 4000 were selected as independent variables. Thirteen formulations were prepared with MDT, T 50% , T 80% , and swelling index as dependent variables. Data were analyzed statistically using linear regression model by IBM SPSS version 22. Effect of varying amount of Methocel K4M and PEG 4000 on the drug content, entrapment efficiency, swelling index, surface morphology (SEM), and in-vitro drug release rate were evaluated. Formulations with high amount of Methocel K4M showed good swelling properties. In SEM studies microspheres appeared rough and spherical in shape. Increasing amount of PEG produced pores on the surface of the microspheres due to the diffusion of polymers towards the external phase thus showing better drug release (B1, B3 & B8). Further clarification on release mechanism from spherical particles were studied using the Kopcha kinetics, Weibull model and Baker-Lonsdale model. In Kopcha kinetics all the formulations had ratio of A/B greater than 1 where release was primarily controlled by a Fickian diffusion. Weibull Model has parameters that are more sensitive to release kinetic data, the value of β was <1 and T d value was low showing enhance drug release. In Baker-Lonsdale model most spherical matrix had uniformly distributed drugs with consistent drug release by diffusion mechanism which was seen as linear graphs.

Methods:-Preparation of carbamazepine microspheres:-
Weighed quantities of polymers and PEG were dissolved in 20 ml of mixed solvent system (MSS) consisting of dichloromethane and ethanol at 3:1 ratio. Then the required amount of carbamazepine was added and dissolved by sonication until a clear solution was formed. This solution was the internal phase. For the preparation of external phase, 50 ml liquid paraffin emulsified with 0.5 ml span 80 was taken in a 250 ml beaker and stirred using an over head stirrer. The internal phase was then slowly poured drop wise to the external phase, while stirring at 450 rpm held by the mechanical stirrer equipped with a three-blade propeller, at room temperature. The whole system was stirred for 3 hours and then the microspheres were separated by filtration, the excess of paraffin oil was eliminated by repeated washing (4 to 5 times) with 50 ml n-hexane and finally dried overnight in desiccators at room temperature to yield free flowing spherical products [3]. After drying, microspheres were kept in a 10 ml vial with proper identification and preserved in the desiccator.

Factorial design of experiments:-
In this present study the formulation of carbamazepine loaded polymeric microspheres was designed by 2 2 factorial central composite design (CCD) using Minitab 17. The independent variables were Methocel K4M and PEG 4000 whereas the dependent variables selected were MDT, T 50% , T 80% , and swelling index. Particle shape and surface analysis by scanning electron microscope (SEM) study:-Shape and surface morphology of microspheres were observed under scanning electron microscopy (SEM). Small amount of microspheres was spread on a glass stub and the stub containing the sample was placed in the scanning electron microscope (JEOL JSM -6490 LA, Japan). Then SEM photographs were taken at an acceleration voltage of 20 kV for 15 minutes. The drug-loaded microspheres were evaluated for their sphericity and surface smoothness under different magnification and photomicrographs of suitable magnifications [5].

In-vitro dissolution study of microspheres containing cabamazepine in 1 % SLS solution:-
The dissolution studies of carbamazepine microspheres were carried out in a USP dissolution 8 station test apparatus type I (Basket type). 50 mg of microspheres were placed in 900 ml of dissolution medium (distilled water containing 1 % sodium lauryl sulfate) and were stirred at 100 rpm at 37 ± 0.5 °C. 10 ml aliquot was withdrawn from the dissolution medium at pre-determined intervals of 30 minute, 1 st hour, 2 nd hour, 3 rd hour, 4 th hour, 5 th hour, 6 th hour, 7 th hour & 8 th hour. At each interval, the withdrawn medium was replaced with an equivalent amount (10 ml) of fresh dissolution medium. Collected samples were filtered and then analyzed by measuring the absorbance through an UV spectrophotometer at 284 nm after suitable dilution to determine the amount of the carbamazepine released from the microspheres. The percentage of drug release was plotted versus time. Each experiment was repeated three times and their average were calculated to find percentage release for each batch.

Data analysis of release studies:-
Eight kinetic models including Zero order, First order, Higuchi plots, Korsmeyer-Peppas, Kopcha kinetics, Hixson Crowell plots, Weibull plots, and Baker and Lonsdale plots were applied to determine the rate and the mechanism of release of carbamazepine from the prepared microspheres.

Successive fractional dissolution time:-
To observe the release of drug from the formulations in different experimental conditions, MDT (mean dissolution time), T 25% , T 50% and T 80% values were determined from dissolution data by the use of following equations: MDT = (n/n+1). K -1/n T 25% = (0.25/k) 1/n T 50% = (0.5/k )1/n T 80% = (0.8/k) 1/n Mean dissolution time (MDT) was measured to characterize the drug release rate from the formulations. Higher value of MDT refers to higher rate retardant efficiency of the polymer. Visual and tactile observations of all the formulations in Fig. 1 confirmed that swelling was dominant in all the formulations due to the presence of Methocel, a hydrophilic polymer as it undergoes swelling in presence of liquid solvent forming a polymeric chains [7]. This mechanical feature of surface hydrated polymer forms a viscous barricade that plays a crucial role in the whole drug release rate.

In-vitro release kinetics of carbamazepine loaded microspheres:-
To determine the release profile of the microspheres, the dissolutions of all the batches were carried out in 1% SLS for 8 hours at 100 rpm and the release rate was calculated. Eudragit RLPO was used as release retardant because it offers pH independent drug release and has highly permeability which could allow the gastriontestinal fluid penetration inside the microspheres. Methocel K4M is a hydrophilic matrix forming polymer which enhances drug release in a controlled rate, moreover it inhibits recrystallization of carbamazepine to carbamazepine dihydrate (CBD) in the gastrointestinal tract, which is one of the major rate-limiting steps in bioavailability of oral dosage forms. Polyethylene glycol (PEG 4000) was used as a channeling agent to improve the drug release properties of the microspheres [8]. From the release profile it was observed that there was a burst release initially from 10-20%. Further analysis of the release profiles also revealed that changes in the amount of Mehocel K4M and PEG 4000 had a great effect on the release rate of carbamazepine from the microspheres. So, the release rate of carbamazepine from the microsphere can be modulated by adjusting the amount of Methocel and PEG (Table 2).          Drug release from matrices may involve processes of diffusion, erosion, and leaching or dissolution. From Table 3 & 4 it can be seen that none of the batches followed zero order, batch B1-B3, B5, B8, B9 and B13 followed first order, Batch B1-B9, B11 and B13 followed Higuchi model, Batch B1, B5 and B8 followed Hixon Crowell cube root law, and Batch B1-B3, B5-B10, and B13 followed Korsmeyer -Peppas model [9,10,11].The values of release exponents (n) for batch B1-B3, B5, B7-B10, B13 was >0.45 which indicate that the drug was released from the formulations by following non-Fickian release mechanism more specifically both diffusion and erosion controlled release mechanism wheras B6 had n values <0.45 which mean the drug release followed Fickian release mechanism (diffusion controlled). As a result the formulation followed both diffusion and erosion mechanism. For further clarification the data were fitted to Kopcha mathematical model [12]. This finding was supported by evaluation of the ratios of the exponents A/B (i.e., diffusional factor A and erosional factor B) derived from the Kopcha model which were greater than 1 in all cases [13]. The data in Table 4 clearly shows that the value of A is far greater than that for B, suggesting that drug release from the microspheres was primarily controlled by a diffusion process. The plots for Kopcha kinetics can be seen in Fig. 6.
Weibull model [14] was used to determine and compare the dissolution release profiles of the formulations as shown in Table 4 and Fig. 8. Graphical representation of Log [-Ln (1 -Q t / Q ∞) ] verses Log time gives a linear relation where the R 2 values were 0.97-0.99 in all the formulations except B11 (R 2 = 0.96). The calculated Weibull parameter β was <1 for all the formulations, indicated a parabolic curve with steeper initial slope that was consistent with the exponential. If the β value was >1 then an S-shaped graph would be seen. The time parameter, T d , can be calculated from α and β parameters (α = (T d ) β ) and represents the time interval necessary to dissolve 63.2% of the drug [14]. From the data in Table 4 the T d value is lowest for Teg CR. Thus drug release was faster in case of the Tegretol compared to the formulation. A higher T d value indicates slower release [15]. Highest T d value was obtained in B7 which shows that the drug release is slowest in this formulation. This could be possible because of less amount of channeling agent (PEG) compared to the other formulations. Formulation B8, B3 and B1 shows quiet similarity with the market product i.e. fast release the possible reason could be the high concentration of PEG that enhanced drug release.
Baker Lonsdale model was used to linearize the controlled release of drug from a spherical matrix [16]. Where formulation B1, B2, B3, B6-B9, and B11-B13 had R 2 values of 0.97-0.99 i.e. the graphical plots were linear, showed in Fig. 9, thus describing release profiles of drugs from matrices with uniform drug distribution. From the Table 5 it is seen that the MDT is highest for formulation B4, B6 and B7 due to comparatively less amount of PEG and more amount of Methocel. The MDT is low in formulations that have high amount of PEG limiting the retarding effect.

Surface morphology study:-
The SEM images in Fig. 10 and 11 revealed the surface morphology and shape of carbamazepine microspheres prepared from Methocel K4M, PEG 4000 and Eudragit RLPO. In the two batches, B8 and B11 the amount of Methocel K4M and Eudragit RLPO were same but the amount of PEG 4000 was 582.80 mg in B8 and 300mg in B11. So changes in the morphology and shape were possibly due to the varying amount of PEG 4000. In Formulation B8, the microspheres were almost spherical but the surface was rough with two pores onit (Fig. 10), the possible reason for this could be the high concentration of PEG 4000 (compared to B11). PEG is partially soluble in the oil phase and thus during the matrix formation the migration of PEG 4000 from the internal phase had led to the formation of pores in the microspheres [17]. Moreover the surface roughness could be due to the slow evaporation of solvent or high permeability of Eudragit RLPO [18]. In Formulation B11 as showed in Fig. 11, the microspheres were almost spherical, the surface was rough but had no holes on it and the size of the microspheres was found to be non-uniform. The possible reasons for surface roughness could be due to the slow evaporation of solvent or high permeability of Eudragit RLPO and Methocel K4M [19]. In case of rough surface there is more chance of wetting and contact of water compared to smoother ones. The uneven texture might cause holding of moisture at crests, cracks, or ridges. This persistence of moisture for more time might cause weakening of the matrix system. Presence of holes enhance drug release since these type of areas facilitate the entrance of dissolution medium to the microsphere. The presence of such holes might cause leaching of the matrix system of the particles. Hence, from the SEM images it can be concluded that the surface morphology of the microspheres vary with changes in polymer type and concentration.

Statistical analysis and model development:-
The regression parameters of the developed model and graphical interpretation for each response with statistical significance were calculated by using IBM SPSS version 22 2013. The F-values were 5.253, 9.11, 4.58 and 6.57 respectively. The p-values were found to be 0.025, 0.006, 0.036 and 0.014 respectively and all these p-values are <0.05 (significance level) indicating that the overall model has significant capacity to explain variation in response variables of MDT (Hours), T 50% (Hours), T 80% (Hours) and swelling index at 8 th (Hours). Insignificant lack of fit (p-value >0.05) also reflects the model adequacy as it can be seen from the

Conclusion:-
The present study was aimed to prepare sustained release microspheres for delivery of carbamazepine that will provide controlled drug release and prevent the re-crystallization of drug in GIT. The W/O emulsion solvent diffusion method was utilized in the preparation of carbamazepine microspheres. The microspheres prepared by this method were found to be of non-uniform in size and provided better controlled release profile of carbamazepine that could prevent the re-crystallization and carbamazepine dihydrate formation. The drug release from microspheres was found to be dependent on the concentration of PEG 4000 and Methocel K4M. The swelling behavior of the formulations were studied where an increase in swelling behavior was observed due to Methocel K4M which could be a good retardant. Methocel K4M and Eudragit RLPO increased the surface roughness due to their permeability characters, and PEG increased the porosity on the microsphere that offered high surface area for dissolution, which could have been the reason for enhanced drug release. Carbamazepine is a high dose drug which requires large quantity of excipient that may be the hurdle for preparation of sizeable dosage form. Further investigations are required to reduce the amount of polymer in the microspheres that can provide maximum drug loading and acceptable dosage form. Moreover, in-vivo behavior of the formulation should be evaluated to prove its clinical efficacy.