Formulation and in vitro/in vivo correlation of a drug-in-adhesive transdermal patch containing azasetron

RESEARCH ARTICLE

Formulation and In Vitro/In Vivo Correlation of aDrug-in-Adhesive Transdermal Patch Containing Azasetron

LIN SUN, DONGMEI CUN, BO YUAN, HONGXIA CUI, HONGLEI XI, LIWEI MU, YANG CHEN, CHAO LIU,ZHONGYAN WANG, LIANG FANG

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, China

Received 24 April 2012; revised 13 July 2012; accepted 17 August 2012

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23317

ABSTRACT: The aim of the present study was to develop a transdermal drug delivery systemfor azasetron and evaluate the correlation between in vitro and in vivo release. The effects ofdifferent adhesives, permeation enhancers, and loadings of azasetron used in patches on thepenetration of azasetron through rabbit skin were investigated using two-chamber diffusioncells in vitro. For in vivo studies, azasetron pharmacokinetic parameters in Bama miniaturepigs were determined according to a noncompartment model method after topical application oftransdermal patches and intravenous administration of azasetron injections. The best perme-ation profile was obtained with the formulation containing DURO-TAK 87-9301 as adhesive, 5%of isopropyl myristate as penetration enhancer, and 5% of azasetron. The optimal patch formula-tion exhibited sustained release profiles in vivo for 216 h. The in vivo absorption curve in Bamaminiature pigs obtained by deconvolution approach using WinNonlin R© program was correlatedwell with the in vitro permeation curve of the azasetron patch. These findings indicated that thedeveloped patch for azasetron is promising for the treatment of delayed chemotherapy-inducednausea and vomiting, and the in vitro skin permeation experiments could be useful to predictthe in vivo performance of transdermal azasetron patches. © 2012 Wiley Periodicals, Inc. andthe American Pharmacists Association J Pharm SciKeywords: azasetron; percutaneous; Bama miniature pig; pharmacokinetics; deconvolution;in vitro/in vivo correlations (IVIVC); drug delivery systems; skin; transdermal

INTRODUCTION

Although there have been great strides in the man-agement of nausea and vomiting in recent years,these two symptoms remain among the most seriousside effects of chemotherapy.1 Chemotherapy-inducednausea and vomiting (CINV) affects 70%–80% ofpeople with cancer, and has a significant impact

Abbreviations used: CINV, chemotherapy-induced nauseaand vomiting; TDDS, transdermal drug delivery system; PSA,pressure-sensitive adhesive; IVIVC, in vitro/in vivo correlation;IPM, isopropyl myristate; MT, L-menthol; DSC, differential scan-ning calorimeter; IS, Internal standard; Q, the cumulative amountof drug; Jss, steady-state flux; F, absolute bioavailability; R(t), re-sponse function; I(t), input function; W(t), weight function; SC, stra-tum corneum.

Additional Supporting Information may be found in the onlineversion of this article. Supporting Information

Correspondence to: Liang Fang (Telephone: +86-24-23986330;Fax: +86-24-23986330; E-mail: [email protected]);Zhongyan Wang (Telephone: +86-24-23986292; Fax: +86-24-23986292; E-mail: [email protected])Journal of Pharmaceutical Sciences© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association

on the patient and their healthcare resources. 5-Hydroxytryptamine3 (5-HT3) receptor antagonistsplay a key role in the management of CINV, the mech-anism of which was mainly associated with the se-lective blockade of 5-HT3 receptors in visceral–vagalafferent fibers and in the chemoreceptor trigger zone.These antagonists are efficacious against all gradesof emetogenic therapy.2

Azasetron (Fig. 1), N-(1-azabicyclo [2.2.2] octan-8-yl)-6-chloro-4-methyl-3-oxo-1,4-benzoxazine-8-carbo-xamide, is a potent and selective serotonin 5-HT3receptor antagonist for the prevention and treatmentof CINV.3 In clinic, it is also used for the treatmentof postoperative nausea and vomiting3 and irritablebowel syndrome.4 Compared with other 5-HT3antagonists, azasetron has a longer duration ofaction5 and a higher affinity to the 5-HT3 receptor.6

However, the drug is currently only available in theform of injections and tablets in spite of its excellentantiemetic activity. Azasetron hydrochloride tabletswere designed to reduce the pain feeling and theinconvenience associated with injection, but it is

JOURNAL OF PHARMACEUTICAL SCIENCES 1

2 SUN ET AL.

Figure 1. The chemical structure of azasetron.

rather difficult to give oral preparations to patientswho show symptoms of nausea and vomiting orpeople who have dysphagia. And more importantly,these two dosage forms of azasetron were onlyeffective for the prevention of acute CINV (occurringwithin 24 h of chemotherapy). They are unresponsiveto delayed CINV (occurring 24 h to several daysafter initial treatment with chemotherapy) unlessadministered repeatedly.2 The required multipledaily administration regimens severely conflict withthe patients’ compliance and increase the costs ofhealthcare. As a consequence, the clinical applicationof azasetron was limited.

To overcome the shortcomings mentioned above,transdermal drug delivery system (TDDS) is designedas an attractive alternative for oral drug delivery andinjections. Boccia et al.7 have confirmed the feasibilityof delivering 5-HT3 antagonists through the skin toobtain extended therapeutic effects, treating delayedCINV, and increasing patients’ compliance. In 2008,as the first transdermal system for the treatment ofCINV, transdermal patch containing granisetron, adrug in the same catalog as azasetron, was approvedby the United States Food and Drug Administration(US FDA).8 As instructed, the patch can be worn forup to 7 days. However, to date, there has been noresearch paper on the delivery of azasetron via thetransdermal route either to animals or to patients.

In the present study, TDDS containing azasetronwas designed and aimed at prolonging the action ofazasetron and benefiting the treatment of delayedCINV. In vitro permeation studies were performedto characterize azasetron permeated through rabbitskin with various pressure-sensitive adhesive (PSA)-based formulations containing different chemical en-

hancers. The pharmacokinetic profile of the optimizedazasetron patch was clarified by permeation studieson Bama miniature pigs in vivo. And an in vitro/invivo correlation (IVIVC) was established by deconvo-lution for speeding up the development of azasetrontransdermal patches in follow-up studies.

MATERIALS AND METHODS

Materials

Azasetron hydrochloride was supplied by NanjingPharmaceutical Company, Ltd. (Nanjing, China);granisetron hydrochloride was purchased fromShanghai FINC Chemical Technology Company, Ltd.(Shanghai, China). Azone, isopropyl myristate (IPM),and L-menthol (MT) were bought from China Na-tional Medicines Company, Ltd. (Shanghai, China);PSAs, DURO-TAK R© adhesives 87-2677, 87-9301, and87-2852 (chemical composition: acrylate) were pur-chased from the National Starch and ChemicalCompany (Bridgewater, New Jersey). Methanol andacetonitrile were of high-performance liquid chro-matography (HPLC) grade and obtained from theYuwang Pharmaceutical Company, Ltd. (Shandong,China) and Fisher Scientific (Fairlawn, New Jersey),respectively. All other chemicals used were of analyt-ical grade or purer and purchased from commercialsuppliers.

Animals

Male rabbits weighing 1.5–2.0 kg were suppliedby the Experimental Animal Center of ShenyangPharmaceutical University (Shenyang, China).Male Bama miniature pigs were purchased fromChongqing Zongshen Biotech Company, Ltd.(Chongqing, China). All experiments were car-ried out in accordance with the NIH Guidelines forthe Care and Use of Laboratory Animals and alsoin accordance with the guidelines for animal usepublished by the Life Science Research Center ofShenyang Pharmaceutical University. All effortswere made to minimize animal suffering and to limitthe number of animals used.

Preparation and Identification of Azasetron

Preparation of Azasetron from Azasetron Hydrochloride

Azasetron free base was prepared from azasetron hy-drochloride, which is commercially available. In brief,a suspension of azasetron hydrochloride in water wastitrated with 0.1 mol L−1 sodium hydroxide solutionto pH 11. Then, the mixture was filtered through aBuchner filter with double-decked filter papers onit, and the blender jar and residue were rinsed withwater continuously until the washing liquid becameneutral. Finally, the filter cake was transferred to an

JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps

FORMULATION AND IVIVC OF THE AZASETRON PATCH 3

amber glass container and kept in a vacuum evapo-rator until the weight loss ceased.

Identification of Azasetron by Differential ScanningCalorimeter

Differential scanning calorimeter (DSC) measure-ments were performed using a Mettler Toledo DSC1 thermal analyzer(Mettler-Toledo AG, Schwerzen-bach, Switzerland).1 Samples were placed in a stan-dard aluminum crucible fitted with a perforated lidfor scanning. An empty pan was used as a reference.The samples were heated at a rate of 10◦C min−1 overa temperature range of 25◦C–330◦C.

Preparation of Azasetron Patches

Azasetron, PSAs, and enhancers were dissolved inethyl acetate and mixed thoroughly with a mechan-ical stirrer to obtain homogeneous coating material.Azasetron patches were prepared by spreading the ob-tained coating material with a laboratory coating unit(SLT200; Kaikai Company, Ltd., Shanghai, China)onto a fluoropolymer-treated polyester release liner(ScotchPakTM 9744; 3M, St. Paul, Minnesota, US) ata thickness of 60 :m. Then the products were ovendried at 50◦C for 10 min to remove the solvent, thenlaminated with a polyethylene monolayer backingfilm (CoTranTM 9720; 3M, St. Paul, Minnesota, US),and finally kept in an aluminum–plastic membrane.

Drug Content Determination

A patch with an area of 1 cm2 (n = 4) was weighedaccurately and added into 100 mL methanol, followedby sonication for 20 min.9 The content of the drugin methanol solution was determined by HPLC afterfiltration and appropriate dilution with mobile phase.

In Vitro Permeation Experiments

Rabbit excised skin was used to evaluate the skinpermeation of azasetron in vitro. The process of skinpreparation was conducted according to a previousreport.10 Rabbits were anesthetized with urethane[20%, w/v, intravenous(ly) (i.v.)]. Hair on the abdom-inal area were carefully removed by trimming witha clipper (TGC model 900, Panasonic Corporation,Hokkaido, Japan) and an electrical shaver. Full thick-ness skin [epidermis with stratum corneum (SC) anddermis] was excised after being sacrificed by injectingair i.v. The integrity of the skin was carefully checkedby microscopic observation, and any skin that wasnot uniform was rejected. The subdermal tissue wassurgically removed and the skin was washed immedi-ately with physiological saline, wrapped in aluminumfoil. And then, the skin samples were stored at −70◦Cuntil required (used within 1 week after preparation).Before starting the experiment, it was thawed to roomtemperature before mounting them within the diffu-sion apparatus.

In vitro permeation experiments were performedusing a two-chamber side-by-side glass diffusion cell(cell capacity of 3.0 mL, effective diffusion area =0.95 cm2) with a water jacket connected to a wa-ter bath at 32◦C. The excised abdominal skin wasmounted between the cell halves with the dermalside of the skin facing the receiver solution. A circu-lar transdermal patch was pressed and stuck on thedermal side of the skin. After securely clamping thecell assembly together, the receptor compartment wasfilled with 3 mL pH 7.4 phosphate buffer solution tomaintain sink conditions and continuously stirred atabout 600 rpm. Care was taken to ensure that no airbubbles remained in the water jacket. Then, samples(2.0 mL) were withdrawn at predetermined intervalsand replaced with the same volume of fresh solutionto maintain sink conditions.

The samples were centrifuged for 5 min at17800 × g and an aliquot of 20 :L of supernatantwas analyzed by HPLC to determine the drug con-tent. Experiments were done in triplicates.

In Vivo Studies

Four male Bama miniature pigs weighing 9–11 kg(15–16 weeks old) were divided into two groups ran-domly (group A and group B) and used for a crossover,single dose administration experiment after being al-lowed to acclimatize for 1 week. A washout period of14 days after the last sampling was respected in be-tween each experiment.

The pigs in group A were given an i.v. administra-tion of azasetron 0.5 mg kg−1 (dissolved in normalsaline) via the abdominal vein. The animals in groupB were treated with an azasetron patch and 2 daysin advance, hair on a certain area of abdominal skinof the pigs in group B were shaved without damagingthe skin. Immediately before the application of thepatch, the skin was cleaned carefully with warm wa-ter and an alcohol swab subsequently, and then pattedto let it dry to avoid any adhesion problems caused byresidues on the skin. A single patch with an area ofabout 50 cm2 and containing 50 mg (correspondingto 5 mg kg−1 per pig) azasetron was applied to theclean skin for 168 h. Blood samples of 1–2 mL werecollected from the abdominal vein and placed in dryheparinized tubes at 0, 0.083, 0.167, 0.333, 0.5, 0.75,1, 2, 4, 8, 12, 24, and 36 h after i.v. administration,and 0, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192,and 216 h after transdermal administration, respec-tively. Plasma samples were separated immediatelyby centrifugation at 1740×g for 10 min, using a Xi-angYi H2050R centrifuge (XiangYi Centrifuge Instru-ment Company Ltd., Changsha, China), and stored at−70◦C until analysis.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES

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The Treatment of Plasma Samples

To a 100 :L portion of each plasma sample, 10 :Lof internal standard (IS) solution (10 ng mL−1

granisetron in methanol) and 10 :L methanol wereadded and vortex mixed for 10 s. After the additionof another 100 :L of methanol for protein precipita-tion, the sample was vortex mixed again for 1 min,followed by centrifugation at 15,600 × g for 5 minat room temperature. The organic layer was sepa-rated and evaporated to dryness at 40◦C under an airstream. The residue was dissolved in 100 :L of themobile phase and 20 :L of the sample was injectedinto the ultra-performance liquid chromatography–tandem mass spectrometric system for analysis.

Analytical Methods

In Vitro Quantitative Analysis

The concentrations of azasetron were measured byHPLC. The HPLC equipment consisted of an L-2420variable-wavelength ultraviolet absorbance detector,an L-2130 pump (Hitachi High-Technologies Corpo-ration, Tokyo, Japan), and an HT-220A column tem-perature controller (Hengao Technology DevelopmentCo.,Ltd, Tianjin, China)5. Analyses were performedon a 5-:m ODS column (200 × 4.6 mm2; DIKMA Tech-nologies, Beijing, China) at 40◦C. The mobile phasewas acetonitrile: 0.03 mol L−1 pH 3.4 KH2PO4–H3PO4buffer solution (8:2, v/v) at a flow rate of 1 mL min−1.The wavelength of the detector was set at 221 nm.

In Vivo Quantitative Analysis

Liquid Chromatography. Liquid chromatographywas performed on an ACQUITYTM UPLC system(Waters Corporation, Milford, Massachusetts) withcooling in an autosampler and a column oven to en-able temperature control of the analytical column. ADiamonsil C18 analytical column (150 × 4.6 mm2,5 :m; DIKMA) was employed. Methanol–ammoniumacetate (5 mmoL L −1)–formic acid (45:55:0.3, v/v/v)was used as a mobile phase delivered at a flow rate of1.0 mL min−1. The outlet of the column was split andonly 0.5 mL min−1 portion of the column effluent wascarried into the mass spectrometer.

Mass Spectrometry. An API 4000 triple quadrupoletandem mass spectrometer (Applied Biosystem/MDSSCIEX, Foster City, California) with electrospray ion-ization was operated in negative ion mode. The quan-tification was performed using multiple reaction mon-itoring method with the transitions of m/z 350.2 →m/z 224.2 for azasetron and m/z 313.4 → m/z 138.1for granisetron (IS). The main working parameterswere set as follows: ion spray voltage, 1.5 kV; ionsource temperature, 600◦C; gas 1, 40 psi; gas 2, 20psi; and curtain gas, 10 psi. The concentrations ofanalyte were determined using the software Ana-

lyst 1.5(Applied Biosystem/MDS SCIEX, Foster City,California).

Statistical Analysis

Data Analysis In Vitro

All in vitro experiments were carried out in triplicate.The amount of drug that penetrated through the skinduring each sampling interval was obtained from themeasured concentration and volume of the receiverphase. The transdermal penetration curve was plot-ted as the cumulative amount of drug permeated perunit area (Q) collected in the receiver compartmentas a function of time. The steady-state flux (Jss) wascalculated from the slope of the linear portion of theplot, using the following equation:

Jss = dQAdt

(1)

All data were calculated and presented as mean ±SD. Statistical analyses of the data were carried outusing analysis of variance and the Student’s t-test.The level of significance was taken as p ≤ 0.05.

In Vivo Pharmacokinetic Analysis

Pharmacokinetic parameters, including the maxi-mal plasma drug concentration (Cmax), time to max-imal plasma drug concentration (Tmax), and meanazasetron concentration after each administration,were obtained directly from the individual bloodconcentration–time profiles. The area under the time–concentration curve from time 0 to time t (AUC0–t)for each administration were calculated by the lin-ear trapezoidal rule, whereas the mean residencetime (MRT) values were obtained by noncompartmen-tal analyses with the help of WinNonlin R© ver. 5.2.1(Pharsight Corporation, San Diego, California) pro-gram.

Absolute bioavailability (F) was calculated from thefollowing equation:

F = AUCtransdermal

AUCi.v.

× dosei.v.

dosetransdermal(2)

The steady-state plasma concentration of azasetronafter the application of patches was calculated fromthe equation11: Css = AUC0–t/time, time is 168 h in ourcase (azasetron transdermal patches were applied onpig skin for a 168 h period). Data are expressed as themean ± SD.

In Vitro/In Vivo Correlation

There were two-stage modeling procedures to evalu-ate the IVIVC for the final formulation of azasetronpatches. First, in vivo permeation data of azasetrontransdermal patches in Bama miniature pigs wereobtained from the data of plasma concentration by



deconvolution method, in which the in vivo resultsof azasetron solution after injection administratedto pigs were used as weight function. In mathemat-ics, deconvolution is an algorithm-based process usedto reverse the effects of convolution on the recordeddata. The convolution technique could be explained byfollowing formula:

R(t) =∫ t

0I(θ )W(t − θ )dθ ≡ I(θ ) ∗ W(θ) (3)

where R(t), I, and W are the plasma concentration as afunction of time, the input into the system (i.e., in vitroskin permeation data), and the weight function (i.e.,i.v. data), respectively, and ∗ is the convolution opera-tor. Convolution was done by solving this formula withrespect to R(t) given I(t) and W(t), whereas deconvo-lution was done by solving the following equation forI(t) given R(t) and W(t).

I(t) = R(θ)//W(θ ) (4)

where the symbol // denotes the deconvolution opera-tion.

Second, the in vivo absorption time profile obtainedin this first stage is connected to the time course ofthe in vitro porcine skin permeation profile.

The correlation analysis was performed with thehelp of the WinNonlin R© with IVIVC Toolkit program(Pharsight), and correlation coefficients were exam-ined for significance (p < 0.05) using Student’s t-test.

RESULTS AND DISCUSSION

Preparation and Identification of Azasetron

Azasetron free base was prepared by neutralizingthe azasetron hydrochloride, which is commerciallyavailable. The yield was over 90% and the preparedproduct was identified to be highly pure (>99.5%)by HPLC using the peak area normalization method.The DSC curves of azasetron (Fig. 2) exhibited char-acteristic sharp endothermic peaks corresponding toits melting point at 181◦C.

Figure 2. DSC curves of azasetron and azasetron hy-drochloride at a heating rate of 10◦C min−1.

The Cumulative Permeation Profiles of Azasetron fromTransdermal Patches

In the present study, the in vitro transdermal perme-ability of azasetron through excised rabbit skin andBama miniature pig skin was investigated. The rab-bit skin was used in the formulation screening stage.The porcine skin was used to provide the in vitro datafor establishing IVIVC.

The azasetron patch formulation was optimizedby investigating the effects of several formulationparameters including the type of PSA, the loadingamount of azasetron, and the type and amount of en-hancers on the determined steady-state flux and Q24of azasetron penetrating through rabbit skin. The set-ting of each parameter and corresponding results arelisted in Table 1.

Acrylic PSAs have been known for a long time be-cause of their favorable properties.12 In this study,the effects of three different acrylic adhesives in-cluding DURO-TAK R© 87-2852 and 87-2677 (NationalStarch and Chemical Company) with carboxylic acidgroups, and DURO-TAK R© 87-9301 (National Starchand Chemical Company) without functional groupswere tested. As shown in Figure 3 and Table 1, whenthe loading of azasetron was set at 5% (w/w, basedon adhesive weight), the cumulative amount of aza-setron permeated from the 87-9301 adhesive was 6.13and 3.63 times higher than that from the 87-2677 and87-2852 adhesives, respectively.

According to the chemical structure of azasetron,it has six acceptors and one donor of H-bonding in it(obtained from SciFinder database). These sites couldinteract with the carboxylic acid group of the PSAs (ifit has any) and produce a significant reduction in pen-etration. Therefore, the PSA of DURO-TAK R© 87-9301(National Starch and Chemical Company), which islack of any functional groups, was ideal for the devel-opment of azasetron transdermal patches.

Adding penetration enhancers in the formulationof patches is the most effective approach to reversiblyovercome the barrier properties of SC.13 In the currentstudy, all the tested enhancer (MT, azone, and IPM)presented a linear penetration curve that indicateda steady-state flux (Fig. 4). Among them, the patchcontaining IPM provided the highest flux and en-hancement. This result suggested that in general, thecumulative amount of drug permeated from theformulations was related to the structures andlipophilicity of enhancers. IPM is known to be safeand has been used to increase the skin perme-ation of a large number of drugs, including proges-terone, estradiol, indomethacin, methyl nicotinate,14

benztropine,15 and amlodipine.16 It is widely acceptedthat IPM could act as a fluidizer of intercellular lipids,and affect the lipid-rich phase in the SC, thereby re-duce the barrier function of SC.17


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Table 1. Summary of Data for the Permeation of Azasetron from the Patches with DifferentFormulations Through Excised Rabbit Skin

Formulation

PSAs Enhancers (w/w) Azasetron Content (w/w) Q24 (:g cm−2) Jss (:g h−1 cm−2) ERa

87-2677 – 5% 22.57 ± 3.09 0.83 ± 0.14 –87-2852 – 5% 38.10 ± 9.59 1.64 ± 0.44 –87-9301 – 5% 138.26 ± 16.22 5.57 ± 0.63 1.0087-9301 – 3% 50.06 ± 10.32 2.33 ± 0.13 0.3687-9301 – 10% 127.54 ± 6.31 5.58 ± 0.34 0.9287-9301 5% MT 5% 320.05 ± 12.74 13.73 ± 0.33 2.3187-9301 5% Azone 5% 354.40 ± 15.87 15.22 ± 1.86 2.5687-9301 5% IPM 5% 430.54 ± 4.88 17.68 ± 0.99 3.1187-9301 3% IPM 5% 282.16 ± 23.77 12.62 ± 2.33 2.0487-9301 10% IPM 5% 412.60 ± 4.10 17.53 ± 0.44 2.98

Data are given as mean ± SD (n = 3).aER is the enhancement ratio calculated as follows: ER = Q (with enhancer)/Q (without enhancer).

Figure 3. The penetration profiles of azasetron patchesin the presence of different adhesives. Data are presentedas the mean ± SD.

Figure 4. The penetration profiles of azasetron throughexcised rabbit skin from the patches containing 5% (w/w)azasetron and 5% (w/w) of different enhancers in PSA87-9301. Each point and bar shows the mean and SD(n = 3).

To investigate the effect of varying the concen-tration of IPM, transdermal patches containing 3%,5%, and 10% IPM were prepared and their in vitropenetration behavior was investigated. As shown inTable 1, significantly higher cumulative amount ofazasetron permeated was achieved when the concen-tration of IPM was increased to 5%. But increasingthe concentration of IPM to 10% did not lead to fur-ther increase of cumulative amount of azasetron asexpected. The fluxes were 5.57 ± 0.63, 12.62 ± 2.33,17.68 ± 0.99, and 17.53 ± 0.44 :g h−1 cm−2 for 0%,3%, 5%, and 10%, respectively. Thus, 5% was chosenas the final concentration of IPM because there is nosignificant difference between 10% and 5% IPM.

On the basis of the above-mentioned experiments,the optimal parameters were identified for the formu-lation of azasetron patch: 5% azasetron, 5% IPM, andusing DURO-TAK R© 87-9301 (National Starch andChemical Company) as PSA. A cumulative amount of430.54 ± 4.88 :g cm−2 and a flux of 17.68 ± 0.99 :gh−1 cm−2 during 24 h was achieved with the optimalformulation.

As the commercially available granisetron patchcould continuously deliver the active ingredient intothe systemic circulation for up to 7 days, the aza-setron patch was also designed for a sustained re-lease of the drug for 7 days in the current study.Rabbit skin was used as a formal barrier in trans-dermal experiments for its convenient permeability.As a natural barrier similar to human skin, porcineskin was widely used in the in vitro and in vivo eval-uations for TDDS. The cumulative permeation pro-files of the optimal formulation through porcine skinwere investigated for a week to provide in vitro datafor further establishment of a correlation relationshipwith in vivo drug permeation data. The experimentalconditions were the same as those used in 24 h per-meation experiments, except sodium azide (0.1%, w/v)was added into the receptor compartment to prevent



skin from rotting. The Q168 through porcine skin was483.34 ± 27.93 :g cm−2.

Pharmacokinetics Study

To reduce the risk and the cost of human study, an-imals were used in bioavailability study of transder-mal delivery systems. It should be noted that animalstudies may not be precisely predictive of human skinpermeation. However, at present many researches in-dicate that porcine skin is not only similar to humanskin, but also less variable than other kinds of skin.Therefore, the porcine skin could be a good alterna-tive for human skin.18 The obtained profiles of meanplasma concentration of azasetron versus time afteri.v. injection or transdermal administration are shownin Figure 5. Pharmacokinetic parameters after i.v. ad-

Figure 5. Plasma concentration–time profiles of aza-setron after intravenous injection of azasetron 0.5 mg kg−1

(dissolved in normal saline) via the abdominal vein (groupA, n = 4) or azasetron transdermal patch (the patch was re-moved after an interval of 168 h) (group B, n = 4). (a) Meanplasma concentration–time profile after i.v. injection; (b)mean plasma concentration–time profile after transdermaladministration. The dotted line (—) indicates the plasmaconcentration after the removal of the patches. Data arepresented as the mean ± SD.

Table 2. Pharmacokinetic Parameters of Azasetron afterTransdermal Administration as a Single Patch (ContainingAzasetron 50 mg, and the Patch was Removed from the Pig afteran Interval of 168 h) and Intravenous (i.v.) Injection of Azasetron0.5 mg kg−1 (Dissolved in Normal Saline) via the Abdominal Vein(n = 4)

Parameters i.v. Transdermal

Cmax (ng mL−1) 918.93 ± 92.75 44.88 ± 7.16Tmax (h) 0.08 ± 0.00 66.00 ± 22.98AUC0–t (h ng mL−1) 548.69 ± 91.96 3335.93 ± 531.99AUC0–∞ (h ng mL−1) 561.64 ± 91.99 3383.40 ± 569.25AUMC (ng h2 mL−1)a 1958.26 ± 334.31 274923.77 ± 63331.62MRT (h) 3.58 ± 0.33 81.87 ± 6.20Css (ng mL−1) – 15.44 ± 2.46CL (mL h−1 kg−1)b 906.71 ± 134.43 –

Data are given as mean ± SD (n = 4).aAUMC is the area under the first moment of the plasma

concentration-time Curve.bCL is the clearance as follows: CL=dose/AUC0–∞/ weight.

ministration of azasetron in pigs were calculated byus because they are not available in literatures. Thesedata are used as weight function to get the in vivo ab-sorption profile of the azasetron patch. The resultsare given in Table 2. After i.v. injection of azasetronat the dose of 0.5 mg kg−1 via the abdominal vein,the mean plasma concentration dropped to minimumat 36 h. Through transdermal administration route,the maximal plasma azasetron concentration (Cmax =44.88 ± 7.16 ng mL−1) was achieved at the time pointof 66.00 ± 22.98 h (Tmax), and the plasma drug con-centration was still detectable at 216 h. Furthermore,the MRT of azasetron was prolonged to 81.87 ± 6.20 h,which was 23 times higher than that of the azasetroninjection. This was believed to be due to the continu-ous replenishment of drug into the systemic circula-tion after constant and controlled release of drug fromthe transdermal patch. Thus, it was demonstratedthat azasetron patches are more efficiency in the termof managing the delayed CINV.

After calculating the dose normalization, the F ofthe transdermal azasetron patch was 60.8%. Thisvalue shows that the transdermal route is an efficientway for azasetron to be absorbed into the systemic cir-culation.

In Vitro/In Vivo Correlation

Defined by the US FDA, IVIVC is a predictive modeldescribing the relationship between in vitro propertyof a dosage form and relevant in vivo response. In thecase of transdermal delivery, the in vitro property isthe rate of permeation through the skin, whereas thein vivo response is the plasma drug concentration.19

On the basis of the in vitro skin permeation data,Qi et al.20 successfully estimated the systemic drugconcentration of 2,3,5,6-tetramethylpyrazine follow-ing transdermal application in rabbit by using convo-lution method. The finding indicated that the in vivo


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Figure 6. Drug permeation profiles of azasetron trans-dermal patch in vivo (—) and in vitro (—).

profile of TDDS was predictable once the IVIVC wasestablished.

The IVIVC Wizard in WinNonlin R© program (Phar-sight) provides an organized environment in which wecan build and manipulate IVIVC models, plus a fea-ture to automatically estimate the W(t) function. Thenoncompartment model of i.v. administration givesthe W(t) function used in the deconvolution after nor-malization to one dose unit, whereas the R(t) functionwas provided using the transdermal pharmacokineticprofile. As presented in Figure 6, the mean deconvo-luted absorption profile in vivo of different pigs wasin agreement with the observed absorption profilein vitro, and the correlation coefficient was 0.9879.This excellent IVIVC relationship demonstrated thatin vitro experiments can be used to screen formula-tions in further studies to develop the final optimalpatch product.

Nevertheless, the process of establishing IVIVC bydeconvolution is complicated, which requires to ob-tain in vitro penetration profiles and in vivo plasmaconcentration profiles from formulations with differ-ent release rates, and then evaluate the internal andexternal predictability. The result shown in this ar-ticle provides a fundament for the extension of thepresent work. Further evidence will be necessary toconfirm and reproduce this IVIVC of azasetron trans-dermal patches, and thus, an operative formulationfor curing the delayed CINV would be developed suc-cessfully.

CONCLUSIONS

Azasetron was formulated into a transdermal patchin an attempt to present a better mode of drug deliv-ery. The optimum formulation for in vitro skin per-meation contained DURO-TAK R© 87-9301 (National

Starch and Chemical Company), 5% of IPM, and 5%of azasetron. The permeation profiles through porcineskin in vitro and in vivo suggested that azasetroncould effectively penetrate through the skin and passinto the systemic circulation. A more effective ap-proach of developing the product was provided by theexcellent IVIVC relationship. The present study indi-cates that the drug-in-adhesive transdermal dosageform of azasetron is promising in the treatment ofdelayed CINV.

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Formulation and in vitro/in vivo correlation of a drug-in-adhesive transdermal patch containing azasetron