Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 598Research Article CODEN: IJPNL6IN-VITRO AND IN-VIVO CORRELATION OF IMMEDIATE RELEASE ACYCLOVIR TABLET USINGWAGNER NELSON METHODSomnath Sakore* and Bhaswat ChakraborthyCadila Pharmaceuticals Ltd. Dholka, Ahmedabad, Gujrat. India*Corresponding author e-mail: email@example.comABSTRACTThe purpose of this study was to establish In-Vitro and In-Vivo correlation of immediate release Acyclovir tablets of800 mg. In vitro and in vivo studies are done on the test product as Acyclovir Tablet USP 800 mg (containingAcyclovir 800 mg) of Cadila Pharmaceuticals Ltd., India versus Zovirax®Tablet 800 mg (containing Acyclovir 800mg) of GlaxoSmithKline, USA. In vivo studies are done in 36 healthy, adult, human subjects under fastingcondition.In vitro dissolution study was done using USP apparatus II at 50 rpm in 0.1N HCL for 45 minutes. The invitro–in vivo correlation of Acyclovir shows R-squared value 0.9794 in excel work sheet, which depicts a successfulcorrelation between in vitro and in vivo Characteristic of the drug. In addition, %PE AUC and %PE Cmax was found tobe –4.604 and -11.19 respectively for each formulation. The present study shows a good correlation between in vivoand in vitro PK profiles of the formulation used as the test drug in the study.Keywords: Acyclovir 800 mg tablets, In Vitro Dissolution, In Vivo absorption, IVIVC.INTRODUCTIONIn vitro in vivo correlations play a key role in thedrug development and optimization of formulation isan integral part of manufacturing and marketingwhich is certainly a time consuming and expensiveprocess. In vitro-in vivo correlation (IVIVC)demonstrates the direct relationships between invitro dissolution / release and in vivo absorptionprofiles. The in vitro property generally is the rate oramount of drug dissolution or release, while in vivoresponse is plasma drug concentration or amount ofdrug absorbed. The in vitro release data of a dosage form containingthe active substance serve as characteristic in vitroproperty, while the in vivo performance is generallyrepresented by the time course of the plasmaconcentration of the active substance. These In vitro& In vivo data are then treated scientifically todetermine correlations. For oral dosage forms, the invitro release is usually measured and considered asdissolution rate. The relationship between the in vitroand in vivo characteristics can be expressedmathematically by a linear or nonlinear correlation.However, the plasma concentration cannot be directlycorrelated to the in vitro release rate; it has to beconverted to the in vivo release or absorption data,either by pharmacokinetic compartment modelanalysis or by linear system analysis. DifferentIVIVC model are used as a tool for formulationdevelopment and evaluation of immediate andextended release dosage forms for setting adissolution specification and as a surrogate forbioequivalence testing. Practically, the purpose ofIVIVC is to use drug dissolution results from two ormore products to predict similarity or dissimilarity ofexpected plasma drug concentration (profiles).Before one considers relating in vitro results to invivo, one has to establish as to how one will establishsimilarity or dissimilarity of in vivo response i.e.plasma drug concentration profiles.As a result, considerable effort goes into theirdevelopment and the main outcome is “the ability topredict, accurately and precisely, expectedbioavailability characteristics for an extended release(ER) drug product from dissolution profilecharacteristics. [2,3]The methodology of establishingsimilarity or dissimilarity of plasma drugconcentrations profile is known as bioequivalencetesting. There are very well established guidancesInternational Journal of PharmacyJournal Homepage: http://www.pharmascholars.com
Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 599and standards available for establishingbioequivalence between drug profiles and products [4,5]. There are four levels of IVIVC that have beendescribed in the FDA guidance, which include levelsA, B, C, and multiple C [6,7].The concept of correlation level is based upon theability of the correlation to reflect the completeplasma drug level-time profile which will result fromadministration of the given dosage form. An IVIVCLevel A correlates the entire in vitro and in vivoprofiles has regulatory relevance. This level ofcorrelation is the highest category of correlation andrepresents a point-to-point relationship between invitro dissolution rate and in vivo input rate of thedrug from the dosage form .The objective of IVIVC evaluation is to estimate themagnitude of the error in predicting the in vivobioavailability results from in vitro dissolution data.This objective should guide the choice andinterpretation of evaluation methods. Any appropriateapproach related to this objective may be used forevaluation of predictabilityPrediction errors areestimated for Cmax and AUC to determine the validityof the correlation. Various approaches of are used toestimate the magnitude of the error in predicting thein vivo bioavailability results from in vitrodissolution data [8,9].It can be calculated by Prediction error that is theerror in prediction of in vivo property from in vitroproperty of drug product. Depending on the intendedapplication of an IVIVC and the therapeutic index ofthe drug, evaluation of prediction error internallyand/or externally may be appropriate. Acyclovir is a synthetic purine nucleoside analoguewith in vitro and in vivo inhibitory activity againstherpes simplex virus types 1 (HSV-1), 2 (HSV-2),and varicella-zoster virus (VZV). The inhibitoryactivity of Acyclovir is highly selective due to itsaffinity for the enzyme thymidine kinase (TK)encoded by HSV and VZV. This viral enzymeconverts Acyclovir into Acyclovir monophosphate, anucleotide analogue. The monophosphate is furtherconverted into diphosphate by cellular guanylatekinase and into triphosphate by a number of cellularenzymes. In vitro, Acyclovir triphosphate stopsreplication of herpes viral DNA. The greater antiviralactivity of Acyclovir against HSV compared to VZVis due to its more efficient phosphorylation by theviral TK . Acyclovir pharmacokinetics has beenextensively investigated during the various phases ofclinical development. Most of the administered drugis eliminated from the body unchanged, via thekidneys by glomerular filtration and tubularsecretion. After intravenous dosing of patients withnormal renal function, 8 to 14% of the dose isrecovered in the urine as the metabolite 9-carboxymethoxymethylguanine.After oral administration, the bioavailability ofAcyclovir was approximately 20%. The plasmaelimination half life of acyclovir is 2.5 to 3.3 hr andprotein binding is 9%-33% .MATERIAL AND METHODSMaterials: All materials used for analysis were ofanalytical grade. The test product for In Vivo studyused Acyclovir Tablet USP 800 mg of CadilaPharmaceuticals Ltd., India reference product usedwas Zovirax®Tablet 800 mg of GlaxoSmithKline.In vitro Evaluation:The dissolution of Acyclovirtablets was carried out using USP Type II Dissolutionapparatus for 45 minutes. The dissolution media usedwas 900 ml 0.1 N HCL at 37ºC ± 0.5ºC and rotatedat a speed of 50 rpm. Analysis of the withdrawnsamples was carried out using UV spectrophotometerat the maximum 254 nm against 0.1 N Hydrochloricacid as a blank.Dissolution procedure: Parameters of the instrumentwere set as mentioned above and the medium wasdegas prior to use. 900 ml of Dissolution media wastransferred into each of the six dissolution vesselsand apparatus was operated as per requirement. Onetablet was dropped into each of six different vessels& dissolution apparatus was started immediately.After 45 min solution was withdrawn, & fiiltered thesolution through whatmann filter paper No.1; discardfirst 2-3 ml of the filtrate. One mL of the filteredsample was diluted to 100 ml with dissolutionmedium and mixed. (Use this solution as samplepreparation)Procedure for analysis: Absorbance was measuredfor the standard preparation and sample preparationon a suitable spectrophotometer at the maximum254nm against 0.1 N Hydrochloric acid as a blank.Percentage of Acyclovir dissolved in 45 minutes inindividual tablets was calculated.In vivo Absorption study: A randomized, open label,two-treatment, two-period, two-sequence, two-waycrossover comparative bioavailability study of asingle oral dose of Acyclovir Tablet USP 800 mg(containing Acyclovir 800 mg) of CadilaPharmaceuticals Ltd., India versus Zovirax®Tablet800 mg (containing Acyclovir 800 mg) of
Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 600GlaxoSmithKline, USA in 36) healthy, adult, humansubjects under fasting condition.Screen Procedure: During screening procedureDemography data, standard physical examinationwith Vital signs, Clinical laboratory tests on bloodand urine samples, Electrocardiogram (ECG) andChest X-ray were done.Study Design; A randomized, open-label, two-treatment, two-period, two-sequence, two-waycrossover, comparative bioavailability study, duringwhich subjects were administered a single dose oftest or reference product under fasting condition withat least 7 days washout period between eachadministration.Sample Size: Minimum of 36 + (04 standby) healthy,adult, subjects was enrolled to allow dosing in bothperiods.Administration: Single a single oral dose of test(Acyclovir Tablet USP 800 mg) or reference product( Zovirax®Tablet 800 mg) products wereadministered along with 240 mL of drinking waterafter an overnight fasting of at least 10 hours in eachPeriod.Bio-analysis of Plasma sample: Samples wereanalyzed for the quantification of Acyclovir inplasma using Liquid Chromatography with MassSpectrometry (LCMS) procedures. Data analysis wascarried out using Win-Nonlin software to get theCmax, AUC 0-t, AUC 0- and kel.Statistical Analysis: Statistical analysis wasperformed on the pharmacokinetic parameters dataobtain from subjects (Completing both the periods)using the SAS Statistical Software version 9.1.3(SAS Institute INDIA Pvt. Ltd).RESULTS AND DISCUSSIONIn Vitro dissolution: The mean percent dissolved iscalculated on the basis of time and it showed thatwithin the first 15 min, 91.0% of Acyclovir drug andwithin the first 20 min, 89 % of Zovirax haddissolved. It was observed that 96.0 % of acyclovirand Zovirax dissolved within 45 min.Percent dissolved versus in vitro dissolution time (inmin) when plotted generates a dissolution profilecurve as shown below in table 1. The amount of drugdissolved over a period of time for test & referenceformulation is given in the figure 1.In vivo absorption: The fraction of drug absorbedwas calculated by Wagner Nelson method usingfollowing equation.0T0ATACdtKCdtKCT)X()X(Above equation relates the cumulative amount ofdrug absorbed after a certain time to the amount ofdrug absorbed. The fraction of drug absorbedcalculated using Wagner Nelson method for test &reference formulation is summarized in the Table 2.The following figure 2 and figure 3 summarizes thefraction of drug absorbed of the Acyclovir moleculeTest and reference respectively at given bloodsampling time points for test drug. It shows that at 1hr almost 90% of the drug was absorbed for both test& reference formulation.Determination of intensity factor : Since the invitro dissolution data was available only for one hourand hence a time scaling factor (i.e., intensity factor)was calculated using Ratio of time of 90 % absorbedand 90 % dissolved & Ratio of time of 50 %absorbed and 50 % dissolved.In vivo observed data: With the help of timescaling factor the in vitro data was compared with Invivo data. Table 3 summarizes fraction of drugabsorbed & percent drug absorbed vs. time data ofZovirax 800 mg tablets obtained using WagnerNelson Method.Development and evaluation of Level A IVIVCModel: The in vitro data was taken based on minutesand the in vivo absorption based on hours. It wasapparent that these two processes occurred overdifferent time scale. To make the time differencebetween in vitro and in vivo data, uniform, a timescaling factor (intensity factor) was calculated. Theintensity factor, I, obtained was 6.66. Thus by usingthis factor we have converted the in vitro time pointsto hours in order to match with the in vivo timepoints. Time scaled normalization of in vitro and invivo data with intensity factor is given in figure 4.Correlation calculation: The correlation graph(Figure 5) was plotted as % absorbed verses %dissolved and there exists an extremely goodcorrelation of formulation between in vitro and invivo data.
Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 601Prediction Error: IVIVC model predictability wasdetermined by the calculating percent Cmax andAUC prediction errors. Prediction error as %PE,AUC and %PE Cmax was found to be – 4.604 and -11.19 respectively for each formulation.An IVIVC should be evaluated to demonstrate thatpredictability of in vivo performance of a drugproduct from its in vitro dissolution characteristics ismaintained over a range of in vitro dissolution ratesand manufacturing process.If in vitro dissolution is shown to be independent ofdissolution conditions such as PH, surfactants,osmotic pressure, agitation intensity, a set ofdissolution data obtain from one formulation iscorrelated with that of in vivo absorption data.To demonstrate a correlation, fraction absorbed invivo should be plotted against fraction dissolved invitro. If this relationship becomes linear with a slopeof 1, then curves are super imposable, and there is a1:1 relationship which is defined as point-to-point orLevel A correlation.Regression analysis was also performed on Excelspread sheet with the same in vitro and in vivo data.R-squared value obtained from graph was 0.9794.According to FDA guidelines and expertise, statisticfrom Level A analysis is r, the correlation coefficient.It’s square, i.e. R-squared, ranges from 0 to 1 and is ameasure of strength of relationship between fractionsabsorbed against fraction dissolved. Often, resultswith sufficient large R-squared (e.g., greater than 0.9)yielded “a successful correlation”.The in vitro–in vivo correlation of Acyclovir showsR-squared value 0.9794 in excel work sheet, whichdepicts a successful correlation between in vitro andin vivo Characteristic of the drug.In a linear correlation, in vitro dissolution and in vivoinput curves may be directly super imposable or maybe made to be super imposable by the use ofappropriate scaling factor (time corrections). Timescaling factor should be the same for all formulationsand different time scales for each formulationindicate absence of an IVIVC.Percent prediction error (PE%) of 10% or less forCmax and AUC establishes the predictability of theIVIVC. In addition, the PE% for each formulationshould not exceed 15%. Here %PEAUC and %PECmaxwas found to be –4.604 and -11.19 respectively.CONCLUSIONThe present study shows a good correlation betweenin vivo and in vitro PK profiles of the formulationused as the test drug in the study. The in vitro–in vivocorrelation of Acyclovir shows R-squared value0.9794 in excel work sheet, which depicts asuccessful correlation between in vitro and in vivoCharacteristic of the drug. In addition, %PE AUC and%PE Cmax was found to be –4.604 and -11.19respectively for each formulation. The concept ofcorrelation level is based upon the ability of thecorrelation to reflect the complete plasma drug level-time profile which will result from administration ofthe given dosage form. An IVIVC Level A correlatesthe entire in vitro and in vivo profiles has regulatoryrelevance. This level of correlation is the highestcategory of correlation and represents a point-to-point relationship between in vitro dissolution rateand in vivo input rate of the drug from the dosageform.TABLE 1: IN VITRO DISSOLUTION DATA OF ACYCLOVIR 800 MG TABLETS (TEST) AND ZOVIRAX 800MG TABLETS (REFERENCE)0.1 N HClTime (min)Acyclovir 800 mg (test)TabletsZovirax 800mg (Reference)tablets0 0 010 86 7415 91 8120 94 8930 95 9445 96 96
Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 602TABLE 2: FRACTION OF DRUG ABSORBED FOR TEST & REFERENCEFraction of Drug AbsorbedTime (hr) Test Reference0 0 00.333 0.208509 0.2835050.667 0.600566 0.8099271 0.902686 0.996561.333 1.086002 1.1958281.667 1.187093 1.3217142 1.13907 1.3277622.5 1.173928 1.3307323 1.257711 1.2811414 1.137136 1.1758275 1.067112 1.0889266 0.956591 0.9815828 0.886052 0.90132110 0.851755 0.87841812 0.835008 0.85113714 0.841072 0.84437416 0.852433 0.85067324 0.876586 0.875232TABLE 3: Fraction of Drug AbsorbedTime (hr) Fraction drug absorbed % Drug absorbed0 0 00.333 0.283505 28.35050.667 0.809927 80.99271 0.99656 99.6561.333 1.195828 119.58281.667 1.321714 132.17142 1.327762 132.7762FIGURE 1: FRACTION OF DRUG DISSOLVED VS TIME GRAPH FOR ACYCLOVIR 800 MG TABLETS
Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848www.pharmascholars.com 603% Drug absorbed vs Time Graph0204060801000 0.5 1 1.5 2 2.5 3Time%Absorbed% FFIGURE 2: FRACTION OF DRUG ABSORBED VS TIME GRAPH FOR ACYCLOVIR 800 MG TABLETS(TEST)% Drug absorbed vs Time Graph0204060801000 0.5 1 1.5 2 2.5 3Time%Absorbed% FFIGURE 3: FRACTION OF DRUG ABSORBED VS TIME GRAPH FOR ZOVIRAX 800 MG TABLETS(REFERENCE)Time scale normalization of IVIVC0204060801000 0.5 1 1.5 2 2.5Time (hr)%DrugAbsorbedorDissolvedIn vivo observed In vitro observed In vitro predictedFIGURE 4: TIME SCALED NORMALIZATION OF IN VITRO AND IN VIVO DATA WITH INTENSITYFACTORFIGURE 5: THE LINEAR REGRESSION PLOT OF % ABSORBED AND % DISSOLVED
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