Formulation and evaluation of a novel capsule-in-a-capsule ...

A.Srinivasa Rao et al., Int J Pharm Biomed Res 2013, 4(3), 170-176 172Table 1Composition of sustained release beads filled capsulesBatchcodeLornoxicam(mg)Sodiumalginate %(w/v)Calciumchloride% (w/v)HPMC Chitosan% (w/v) % (w/v)Pectin% (w/v)BFC-1 8 2 3 -- -- --BFC-2 8 3 5 -- -- --BFC-3 8 4 7 -- -- --BFC-4 8 3 5 0.5 -- --BFC-5 8 3 5 1.0 -- --BFC-6 8 3 5 1.5 -- --BFC-7 8 3 5 0.5 --BFC-8 8 3 5 1.0 --BFC-9 8 3 5 1.5 --BFC-10 8 3 5 0.5BFC-11 8 3 5 1.0BFC-12 8 3 5 1.5Each batch contains100mg of lornoxicam. Drug equivalent to 8 mg is takenfor single sustained release dose.where, ‘h’ is the cone height of beads, ‘r’ is the radius ofcircular base formed by the beads on the ground.2.4.2. Entrapment efficiencyAccurately weighed quantities of beads equivalent to 8mg of lornoxicam were placed in 25mL of 0.1N HCl. Thesolution was centrifuged using the centrifuge at 4200 rpm for30min; the supernatant layer of the liquid was assayed byUV-spectroscopy at 374nm. The encapsulation efficiencywas determined by the following equation.Encapsulation efficiency =% Drug of formulation x Total weight of the dried beads----Eq.(2)Amount of drug loaded - Drug loss in the gelation mediaThe batches, BFC-7 to BFC-9 were prepared using fixedconcentration of sodium alginate and different concentrationsof chitosan, as follows: The mixture of lornoxicam andsodium alginate dispersion was dropped through a syringewith a needle of size no.18 into 100mL of chitosan solutioncontaining 5% calcium chloride (Chitosan dissolved in 10mLof 5% (w/v) acetic acid) and stirred at 100rpm. After stirringfor 30 min, the coated beads were separated by filtration,washed with water and dried at 60°C or 6h in an oven.Similarly, BFC-10 to BFC-12 were prepared using fixedconcentration of sodium alginate and different concentrationsof pectin.2.3.3. Preparation of capsule-in-a-capsuleSpecial leak proof capsules for both smaller and biggersize was used in this formulation. To prepare a novel capsulein-a-capsuletechnology the prepared optimized sustainedrelease beads equivalent to 8 mg of lornoxicam were filled insize 2 hard gelatin capsule and was sealed with 15% (m/m)warm gelatin solution. This prepared sustained releasesmaller capsule was filled into a bigger capsule body size 0which was further filled with the liquid dispersion oflornoxicam equivalent to 3.25mg as loading dose usingmedicine droppers. After closing with cap the bigger capsulewas also sealed with 15% (m/m) warm gelatin solution. Thefilled capsules were stored at room temperature until testing.2.4. Evaluation parameters2.4.1. Flow propertyAngle of repose method was employed to assess theflowability of the beads. Beads were allowed to fall freelythrough the funnel fixed at 2cm above the horizontal flatsurface until the apex of conical pile just touched the tip ofthe funnel. The angle of repose (θ) was determined by theformula,θ = tan -1 (h/r)------------------- Eq.(1)2.4.3. Drug content uniformityLiquid dispersion and beads equivalent to 3.25mg and8.0mg of lornoxicam respectively was extracted in phosphatebuffer of pH 6.8. The solution was filtered through aMillipore filter (0.45μm pore size) and the drug content wasdetermined spectrophotometrically at λ max of 378nm aftersuitable dilution. The studies were carried out in triplicateand the mean values were noted indicating the reproducibilityof the results.2.4.4. In vitro release studiesA. Immediate release liquid dispersion of lornoxicamDissolution studies were carried out using USP XXIIIdissolution test apparatus II basket type (Electrolab TDL-08L) at a rotation speed of 100rpm and at 37 ± 0.5°C using900mL of 0.1N HCl (pH 1.2) for two hours. A 5mL samplewas withdrawn at 30min time intervals and replaced by anequal volume of pre-warmed 0.1N HCl (pH 1.2). Sampleswithdrawn were filtered through whatmann filter paper(0.45µm). The amount of lornoxicam released was analyzedat 374nm using a Shimadzu UV-spectrophotometer. Thestudies were carried out in triplicate and the mean valuesplotted verses time with standard error of mean, indicatingthe reproducibility of the results.B. Sustained release beads and capsule-in-capsuleformulationsDissolution studies were carried out using USP XXIIIdissolution test apparatus II basket type (Electrolab TDL-08L) at a rotation speed of 100rpm and at 37 ± 0.5°C using900 ml of 0.1N HCl (pH 1.2) for two hours and remaininghours in pH 6.8 phosphate buffer. A 5 ml sample waswithdrawn at 30min time intervals and replaced by an equalvolume of pre-warmed 0.1N HCl (pH 1.2) and phosphatebuffer pH 6.8, respectively. Samples withdrawn were filtered

A.Srinivasa Rao et al., Int J Pharm Biomed Res 2013, 4(3), 170-176 173through whatmann filter paper (0.45 micron). The amount oflornoxicam released was analyzed at 374nm and 376nm forsamples tested in 0.1N HCl and the phosphate buffer pH 6.8respectively, using a Shimadzu UV-spectrophotometer. Thestudies were carried out in triplicate and the mean valuesplotted verses time with standard error of mean, indicatingthe reproducibility of the results.2.4.5. Release kinetics studiesTo study the release kinetics in vitro release data wasapplied to kinetic models such as zero-order, first order,Higuchi and Korsemeyer-Peppas [12,13].3.1. Interaction studiesLornoxicam has got aromatic CH-stretch by the presenceof characteristic peak at 3061cm -1 . It has also shown thepeaks at 1157cm -1 and 1143cm -1 which corresponds to SO 2 Nstretch. CH-bending (aliphatic) is also observed at 1384cm -1 .The peaks at 1326cm -1 and 828cm -1 corresponds to CN stretchand CCL stretching, respectively. The IR spectra oflornoxicam with PEG 6000, Sodium alginate and HPMC, didnot reveals any extra peaks which confirms the absence ofchemical interactions between Lornoxicam and excipientsused (Fig.2).2.4.6. Stability studiesThe optimized formulation capsules were stored in glassbottles and subjected to accelerated stability studies as perICH guidelines i.e. 40°C/75%RH. Sampling was done atpredetermined time intervals of 0, 2, 4 and 6 months.Capsules were evaluated for the drug content and in vitrorelease profile. It was also noted that no leakage or visiblechange in appearance was apparent during the time of storageunder ambient temperature [14].3. RESULTS AND DISCUSSIONLornoxicam capsule-in-a-capsule technology designed inthis study consists of two phases; immediate and sustainedreleasing phase. The immediate dose (i.e loading dose) willbe useful for quicker onset of action to relieve headache andthe sustained dose (i.e maintenance dose) will be helpful toreduce the frequency of doses, throughout the day. Thedevice was formulated into two steps: First lornoxicam wasprepared as sustained release beads with hydrophilic polymerand filled into a size 2 leakage proof capsule which wasfurther filled into a size 0 leakage proof capsule body; secondlornoxicam was prepared as a liquid dispersion withsolubilising solvent and filled into the bigger capsule bodycontaining smaller capsule filled beads.Table 2Results of angle of repose, drug content and drug entrapment (n = 6)BatchesAngle of repose(θ ± SD)Drug content(%)Drug entrapmentefficiency (%)LFC-1 -- 98.58±0.72 --LFC-2 -- 98.15±0.90 --BFC-1 29°14’±0.15 97.22±0.51 73.6±0.67BFC-2 27°30’±0.25 98.45±0.74 75.8±0.46BFC-3 27°45’±0.19 98.43±0.66 79.2±0.84BFC-4 19°78’±0.28 96.41±0.70 90.66±1.08BFC-5 21°44’±0.35 97.22±0.93 92.71±1.88BFC-6 23°54’±0.38 98.87±0.58 96.50±1.34BFC-7 26°75’±0.20 96.32±0.65 91.30±0.65BFC-8 25°48’±0.22 97.10±0.72 93.14±0.78BFC-9 25°30’±0.16 97.86±0.55 94.06±0.55BFC-10 28°36’±0.32 97.23±0.74 87.12±0.40BFC-11 27°72’±0.25 98.42±0.34 90.56±0.56BFC-12 26°64’±0.18 98.10±0.40 93.20±0.77LFC- Liquid filled capsules; BFC- Beads filled capsulesFig.2. FTIR spectra of (a) Pure drug lornoxicam (b) lornoxicam+PEG 6000(c) Lornoxicam+ sodium alginate and (d) Lornoxicam+ HPMCMean % cumulative drug release1008060402000 5 10 15 20Time ((h))LFC-1LFC-2Fig.3. In vitro release profile of liquid dispersion filled capsules oflornoxicam

A.Srinivasa Rao et al., Int J Pharm Biomed Res 2013, 4(3), 170-176 174Fig.4. In vitro release profile of sustained release beads-filled-capsules of lornoxicam3.2. Flow properties, drug content and entrapment efficiencyThe angle of repose values ranged from 19°78’±0.28 to29°14’±0.15 which indicates good flow properties of thebeads. The mean drug content of liquid dispersion and beadswas studied and the values were found to be more than 95%.The drug entrapment efficiency of the sustained release beadswas also studied and the values were found to range between73.6±0.67 to 96.50±1.34 and the maximum drug entrapmentwas found to be in BFC-6 batch i.e. 96.50±1.34 as shown inTable 2. Each value represents mean ± SD of threedeterminations.3.3. In vitro release studiesIn vitro release studies of immediate release phase reveals93.73±0.72 and 98.50±0.88% of drug release from LFC-1and LFC-2, respectively at the end of 20min (Fig.3). Thus,LFC-2 was considered to be the best batch to getincorporated in a capsule-in-a-capsule formulation. Thebatches BFC-1, BFC-2 and BFC-3 prepared with sodiumalginate have released 97.79±0.68, 96.83±0.95 and92.42±0.84% of lornoxicam, respectively at the end of 10h.BFC-4, BFC-5 and BFC-6 prepared with sodium alginate incombination with HPMC have released 98.38±0.76,Mean % cumulative drug release1008060402000 2 4 6 8 10 12Time (h)Fig.5. In vitro release profile of biphasic release capsule-in-a-capsuleformulation of lornoxicam94.21±0.92 and 91.82±0.80% of lornoxicam, respectively atthe end of 12h. BFC-7, BFC-8 and BFC-9 prepared withsodium alginate in combination with chitosan have released97.19±0.67, 95.52±0.80 and 93.25±0.93% of lornoxicam,respectively at the end of 12h. BFC-10, BFC-11 and BFC-12prepared with sodium alginate in combination with pectinhave released 96.95±0.91, 92.06±0.72 and 88.60±0.60% oflornoxicam, respectively at the end of 12h.From the results (Fig.4) of separate in vitro dissolutionstudy of beads it was found that release was found to be moresustained when sodium alginate was used in combination

A.Srinivasa Rao et al., Int J Pharm Biomed Res 2013, 4(3), 170-176 175Fig.6. Graphical representation of release kinetics of capsule-in-a-capsule technologyTable 3Results of stability studiesParameters Initial values After 2 months After 4 months After 6 monthsAppearance Normal No signs of leakage No signs of leakage No signs of leakage butcapsules became littlesofter% Drug content of liquid-filled-capsules (LFC-2) 98.15±0.90 98.05±0.94 97.80±0.67 97.48±0.82% Drug content of beads-filled-capsules (BFC-6) 98.87±0.58 98.39±0.63 98.15±0.46 97.96±0.74% Drug release at the end of 30min and 12h 22.65±0.74% of drug atthe end of 30min and95.04±0.88% of drug atthe end of 12h22.40±0.52% of drug atthe end of 30min and95.01±0.79% of drug atthe end of 12h22.16±0.97% of drug atthe end of 30min and94.78±0.55% of drug atthe end of 12h22.12±0.49% of drug atthe end of 30min and94.72±0.90% of drug atthe end of 12hwith other release retardant polymers instead of using italone. It was also found that as the concentration of sodiumalginate alone or in combination with other polymers wasincreased, the release rate of lornoxicam was found to bedecreased. It is due to increased concentration of hydrophilicpolymers leads to increased gelling consistency whichdecreased the drug release rate. Thus, BFC-6 found be themost suitable to get incorporated in capsule-in-a-capsuleformulation as it contains maximum entrapment efficiencyand optimum sustained release profile.Finally, both the sustained release beads (BFC-6) andimmediate release liquid dispersion (LFC-2) of lornoxicamwere combined in a smaller and a bigger capsule,respectively in the form of capsule-in-a-capsule technologyand subjected to in vitro dissolution studies. From the resultsof in vitro dissolution studies, it was found that our capsulein-a-capsuleformulation released 22.65±0.74% of drug at theend of 30min and 95.04±0.88% of drug at the end of 12h(Fig.5).3.4. Release kineticsIn order to determine the mechanism of drug release formthe formulation, the in vitro dissolution data was fitted toZero order, First order, Higuchi plot and Korsemeyer-peppa’splot (Fig.6). The drug release from capsule-in-a-capsuleformulation fits well with Higuchi model followed by zeroorder, first order and Korsemeyer-peppa’s model. The

A.Srinivasa Rao et al., Int J Pharm Biomed Res 2013, 4(3), 170-176 176in vitro release data was further fitted to Korsmeyer-Peppasmodel which is generally used to analyze the releasemechanism when more than one type of release phenomenonis operational. Good linearity was observed with high ‘R 2 ’value. The value of release exponent ‘n’ is an indicative ofrelease mechanism. The value of ‘n’ obtained for theoptimized formulation was found to be 0.79 suggestingprobable release by non-Fickanian or anamolous diffusion.The analysis of experimental data in the light of theKorsmeyer-Peppas equation, as well as the interpretation ofthe corresponding values of n, leads to a better understandingof the balance between these mechanisms. The value of ‘n’obtained for the optimized formulation was found to be 0.79,which indicates that the release mechanism of lornoxicamfrom capsule-in-a-capsule formulation is non-Fickiantransport, which suggests that both dissolution and diffusionof the drug in matrices and also its own erosion modulatedrug release.3.5. Stability studiesThe results of accelerated stability studies reveal that theoptimized capsule-in-a-capsule formulation did not show anychange in appearance, drug content and in vitro releasecharacteristics during the study period (Table 3).4. CONCLUSIONSA novel biphasic drug delivery system was successfullydeveloped by filling smaller beads-filled-capsule into abigger liquid dispersion-filled-capsule body. The biggercapsule body was sealed with 15% (m/m) warm gelatinsolution. The best fast releasing liquid dispersion (BFC-6)and slow releasing beads (LFC-2) of lornoxicam wereselected through in vitro dissolution studies. Our optimizedcapsule-in-a-capsule formulation released 22.65±0.74% ofdrug at the end of 30min and 95.04±0.88% of drug at the endof 12h. It was also found to be stable at 40°C/75% RH for aperiod of 6 months as per ICH guidelines.ACKNOWLEDGMENTThe authors are very much thankful to the Chairman ofJB group of Educational Institutions Sri. J. Bhaskar Rao Garufor his constant help, support and encouragement to theacademics generally and research particularly. The authorsare also thankful to him for providing suitable research labfacility at Bhaskar Pharmacy College, R.R.District,Hyderabad.REFERENCES[1] Collins, J.G., Vital Health Statistics 1986, 10, 1-66.[2] Lipton, R.B., Stewart, W.F., Simon, D., Headache 1998, 38, 87-96.[3] Nabih Ramadan, M., Stephen Silberstein, D., Frederick Freitag, G.,Thomas Gilbert, T., Benjamin Frishberg, M. Evidence-BasedGuidelines for Migraine Headache in the Primary Care Setting:Pharmacological Management for Prevention of Migraine. AmericanAcademy of Neurology 2013, 1-55.[4] Carla Martins Lopes, Jose Sousa Lobo, M., Joao Pinto, F., Paulo Costa,C. AAPS Pharm Sci Tech 2007, 8, Article 76.[5] Biphasic release: colorcon, Available at http://www.colorcon.com/formulation/app/tailoring-release-profiles/biphasic-release[6] Mohd Abdul Hadi, Raghavendra Rao, N.G., World J Pharm Res 2012,1, 415-431.[7] Julia, A., Balfour, A.F., Barradell, L.B., Drugs 1996, 51, 639-657.[8] Welte, S.R., Rabbeseda, X., Drugs of Today 2000, 36, 55-76.[9] Buritova, J., Besson, J.M., Inflammopharmacol 1997, 5, 331-341.[10] Cooper, S.A., Fielding, A.F., Lucyk, D., Hersh, E.V., Quinn, P.D.,Betts, N. et al., Adv Ther 1996, 13, 67-77.[11] Hariprasanna, R.C., Qamar Jamal Ahmad, Upendra kulkarni, Asian JBiochem Pharm Res 2011, 2, 552-561.[12] Londhe, S., Gattani, S., Surana, S., J Pharm Sci Tech 2010, 2, 361-367.[13] Varahala Setti, M.L., Vijaya Ratna, J., Asian J Pharm 2009, 3, 252-256.[14] Harris Shoaib, M., Jaweria Tazeen., Hamid Merchant, A., Rabia IsmailYousuf, Pak J Pharm Sci 2006, 19, 114-118.