Pharma Science Monitor 5(2), Sup-1, Apr-Jun 2014 PHARMA SCIENCE MONITOR

Impact factor: 0.3397/ICV: 4.10 153 Pharma Science Monitor 5(2), Sup-1, Apr-Jun 2014 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: http://www.pharmasm.com FORMULATION, EVALUATION AND OPTIMIZATION OF PRESS COATED PULSATILE TABLET OF ZALTOPROFEN FOR THE TREATMENT OF RHEUMATOID ARTHRITIS Chetan G Kukadiya*, Kesha Desai, S. M. Vijayendra Swamy Bhagwan Mahavir College of Pharmacy, Nr. Ashirwad Villa, New City Light Road, B/H Heena Bunglow s, Vesu, Bharthana, Surat-395017 ABSTRACT The aim of the present study was to formulate, evaluate and optimize press coated pulsatile tablet of Zaltoprofen for the treatment of rheumatoid arthritis. The drug delivery system is based on the concept of chronotherapeutics. Dosage form provide delayed release up to 5 hour and after completion of lag time up to 5 hour dosage form provides burst release of drug therefore highest blood level of the drug coincide with peak pain and stiffness. Formulation comprising a core containing active ingredient, surrounded by the coating layer containing enteric ph dependent polymer eudragit L 100 and Ethyl cellulose. Core tablet was prepared by direct compression using crospovidone as a superdisintegrant and microcrystalline cellulose as a diluent. Core tablet was compression coated with barrier layer containing eudragit L 100 and ethyl cellulose using direct compression method. A 3 2 full factorial design was used to optimize barrier layer. The optimized check point formulation A1 was selected from overlay plot using Design expert 9.0. The press coated tablet with weight ratio of ethyl cellulose: eudragit L 100 76.48: 23.52 (%) with coating weight 356 mg is most satisfactory to provide desired pulsatile delivery of Zaltoprofen for effective treatment of rheumatoid arthritis. KEYWORDS: Zaltoprofen, Rheumatoid arthritis, Chronotherapy, Pulsatile release, Press coated tablet, Crospovidone, Eudragit L 100, Ethyl cellulose, Lag time. INTRODUCTION For the treatment of various diseases oral route is the most preferred route and conventional dosage forms are widely used for treatment. In conventional therapy drug is released immediately after medication. So, the drug concentration in the plasma is raised and sometimes it is more than the toxic level. The target of drug discovery is to obtain maximum drug efficacy and minimum side effect. Although sustained and constant release systems have been developed biological systems are not so responsive to these release systems. Several controlled release preparation present numerous problems such as resistance and drug tolerance, and activation of the physiological system due to long term constant drug concentrations in the blood and tissues. The diseases currently targeted for chronopharmaceutical formulations are those for which have

Impact factor: 0.3397/ICV: 4.10 154 enough scientific backgrounds to justify their Chronopharmaceutical drug delivery compared to the conventional drug administration approach. These include asthma, arthritis, duodenal ulcer, cancer, diabetes, cardiovascular diseases, hypercholesterolemia, ulcer and neurological diseases. The efficacy and side effect of many drugs vary depending on dosing time associated with circadian rhythms of biochemical, physiological, and behavioral processes under the control of circadian clock. It has been found out that circadian rhythm is useful for the treatment of various pathophysiological conditions of human body. Rheumatoid arthritis (RA) is a chronic disease that causes pain, stiffness, swelling and limited motion and function of many joints. The stiffness seen in active RA is most often worst in the morning. These Symptoms closely follows the circadian rhythms and mainly result of imbalance between anti-inflammatory effects of cortisol and pro-inflammatory effect of melatonin (MLT) in RA during night and early morning. Moreover, typical circadian rhythm of melatonin exhibits a maximum at 3.00 AM. The pulsatile drug delivery system (PDDS) is intended to deliver a rapid, or transient, and quantified medication release after a predetermined off-release period (lag time). PDDS can deliver the correct amount of medication at the desired location at the optimal time for maximum effect against disease, thereby enhancing therapeutic efficacy and improving patient compliance. Zaltoprofen is a novel NSAID S with powerful anti inflammatory and analgesic effects on inflammatory pain. Zaltoprofen is a drug with high efficacy contributing to the improvement of daily activities in patient of chronic rheumatoid arthritis. Press coating is a novel, simple and less time consuming technique of coating thereby Press coating technique is suitable to formulate pulsatile release tablet. This system can be administered at night (before sleep) and gives drug release in early morning that would be a promising chronopharmaceutic system 1-8 MATERIALS AND METHODS Materials: Materials used in the present investigation were obtained from the following sources: Zaltoprofen was obtained from ZCL chemical, Mumbai. Crospovidone, Microcrystalline cellulose, Eudragit L 100, Etheyl cellulose, Talc, Magnesium stearate were obtained from the chemdyes corporation, Ahmedabad. Methods: 1. Preformulation study 1.1) Organoleptic evaluation 9 The color, odor, and taste of the drug were characterized and recorded using descriptive terminology.

Impact factor: 0.3397/ICV: 4.10 155 1.2) Drug excipients compatibility study The drug excipients interaction studies were carried out using Fourier Transform Infrared Spectrophotometer (FTIR). 1.3) Solubility study of Zaltoprofen 10 Solubility of Zaltoprofen in phosphate buffer ph 7.4, ph 6.8 and in 0.1N HCL ph 1.2 was determined by equilibrium solubility method. Sufficient excess amount of Zaltoprofen was added to 5 ml stoppered glass vials containing ph 7.4, ph 6.8 and ph 1.2 buffer solutions separately. The vials were shaken reciprocally for 72 h on mechanical shaker to reach equilibrium at R.T. The solutions were transferred into tubes and centrifuged for 30 min at 2500 rpm. Solutions were filtered using whatmann filter paper and the filtrate was analyzed for drug content by UV visible spectrophotometer at 340nm and 338 nm after appropriate dilutions. The study was performed in triplicate. 2. Preparation of press coated pulsatile tablet of zaltoprofen 11 Two steps are involved in preparation of press coated pulsatile tablet of zaltoprofen: 2.1) Preparation of core tablet of zaltoprofen The core tablets of Zaltoprofen were prepared by direct compression method. As shown in below table. Core tablets of Zaltoprofen were prepared by using fixed concentration of superdisintegrant crospovidone and diluent i.e. microcrystalline cellulose. Calculated quantities of Zaltoprofen, super disintegrants, and diluents were accurately weighed and blended in a mortar. All ingredients were passed through sieve no. 60 # and throughly mixed. Then the talc and magnesium stearate were added to the mixture. The mixture was compressed into tablet on a rotary tablet punching machine using 8 mm punch. Table 1: Composition of core tablet Ingredients Zaltoprofen Crospovidone Microcrystalline Magnesium Talc cellulose stearate Quantity 80 5 70 2 1 (mg) 2.2) Preparation of Zaltoprofen press-coated tablet The press-coated tablets of Zaltoprofen were prepared by direct compression method. Calculated quantities of ethyl cellulose and eudragit L 100 were accurately weighed and blended in a mortar. All ingredients were passed through sieve no. 60 # and thoroughly mixed. Then this coating material was used as barrier layer to prepare press-coated tablet. Half the quantity of the

Impact factor: 0.3397/ICV: 4.10 156 coating material was placed in the die cavity; the core tablet was carefully positioned in the center of the die cavity and was filled with other half of the coating material. The coating material was compressed using 12 mm punch on a rotary tablet compression machine. 3. Optimization by 3 2 full factorial design In the present study, a 3 2 full factorial design by response surface methodology was used to optimize press coated pulsatile tablet formulation of Zaltoprofen. In this design, two independent variables were evaluated, each at three levels, and experimental trials were performed at all nine possible combinations. Design-Expert 9.0 software (State-Ease Inc., USA) was used for mathematical modeling, evaluation of the ability to fit to the model and response surface modeling. Two independent variables, weight ratio of ethyl cellulose to eudragit L 100 (%) (X 1 ) and coating level (mg) (X 2 ) were set at three different levels. High and low levels of each factor were coded as 1 and -1 respectively and the medium level as zero. The levels of these formulation variables were chosen on the basis of results obtained from the preliminary studies and literature survey. In addition to factors and levels, dependent variables were also selected for the evaluation of the factorial design batches. The dependent variables measured were lag time (t 10 %) (min) (Y 1 ), Cumulative percentage Drug release at 7 hour (Y 2 ). Table 2: Coded and decoded values for all the formulations Batch code Coded value for Coded value for Decoded value for Decoded value for X1 factor X2 factor X1 factor (%) X2 factor (mg) F1-1 -1 75:25 300 F2-1 0 75:25 350 F3-1 1 75:25 400 F4 0-1 80:20 300 F5 0 0 80:20 350 F6 0 1 80:20 400 F7 1-1 85:15 300 F8 1 0 85:15 350 F9 1 1 85:15 400

Impact factor: 0.3397/ICV: 4.10 157 Table 3: Composition of factorial batches press coated tablet Ingredients F1 F2 F3 F4 F5 F6 F7 F8 F9 (mg) Zaltoprofen 80 80 80 80 80 80 80 80 80 Crospovidone 5 5 5 5 5 5 5 5 5 Microcrystalline cellulose 70 70 70 70 70 70 70 70 70 Mg. Stearate 2 2 2 2 2 2 2 2 2 Talc 1 1 1 1 1 1 1 1 1 Ethyl cellulose 225 262.5 300 240 280 320 255 297.5 340 Eudragit L 100 75 87.5 100 60 70 80 45 52.5 60 Total 458 508 558 458 508 558 458 508 558 4. Evaluation of tablets All the prepared press coated tablets were evaluated for pre compression and post compression parameters 4.1) Pre compression evaluation parameters 12 Angle of repose ( ): The angle of repose of powder blend was determined by the funnel method. The accurately weight powder blend was taken in the funnel. The height of the funnel was adjusted in such a way the tip of the funnel just touched the apex of the powder blend. The powder blend was allowed to flow through the funnel freely on to the surface. The diameter of the powder cone was measured and angle of repose was calculated using the following equation. tan = h/r Where, h and r are the height and radius of the powder cone. Bulk density It is the ratio of total mass of powder to the bulk volume of powder. It was measured by pouring the weighed powder into a measuring cylinder and initial weight was noted. This initial volume was called the bulk volume. From this the bulk density was calculated according to the formula mentioned below. It is expressed in gm/ml and is given by Weight of powder Bulk density = Volume of bulk powder in cylinder

Impact factor: 0.3397/ICV: 4.10 158 Tapped density: It is the ratio of total mass of the powder to the tapped volume of the powder. Volume was measured by tapping the powder for 100 times and the tapped volume was noted. It is expressed in gm/ml and is given by Tapped Density = Mass of powder Tapped volume of the powder Compressibility index (Carr s index): It indicates powder flow properties. It is expressed in percentage and is given by Tapped density Bulk density Carr s consolidation index = 100 Tapped density Hausner s Ratio: It was calculated by the following formula. Tapped density Hausner s ratio = Bulk density 4.2) Post compression evaluation parameters Uniformity of weight 13 Weigh individually 20 units selected at random and calculate the average weight. Not more than two of the individual weights deviate from the average weight by more than the percentage shown in the table and none deviates by more than twice that percentage. Table 4: Uniformity of Weight Average weight of tablet % deviation 80 mg or less 10 More than 80 mg but less than 250 mg 7.5 250 or more 5

Impact factor: 0.3397/ICV: 4.10 159 Thickness and diameter 11 Thickness and diameter of tablets was determined using vernier caliper. Three tablets were evaluated and an average value was calculated. The thickness and diameter were measured in mm. Hardness test 11 Hardness was measured using Monsanto hardness tester. The force required to break the tablet is recorded. The hardness of tablets of each batch was measured in kg/cm 2 Friability test 11 Tablets require certain amount of strength or hardness and resistance to withstand mechanical shock of handling in manufacturing, packaging, and shipping. A pre weighed tablets were placed in the Roche friabilator and apparatus was rotated at 25 rpm for 4 minutes. After revolutions the tablets were dedusted and weighed again. The percentage friability was measured using the formula, Initial weight Final weight % Friability = X 100 Initial weight Drug content 14 Five tablets were taken and powdered. Tablet powder equivalent to 100 mg of Zaltoprofen was weighed, sufficient volume of phosphate buffer was added and volume was made up to 100 ml with phosphate buffer ph 6.8. Then the solution was filtered and the filtrate was further diluted with phosphate buffer ph 6.8 to get require concentration. The absorbance of resulting solution was measured by UV spectrophotometer at 340 nm. In vitro disintegration time for core tablet 15 Disintegration time was determined using USP disintegration apparatus with phosphate buffer of ph 6.8. The volume of medium was 900 ml and temperature was 37±0.5 C. The time in seconds taken for complete disintegration of the tablet with no palatable mass remaining in the apparatus was measured. In vitro dissolution study of core tablet 15 In-vitro dissolution study of core tablet was performed using USP Type II dissolution apparatus (Paddle type) at speed of 50 rpm. 900 ml of phosphate buffer ph 6.8 was utilized as dissolution medium. The temperature of the medium was maintained at 37 ± 0.5 C. Aliquot of dissolution

Impact factor: 0.3397/ICV: 4.10 160 medium 5 ml were withdrawn at specific time intervals (5, 10, 15, 20, 30, 45, 60 & 90 min) and filtered each with whatman filter paper. Equal amount of fresh dissolution medium was replaced immediately after each withdrawal. The amount of drug present in each sample was determined by UV-Visible spectrophotometer at 340 nm. 15, 16 In vitro dissolution study of press coated tablet The in-vitro drug release studies of press-coated tablets of prepared formulations were carried out using USP dissolution test apparatus type-ii (Paddle type) using 900 ml of 0.1N HCL for 2 hrs and then replaced with phosphate buffer ph 6.8 at speed of 50 rpm at 37 ± 0.5 ºC and the aliquot of dissolution medium 5 ml were withdrawn at specific time intervals and filtered each with whatman filter paper. Equal amount of fresh dissolution medium was replaced immediately after each withdrawal. The absorbance of the resulting solution was measured at the 338nm (0.1N HCL ph 1.2) and 340nm (phosphate buffer 6.8 ph) using UV spectrophotometer. 5. Statistical analysis and validation of design Statistical analysis and validation of model were performed using design expert 9.0 software (Stat-Ease Inc., USA). The responses were analyzed using ANOVA, the individual response parameters were evaluated using F test and polynomial equation was generated for each response using multiple linear regression analysis. Counter plot and 3D surface plot were constructed using design expert software. By utilizing design expert software, one final formulation corresponding to the predicted optimum polymer ratio and coating level were prepared to determine the validity of the model generated. Afterward, the observed experimental data of the response properties were quantitatively compared with those of the predicted values. 6. Stability study of optimized formulation Accelerated stability study of optimized press coated pulsatile tablets was performed as per the ICH guideline Q1C. Optimized PCPT of Zaltoprofen was wrapped in aluminum foil and stored in stability chamber at 40 ± 2 C/ 75 ± 5 % RH for a period of 1 month. After a period of one month tablets were withdrawn from chamber and evaluated for uniformity of weight, friability, hardness, drug content and in vitro drug release study. RESULT AND DISCUSSION 1. Preformulation study 1.1) Organoleptic evaluation: The color, odor, and taste of the drug were characterized and recorded using descriptive terminology; the results were shown in the below table 5.

Impact factor: 0.3397/ICV: 4.10 161 Table 5: Organoleptic evaluation Properties Results Description Crystalline Taste Bitter Odor Odorless Color White to light yellow 1.2) Drug excipients compatibility Study: The FTIR spectra of pure drug and mixture of press coated tablet blend were shown in below figure 1 and 2. From the result it can be concluded that functional group peaks remain same even after physical mixture was prepared using excipients and APIs. From the observation peaks it can be established that both APIs and excipients are compatible with each other without any significant interaction Figure 1: FTIR spectra of pure drug

Impact factor: 0.3397/ICV: 4.10 162 Figure 2: FTIR spectra of press coated pulsatile tablet powder blend 1.3) Solubility study of zaltoprofen: The result of solubility study of zaltoprofen was shown in below table 6. In ph 7.4 solubility (1.690 ± 0.0629) of Zaltoprofen was higher as compare to ph 6.8 (1.325 ± 0.0320) and ph 1.2 (0.0047 ± 0.0001), Moreover solubility of Zaltoprofen in ph 6.8 was higher as compare to ph 1.2 So, it was concluded that as the ph increases the solubility of Zaltoprofen also increases with it and Zaltoprofen has a ph dependent solubility profile. Table 6: Solubility study of zaltoprofen Batch ph Solubility*(mg/ml) code S1 1.2 0.0047 ± 0.0001 S2 6.8 1.325 ± 0.0320 S3 7.4 1.690 ± 0.0629 2. Evaluation of tablet: The result of pre and post compression evaluation parameters of core tablet of zaltoprofen was shown in below table 7 and 8. 2.1) Evaluation of core tablet

Impact factor: 0.3397/ICV: 4.10 163 Table 7: Pre and post compression evaluation parameters of core tablet Pre Angle of Bulk Tapped Compressibility Hausner s compression Repose Density Density Index Ratio evaluation parameters Result 30.31 ± 0.56 0.40 ±0.0050 0.48 ±0.0073 16.66 ± 0.21 1.20 ± 0.0058 Post compression Uniformity of Thickness Hardness Friability test Disintegration time evaluation parameters Weight Result 158.1 ± 1.04 2.73 ± 0.058 3.57 ± 0.058 0.56 56.33 ± 3.49 Table 8: In vitro dissolution study of core tablet Time (min) 0 5 10 15 20 % CDR 0 85.78 ± 2.07 91.9 ± 1.55 94.87 ± 1.41 98.04 ± 1.32 Figure 3: In vitro drug release study of core tablet 2.2) Evaluation of press coated tablet: The result of pre compression evaluation parameters of powder blend were shown in below table 9. From the result of pre compression evaluation parameters it can be concluded that powder blend has a good flow property.

Impact factor: 0.3397/ICV: 4.10 164 2.2.1) Pre compression evaluation of press coated tablet Table 9: Pre compression evaluation of press coated tablet Batch code Angle of Bulk Tapped Compressibility Hausner s Repose* Density* Density* Index*(%) Ratio* (ѳ) (g/cm3) (g/cm3) F1 21.98±0.56 0.34±0.011 0.41±0.016 16.99±0.56 1.20±0.01 F2 22.61±0.29 0.28±0.0067 0.34±0.0096 17.95±2.01 1.21±0.03 F3 22.52±0.33 0.28±0.0057 0.33±0.013 15.31±2.21 1.18±0.03 F4 21.29±0.66 0.35±0.011 0.42±0.016 17.32±0.56 1.21±0.01 F5 21.72±0.81 0.30±0.0073 0.36±0.010 17.15±0.41 1.21±0.01 F6 21.90±0.87 0.29±0.0061 0.35±0.015 16.96±1.84 1.20±0.02 F7 19.95±0.57 0.37±0.012 0.45±0.019 18.37±0.64 1.22±0.01 F8 19.72±0.34 0.29±0.010 0.35±0.017 20.18±1.39 1.19±0.05 F9 20.48±0.36 0.29±0.0061 0.35±0.015 18.08±0.39 1.20±0.02 2.2.2) Post compression evaluation of press coated tablet Table 10: Post compression evaluation of press coated tablet Batch Uniformity Thickness* Hardness* Friability Drug code of (mm) (kg/cm 2 ) test content* Weight (%) (%) (mg) F1 458.65±1.18 4.65±0.05 5.90±0.10 0.26 99.27±0.32 F2 508.75±1.65 5.08±0.08 5.93±0.06 0.23 98.84±0.14 F3 558.35±1.26 5.47±0.06 6.07±0.12 0.21 99.39±0.50 F4 458.45±1.05 4.67±0.03 6.13±0.15 0.17 100.06±0.14 F5 508.6±1.09 5.03±0.06 6.13±0.06 0.15 99.78±0.68 F6 558.5±1.05 5.43±0.06 6.20±0.17 0.10 98.42±0.19 F7 458.5±1.23 4.63±0.06 5.83±0.06 0.30 98.67±0.32 F8 508.7±1.21 5.57±0.08 6.17±0.15 0.15 99.97±0.14 F9 558.9±1.33 5.43±0.06 6.07±0.23 0.21 98.97±0.30 The results of post compression evaluation parameters of press coated tablets were shown in above table 10. From the result it can be concluded that

Impact factor: 0.3397/ICV: 4.10 165 Weight variation: Deviation in weight of factorial batches tablets are within the limit described in table 4 indicated that there was no significant weight variation in the prepared press coated tablets. Hence, all the tablets formulations passed the weight variation test. Thickness: Thickness of tablet was found to be in the range from 4.63 ± 0.06 to 5.57 ± 0.08 Hardness: Hardness of all formulation prepared by direct compression was found to be 5.83 ± 0.06 to 6.20 ± 0.17 kg/cm 2. Friability: The percentage friability was less than 1% in all the formulations, indicating that the friability is within the prescribed limits. The results of friability indicates that the tablet posses good mechanical strength. Drug content: Drug content in press coated tablets was found to be in the range of 98.42 ±0.19 to 100.06±0.14 that is within the acceptable limit. 2.2.3) In vitro dissolution study of press coated tablet Table 11: Cumulative percentage drug release of factorial batches F1 to F9 % Cumulative drug release Time (min) F1 F2 F3 F4 F5 F6 F7 F8 F9 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 60 0 0 0 0 0 0 0 0 0 90 0 0 0 0 0 0 0 0 0 120 0 0 0 0 0 0 0 0 0 150 0 0 0 0 0 0 0 0 0 180 0 0 0 0 0 0 0 0 0 210 0 0 0 0 0 0 0 0 0 240 2.56 0 0 0 0 0 0 0 0

Impact factor: 0.3397/ICV: 4.10 166 ±0.79 270 42.53 ±0.66 0 0 15.89 ±1.57 0 0 0 0 0 300 69.21 ±1.12 22.94 ±1.18 0 53.47 ±2.87 2.75 ±1.71 0 26.1 ±1.70 0 0 330 80.14 ±1.68 62.02 ±2.19 0 75.98 ±1.32 42.7 ±1.32 0 63.45 ±1.64 6.84 ±1.34 0 360 87.42 ±0.86 79.56 ±1.41 29.27 ±1.57 85.47 ±2.61 66.75 ±1.13 10.6 ±0.92 79.85 ±1.32 45.31 ±1.27 0 93.39 86.45 64.35 89.96 80.22 49.08 86.78 69.71 17.64 390 ±0.91 ±1.48 ±1.13 ±2.24 ±1.36 ±1.00 ±1.02 ±0.95 ±1.09 97.72 90.47 78.94 94.88 87.62 70.67 91.12 80.43 59.47 420 ±1.15 ±1.15 ±1.44 ±1.34 ±0.85 ±1.17 ±1.14 ±1.47 ±1.88 95.45 87.21 98.39 93.66 83.18 96.23 89.31 78.54 450 ±1.16 ±1.37 ±1.09 ±1.28 ±1.04 ±1.00 ±1.23 ±1.31 98.16 91.79 97.59 89.52 98.69 94.36 85.75 480 ±0.65 ±1.32 ±0.98 ±0.72 ±1.16 ±1.33 ±1.56 96.18 94.60 98.32 90.04 510 ±1.1 ±1.05 ±1.21 ±1.05 98.24 98.43 95.10 540 ±0.82 ±1.02 ±1.04 98.32 570 ±1.18 2.2.3.1) Lag time of factorial batches press coated tablets: Table 12: Lag time of factorial batches Batch code F1 F2 F3 F4 F5 F6 F7 F8 F9 Lag Time* (t 10% ) 246 290 343 265 306 360 287 333 385 (min)

Impact factor: 0.3397/ICV: 4.10 167 Figure 4: In vitro drug release profile of factorial batches F1 to F9 The result of in vitro drug release study was shown in above table 11. Formulation F1, F4 and F7 contain different percentage weight ratio of EC: EL (75:25, 80:20, 85:15) and has shown lag time of 246, 265 and 287 respectively, whereas formulation F1, F2, F3 contain different coating level (300,350,400) and has shown lag time of 246, 290 and 343 respectively. Formulation F2, F5 and F8 contain different percentage weight ratio of EC: EL (75:25, 80:20, 85:15) and has shown lag time of 290, 306 and 333 respectively, whereas formulation F4, F5, F6 contain different coating level (300,350,400) and has shown lag time of 265, 306 and 360 respectively. Formulation F3, F6 and F9 contain different percentage weight ratio of EC: EL (75:25, 80:20, 85:15) and has shown lag time of 343, 360 and 385 respectively, whereas formulation F7, F8, F9 contain different coating level (300,350,400) and has shown lag time of 287, 333 and 385 respectively. From the result it was concluded that as the ratio of ethyl cellulose to eudragit L 100 and coating level increased lag time was also increased with it. Moreover from the result it can be conclude that coating level has more significant effect on lag time compare to percentage weight ratio of EC: EL. 2.2.4) In vitro ruptured behavior of press coated pulsatile tablet:

Impact factor: 0.3397/ICV: 4.10 168 Figure 5: Rupture behavior of press coated pulsatile tablet in dissolution media A: Press coated tablet in 1.2 ph B: Press coated tablet started to rupture in 6.8 ph after lag time C: Completely ruptured press coated tablet 3) Statistical analysis of factorial design: The results summarized in table 13 clearly indicate that both the dependent variables lag time (t 10 %) (min) and Cumulative percentage drug release at 7 hour (%) are strongly affected by the selected independent variable. The selected independent variables show a wide variation among the 9 batches (F1 to F9) Table 13: 3 2 design layout with respective observed responses Batch Code X 1 X 2 Y 1 Y 2 (% weight ratio of EC:EL) (%) (Coating level) (Mg) (Lag time) (t 10 %) (Min) (Cumulative percentage drug release at 7 hour) (%) F1 75:25 300 246 97.72 F2 75:25 350 290 90.47 F3 75:25 400 343 78.94 F4 80:20 300 265 94.88 F5 80:20 350 306 87.62 F6 80:20 400 360 70.67 F7 85:15 300 287 91.12 F8 85:15 350 333 80.43 F9 85:15 400 385 59.47

Impact factor: 0.3397/ICV: 4.10 169 3.1) Summary output of multiple regression analysis for effect of X 1 and X 2 on response Y 1 and Y 2 : The result of the analysis of variance (ANOVA) for responses Y1 and Y2 (P > 0.05) were shown in table 14. The F value in the ANOVA table was the ratio of model mean square (MS) to the appropriate error (i.e. residual) mean square. The larger the F value and the more likely that the variance contributed by the model was significantly larger than random error. In the table model F-value and high R square values suggested that these models were significant. The results of multiple linear regression analysis (table 15) reveal that both the coefficient b1 and b2 bear a positive sign for lag time (Y1). Therefore, increasing the ethyl cellulose content and coating level was expected to prolong lag time. For response cumulative drug release at 7 hr (Y2), both the coefficient b1 and b2 bear a negative sign; indicate antagonistic effect of both independent variables (X1 & X 2). Therefore, an increase in ethyl cellulose content and coating level leads to decrease in cumulative drug release in 7 hr. The polynomial equation for each response variable was as follow: Y 1 = 307.22+21.00 X 1 +48.33X 2 + 3.67 X 2 2 1 +4.67X 2 2 Y 2 = 87.08-6.02 X 1-12.44 X 2-3.22 X 1 X 2-4.04 X 2 Table 14: Result of Analysis of variance For Lag Time Regression DF SS MS F R 2 4 16733.11 4138.28 2596.52 0.9996 Residual 4 6.44 1.61 For cumulative drug release at 7 hour Regression DF SS MS F R 2 4 1219.90 304.97 158.40 0.9937 Residual 4 7.70 1.93

Impact factor: 0.3397/ICV: 4.10 170 Table 15: Summary of result of regression analysis For Lag Time Co-efficient b 0 b 1 b 2 b 12 b 11 b 22 Co-efficient + 307.22 + 21.00 + 48.33 + 0.25 + 3.65 + 4.67 value P- Value 0.0001 0.0001 0.0001 0.7509 0.0365 0.0194 For cumulative drug release at 7 hour Co-efficient b 0 b 1 b 2 b 12 b 11 b 22 Co-efficient + 87.08-6.02-12.44-3.22-1.36-4.04 value P- Value 0.0006 0.0010 0.0001 0.0113 0.1921 0.0157 4) Optimization of compression coated tablet: The application of desirability function gives possibility to predict the optimum levels of the independent variables. Optimized checkpoint formulation was designed accordance to the ramp plot and overlay plot as shown in table 16 (solution). Table 16: Optimization of compression coated tablet Constraints Name Goal Lower Limit Upper Limit Weight ratio of EC: In range 0 20 EL (%) Coating Level (mg) In range 300 400 Lag time (min) Targeted to 300 Cumulative drug In range 80 90 release at 7 hr (%) Solution Weight ratio of EC: Coating Level Lag time (min) Cumulative Desirability EL (%) (mg) drug release at 7 hr (%) 76.48: 23.52 356 300 89.15 1.0

Impact factor: 0.3397/ICV: 4.10 171 4.1) Generation of Contour Plot and Response Surface Plot for Response Y 1 Figure 6: Counter plot showing the effect on lag time using different combination of percentage weight ratio of ethyl cellulose: eudragit L 100 and coating level Figure 7: Response surface plot showing the effect of percentage weight ratio of ethyl cellulose: eudragit L 100 and coating level on lag time

Impact factor: 0.3397/ICV: 4.10 172 4.2) Generation of Contour Plot and Response Surface Plot for Response Y 2 Figure 8: Counter plot showing the effect on cumulative percentage drug release at 7 hour using different combination of percentage weight ratio of ethyl cellulose: eudragit L 100 (X 1 ) and coating level (X 2 ) Figure 9: Response surface plot showing the effect of percentage weight ratio of ethyl cellulose: eudragit L 100 (X 1 ) and coating level (X 2 ) on cumulative

Impact factor: 0.3397/ICV: 4.10 173 4.3) Generation of Contour plot and Ramp plot for optimized formula Figure 10: Ramp plot of optimized checkpoint formulation Figure 11: 3D surface plot of optimized checkpoint formulation 4.5) In vitro drug release of optimized formula: Optimized checkpoint formulation was prepared and the result of in vitro drug release study was shown in below table 17. Result of the in vitro drug release study of optimized checkpoint formulation suggests pulsatile release from the press coated tablet with a lag time of 302 min.

Impact factor: 0.3397/ICV: 4.10 174 Table 17: Cumulative percentage drug release of optimized checkpoint formulation Time (min) % CDR 0 0 30 0 60 0 90 0 120 0 150 0 180 0 210 0 240 0 270 0 300 7.75 ± 0.89 330 47.37 ±1.44 360 71.01 ± 1.30 390 80.75 ± 0.70 420 87.80 ± 0.53 450 93.27 ± 0.50 480 97.68 ± 0.92 Figure 12: In vitro drug release profile of optimized checkpoint formulation

Impact factor: 0.3397/ICV: 4.10 175 5) Stability study of optimized formulation: The result of accelerated stability study was shown in below table and it suggest that there were no significant changes in percentage cumulative drug release and lag time of the optimized formulation after a period of one month stored in stability chamber at 40 ± 2 C/ 75 ± 5 % RH. Hence the press coated tablets were found to be stable after one month accelerated stability study. 5.1) Post compression evaluation parameters of optimized formulation before and after stability study Table 18: Post compression evaluation parameters of optimized formulation before and after stability study Parameter Before After 1 month Accelerated condition 40 C ± 75% RH Uniformity of weight (%) 514.10 ± 2.00 513.85 ± 1.78 Hardness (kg/cm 2 ) 6.33 ± 0.20 6.40 ± 0.1 Friability (%) 0.19 0.23 Drug content (%) 99.65 ± 0.46 98.60 ± 0.41 5.2) Comparison of in vitro drug release of optimized formulation (A1) before and after stability study Table 19: Comparison of cumulative percentage drug release of optimized formulation (A1) before and after stability study Time (min) %CDR Before stability study %CDR After 1 month Accelerated condition 40 C ± 75% RH 0 0 0 30 0 0 60 0 0 90 0 0

Impact factor: 0.3397/ICV: 4.10 176 120 0 0 150 0 0 180 0 0 210 0 0 240 0 0 270 0 0 300 7.75 ± 0.89 14.34 ± 1.09 330 47.37 ±1.44 52.83 ± 1.01 360 71.01 ± 1.30 75.22 ± 0.88 390 80.75 ± 0.70 84.51 ± 0.75 420 87.80 ± 0.53 89.98 ± 1.36 450 93.27 ± 0.50 94.36 ± 0.53 480 97.68 ± 0.92 97.28 ± 0.31 Figure 13: Comparison of in vitro drug release profile of optimized formulation (A1) before and after stability study

Impact factor: 0.3397/ICV: 4.10 177 CONCLUSION The present investigation was aimed to develop press coated pulsatile tablet (PCPT) of Zaltoprofen for the treatment of rheumatoid arthritis. The result of FTIR analysis confirmed presence of Zaltoprofen and showed compatibility between drug and polymer without any significant interaction. Solubility study of Zaltoprofen was carried out by equilibrium solubility method. The result of solubility study suggested that Zaltoprofen is a drug with ph dependent solubility profile. Zaltoprofen pulsatile release tablets were prepared by compression coating technique. Initially core tablets were prepared by direct compression, tablets were found satisfactory in terms of hardness, thickness, uniformity of weight, drug content, disintegration time and in vitro drug release study. Press coated pulsatile tablet was optimized using 3 2 full factorial design. Percentage weight ratio of ethyl cellulose: eudragit L 100 (X 1 ) and coating level (X 2 ) were selected as an independent variable. Lag time (Y 1 = t 10% ) and cumulative percentage drug release at 7 hour (Y 2 ) were selected as a dependent variable. All the factorial batches press coated tablets were prepared by direct compression method and tablets were evaluated for uniformity of weight, hardness, thickness, drug content, friability and in vitro drug release study. From the result of in vitro drug release study it can be concluded that as the concentration of ethyl cellulose to eudragit L 100 (%) and coating level (mg) increased lag time increased and cumulative percentage drug release at 7 hour decreased. Optimized check point formulation was design according to the result of overlay plot and desirability function and characterized under same condition as outlined for factorial batches. The results of stability study of optimized batch were confirmed good compatibility and stability with selected excipients. In conclusion, the novel PCPT developed for Zaltoprofen could be a promising chronomodulated therapeutic system for the relief of morning pain and stiffness in patients with rheumatoid arthritis. REFERENCES 1. Asim Sattwa Mandal, Biswas Nikhil, Kazi Masud Karim: Drug delivery system based on chronobiology. Journal of Controlled Release. 2010; 147: 314-325. 2. Shan-Yang Lin and Yoshiaki Kawashima: Current status and approaches to developing press-coated chronodelivery drug systems. Journal of Controlled Release. 2012; 157: 331-356. 3. Walter grassi, Rossella De Angelis, Gianni Lamanna, Claudio Cervini: The clinical features of rheumatoid arthritis. European Journal of Radiology 1998; 27: 18-S24.

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Impact factor: 0.3397/ICV: 4.10 179 16. Dr. S.S. Khadabadi, Chishti NH, Khan Farhan, Tadvee Akeel: Formulation and evaluation of press coated tablet of ketoprofen A chronotherapeutic approach. International Journal of Pharmacy and Pharmaceutical Sciences 2013; 5: 733-738. For Correspondence Chetan G. Kukadiya Email: kukadiya.chetan@yahoo.in