Pectin based multiparticulate formulation of Ketoprofen for bacterial enzyme dependent drug release in colon.
Abstract: The main objective in relation to this study was development of an oral controlled release multiparticulate colon-specific formulation of Ketoprofen by Powder Layering Technology. The intention was to prepare a formulation, using a combination of polymers as excipients that allowed drug liberation by depending on the bacterial enzyme in colon and absorption after a lag time of about 5-6 hours in the fasting state. Because of its rapid absorption and metabolism at the upper GI tract, development of a colon-specific delivery system of Ketoprofen becomes important for the treatment of colonic diseases such as colonic inflammation, Corn's disease etc. Hence, Ketoprofen-loaded Pectin/EC beads were developed as multi-particulate colon- specific delivery system. Beads prepared with 64.06%w/v in ethyl alcohol and 35.94% Pectin HM (59% methoxilation) in acetone: isopropyl alcohol (40:60) with 18.63% coating level were observed to be the best formulations as they could encapsulate more than 80 % of the drug and in-vitro investigation proved desire drug release pattern of the statistically optimized formulation. Very high amount of Ketoprofen can be encapsulated into these beads without altering the Ketoprofen retention pattern in simulated GI conditions. Our study showed that the beads were able to prevent release of Ketoprofen in simulated upper GI conditions and release at simulated colonic condition.
Article Type: Report
Subject: Pectin (Usage)
Pectin (Properties)
Ketoprofen (Production processes)
Controlled release preparations (Production processes)
Pharmaceutical chemistry (Research)
Drugs (Controlled release)
Drugs (Production processes)
Authors: Majumdar, S.
Lukka, Vaswani K.
Pub Date: 10/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Oct, 2011 Source Volume: 25 Source Issue: 4
Topic: Event Code: 320 Manufacturing processes; 310 Science & research
Product: Product Code: 2099950 Pectin NAICS Code: 311999 All Other Miscellaneous Food Manufacturing SIC Code: 2099 Food preparations, not elsewhere classified; 2834 Pharmaceutical preparations
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842729
Full Text: Introduction

A number of oral systems with variety of approaches have been designed for the drug release into the colon which include a) taking advantages of the apparent consistency of small intestine transit time, b) the utilization of the pH changes within the G.I. tract and c) the exploitation of bacterial enzyme localized in the colonic region of G.I. tract [1]. Amongst all the approaches used for colon targeting, a microbial controlled delivery system is the one of the most appealing as it relies on the unique enzymatic ability of the colonic micro flora and enables a more specific targeting, independent of pH variations along the GI tract. Many natural polysaccharides such as chondroitin sulphate, pectin, dextran, guar gum etc. have been investigated for their potential in designing colon specific drug delivery [2].

The delivery of drugs to colon for systemic action or a local effect is valuable in a variety of circumstances. These includes the topical treatment of disease such as ulcerative colitis, Crohn's disease and colon carcinoma and the potential for the oral delivery of peptide and other labile drugs which are unstable in the upper part of gastrointestinal (GI) tract [3, 4]. The requirement for an oral colonic drug delivery system is to reduce the drug release to a minimum prior to the ceacum. If a systemic absorption from colon is desired, the drug should thereafter be released rapidly [5].

Multiparticulate formulations may be prepared by several methods. Different methods require different processing conditions and produce multiparticulates of distinct qualities. Some of these methods may be broadly classified as pelletization, granulation, spray drying, and spray congealing [6]. The layering process comprises the deposition of successive layers of drug entities from solution, suspension or dry powder on nuclei which may be crystals or granules of the same material or inert starter seeds. In solution/suspension layering, drug particles are dissolved or suspended in the binding liquid [7, 8]. Sugar spheres were poured into the coating pan, and then intermittently treated with a nebulized binder solution applied by spray guns and with a finely dispersed drug powder applied by a specially designed powder feeding unit. The most attractive features of this powder layering system are the uniform distribution of the binder solution, as well as the easy-to-clean pan and the possibility of applying the successive functional film coating using the same equipment [9].

Ketoprofen (RS)2-(3-benzoylphenyl)-propionic acid (chemical formula [C.sub.16][H.sub.14][O.sub.3]) is one of the propionic acid derivatives of non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects [10]. Ketoprofen , unlike many NSAIA'S, inhibits the synthesis of leukotrienes and leukocytes migration into inflamed joints in addition to inhibiting the biosynthesis of prostaglandins. It is metabolised by glucuronidation of the carboxylic acid, hydroxylation of the benzoyl ring, and reduction of the keto function [11].

Materials and Methods

Ethyl Cellulose (45cps) was kindly provided by Dow Chemical Company Ltd, Middlesex, England. Pectin (59%) esterification was generous gift sample of Herbstreith & Fox, Germany. Pectinex Ultra SP-L (Pectinolytic Enzyme) obtained from Novo Ferment, Switzerland. 1 ml of enzyme has an activity of 9500 PG/ ml. The PG being the milli equivalence of reducing (carbonyl) groups liberated from pectin per minutes per unit of enzyme. The plasticizer TEC (triethyl citrate)-K PH EPs kindly provided by Polynt SPA, Italy and Ketoprofen by Aarti Drugs Limited, Mumbai, India. All other chemicals are of analytical grade.

Preparation of Ketoprofen loaded pellets by Powder Layering Technology

Drug containing multiparticulates were prepared by using conventional coating pan attached with air compressor with Spray Gun. Accurately weighted the non-pareil seed (inert sugar spheres) of 30 mesh size (600[micro]m) were taken and dried at 40[degrees]C for removal of moisture present. Composition of the formulation was described in Table 1. These dried non-pareils were charged into the coating pan, which has the bed temperature of 40[degrees]C. 5% Poly Vinyl Pyrrolidone (PVP) as a binder solution in 70:30 ratio of Isopropyl alcohol : Water was sprayed with the help of spray gun till the bed become wet. Immediately the require amount of powder drug i.e Ketoprofen was layered on to the wet bed of pellets. Pan rotation continued till the dry powder adheres onto the wetted pellets properly. Drying bed temperature and blowing air temperature maintain properly to avoid over heating of drug loaded pellets, which may cause the separation of drug from the pellets after several pan rotation. After layering the drug loaded multiparticulate formulation were kept in an oven for 2 hrs at 40[degrees]C.

Experimental Design

The central composite design was used in this study. The Ratio of EC ([X.sub.1]) and Coating Level ([X.sub.2]) were selected as the independent variables. The Table 2 summarizes an account of the 9 experimental runs studied, their factor combination, and the translation of the coded level to the experimental units employed during the study. Percentage drug release at 6 hour ([Y.sub.1]), Time at which 50% drug release ([T.sub.50]) ([Y.sub.2]) and Time at which 80% drug release ([T.sub.80]) ([Y.sub.3]) were taken as the response variables.

Statistical Analysis

Statistical validity of the polynomials was established on the basis of ANOVA provision in the Design expert Software. Three-dimensional (3D) response surface plots and two dimensional (2-D) contour plots were constructed based on the model polynomial functions using Design Expert software. These plots are very useful to see interaction effects on the factors on the responses. Eight optimum checkpoints were selected by intensive grid search, performed over the entire experimental domain, to validate the chosen experimental design and polynomial equations. The formulations corresponding to these checkpoints were prepared and evaluated for various response properties. Subsequently, the resultant experimental data of response properties were quantitatively compared with that of their predicted values. Also, linear regression plots between observed and predicted values of the response properties were drawn using MS-Excel, forcing the line through origin.

Formation of Coating Layer

Two polymers, Pectin HM (59% esterification) and Ethyl cellulose 45-CPS in different ratio and in different coating level were investigated for multiparticulate formulation shown in Table 2. A measured quantity of pectin was first added to a mixture of 100 ml acetone and isopropyl alcohol (40:60) and mixed properly using magnetic stirrer for 20 minuets. An anti sticking agent Talc was added to the solution based on the solid dry weight (5%w/w) of pectin present and mixed properly for 30 minutes. The require quantity of ethyl cellulose was dissolved in ethyl alcohol containing 10%w/w (based on solid dry weight of ethyl cellulose) TEC as plasticizer. The plasticizer--ethyl cellulose solution was then added to non-aqueous pectin solution and stirred for another 30 minuets using magnetic stirrer to produce coating formulation containing 1:1, 1:2 and 1:4 ratio of Pectin : Ethylcellulose. The coating specifications were given in the Table 3. The coating solution was sprayed onto the drug loaded multiparticulates by using Spray Gun attached with air compressor. Sample of coated formulations were removed from the coating pan when the coating level reached 5%, 10% & 20%. All batches of formulations were kept in an oven for 2 hours at 40[degrees]C.

Coating level

Coating level is expressed as the theoretical percentage of the weight gained (TWG %) used relative to the weight of the coated pellets. It is calculated using the following formula:

Coating level (%) = (X/Y - 1) x 100

X = Weight of uncoated pellets Y = Weight of coated pellets

Particle Size Distribution

The particle size distribution of Ketoprofen containing multiparticulate was carried out by sieve analysis using a set of US standard sieves. Sieves of size #14, 16, 18 and 20 were used along with a pellet load of 10 g. The sieve set was then shaken for 10 min. The net weight retained on each sieve was determined and these values were used for calculation of the particle size distribution. The amplitude was 6 .The determinations were performed in triplicate [12].

Bulk and Tapped Density

The bulk density was determined by pouring 50 g of materials into a 250-ml graduated glass cylinder which was kept at an angle of 450 to horizontal while pouring. The cylinder was straightened up and the volume occupied by the materials read to the nearest 1 ml. The bulk density was calculated by dividing the weight by the occupied volume. The tapped density was determined by a tapped density tester (Testing instruments, Kolkata) in which the glass cylinder was tapped 750 times. The tapped density was calculated in the same way as the bulk density. The Hausner ratio and Carr's Index were then calculated from them.

Fourier Transform Infrared Spectroscopy (FT-IR)

In order to assess the possible drug interactions IR spectroscopy of Ketoprofen loaded polymer coated multiparticulate formulation were performed on Fourier Transformed Infrared Spectrophotometer (840, Shimadzu, Japan). The conventional KBr pellet technique was used to prepare sample for FTIR and was scanned in the wave number range of 2000-500 [cm.sup.-1] coupled to a personal computer. The characteristic peaks of IR transmission spectra of Ketoprofen pure drug and Ketoprofen formulation were recorded.

Differential Scanning Calorimetry (DSC)

Thermograms of Ketoprofen pellets were obtained using a Perkin Elmer-Jeda DSC instrument equipped with an intra-cooler. Powder samples were hermetically sealed in perforated aluminum pans and heated at a constant rate. Purge gas-nitrogen at a flow rate 20 ml/min and heating temperature of 100[degrees]C was used to maintain inert atmosphere.

Scanning Electron Microscopy (SEM)

The surface morphology of pellets of optimized formulation was examined before and after dissolution using scanning electron microscope. The samples were fixed on a brass stub using double-sided tape and then gold coated in vacuum by a sputter coater. The pictures were taken at excitation voltage of 10 KV and at 400X magnification by using JSM-840A scanning Microscope; Jeol-Japan.

Loose Surface Crystal Study (LSC)

The formulation of equivalent to 100mg of pure drug was suspended in a 100 ml volumetric flask containing simulated colonic fluid pH 6.0 was shaken vigorously for 5 minuets. The drug was leached out from the surface of the multiparticulates into pH 6.0 medium in presence of Pectinolytic enzyme was analyzed spectrophotometrically at 260.5nm. The absorbance found from UV-Visible Spectrophotometer was plotted on the standard graph to get the concentration of loose drug present on the multiparticulates. Percentage of drug released with respect to entrapped drug in the sample was recorded [13, 14, 15].

(LSC (%) = (Drug leached out from the multiparticulates / Drug content in the experimental multiparticulate) x 100

Drug Entrapment Efficiency

The total mass of dried beads obtained from a batch was considered as practical yield of the process. (30) The equivalent amount of multiparticulates in which 100mg of Ketoprofen was present. Then the multiparticulates were crushed and suspended on 100 ml of simulated colonic fluid 6.0 pH with Pectinolytic enzyme shaken vigorously until the entire drug comes into the solution and it was also kept for 24 hours at room temperature, later it was stirred for 5 minuets and filtered. From the filtered solution 10 ml was taken and diluted with the buffer solution. Then the absorbance was measured spectrophotometrically at 260.5nm.The absorbance values were plotted on the standard curve to get the concentration of the entrapped drug.

Drug entrapment efficiency (%) = (Actual quantity of drug present / Theoretical quantity of drug that must be present) x 100

In-Vitro Drug Release Study

Drug release study was carried out in USP-1 apparatus at 100 rpm and temperature of 37 [+ or -] 0.5 [degrees]C. The Simulated Gastric Fluid (SGF) was employed as dissolution medium for 2 hours later replaced with Simulated Small Intestinal Fluid (SSIF) and Simulated Caecal Fluid (SCF) for 3 hours and rest of study i.e., until the multiparticulates dissolve completely, respectively.5ml of sample was withdrawn at regular intervals were analyzed spectrophotometrically at 259nm for SGF and at 260.5nm for SSIF, SCF and were replaced with the respective simulated fluid.

Data analysis

To assess the drug release rate and mechanism the in vitro release data obtained was fitted to different models viz. zero order, first order, Higuchi, Hixson-Crowell cube root by employing the following equations.

Zero-order rate equation - [Q.sub.0] - [Q.sub.t] = [k.sub.0]t

First order rate equation - log[Q.sub.0] - log[Q.sub.t] = [k.sub.1]t/2.303

Higuchi model - [Q.sub.t] = k [t.sup.1/2]

Korsmeyer-Peppas model - [Q.sub.t] /[Q.sub.n] = K[t.sup.n]

Where, [Q.sub.0], [Q.sub.t], Q, corresponds to amount of the drug at zero time, at time t, at infinite time respectively. [k.sub.0], [k.sub.1], k, K were zero-order rate constant, first order constant, constant for Higuchi model and Korsmeyer-Peppas model constant respectively. n is the release exponent [16]. Diffusion exponent and solute release mechanism for Korsmeyer-Peppas model were given in Table 4.

Results and Discussion

Particle Size and Size Distribution

From the data obtained (Table 5) it was found that about 72.34 [+ or -] 0.04% to 79.79 [+ or -] 0.17% of multiparticulate formulation were of 12# mesh size that is the particle size ranges between 1300-1000[micro].m, which proves the flexibility of the powder layering method i.e. particle size can easily be controlled by adjusting the size of non-pareil seed and coating level. Higher the coating level, size of the multiparticulates increases gradually.

Bulk and Tapped Density

Table 6 reveals that the Bulk Density, Tapped Density and Hausner Ratio of formulation K-P-EC F1 to K-P-EC F9 ranges from 0.810 [+ or -] 0.04 to 0.678 [+ or -] 0.08 gm/[cm.sup.3], 0.851 [+ or -] 0.07 to 0.726 [+ or -] 0.03 gm/[cm.sup.3], and 1.105 [+ or -] 0.02 to 1.050 [+ or -] 0.02 respectively that indicates the ease of tabletabbility and a good uniform flow of pellets to gain uniform weight of tablets or capsules. The Carr's index ranges between 9.299 [+ or -] 0.10 to 4.817 [+ or -] 0.12 %. The values of Carr's index are below 10 which indicate excellent compressible properties. This shows that the pellets can be readily made into a tablet.

Fourier Transform Infrared Spectroscopy

In FTIR spectrum of pure drug (Figure 1A) the characteristics peaks correspondence to -COOH group (1659 [cm.sup.-1] for the symmetric stretch), an aromatic--CH = CH-group (1284 [cm.sup.-1] for symmetric bend), a C[H.sub.2] group (2975 [cm.sup.-1] for scissors stretch) and - C=O- group (1693 [cm.sup.-1] for symmetric stretch) was identified. The spectrum of ketoprofen formulation (Figure 1B) and ketoprofen pure drug shows that there was no significant interaction between drug and other polymers and plasticizer. From the FTIR spectra it was confirmed that Ketoprofen and formulation components were compatible with each other.

[FIGURE 1 OMITTED]

Differential Scanning Calorimetry (DSC)

DSC scan for ketoprofen and ketoprofen pellets was depicted in Figure 2. The pure ketoprofen displayed (Figure 2A) a single sharp endothermic peak at 96.47[degrees]C corresponding to the melting point of the drug and an identical peak was also observed in the pellet formulations. Drug-polymer physical mixture (Figure 2B) shows a sharp endothelium peak at 91.69[degrees]C. This is indicative of a very slight interaction in drug-polymer physical mixture but not necessarily corresponds to an incompatibility. The thermographic result shows that the drug retains its identity in the coated pellet formulations. The additional peak in the formulation is due to other components present in the pellets.

Shape and Surface Characteristics by SEM

SEM photographs of the multiparticulates were taken before (Figure 3A) and after dissolution (Figure 3B). SEM images of the multiparticulates showed that they possess a spheroid form, when compared to the active drug particles. The higher the viscosity and concentration of the selected polymer, the more spherical in shape pellets can be obtained with less micro pores on the surface. Comparing the surfaces of multiparticulates 1:2 ratio and 20% coating level of Pectin: Ethyl Cellulose with 1:1 ratio of Pectin: Ethyl Celluloses at 20% coating level, it could be observed that shape and porosity of pellets were influenced by Pectin concentration. For this reason, K-P-EC F3 pellets prepared with the highest amount of pectin have more regular particles.

Loose Surface Crystal Study (LSC)

The study was executed with various prepared formulations and the results were tabulated in Table 7. The percentage of LSC in case of low concentration of ethyl cellulose with less coating level i.e. 1:1 ratio of Pectin-Ethyl cellulose with 10% coating level is slightly higher as compared to the formulation containing 1:2 and 1:4 ratio. As the ethyl cellulose concentration increases with increase in coating level, leaching was negligible. It indicates that drug leaching was controlled by the ethyl cellulose network. But the average % LSC significantly low in all the formulation indicates that powder layering technology is able to produce successive coating by using conventional coating pan.

Drug Entrapment Efficiency

The encapsulation efficiency of Ketoprofen multiparticulates were tested under different processing parameters and the results were shown in Table 7. The encapsulation efficiency was increased from 86.3% to 97.8% when the polymer ratio increases from 1:1 to 1:4. However, coating level plays a significant role on drug entrapment efficiency. Formulation K-P-EC F1 which consists of 1:1 ratio with 10% coating level of Pectin-Ethyl cellulose shows a low entrapment efficiency as the less amount of ethyl cellulose was applied with less percentage of coating level. Further increasing of ethyl cellulose ratio from 1:1 to 1:2 with the same coating level i.e. 10% improves the encapsulation efficiency indicates that ethyl cellulose concentration influence the efficiency. Highest encapsulation efficiency was observed K-P-EC F9 formulation as it consist of 1:4 ratio of Pectin-Ethyl cellulose with 20% coating level. It is because once the higher coating level applied on to the non-pareil, time required to rotate the coating pan need to increase causes more impaction of coated multiparticulates with the pan surface cause tight bonding between drug and polymer resulted prominent entrapment efficiency. At the same time increasing the polymer concentration and coating level minimize the chances of drug leaching from the surface of the multiparticulates. During the preformulation study it was observed that PVP-K30 concentration less than 5% produces the multiparticulates with higher leaching of drug indicates the importance of PVP concentration as a binder in powder layering process. This is due to that more viscous external aqueous phase caused by raising PVA concentration resulted in the reduction of diffusing rate from the inner water phase to the outer water phase, consequently improved the entrapment efficiency.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Experimental Design

Various RSM computations for the current optimization study were performed employing Design Expert Software (design expert 8.0.3 trial version) State-Ease Inc. Polynomial models including interaction and quadratic terms were generated for all the response variables using multiple linear regression analysis (MLRA) approach. The general form of the MLRA model is represented as the following equation:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

here, [[??].sub.0] is the intercept representing the arithmetic average of all quantitative outcomes of 9 runs; [[??].sub.1] to [[??].sub.7] are the coefficients computed from the observed experimental response values of Y; and [X.sub.1] and [X.sub.2] are the coded levels of the independent variable(s). The terms [X.sub.1] [X.sub.2] and [Xi.sup.2] (i = 1 to 2) represent the interaction and quadratic terms, respectively. Mathematical relationships in the form of polynomial equation for the measured response (% Drug release at 6 hour, [T.sub.50] and [T.sub.80] were taken as the response variables obtained with the stat-ease software. The polynomial equation relating the different response and independent variable is given below:

Y1 (% Drug release at 6 hr) = 14.14-8.60[X.sub.1] - 3.25[X.sub.2] + 1.99[X.sub.1][X.sub.2] + 9.62 [X.sub.1.sup.2] - [X.sub.2.sup.2]

Y2 ([T.sub.50]) = 8.95 + 4.84[X.sub.1] + 0.93[X.sub.2] + 0.58[X.sub.1][X.sub.2] + 2.26[X.sub.1.sup.2] + 0.039 [X.sub.2.sup.2]

Y3 ([T.sub.80]) = 16.92 + 12.27 [X.sub.1] + 2.77 [X.sub.2] + 1.46[X.sub.1][X.sub.2] + 5.37[X.sub.1.sup.2] + 2 + 0.53[X.sub.2.sup.2]

The above equation represents the quantitative effect of process variables and their interaction on the response. For estimation of the significance of the model, the analysis of variance (ANOVA) was determined as per the provision of design expert software as shown below. Using 5% significance level, a model is considered significant if the p-value (significance probability value) is less then 0.05. Figure 4 represents the 3D analysis for the studied response properties i.e. % drug release at 6 hr. From the contour plot it was concluded that release in 6 hr decreased with augmentation of both the variables. The response changes the variables in a linear and descending manner. But the contour plot shows that both the variables i.e. Ratio of EC and Coating level has greater influence on drug release at 6 hr. Although at 6 hr, % drug release is less but still they are influenced by the 2 independent variables. Ratio of EC at certain level improves the drug release, but above that ratio there is no significant improvement of drug release observed. However a very little effect of coating level was observed on drug release at 6 hr. Figure 5 demonstrates the relation of [T.sub.50] with ratio of EC and coating level. The effect of ratio of EC and coating level was found to be ascending manner i.e. increasing the amount of the both increases the response. The effect of coating level found to be linear fashion throughout different level; increase in coating level increase the [T.sub.50] i.e. delayed the drug release. Effect of ratio of EC also found to be linear i.e. at higher ratio [T.sub.50] increases. From the contour and 3D plot it was quite evident that both ratio of EC and coating level has completely greater influence on the response variables. Figure 6 shows the 3D analysis for the studied response properties for [T.sub.80]. From the contour plot it can be concluded that [T.sub.80] increases with augmentation of both the variables. The response changes the variables in a linear and ascending manner. Increase in ratio of EC increase the [T.sub.80] that is the time requires releasing 80% of drug increase, but further increase in ratio increase [T.sub.80] level beyond the desire range.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

In-Vitro Drug Release Study

The data for the various regression parameters obtained after fitting them into different kinetic models were depicted in Table 8.

It was found that the release of multiparticulate formulation of Ketoprofen coated with pectin was dependent on the polymer (ethyl cellulose) concentration. In the formulations K-P-EC F1

where 1:1 ratio of pectin/ethyl cellulose with 10% coating level was used, before 6 hr i.e. before reaching in colon there is significantly less amount of drug released (6.12%) but after 6 hour at pH 6.0 a sudden increase in drug release (38.244%) was observed and 88.016% drug released within 10 hour. In the same formulation with 15% and 20% coating level (K-P-EC F2 & F3) there is slight improvement in drug release i.e. after 6 hour they released 32.77% and 25.55% and after 10 hour they released 75.07% and 65.79% respectively. So increase in coating level % can influences the drug release kinetics. In the formulation K-P-EC F4 with 10% coating level, where ethyl cellulose ratio increased to 1:2, drug released was observed 15.19% at 6th hour, 78.39% at 16th hour and 90.41% at 20th hour. But increase in coating level % to 15 and 20% shown significantly improves drug release. In formulation K-P-EC F6 i.e. 1:2 ratio with 20% coating level showed 16.90% drug release at 6th hour, 79.51% at 20th hour and 86.54% at 24th hour, which is quite identical as a controlled release colon specific formulation. Because it released only 4.48% drug at 5th hour i.e. before reaching to colon. In colon they also released the drug at a controlled manner and able to delay the drug release more then 24 hours.

Further increase in ethyl cellulose ratio to 1:4 with 10%, 15% & 20% coating level, the release was found 17.10%, 63.60% & 70.18% in K-P-EC F7, 15.40%, 57.41% & 65.74% in K-P-EC F8 & 12.41%, 53.95% & 61.58% in K-P-EC F9 formulation which is significant less, indicated that at higher concentration ethyl cellulose hinder the drug release profile.

The in-vitro release profile of all the multiparticulate formulation was best expressed by first order rate kinetics (Figure 7) with best value of 0.9830 of K-P-EC F6 formulation. The data were best fitted into Korsmeyer-Peppas model with the slope or exponential value (n) ranging from 0.842 to 0.919. So n value of K-P-EC F6 formulation was 0.896 indicating super case II transport which reflect greater relaxation contribution. Case-II relaxation release is the drug transport mechanism associated with stresses and state-transition in hydrophilic glassy polymers which swell in water or biological fluids. This term also includes polymer disentanglement and erosion [17].

Conclusion

Beads prepared with 64.06%w/v in ethyl alcohol and 35.94% Pectin HM (59% methoxilation) in acetone: isopropyl alcohol (40:60) with 18.63% coating level were observed to be the best formulations as they could encapsulate more than 80 % of the drug and at the same time showed desirable drug release pattern. Very high amount of Ketoprofen can be encapsulated into these beads without altering the Ketoprofen retention pattern in simulated GI conditions. Moreover, a small amount of drug is required to prepare the beads with desired release profile. Though our study showed that the beads were able to prevent release of Ketoprofen in simulated upper GI conditions and can release at simulated colonic condition. Further in-vitro release pattern of the statistically optimized formulation.

Acknowledgements

The authors are indebted to Dr. Santanu Ganguly, Head of section, Regional Radiation medicine centre, Department of Atomic energy, Govt. of India for his technical advice and kind support.

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S Majumdar *, Vaswani K. Lukka

Department of Pharmaceutics, Calcutta Institute of Pharmaceutical Technology and Allied Health Sciences, Uluberia, 711316, India

* Corresponding author, (sanku_20011@rediffmail.com) Dr. S. Majumdar

Received 24 November 2010; Accepted 18 August 2011; Available online 8 September 2011
Table 1: Composition of the formulations

formulation    Non pareil      Ketoprofen        Ratio of
                  (mg)            (mg)        [pectin: ethyl-
                                                 cellulose

K-P-EC Fl        400 mg          100 mg            1 : 1
K-P-EC F2        400 mg          100 mg            1 : 1
K-P-EC F3        400 ms          100 mg            1 : 1
k-P-LC 14        400 mg          100 mg             1:2
K-P-EC F5        400 mg          100 mg             1:2
K-P-EC F6        400 mg          100 mg             1:2
K-P-EC F7        400 mg          100 mg             l:4
K-P-EC F8        400 mg          100 mg             l:4
K-P-EC F9        400 mg          100 mg             l:4

formulation      Coating          Talc             TEC
                level (%)        (%w/w)          (%w/w)

K-P-EC Fl          10%           5% w/w          10% w/w
K-P-EC F2          15%           5% w/w          10% w/w
K-P-EC F3          20%           5% w/w          10% w/w
k-P-LC 14          10%           5% w/w          10% w/w
K-P-EC F5          15%           5% w/w          10% w/w
K-P-EC F6          20%           5% w/w          10% w/w
K-P-EC F7          10%           5% w/w          10% w/w
K-P-EC F8          15%           5% w/w          10% w/w
K-P-EC F9          20%           5% w/w          10% w/w

Table 2: Formulations trial chart

Batches    X1    [X.sub.2]   [Y.sub.1]   [Y.sub.2]   [Y.sub.3]

F*1        0         0        13.558       9.055      16.277
F*2        1        -1        17.104      14.375      30.960
F*3        0         1        13.068      10.390      20.090
F*4        -1        1        25.524       7 010      11.433
F*5        1         0        15 367      15 952      34.904
F*6        1         1        12.379      17.894      38.842
F*7        -1        0        32.742       6 366      10.307
F*8        0        -1        15 163       8.409      14.439
F*9        -1       -1        38.210       5.821       9.375

[X.sub.1] is percentage of EC replacing pectin and [X.sub.2] is
coat weight as % w-w. [Y.sub.1] corresponds to percentage drug
release at 6th hour, [Y.sub.2] and [Y.sub.3] were [T.sub.50]
(Time at which 50% drug release) and [T.sub.80] (Time at which
80% drug release) respectively. Translation of coded levels into
actual units was for [X.sub.1], -1, 0 and 1 were 50, 65 and 80,
and for [X.sub.2], -1, 0 and 1 were 10, 15 and 20 respectively.

Table 3: Coating specification of formulation

Coating equipment                Specification

Pan diameter                     12 inch
Heating temperature              40[degrees]C
Diameter of Blower               40mm
An flow                          10 CFM
Pan rotation                     20 rpm
Bed temperature                  35[degrees]C
Pressure from air compressor     15kg/[cm.sup.2]
Powder drug application rate     20gm/min
Spray gun position to the bed    90[degrees] to the bed
Spray nozzle diameter            1.2 mm
Rolling time                     15 min

Table 4: Diffusion exponent values with mechanism of
drug release

Diffusion exponent (n)   Overall solute diffusion mechanism

0.43                     Fickian diffusion
0.43 < n < 0.85          Anomalous (non-Fickian) diffusion
0.85                     Polymer swelling
n > 0 85                 Super case-II transport

Table 5: Sieve analysis data of different formulations

Sieve Number                    #12                   #16

Size Range ([micro]m)        1300-1000             1000-850

                       Fur mutation (% retained)

K-P-EC Fl               72.34 [+ or -] 0.04   20.31 [+ or -] 0.01
K-P-EC F2               75.82 [+ or -] 0.08   18.87 [+ or -] 0.17
K-P-EC F3               79.10 [+ or -] 0.04   17.37 [+ or -] 0.13
K-P-EC F4               73.51 [+ or -] O.01   19.45 [+ or -] 0.02
K-P-EC F5               74.2i [+ or -] 0.07   19.04 [+ or -] 0.09
K-P-EC F6               79.79 [+ or -] O.17   16.96 [+ or -] 0.06
K-P-EC F7               73.11 [+ or -] 0.16   21.34 [+ or -] 0.19
K-P-EC F8               76.07 [+ or -] 0.03   20.45 [+ or -] 0.16
K-P-EC F9               78.83 [+ or -] 0.22   16.62 [+ or -] 0.10

Sieve Number                    #20                   #30

Size Range ([micro]m)         850-600               600-450

                       Fur mutation (% retained)

K-P-EC Fl               5.34 [+ or -] 0.21    2.01 [+ or -] 0.10
K-P-EC F2               3.47 [+ or -] 0.08    1.84 [+ or -] 0.14
K-P-EC F3               3.01 [+ or -] 0.06    0.52 [+ or -] 0.10
K-P-EC F4               6.45 [+ or -] 0.05    0.59 [+ or -] 0.09
K-P-EC F5               6.03 [+ or -] 0.17    0.68 [+ or -] 0.0S
K-P-EC F6               3.18 [+ or -] 0.19    0.07 [+ or -] 0.14
K-P-EC F7               4.99 [+ or -] 0.14    0.56 [+ or -] 0.06
K-P-EC F8               3.07 [+ or -] 0.04    0.41 [+ or -] 0.11
K-P-EC F9               4.06 [+ or -] 0.15    0.49 [+ or -] 0.10

* All values are expressed as Mean  [+ or -] SD, n=3.

Table 6: Micromeritic properties of different formulations

Formulation Code          Bulk Density         Tapped Density

K-P-EC Fl             0.810 [+ or -] 0.04    0.851 [+ or -] 0.07
K-P-EC F2             0.704 [+ or -] 0.06    0.755 [+ or -] O.O6
K-P-EC F3             0.712 [+ or -] 0.10    0.785 [+ or -] O.O2
K-P-EC F4             0.678 [+ or -] 0.08    0.726 [+ or -] 0.03
K-P-EC F5             0.723 [+ or -] 0.08    0.768 [+ or -] 0.06
K-P-EC F6             0.711 [+ or -] 0.0l    0.786 [+ or -] O.C4
K-P-EC F7             0.684 [+ or -] 0.03    0.726 [+ or -] 0.05
K-P-EC FS             0.700 [+ or -] 0.09    0.742 [+ or -] 0.0l
K-P-EC F9             0.688 [+ or -] 0.10    0.7S4 [+ or -] 0.05

Formulation Code         Carr's Index          Hausner Ratio

K-P-EC Fl             4.817 [+ or -] 0.12   1.050 [+ or -] 0.02
K-P-EC F2             6.755 [+ or -] 0.14   l.072 [+ or -] 0.04
K-P-EC F3             9.299 [+ or -] 0.10   1.102 [+ or -] 0.07
K-P-EC F4             6.616 [+ or -] 0 17   1.070 [+ or -] 0.04
K-P-EC F5             5.859 [+ or -] 0.13   1.062 [+ or -] 0.03
K-P-EC F6             9.542 [+ or -] 0.15   0.105 [+ or -] 0.02
K-P-EC F7             5.785 [+ or -] 0.16   1.061 [+ or -] 0.04
K-P-EC FS             5.660 [+ or -] 0.14   1.060 [+ or -] 0 06
K-P-EC F9             8.753 [+ or -] 0.13   1.095 [+ or -] 0.07

Table 7: Drug Entrapment Efficiency & Loose Surface Crystal Study

                 Weight of                            Drug
             multiparticulates   Absorbance        Entrapment
Batch No          in mg.             nm            Efficiency

K-P-EC Fl           100            0.6756     86.30 [+ or -] 10.43
K-P-EC F2           100            0.6883     88.4l [+ or -] 0.37
K-P-EC F3           100            0.7048     90.53 [+ or -] 0.78
K-P-EC F4           100            0.6931     89.05 [+ or -] 0.65
K-P-EC F5           100            0.7228     92.83 [+ or -] 0.1B
K-P-EC F6           100            0.7431     95.45 [+ or -] 0.54
K-P-EC F7           100            0.7261     93.29 [+ or -] 0.79
K-P-EC F8           100            0.7441     95.58 [+ or -] 0.47
K-P-EC F9           100            0.7620     97.87 [+ or -] 0,99

             Multiparticulate
              equivalent to
Batch No      100mg of Drug            LSC %

K-P-EC Fl         115.87        1.354 [+ or -] 0.04
K-P-EC F2         113.11        0.927 [+ or -] 0.07
K-P-EC F3         110.46        0 744 [+ or -] 0 04
K-P-EC F4         112.3         9.835 [+ or -] 0.06
K-P-EC F5         107.72        0.605 [+ or -] 0.03
K-P-EC F6         104.77        0.399 [+ or -] 0.04
K-P-EC F7         107.19        0.552 [+ or -] 0.07
K-P-EC F8         104.63         0.22 [+ or -] 0 08
K-P-EC F9         102.18        0.103 [+ or -] 0.05

Table 8: Statistical data

              Ration of        ZERO ORDER              FIRST ORDER
               pectin
Formulation     to EC     [r.sup.2]     slope     [r.sup.2]     slope

K-P-EC F1       1 : 1       0.7740      5.3420      0.9780     -0.1120
K-P-EC F2       1 : 1       0.8191      5.3460      0.9650     -1.1010
K-P-EC F3       1 : 1       0.8070      5.3310      0.9590     -1.0900
K-P-EC F4       1 : 2       0.9136      4.9570      0 9010     -1.0660
K-P-EC F5       1 : 2       0.9216      4.7810      0.9420     -1.0540
K-P-EC F6       1 : 2       0.9220      4.4590      0.9830     -1.0410
K-P-EC F7       1 : 4       0.9260      3.3840      0.9750     -1.0240
K-P-EC F8       1 : 4       0.9212      3.1320      0.9670     -1.0210
K-P-EC F9       1 : 4       0.9489      2.9970      0.9790     -0.0190

                      HIGUCHI               PAPPAS-
                      MATRIX                KORSMEYER

Formulation   [r.sup.2]     slope     [r.sup.2]       n

K-P-EC F1       0.8600     33.9250      0.8420      1.6820
K-P-EC F2       0.8900     33.5710      0.8650      1.7910
K-P-EC F3       0.9170     33.0090      0.8770      1.7400
K-P-EC F4       0.9360     30.2210      0.8890      1.7130
K-P-EC F5       0.9380     29.0600      0.8980      1.7690
K-P-EC F6       0.9410     27.0780      0.8960      1.7710
K-P-EC F7       0.9509     20.6520      0.8930      1.5570
K-P-EC F8       0.9480     19.1290      0.8960      1.5460
K-P-EC F9       0.9560     18.1210      0.9190      1.5800
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