DEVELOPMENT AND CHARACTERIZATION OF TERBUTALINE SULPHATE MICROSPONGE AND ITS COLONIC DELIVERY BY COMPRESSION COATED TABLETS

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1 Author s Accepted Manuscript: Basu et al, WRJPT, 2017, 3(3): 1-75 Available at: Author s Accepted Manuscript DEVELOPMENT AND CHARACTERIZATION OF TERBUTALINE SULPHATE MICROSPONGE AND ITS COLONIC DELIVERY BY COMPRESSION COATED TABLETS Biswajit Basu, Mori Ankita S, Partha Pratim Mahata, Suparna Ghosh, Ritu Gaur, Boundugulapati Murali Krishna To appear in: World Research Journal of Pharma Technology Received date: 18 March 2017 Revised date: 28 March 2017 Accepted date: 22 April 2017 Cite this article as: Basu B, Mori Ankita S, Mahata P P, Ghosh S, Gaur R, Krishna BM, Development and characterization of terbutaline sulphate microsponge and its colonic delivery by compression coated tablets, WRJPT, 2017, 3(3): This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. For any copyright content the authors will be responsible by them self. 1

2 DEVELOPMENT AND CHARACTERIZATION OF TERBUTALINE SULPHATE MICROSPONGE AND ITS COLONIC DELIVERY BY COMPRESSION COATED TABLETS Biswajit Basu 1 *, Mori Ankita S 1, Partha Pratim Mahata 2, Suparna Ghosh 2, Ritu Gaur 3, Boundugulapati Murali Krishna 4# 1 Department of Pharmaceutics, Atmiya Institute of Pharmacy, Yogidham Gurukul, Kalawad Road, Rajkot , Gujarat State, India, 2 BCDA College of Pharmacy & Technology, Hridaypur, Barasat, Kolkata, India, 3 Panacea Biotec Ltd, Baddi, Himachal Pradesh, India, 4 Birla Institue of Technology Mesra, Ranchi, Jharkhand , India # ACG Pharma Technologies Pvt Ltd., India Corresponding Author: Biswajit Basu Department of Pharmaceutics, Atmiya Institute of Pharmacy, Yogidham Gurukul, Kalawad Road, Rajkot , Gujarat State, India ABSTRACT The objective of this study was to develop compression coated tablets (CCT) of terbutaline sulphate (TBS) microsponges for potential colonic drug delivery with improved gastric resistance and pulsatile release based on coatings of powder blends of the swelling polymer HPMC K100M and the extended release polymer EC. 3 2 full factorial design was employed to study the effect of drug:polymer ratio and percentage of magnesium stearate on particle size as well as drug release of microsponge and effect of total polymer amount as well as HPMC:EC ratio on the targeted drug release of TBS. Microsponges containing TBS were prepared by oil in oil solvent diffusion method using eudragit RS 100. The effects of stirring time and speed on the physical characteristics of microsponges were investigated. SEM characterized the porous structure of microsponges and they were found spherical in shape. The lag phase in drug release was dependent on the coat weight of HPMC and EC and HPMC:EC ratio in the outer shell. In-vitro studies exhibited that tablet formulations releases less than 20 % of drug in lower intestinal track. The DSC & FTIR study showed that TBS did not interact with the excipients. The release kinetics showed that the mechanism of drug release was dissolution controlled. CCT were evaluated for hardness, friability, weight variation and thickness. CCT of TBS microsponge shows no noticeable change in appearance and drug release after 1 month. Thus, CCT is promising approach for colon targeted delivery of TBS in relieving asthma attacks. Keywords: Terbutaline Sulphate, Hydroxylpropylmethylcellulose, Ethylcellulose, Compression-coating, Pulsatile release, Colonic drug delivery. 2

3 INTRODUCTION Asthma is characterized by airway inflammation resulting in hyper responsiveness of lower respiratory tract to various environmental stimuli 1. Airway resistance increases progressively at night in asthmatic patient. Risk of asthma attacks is 100 fold greater at night. This asthma known as nocturnal asthma which is an exacerbation of asthma with increase in symptoms, airway responsiveness and/or lung function. It is a good target for chronotherapy because broncoconstriction and exacerbation of symptoms vary on circadian fashion. Thus, it requires to prepare a dosage form that delivers drug when the disease progresses. This condition demands release of drug as a "pulse" after a time lag and such system has to be designed in a way that complete and rapid drug release should follow the lag time. Such systems are known as pulsatile drug delivery systems. Single unit colon targeted drug delivery systems may suffer from the disadvantage of unintentional disintegration of the formulation due to manufacturing deficiency or unusual gastric physiology that may lead to drastically compromised systemic drug bioavailability or loss of local therapeutic action in the colon. Recently, much emphasis is being laid on the development of multiparticulate dosage forms in comparison to single unit systems because of their potential benefits like increased bioavailability, reduced risk of local irritation and predictable gastric emptying. A microsponge drug delivery system (MDDS) is, highly cross-linked, porous, polymeric system consisting of porous microspheres that can entrap wide range of actives and then release them over a time and in response to trigger. Moreover, they may enhance stability, reduce side effect and modify drug release favourably. The MDDS would also be advantageous when a delay in absorption is desirable from a therapeutic point of view as for the treatment of diseases that have peak symptoms in the early 3

4 morning and that exhibit circadian rhythm, such as nocturnal asthma, angina pectoris and rheumatoid arthritis. So by developing the pulsatile device for specific colonic delivery, plasma peak is obtained at an optimal time, number of doses can be reduced; saturable first pass metabolism and tolerance development can also be avoided. The aim of this work is to prepare a polymeric drug delivery system for a drug, intended to be administered orally which is capable of retaining its integrity through the gastric and intestinal ph along with many applications in local and systemic delivery of drugs. Terbutaline is a synthetic β2-adrenoceptor stimulant that is used as a bronchodilator in the treatment of bronchial asthma. The usual oral dose of TBS for adults is 5 mg, every 6 hours 3 times a day. In children 12 to 15 year age the does is 2.5 mg 3 times a daily. After inhalation, only about 10% 20% of inhaled dose reaches the lungs and the rest is swallowed. There are also reports about the harmful effects of aerosol bronchodilator therapy. Its short biological half life and thus frequent administration create necessity to develop long acting formulation which is desirable to improve not only the treatment of lung disorder but also the patient compliance 2,3,4,5. In present work, it has been tried to prepare compression coated tablets containing terbutaline sulphate microsponges, to release drug chronobioilogically with advantages of multiparticulate drug delivery system. 4

5 MATERIALS AND METHODS Materials The drug, Terbutaline sulphate was gifted from Zeenish Pharma, Ahmedabad. Eudragit RS 100 was purchased from Evonic industries. All HPMC grade were gifted from Colorcon Asia Pv. Ltd., Goa. Ethyl cellulose was obtained from Himedia laboratories pvt. Ltd, Mumbai. Acetone was obtained from Rankem Ltd. Delhi. Avicel PH 101 was gifted from Maple Biotech Pvt. Ltd., Pune. All other reagents were of analytical grades. Analytical Methods Preparation of terbutaline sulphate Standard Stock Solution in 0.1 N HCl, ph 6.8 and 7.4 phosphate buffer 1000 μg/ml stock solution of drug was prepared by dissolving 25 mg of drug in 25 ml of 0.1 N HCl, ph 6.8 and 7.4 phosphate buffers (stock-i, stock-ii and stock-iii respectively). 100 μg/ml of secondary stock solution is prepared by diluting 2.5 ml of primary stock solution to 25 ml by 0.1 N HCl, ph 6.8 and 7.4 phosphate buffers (stock-iv, stock-v and stock-vi respectively). Spectrophotometric scanning of terbutaline sulphate Determination of analytical wavelength of terbutaline sulphate in 0.1N HCl, ph 6.8 and 7.4 phosphate buffer From the standard stock solution (IV, V and VI) 6 ml was pipetted in to 10 ml volumetric flask. The volume was made up to 10 ml with 0.1N HCl, ph 6.8 and 7.4 phosphate buffer solutions respectively. The resulting solutions containing 60 mcg/ml were scanned between 200 and 400 nm. 5

6 Calibration curve of terbutaline sulphate in 0.1 N HCl, ph 6.8 and 7.4 phosphate buffer From the standard stock solution (IV, V and VI), the series of solutions having concentration 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/ml were prepared from stock solution by diluting 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml of stock solution with 0.1 N HCl, ph 6.8 and 7.4 phosphate buffer to make final volume 10 ml respectively. Preformulation studies Preformulation testing is the first step in the rational development of dosage forms of a drug substance. It can be defined as an investigation of physical and chemical properties of a drug substance alone and when combined with excipients. The overall objective of preformulation testing is to generate information useful to the formulator in developing stable and bioavailable dosage forms which can be mass produced. 1. Determination of melting point: Melting point was determined by taking small amount of TBS in a capillary tube closed at one end. The capillary tube was placed in an electrically operated digital melting point apparatus and the temperature at which the drug melts was recorded. This was performed thrice and average value was noted. 2. Drug excipient compatibility studies: In the preparation of in situ gelling formulation, drug and polymer may interact as they are in close contact with each other, which could lead to the instability of drug. Preformulation studies regarding the drug-polymer interaction are therefore very critical in selecting appropriate polymers. FT-IR spectroscopy: It was employed to ascertain the compatibility between TBS and the selected polymers. The pure drug and drug with excipients were scanned separately. The powdered sample is pressed in the Zinc-Selanide lense without any atmospheric compensation. FT-IR spectrum of TBS was compared with FTIR spectra of optimized formulation and physical 6

7 mixture of drug and polymers. Disappearance of TBS peaks or shifting of peak in any of the spectra was studied 6. Differential scanning calorimetry (DSC) Study DSC analysis of pure drug, and optimized formulation was performed with Shimadzu DSC-60 thermal analyser at the heating flow rates of 10 C per min between 50 and 300 C under static air using aluminium pans 6. Formulation of microsponges Preparation of Terbutaline sulphate Microsponges Microsponges were prepared by oil in oil solvent diffusion method. A specific weight of Eudragit RS-100 was dissolved in acetone. Once a clear solution was obtained, 0.2 g of the drug was added in addition to magnesium stearate (2% w/v of solvent), and the whole mixture was kept in the ultrasonic bath of 70-kHz frequency for 2 min (Life care equipment pvt. Ltd) where homogenous dispersion was obtained. The mixture was then poured into 150 ml of liquid paraffin while it was stirred by a mechanical stirrer for 3 hours. During this time, the acetone was completely removed by diffusion into liquid paraffin and evaporation through the air/liquid interface. The solidified microsponges were filtered, washed six times with 50 ml of n-hexane, air-dried at room temperature for 12 h, and stored in a desiccator for further investigations. Application of 3 2 full factorial design for optimization of Microsponges Microsponges were prepared by applying 3 2 full factorial design. Drug:polymer ratio and % magnesium stearate are independent variables whereas drug release and size of the microsponges are dependant variables (table 1). Table 1 shows the experimental design and composition of corresponding formulations in table 2. 7

8 Formulation of core and compression coated tablets Preparation of core tablets 7,8,9 Core tablets of microsponges equivalent to 7.5 mg TBS were prepared by direct compression method. According to the table 3, all ingredients, microsponge, avicel PH 101, talc were passed from the 60 # sieve to ensure uniform mixing and dry blended for 20 minutes followed by addition of magnesium stearate and talc. The mixture was then further blended for 10 minutes to ensure complete mixing. 100 mg of the resultant mixture was directly compressed using 3 mm punch and die on a rotary tablet compression machine (Hardik Eng. Pvt. Ltd, Ahmedabad, India). Preparation of compression coated tablet 7,8,9 Compression coated tablets were prepared by using different ratios of HPMC and EC. Previously prepared core tablet was compression coated with different quantities of coating material containing different polymers. Different quantities of HPMC and EC according to the table 6 were well blended to ensure uniform mixing. Half the quantity of the coating material was placed in the die cavity. The core tablet was carefully placed in the centre of the die cavity and remaining half quantity of the coating material will be placed over it. It was compressed using 9 mm punches by rotary tablet compression machine (Hardik Eng. Pvt. Ltd, Ahmedabad, India). Hundred tablets were prepared for each batches. Optimization of polymers and polymers amount for compression coted tablets For the optimization of polymers, Ethyl cellulose, HPMC K 100M, HPMC K 15M and HPMC K 4M were selected. Compression coated tablets were prepared by selecting different ratios of these polymers, HPMC K 4M: EC (F3), HPMC K 15M: EC (F4), HPMC K 100M: EC (F5) as shown in table 4. Tablets of HPMC (F1) and EC (F2) alone were also prepared. Compression 8

9 coated tablets with core tablet and five different amounts of polymers (200,225, 250, 275, 300) were also prepared to evaluate the effect of coat weight on drug release. Application of 3 2 full factorial design for optimization of compression coated tablets Compression coated tablets were prepared by applying 3 2 full factorial design. HPMC:EC ratio and total polymer amount are independent variables (table 5). Whereas drug release at 5h (Q5), 10h (Q10), and 24h (Q24) are dependant variables. Table 6 shows the experimental design and composition of corresponding formulations. Evaluation of Microsponges 1. Morphology of Microsponges 7 i. Determination of particle size distribution of microsponge The particle size distribution was studied by the microscopic method. The shape of the crystals was observed under trinocular microscope (10X) attached to a computer. ii. Scanning electron microscopy (SEM) To evaluate the surface characteristics and morphology of microsponges SEM (JSM-5610, Japan) was carried out. 2. Determination of drug content and drug entrapment efficiency (%) (DEE) 7 The drug content of the formulation was determined by dissolving microsponges equivalent to the 40 mg of drug in 100 ml of ph 7.4 phosphate buffer. From the primary stock solution, 1 ml was pipetted out and it diluted in 10 ml volumetric flask followed by sonication for 30 min. Then volume was makeup to the mark. The resulting solution was filtered and the maximum absorbance was measured at 276 nm using UV-Visible Spectrophotometer (Shimadzu 1700, Tokyo, Japan). The encapsulation efficiency was calculated as given below. 9

10 In-Vitro drug release studies of terbutaline sulphate loaded microsponges 10 The microsponges containing 100 mg of TBS ware subjected to in vitro drug release studies. In vitro release studies were carried out in USP basket apparatus with stirring rate of 75 rpm at 37 ± 0.5 C. Drug release was carried out in 900 ml of ph 7.4 phosphate buffer for 24 h. Samples were withdrawn at regular interval of time. The sink condition was maintained by adding equal amount of dissolution medium. The samples were analysed spectrophotometrically (Shimadzu UV-1700) at a wavelength of 276 nm. Dissolution tests were performed in triplicate for each sample Evaluations of core and compression coated tablets Precompressional parameters 11 The bulk density and tapped density of plain TBS and microsponge - core powder blend was determined using a digital bulk density apparatus. Carr s index and Hausner s ratio was calculated using bulk density and tapped density. The angle of repose was assessed by the fixed funnel method. Physiochemical Parameters of core and compression coated Tablets A. Weight Variation 12 The weight variation test was performed by weighing 20 tablets individually and collectively, calculating the average weight, and comparing the individual tablet Weight to the average (Pharmacopoeia of India 1996). B. Thickness 12 Thickness of the tablets was determined using verniar calipers. Thickness of 10 tablets was individually measured and average thickness was calculated. 10

11 C. Hardness 12 Hardness of the tablet was determined with the help of Monsanto hardness Tester. The tester consists of a barrel containing a compressible spring held between two plungers. The lower plunger is placed in contact with the tablet, and a zero reading is taken. The upper plunger is then forced against a spring by turning a threaded bolt until the tablet fractured. The force of fracture was recorded by monitoring a pointer which rides along a gauge in the barrel to indicate the force. D. Friability 12 The friability of the tablets was determined using Roche friabilator. It was expressed in percentage (%). 10 tablets were accurately weighed and transferred to the friabilator. The friabilator was operated at 25 rpm for four minutes. After four minutes the tablets were weighed again. The % friability was then calculated using the formula; E. In vitro drug release study of core tablets 13 In vitro dissolution studies were carried out using USP Type II (paddle method) apparatus (Electrolab TDT-08L). 7.4 phosphate buffer was used as dissolution medium. Release pattern was studied visually by taking sample of 5 ml at the specific time intervals. Also the sample was analyzed at appropriate wavelength using a UV spectrophotometer. F. Position of core tablet Compression coated tablet is cut vertically and cross sectional photographs were taken to evaluate the position of core tablet in the compression coated tablet. G. In Vitro Drug Release Studies of coated tablets 13 In vitro drug release studies from the press coated tablets were performed according to the USP paddle method (Apparatus II) at 37±0.5 C and stirred at 50 rpm. Tablets was placed in 900 ml 11

12 0.1N HCL (ph 1.2) for 2 h, in ph 6.8phosphate buffer for 3 h and finally in ph 7.4 phosphate buffer for 10 h. At time intervals of 1 h, samples were withdrawn. The amount of drug present in each sample was determined using UV-visible spectrophotometer (Shimadzu 1700, Tokyo, Japan) at appropriate wavelength. H. Statistical analysis 14 The statistical analysis of the factorial design batches were performed by multiple regression analysis using Microsoft Excel. To evaluate contribution of each factor with different levels on responses, analysis of variance (ANOVA) was performed using design expert-8. To graphically demonstrate the influence of each factor on responses, the response surface plots were generated using design expert-8. I. Drug release kinetic studies 13,15 Data obtained from in vitro release studies was fitted to various kinetic equations to find out the mechanism of TBS release from press coated tablets. Various models are available for explaining the kinetics of drug release. They are listed below: Zero order model, First order model, Higuchi model, Korsmeyer Peppas model, and Hixon-Crowell model. J. Stability Study 15 After determining the drug content, the optimized batches of tablet was monitored for stability as per ICH guidelines. The final formulation was sealed in aluminum foil and kept in the stability chamber (REMI) maintained at 400 C ± 20 C and 75 % ± 5% RH for 1 month. 12

13 RESULTS AND DISCUSSION Analytical Methods Spectrophotometric scanning and calibration curve of terbutaline sulphate in 0.1N HCl & 7.4 phosphate buffer The solution containing 60 g/ml of TBS in 0.1 N HCL was scanned between 200 and 400 nm using double beam UV visible spectrophotometer. The max was found to be nm. max, 276nm was taken as analytical wavelength of TBS in 0.1 N HCl & 7.4 phosphate buffer. Calibration curve of terbutaline sulphate in 0.1N HCl & 7.4 phosphate buffer are shown in figure 1 & 2. Preformulation Studies The following preformulation studies were performed on terbutaline sulphate and excipients. Melting Point Melting point of TBS was determined by capillary tube method and it was found to be 120 ± C (n = 3). This value is similar as that of the literature citation C. Drug Excipients Compatibility Studies FT-IR spectroscopy: The FTIR spectra of pure TBS and its physical mixture with other excipients are shown in figure 3. Pure TBS showed major peaks at 3335cm-1 (OH stretch), 3057cm 1 (aromatic CH stretch), 2974cm 1 (methyl asymmetric stretch), 1608 & 1485cm 1 (aromatic ring stretch), 1380cm 1 (t-butyl symmetric bend), 1068cm 1 (secondary alcohol stretch. The results revealed no considerable changes in the IR peak of TBS in the prepared formulation when compared to pure drug, thereby indicating the absence of any interaction. Differential scanning calorimetry (DSC) Study DSC studies were carried out with drug, and microsponge. The melting point of TBS was estimated by the open capillary method and found to be 120 C which agreed with the DSC thermogram. The DSC thermograms of TBS and microsponge showed sharp distinct endothermic peaks for TBS which 13

14 corresponds to individual drug without exhibiting any modification, which indicates that TBS and polymers are compatible in microsponge (figure 4). Characterization of microsponges By using the oil in oil emulsion solvent diffusion method, free flowing microsponges were prepared. The microsponges had regular, spherical shape with roughness on the surface and several pores. Optimization of microsponge formulations The effect of various variables was studied like i) Effect of stirring speed on the size of microsponges. ii) Drug to polymer ratio. iii) Effect of amount of magnesium stearate on the nature of microsponges. i) Effect of Stirring Speed on the Size of Microsponges The effect of stirring rate on the physical characteristics of the formulated microsponges was examined. A suitable stirring rate to optimize particle size, size distribution and subsequent drug release from microsponges was needed. Our study showed that an increase in the stirring rate resulted in a reduction in mean particle size. Any increase in mean particle size at lower stirring rates can be attributed to the increased tendency of globules to coalescence and aggregate. On the other hand, at higher stirring rates, a vigorous, uniform, increased mechanical shear is imposed and this results in a rapid dispersion of the formed droplets which may have less chance of coalescing into bigger droplets. This suggests that the size of the droplets formed during the encapsulation process may therefore be closely related to the size of the final microsponges produced. 16 Microsponges were prepared with 500 & 600 rpm speed shows agglomeration. 14

15 While microsponges prepared with 800, 900 & 1000 rpm shows reduction in particle size. Spherical and uniform particles were obtained at 700 rpm. So, 700 rpm speed was selected for the formulation. (table 7) ii. Effect of Drug to Polymer Ratio on the Size of Microsponges Eudragit RS 100 was used to sustain the drug release upto 24 hours. Nine additional concentrations of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5 drug to polymer ratios were selected to prepare the microsponges. From the above 9 different ratios only 3 different levels 1:2, 1:2.5, 1:3 were selected for factorial design. Below these three levels, the microsponges prepared were of very small sizes and above these levels the microsponges formed were not of micron sizes. iii. Effect of Changing the Percentage of Magnesium Stearate Magnesium stearate was added to the formulations as droplet stabilizer to overcome the problem of coalescence during solvent evaporation as it reduces the phase tension between Eudragit microcapsules and liquid paraffin and prevents flocculation of microsponges. Three additional concentrations 1%, 2% and 3% w/v of magnesiumg stearate were used in preparing the microsponges possessing (F5, F6) 1:1 and (F7, F8) 1:2 drug/polymer ratio. Low concentration of magnesium stearate (1%) resulted in aggregation of microsponges. It has been reported that metal stearate, such as magnesium and aluminum, reduces the interfacial tension and prevents electrification and flocculation during the preparation of microspheres in general. However, increasing its percentage to 3% neutralized the electric charge of eudragit RS-100 due to accumulation of magnesium stearate on the surface, leading to aggregates as those previously reported by Kim et al. 17. Magnesium stearate was previously reported to produce smooth nonporous surfaces when dissolved in the dispersed phase solvent. However, the porous 15

16 microsponges which resulted in the study were due to its insolubility and just its dispersion in the solvent of the dispersed phase. 18 A. Morphology of microsponges i. Determination of particle size distribution of microsponges Preliminary trials were undertaken to establish the effect of drug: polymer ratios and stirring speed on the physical characteristics of microsponge. The shape of microsponge of optimized formulation was spherical (figure 5). Particle size distribution was uniform having 80.4 μm mean particle size (figure 6). Particle size of all the formulations is as shown in table 8. i. Scanning electron microscopy (SEM) SEM of microsponges according to the (C) of Figure 7, shows the presence of pores on the surface. Compressibility of the microsponges is quite good as SEM shows no deformation of microsponges. (A) of figure 7 (A) is the microsponges before compression and (B) is the microsponges after compression at magnification 100x. So, from SEM study it was confirmed that there will be no problem to the microsponge structure when compressed. B. Drug Content and Drug Entrapment Efficiency of Microsponges Microsponges were evaluated for drug content. Table 8 shows the results of drug content of M1- M9 batches of microsponges. Optimized batch M5 shows % drug content. According to the results shown in table 8, formulation M5 shows % drug entrapment. Formulations M1- M3 shows lower amount of drug entrapment may be because of lower amount of polymer available to entrap the drug. Whereas formulations M7-M9 also shows lower drug entrapment efficiency. It may be due to high polymer amount which forms microsponges before drug loading. 16

17 C. In-vitro dissolution study In vitro release profile of optimized TBS microsponge is as shown in figure 8. The cumulative percentage release from in vitro release study of optimized formulation was 90% 12 h. The in vitro release profile from the microsponge shows sustained effect at ph 7.4. Characterization of core and compression coated tablets Optimization of polymers for compression coated tablets Effect of HPMC and EC on the drug release of compression coated tablets Figure 9 shows the dissolution profile of TBS core tablets compression coated with HPMC and EC alone. As EC is erodible polymer it is found from the dissolution profile that TBS core tablets compression coated with EC allows the dissolution medium to diffuse inside and erodes the tablet releasing drug in lower track of GIT. This premature drug release makes it unfavourable to use alone for compression coated tablets. Core tablets coated with HPMC alone reduces the drug release for very long time. HPMC forms viscous layer around the tablet making drug diffusion very slow from the thick layer. The viscous layer of HPMC gel formed around the core tablet reduces the dissolution medium to diffuse inside. Comparison of drug release between the HPMC grades Three different grades of HPMC can be used to target the colon. The compression coated tablet should not show any premature drug release in stomach or small intestine. Figure 10 shows that HPMC K4 M and K15 M releases more than 20 % of drug before reaching to the colon. Whereas HPMC K100 M releases less than 15 % drug in stomach and small intestine. From the comparison of drug release profiles, HPMC K 100 M is found more suitable to prepare compression coated tablets. Effect of coat weight on the drug release from compression coated tablets From the figure 11 it is found that percentage of drug release is inversely proportional to the increase in amount of coating polymer used to coat the core tablet. Tablets prepared with

18 and 300 mg coat weight showed that only 11.8 and 12.2 % of drug is released after 5 h respectively. And tablets prepared with 200 and 225 mg of coat weight showed more than 20 % of drug release in lower intestinal track. Whereas tablet prepared with 250 mg coat weight showed 20 % of drug release after reaching the colon. From the results obtained three different levels of coat weight selected were 225, 250 and 275 mg. A. Micromeritic properties Prepared core powder blend was showing good micromeritic properties and flowability compared to pure drug shown in table 9. B. Physiochemical parameters of core tablets Core tablet exhibited low weight variation. Friability of tablet was found below 1% indicating good mechanical resistance. The thickness, hardness was found within the acceptable limits. The result of physiochemical parameters of core tablet is as shown in table 10. C. In vitro drug release study of core tablets In vitro drug release study of core tablets shows the sustained release behaviour in ph 7.4 phosphate buffer at 24 hours (figure 12). It releases 90% of drug at 12 hours. Cumulative percentage drug release of optimized core tablet is shown in table 11. D. Position of core tablet Compression coated tablet is cut vertically and cross sectional photographs (figure 13) were taken to evaluate the position of core tablet in the compression coated tablet. Core tablet was found in the centre of coating. E. Physiochemical parameters of compression coated tablets All the formulations exhibited low weight variation, friability of tablet was found below 0.8% indicating good mechanical resistance. The drug content, thickness and hardness of all the 18

19 formulations was found within the acceptable limits. The result of physiochemical parameters of compression coated tablet is as shown in table 12. F. In-vitro dissolution study of compression coated tablets In vitro release studies of TBS tablets were carried out in1.2 ph acid buffer, ph 6.8 and ph 7.4 phosphate buffers respectively. Cumulative drug release and cumulative % drug retained were calculated on the basis of drug content of TBS present in the tablets. The results obtained in the in vitro drug release for the formulations F1 to F9 is tabulated in table 12. Rapid drug dissolution was observed in F1, F4 and F7. It may be because of the lower amount of polymers used to coat the tablets. Slow drug dissolution was observed in F3, F6 and F9 with release 96.48%, % and 80.17% respectively at 24 hours. As the concentration of the polymer increased, the drug release was found to be decreased due to the increase in the time required for wetting and dissolving the drug molecules present in the polymer matrices. Dissolution profile is shown in figure 14. Higher polymer amount used to coat the core tablets may be the reason behind this sustain release pattern, releasing the drug after 5 hours in colon. From all the evaluation parameters, it has been seen that F6 formulation fulfill all the characteristics of compression coated tablets, so F6 formulation was selected as best formulation. From the in-vitro dissolution study it was observed that formulation F6 shows 100 % of drug release at 24 hour. G. Experimental Design 3 2 Factorial design has often been applied to optimize the formulation variables with basic requirement of understanding interaction of independent variables. Preliminary investigations of the process parameters revealed that independent factors like drug to polymer ratio (X1) and percentage of magnesium stearate (X2) showed significant influence on dependant factors like particle size as well as drug release of microsponge. Independent factors like total polymer 19

20 amount (Y1) as well as HPMC:EC ratio (Y2) has profound effect on the targeted drug release of TBS from the compression coated tablets. Hence, they were utilized for further systematic studies. For all 9 batches, both the selected dependent variables (X1 and X2) and (Y1 and Y2) showed a wide variation in particle size and drug release of the microsponge, and colonic targeting of the terbutaline sulphate from the compression coated tablets. The data clearly indicated strong influence of X1 and X2 on selected responses (R1, R2) and influence of Y1 and Y2 on the R3, R4, R5. The polynomial equations can be used to draw conclusions after considering magnitude of coefficients and mathematical sign it conveys either positive or negative. Results for experimental design batches and its ANOVA were shown below. Response 1: Drug release (R1) The Model F-value of implies the model is significant. "Adeq Precision" measures the signal to noise ratio. Ratio of indicates an adequate signal. This model can be used to navigate the design space. Effect of design factors on drug release The ANOVA results, contour plot and 3d surface plot for the CPR of microsponge showed the strong effect of the two factors (percentage of magnesium stearate and drug to polymer ratio. Polynomial equation of the CPR was indicated that the both, percentage of magnesium stearate and drug to polymer ratio, have positive effect on CPR. Drug release of the microsponge was found to increase with increase in the amount of the magnesium stearate due to decrease in particle size. It was observed that CPR varies from ± 2.15 to ± 2.5 for all the formulations. CPR of optimized formulation F5 was found ± Reduced model for drug release The significance level of coefficients b22 and b12 was found to be p = and respectively, hence it was omitted from the full model to generate the reduced model. The results 20

21 of statistical analysis are as shown in table 13. Coefficients b1, b2 and b11 were found to be significant at p < 0.05; hence they were retained in the reduced model. The reduced model was tested in portions to determine whether the coefficients b22 and b12 contributed significant information for the prediction of CPR. The results of testing the model in portions are shown in table 14. The critical value of F for α= 0.05 is equal to 9.55 (df = 2, 3) is higher than the calculated value (F=6.348). It may be concluded that the interaction terms b22 and b12 do not contribute significantly to the prediction of CPR and therefore can be omitted from the full model. Contour plots are shown in figure 15. Response 2: Particle size (R2) The Model F-value of implies the model is significant. The "Pred R-Squared" of is in reasonable agreement with the "Adj R-Squared" of "Adeq Precision" measures the signal to noise ratio. The ratio of indicates an adequate signal. This model can be used to navigate the design space. Effect of design factors on particle size The ANOVA results, contour plot and 3d surface plot for the particle size of microsponge showed the strong effect of the two factors (percentage of magnesium stearate and drug to polymer ratio). Polynomial equation of the CPR was indicated that, drug to polymer ratio, have positive effect on particle size, whereas increase in percentage of magnesium stearate decreases the particle size. Particle size of the microsponge was found to increase significantly with increase in the drug to polymer ratio. Formulation F7 shows the larger particle size because of the lower percentage of magnesium stearate and F3 shows smaller particle size because of higher percentage of magnesium stearate. It was observed that particle size varies from ± 2.60 to ± 1.92 for all the formulations. Particle size of optimized formulation F5 was found 81.1 ±

22 Reduced model for particle size The significance level of coefficients b22 and b12 was found to be p = and respectively, hence it was omitted from the full model to generate the reduced model. The results of statistical analysis are as shown in table 13. Coefficients b1, b2 and b11 were found to be significant at p < 0.05; hence they were retained in the reduced model. The reduced model was tested in portions to determine whether the coefficients b22 and b12 contributed significant information for the prediction of particle size. The results of testing the model in portions are shown in table 14. The critical value of F for α= 0.05 is equal to 9.55 (df = 2, 3) is higher than the calculated value (F=1.509). It may be concluded that the interaction terms b22 and b12 do not contribute significantly to the prediction of particle size and therefore can be omitted from the full model. Contour plots are shown in figure 16. Response 3: Q5 (R3) The Model F-value of implies the model is significant. The "Pred R-Squared" of is in reasonable agreement with the "Adj R-Squared" of "Adeq Precision" measures the signal to noise ratio. The ratio of indicates an adequate signal. This model can be used to navigate the design space. Effect of design factors on CPR Q5 The ANOVA results, contour plot and 3d surface plot for the amount of drug released in 5 hours (CPR Q5; fig. 2) showed the strong effect of the two factors (HPMC:EC ratio and polymer amount). Polynomial equation of the CPR Q5 was indicated that Polymer amount have positive effect on the CPR Q5 whereas HPMC:EC ratio has negative effect on drug release. CPR Q5 of the tablets was found to decrease with increase in the amount of the polymer. It was observed that CPR Q5 varies from 10.6 to 25.4 % for all the formulations. 22

23 Reduced model for Q5 The significance level of coefficients b2 was found to be p = , hence it was omitted from the full model to generate the reduced model. The results of statistical analysis are as shown in table 16. Coefficients b1 was found to be significant at p < 0.05; hence it was retained in the reduced model. The reduced model was tested in portions to determine whether the coefficients b2 contributed significant information for the prediction of CPR at 5th hour. The results of testing the model in portions are shown in table 17. The critical value of F for α= 0.05 is equal to 5.99 (df = 1, 6) is higher than the calculated value (F=2.177). It may be concluded that the interaction terms b2 do not contribute significantly to the prediction of Q5 and therefore can be omitted from the full model. Contour plots are shown in figure 17. Response 4: Q10 (R4) The Model F-value of implies the model is significant. The "Pred R-Squared" of is in reasonable agreement with the "Adj R-Squared" of "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. The ratio of indicates an adequate signal. This model can be used to navigate the design space. Effect of design factors on CPR Q10 The ANOVA results, contour plot and 3d surface plot for the amount of drug released in 10 hours (CPR Q10; figure 18) showed the strong effect of the two factors (HPMC:EC ratio and polymer amount). Polynomial equation of the CPR Q10 was indicated that the both HPMC:EC ratio and polymer amount have negative effect on the CPR Q10. CPR Q10 of the tablets were found to decrease with increase in the HPMC:EC ratio. It was observed that CPR Q10 varies from to % for all the formulations. 23

24 Reduced model for Q10 The significance level of coefficients b22 and b12 was found to be p = and respectively, hence it was omitted from the full model to generate the reduced model. The results of statistical analysis are as shown in table 16. Coefficients b1, b2 and b11 were found to be significant at p < 0.05; hence they were retained in the reduced model. The reduced model was tested in portions to determine whether the coefficients b22 and b12 contributed significant information for the prediction of CPR at 10th h. The results of testing the model in portions are shown in table 17. The critical value of F for α= 0.05 is equal to 9.55 (df = 2, 3) is higher than the calculated value (F=0.0388). It may be concluded that the interaction terms b22 and b12 do not contribute significantly to the prediction of Q10 and therefore can be omitted from the full model. Contour plots are shown in figure 18. Response 5: Q24 (R5) The Model F-value of implies the model is significant. The "Pred R-Squared" of is not as close to the "Adj R-Squared" of as one might normally expect. This may indicate a large block effect or a possible problem with model and/or data. Things to consider are model reduction, response transformation, outliers, etc. Effect of design factors on CPR Q24 The ANOVA results, contour plot and 3d surface plot for the amount of drug released in 24 hours (CPR Q24; Figure 19) showed the strong effect of the two factors (HPMC:EC ratio and Polymer amount). Polynomial equation of the CPR Q24 was indicated that HPMC:EC ratio total polymer amount have negative effect on the CPR Q24. CPR Q24 of the tablets were found to decrease with increase in the HPMC:EC ratio and polymer amount. It was observed that CPR Q24 varies from to % for all the formulations. 24

25 Reduced model for Q24 The significance level of coefficients b2, b22 and b12 was found to be p = , and respectively, hence it was omitted from the full model to generate the reduced model. The results of statistical analysis are as shown in table 16. Coefficients b1 and b11 were found to be significant at p < 0.05; hence they were retained in the reduced model. The reduced model was tested in portions to determine whether the coefficients b2, b22 and b12 contributed significant information for the prediction of CPR at 24th h. The results of testing the model in portions are shown in table 17. The critical value of F for α= 0.05 is equal to 9.28 (df = 3, 3) is higher than the calculated value (F=0.899). It may be concluded that the interaction terms b2, b22 and b12 do not contribute significantly to the prediction of Q24 and therefore can be omitted from the full model. Design summary are shown in table 15. Contour plots are shown in figure 19. Optimization of final batch Optimization of dependant variables for microsponge was done on the basis of in-vitro dissolution and particle size of microsponge. F5 formulation was optimized on the basis of CPR , and particle size μm. Scanning electron microscopy of microsponges shows the presence of pores on the surface of microsponges. Other parameters of the microsponges were also implying to the optimized formulation. For the compression coated tablets, optimization of dependant variables was done on the basis of in-vitro dissolution. F6 formulation was optimized on the basis of CPR Other physiological parameters were also implying to the optimized formulation. H. Drug Release Kinetic Studies Data obtained from dissolution studies were fitted to various kinetic equations to examine the kinetic behaviour of compression coated tablets of TBS. The kinetic models used were first order 25

26 (log cumulative percentage of drug remaining vs time), Higuchi s (cumulative percentage of drug released vs square root of time) and Korsmeyer (log cumulative percentage of drug released vs log time) equation. The data of average values were processed as per Hixon Crowell cube root law, Higuchi s equation, Korsmeyer-Peppas model and are given in the tables 19 and 20 and the figure 20. The equations were generated through statistical procedures and reported in the table 19 and 20. The release data of terbutaline sulphate from all the tablets were given in table 19 and 20. Data of the in vitro release were fit into different equations and kinetic models to explain the release kinetics of TBS from these tablets. The release kinetics of TBS followed first order from all the films F1 to F9 (table 18). The better fit (highest R 2 values) was observed in case of Hixon Crowel model than Higuchi s model in all the tablets. Hence mechanism of drug release from the TBS tablets F1 to F9 followed are dissolution controlled. The mechanisms of drug release are non-fickian diffusion (super case-ii), since they fitted well with Korsmeyer Peppas models (n value above 1). The n value could be used to characterize different release mechanisms. This indicates that drug release depends on diffusion through polymer with first order release kinetics. Application of Korsmeyer Peppas model provides information about the release mechanism, namely case-ii transport mechanism. Application of Korsmeyer Peppas model provides information about the release mechanism, namely case-ii transport mechanism. Stability Study From the results of stability study, it was observed that the optimized formulation was stable for the one month at 40 0 C, 75% RH as per the ICH guidelines. The in vitro drug release profiles of the formulation obtained before and after stability studies were compared (table 21 & figure 21). The drug release profiles are almost similar. The physical appearance is also unchanged after 1 month. 26

27 Conclusion The compression coated targets the drug, TBS to the colon, that can relieve asthma attack during early morning hours and microsponge drug delivery system helps in sustaining drug release for more than 12 hours. Thus, compression coated tablets of TBS microsponge is promising approach for colon targeted delivery of TBS. Acknowledgement We would like to thank the company Zeenish Pharma, Ahmedabad for giving us the free gift samples of terbutaline sulphate. We are also thankful to Colorcon Asia Pvt. Ltd., Goa, for the gift sample of all HPMC grade and thankful to Saurashtra University, Rajkot for providing us the facility of IR. We are also thankful to Atmiya Institute of Pharmacy, Rajkot for providing us the facility for carrying out the project successfully. 27

28 References: 1. Smolensky MH, Lemmer B, Reinberg AE. Chronobiology and chronotherapy of allergic rhinitis and bronchial asthma, Adv. Drug Deliv. Rev. 2007; 59: Indian Pharmacopoeia. The Controller of Publication, New Delhi, India; Karabey Y, Sahin S, Oner L, Hincal AA. Bioavailability File: Terbutaline. Journal of Pharmaceutical Sciences. 2003; 28: Pramod kumar TM, Shivakumar HG. Novel core in cup buccoadhesive systems and films of terbutaline sulphate.asian Journal of Pharmaceutical Sciences. 2006; 1(3-4): Orlu M, Cevher E, Araman A. Design and evaluation of colon specific drug delivery system containing flubiprofen microsponges. International Journal of Pharmaceutics. 2006; 318: Krishnaiah YSR, Satyanarayana V, Dinesh Kumar B, Karthikeyan RS. In vitro drug release studies on guar gum-based colon targeted oral drug delivery systems of 5- fluorouracil. European Journal of Pharmaceutical Sciences. 2002; 16: Watanabe Y, Mukai B, Kawamura KI, Ishikawa T, Namiki M, Utoguchi N, Fujii M. Preparation and evaluation of press-coated aminophylline tablet using crystalline cellulose and polyethylene glycol in the outer shell for timed-release dosage forms. The Pharmaceutical Society Japan. 2002; 122(2): Mahajan AN, Pancholi SS. Formulation and evaluation of timed delayed capsule device for chronotherapeutic delivery of terbutaline sulphate. Ars Pharm, 2010; 50(4):

29 11. Gupta VR, Srikanth K, Prasad BS, Reddy N, Sudheer B. Formulation and evaluation of directly compressible agglomerates of celecoxib. International Journal of Pharmaceutical Sciences and Nanotechnology. 2011; 3(4): Moon A, Kondawar M, Shah R. Formulation and evaluation of press coated indomethacin tablets for pulsatile drug delivery system. Journal of Pharmaceutical Research. 2011; 4(3): Elshafeey AH, Sami EI. Preparation and in-vivo pharmacokinetic study of a novel extended release compression coated tablets of fenoterol hydrobromide. AAPS Pharm Sci Tech. 2008; 9(3): Patel MR, Soniwala MM, Patel KR, Patel SS, Patel NM. Statistical development of colon targeted delivery system containing 5-fluorouracil. International Journal of Pharmaceutical Sciences. 2009; 1(1): VeeraReddy PR, Manthri RP. Formulation and evaluation of compression coated piroxicam tablets for colon specific drug delivery. Acta Pharmaceutical Sciences. 2010; 52: Perumal D. Microencapsulation of ibuprofen and Eudragit RS 100 by the emulsion solvent diffusion technique. International Journal of Pharmaceutics. 2001; 218: Kim CK, Kim MJ, Oh KH. Preparation and evaluation of sustained release microspheres of terbutaline sulfate. International Journal of Pharmaceutics. 1994; 106: Rizkalla CMZ, Aziz RL and Soliman II. In Vitro and In Vivo evaluation of hydroxyzine hydrochloride microsponges for topical delivery. AAPS Pharm. Sci. Tech. 2011; 12(3):

30 Figure legends Figure 1: Calibration curve of terbutaline sulphate in 0.1 N HCL at 276 nm Figure 2: Calibration curve of terbutaline sulphate in ph 7.4 phosphate buffer at 276 nm Figure 3: FTIR spectra of (A) TBS (B) Microsponge (C) TBS + HPMC K100 M + EC; (D) TBS+ HPMC K100 M + EC Figure 4: DSC thermogram of TBZ and microsponge Figure 5: Trinocular microscopic observation of optimized formulation Figure 6: Plot of particle size of Microsponges Figure 7: Scanning electron microscopy of microsponges [A] Before compression, [B] After compression and [C] Pores on the surface of microsponge Figure 8: In-Vitro drug release studies of terbutaline sulphate loaded Microsponges Figure 9: Compression coated tablets of EC and HPMC Figure 10: Comparison of drug release between the HPMC grades Figure 11: Comparison of drug release from different amount of coat weight Figure 12: In vitro drug release study of core tablets in ph 7.4 phosphate buffer Figure 13: Cross sectional view of compression coated tablets Figure 14: In Vitro Drug Release Studies of compression coated tablets Figure 15: Contour plot and 3D Surface Plot of CPR against % of magnesium stearate and Drug/polymer ratio 30

31 Figure 16: Contour plot and 3D Surface Plot of particle size (µm) against % of magnesium stearate and Drug/polymer ratio Figure 17: Contour plot and 3D Surface Plot of Q5 (5 h) against HPMC: EC ratio and total polymer amount Figure 18: Contour plot and 3D Surface Plot of Q8 (8 h) against HPMC: EC ratio and total polymer amount Figure 19: Contour plot and 3D Surface Plot of Q24 (24 h) against HPMC: EC ratio and total polymer amount Figure 20: Drug release kinetics of optimized formulation Figure 21: Dissolution profile of optimized formulation before and after stability study 31

32 Table 1: Variables in a 3 2 Factorial Design for microsponges Coded values Actual values X 1 = (Drug:Polymer ratio) * X 2 : % Magnesium Stearate* -1 1:2 1% 0 1:2.5 2% 1 1:3 3% *X 1 indicates drug to polymer ratio (mg); X 2, % magnesium stearate. 32

33 Table 2: Composition of Microsponges Formulation F1 F2 F3 F4 F5 F6 F7 F8 F9 Ingredients(g) 1:2 1:2 1:2 1:2.5 1:2.5 1:2.5 1:3 1:3 1:3 Drug Polymer Sucrose Magnesium stearate

34 Table 3: Formulation of core tablet Ingredients Quantity (mg) Microsponge Avicel ph Talc 2 Magnesium stearate 1 34

35 Table 4: Composition of polymers for optimization Ingredients (mg) Batch code F1 F2 F3 F4 F5 HPMC K4M HPMC K15M HPMC K100M Ethyl cellulose

36 Table 5: Variables in a 3 2 Factorial Design for compression coated tablets Coded values Actual values X 1 = (HPMC:EC)* X 2 :Polymer weight* -1 1: : :1 250 *X 1 indicates polymer ratio (mg); X 2, amount of total polymer (mg). 36

37 Table 6: Formulation of compression coated tablet Batch code X1 X2 X1(HPMC:EC) Polymer weight Total weight of the tablet (mg) F : F : F : F : F : F : F : F : F :

38 Table 7: Optimization of speed Batch code Speed (rpm) Result M1 500 Agglomeration M2 600 Agglomeration M3 700 Spherical and Uniform particles M4 800 Size reduction M5 900 Size reduction M Size reduction 38

39 Table 8: Drug content & drug entrapment efficiency of microsponges Microsponge Drug content a Drug entrapment efficiency a Particle size (µm) M ± ± ±1.25 M ± ± ±1.96 M ± ± ±2.60 M ± ± ±3.56 M ± ± ±2.64 M ± ± ±3.45 M ± ± ±1.92 M ± ± ±1.84 M ± ± ±2.65 a All results are shown in mean± S.D. (n=3) 39

40 Table 9: Micromeritic properties of optimised batch of microsponge core powder blend and pure drug Sample Bulk density Tapped density (g/ml) a (g/ml) a Carr's index a Hausner's Angle of ratio a repose a Core powder blend 0.52± ± ± Terbutaline 0.50 ± ± ± Sulphate a All results are shown in mean ± S.D. (n=3) 1.22 ± ± ± ±

41 Table 10: Physiochemical parameters of core tablets Formulation Thickness a Weight variation a Hardness a Friability (mm) (kg/cm 2 ) (%) F ± ± ± F ± ± ± F ± ± ± F ± ± ± F ± ± ± F ± ± ± F ± ± ± F ± ± ± F ± ± ± a All results are shown in mean± S.D. (n=3) 41

42 Table 11: Cumulative percentage drug release of optimized core tablet Time (min) CPR a

43 Table 12: Results of Physiochemical parameters & in-vitro drug release of Compression coated tablets Thickness a Hardness a Friability In-vitro Formulation Weight drug (mm) variation a (kg/cm 2 ) (%) release (%) F ± ± ± ±2.3 F ± ± ± ±2.24 F ± ± ± ±1.11 F ± ± ± ±1.2 F ± ± ± ±1.0 F ± ± ± ±2.1 F ± ± ± ±3.19 F ± ± ± ±3.20 F ± ± ± ±1.28 a All results are shown in mean± S.D. (n=3) 43

44 Table 15: Design Summary Formulation Code R3(Q 5 ) R4(Q 10 ) R5(Q 24 ) F F F F F F F F F

45 Table 16: Summary of results of regression analysis* Response Q 5 b 0 b 1 b 2 b 11 b 22 b 12 For drug release FM P values RM P values Response Q 10 b 0 b 1 b 2 b 11 b 22 b 12 For drug release FM E E P values RM P values < < < Response Q 24 b 0 b 1 b 2 b 11 b 22 b 12 For drug release FM P values RM P values < *FM: Full model; RM: Reduced model 45

46 Table 17: Calculations for testing the model in portions* For Q 5 DF SS MS F R 2 Regression F calc =2.177 FM F table =5.99 RM DF =(1, 6) Error FM RM For Q 10 DF SS MS F R 2 Regression F calc = FM F table =9.55 RM DF =(2, 3) Error FM RM For Q 24 DF SS MS F R 2 Regression F calc =0.599 FM F table =9.28 RM DF =(3, 3) Error FM RM *DF: Degree of freedom; SS: Sum of squares; MS: Mean of squares; F: Fischer s ratio; R 2 : Regression coefficient; FM: Full model and RM: Reduced model 46

47 Table 18: Comparison of orders of in vitro release of terbutaline sulphate from all the formulations Formulation Zero Order First Order F1 y = x y = x R² = R² = F2 y = -3.96x R² = y = x R² = F3 y = x y = x R² = R² = F4 y = x y = x R² = R² = F5 y = x y = x R² = R² = F6 y = x y = x R² = R² = F7 y = x y = x R² = R² = F8 y = x y = x F9 R² = y = x R² = R² = y = x R² =

48 Table 19: Regression equations of in vitro release of terbutaline sulphate from all the formulations Formulations Hixon Crowell Higuchi Korsmeyer Peppas F1 y = 0.100x R² = y = 20.25x R² = y = 1.487x R² = F2 y = 0.094x R² = y = 19.36x R² = y = 1.141x R² = F3 y = 0.088x R² = y = 18.77x R² = y = 1.126x R² = F4 y = 0.158x R² = y = 24.69x R² = y = 1.491x R² = F5 y = 0.144x R² = y = 23.86x R² = y = 1.739x R² = F6 y = 0.208x R² = y = x R² = y = 1.370x R² = F7 y = 0.249x R² = y = 24.95x R² = y = 1.312x R² = F8 y = 0.162x y = 24.34x y = 1.357x R² = F9 y = 0.140x R² = R² = y = 23.70x R² = R² = y = 1.279x R² =

49 Table 20: Slope of Korsmeyers-Peppas equation and proposed release mechanism Slope (n) Mechanism < 0.5 Fickian diffusion (Higuchi Matrix) 0.5 < n < 1 Non-Fickian diffusion 1 Case-II transport 49

50 Table 21: Cumulative percentage drug release of optimized formulation before and after stability study Time (h) CPR Before After

51 Figure 1: Calibration curve of terbutaline sulphate in 0.1 N HCL at 276 nm 51

52 Figure 2: Calibration curve of terbutaline sulphate in ph 7.4 phosphate buffer at 276 nm 52

53 53

54 Figure 3: FTIR spectra of (A) TBS (B) Microsponge (C) TBS + HPMC K100 M + EC; (D) TBS+ HPMC K100 M + EC 54

55 Figure 4: DSC thermogram of TBZ and microsponge 55

56 Figure 5: Trinocular microscopic observation of optimized formulation 56

57 Figure 6: Plot of particle size of microsponges 57

58 (A) (B) 58

59 Figure 7: Scanning electron microscopy of microsponges [A] Before compression, [B] After compression and [C] Pores on the surface of microsponge (C) Figure 8: In-Vitro drug release studies of terbutaline sulphate loaded microsponges 59

60 Figure 9: Compression coated tablets of EC and HPMC 60

61 Figure 10: Comparison of drug release between the HPMC grades 61

62 62

63 Figure 11: Comparison of drug release from different amount of coat weight 63

64 Figure 12: In vitro drug release study of core tablets in ph 7.4 phosphate buffer 64

65 Over view Side view Figure 13: Cross sectional view of compression coated tablets 65

66 Figure 14: In Vitro Drug Release Studies of compression coated tablets 66

67 Figure 15: Contour plot and 3D Surface Plot of CPR against % of magnesium stearate and Drug/polymer ratio 67

68 68

69 Figure 16: Contour plot and 3D Surface Plot of particle size (µm) against % of magnesium stearate and Drug/polymer ratio 69

70 Figure 17: Contour plot and 3D Surface Plot of Q5 (5 h) against HPMC: EC ratio and total polymer amount 70

71 Figure 18: Contour plot and 3D Surface Plot of Q8 (8 h) against HPMC: EC ratio and total polymer amount 71

72 Figure 19: Contour plot and 3D Surface Plot of Q24 (24 h) against HPMC: EC ratio and total polymer amount 72