Effect of P-glycoprotein inhibitor, verapamil

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Effect of P-glycoprotein inhibitor,verapamil,on oral bioavailability and pharmacokinetics of irinotecan in rats Tripta Bansal a,b,?,Gautam Mishra b,Manu Jaggi b,Roop K.Khar a,

Sushama Talegaonkar a,?

a Dept.of Pharmaceutics,Faculty of Pharmacy,Jamia Hamdard(Hamdard University),Hamdard Nagar,New Delhi110062,India

b Dabur Research Foundation,22Site IV,Sahibabad,Ghaziabad,U.P.201010,India

a r t i c l e i n f o

Article history:

Received15March2008

Received in revised form

17November2008

Accepted11December2008

Published on line24December2008

Keywords:

Irinotecan(CPT-11)

Verapamil

Bioavailability

P-glycoprotein

Biliary excretion

a b s t r a c t

The objective of present investigation was to study the effect of verapamil on the pharma-

cokinetics of irinotecan in order to evaluate the role of P-glycoprotein(P-gp)in irinotecan

disposition.An in vitro study using Caco-2intestinal cell monolayer was?rst carried out to

determine the effect of verapamil on the function of intestinal P-gp.Verapamil(25mg/kg)

was administered orally2h before irinotecan oral(80mg/kg)or intravenous(20mg/kg)dos-

ing in female Wistar rats.Plasma and biliary samples were collected at speci?ed time

points from control and treated animals to determine irinotecan and its metabolite,SN-

38concentrations.Bi-directional transport and inhibition studies in Caco-2cells indicated

irinotecan to be a P-gp substrate and the function of intestinal P-gp was signi?cantly

inhibited in presence of verapamil.After oral irinotecan dosing,the mean area under

the plasma concentration–time curve(AUC)was found to be14.03±2.18?g h/ml which

was increased signi?cantly,i.e.61.71±15.0?g h/ml when verapamil was co-administered

(P<0.05).Similarly,the mean maximum plasma concentration of irinotecan increased from

2.93±0.37?g/ml(without verapamil)to10.75±1.0?g/ml(with verapamil)(P<0.05).There

was approximately4–5-folds increase in apparent bioavailability.On the other hand,the

intravenous irinotecan administration with verapamil resulted in small but statistically

signi?cant effect on AUC(10.76±2.0to23.3±3.8?g h/ml;P<0.05)and systemic clearance

(1206.4±159.7to713.5±78.2ml/(h kg)).In addition,SN-38showed signi?cant change in oral

pharmacokinetic parameters and minor changes in intravenous pharmacokinetic pro?le.

Biliary excretion curves of both irinotecan and SN-38were lowered by verapamil.The mean

percent of irinotecan excreted into bile over5h following intravenous and oral adminis-

tration was found to be8%and1%,respectively,which was further reduced to half when

treated with verapamil.These results are quite stimulating for further development of a

clinically useful oral formulation of irinotecan based on P-gp inhibition.

?2009Elsevier B.V.All rights reserved.

1.Introduction

Irinotecan(CPT-11),a water-soluble camptothecin,is the agent

of choice for treatment of metastatic carcinomas of colon

?Corresponding authors at:Dabur Research Foundation,22Site IV,Sahibabad,Ghaziabad,U.P.201010,India.Tel.:+919212118120;

fax:+911204376902.

and rectum.It exerts anti-tumor activity by inhibiting the

intranuclear enzyme topoisomerase I.Irinotecan itself is a

prodrug and gets converted in vivo to100–1000times more

potent active metabolite,7-ethyl-10-hydroxy camptothecin

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(SN-38)by carboxylesterase enzyme.Majority of the studies performed with irinotecan are using intravenous administra-tion(Chu et al.,1997;de Forni et al.,1994;Gupta et al.,1996;Iyer et al.,2002).However,it has been given orally in early clinical studies and its pharmacokinetic pro?le is characterized by rel-atively poor and highly variable oral bioavailability(Drengler et al.,1999;Schoemaker et al.,2005;Soepenberg et al.,2005; Zamboni et al.,1998).The main adverse effects of irinotecan in humans are gastrointestinal toxicity and myelosuppression which limits its usage and administration(Mathijssen et al., 2001;Yang et al.,2005).Since biliary excretion of irinotecan and its metabolites is a major elimination pathway,account-ing for about60%of the administered dose,several hypotheses for its toxicity directly involve the biliary excretion of these compounds(Chu et al.,1998).

The involvement of active transporters particularly P-glycoprotein(P-gp)in the transport of both irinotecan and SN-38is demonstrated by various researchers(Itoh et al., 2005;Iyer et al.,2002;Luo et al.,2002;Takemoto et al., 2006;Yamamoto et al.,2001).P-gp is the major ef?ux trans-porter protein responsible for poor absorption of many drugs. Presence of P-gp transporter in intestinal epithelial mem-brane and biliary canalicular membrane makes its inhibition a logical strategy to enhance irinotecan oral bioavailability and ameliorating diarrhoeal toxicities.Verapamil is the most extensively characterized P-gp inhibitor and multi-drug resis-tance(MDR)reversal agent that has entered clinical trials (Perez-Tomas,2006).It is also reported that verapamil has increased the AUC of doxorubicin and paclitaxel in rodents (Candussio et al.,2002;Choi and Li,2005).Therefore,the effect of co-administration of verapamil on the oral bioavail-ability and pharmacokinetics of irinotecan is the subject of current investigation.Cyclosporin,PSC-833,ketoconazole, loperamide,probenecid and imatinib as the inhibitors of active transporters and cytochrome enzymes have modulated pharmacokinetics of irinotecan(Arimori et al.,2003;Gupta et al.,1996;Horikawa et al.,2002;Iyer et al.,2002;Kehrer et al., 2002;Liu et al.,1996;Stewart et al.,2004;Tobin et al.,2005). The main objective of the study is to evaluate the scope of improvement in oral delivery of irinotecan via co-treatment with oral P-gp inhibitor.P-gp inhibitors are generally admin-istered intravenously and no suggestion is made regarding their oral intake.Parenteral administration of P-gp inhibitors in therapeutic doses into humans may cause severe clinical consequences.This is because in addition to intestine,P-gp is present in hepatocytes,bile canaliculi,brain and kidney suggesting its role in distribution,metabolism and excretion. Consequently,clinical pharmacokinetic interactions could be anticipated when P-gp inhibitors are co-administered.An ideal P-gp inhibitor should give nearly maximal inhibition of P-gp transport locally at intestine with a minimal systemic effect and whole body burden(Dantzig et al.,2003;Varma et al.,2003).

The design of present study includes an initial in vitro investigation on Caco-2cells to evaluate the effect of verapamil on the function of intestinal P-gp.This was followed by determination of irinotecan pharmacokinetics these studies,it might be feasible to develop an oral irinote-can formulation which is much more convenient than the i.v. dosage forms.

2.Materials and methods

2.1.Chemicals and apparatus

Irinotecan(>99%),SN-38(>96%)and topotecan(>98%)orig-inated from Dabur Pharma Limited(U.P.,India).Verapamil, disodium ethylene diamine tetra acetic acid and non-essential amino acids were purchased from Sigma–Aldrich(St.Louis, MO,USA).Acetonitrile of HPLC grade was obtained from J.T. Baker(USA).O-phosphoric acid and DMSO were from Merck Ltd.(India).Ketamine(Aneket)and xylazine HCl(Xylaxin) were obtained from Neon Labs Ltd.(Mumbai,India)and Indian Immunologicals Ltd.(A.P.,India),respectively.Dul-becco’s modi?ed Eagle’s medium(DMEM)was from Caisson Labs.(North Logan,UT,USA),fetal bovine serum(FBS) from Cansera(Rexdale,Ontario,Canada),trypsin ethylene-diaminetetraacetic acid from Amresco(Solon,OH,USA), penicillin and streptomycin were from HyClone(Logan,UT, USA).All other chemical and reagents were of analytical or HPLC grade as appropriate and procured locally.Transport buffer refers to9.80g of Hank’s balanced salt solution(HBSS), 0.37g of sodium bicarbonate,3.50g of glucose,5.69g of N-[2-monohydroxyethyl]piperazine-N9-[4butanesulfonic acid] (HEPES),1.16g of sodium chloride and made up to1l with Milli-Q water and pH was adjusted to7.4using1M NaOH or1M HCl. Transport buffer was sterilized by?ltering through a0.2-?m ?lter.All other chemicals and reagents were of analytical or HPLC grade as appropriate and procured locally.

The apparatus used consisted of liquid chromatographic system(LC-2010C HT series,gradient?ow control pump,on-line solvent degasser,autosampler,diode array detector and column oven controlled by LC solutions software version1.21 SP1,Shimadzu Corp.,Nakagyo-ku,Kyoto,Japan),vortexer (MultiReax Heidolph,The Labware House,London,UK),vac-uum dryer(Savant DNA SpeedVac,NJ,USA),microcentrifuge (Heraeus Labofuge,Thermo Scienti?c,MA,USA)and bath son-icator(Branson,USA).

2.2.In vitro studies

2.2.1.Cell culture

The Caco-2cells(HTB-37)originating from a human colorec-tal carcinoma was obtained from the American T ype Culture Collection(Rockville,MD,USA)at passage number17.Cells were cultured in DMEM supplemented with20%FBS,1.0mM sodium pyruvate,0.1mM non-essential amino acids,and 100U ml?1penicillin and streptomycin.Cells were maintained by serial passaging in T-75tissue culture?asks(Nalge Nunc Int.,NY,USA)when reached70–80%con?uence at a split ratio of1–5to1–7.Cells were grown in an atmosphere of5%CO2 and90%relative humidity at37?C and given fresh medium every3–4days.Viable cells were counted using the trypan blue

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2.2.2.Bi-directional transport studies

For transport studies,cells were seeded in6-well plates at a density of3.0×105cells/insert in3.0-?m pore-size25mm i.d.polycarbonate tissue culture inserts(Nalge Nunc Int.,NY, USA).The culture medium(1.5ml in the insert and2.6ml in the well)was replaced every3days for?rst7days and every2days thereafter.Cells were used for transport exper-iments at passages21–27at21–25days after seeding.The trans-epithelial electric resistance(TEER),expressed in cm2, was measured using a Milli-cell-ERS apparatus(Millipore,Bed-ford,MA,USA)at room temperature.In addition to routine TEER measurements,the paracellular transport marker lucifer yellow was used to con?rm the integrity of Caco-2monolay-ers.Lucifer yellow(excitation485nm and emission530nm) and rhodamine123(excitation498nm and emission525nm) was quanti?ed by spectro?uorimetry using Varioskan Instru-ment(Thermo Electron Corp.,Waltham,MA,USA)controlled by Skan-It software version2.2.1.The monolayers used for the transport experiments had TEER values greater than400 cm2 and the leakage rate of lucifer yellow was less than1% per h.

On the day of experiment before performing the trans-port experiments,the cells monolayers were washed twice with phosphate buffer saline pH7.4to remove traces of culture media.After washing,the plates were incubated at37?C for30min and TEER was measured.TEER values measured for the inserts with cells were corrected by sub-tracting the values with bare?lter inserts.Transport buffer on both sides was then removed gently by aspiration.For the measurement of apical-to-basolateral transport(A–B), 1.5ml of transport buffer containing drug solution(irinote-can,1,10and100?M)was added to the apical side and 2.6ml of blank transport medium to basolateral compart-ment.For the investigation of basolateral-to-apical transport, drug was placed in the basolateral side and blank media on the other side.Drug containing compartment is referred to as donor compartment whereas samples withdrawing compartment is the receptor/acceptor compartment.Sam-ples of200?l were withdrawn from acceptor compartment at respective time points of30,60,90and120min.The vol-ume drawn was replenished with the blank transport buffer every time.Rhodamine123is a typical P-gp substrate that is subjected to P-gp dependent extrusion through the api-cal membrane.It is a model compound for studying P-gp mediated transport due to its excellent?uorescent properties and transport characteristics(Troutman and Thakker,2003). Therefore,before conducting experiments with irinotecan,the P-gp expression in cell monolayer was ascertained by carry-ing out bi-directional studies using rhodamine123at1?M concentration.

2.2.

3.Inhibition studies

For inhibition studies,verapamil(100?M)was added to the transport buffer in both apical and basolateral sides.Vehicle was used for the control inserts.Experiment was performed in shaker incubator at37?C and50–60rpm(Irvine et al.,1999). The samples collected from each time point were stored at

Table1–Experimental groups(n=5in each group).

Group Irinotecan

administration

Verapamil administration

I20mg/kg,i.v.bolus Control

II20mg/kg,i.v.bolus Pre-treatment,25mg/kg,PO

III80mg/kg,PO Control

IV80mg/kg,PO Pre-treatment,25mg/kg,PO 2.3.In vivo studies

2.3.1.Animals and drug administration

Healthy female Wistar rats(180–200g,n=5)used for pharma-cokinetic studies were obtained from breeding stock of Dabur Research Foundation(Ghaziabad,U.P.,India).Rats housed in cages were kept in a room under controlled temperature (20–22?C)and12h day–night cycle.Animals were used for pharmacokinetic studies after1-week acclimatization with free access to water and feed.All animal procedures were approved by Institutional Animal Ethics Committee(Dabur Research Foundation,U.P.,India).

For pharmacokinetic studies,irinotecan hydrochloride injection(20mg ml?1)manufactured by Dabur Pharma Lim-ited(U.P.,India)was used and the dose was selected on the basis of existing reports(Gupta et al.,1996;Mathijssen et al., 2001).Pilot studies after intravenous and oral dosing,were also carried out to ensure that the drug levels are above quantita-tion limits.Verapamil dose(25mg/kg)is selected on the basis of the nature of study in reference to reported values(Varma and Panchagnula,2005).Verapamil solutions were prepared in distilled water containing5%DMSO.The potency of both irinotecan and verapamil solutions was>96%and was checked before administration into rats by HPLC.Rats were divided into four groups and treated as per Table1.Separate sets of animals were used for biliary excretion studies.

2.3.2.Intravenous studies

The control group received the target dose of irinotecan (20mg/kg)through intravenous via the lateral tail vein.The pre-treatment group received oral verapamil(25mg/kg),2h prior to irinotecan(20mg/kg,i.v.)administration.Control groups received vehicle without verapamil in the similar way. Blood samples were withdrawn prior to dosing and at0.083, 0.25,0.5,1,2,4,6,8and10h post-dosing from retro-orbital plexus into microtubes containing disodium ethylene diamine tetra acetic acid.Plasma was obtained by immediate centrifu-gation at1500×g for10min at4?C.A100-?l aliquot of the plasma was processed to determine levels of irinotecan and SN-38,as described in the following sections.Supernatant after protein precipitation was stored at?80?C until HPLC analysis.

2.3.3.Oral studies

Rats were fasted overnight before oral dose administration and approximately3h post-dose.The control group received the target dose of irinotecan(80mg/kg)orally2h after blank vehicle,via gavage(80mg/kg,PO)using a ball-tipped needle. The pre-treatment group received oral verapamil(25mg/kg),

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4,6,8,10,12and24h post-dosing from retro-orbital plexus into microtubes containing the anti-coagulant.Plasma was obtained immediately and processed by protein precipitation. Samples were stored at?80?C until HPLC analysis.

2.3.4.Biliary excretion studies

Verapamil(25mg/kg,PO)was given to pre-treatment groups and control groups received vehicle2h before cannulation. Bile cannulation experiments were carried out as described previously(Chu et al.,1997).Brie?y,after induction of anes-thesia with ketamine(90mg/kg,i.p.)and xylazine(10mg/kg, i.p.),the rat was placed in dorsal recumbency and a midline abdominal incision made starting at the xiphoid cartilage and extending caudally about halfway down the abdomen(≈4-cm long in a rat).The duodenum and a small part of the intestine is pulled out and placed on a saline-moistened gauze pad.The course of the bile duct through the pancreatic tissue is traced. The major lobes of the liver were moved cranially against the diaphragm.A portion of the bile duct near the hilum of the liver was identi?ed.The duct was catheterized near the hilum in order to collect pure bile without contamination of pancre-atic juices.Forceps were used to carefully clear the bile duct of surrounding connective tissue for a distance of approxi-mately1cm.A doubled-up length of suture under the bile duct is passed and cut to create two separate threads.One suture is tied to create a ligature just cranial to the pancreatic tis-sue to obstruct bile?ow.A PE10polyethylene tubing(inner diameter,0.28mm;outer diameter,0.61mm;Harvard Appara-tus,Holliston,MA,USA),was cut so that a short beveled point is obtained.The bile duct near to the?rst ligature was held with?ne forceps and partially transected with micro-scissors. The catheter is introduced into the duct towards the liver and advanced until several mm past the cranial suture.This suture is tightened and the knot completed to secure the catheter in the bile duct.Bile should be visible within the lumen of the catheter.During the experimental period body temperature of the rats was maintained at37?C with a heating pad.Bile (～1ml at each time interval)was collected at0–1,1–2,2–3,3–4 and4–5h intervals after irinotecan oral and i.v.administra-tion and at5h,the rats were sacri?ced.A100-?l aliquot of the bile was processed as described and the sample was stored at ?80?C until analysis.

2.4.Determination of irinotecan and SN-38by HPLC

A new,simple,sensitive and speci?c reversed-phase high performance liquid chromatographic(HPLC)method using ultraviolet detection was developed and validated for the analysis of irinotecan( max=254and365nm)and its major active metabolite,SN-38( max=380nm),in rat plasma and bile(Bansal et al.,in press a,b).The sample pre-treatment from plasma involved a single protein precipitation step with cold acetonitrile.In case of bile,liquid–liquid extrac-tion with dichloromethane:tert-butyl methyl ether(3:7)was carried out.Topotecan,a structurally related camptothecin, was used as an internal standard.An aliquot of50?l was injected onto a C18column.The chromatographic separation was achieved by gradient elution consisting of acetonitrile 30min.All the analytes viz.topotecan,irinotecan and SN-38 were well separated with retention times of11.4,13.4and 15.5min,respectively.Method was found to be selective,linear (R2≈0.999),accurate(recovery±15%)and precise(<5%C.V.) in the selected concentration ranges for both the analytes. The quanti?cation limit for irinotecan was40ng ml?1and for SN-38was25ng ml?1.The percent extraction ef?ciencies of irinotecan and SN-38were92–97%for plasma and57–70%for bile.

Plasma proteins were precipitated by the addition of ice cold10?g ml?1topotecan(internal standard)in acetonitrile containing0.1%glacial acetic acid(1:1ratio).After rigorous vortex mixing for5min,the mixtures were centrifuged at 3000×g for10min at4?C.An aliquot of150?l supernatant was transferred to a fresh tube and50?l was injected onto column for analysis.

A100-?l bile sample was transferred to an eppendorf tube and spiked with10?l of100?g ml?1topotecan(inter-nal standard).Feasibility of various solvent mixtures such as ethyl acetate,dichloromethane(DCM),tert-butyl methyl ether(TBME),diethyl ether,n-hexane was tested and?nally DCM:TBME(3:7)mixture provided adequate speci?city and sensitivity.1ml of DCM:TBME(3:7)was added and the mixture was vortexed followed by shaking for10min.Subsequently, centrifugation(3000×g for10min)was performed to separate the aqueous and organic layer.A900-?l of organic layer was transferred to a clean microtube and evaporated to dryness under vacuum at40?C.The dried extracts were redissolved by vortexing and sonication in150?l20:80(v/v)acetonitrile:water (pH3.0),of which50?l was injected onto the HPLC system. The percent extraction ef?ciency of irinotecan and SN-38in plasma was found to be in the range of92–97%and that in bile was found to be in the range of57–70%.The method was successfully used to determine plasma and biliary excretion time pro?les of irinotecan and SN-38,following oral and intra-venous irinotecan administration in rats.

2.5.Data treatment

2.5.1.Calculation of P app across Caco-2cells

Results of bi-directional transport and inhibition studies are expressed as permeability coef?cient(nm s?1),which was cal-culated using the equation:

P app=d Q

d T

CA(1)

where d Q/d T is the rate of appearance of compound in the receptor chamber in nmoles s?1,C is the substrate concen-tration in the donor chamber in micromolar and A is the cross-sectional area of the cell monolayer in cm2.

2.5.2.Pharmacokinetic calculations

Pharmacokinetic parameters were calculated by non-compartment model using WinNon-Lin 5.0programme (Pharsight,Mountain View,CA,USA).The plasma irinotecan and SN-38concentration versus time curves were used to determine maximum plasma concentration(C max),time to

584e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s36(2009)580–590

the respective sampling point(AUC0–t),volume of distribution

(V d),elimination rate constant(K el),half-life(t1/2)and total

body clearance.C0was the initial plasma concentration of

drugs obtained by back-extrapolation to y-axis.The absolute

bioavailability(F)of irinotecan after the oral administration

(80mg/kg)compared to the intravenous(i.v.)administration

(20mg/kg)was calculated as follows:

F=AUC oral i.v.dose

AUC IV oral dose

×100(2)

The relative bioavailability(RB%)of irinotecan was calculated

as follows:

RB%=AUC verapamil treated

AUC control

×100(3)

The amounts of irinotecan and its metabolite,SN-38excreted in bile over4h were expressed as percentages of total dose of irinotecan administered in each rat.Biliary excretion clear-ance(CL bile)de?ned with respect to the plasma concentration of drug was determined using following equation:

CL bile=X bile(0?4h)

AUC(0?4h)

(4)

where X bile,0–4h is the cumulative amounts of drug excreted in bile over4h.

2.6.Statistical analysis

Data of?ve different experiments/animals were reported as mean±standard deviation(S.D.)unless otherwise noted. Statistical analysis was performed using GraphPad prism soft-ware version4.0(San Diego,CA,USA).Student’s unpaired t-test was used to test the signi?cance of differences between the controls and treated groups.Differences between the con-centration time pro?les over the entire range tested were analyzed by two-way ANOVA(Bonferoni post-test).The dif-ferences were considered to be signi?cant at P<0.05.

3.Results

3.1.In vitro studies

3.1.1.Bi-directional transport of irinotecan in Caco-2cells

No signi?cant change was observed in the TEER value (>400 cm2)measured before(0h)and after(2h)completion of transport studies.The transport rate of lucifer yellow was also not changed in the presence of various P-gp modulators. The results obtained with rhodamine123indicated its polar-ized transport(P appB–A:128±12nm s?1;P appA–B:5±2nm s?1) similar to that reported earlier(Troutman and Thakker,2003), enabling the complete standardisation of Caco-2cell mono-layer(Irvine et al.,1999).Absorptive and secretory transport of irinotecan was determined as a function of concentration. Determination of the ratio of apical permeability to basal per-meability(known as,ef?ux ratio)provides the most direct way of identifying P-gp substrates where the ratio is compared

Fig.1–Bi-directional transport of irinotecan(IRI)across Caco-2cells at different concentrations.The apparent apical-to-basal permeability coef?cient(P appA–B,black bars) of irinotecan(10?M)was increased and the basal-to-apical permeability(P appB–A,open bars)was decreased as the P-gp inhibitor verapamil(100?M)was added.Each value is mean±S.D.of three determinations.Statistical signi?cant difference between irinotecan alone P app value and that obtained after co-treatment with P-gp inhibitor;*P<0.05,

**P<0.01.

entire range of1–100?M,indicating the apically directed ef?ux of irinotecan.The ef?ux ratio was much higher(i.e.close to16) at1and10?M as compared to that at100?M concentration.

3.1.2.Inhibition studies of irinotecan with verapamil

across Caco-2cells

Addition of verapamil(100?M)to both compartments dimin-ishes the P-gp mediated ef?ux.Inhibitory effect produced is seen through a decrease in irinotecan B→A transport and an increase in A→B transport.Verapamil(100?M)signi?cantly inhibited the irinotecan B→A transport and improved A→B transport.The change in P app values is depicted in Fig.1.A–B and B–A P app values for irinotecan transport after addition of verapamil was found to be25±6and70±12nm s?1,respec-tively,giving the net ef?ux ratio to2.8.

3.2.In vivo studies

3.2.1.Pharmacokinetics of irinotecan in the absence and presence of verapamil in female Wistar rats

Plasma concentration versus time curves of irinotecan follow-ing i.v.(20mg/kg)and oral(80mg/kg)administration in the absence and presence of a concomitant verapamil oral dose (25mg/kg)are depicted in Fig.2.Pharmacokinetic parameters of irinotecan are mentioned in Tables2and3.Irinotecan was absorbed rapidly after oral administration and the observed time to peak irinotecan and SN-38levels was within2h of administration.After an initial absorption phase,plasma concentrations of irinotecan declined monoexponentially. Verapamil administration was associated with an increase in irinotecan and SN-38plasma concentrations following both i.v.and oral administration.Oral irinotecan concentra-

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585

Fig.2–Plasma concentrations time curves of irinotecan (CPT-11)following its(A)intravenous administration at

20mg/kg and(B)oral administration at80mg/kg in control (-?-)and pre-treated(- -)female Wistar rats.The

pre-treated groups were administered an oral dose of

25mg/kg verapamil,2h before the irinotecan dose.Data are expressed as mean±S.E.M.in n=5rats.**Statistical

signi?cant difference between control and

verapamil-treated rats,P<0.01.

Table2–Pharmacokinetic parameters of irinotecan after intravenous administration with and without

pre-treatment with oral verapamil(25mg/kg)in Wistar rats.

PK parameter of irinotecan

Irinotecan,IV,20mg/kg Control With verapamil

AUC0–last(h?g/ml)10.76±2.023.2±3.8*

C0(?g/ml)9.51±1.48.02±1.34 MRT(h) 2.47±0.41 3.45±0.86 t1/2(h) 3.1±0.60 3.7±0.44 CL obs(ml/(h kg))1206.4±159.7713.5±78.2*

V ss,obs(ml/kg)4852.2±703.83679.1±495.1* K el(h?1)0.232±0.050.174±0.01* RB(%)100216

Each value represents the mean±S.E.M.of?ve rats.

Table3–Pharmacokinetic parameters of irinotecan after oral administration in rats.

PK parameter of

irinotecan

Irinotecan,PO,80mg/kg

Control With verapamil AUC0–last(h?g/ml)14.03±2.1861.71±15.0**

C max(?g/ml) 2.93±0.3710.75±1.0*

t max(h) 2.6±0.89 1.75±1.8

MRT(h) 3.6±0.69 5.13±1.48

t1/2(h) 2.24±0.51 4.18±1.2*

CL obs(ml/(h kg))5613.8±1126.3897.09±177.9*

V ss,obs(ml/kg)32197.0±8067.17571.1±151.3*

K el(h?1)0.293±0.090.17±0.05*

F(%)33143

RB(%)100440

Each value represents the mean±S.E.M.of?ve rats.

?Statistical signi?cant difference between control and verapamil-treated rats,P<0.05.

??Statistical signi?cant difference between control and verapamil-treated rats,P<0.01.

14.03±2.18to61.71±15.0?g h/ml paralleled with a reduction in irinotecan CL from5613.8±1126.3to897.09±177.9ml/(h kg) and increase in half-life from2.24±0.51to4.18±1.2h.

The pharmacokinetic data for i.v.irinotecan with vera-pamil showed a relatively small(when compared against oral irinotecan)but signi?cant increase in AUC(10.76±2.0 to23.3±3.8?g h/ml;P<0.05)and decrease in clearance (1206.4±159.7to713.5±78.2ml/(h kg)),without affecting the C0(Table2).The oral bioavailability of irinotecan calculated from the ratio of AUC after oral and i.v.administration with the correction in difference of dose was33%with-out verapamil,which increased to144%when given with verapamil.

The plasma concentration versus time curves of irinotecan metabolite SN-38,after oral and i.v.administration are shown in Fig.3.There was no signi?cant difference between C0, MRT and elimination half-life following verapamil treatment. However,AUC last was increased8.7±0.49to10.3±1.3?g h/ml, though the change was not statistically signi?cant(Table4). Oral pharmacokinetic parameters of SN-38were signi?cantly modi?ed by verapamil(Table4).SN-38showed biphasic plasma disposition after oral administration,with a terminal half-life of about1–2h.Verapamil had no effect on SN-38con-centrations till4h but beyond that an increase in secondary peak area is noticed(Fig.3).This could be attributed to the saturation of carboxylesterase enzymes due to high irinote-can concentrations in the initial time points leading to lesser conversion of irinotecan to SN-38.There is signi?cant increase in AUC0–last(1.81±0.30to2.99±0.34?g h/ml;P<0.05),C max (0.48±0.07to0.62±0.06?g/ml;P<0.05)and MRT(3.84±0.67 to5.26±0.26h;P<0.05)(Table4).

3.2.2.Biliary excretion of irinotecan in the absence and presence of verapamil in female Wistar rats

The biliary excretion study of irinotecan and its metabolite, SN-38was conducted up to5h in female Wistar rats after i.v.and oral irinotecan administration.Verapamil decreases

586e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s36(2009)

580–590

Fig.3–Plasma concentrations of SN-38following irinotecan administration in control and pre-treated female Wistar rats.The control group were given(A)an i.v.bolus dose of20mg/kg irinotecan,(B)an oral dose of80mg/kg irinotecan,only.The pre-treated groups were administered an oral dose of25mg/kg verapamil,2h before the irinotecan dose.Data are expressed as mean±S.E.M.in

n=5rats.*Statistical signi?cant difference between control and verapamil-treated rats,P<0.05.

SN-38over5h after irinotecan administration,expressed as percentages of irinotecan dose are shown in Fig.4a–d.

Following i.v.administration,about8–9%of irinotecan dose was recovered unchanged in bile in control rats which got reduced to4%when treated with verapamil.Recov-ery of SN-38was minor(0.1–0.2%)in control as well as treated group.However,the amount of SN-38further showed a decrease in verapamil-treated rats.The pharmacokinetic parameters obtained are mentioned in Table5.AUC0–4h, cumulative amounts excreted(X bile,0–4h)and the biliary clear-ance(CL bile,plasma)for irinotecan was signi?cantly altered (P<0.05)in treated group as compared to the control group. In contrast,only the cumulative amount of SN-38excreted in bile showed statistical difference.Verapamil lowers the biliary excretion curves in both irinotecan and SN-38.

Following oral administration,about1%of dose is excreted into bile which gets further reduced to0.5%in presence of verapamil.SN-38levels also get decreased after vera-pamil treatment but to a lesser extent as evaluated against irinotecan.The pharmacokinetic parameters obtained are mentioned in Table5.Cumulative amounts of irinote-can excreted in bile over4h(X bile,0–4h)was decreased from1295.7±110.8to596.7±193.8?g/kg after i.v.dos-ing and597.0±79.4to353.5±37.1?g/kg after oral dosing. The biliary clearance expressed as CL bile,plasma declined from149.6±8.04to37.88±7.81ml/(h kg)and68.60±6.7to 11.01±3.5ml/(h kg)following i.v.and oral administration, respectively.X bile,0–4h and the biliary clearance(CL bile,plasma) for SN-38also decreased in treatment group versus the control group after oral and i.v.administration.

4.Discussion

Irinotecan,a FDA approved anti-cancer agent,is widely indi-cated for the treatment of various malignancies.It is currently marketed for intravenous use although few reports of its oral drug administration exist(Drengler et al.,1999;Schoemaker et al.,2005;Soepenberg et al.,2005;Stewart et al.,1997).Data available on its absorption and disposition showed encourag-ing results with variable absorption,poor ef?cacy and toxicity pro?les.As a result novel formulations of irinotecan exhibiting better oral absorption and bioavailability need to be devel-oped.Oral bioavailability is re?ected by the amount of drug absorbed,which is a function of solubility of the drug and its intrinsic permeability across the intestinal wall.Since the drug is having good water solubility,its physico-chemical param-eters are expected to play a minor role in determining oral bioavailability.However,several compounds of great thera-peutic relevance have been shown to have low bioavailability due to the P-gp mediated ef?ux occurring in small intestine (Breedveld et al.,2006;Choi and Li,2005;Jonker et al.,2000;

Table4–Pharmacokinetic parameters of SN-38,major metabolite of irinotecan,with and without pre-treatment with oral verapamil(25mg/kg),after oral(80mg/kg)and intravenous(20mg/kg)administration of irinotecan in rats.

PK parameter of SN-38Irinotecan,IV,20mg/kg Irinotecan,PO,80mg/kg

Control With verapamil Control With verapamil

AUC0–last(h?g/ml)8.7±0.4910.3±1.3 1.81±0.30 2.99±0.34*

C0(?g/ml) 2.10±0.61 1.92±0.60––

C max(?g/ml)––0.48±0.070.62±0.06*

t max(h)–– 2.8±0.45 4.2±1.64 MRT(h) 2.17±0.24 2.25±0.23 3.84±0.67 5.26±0.26*

t1/2(h) 1.1±0.16 1.1±0.34 1.47±0.35 1.28±0.26

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587

Fig.4–Biliary excretion of irinotecan(CPT-11)(A and C)and its metabolite SN-38(C and D),expressed as percent dose of irinotecan,following intravenous(A and B)irinotecan at20mg/kg and oral(C and D)irinotecan administration at80mg/kg in control rats(black bars)and rats pre-treated with25mg/kg verapamil(grey bars).Each bar represents the mean±S.D.of three different rats.

Kruijtzer et al.,2002;Luo et al.,2002).Therefore,an attempt was made to improve low and erratic absorption of irinotecan by inhibiting P-gp mediated ef?ux.

As shown in Fig.1,irinotecan P app,A–B remained more or less constant across the concentration range studied in contrast to P app,B–A.This could be due to the fact that P-gp mediated transport involves active carriers which are saturable at high concentrations.

Verapamil diminishes the secretory transport of irinotecan in Caco-2monolayer assay indicating that P-gp inhibition may result in increased bioavailability of irinotecan.Subsequently, pharmacokinetic studies of oral and i.v.administered irinote-can were conducted with verapamil in Wistar rats.

Rat is the best F a,human(fraction of drug absorbed in humans)predictor animal model for passive permeability and further there exists a similar level of P-gp expression (Stephens et al.,2001)and overlapped substrate speci?city with quantitatively same af?nity for a large number of P-gp substrates in rat MDR1a and human MDR1(Yamazaki et al., 2001).Therefore,pharmacokinetic studies in rat provide more

Table5–Pharmacokinetic parameters of irinotecan and SN-38pertaining to biliary excretion after oral(80mg/kg)and intravenous(20mg/kg)administration of irinotecan in rats.

Groups Parameter Unit CPT-11SN-38CPT-11SN-38

20mg/kg,IV80mg/kg,PO

Control AUC0–4h?g h/ml8.66±1.387.77±0.428.70±2.15 1.19±0.11 X bile,0–4h?g/kg1295.7±110.828.26±6.9597.0±79.420.36±1.67

CL bile,plasma ml/(h kg)149.6±8.04 3.64±0.9268.60±6.717.08±2.45 Verapamil pre-treatment AUC0–4h?g h/ml15.75±2.22*8.46±0.2332.10±4.89* 1.23±0.04* X bile,0–4h?g/kg596.7±193.8*12.85±3.27*353.5±37.1*16.98±2.47*

CL bile,plasma ml/(h kg)37.88±7.81* 1.52±0.28*11.01±3.5*13.84±3.12

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meaningful forecast on human absorption of P-gp substrates. It may bring new and interesting observations concerning hepatic extraction,biliary excretion,and lactone/acid inter-conversion.The selection of doses of irinotecan and verapamil was based on their pharmacokinetic/toxicological properties and the nature of study(Lokiec et al.,1995;Mathijssen et al., 2001;Varma and Panchagnula,2005).Verapamil increased the plasma concentration and AUC with a concomitant decrease in plasma clearance and volume of distribution(Fig.2and Tables2and3).The absolute bioavailability(F)of irinote-can was33%,which was increased to143%(4.3-fold)in the presence of verapamil administration.The relative bioavail-ability(RB%)of irinotecan co-administered with verapamil was very much higher(440%)than the same dose of irinotecan alone.The increased rate of absorption observed for irinote-can with verapamil treatment(Table3)could be attributed to its inhibitory effect on the P-gp present in intestine.The sig-ni?cant change in MRT and K el suggests the increased time for which the drug remains in the body.Monitoring plasma concentrations alone is insuf?cient for predicting the P-gp inhibitory effect on elimination processes(hepatic or renal); hence biliary excretion studies were performed.

In experiments,the bile?ow rate was not signi?cantly affected by drug administration.Biliary excretion of irinote-can was lower after oral administration as compared to i.v.administration indicating incomplete absorption of orally administered irinotecan(Fig.4and Table5).The low percent-ages of drug being excreted in bile could be due to species difference(mice,humans,etc.)and also the short duration of study(5h)as against24–48h(Iyer et al.,2002;Lokiec et al.,1995,1996).Verapamil further decreased biliary excre-tion,contributed by the inhibition of P-gp mediated transport in the liver.This resulted in increased AUC0–4h as evident from decrease in cumulative amounts of irinotecan and SN-38 eliminated over4h.It appears that the concomitant and syn-ergistic inhibition of P-gp present in rat intestine and liver is a plausible explanation for prominent increase in oral bioavail-ability of irinotecan.The possible mechanisms responsible for irinotecan-associated diarrhea and identi?cation of effec-tive agents for alleviating irinotecan-induced diarrhea have received a great deal of attention.One of the reasons behind this toxicity is high levels of SN-38and/or irinotecan retained in the intestine for prolonged periods of time and their bil-iary excretion as SN-38-glucuronide mediated by transporters such as P-gp(Araki et al.,1993).A biliary index(the prod-uct of the relative area ratio of SN-38to SN-38-glucuronide multiplied by that of irinotecan AUC)has been de?ned and found to be correlated with the degree of diarrhea in humans (Gupta et al.,1994).Our studies are in agreement with the hypothesis that the irinotecan-associated diarrhea may be ameliorated by inhibition of biliary excretion of irinotecan and/or SN-38caused by verapamil,a potent P-gp inhibitor. Therefore,co-treatment with P-gp inhibitor is a useful strat-egy for both bioavailability enhancement and prevention of diarrhea following irinotecan treatment.Oral formulations of irinotecan having better ef?cacy and less toxicity could be developed using appropriate P-gp inhibitor.However,fur-ther studies are required to compare the ef?cacy of verapamil ferences mediated by P-gp in both hepatic metabolism and intestinal transport may be an important contributing fac-tor in the wide interpatient variability in the disposition and toxicity of irinotecan.In addition,treatment with irinotecan should be monitored closely when drugs that are known to affect the biotransformation of SN-38are co-administered. The possibility of in?uence of CYP3A4drug substrates on irinotecan metabolism and its treatment outcome could also be explored.Our in vitro studies focuses on P-gp being one of the various possible mechanisms observed behind bioavail-ability enhancement because Caco-2cells is the most suitable human intestinal cell line expressing P-gp.However,other mechanisms could also exist.In order to identify the exact proteins involved in the active transport of irinotecan and SN-38,transfected cell monolayers,knock-out or mutant animals could be used.All this information would enable irinotecan administration and its use by different routes and schedules.

5.Conclusions

The simultaneous administration of verapamil signi?cantly modulates the oral bioavailability and pharmacokinetics of irinotecan.Oral absorption and bioavailability of irinotecan are markedly increased(4.3-fold)suggesting that its low sys-temic exposure after oral administration is,at least in part, due to its high af?nity for P-gp ef?ux pump.P-gp in the gastro-intestinal mucosa limits the absorption of orally administered xenobiotics and mediates its excretion into the bile.The effect of co-administration of verapamil on hepatic elimina-tion is evident from the decreased levels of irinotecan and its metabolite,SN-38in bile.The observed effect may be bene-?cial in a way to develop oral irinotecan dosage forms using safe P-gp inhibitors to improve its oral bioavailability.Clini-cally,concomitant verapamil and irinotecan treatment would allow dose reduction(about50–80%)and still achieve com-parable exposure of irinotecan.More importantly,the risk of intestinal toxicity could be substantially reduced because of reduced dose and lowered biliary secretion and accumulation. The results of this study could be utilized to evaluate different dosing strategies and methods of administration for irinote-can in humans.Such dose-optimization studies of irinotecan in combination with verapamil are currently underway.

Acknowledgements

One of the authors(T.B.)is grateful to the Council of Scienti?c &Industrial Research(CSIR),New Delhi,India for providing senior research fellowship.

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