pharmacokinetics and biliary excretion of mitoxantrone in rats

Pharmacokinetics and Biliary Excretion of Mitoxantrone in Rats

XINNING YANG, MARILYN E. MORRIS

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University ofNew York, Amherst, New York 14260-1200

Received 19 July 2009; revised 16 September 2009; accepted 12 October 2009

Published online 9 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22011

Abbreviationbiliary clearancdrug; P-gp, P-gmacokinetic rela

Corresponde4839; Fax: 1-71

Journal of Pharm

� 2009 Wiley-Liss

2502 JOURN

ABSTRACT: The objective of this investigation was to compare the observed biliary clearance(CLb) and % of dose excreted in the bile (PDb) of mitoxantrone with the predicted values obtainedfrom quantitative structure pharmacokinetic relationship (QSPKR) models. Blood and bilesamples were collected from bile duct cannulated rats after an intravenous bolus dose of 0.5 or2 mg/kg mitoxantrone, and the concentrations were measured by HPLC. Mitoxantrone plasmaconcentrations exhibited a tri-exponential profile with systemic clearance of 118� 6.8 mL/min/kg. After dosing, 6.08� 2.32% and 5.69� 0.59% of the dose were excreted into bile in unchangedform after a 3-h collection. CLb was 7.20� 4.54 and 7.46� 0.62 mL/min/kg after the two doses.With the co-administration of 10 mg/kg GF-120918, a P-glycoprotein and BCRP inhibitor, PDb

was reduced to 0.69� 0.07%, suggesting that BCRP or P-glycoprotein may play an importantrole in the biliary elimination of mitoxantrone. Using QSPKR models developed for the biliaryexcretion of cations/neutral compounds in rats, CLb and PDb of mitoxantrone were predicted as5.18 mL/min/kg and 7.21%, respectively, suggesting that the models could be used to predict thebiliary excretion of mitoxantrone. � 2009 Wiley-Liss, Inc. and the American Pharmacists Association J

Pharm Sci 99:2502–2510, 2010

Keywords: active transport; biliary cleara
nce; biliary excretion; breast cancer resistanceprotein; mitoxantrone; pharmacokinetics; P-glycoprotein; QSAR; QSPKR; rat
INTRODUCTION

Mitoxantrone is an anti-cancer drug used to treatbreast cancer, acute leukemia, and non-Hodgkin’slymphoma.1 It is a substrate of an efflux transporter,Breast Cancer Resistance Protein (BCRP), althoughit has been reported also to be transported by P-glycoprotein (P-gp) to a minor extent.2–5 Both BCRPand P-gp are members of the ATP binding cassette(ABC) family of efflux transporters and involved inbiliary excretion of compounds.6–13 After intravenousadministration of 14C-mitoxantrone in humans,18.3% of the radioactivity was excreted in the feceswithin 5 days, suggesting that there is biliaryexcretion of mitoxantrone or its metabolites.14 Thebiliary excretion of the parent drug, mitoxantrone,has only been directly measured using collectionsfrom a T-tube inserted during surgery in one patientwho had jaundice and hepatic impairment.15 At 96 h

s: BCRP, breast cancer resistance protein; CLb,e; PDb, % of dose excreted in the bile as parentlycoprotein; QSPKR, quantitative structure phar-tionship.

nce to: Marilyn E. Morris (Telephone: 1-716-645-6-645-3693; E-mail: [email protected])

aceutical Sciences, Vol. 99, 2502–2510 (2010)

, Inc. and the American Pharmacists Association

AL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 201

after dosing, 2.7% of the administered radioactivitywas eliminated in bile. However, the systemicclearance of mitoxantrone in this patient wassignificantly lower than that in patients with normalliver function (150 mL/kg/h vs. 239 mL/kg/h). Thedecreased systemic clearance in this patient may bepartially explained by impaired biliary excretion ofmitoxantrone due to abnormal liver function. There-fore, biliary excretion of mitoxantrone in the generalpopulation of patients may be higher than thisreported value.

The biliary excretion of mitoxantrone in rats hasnot been fully investigated. The percentage of thedose of mitoxantrone excreted in bile has beenreported in one published article and twoabstracts.16–18 In one study using isolated perfusedrat livers, 25.8� 2.6% of a 0.2 mg/kg dose wasexcreted into bile during 4 h, with the parent drugaccounting for about 5.5%. Recovery of drug in thebile decreased to 10.5� 3.1% following a 0.4 mg/kgdose and 2.5% of dose was eliminated in unchangedform.18 In another study using whole animals, 10.5–20.6% of the radiolabeled dose was found in bile overa 3-h period.16 A higher percentage (23.9%) wasreported in a study in bile-duct cannulated rats whenthere was a longer bile collection period (6 h);17

however, the percentage of unchanged drug in thebile was not determined.

0

PHARMACOKINETICS AND BILIARY EXCRETION OF MITOXANTRONE IN RATS 2503

We have previously reported an in silico method,quantitative structure pharmacokinetic relationship(QSPKR) model, for the prediction of biliary clearance(CLb) and % of dose excreted in bile (PDb) for bothhumans and rats.19 The models were based on anumber of physicochemical properties of compoundswhich can be easily calculated using softwareprograms, such as QSARis� (SciVision-AcademicPress, San Diego, CA), and thus can be applied atan early stage of drug discovery. The predictionperformance of the models was supported by internalvalidation using the ‘‘leave-one-out’’ method and byexternal validation through the use of test groupswhich were not used during the construction of themodels. To further verify the predictability of ourmodels, both CLb and PDb of unchanged mitoxan-trone were determined in bile duct-cannulated rats inthe present study and the experimental values werecompared with the predicted values from our QSPKRmodels.

MATERIALS AND METHODS

Materials

Mitoxantrone dihydrochloride, 5-sulfosalicylic aciddihydrate (5-SSA), triethylamine, sodium dihydrogenphosphate was purchased from Sigma–Aldrich (St.Louis, MO). Ametantrone (AMT, NSC 287513) wasobtained from the Drug Synthesis and ChemistryBranch, Developmental Therapeutics Program, Divi-sion of Cancer Treatment and Diagnosis, NationalCancer Institute, NIH (Bethesda, MD). GF-120918was purchased from API Services, Inc. (Westford,MA). All other reagents or solvents used wereanalytical or high-performance liquid chromatogra-phy (HPLC) grade.

Animals

Male Sprague–Dawley (SD) rats (300–430 g) werepurchased from Harlan (Indianapolis, IN). Animalswere housed in a temperature-controlled environ-ment with a 12-h light/dark cycle and with free accessto tap water and a standard diet. Animals wereacclimatized to this environment for at least 1 week.All the animal studies were approved by theUniversity at Buffalo Institutional Animal Careand Use Committee.

Pharmacokinetic Studies

Jugular vein cannulation was first carried out in ratsfollowing an intramuscular injection of 90 mg/kgketamine and 10 mg/kg xylazine (Henry Schein,Melville, NY). Bile duct cannulation was thenperformed. After the surgery, bile samples werecollected for 15 min before mitoxantrone administra-tion. A dose of 0.5 or 2 mg/kg mitoxantrone dissolved

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in saline at a concentration of 0.5 or 2 mg/mL wasthen administered to rats via a jugular vein cannula.Blood samples (200mL) were obtained from a jugularvein cannula at 0 (predose), 2, 7, 15, 30, 60, 90, 120,180, and 240 min after administration of mitoxan-trone. Bile samples were collected over 15-minintervals for 180 min after dosing. For the 2 mg/kggroup, bile was also collected over 5-min intervalsduring the first 15 min. For the inhibition study,10 mg/kg GF-120918 dissolved in ethanol: PEG-200:5% glucose (2:6:2) at a concentration of 3.3 mg/mL orvehicle was given to rats 5 min before the adminis-tration of 2 mg/kg mitoxantrone. Bile samples werecollected in preweighted Eppendorf tubes which wereweighted again after collection. The volumes of bilesamples were calculated based on the difference ofweight of the tubes before and after collection,assuming the density of bile is the same as water.The tubes were covered with aluminum foil to avoidlight exposure. Blood and bile samples were stored at�808C until HPLC analysis. Three animals were usedfor each group. The animals were kept underanesthesia throughout the whole experiment.

HPLC Analysis

The concentrations of mitoxantrone in rat plasma andbile samples were analyzed by a previously publishedreverse-phase HPLC method with modifications,20 asdescribed by An and Morris.21 In brief, 5mL of 5mg/mL ametantrone (internal standard) preparedin H2O was added to a 85mL plasma sample. Thetotal volume was made up to 100mL with H2O. Forthe standard curve, blank rat plasma was used and10mL of mitoxantrone at different concentrationswere added. Then, 25mL 5-SSA and 75mL acetonitrilewere added to precipitate proteins. The mixture wasvortexed vigorously and then centrifuged for 10 minat 14,000g at 48C. Seventy microliters of supernatantsamples were analyzed. The system was composed ofa 250 mm� 4 mm NUCLEOSIL1 C18 reversed phasecolumn (Macherey-Nagel Co., Bethlehem, PA), aWaters 1525 pump, a 2847 UV detector, a 717 plusautosampler and a Waters Breeze workstation. Thecolumn was preconditioned with mobile phaseisocratically at a flow rate of 1.0 mL/min. The mobilephase was a mixture of 10 mM sodium phosphatebuffer (pH 2.3, supplemented with 0.1% triethyla-mine) and acetonitrile (82:18). Absorbance of mitox-antrone and ametantrone was monitored at 610 nm.Retention times for mitoxantrone and ametantronewere �8.5 and 6 min, respectively. The standardcurve was linear over the concentration range of 5–2500 ng/mL. The intra-day and inter-day coefficientsof variation (CV%) were <15%.

The procedure was slightly modified for theanalysis of bile samples. Two to 95mL of bile werespiked with 5mL internal standard. The total volume

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

Figure 1. Plasma concentration versus time profiles ofmitoxantrone in rats. Data were simultaneously fitted witha three-compartment model using WinNonlin 5.0 software.Open and closed circles represent observed plasma concen-trations after intravenous dosing of 0.5 mg/kg (N¼ 3) and2 mg/kg (N¼ 3) mitoxantrone, respectively. Solid anddashed lines indicate plasma concentration curves pre-dicted by a three-compartment model used to simulta-neously fit the observed data altogether.

2504 YANG AND MORRIS

was made up to 100mL with mobile phase. Thecomposition of mobile phase was adjusted by chan-ging the ratio of sodium phosphate buffer toacetonitrile to 85:15. The retention times for mitox-antrone and ametantrone were �9.5 and 6 min. Thelowest limit of quantification (LLOQ) was 20 ng/mL,with a linear standard curve covering a range ofconcentrations from 20 to 2000 ng/mL.

Pharmacokinetic Analysis

The plasma concentration-time profiles of all the ratsreceiving 0.5 or 2 mg/kg doses were simultaneouslyfitted with two-compartment and three-compartmentmodels with first-order elimination using WinNonlinversion 5.0 (Pharsight, Mountain View, CA). Theinitial values for the pharmacokinetic parameterswere obtained by performing compartmental analysisfor each rat and then taking the average values.Iterative reweighting (reciprocal of the square ofpredicted concentration) was chosen as the weightingscheme. The selection of the final model was based ongoodness of fit, Akaike Information Criterion (AIC),and Schwartz Bayesian Criterion (SBC).

The amount of mitoxantrone excreted into bile(Xt1� t2) during each interval was calculated bymultiplying the bile concentration with the volumeof bile sample. Cumulative amount of mitoxantroneexcreted into bile (Xbile) over a certain time period wascalculated by adding all the Xt1� t2 within the period.The same data was also expressed in the form of % ofdose by dividing Xbile with the total dose admini-strated. The area under the plasma concentrationversus time curve to 180 min (AUC0–180 min) wascalculated for mitoxantrone using the trapezoidalmethod. Biliary clearance (CLb) was determinedusing the following equation:

CLbile ¼ Xbile;0�180 min

AUC0�180 min

QSPKR Analysis

Biliary clearance (CLb) and % of dose of mitoxantroneexcreted in bile (PDb) in rats were predicted usingQSPKR models previously developed.19 Mitoxantroneitself was not included in the training sets used toderive the QSPRK models. Briefly, the structure ofmitoxantrone was obtained from SciFinder scholardatabase (American Chemical Society, Washington,DC) and drawn using ChemDraw Ultra 8.0 software(CambridgeSoft, Cambridge, MA). A SMILES stringwhich was a unique code representing the compoundwas created based on its structure. A total of 116structural descriptors were calculated for mitoxan-trone based on the SMILES string, among which log Pand molecular weight values were calculated usingthe online version of KowWin program developed by


Syracuse Research Corporation (http://www.epa.gov/oppt/exposure/pubs/episuite.htm) and the other 114structural descriptors were calculated using theQSARis� version 1.2 software (SciVision-AcademicPress). QSPKR equations were derived by multiplelinear regression (MLR) using all compounds in thetraining sets and also using the subsets of cation/neutral compounds. In this study, these previouslydeveloped models were then used to predict the CLb

and PDb values of mitoxantrone, by applying thevalues of those structural descriptors of mitoxantroneinto the equations.

RESULTS

Pharmacokinetics of Mitoxantrone in Rats

After i.v. administration of a 0.5 or 2 mg/kg bolusdose, plasma concentrations of mitoxantrone exhib-ited multiexponential profiles, decreasing rapidlywithin the first 15 min and then declining slowlywith a long terminal phase (Fig. 1). After normalizedwith doses, the concentration profiles after 0.5 and2 mg/kg doses overlapped with each other and nosignificant difference was observed at any time point,suggesting proportional increase of concentrationsalong with doses. In addition, compartmental analy-sis with either two-compartment or three-compart-ment models revealed no difference in the values ofpharmacokinetic parameters between 0.5 and 2 mg/kg groups, further supporting linear pharmacoki-netics within this range. Therefore, all the plasmaconcentration data were simultaneously fitted. A

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Table 1. Summary of the Estimates for the Parameters of Two-Compartment or Three-Compartment Models Used toSimultaneously fit all the Plasma Concentrations of Mitoxantrone After Intravenous Dosing of 0.5 and 2.0 mg/kg

Two-Compartment Model Three-Compartment Model

Parameters Estimated CV% Parameters Estimated CV%

CL (mL/min/kg) 118 5.43 CL (mL/min/kg) 118 5.75

VC (mL/kg) 1300 15.8 VC (mL/kg) 1100 17.0

CLD (mL/min/kg) 103 14.4 CLD1 (mL/min/kg) 88.8 24.0

VP (mL/kg) 5400 12.8 VP1 (mL/kg) 1500 46.9

CLD2 (mL/min/kg) 47.8 31.1

VP2 (mL/kg) 5000 27.1

AIC 91.6 AIC 67.7

SBC 99.3 SBC 79.3

VC and VP (mL/kg) represent the volume of distribution of central compartment and peripheral compartment(s). CLD, CLD1, CLD2 (mL/min/kg) indicatedistribution clearances. Akaike Information Criterion (AIC) and Schwartz Bayesian Criterion (SBC) were used as the standards for diagnostics.


three-compartment model captured the concentra-tion profiles better than a two-compartment modeland had lower values of AIC or SBC, indicating thatpharmacokinetic profile of mitoxantrone was tri-exponential (Table 1, Fig. 1). Estimated values forCL, CLD1, CLD2, VC, VP1, and VP2 were 118� 6.8,88.8� 21.3 and 47.8� 14.9 mL/min/kg, and 1100�187, 1500� 703 and 5000� 1350 mL/kg, respectively.HPLC analysis did not detect the presence ofany metabolites of mitoxantrone in plasma(Fig. 2A). AUC(0–180 min) was calculated as 4.15�0.17 and 15.3� 1.83mg/mL min after dosing of 0.5 and2.0 mg/kg. Using the AUC(0–180 min) values andmeasured % of dose excreted into bile as the parentdrug during this interval, CLb of mitoxantronewas determined to be 7.20� 4.54 and 7.46�0.62 mL/min/kg, respectively.

Figure 2. HPLC chromatographs of rat plasmafor mitoxantrone (MX) and its metabolites were:(mitoxantrone in bile), 7.64 min (M1 in bile), and

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Biliary Excretion of Mitoxantrone in Rats

After a dose of 2 mg/kg, the concentration ofmitoxantrone in bile initially increased and reacheda peak concentration of 68.3� 13.4mg/mL around 10–15 min. Then, the concentrations gradually decreasedand the profile appeared to be bi-exponential with theterminal phase exhibiting a similar slope as that ofthe plasma concentration versus time profile(Fig. 3A). The concentrations in bile were muchhigher than those in plasma during the whole periodof study. The bile/plasma ratio ranged from 475 at30 min to 73 at 120 min, indicating that mitoxantronewas highly concentrated in bile and active effluxtransporters were involved. The cumulative amountof unchanged mitoxantrone excreted into bile over a180 min period after dosing, was 50.4� 4.45mg and

(A) and bile (B) samples. The retention times8.68 min (mitoxantrone in plasma), 9.69 min4.51 min (M2 in bile).


Figure 3. Bile and plasma concentrations of mitoxan-trone in rats after intravenous doses of 2 mg/kg (A) or0.5 mg/kg (B) mitoxantrone. Circles and triangles representsamples after 0.5 and 2.0 mg/kg dosing, respectively. Openand closed symbols indicate plasma and bile concentrations,respectively. The bars represent SD (N¼ 3).

Figure 4. Cumulative excretion of mitoxantrone in bile(PDb) in rats after administration of 0.5 mg/kg (circles) or2 mg/kg mitoxantrone (triangles) intravenously. The barsrepresent SD (N¼ 3).


accounted for 5.69� 0.59% of the administered dose(Fig. 4). Most of the drug appearing in the bile wasexcreted within the first hour after dosing, duringwhich 5.29� 0.55% of the total dose was eliminatedvia bile. The collection period of bile was extended to240 min in two rats, in which only an additional 0.07%and 0.08% of the dose were excreted into bile beyond180 min. Therefore, in the following studies, bile wascollected for 3 h.

Biliary excretion profiles were similar after the0.5 and 2 mg/kg doses. Peak concentrations (12.2�6.52mg/mL) were observed around 15 min after the0.5 mg/kg dose, with the terminal elimination phaseparalleling that of plasma concentrations (Fig. 3B).The bile/plasma concentration ratio varied from 32 at180 min to 337 at 30 min. The cumulative amount ofunchanged mitoxantrone excreted into bile reached a


plateau at �2 h after dosing and represented6.08� 2.32% of the total dose at 180 min (Fig. 4). Incontrast to plasma samples, metabolites of mitoxan-trone were present in bile. HPLC analysis indicatedthat there were two metabolites with retention timesof 7.6 min (metabolite 1, M1) and 4.5 min (metabolite2, M2), suggesting that the metabolites may be morepolar than the parent drug (Fig. 2B). However, ourdetermination of biliary excretion and clearance isbased on unchanged mitoxantrone.

Effect of GF-120918 on Biliary Excretion ofMitoxantrone in Rats

Involvement of efflux transporters in biliary excretionof mitoxantrone was further investigated in ratsco-administered GF-120918, a potent inhibitor ofBCRP and P-gp. In the presence of 10 mg/kg GF-120918, biliary excretion of mitoxantrone (2 mg/kg)was significantly inhibited during the whole studyperiod and the peak concentration dropped to2.95� 0.19mg/mL. The cumulative amount of intactmitoxantrone excreted into bile was only 0.69� 0.07%of the dose administered, which was about 12% of thevalue in the absence of GF-120918, suggesting thatBCRP or P-gp, or both, play a very important role inthe biliary excretion of mitoxantrone (Fig. 5). Bile flowrates varied from 25.4� 5.39mL/min at 5 min to14.5� 3.34mL/min at 180 min after dosing, whichwere in the normal range and similar with those inthe rats receiving mitoxantrone alone.

Prediction of Biliary Excretion of Mitoxantrone in Rats

The QSPKR model previously developed for predic-tion of % of dose excreted in bile (PDb) for cation/neutral compounds in rats is given below.19 Mitox-antrone was not included in the training set for

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Figure 5. Cumulative excretion of mitoxantrone in bile(% of dose administered) of mitoxantrone in rats afterintravenous dosing of 2 mg/kg mitoxantrone with (squares)or without (triangles) co-administration of 10 mg/kg GF-120918. The bars represent SD (N¼ 3).


QSPKR development for either PDb or CLb

PDb ¼ 1:17 � SHBint9 Acnt � 8:82 � SddssS

þ 100 � xvch5 þ 73:5 � MaxNeg þ 835:5

� xvch7 þ 0:351 � SsOH þ 16:0

� SaaNH acnt þ 25:1

ðR2 : 0:828;p < 0:01;Q2 : 0:731;p < 0:01Þ

(1)

Utilizing this equation, PDb of mitoxantrone waspredicted to be 7.21%, which was very close to theexperimental values, 6.08� 2.32% at 0.5 mg/kg and5.69� 0.59% at 2.0 mg/kg.

CLb of mitoxantrone was predicted to be 14.1 mL/min/kg based on the QSPKR model previouslydeveloped for all compounds in rats as following:

CLb ¼ 521:7 � xch9 þ 0:712 � Q � 2:47

� SsssN acnt � 11:4 � Hmin þ 8:37

ðR2 : 0:689;p < 0:01;Q2 : 0:618;p < 0:01Þ

(2)

A QSPKR equation (not previously published) wasalso developed for prediction of CLb for the subset ofcation/ neutral compounds in rats

CLb ¼ �5:84 � SssssC � 0:336 � SHBint3

þ 1:10 � SaasC acnt þ 1:37

ðR2 : 0:956;p < 0:01;Q2 : 0:889;p < 0:01Þ

(3)

Prediction of CLb for mitoxantrone from this modelwas 5.18 mL/min/kg, very close to the observedvalues (7.20� 4.54 mL/min/kg at 0.5 mg/kg and7.46� 0.62 mL/min/kg at 2 mg/kg). Overall, QSPKR

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models provided good prediction for PDb and CLb ofmitoxantrone.

DISCUSSION

Biliary excretion is an important excretory pathwayfor mitoxantrone and its metabolites. In a clinicalstudy, 18.3% (13.6–24.8%) of a radiolabeled dose(12 mg/m2) was recovered in the feces of patients.22 Inrabbits receiving i.v. bolus doses of 0.04, 0.20, and1.0 mg/kg of [14C] mitoxantrone, 29.5� 9.3%,27.6� 7.9%, and 28.3� 3.8% of the doses wereexcreted into bile within 6 h. The majority of theradioactivity in bile was found to be parent drug,which accounted for about 20% of the total dose.23 In astudy in rats, 23.9% of the radiolabeled mitoxantronewas excreted into bile over a 6-h period afterintravenous dosing of 1.0 mg/kg. When the collectionperiod was extended to 51 h, another 8.5% of the dosewas recovered in bile.17 These values representedboth unchanged drug and metabolites, since onlyradioactivity was measured. In the current study, wefocused on parent drug itself and found that biliaryexcretion of intact drug accounted for 5.69� 0.59% ofthe dose following a 2 mg/kg i.v. dose; the value wassimilar after dosing of 0.5 mg/kg mitoxantrone.Biliary excretion of parent drug occurred veryrapidly, with more than 90% of mitoxantrone-relatedmaterial excreted in bile appearing within the firsthour after dosing. Our results were in agreement witha previous study conducted in isolated perfused ratlivers, in which about 5.5% of a 0.2 mg/kg dose wasfound in bile in unchanged form.18 Recovery of parentdrug in bile decreased to 2.5% following the admin-istration of a higher dose (0.4 mg/kg) in that perfusedrat liver study, although the decrease might beattributed to impairment of hepatic function result-ing from liver toxicity occurring at the higher dose. Inour study, no toxicity was observed after i.v.administration of 0.5 and 2 mg/kg doses, and bileflow rates remained in the normal range during thewhole study period. This is likely due to the fact thatmitoxantrone also undergoes renal excretion and hasextensive tissue distribution,1,17 resulting in less liverexposure to the drug and thus less toxicity after its i.v.administration.

Throughout the study period, the concentrations ofmitoxantrone in bile were significantly higher thanthose in plasma with bile/plasma ratios ranging from32 to 475, suggesting the involvement of activetransport. In the current study, we found that almost90% of the biliary excretion of unchanged mitoxan-trone was inhibited by GF-120918 indicating thatBCRP or P-gp, or both, play an important role in thebiliary elimination of mitoxantrone. It is known thatmitoxantrone is a well-characterized BCRP substrate



but only a weak P-gp substrate.2–5 The involvement ofthese efflux transporters in the transport of mitox-antrone across the brain–blood barrier (BBB) hasbeen investigated using in situ perfused mouse brainpreparations.24 The study indicated that Bcrp was themajor transporter limiting permeability of mitoxan-trone across the BBB. PSC-833, a potent and specificinhibitor of P-gp, had no effect on the brain uptake ofmitoxantrone, while GF-120918, a P-gp, and BCRPinhibitor, exhibited a significant inhibitory effect onKin (brain transport coefficient) of mitoxantrone inwild type mice and an even stronger effect in mdr1aknock-out mice. Further investigations are necessaryto elucidate the transporters important in the biliaryexcretion of mitoxantrone.

Besides parent drug, there was also substantialbiliary excretion of mitoxantrone metabolites. HPLCanalysis revealed that there were two metabolitespresent in bile with shorter retention times thanmitoxantrone itself, suggesting that the metabolitesmay be more polar than the parent compound. Sincethe primary goal of this study was to compareexperimentally measured biliary excretion of mitox-antrone with that predicted by QSPKR models, nofurther investigations were conducted to identify theproperties of these metabolites. It is known thatmitoxantrone could be metabolized to mono- and di-carboxylic acid metabolites, as well as glucuronideconjugates of these acids.1 In an in vitro study usingrat hepatic microsomes, mitoxantrone was found to bedirectly metabolized to a glucuronide conjugate or toglutathione conjugates of its metabolites.25 Interest-ingly, in the present study, biliary excretion of M1was not affected by GF-120918, suggesting thatneither Bcrp nor P-gp was involved. It is well knownthat Multidrug resistance-associated protein 2(MRP2) often mediates the transport of anionsincluding glucuronide and glutathione conjugatesand is important for the biliary excretion of theseconjugates.26 Hence, we speculate that the metabo-lite(s) found in bile might be glucuronide or glu-tathione conjugates of mitoxantrone or itsmetabolites. The identities of these metabolites,and whether MRP2 is involved in their biliaryexcretion require further investigation.

In the current study, we also measured the plasmaconcentrations of mitoxantrone and determinedits CLb in rats, which has not been reportedpreviously. The pharmacokinetics of mitoxantronewas linear in the dose range we used, which was inagreement with the literature.16,17 The concentra-tions of mitoxantrone in plasma after a 0.5 mg/kg dosewere similar to those reported in an earlier studyusing a different analytical procedure, radioimmu-noassay, to quantitate mitoxantrone.27 The plasmaconcentrations of mitoxantrone exhibited a tri-expo-nential profile and were best described by a three-


compartment model, also consistent with previousfindings in rats, rabbits, dogs, and humans.16,28–31

The peripheral compartment with the larger dis-tribution clearance (CLD1) may represent highlyperfused organs such as liver, kidney, and spleen,while the compartment with the smaller distributionclearance (CLD2) may reflect tissues with less bloodperfusion, including fat and muscle.14,28 The rapiddecrease of mitoxantrone concentration within thefirst 15 min after dosing may result from extensiveuptake of the drug by tissues, in accordance with thevery large volumes of distribution of peripheralcompartments. The presence of a deep compartmentalso explained the flat terminal phase observed,which was probably due to the slow re-distribution ofdrug from tissue back to plasma.1

We have previously developed an in silico method,QSPKR models, to predict CLb and % of dose excretedin bile (PDb) of compounds in rats and humans.19 AQSPKR model for the prediction of PDb in rats wasdeveloped using a training group of 46 cation orneutral compounds with diversified structures. Therelationship derived was significant (R2¼ 0.828,p< 0.01) and only based on seven physicochemicaldescriptors. The prediction performance of the modelwas supported by a good Q2 value (0.731) generatedfrom a commonly used internal validation method,the ‘‘leave-one-out’’ method.32,33 Moreover, the modelexhibited good predictability when it was applied to24 cation or neutral compounds not included in thetraining group. Root mean square error reflectingaccuracy of prediction and mean error representingprecision of prediction were 11.2 and 4.39, respec-tively, suggesting that the model could be used forprediction purposes.19 The excellent prediction per-formance of the model was further confirmed in thepresent study comparing the predicted value (7.21%)with the experimental values (5.69% and 6.08%) ofmitoxantrone, which is a weak base under physiolo-gical conditions (pH 7.4).34 A QSPKR model devel-oped for CLb in rats based on 37 compounds was alsovalidated by the ‘‘leave-one-out’’ method and a testgroup having 18 compounds. The model provided apredicted CLb value of 14.1 mL/min/kg, which fellwithin a twofold error range of the measured value(7.20 and 7.46 mL/min/kg for the two doses ofmitoxantrone). A QSPKR model was also developedfor CLb of the subset of cation/neutral compounds andprovided an even better prediction for mitoxantrone(5.18 mL/min/kg). It was recognized that nonlinearpharmacokinetics of compounds may add complex-ities to the prediction of PK parameters using QSPKRmodels. Usually the parameter values representingthe most typical values in the population were usedand also the values obtained at lower doses werepreferred.35 In our study, the dose of 2 mg/kg in rats isequivalent to 12 mg/m2, which is the recommended

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dosing regimen for the treatment of breast cancerpatients, when mitoxantrone is administered as asingle intravenous infusion dose.1 The possibility ofsaturated biliary excretion of mitoxantrone in rats athigher doses than 2 mg/kg can not be excluded.

In conclusion, biliary excretion of unchangedmitoxantrone was measured in the present studyand this is the first report determining the biliaryclearance of this drug. The predicted biliary excretionby our previously developed QSPKR models was ingood agreement with the measured values, furtherdemonstrating that the biliary excretion of com-pounds can be predicted using this in silico methodwhich purely relies on the structures of compounds.In addition, we found that biliary excretion ofunchanged mitoxantrone was significantly inhibitedby GF-120918, a potent inhibitor of Bcrp and P-gp,suggesting that these transporters may be involved inthe active efflux of mitoxantrone across the livercanalicular membranes.

ACKNOWLEDGMENTS

This work was supported in part by Pfizer, Inc. Wethank Dr. Lisa J. Benincosa from Hoffman-La RocheInc and Dr. David B. Duignan from the Groton/NewLondon Laboratories at Pfizer, Inc. for their supportand suggestions regarding this research.

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