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Available online at www.sciencedirect.com

journa l homepage: www.e lsev ier .com/ locate /e tap

hort communication

ransepithelial transport of biperiden hydrochloride inaco-2 cell monolayers

vana S. Abalos ∗, Yanina I. Rodríguez, Verónica Lozano, Marina Cereseto,aria V. Mussini, Marta E. Spinetto, Carlos Chiale, Guido Pesce

nstituto Nacional de Medicamentos (INAME), Departamento de Farmacología, Av. Caseros 2161 (1264), Buenos Aires, Argentina

r t i c l e i n f o

rticle history:

eceived 25 April 2011

eceived in revised form

January 2012

ccepted 3 April 2012

vailable online 10 April 2012

eywords:

iopharmaceutics Classification

ystem (BCS)

aco-2 cells

ermeability

a b s t r a c t

The aim of this research has been to determine the biperiden hydrochloride permeability in

Caco-2 model, in order to classify it based on the Biopharmaceutics Classification System

(BCS).

The World Health Organization (WHO) as well as many other authors have provisionally

assigned the drug as BCS class I (high solubility–high permeability) or III (high solubility–low

permeability), based on different methods.

We determined biperiden BCS class by comparing its permeability to 5 pre-defined com-

pounds: atenolol and ranitidine hydrochloride (low permeability group) and metoprolol

tartrate, sodium naproxen and theophylline (high permeability group).

Since biperiden permeability was higher than those obtained for high permeability drugs,

we classified it as a BCS class I compound.

On the other hand, as no differences were obtained for permeability values when apical to

egulatory science

iperiden hydrochloride

basolateral and basolateral to apical fluxes were studied, this drug cannot act as a substrate

of efflux transporters.

As a consequence of our results, we suggest that the widely used antiparkinsonian drug,

biperiden, should be candidate for a waiver of in vivo bioequivalence studies.

© 2012 Elsevier B.V. All rights reserved.

release solid oral dosage forms (ANMAT, 2009), has alsoadopted the BCS criterion.

. Introduction

he Biopharmaceutics Classification System (BCS) (Amidont al., 1995) is a regulatory tool developed to replacen vivo bioequivalence studies for immediate-release solid oralosage forms by in vitro dissolution tests. Both FDA (Food and

rug Administration) and EMA (European Medicines Agency)

ssued guidelines for industry under which the pharmaceuti-al companies can request a waiver of in vivo bioequivalence

∗ Corresponding author.E-mail address: iabalos@anmat.gov.ar (I.S. Abalos).

382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2012.04.004

studies (CDER/FDA, 2000; EMA, 2008). Recently, in Argentina,the Administración Nacional de Medicamentos Alimentos y Tec-nología Médica (ANMAT – National Administration of Drugs,Foods and Medical Devices), by means of its 758/09 Provision:Biowaiver criteria of bioequivalence assays for immediate

The BCS classifies the drug substances in four differentcategories:

d p h

224 e n v i r o n m e n t a l t o x i c o l o g y a n

CLASS I: high solubility–high permeability;CLASS II: low solubility–high permeability;CLASS III: high solubility–low permeability;CLASS IV: low solubility–low permeability.

A drug product is considered highly permeable when it isabsorbed to an extent of 85% or more. When the highest dosestrength is dissolved in 250 ml buffer at 37 ◦C over a pH rangeof 1.2–6.8, the drug is classified as highly soluble.

The World Health Organization (WHO), in its essen-tial medicines model list of minimum medicines requiredfor basic health care (WHO, 2006), summarizes molecularproperties and provides provisional biopharmaceutical clas-sification. In some cases, either the solubility or permeabilitydata (or both) were not completely satisfactory, because of thatthe WHO assigned the drug to one (or two) specific BSC class.

Biperiden is an antiparkinsonian drug of the anticholiner-gic type. Also, it is used to control extrapyramidal disorderssecondary to neuroleptic drug therapy (e.g., phenotiazines)(Martindale, 2005).

Despite the recognized efficacy of biperiden for the treat-ment of symptoms in Parkinson’s disease, only limitedpharmacokinetic studies are available, particulary in humans(Grimaldi et al., 1986).

The WHO provisionally has classified biperiden as BCSclass I/III. According to this fact, the prediction of its classi-fication with certainty is an unsolved issue.

After literature searching we found there is no consensuson the BCS biperiden class. From in vitro results, Varma et al.(2005) classified it as BCS class I. The same classification wasmade by Kasim et al. (2004), based on a correlation of the n-octanol/water partition coefficient of the uncharged form ofbiperiden molecule (Log P). On the other hand, Lindenberget al. (2004), reported biperiden as BCS class III, based on bib-liographic data.

Due to this situation, the purpose of our research has beento determine the biperiden permeability in Caco-2 model, acolon adenocarcinoma cell line recommended by FDA and theWHO (Fogh et al., 1977; WHO, 2005) to classify this drug basedon the BCS. If biperiden is incorporated as a BCS class I drug,it will become a granted biowaiver.

2. Materials and methods

2.1. Chemicals

The chemicals used were obtained from the followingsuppliers: biperiden hydrochloride (Abbot S.A.), metoprololtartrate (Novartis S.A.), sodium naproxen (Elea S.A.C.I.F y A.).Ranitidine hydrochloride, theophylline and atenolol were pur-chased from the Instituto Nacional de Medicamentos (INAME,National Institute of Drugs, Argentina).

All chemical solutions were used immediately after prepa-

ration.

The permeability assay consists of different phases: cellculture, transport experiment and data analysis, previouslystandardized in our laboratory (Rodríguez et al., 2011).

a r m a c o l o g y 3 4 ( 2 0 1 2 ) 223–227

2.2. Cell culture

The Caco-2 cells were cultured using Dulbecco’s modi-fied Eagle’s culture medium (DMEM) supplemented with10% of fetal bovine serum (FBS), 1% of non-essentialamino acids, 1% penicillin–streptomycin and 1% glutamineand incubated at 37 ◦C in an atmosphere of 5% CO2

at 95% relative humidity. The cells were tripsinized andseeded onto Snapwell® semi-permeable supports, 1.13 cm2

detachable inserts (Corning-Costar) containing 6 wells with0.4 �m pore size polycarbonate membranes at a density of7 × 104 cells/insert in culture medium for 23 days.

The monolayer growth was tested by measuring thetransepithelial electrical resistance (TEER) at different culturedays (9, 18 and 23) using a Millicell-ERS® voltimeter (Millipore).

To check the monolayer integrity, the TEER was measuredbefore starting and after finishing every permeability assay(pre- and post-diffusion, respectively) by means of VCC MC6(Physiologic Instruments) equipment coupled with a verti-cal diffusion chamber system Navicyte® (Harvard Apparatus).For the assays performance, monolayers with TEER valuesexceeded 550 � cm2 on day 23 and pre-diffusion values higherthan 200 � cm2 were employed.

2.3. Transport experiment

The biperiden hydrochloride permeability class was deter-mined by comparing it to 5 pre-defined compounds: atenolol(marker of monolayer integrity (Rodríguez et al., 2011;Hubatsch et al., 2007)) and ranitidine hydrochloride (P-glycoprotein (P-gp) substrate (Collett et al., 1999)) of lowpermeability and metoprolol tartrate, sodium naproxen andtheophylline of high permeability. The chemicals are listed inthe CDER/FDA Waiver Guidance (2000) as recommended drugsfor permeability classification and were tested at least in 12independent inserts.

The permeability assays were performed, at day 23, in api-cal (AP) to basolateral (BL) direction to evaluate compoundabsorption. Ranitidine and biperiden were studied in both APto BL, or BL to AP direction, to evaluate compound efflux. Effluxratios (BL–AP/AP–BL) were calculated.

The highest dose strength dissolved in 250 ml of transportbuffer (Hank’s balanced salt solution), or initial concentration,was applied to the donor site (i.e. either the AP or BL compart-ment). During the experiments, samples were taken from thereceiver compartment at 20, 40, 60, 80, 100 and 120 min, andreplaced by an equal volume of fresh buffer.

2.4. Quantification and data analysis

All sample drugs were analyzed with a reversed-phaseHPLC system (Shimadzu LC-10Avp) equipped with a vari-

able wavelength UV detector (Shimadzu SPD-10Avp). Allanalytical methods were validated for each compound accord-ing to international recommendations (USP 32-NF 27, 2009;CDER/FDA, 2001).

p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 223–227 225

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The apparent permeability coefficients (Papp × 10−6 cm/s)ere calculated from the following equation:

app = dc

dt× VR

A × C0

here dC/dt is the drug flux (�g cm3/s), VR is the volume ofeceiver chamber (ml), A is the support membrane area (cm2),nd C0 is the initial drug concentration in the donor chamber

�g/ml). The flux was calculated by means of a linear regres-ion analysis based on the slope obtained from the graphicelated to the amount of drug accumulated in the receiverhamber over time.

. Results

.1. Reference drugs

able 1 shows initial concentrations and apparent permeabil-ty results for the five selected drugs in our Caco-2 model

ith their respective human absorption fraction (HIA) and BCSlass obtained from literature. The mean values were similaro previous results, which had established method suitabil-ty of Caco-2 assay. As it has been reported (Artursson andarlsson, 1991; Volpe, 2008) at least two groups of permeabil-

ty values could be clearly divided: the high permeability grouptheophylline, metoprolol tartrate and sodium naproxen), withIA between 90% and 100% and Papp values of approximately1 × 10−6 cm/s and the low permeability group (atenolol andanitidine hydrochloride) with HIA between 0% and 89% and

app values of <1 × 10−6 cm/s in AP to BL transport direction.Also, taking into account that secretive-transport com-

ounds show an efflux ratio (BL–AP/AP–BL) > 2 (Yee, 1997) weonfirmed that, in our model, ranitidine is subjected to thisind of transport (BL–AP/AP–BL = 2.79), possibly acting as P-gpubstrate (Collett et al., 1999). Compounds passively trans-orted by the paracellular or transcellular route exhibit aimilar flux in both directions.

As our permeability results agreed with other author’s pub-ications and a good correlation was obtained between data onral absorption in human and the results in our model, we con-idered that with the 5 selected substances we could certainlyetermine biperiden permeability.

.2. Biperiden

iperiden’s TEER values are shown in Table 2. As mentionbove, monolayers with TEER values exceeded 550 � cm2 onay 23 and pre-diffusion values higher than 200 � cm2 weresed.

In the same table, it can be seen that TEER values kepttable along the assay (by Student’s t test, there was no sig-ificant differences between pre and post-diffusion values),hich indicates that the monolayer maintained its integrity.

From our experimental conditions, the calculated biperi-en Papp was 82.9 ± 5.6 (×10−6 cm/s).

Taking into account that AP–BL biperiden permeabilityalue was higher than those obtained for high permeabilityre-defined drugs (Table 1), we can conclude that, in our sys-em, biperiden belongs to the high permeability group.

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226 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 223–227

Table 2 – Monolayer parameters used for biperiden assays and permeability values.

TEER (� cm2) Papp (×10−6 cm/s)Cc0 = 0.02 mM

Ratio BL–AP/AP–BL

Day 23 Pre-diffusion Post-diffusion

AP–BL (n = 11) 912 ± 139 528 ± 160 410 ± 122 82.9 ± 5.6 1.17BL–AP (n = 10) 849 ± 73 389 ± 126 391 ± 123 96.6 ± 10.4

Millicell-ERS® TEER values at 23 days of culture and VCC MC6 TEER pre and post-diffusion assays. Biperiden apparent permeability (Papp) anddevia

r

its BL–AP/AP–BL ratio. All results are expressed as mean ± standardassayed.

On the other hand, as no significant differences wereobtained for biperiden permeability values when AP–BL andBL–AP fluxes were studied, as other authors have been pro-posed (Mahar Doan et al., 2002; Varma et al., 2005), this drugdid not act as efflux transporters substrate such as P-gp(Table 2).

4. Discussion

Considering our conditions, biperiden is a high permeabilitycompound and beside the fact that it is “highly soluble” (WHO,2006), it would be classified as BCS class I.

This result agrees with Kasim et al. (2004) and Varma et al.(2005) BCS classification. As mention before, Kasim classi-fied biperiden as BCS class I based on the Log P data. Varma,however, collected permeability data from Mahar Doan whoconducted in vitro assays in Multidrug Resistance TransfectedMadin Darby canine kidney type II (MDRI-MDCKII) cell line.Despite the fact that Caco-2 cells are the most popular cel-lular model in transport studies, this alternative cell line hasgained popularity in recent years mainly because they takeonly 3–7 days to grow into a suitable monolayer (Irvine et al.,1999). Both in vitro Caco-2/MDCK methods are acceptable toFDA for the purpose of BCS classification since similar Papp val-ues have been noted between them (Irvine et al., 1999; Spiceret al., 2002).

On the other hand, our result was different fromLindenberg et al. (2004) who classified this drug as BCS classIII. As they mention in the paper, this drug was one of whichcomplete solubility and/or permeability data were lacking andhighlighted that biperiden has a large first-pass metabolism.Analyzing the references that they used to assigned this provi-sional classification (Grimaldi et al., 1986), it could be observedthat the information was mainly collected from a pharma-cokinetic and pharmacodynamic study made in 6 volunteers.As the aim of this scientific research was to obtain the sys-temic availability they did not performed a permeability assayto confirm the BCS class.

To summarize, according to the information we have, thisarticle is the first one about biperiden permeability carried outon Caco-2 model.

In South America, the incidence of Parkinson’s disease is31.16 in every 100,000 people (Bauso et al., 2009). Anticholiner-

gic monotherapy is used early in the course of the disease todelay the need for later L-dopa treatment, which may permita reduction in its dose required for more advanced cases. Theincorporation of biperiden as a BCS class I drug would allow

tion, Cc0 is the initial concentration and n is the number of insert

a biowaiver, according to 758/09 ANMAT Provision (ANMAT,2009), which would possibly eliminate unnecessary drug expo-sure to healthy subjects, reduce regulatory burden, and godown the cost of bringing a product onto the market.

Conflict of interest statement

The authors declare that are no conflicts of interest.

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