pharmacokinetics and in vitro and in vivo correlation of...

578Current Trends in Biotechnology and PharmacyVol. 4 (1) 578-588 January 2010. ISSN 0973-8916

Pharmacokinetics of Microspheres in Rats

Pharmacokinetics and In Vitro and In Vivo Correlation of NN-dimethylaminocurcumin (NNDMAC) Loaded Polycaprolactone

Microspheres in Rats

Jithan Aukunuru* and Vidyavathi SankavarapuVaagdevi College of Pharmacy, Ramnagar, Hanamkonda-506001, India

*For correspondence - [email protected]

AbstractN N - d i m e t h y l a m i n o c u r c u m i n

(NNDMAC), a novel curcumin analogue, hasdemonstrated significant hepatoprotective activityafter oral administration. The objective of thisinvestigation was to determine thepharmacokinetics of NNDMAC after theadministration of its microsphere formulation.Additionally, it was aimed to determine the invitro in vivo correlation (IVIVC) with themicrosphere formulation. NNDMACbiodegradable microspheres were prepared usingsolvent evaporation technique by takingpolycaprolactone as the polymer. A suitablerelease study based on the volume of distributionof NNDMAC was selected. In vitro release ofthe drug was determined. For in vivo studies,the microsphere formulation was injected by IProute. Pharmacokinetic properties ofmicrosphere-encapsulated NNDMAC weredetermined and a comparison with i.v. solutionform of NNDMAC was made. Pharmacokineticanalysis was performed using KINETICA andnon-compartmental parameters were determined.Concentrations of the drug in plasma weredetermined by HPLC. IVIVC was establishedaccording to Drewe and Guitard (degree A). Invivo drug release into the systemic circulationwas determined using Wagner-Nelson method.Results indicated that, when NNDMAC

formulations were administered by IP route,mean residence time (MRT) and the area underthe curve (AUC) were significantly higher(P<0.05) and maximum concentration (C

max) of

NNDMAC was lower than that of the free form.T

max was same with both the administrations. The

results obtained in the present study showed thatmicrosphere encapsulated NNDMAC providesprolonged and effective plasma concentrationafter IP administration. The microsphereformulation sustained the release of the activefor 9 days in vitro as well as in vivo in this ratmodel. Good IVIVC was achieved when therelease medium selected was based on thevolume of distribution of the drug.

Key words: NNDMAC, microspheres, sutainedrelease, pharmacokinetics, IVIVC

IntroductionCurcumin and its analogues have been

the subject of several pharmacological studies.Most of these studies were conducted with anintention to unravel their therapeutic potential andexploit the chemical structure for clinical use.Several bioactivities for curcumin and itssynthetic analogues including their use in thecancers, tumors, alzheimers disease,inflammation, malaria, bacterial infections,neurological disorders, etc. have been reported(1). The analogues of curcumin were mainly

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synthesized to increase the poor bioavailabilityof curcumin, its stability as well as solve theproblems associated with its synthesis (2).Curcumin is of particular interest for a variety ofpharmacological applications. Most studiesinvolving its use do not obtain pure samples.Extractions from the natural product, turmeric,are the most common sources of curcumin.Purification is accomplished using extensivechromatographic extraction and is a very laborintensive endeavor that does not provide a verypure material suitable for pharmacological use.As an alternative to the extraction, synthesis ofcurcumin has been attempted. The synthesis ofcurcumin involves the use of relatively expensivecomponents that require intensive removal ofimpurities that require treatment for their disposal(3). Other methods for the synthetic productionof curcumin from the starting products vanillinand 2,4-pentanedione involve the use of tri-butylborate, boron oxide, and butylamine in a hydrolysisreaction with N,N-dimethylacetamide as a solventand recrystallization using acetonitrile. In thissecond approach, there are problems associatedwith the recovery, waste disposal and toxicity.Because of these problems with the synthesis ofcurcumin, synthesis of curcumin analogues wasalso accomplished as an alternative. Analoguesof curcumin were synthesized using a variety ofapproaches (2). After synthesis, thesecompounds were screened for a variety ofactivities. Our group has synthesized several of1,7-diaryl-1,6-heptadiene-3,5-diones, and inparticular curcumin and its analogues.NNDMAC is one such analogue whichpossessed hepatoprotective, antidepressant andanti-inflammatory activities (4,5,6). Its chemicalstructure is shown in the Figure 1. We previouslydeveloped a biodegradable microsphereformulation, a parenteral depot system, forNNDMAC (6). The purpose of this study wasto investigate the pharmacokinetics of

NNDMAC microspheres in a rat model and alsodetermine the IVIVC with the formulation. Thedata from this study adds knowledge to our questto improvise therapy against several diseaseswhere in curcumin analogues are useful. Thereare several advantages with biodegradableparenteral depot system (7). With these types ofbiodegradable parenteral depot systems, there isa possibility of patenting clinically successfuldrugs after incorporating them into newer drugdelivery systems without infringing the originaldrug or formulation patents. Further, thedevelopment in the concepts and techniques ofcontrolled release drug delivery systems coupledwith the increasing expense bringing new drugentities to market, has encouraged thedevelopment of this new drug delivery system.It is also easy to deliver the novel, geneticallyengineered pharmaceuticals, i.e. peptides andproteins to their site of action without incurringsignificant immunogenicity or biologicalinactivation with this new drug delivery system.The basic rationale for controlled drug deliveryis to alter the pharmacokinetics andpharmacodynamics of pharmacologically activemoieties by using novel drug delivery systems orby modifying the molecular structure andphysiological parameter inherent in selected routeof administration. Thus, parenteral biodegradabledepot microspheres is the attractive dosage formto be tested to enhance the pharmacokineticproperties as well the pharmacodynamic activitywith selected curcumin analogues.

Current Trends in Biotechnology and PharmacyVol. 4 (1) 578-588 January 2010. ISSN 0973-8916

Jithan Aukunuru and Vidyavathi Sankavarapu

N(CH3)2

(CH3)2 N

Fig. 1: Chemical Structure of NNDMAC

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Materials and MethodsThe required aromatic aldehyde p N N

dimethylamino benzaldehyde was obtained fromMerck. Benzene was purchased from Universallaboratories. Column silica gel was purchasedfrom Finar chemicals limited. HPLC grademethanol and acetonitrile were purchased fromMerk specialties. Methanol, ethyl acetate, andn-Hexane were purchased from Finar reagents.Acetone, benzene and toluene were purchasedfrom Universal laboratories. Polycaprolactonewas purchased from Sigma-aldrich, Germany.Ethanol LR, ethyl acetate, tween 80 anddichloromethane were purchased from Finarreagents. Benzene purchased from Universallaboratories was used. To conduct in vitro drugrelease studies, magnetic stirrer and cyclo mixerfrom Remi Equipments Pvt. Limited were used.A SL 164 Elico Double Beam UV-VisSpectrophotometer was used to analyze thesamples. HPLC from Cyberlabs was used foranalysis of all the plasma and serum samples.Male Wister rats (100 to 150 gms, 5 to 6 weeksold) purchased from animal center of Mahaveeraenterprises, Hyderabad were used in this study.A pharmacokinetic software KINETICA wasused in the data analysis and the determinationof pharmacokinetic parameters.

Synthesis and Characterization ofNNDMAC

A mixture of acetyl acetone (0.01 mole), pN N dimethyl benzaldehyde (0.02 moles), boricacid (0.01 mole), in dimethyl formamide (10 ml),was taken into a round bottom flask (RBF) andfew drops of diethanolamine and acetic acidmixture was added. The mixture was thenrefluxed in a mantel for 16 hours at 1500Ctemperature. The reaction was monitored byTLC (Thin Layer Chromatography) for theconfirmation of the product. After 16hrs of refluxthe reaction mixture was poured into a 10% acetic

acid solution and stirred for one hour to get asolid mass. Thus obtained mass was filtered andwashed with water. This crude drug was purifiedand separated by column chromatography using60-120 mesh TLC grade silica gel. The columnwas filled with silica gel of mesh size 60 to 120and wet packing method was followed. Thereaction product mixed with silical gel was loadedon top of the column and column was run with amixture of n hexane and ethyl acetate (75:25) of500 ml volume. The pure product wassubsequently eluted by running the column witha mixture of methanol and benzene (50:50). Theelutant was allowed to air dry. It wasrecrystallized by subsequent solubilization inbenzene followed by methanol to get pureproduct. The purity of the compound obtainedwas confirmed using HPLC. A HPLC procedureemploying a C-18, 100 X 4.6 column, SPD-10AUV-Vis detector, LC-10 AD pump and C-R7APlus integrator was used. HPLC grade methanoland water in the ratio of 70:30 was taken as themobile phase. The detection wavelength was 425nm. Further, the structure was confirmed usingNMR.

Fabrication of NNDMAC MicrospheresMicrospheres of NNDMAC using

biodegradable polycaprolactone as the polymerwere fabricated using emulsion-solventevaporation method. Dichloromethane was takenas organic phase in which polymer (400mg) anddrug (200mg) in a ratio of 2:1 were dissolved(20ml). This organic phase was added to theaqueous phase containing tween 80 as surfaceactive agent (1% w/v solution) drop by drop whilethe aqueous phase was kept for stirring on amagnetic stirrer. Stirring was continued tillcomplete evaporation of dichloromethaneoccurred. As the organic phase evaporatesprecipitation of the polymer and drug occurs dueto which drug gets entrapped in the polymer and



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stirring results in size reduction as well asspherical particle formation.

In vitro Drug Release StudiesA dialysis membrane was used for the

release study. The release medium, PBS (7 ml)was taken into the receiver compartment. Releasemedium was designed based on the volume ofdistribution. The volume of distribution of this drugin rats was ~ 7 ml (5). This has been selected soas to obtain good in vitro – in vivo correlation.The donor compartment was immersed into thereceiver compartment so that the edge justtouches the receiver compartment. A 100mg ofthe microparticles were dispersed in 2 ml of PBSand placed in the donor compartment and of thissuspension 1 ml was used in the release studies.The percentage loading of the drug was found to70% and as a reason, the 50 mg of microspheresuspension used in the release studies contained35 mg of the drug. The rpm of the system wasmaintained using magnetic stirrer and bead.Samples (1 ml) were removed from the receptorcompartment and replaced with fresh mediumimmediately. The samples were then analyzedfor the drug. A n=3 was used in the study and thedata is reported as mean ± S.D. The releasestudies were also conducted with the pure drugso as to show the sustained release of the drugfrom the microspheres. This set of release studieswere exactly similar to that conducted using themicrospheres, excepting the use of pure drug inthis case. The amount of the drug taken in therelease studies was also the same (35 mg).Before selecting the wavelength to be used inthe analysis of the compound, a UV spectrum ofthe compound in PBS and methanol wasgenerated. The UV spectrum of the compoundin the release medium was also generated. Basedon the results and the sensitivity, the sampleswere analyzed using a UV-visiblespectrophotometer at 425 nm wavelength.

Pharmacokinetic StudyThe study was conducted in rats after

getting approval from ethical committeeconstituted for this project in Vaagdevi Collegeof Pharmacy, Warangal, AP, India. Male wistarrats (250 g) were purchased from MahaveerEnterprises, Hyderabad. Animals weremaintained in an air-conditioned room at 22 ± 2ºC and relative humidity of 45-55% under a 12 hlight:12 h dark cycle. The animals had free accessto standard food pellets and water was availablead libitum. The experimental protocol wasapproved by the Institutional Animal EthicsCommittee (IAEC) of Vaagdevi College ofPharmacy, Warangal (Registration No: 1047/ac/07/CPCSEA) and constituted in accordance withthe rules and guidelines of the Committee for thePurpose of Control and Supervision onExperiments on Animals (CPSEA), India. Afterquarantine period, rats were divided into twogroups (n=4). One group was administered drugsolution via i.v. route while other group wasinjected with biodegradable microspheresintraperitoneally. A 35 mg of the drug dissolvedin sterile PEG 400 was used in the i.v. solutionadministration. A 400 mg of the NNDMACmicrospheres were dispersed in 4 ml of normalsaline and a 500 l of this suspension containing35 mg of the drug were injected into each rat.Blood samples (0.5 ml) were collected from retroorbital sinus of rat eye under anesthesia atintervals of 0.25, 0.5, 0.75, 1, 3, 6, 12, and 24 hrsin case of i.v. solution. For microparticular systemalong with above time intervals samples werealso collected after 4 and 9 days. The bloodsamples so collected were added to a series ofgraduated micro centrifuge tubes containing 0.3ml of sodium citrate solution (4% w/v in water).All the samples were centrifuged at 3000 rpmfor 10 minutes and plasma was separated intoother micro centrifuge tube by using micro pipetteand stored in deep freeze. The drug was extracted



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from the plasma by adding 500 µl of ethyl acetate,and vortexed on cyclo mixer for 20 min. Theorganic phase was separated and collected intoanother micro centrifuge tube and allowed to airdry by keeping the lid of the tube open for 24hours. These dried tubes were stored in deepfreeze until HPLC analysis was performed.HPLC analysis samples were reconstituted with50 µl of mobile phase (methanol: water, 70:30)and analyzed at 230nm wavelength. Thewavelength was selected based on the sensitivityand specificity of the compound in a HPLC UV-Vis detection. The HPLC method was validatedfor inter day and intraday variability’s. From theplasma data various pharmacokinetic parameterswere determined.

In Vitro In Vivo CorelationThere are four levels of IVIVC that have

been described in the FDA guidance, whichinclude levels A, B, C, and multiple C (8). Herethe correlation was established according toDrewe and Guitard basing on degree A. Theparameters compared were cumulativeabsorption profile to that of in vitro dissolutionprofile i.e. correlation of the amount of drugdissolved to that of respective fraction of doseabsorbed (T

50). Cumulative amount of the drug

absorbed was calculated using Wagner-Nelsonmethod approximating the kinetics of the drug toone compartment open model. According toWagner-Nelson method, the cumulative amountof drug released from the microspheres into thesystemic circulation in a rat was calculated asgiven below:

Ab /A

b = (C

p + K[AUC]

0t ) /K[AUC]

0

Where Ab is the cumulative amount released

at any time, Ab is the dose administered, C

p is

the plasma concentration at any time t, K is the

elimination rate constant and AUC is the areaunder the curve. K was also determined in thisstudy in another set of rats where NNDMACwas administered via i.v. route. To determine theIVIVC with the formulation, the percentage ofNNDMAC dissolved from microspheres wasplotted on X-axis and the correspondingNNDMAC absorbed in the rats from themicrospheres was plotted on the Y-axis and thisgraph was fitted to a straight line and thecorrelation coefficient was determined. Thiscorrelation indicates the strength of IVIVC ofthis study.

Data AnalysisAll data in this study were presented as

mean or mean±SEM. Data were analyzed by t-test. Significance was recognized at P < 0.05.

ResultsThe drug was synthesized successfully

using protocol followed in this study. Aftersynthesis, the structure of NNDMAC wasconfirmed using NMR. The NMR results are asfollows: 7.6-7.8(m, 8H, Ar); 6.1(d, 2H, HC=CH);4.9(d, 2H, HC=CH); 3.3(s, 2H, CH

2); 2.2(s, 12H,

N(CH3)

2). A UV-Vis spectrum was generated

so as to identify the wavelength to be used in theassay of the compounds. The spectrum indicatedtwo different λ

max values which were 230 nm and

425 nm. The drug assay at 425 nm was used toanalyze the in vitro release samples while theassay at 230 nm was used in the HPLC. This isbecause the sensitivity of the assay was higherat 230 nm in a UV-Vis detector used in theHPLC and the specificity of the assay was higherat 425 nm when a UV-Vis spectrophotometerwas used. At 230 nm, there was a significantinterference from the degradation products ofthe polymers in the in vitro release studies with


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that of the drug and this interference was notnoted at 425 nm, suggesting more specificity ofthe assay at 425 nm in a UV-Visspectrophotometer. Thus, this wavelength wasalso used in the assay of the drug after synthesis.In this case, the starting material used in thesynthesis also demonstrated a λ

max at 230 nm.

The assay at 230 nm in HPLC for plasmasamples was used because of the highersensitivity when compared to that at 425 nm. TheHPLC method used was validated for interdayand intraday variabilitys. The results of intra andinter-day variation of NNDMAC at threedifferent concentrations levels (Level 1, Level 2and Level 3) were determined. The data indicatesthat the maximum %relative error at Level 1,Level 2 and Level were 1.8, 1.72 and -1.99,respectively while the maximum % relativestandard deviation was 1.5, 1.9 and 1.4respectively indicating that the method hasacceptable accuracy and precision. Also, thecalculated t-values were lesser than the tabulatedt-value of 4.3 for α = 0.05 at two degrees offreedom. This indicated that the experimentalvalues were not significantly different from thenominal which reflected the accuracy of themethod. Further, one way ANOVA was

Fig. 2: In Vitro Drug Release from NNDMACMicrospheres

performed to get estimates of within and betweenday variability. The calculated F value was lesserthan the tabulated F

2,6 (α = 0.01) of 10.92

indicating that the inter day variability was notsignificantly different from the intra day variabilityat 1% level of significance.

The drug release in vitro from themicrospheres was sustained for 9 days (Figure2). A 99.8% drug was released during this time.The rest of the drug could be the drug irreversiblybound to the polymer. The release of the drugfrom pure samples was also investigated. A100% of the entire pure drug taken at similarquantities was released within 48 hours undersimilar conditions. This confirms the sustainedrelease of the drug from the microsphereformulations. Plasma concentration vs time curveafter single IV bolus solution and IP formulation(microsphere) administration are shown in theFigure 3. Drug concentration started to bedetected in the plasma from 1 hour onwards.Drug release was sustained in vivo after themicrosphere administration. After IPadministration of the microspheres, thenoncompartmental parameters, area under theconcentration time curve (AUC), MRT, t

max, and

Cmax

were determined using KINETICA. All thenoncompartmental PK parameters for the i.v.bolus administration and microsphereadministration are shown in Table 1. Afteradministration NNDMAC microsphereformulation via the IP route, mean residence time(MRT) and the area under the curve (AUC) weresignificantly higher (P<0.05) and maximumconcentration (C

max) of NNDMAC was lower

than that of the free form. Tmax

for both theadministrations was the same. To determinedegree A IVIVC, the amount of the drugabsorbed was determined using Wagner-Nelson.The cumulative percentage of drug dissolved and



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PK Parameter IV Solution Administration IP MicrosphereAdministration

Cmax (g/ml) 2.4 ± 0.2 3.2±0.3

Tmax (h) 1 ±0.2 1 ±0.1

AUC (g*h/l) 0.025 ±0.01 0.055 ±0.005

MRT (h) 9.3 ±2 131.47 ±3

R2 0.997 0.954

Fig. 3: Plasma Log(Conc) vs Time Profile ofNNDMAC After IV Administration of Solution(—- —— ) and IP Administration of theMicrosphere Formulation (—— ——-)

Fig. 4: In vitro In Vivo Correlation of Drug

Release From the Microspheres

Table 1 Pharmacokinetic Variables of NNDMAC after single IP and IV Administration ofMicrospheres and Solution (Mean±SEM)

cumulative fraction of drug absorbed werecompared. The graphical analysis confirms a gooddegree of correlation (r2 = 0.982) (Figure 4). Thus,it can be concluded that good IVIVC wasobtained when the volume of distribution was usedas the volume of the release study.

DiscussionOptimal design of controlled release

systems requires a through understanding of

pharmacokinetics of drug with and without thedelivery system (9). Also the comprehension ofin vitro in vivo correlation (IVIVC) of the deliveryis essential to better tailor the delivery systemfor the future needs (8,10). The development ofnew injectable drug delivery systems has receivedconsiderable attention over the past few years.This interest has been sparked by the advantagesthis delivery system possess, which include easeof application, localized delivery for a site specific


585

action, prolonged delivery periods, decreasedbody drug dosage with concurrent reduction inpossible undesirable side effect common to mostforms of systemic delivery and improved patientcompliance and comfort. Thus, we developedNNDMAC microspheres and in this study,investigated the pharmacokinetics and IVIVC ofthe active. The developed formulation can be usedwhere in the curcumin analogues can be usedfor therapeutic purposes. The pharmacology ofcurcumin and its analogs is complex, withextensive metabolic conversions involved in theactivation, inactivation and elimination of the drug.It is cleared via glucuronidation. These drugproperties also contribute to the markedheterogeneities in efficacy observed withcurcumin and its analogs. Hence, drug carriertechnologies represent a rational strategy toimprove pharmacokinetics thereby enhancing thepharmacodynamics of the drug. After IPadministration of the microspheres, drugconcentration reached C

max within a longer time

(P<0.05) than that of solution forms. The resultsare similar to studies performed with other drugs,administration routes and species(11). The resultssuggest that the absorption of NNDMAC aftermicrosphere administration from injection siteswas slower. In this study, it was determined thatthe C

max of NNDMAC with microsphere

administration was lower than that obtained outof injecting solution. After IP administration ofmicrosphere encapsulated drugs, which acted asa local depot, there was a slower release, lowerC

max, and long-lasting concentrations of active

agent in the plasma compared with administrationof the free form. After IP administration ofmicrosphere formulation, the low C

max and plasma

concentrations may cause a reduction in dose-dependent side effects of the drug (12). In thepresent study, MRT of NNDMAC frommicrospheres was longer than those of the drugobtained out of injecting a solution. These results

suggest that microsphere encapsulated drugformulations provide longer effectiveconcentrations in plasma. The microspheres ofthis study were prepared using a biodegradablepolymer polycaprolactone. Polycaprolactone(PCL) is a biodegradable polyester with a lowmelting point of around 60°C and a glass transitiontemperature of about “60°C (13). PCL isdegraded by hydrolysis of its ester linkages inphysiological conditions (such as in the humanbody) and has therefore received a great deal ofattention for use as an implantable biomaterial.In particular it is especially interesting for thepreparation of long term implantable devices,owing to its degradation which is even slowerthan that of polylactide. PCL is an Food and DrugAdministration (FDA) approved material that canbe used in the human body as (for example) adrug delivery device. A variety of drugs have beenencapsulated within PCL beads for controlledrelease and targeted drug delivery.

The second objective of this study wasto evaluate the IVIVC with the microsphereformulation. A predictive IVIVC can empowerin vitro dissolution as a surrogate for in vivobioavailability/biotheraequilance (8,10). IVIVCscan decrease regulatory burden by decreasingthe number of biostudies required in support of adrug product. Additionally, IVIVC is also helpfulin the product development including thedevelopment of depot microspheres. Thedevelopment of an IVIVC is a dynamic processstarting from the very early stages of developmentprogram through the final step. Different typesof IVIVCs are used in the regulatory terminology.These include assumed IVIVC, retrospectiveIVIVC, and prospective IVIVC. An assumedIVIVC is essentially one that provides the initialguidance and direction for the early formulationdevelopment activity. Thus, during stage 1 andwith a particular product concept in mind,



586


appropriate in vitro targets are established tomeet the desired in vivo profile specification. Thisassumed model can be the subject of revision asprototype formulations are developed andcharacterized in vivo, with the results oftenleading to a further cycle of prototype formulationand in vivo characterization. Out of this cycleand in vivo characterization and, of course,extensive in vitro testing is often developed whatcan be referred to as retrospective IVIVC. Witha defined formulation that meets the in vivospecification, Stage 2 commences. At this stagebased on a greater understanding and appreciationof defined formulation and its characteristics, aprospective IVIVC is established through a welldefined prospective IVIVC study. Once theIVIVC is established and defined it can be thenused to guide the final cycle of formulation andprocess optimization leading into Stage 3 activitiesof scale-up, pivotal batch manufacture, andprocess validation culminating in registration,approval and subsequent post-approval scale-upand other changes. In this study, a part ofretrospective IVIVC was accomplished basedon the volume of the distribution to be the volumeused in the release studies. It is taken under theassumption of one definition of volume ofdistribution and the definition is “It is thehypothetic volume of the body compartment inwhich the drug is distributed”. Although thisdefinition is still controversial it would most likelybenefit our study in establishing the retrospectiveIVIVC we aimed at. Thus, the release wasperformed in 7 ml of PBS based on the volumeof distribution (5). The aim of such a study is toobtain good in vitro – in vivo correlation. Ideally,physiological conditions at the site ofadministration should be taken into account whenselecting the in vitro dissolution/release testconditions. The complexity of the releasemechanism of some novel/special dosage formsand the lack of knowledge about the conditions

under which release occurs in vivo make itdifficult to design physiologically based tests inall cases, but it should be possible to conceive atest that can detect the influence of criticalmanufacturing variables, differentiate betweendegrees of product performance, and to someextent characterize the biopharmaceutical qualityof the dosage form. As the release mechanismand site of application vary dramatically amongthe novel/special dosage forms, the experimentaltest conditions have to be tailored according tothe conditions at the site of administration (eg,temperature of the test) and the releasemechanism (eg, chewing gums will requiredifferent agitation rates than suspensions). Withina given category, it may be necessary to haveproduct type-specific dissolution tests (eg,separate tests for lipophilic and hydrophilicsuppositories), and in some cases for productscontaining the same drug and administered in thesame type of novel/special dosage form but witha different release mechanism (analogous to therange of tests available in the USP fortheophylline extended release dosage forms).Several studies also used volume of distributionto be volume to be used in the release mediumespecially for sustained release dosage forms.In this study also, we used similar approach.

The correlation was established accordingto Drewe and Guitard basing on (degree A) i.e.,the comparison of cumulative absorption profileand cumulative in vitro release profile was made.A level A correlation of in vitro release and invivo absorption could be obtained for individualplasma level data by means of the Wagner andNelson method. This type of evaluation of in vivoabsorption was previously applied for drugdelivery systems. To develop level A correlationthe estimation of the in vivo absorption ordissolution time course is performed using anappropriate deconvolution techniques such asWagner-Nelson procedure or Loo-Riegelman


587

method or numerical deconvolution for eachformulation and subject. Wagner-Nelson andLoo-Riegelman methods are both modeldependent in which the former is used for a one-compartment model and the latter is for multi-compartment system. However, Wagner-Nelsonmethod is less complicated than the Loo-Riegelman as there is no requirement forintravenous data. However, misinterpretation onthe terminal phase of the plasma profile may bepossible in the occurrence of a flip-flopphenomenon in which the rate of absorption isslower than the rate of elimination. To avoidcomplicated calculations which are bound byregression parameters and make the analysissimpler, we used Wagner-Nelson method in thisstudy. Further, the presence or absence of flip-flop in the pharmacokinetic data was also verifiedusing i.v. bolus data. Additionally, these techniquesrepresent a major advance over the single-pointapproach in that these methodologies utilize allof the dissolution and plasma level data availableto develop the correlations. Good IVIVC wasobserved in this retrospective IVIVC study. Thecorrelation made according to the volume ofdistribution is suitable for NNMDACmicrosphere. The same methodology should beinvestigated for other drugs so as to further adda speck of knowledge to this type of establishmentof IVIVC.

ConclusionIn conclusion, when microsphere

formulations are compared to solution, formulationdemonstrated a lower Cmax, higher MRT andprovides effective and prolonged plasmaconcentration in the body after IP administration.In addition, when NNDMAC was administeredin a microsphere entrapment form it had longduration of activity threshold, this result may bebeneficial for the diseases in which NNDMACis useful. Good IVIVC was obtained when thevolume of distribution was used as the volume ofthe release study.

Acknowledgements

The authors of this work would like toacknowledge the management of VaagdeviCollege of Pharmacy, Warangal, for providinginfrastructure useful in the conduction of thiswork. One of the Authors Dr. Jithan Aukunuruwould like to acknowledge the Department ofScience and Technology, India for providingfinancial assistantship for this project. This workwas funded under a SERC-DST YoungInvestigator project to Dr. Jithan Aukunuru.

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