Acceleration of in vitro dissolution studies of sustained release dosage form of theophylline and in vitro–in vivo evaluations in terms of correlations

The aim of the study was to accelerate the dissolution of the sustained release dosage forms using both elevated temperature and high rpm rates. Teokap® SR 200 mg pellets were tested by in vitro sustained and accelerated dissolution studies using USP

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  ORIGINAL PAPER Acceleration of in vitro dissolution studies of sustained releasedosage form of theophylline and in vitro–in vivo evaluationsin terms of correlations Go ¨khan Ertan  • Ercu ¨ment Karasulu  • Is¸ ı k O ¨zgu ¨ney  • Yes¸im Karasulu  • S¸ebnem Apayd ı n  • Gu ¨lten Kantarc ı  • Aysu Yurdasiper  • Mehmet Ali Ege Received: 9 February 2011/Accepted: 6 June 2011/Published online: 8 July 2011   Springer-Verlag France 2011 Abstract  The aim of the study was to accelerate thedissolution of the sustained release dosage forms usingboth elevated temperature and high rpm rates. Teokap  SR200 mg pellets were tested by in vitro sustained andaccelerated dissolution studies using USP XXIII rotatingpaddle method. Sustained dissolution studies were carriedout for 12 h in phosphate buffer at 37  ±  0.5  C and 50 rpm.Accelerated dissolution studies were performed for 48 minin distilled water at 90  ±  1  C and 250 rpm. The resultsobtained from accelerated and sustained dissolution studieswere correlated using both linear and linear kinetic corre-lation methods by a computer program. While  r  2 andmaximum error between calculated and observed drugrelease rates were found to be 0.9129 and 15.9%, respec-tively, in linear correlation method, these values wereobserved as 0.9938 and 3.6%, respectively, in linear kineticcorrelation method. In vivo plasma concentration data wereobtained from six New Zealand rabbits after administrationof a single dose of Teokap  SR 200 mg pellet. Then, theresults of in vivo study were evaluated with in vitroaccelerated and sustained dissolution results by applyingthem to in vitro–in vivo linear correlations. As a result of these correlations, it was shown that the in vitro correlationplots were very similar to the plot which was obtained bythe in vivo study (  f  2  =  73.81–77.11). This study suggesteda way to prevent the loss of time for routine dissolutionstudies of sustained release preparations for quality controlprocedures using in vitro accelerated dissolution tests. Thestorage and quarantine periods of the product in processand process controls in the manufactories could be short-ened by this method. Calculation of the in vivo perfor-mance of sustained release dosage forms using the resultsof the accelerated dissolution studies may be counted asanother advantage of the method. Keywords  Theophylline    Sustained release   Accelerated release    Correlation    Similarity 1 Introduction In the last decades, numerous investigations have beenconducted on sustained release dosage forms. They canachieve therapeutically effective concentrations of drug inthe systemic circulation over an extended period of time,thus reducing the doses of the drugs. There are nearly 170sustained release dosage forms used in therapy accordingto an electronic drug index (The internet drug index forprescription drugs 1995). Dissolution procedures of thesedosage forms take generally 8–12 h. Moreover, the disso-lution period may increase to 20, 30 h for ultra-sustained G. Ertan ( & )    I. O¨zgu¨ney    Y. Karasulu    A. Yurdasiper   M. A. EgeDepartment of Pharmaceutical Technology,Faculty of Pharmacy, Ege University, Bornova,35100 Izmir, Turkeye-mail: KarasuluDepartment of Biopharmacy and Pharmacokinetics,Faculty of Pharmacy, Ege University, Bornova,35100 Izmir, TurkeyE. Karasulu    S¸. Apayd ı nCenter for Drug R&D and Pharmacokinetic Applications,Ege University, Bornova, 35100 Izmir, TurkeyG. Kantarc ı Department of Pharmaceutical Biotechnology,Faculty of Pharmacy, Ege University, Bornova,35100 Izmir, Turkey  1 3 Eur J Drug Metab Pharmacokinet (2011) 36:243–248DOI 10.1007/s13318-011-0049-6  dosage forms (Harrison et al. 1993; Karasulu et al. 2003) and 1–5 months for some biodegradable implants con-taining antibiotics, antineoplastics, enzymes or antiin-flammatories and etc. (Witt and Kissel 2001; Woo et al.2001; Zhang et al. 2003). Because of this reason, automatic equipments are started to be used in these studies. Auto-matic equipments are well suited for use in process controlas well as in the early stages of the formulation develop-ment. The dissolution tests improved quality and cycletimes. The disadvantages of these equipments can beexplained as loss of money and some problems in stan-dardization procedures.Furthermore, long dissolution procedures may causesome problems to drug manufacturers such as the storageand distribution of the batch. Production and distributiontime may decrease by performing accelerated studies. Anaccelerated dissolution method could be helpful for a fastassessment of the formulation and processing variables(Zackrisson et al. 1995). The accelerated dissolutionmethod is desirable for the quality control, especially in thepreparation of specifications for all product batches. Theaccelerated dissolution test is important for effective con-trol of the production and reducing the spending time fordissolution tests. On the other hand, to get a rapid qualityfeed-back to the operators in production is very importantas well. It is noticed that quality of the megabrands hasbeen continuously increasing with the quality feed-back although the volumes and number of operators involvedhave increased dramatically.Recently, it was reported theoretically that the long-termdissolution studies could be completed in 1–2 h whendissolution rate was accelerated by increasing the temper-ature, increasing the rotation rate of the paddle and thebasket and changing the pH of the dissolution medium etc.(Ertan et al. 2000). It was declared that the dissolution ratesof drugs also increase with the agitation of dissolution(Breier et al. 2005; Costa and Sousa Lobo 2001; Qiu et al. 2003; Wu et al. 2004), addition of alcohol (Billich et al. 2004; Faergemann et al. 2005) organic solvents (Dash and Cudworth II 2001; Souto et al. 2004) and surface-active agents (Lee et al. 2005; Qiu et al. 2003) to the dissolution medium.The first aim of this study was to accelerate the disso-lution of the sustained release Teokap  SR 200 mg pelletsusing both elevated temperature and high rpm rates in USPXXIII rotating paddle method in order to decrease thedissolution time significantly. The second aim was toestablish correlations between (The internet drug index forprescription drugs 1995) in vitro dissolution profiles of sustained and accelerated releases and also between(Harrison et al. 1993) these in vitro release profiles and invivo plasma profiles of the rabbits which were adminis-tered the same dosage form. By performing thesecorrelations, it was aimed to predict both the results of invitro sustained release and in vivo release profiles. 2 Materials and methods 2.1 MaterialsTeokap  SR 200 mg pellets (Nobel Co, Istanbul, Turkey),Theophylline (Dolder Ltd., Switzerland). The otherchemicals used were all in pharmaceutical grade.2.2 Methods 2.2.1 In vitro routine (sustained) dissolution studies(long-time release) In vitro dissolution studies of Teokap  SR 200 mg pelletswere carried out with the rotating paddle method (appara-tus 2 of USP XXIII, Sotax AT 7). 900 mL phosphate buffersolution (PBS) was used as a dissolution medium at pH 6.2.The experiment was maintained at 37  ±  0.5  C and stirringrate of paddle was fixed to 50 rpm. 1 mL sample waswithdrawn from dissolution medium at every hour with theaid of an injector fitted with 0.45- l m filter paper, a Milli-pore HA. An equal volume of buffer was returned to thesystem after each withdrawal. All experiments were repe-ated 12 times. Sampling was performed over 12 h at pre-determined time intervals. The amount of theophyllinereleased from each sample was measured by UV detectionat 272 nm (HP 8453, Germany). Finally, the amount of drug release was plotted versus time. 2.2.2 In vitro accelerated dissolution studies(short-time release) USP XXIII rotating paddle method (apparatus 2 of USPXXIII, Aymes, Istanbul, Turkey) was also used for in vitroaccelerated dissolution studies and the vessel of the dis-solution was heated by an external water bath with athermostat.The preliminary dissolution studies were performed at 45,60, 80, 85, 90 and 95  C between 250 and 500 rpm to deter-mine the appropriate temperature of dissolution medium andstirring rate. Sampling interval was 15 min for the pre-liminary studies. According to the best results of the pre-liminary studies, the ideal temperature was selected as90  ±  1  Candtheidealstirringrateas250 rpm.1 mLsamplewas withdrawn fromdissolution mediumatevery 4 min withthe aid ofaninjectorfittedwith a Millipore HA0.45 l m filterpaper. An equal volume of distilled water was returned to thesystemaftereachwithdrawal.Thesampleswereimmediatelyanalysed spectrophotometrically at 272 nm. 244 Eur J Drug Metab Pharmacokinet (2011) 36:243–248  1 3  2.2.3 In vivo experiments The in vivo data for Teokap  SR 200 mg pellets used inthis study were abstracted from reports developed in ourlaboratory by Karasulu et al. (2006). Six male New Zea-land rabbits weighing 2–3.5 kg were used throughout thestudy. The study was approved by the Animal EthicalCommittee in University of Ege, Faculty of Medicine,(Protocol no: 24). Rabbits were kept in well-ventilatedcages and fed with dry food and water ad libitum. Rabbitswere allowed 7 days for acclimatization before they wereincluded in the experiment.At time zero, rabbits received a single dose of Teokap  SR 200 mg pellet by oral route. Blood samples werewithdrawn by a 22G cannula inserted in the marginal veinof an ear before and at 1, 2, 3, 4, 5, 6, 8, 10, 24, 27, 30, 33,and 48 h after theophylline administration. Blood wasimmediately centrifuged for 10 min at 4,000 rpm and theplasma was stored at  - 20  C until analysis. 2.2.4 Analysis of theophylline levels in plasma Reversed-phase high performance liquid chromatography(HPLC) was used for the determination of theophylline inplasma using caffeine as an internal standard. The appa-ratus consisted of Hewlett-Packard (HP) 1090 pump con-nected to a HP 1090 UV/VIS detector and a HP 1090 autoinjector. A reversed-phase column (l C18, 5  l m,4.6  9  150 mm) was used at room temperature. The mobilephase consisted of a 5:95 mixture of acetonitrile and0.023 M sodium phosphate monobasic buffer, flowing at arate of 1 mL/min. The wavelength for determination was273 nm.Prior to analysis, the frozen samples were thawed out atroom temperature and any precipitants were removed. A250- l L aliquot was placed in a centrifuge tube and a2.5 mL of a mixture of 97% chloroform containing 3%isopropyl alcohol was added. The mixture was vigorouslymixed for 5 min and then centrifuged at 1,500 rpm for15 min. Organic phase was removed and placed in anothertube. Then 2.5 mL of 97% chloroform containing 3%isopropyl alcohol was placed in an aliquot, and the mixturewas vigorously mixed for 5 min and then centrifuged at1,500 rpm for 15 min. The organic phase was removed,placed in a tube and evaporated by a nitrogen tube. Theresidue was dissolved in 500  l L mobile phase. Finally, a25  l L aliquot was introduced into HPLC. 2.2.5 Stability studies on pure theophylline The dissolution studies of pure theophylline were repeatedat both 37  ±  0.5  C and 90  ±  1  C using USP rotatingpaddle method in 900 mL distilled water at 250 rpm. 1 mLsample was withdrawn from dissolution medium at every4 min with the aid of an injector fitted with a Millipore HA0.45  l m filter paper. An equal volume of distilled waterwas returned to the system after each withdrawal. Thesamples were immediately analysed for drug concentrationspectrophotometrically at 272 nm. 2.2.6 Evaluation of kinetics of the dissolution results Hixson–Crowell (cube-root law), Higuchi (square root of time), zero-order, Langenbucher, modified Langenbucher,Hopfenberg, RRSBW (Rosin–Rammer–Sperling–Bennet–Weilbull), first-order and (Bt) a kinetic models were usedfor the evaluation of the sustained and accelerated disso-lution results. The release rate constants ( k  ), correlationcoefficients ( r  ) and determination coefficients ( r  2 ) werecalculated by a computer program (Ertan et al. 2000). 2.2.7 Correlations between accelerated (short-time)and sustained (real time) release rates The kinetic linear correlation method which was indepen-dent of time was established between the kinetics of accelerated and sustained in vitro percentage release ratesusing a computer program (Ege et al. 2001; Ertan et al.2000). The best regression equation was found betweenthese kinetic values of release rates using a linear regres-sion analysis and the figure of the best correlation wasplotted. Afterwards, the kinetics of percentage releasevalues obtained from accelerated dissolution was applied tothe best regression equation. Then, calculated percentagerelease rates of sustained dissolution were compared withthe observed release rates of sustained dissolution. 2.2.8 Calculation of the sustained release time from accelerated release time The equation below shows the equivalent sustained releasetime according to accelerated time. t  s  ¼ t  a   f   ð 1 Þ where,  t  s  was sustained release time (1–12 h),  t  a  wasaccelerated release time (4–48 min) and  f   was calculationcoefficient.For example, if sustained release time ( t  s ) was 1 h(60 min) and accelerated release time ( t  a ) was 4 min, cal-culation coefficient (  f  ) could be calculated with Eq. 2. as15. If the calculation coefficient was put on the Eq. 1,sustained release time could be calculated easily.  f   ¼ t  s t  a ð 2 Þ Eur J Drug Metab Pharmacokinet (2011) 36:243–248 245  1 3  2.2.9 In vitro–in vivo correlations The sustained, accelerated and calculated-sustained in vitrodissolution results obtained from correlation procedureswere applied separately to in vitro–in vivo linear correla-tion procedure using a computer program (Ege et al. 2001). 2.2.10 Similarity tests The dissolution profile comparison was carried out using  f  2 similarity factor. The similarity factor is a logarithmicreciprocal square-root transformation of the sum of squarederror and is a measurement of the similarity in the per-centage of dissolution between the two curves:  f  2  ¼ 50log 1 þ 1 n X nt  ¼ 1  R t   T  t  ð Þ 2 " #  0 : 5  100 8<:9=; ð 3 Þ where  n  was the number of time points,  R t   and  T  t   were thecumulative percentages dissolved and collected at each of the selected  n  time points of the dissolution system (Eq. 3).Two dissolution profiles were considered similar whenthe  f  2  value was greater than or equal to 50. When 85% ormore of both test and reference products were dissolved inno more than 15 min, the profile comparison with a  f  2  testwas considered as unnecessary (Moore and Flanner 1996). 2.2.11 Statistical analysis Data were expressed as mean  ±  SD and the differences inthe results of in vitro and in vivo studies were evaluatedusing one-way ANOVA followed by Duncan’s test. Dif-ferences were considered statistically significant when P \ 0.05. 3 Results and discussion Thedissolutionprofilesofthepreliminarystudiesconductedat 250 rpm at elevated temperatures were shownin Fig. 1.Itwas clearly seen from the figure that when the temperaturewas increased, the drug release was significantly increased.The reason of the fast release at elevated temperatures maybe the softening of the gums or resins used in the productionofthepellets.Theresultsofthedissolutionsat95  Cwerenotevaluated due to the high evaporation of the dissolutionmedium (water). Slow releases were observed in studiesperformed at \ 90  C (Fig. 1).The results of the release conducted at 500 rpm couldnot be evaluated because of the spill-over of the mediumout of the vessel. According to the results of the pre-liminary studies, 250 rpm and 90  C parameters werechosen for the correlation studies (Fig. 2). The acceleratedand sustained in vitro dissolution results were shown inFig. 2. While the sustained dissolution was completed in12 h, accelerated dissolution was completed in 48 min.The samples of sustained dissolution were taken at everyhour and the samples of accelerated dissolution were takenat every 4 min. Thus, 12 samples were taken both forsustained and accelerated release studies. Sustained disso-lution time may be calculated by multiplying the acceler-ated time by 15. For example when 4 min were multipliedwith the calculation coefficient (  f  ) which was 15, sustaineddissolution time (hours) could be obtained as 60 min (1 h).The results of our accelerated and sustained dissolutionstudies were correlated using both linear and kinetic linearcorrelation methods. Briefly, kinetic linear correlation wastheonlycorrelationshowingthekineticresultsofthepercentdissolutions of two dissolution methods (Ertan et al. 2000)anditwasfoundtobevery usefultoincreasetheaccuracyof the linear correlation (Karasulu et al. 2003). Thus, correla-tion figures were plotted by correlating the sustained releaseand accelerated release values to obtain the correlationequations (Figs. 3, 4). The best correlation was obtained by (Bt) a and Langenbucher kinetics for accelerated and sus-taineddissolutionprofiles,respectively. r  2 ofthiscorrelation 0102030405060708090100 020406080100120140160 Time (minute)    R  e   l  e  a  s  e   d   % 90°C85°C80°C60°C45°C Fig. 1  Dissolution profiles of the preliminary studies at 250 rpm.Values are mean  ±  SD; in some cases the error bars are smaller thanthe symbols ( n  =  3) 01020304050607080901000 2 4 6 8 10 12 Time (h)    D   i  s  s  o   l  v  e   d   (   %   ) 0204060801000 10 20 30 40 50 Time (minute)    D   i  s  s  o   l  v  e   d   (   %   ) Fig. 2  The accelerated and sustained in vitro dissolution profiles.( n  =  12)246 Eur J Drug Metab Pharmacokinet (2011) 36:243–248  1 3  equation was calculated as 0.9938. Moreover, the calculatedvalues obtained using this correlation equation were com-pared with the values obtained from sustained dissolution(Fig. 4). This comparison showed that there was only amaximum3.6%differencebetweencalculatedandobservedsustained drug release rates (  f  2  =  82.73). As to the linearcorrelation,the r  2 was0.9129andtherewasabig(maximum15.9%,  f  2  =  51.8) difference between calculated andobserved drug release rates (Fig. 5). There were only tworeports related to the correlations of the accelerated and realdissolutions of microspheres published by the Deluca’sresearch groups (D’Souza et al. 2005; Shameed et al. 1999). These studies reported that the correlations between thepercentreleasesofrealtimedissolutionsasdaysat37  Candthe correlations with accelerated times as hours at 50 and60  C for peptide release from PLGA microspheres (Sham-eed et al. 1999).To understand whether theophylline was degraded instress conditions, comparative stability studies were per-formed.Dissolutionresultsobtainedat37  Cwerefoundtobenearly the same with the results obtained at 90  C ( P [ 0.05).It was almost certain that, there was no degradation of the-ophylline and total drug release rate was nearly 100%.In vivo profile of the Teokap  SR 200 mg pellets wereshown in Fig. 6. The linear in vitro–in vivo correlationresults obtained between accelerated, calculated-sustainedand sustained percent release rates with the percentabsorption rates were shown in Figs. 7, 8. It was clearly seen that the correlation plots and in vivo plots were verysimilar and the similarity factors (  f  2 ) were 73.81, 77.11,and 73.89 for the accelerated, calculated-sustained, andsustained release rates, respectively. It was surprising thatthe similarity factors and in vitro–in vivo correlationgraphics of accelerated and sustained release products werenearly similar (Fig. 7). The maximum error was below 5%for three plots. Finally, the in vivo results could be esti-mated both from sustained and accelerated dissolutionresults successfully. 4 Conclusions Accelerated dissolution seems to be an acceptable andapplicable method for sustained release preparations. Theresults of the accelerated dissolution may be used for theestimation of the percent of the sustained dissolutionresults and for the prediction of in vitro-in vivo y = 1,4105x - 30,315R 2  = 0,9131 020406080100120 40 50 60 70 80 90 100 Accelarated (%)    S  u  s   t  a   i  n  e   d   (   %   ) Fig. 3  The linear correlation plot of the accelerated and sustained invitro dissolution profiles y = 0,5202x + 0,9693R 2  = 0,9938 00,10,20,30,40,50,60,70,80,9 -2-1,5-1-0,50 (Bt) a Accelerated    L  a  n  g  e  n   b  u  c   h  e  r   S  u  s   t  a   i  n  e   d Fig. 4  The kinetic linear correlation plot of the accelerated andsustained in vitro dissolution profiles Fig. 5  Comparison of sustained percent values with the calculatedpercent results obtained by linear and kinetic linear correlations 0481216200 6 12 18 24 30 36 42 48 Time (h)    P   l  a  s  m  a   l  e  v  e   l   (  µ  g   /  m   l   ) Fig. 6  Plasma levels of Teokap  SR 200 mg pellets after adminis-tration to rabbits ( n  =  6)Eur J Drug Metab Pharmacokinet (2011) 36:243–248 247  1 3
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