nonlinear stereoselective pharmacokinetics of ketoconazole in rat after administration of racemate

Nonlinear Stereoselective Pharmacokinetics of Ketoconazolein Rat After Administration of Racemate

DALIA A. HAMDY AND DION R. BROCKS*

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

ABSTRACT The stereoselective pharmacokinetics of ketoconazole (KTZ) enantio-mers were studied in rat after iv and oral administration of (6)-KTZ. Sprague-Dawley ratswere administered racemic KTZ as 10 mg/kg iv or orally over the range 10–80 mg/kgas single doses. Serial blood samples were collected over a 24-h period via surgicallyplaced jugular vein cannulae. Plasma was assayed for KTZ enantiomer concentrationsusing stereospecific HPLC. Enantiomeric plasma protein binding was determined usingan erythrocyte partitioning method at racemic concentrations of 10 and 40 mg/L. Ste-reoselective metabolism was tested by incubating the racemate (0.5–250 lM) with ratliver microsomes. In all rats, (1)-KTZ plasma concentrations were higher (up to 2.5-fold) than (2)-KTZ. The clearance and volume of distribution of the (2) enantiomerwere approximately twofold higher than antipode. Half-life did not differ between theenantiomers. After oral doses the tmax was not stereoselective. For both enantiomerswith higher doses the respective half-life were found to increase. The mean unboundfraction of the (2) enantiomer was found to be up to threefold higher than that of the(1) enantiomer. At higher concentrations nonlinearity in plasma protein binding wasobserved for both enantiomers. There was no evidence of stereoselective metabolismby liver microsomes. Stereoselectivity in KTZ pharmacokinetics is attributable to plasmaprotein binding, although other processes such as transport or intestinal metabolismmay also contribute. Chirality 21:704–712, 2009. VVC 2008 Wiley-Liss, Inc.

KEY WORDS: enantioselective; HPLC; antifungals; metabolism

INTRODUCTION

Ketoconazole (KTZ), cis-1-acetyl-4-[4-[[2-(2,4-dichloro-phenyl)-2-(1H imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]me-thoxy]phenyl]-piperazine, is a broad spectrum antifungalagent used for systemic and local infections. As with otherazoles, its antifungal activity is attributable to the inhibitionof cytochrome P450 (CYP)-mediated 14-a-demethylation oflanosterol in ergosterol biosynthesis. This results in fungalergosterol depletion, interruption of membrane integrity/activity, and inhibition of cell growth.1 These CYP inhibi-tion properties extend to mammalian CYPs and stronginteractions are possible with both hepatic drug metaboliz-ing and steroidogenic enzymes.2–4 Owing to the risk ofKTZ-induced hepatotoxicity and serious drug–drug inter-actions, the use of KTZ as an antifungal agent tends to berestricted to more serious infections that are resistant toother safer azoles.5,6

Because of its CYP inhibitory properties, KTZ is exten-sively used as a CYP and P-glycoprotein modulator for invitro and in vivo drug interaction studies.7–11 Surprisingly,the pharmacokinetic information pertaining to KTZ is rela-tively limited. Human studies have shown that dispropor-tionate changes in area under the plasma concentrationvs. time curve (AUC) can occur with changing dose levels,in conjunction with alterations in terminal elimination half-life (t[1/2]) thereby suggesting nonlinear kinetics.12,13

Human studies have been limited primarily to oral admin-istration due to the lack of intravenous (iv) formulations.The primary pharmacokinetic parameters of clearance(CL) and volume of distribution (Vd), have been deter-mined in rats and dogs.14–17 It is reported that a one com-partment open model fits well to KTZ plasma concentra-tion vs. time data with oral absolute bioavailability (F)ranging from 32% to 37% in rats and 12% to 88% in dogs.14–16

After KTZ iv bolus and infusion over a range of doses, ratsexhibited dose-dependent changes in the volume of distri-bution, and a disproportionate increase in area under theplasma concentration vs. time curves (AUC) with escalatingdose.16,17 Over the concentration range of 0.1 to 10 mg/L,KTZ showed strong linear plasma protein binding with anaverage unbound fraction of 0.037.16

Although some information is available regarding KTZpharmacokinetics, it should be recognized that it is chiral

Contract grant sponsor: Canadian Institutes of Health Research;Contract grant number: MOP 87395.*Correspondence to: Dion R. Brocks, Ph.D., Associate Professor, Facultyof Pharmacy and Pharmaceutical Sciences, 3118 Dentistry/PharmacyCentre, University of Alberta, Edmonton, AB, Canada, T6G 2N8.E-mail: [email protected] for publication 13 June 2008; Accepted 17 September 2008DOI: 10.1002/chir.20669Published online 5 November 2008 in Wiley InterScience(www.interscience.wiley.com).

CHIRALITY 21:704–712 (2009)

VVC 2008 Wiley-Liss, Inc.

and is administered clinically as a racemate of the cis-con-figuration (see Fig. 1). All previous pharmacokinetic datawere generated using nonstereospecific assay methodolo-gies and as such reflected the combined kinetic propertiesof both KTZ enantiomers. The enantiomeric absolute con-figuration of KTZ has been reported to be the (1)-(2R,4S)and the (2)-(2S,4R) enantiomer.18 It has been previouslyshown for many enantiomeric pairs of drugs, that the phar-macological activity of one enantiomer may differ fromthat of its antipode.19,20 In fact in vitro studies applyingKTZ enantiomers to human CYP3A4 supersomes usingtestosterone and methadone as substrates suggested ste-reoselective inhibition, with the (2)-KTZ enantiomer dis-playing approximately twofold more inhibitory potency.21

In many cases as well, the pharmacokinetic behavior ofone enantiomer may differ from its antipode.22,23 Recently,the first HPLC method for the determination of KTZ enan-tiomers in biological specimens was published.24 Theassay has now been applied to a pharmacokinetic study of(6)-KTZ in the rat and pharmacokinetic data of KTZ enan-tiomers after oral and iv administration of the drug arereported here.

EXPERIMENTALMaterials and Reagents

Ketoconazole, nicotinamide adenine dinucleotide phos-phate tetrasodium (NADPH), and amiodarone HCl wereobtained from Sigma (St. Louis, MO). Hexane, isopropylalcohol, absolute ethanol, tert-butylmethyl ether, metha-nol, acetonitrile (all HPLC grade), diethylamine, propyleneglycol, and polyethylene glycol (PEG) 400 were purchasedfrom Fisher Scientific (Fair Lawn, NJ). KTZ oral tablets foradministration to rat (Nizoral1 McNeil Consumer Health-care, Guelph, Ontario, Canada) were purchased from theUniversity of Alberta Hospitals (Edmonton, Alberta, Can-ada). Potassium dihydrogen orthophosphate, dipotassiumhydrogen orthophosphate, magnesium chloride hexahy-drate, potassium chloride, sucrose, and calcium chloridedihydrate (all analytical grade) were obtained from BDH(Toronto, ON, Canada). Heparin sodium for injection was

obtained from Leo Pharma Inc. (Thornhill, Ontario,Canada).

In Vivo Studies

Animals and pre-experimental procedures. Experi-mental protocols involving animals were approved by theUniversity of Alberta Health Sciences Animal Policy andWelfare Committee. A total of 27 male Sprague-Dawleyrats (Charles River Canada) were included in the pharma-cokinetic study. The rats weighed between 250 and 350 gand were housed in temperature-controlled rooms with12 h of light per day. The animals were fed a standardrodent chow containing 4.5% fat (Lab Diet

1

5001; PMINutrition LLC, Richmond, IN). Free access to food andwater was permitted prior to experimentation. Rats wereallocated into six groups: one group iv dosed with 10 mg/kgracemic KTZ (n 5 7) and five groups orally administered10, 20, 40, 50, or 80 mg/kg racemic KTZ (n5 3–4 each).

The right jugular vein of all rats was catheterized withMicro-Renathane tubing (Braintree Scientific, Braintree,MA) under isoflurane anesthesia the day before the phar-macokinetic study. The cannula was filled with 100 U/mLheparin in 0.9% saline. After implantation, the rats weretransferred to their regular holding cages and allowed freeaccess to water, but food was withheld overnight so thatdrug would be administered in the fasted state. The nextmorning, rats were transferred to the metabolic cages anddosed with (6)-KTZ.

Drug administration and sample collection. TheKTZ iv dosing solution was prepared by dissolving pow-dered racemic compound in 9:1 polyethylene glycol 400:propylene glycol (10 mg/mL).16 The iv dose was injectedover 2 min via the jugular vein cannula, immediately fol-lowed by injection of �1 mL of sterile normal saline solu-tion over the next 2 min. At the time of first sample with-drawal, the first 0.2 mL volume of blood was discarded.For oral dosing, KTZ (44 mg/mL) suspension was pre-pared. The tablets of (6)-KTZ were ground to a powderusing a mortar and pestle then dispersed in 1% methylcel-lulose. On the morning of the pharmacokinetic study, therat groups received the desired dose by oral gavage. Forboth routes of administration, food was provided to ani-mals 2 h after the dose administration.

Serial blood samples (0.15–0.25 mL) were collected at0.5, 1, 2, 3, 4, 6, 8, 10, and 24 h postdose for oral dosingand at 0.08, 0.25, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, and 10 h post-dose for iv dosing into polypropylene microcentrifugetubes. Heparin in normal saline (100 U/mL) was used toflush the cannula after each collection of blood. Plasmawas separated by centrifugation of the blood at 2500g for3 min. The samples were kept at 2308C until assayed forKTZ enantiomers.

In Vitro Studies

Microsomal incubations. Four male Sprague-Dawleyrats (Charles River Canada, Montreal, QC, Canada) weresacrificed under halothane anesthesia, and their liverswere excised. The livers were washed in ice-cold KCl(1.15% w/v), cut into pieces, and homogenized separately

Fig. 1. Structure of ketoconazole. Asterisks denote centers ofassymmetry.

705STEREOSELECTIVE KETOCONAZOLE KINETICS

Chirality DOI 10.1002/chir

in cold sucrose solution (5 g of tissue in 25 mL of 0.25 Msucrose). Microsomal protein was separated by differentialultracentrifugation. The final pellets were collected in coldsucrose and stored at 2808C.25 The microsomes from fourrats were pooled. The Lowry method was used to measurethe total protein concentration in each microsomal prepa-ration.26

Each 0.5 mL incubate contained 1 mg/mL protein of thepooled liver microsomal preparation, 0.5–250 lM (0.27–133 mg/L) of (6)-KTZ (dissolved in methanol), 1 mM ofNADPH, and 5 mM of magnesium chloride hexahydratedissolved in 0.5 M potassium phosphate buffer (pH 5 7.4).The volume of methanol added to each incubate with theaddition of KTZ racemate was 0.8% v/v. The substrate wasadded to the liver microsomal suspensions and the oxida-tive reactions were started upon the addition of NADPHafter a 5-min pre-equilibriation period. All incubations wereperformed in quadruplicate at 378C in a shaking waterbath for 15 min. Incubation conditions were optimized sothat the rate of metabolism was linear with respect to incu-bation time and microsomal protein concentration. Allmicrosomal incubations were stopped by the addition ofthree volumes of acetonitrile. The samples were then keptat 2308C until assayed for KTZ enantiomers. The rate ofmetabolism was calculated by measuring the rate of reduc-tion in parent drug (substrate depletion).

Plasma Protein Binding Study. An erythrocyte vs.buffer or diluted plasma partitioning method was used forthe determination of plasma protein binding of KTZ enan-tiomers.27 Briefly, four rats were anesthetized and bloodwas collected into heparinized tubes by cardiac puncture.The blood obtained from each rat was equally splitbetween two tubes. Plasma was separated by centrifuga-tion of the whole blood at 2500g for 10 min. After removalof the plasma and buffy coat layer, the cells were washedwith an equal volume of isotonic Sorensen’s phosphatebuffer (pH 5 7.4) and recentrifuged at 2500g for 8 min.The washing step was repeated two more times afterwhich the volume of erythrocytes in each of the tubes wasmeasured. Either isotonic phosphate buffer (pH 5 7.4) ordiluted plasma (1:10 in isotonic phosphate buffer) wasthen added to the blood cells to provide a hematocrit of0.3 (buffer) or 0.4 (diluted plasma).

Stock solutions of (6)-KTZ (25 and 100 mg/mL) wereprepared in methanol and added to the buffer-containingand diluted plasma-containing mixtures to allow a finalconcentration of �10 or 40 mg/L of racemate. For thisexperiment blood and plasma from two rats were spikedwith the lower concentration, and specimens from theother two rats were spiked with the higher concentration.The volume of methanol added to each tube did notexceed 0.05% v/v. Tubes were incubated for 1 h in a 378Cwater bath shaker.

At the conclusion of the incubation, replicates of fiveblood samples (50 lL each) were set aside for assay withan additional 50 lL of water added to each tube beforefreezing. For the remainder of the blood, the plasma andbuffer were isolated by centrifugation for 10 min at 2500g.Replicates of five samples of 100 lL from each sample was

set aside for assay. All samples were frozen at 2308C untilassayed for KTZ enantiomer.

Blood:plasma ratio. Known amounts of (6)-KTZ inmethanol were added to heparinized tubes containingfreshly obtained rat blood to provide a final concentrationof 18 lg/mL of racemate. The volume of methanol addedwas 5 lL/mL of blood. The tubes were placed in a shakingwater bath at 378C for 1 h. At that time, the tubes wereremoved and 100 lL of blood was transferred to microcen-trifuge tubes (n 5 5) containing 100 lL of water. Theremaining blood was centrifuged at 2500g for 10 min. Avolume of 100 lL of the plasma layer was transferred tomicrocentrifuge tubes (n 5 5). Samples were kept frozenat 2308C until being assayed for KTZ enantiomer concen-trations.

Assay

A validated stereospecific HPLC method was used formeasurements of KTZ enantiomers concentrations ineach blood or plasma sample.24 The validated lower limitof quantitation was 62.5 ng/mL for both enantiomersbased on 0.1 mL of blood or plasma. Volumes of plasmaassayed in samples from the pharmacokinetics studyranged between 75 and 150 lL. For the assay of wholeblood, buffer, blood-buffer, and blood-diluted plasma mix-tures from the plasma protein binding and blood: plasmaratio determinations, standard curves were prepared fromsimilar drug-spiked matrices.

The method was modified to assay KTZ enantiomers inliver microsomal preparations. Denatured microsomalmedia was used and spiked for standard curve prepara-tion. Briefly, to the tubes containing 500 lL of microsomalincubation mixture and 1.5 mL of acetonitrile, 100 lL of in-ternal standard in methanol (100 lg/mL amiodarone HClstock) and 125 lL methanol was added. The tubes werebriefly vortex mixed for 4 s at high speed and then subse-quently centrifuged for 3 min at �2500g. The supernatantwas carefully transferred to new glass tubes. The tubeswere rendered acidic using 1M HCl and then 2 mL of hex-ane was added. The tubes were vortex mixed for 30 s andcentrifuged for 3 min. The organic supernatant was care-fully aspirated, followed by the addition of 1M NaOH and7-mL tert-butyl methyl ether. The tubes were then vortexmixed again for 30 s and centrifuged at �2500g for 3 min.The organic layer was transferred to new glass tubes andevaporated to dryness in vacuo. The residues were recon-stituted in 150-lL mobile phase of which 75–125 lL vol-umes were injected into the HPLC. For standard curveconstruction, drug-free microsomal preparations wereused and spiked with appropriate amounts of (6)-KTZ.

Data and Statistical Analysis

Noncompartmental methods were used to calculate thepharmacokinetics parameters. The elimination rate con-stant (kz) was calculated by subjecting the plasma concen-trations in the terminal phase to linear regression analysis.The t[1/2] was calculated by dividing 0.693 by kz. TheAUC0-1 was calculated using the combined log-linear trap-ezoidal rule from time 0 h postdose to the time of the last

706 HAMDY AND BROCKS


measured concentration, plus the quotient of the lastmeasured concentration divided by kz. The concentrationat time 0 h after iv dosing was estimated by extrapolationof the log-linear regression line using the first three meas-ured plasma concentrations to time 0. The CL was calcu-lated as the quotient of dose to AUC0-1 and the steadystate Vd (Vdss) as CL 3 AUMC/AUC, where AUMC isthe area under the first moment plasma concentration vs.time curve, from time of dosing to infinity. The mean resi-dence time (MRT) was determined as the quotient ofAUMC to AUC. The oral F was calculated as follows:

F ¼ mean AUC oral

mean AUC iv3

Dose iv

Dose oral

The maximum plasma concentration (Cmax) and thetime at which it occurred (tmax) were determined by visualexamination of the data. The calculation of the plasmaunbound fraction (fu) was determined using a series ofequations outlined by Schuhmacher et al.27 The erythro-cyte concentration of enantiomer in the erythrocyte-dilutedplasma was determined by the following equation:

CE ¼CB� Cp 1�HCTð ÞHCT

Where CB is the concentration of enantiomer in the bloodcell-diluted plasma suspension, Cp is the concentration ofenantiomer in the diluted plasma, and HCT is the hemato-crit in the erythrocyte-diluted plasma sample. To estimatethe erythrocyte concentration of enantiomer in the eryth-rocyte-buffer samples (CE*), the concentration of enan-tiomer in the blood cell-buffer suspension was substitutedfor CB, and the buffer concentration of enantiomer wassubstituted for Cp. The fu of enantiomers was determinedby

fu ¼ a � PpPb1� Pp

Pb � 1� að Þ� �

Where a is the plasma dilution factor. The partition coeffi-cients for erythrocyte: diluted plasma (Pp) or buffer (Pb)are represented by the quotients CE/Cp and CE*/Cbuffer,respectively.

The mean blood CL of (1) and (2)-KTZ were estimatedby dividing the respective mean plasma CL by the corre-sponding blood to plasma ratio. The hepatic extraction ra-tio (E) was estimated, assuming negligible extrahepaticCL, by taking the quotient of iv blood CL divided byaverage hepatic blood flow of 55.2 mL/min/kg.28 The gas-trointestinal availability (fg) in turn was calculated as thequotient F divided by (1 2 E), where 1 2 E represents thehepatic availability.

All data are reported as mean 6 SD, unless otherwiseindicated. Means were compared by one-way ANOVA andStudent’s paired or unpaired t-tests as appropriate. Thelevel of significance was set at P 5 0.05.

RESULTS

In general, after iv doses of 10 mg/kg (6)-KTZ, theplasma concentrations appeared to decline monoexponen-tially for both enantiomers, with mean t[1/2] of �40 min(Fig. 2, Table 1). However, in examining the individualconcentration vs. time plots, in some rats there was evi-dence of apparent convexity in the latter concentrationtime points (Fig. 2c). In all rats, the differences betweenenantiomers were significant with respect to AUC0-1, CLand Vdss with overall mean (1):(2) KTZ ratios of 2.05,0.47, and 0.56, respectively (Table 1).

As for iv doses, in all rats given oral doses (1)-KTZplasma concentrations were significantly higher than (2)-KTZ with overall mean (1):(2) AUC0-1 and Cmax ratio of2.36 and 2.19, respectively (Fig. 3 and Table 1). For all ofthe doses, the median tmax was very similar between thetwo enantiomers. However, there seemed to be a trendtowards longer tmax with higher dose levels. There wereno significant differences between enantiomers withindoses for the t[1/2], although the slope of log plasma con-centration vs. time for both enantiomers seemed tobecome shallower (ANOVA P < 0.05) as the doseincreased (Table 1). Because of dose and perhaps a longert[1/2], the KTZ enantiomers were detected for longer athigher doses (see Fig. 3). In all rats an AUC was assessedover at least 4 h, so the AUC0-4h are displayed in Table 1for comparison with AUC0-1. In conjunction with the ivdata, the F for the 10 mg/kg oral dose group were calcu-lated to be 31.1% and 28.7% for the (1)- and (2)-KTZ,respectively.

The correlation between Cmax or AUC0-1 and doseshowed a disproportional increase in plasma levels withescalating dose (Fig. 4, Table 1). Three phases seemed tobe present in the Cmax vs. doserelationship, and twophases in the AUC0-1 vs. dose profile. For both Cmax andAUC0-1, with the initial increases in dose, the plasma con-centrations increased in an approximately linear fashion.Above these doses levels the nature of the relationshipappeared to change. From 20 to 40 mg/kg the twofoldincrease in dose resulted in 3.6- and 5.9-fold increases ofthe (1)-KTZ Cmax and AUC0-1 and 3.7- and 5.5-foldincreases for the equivalent (2)-KTZ, respectively. Abovethese doses the mean AUC0-1 appeared to increase line-arly with a different slope than lower doses. In contrast toAUC, the Cmax between 40 and 80 mg/kg doses tended toreach a plateau. For the dose-normalized AUC, a signifi-cant difference was noted between dose groups (P < 0.05,ANOVA).

Unlike the plasma concentrations, there was no evi-dence of stereoselectivity in metabolism by hepatic micro-somes. There were virtually superimposable velocities ofdisappearance of enantiomers observed across the concen-tration ranges tested (see Fig. 5). Because this experimentwas designed to examine only stereoselective metabolismusing replicate samples of the same pooled microsomes,kinetic constants of metabolism are not reported.

Extensive plasma protein binding (>97%) was observedwith both (1)-and (2)-KTZ in rat plasma. At lower KTZconcentrations (10 lg/mL of racemate), the unbound



fractions for (1)-KTZ were found to be 0.64% 6 0.12% and0.61% 6 0.10% for the two rats. In contrast, the (2)-KTZunbound fractions were 1.73% 6 0.30% and 1.90% 6 0.32%.At 40 lg/mL of racemate, the fu of (1)-KTZ in plasma

TABLE1.Pharmac

okinetic

param

eters(m

ean6

SD)ofketoco

nazole

enan

tiomers(K

TZ)afteriv

andoraldose

sofrace

mate

Racem

ate

(mg/k

g)

KTZ

AUC0-4h(m

g�h

/L)

AUC

0-1

(mg�h

/L)

t½(h)

Cmax

(mg/L

)t m

ax(h)

Vdss

(L/K

g)

CLb(L/h

/Kg)

MRT(h)

Intraven

ousdose

10(1

)17

.296

4.25

a17

.946

4.91

a0.66

16

0.25

9_

_0.70

76

0.07

5a0.59

06

0.14

4a1.25

60.25

9a

(2)

8.45

62.67

a8.72

62.90

a0.61

56

0.25

8_

_1.27

60.19

6a1.26

60.39

0a1.07

60.24

6a

Oraldoses

a

10(1

)5.46

63.57

a5.59

63.63

a0.58

06

0.06

02.72

61.72

0a0.93

0(0.900

20.95

0)_

2.36

61.320a

1.54

60.09

5a

(2)

3.19

61.72

a2.51

61.43

a0.67

26

0.25

21.26

60.80

0a0.93

0(0.900

20.95

0)_

5.28

63.381a

1.63

60.23

4a

20(1

)8.74

64.39

a9.70

65.21

a0.67

46

0.33

64.23

61.99

a 01.87

(1.152

1.95

)_

3.22

63.021a

1.90

60.61

6a

(2)

3.72

62.14

a4.11

62.55

a0.64

66

0.35

51.79

60.93

2a1.87

(0.333

21.95

)_

8.91

69.531a

1.82

60.66

0a

40(1

)38

.96

3.84

a56

.86

18.4a

1.04

60.04

115

.16

2.15

a 01.53

(0.883

22.88

)_

0.80

26

0.23

7a2.86

60.59

0a

(2)

16.826

2.44

a22

.76

7.71

a0.94

26

0.20

66.52

61.03

0a1.53

(0.883

22.88

)_

1.88

60.48

7a2.65

60.46

8a

50(1

)44

.76

11.0a

71.8

618

.7a

1.23

60.41

218

.26

2.10

a 01.91

(0.966

22.08

)_

0.73

86

0.21

7a3.16

60.31

1a

(2)

20.1

64.64

a27

.06

13.0a

1.07

60.27

98.22

60.88

9a1.91

(0.966

22.08

)_

2.24

61.081a

3.71

60.49

3a

80(1

)56

.36

8.49

a11

26

37.2a

2.02

60.89

220

.76

3.11

a 02.05

(1.952

2.07

)_

0.77

16

0.26

4a5.22

62.340a

(2)

27.2

63.19

a53

.96

19.5a

1.92

60.93

510

.06

1.30

0a2.05

(1.952

2.07

)_

1.62

60.59

5a4.77

62.520a

aSignificantdifference

from

correspo

nding(2

)en

antiom

er.

bCL/F

fortheoral

doses.

Fig. 2. Plasma concentration vs. time plots of KTZ enantiomers afteradministration of 10 mg/kg of the racemate as iv doses. (a) mean 6 SD ofall 7 rats, (b) an individual rat displaying monoexponential decline, (c) anindividual rat displaying apparent nonlinearity in decline.



from the rats were 0.97% 6 0.29% and 0.92% 6 0.30%,respectively. In comparison the (2)-KTZ unbound frac-tions were 2.38% 6 0.65% and 2.32% 6 0.88%. For bothenantiomers at the higher concentrations of 40 lg/mL,the unbound fractions were significantly higher comparedto the lower concentration tested (10 lg/mL). Stereoselec-tivity was similar at both concentrations; the mean ratiosof (2):(1) unbound enantiomer were 2.9 and 2.5 at 10and 40 lg/mL of racemate, respectively.

The mean blood:plasma ratios of (1)-and (2)-KTZwere 0.61 6 0.039 and 0.64 6 0.065, respectively. This

indicated minimal blood cell partitioning for KTZ enan-tiomers and its restriction to plasma within the blood ma-trix. The blood CL was calculated by dividing the plasmaCL by the blood:plasma ratios, yielding mean blood CL of0.976 and 1.97 L/h/kg for the (1)-and (2)-KTZ enantiom-ers, respectively. Using the reported mean hepatic bloodflow in rat28 and assuming that the majority of the clear-ance of KTZ enantiomers occurs in the liver, E was esti-mated to be 0.30 and 0.60 for the (1)-and (2) enantiom-ers, respectively. The fg was calculated to be 0.44 and 0.72for the (1) and (2) enantiomers, respectively.

Fig. 3. Mean (6S.D.) plasma concentration vs. time curves of ketoconazole enantiomers after oral racemic doses of 10, 20, 40, 50, or 80 mg/kg (n 5three to four rats per dose group).



DISCUSSION

Although KTZ has been in clinical use for many years,and is extensively used as a presumed inhibitor of CYP3Aisoenzymes,29–31 it has been similarly widely overlookedthat it is chiral and administered as the racemate of thetwo cis-isomers. Thus far the only stereoselective kineticdata that we know of in the literature is based on two ratswhich were used to illustrate applicability of the assaymethod. Similar to those rats,24 all rats examined here,regardless of route of administration and dose, showed ahigh level of stereoselectivity in the plasma concentrations(Table 1, Figs. 2 and 3).

As reported for the sum of the two enantiomers, after ivadministration a one compartment model was found toconform best to the data of each individual enan-tiomer.15,16 Each enantiomer had a Vdss reflective of adrug with substantial distribution to tissues. In addition,assuming that (6)-KTZ is mostly eliminated by the liver,the enantiomers had an estimated extraction ratios placingthem in the low to moderate range. However, in each ofthese primary pharmacokinetic indices, marked stereose-

lectivity was noted. The CL and Vdss of the (2) enantiomerwere significantly higher than that of antipode. It is knownthat (6)-KTZ is strongly bound to rat plasma proteins overthe concentration range 0.1 to 10 mg/L,16 and extensivelymetabolized.15,17,32,33 The cause of enantioselectivity maybe at the level of protein binding and/or hepatic metabo-lism. In the plasma protein binding experiment, it wasobserved that the unbound fraction of the (2) enantiomerwas higher than that of its (1) antipode. Although therewas no evidence of stereoselective microsomal hepatic me-tabolism (see Fig. 5), this must be viewed with some cau-tion as this is not a cellular system, and is devoid of func-tional transport proteins. Nevertheless, the extent of ste-reoselectivity in the (2):(1) unbound fraction ratios(range 2.5–2.7 at 10 and 40 lg/mL of the racemate) was inline with the mean enantiomer ratios observed for Vdss

and CL (range, 1.8–2.1). Our findings for the enantiomerunbound fractions, blood:plasma ratio and moderate he-patic extraction ratio are in agreement with previouslyreported data for the sum of both enantiomers.16 Thedegree of stereoselectivity in CL and Vdss was similarleading to essentially the same mean t[1/2] for eachenantiomer.

After oral administration, the KTZ enantiomers showedsimilar tmax suggesting nonstereoselectivity in absorptionrate (Table 1). The Cmax and AUC0-1 values for the enan-tiomers were consistent with the stereoselectivityobserved in the iv dosed rats. The absolute F calculatedfor the two KTZ enantiomers after 10 mg/kg (6)-KTZorally coincided with the racemate values reported byRemmel et al. with oral and iv doses of 5 mg/kg.15 How-ever, both of these estimates of F, especially for the (1)enantiomer, were lower than expected based on the valuescalculated from the iv data assuming complete hepaticelimination of drug.15,33 This suggests that a combination

Fig. 4. Relationship between the racemic ketoconazole oral doses andenantiomer (a) Cmax, and (b) AUC.

Fig. 5. Mean rates of disappearance of ketoconazole enantiomers vs.initial enantiomer concentration in the presence of rat liver microsomes.Incubations were run over 15 min after being spiked with the racemate(n 5 2–4 per concentration).



of other factors, including possibly incomplete absorptionof parent drug, intestinal presystemic metabolism and per-haps transport of the enantiomers, occur.

The mean t[1/2] values reported for both KTZ enan-tiomers in this study were within the range of thosereported previously for the sum of both enantiomers afterracemic doses.15–17,33 In some of the rats given iv doseshere, there was evidence of a greater slope of declinein the later parts of the concentration vs. time curves(Fig. 2c). This profile is consistent with a saturable elimi-nation process. Similar results were reported by Sjoberget al. for the racemate.17 This was also reflected in the oraldose ranging study. Upon increasing the dose above20 mg/kg, the dose vs. Cmax and AUC 0-1 slopes of thetwo enantiomers tended to increase to a level higher thanthat at the dose range below 20 mg/kg. It is known thatKTZ undergoes several metabolic biotransformations,including oxidation, scission, and degradation of the imid-azole ring, scission and degradation of the piperazine anddioxolane rings and oxidative O-dealkylation.32,33 There-fore, the initial change in slope from 20 mg/kg may beexplained by saturation of one or more of these metabolicpathways. The plateau in Cmax with the highest oral doselevels seems to be due to saturable, nonlinear, plasma pro-tein binding of KTZ enantiomers. Confirmation of satura-tion of this process was provided by the increases inunbound fraction of both enantiomers between 10 and40 lg/mL of racemate.

The mean F of the enantiomers were virtually identical.This was of interest because based on the iv dosing andassuming that most of the CL of KTZ occurs in the liver, itwas suggested that the drug undergoes stereoselective he-patic extraction, and that the (2) enantiomer shows amoderate hepatic E. Hence, stereoselectivity in bioavaila-bility would have been expected if the absorption of thedrug was passive and little extrahepatic metabolismoccurred. Calculation of the fg, however, indicated thatthere was stereoselectivity in the fraction of the drug avail-able from the intestinal tract, which was opposite in direc-tion to that imparted by the liver. This suggests possiblestereoselectivity in extrahepatic mechanisms, such as in-testinal metabolism or transport protein activity. It hasbeen shown that stereoselectivity in metabolism may beopposite between CYP isoenzymes. One recent example ofthis was seen in rat for CYP3A1 and 2C11, where thedirection of stereoselectivity for formation of desbutylhalo-fantrine enantiomers from halofantrine was reversed.34

Given that KTZ can inhibit a number of CYPs,11 it is possi-ble that the drug is metabolized by CYPs other than the3A isoforms. Also although most reports have used KTZonly as a probe for inhibition of P-glycoprotein, it has beenshown to be an effective substrate for efflux at the level ofthe blood–brain barrier.35 Further studies are required todefinitively explain this issue.

The clinical use of (6)-KTZ has been more recentlyovershadowed by other azole antifungals because of itsdrug–drug interactions and reported hepatotoxicity.36,37

Nevertheless, in drug development it is still frequentlyused as a probe to study the possibility of drug interac-tions due to its presumed, but not necessarily spe-

cific,11,38,39 ability to inhibit CYP3A isoforms. It is knownthat the enantiomers differ in their ability to bind to CYPisoforms, and that the (–) enantiomer is more potentagainst some strains of fungi.21 Whether the enantiomersdiffer in their ability to cause toxicities remains to bedetermined.

In conclusion, this study is the first to report the stereo-selective pharmacokinetics of KTZ enantiomers in ratplasma. Stereoselectivity in plasma concentrations of KTZenantiomers can be attributed to enantiospecificity inplasma protein binding although other unknown mecha-nisms appear to be involved as well. In agreement withprevious reports for (6)-KTZ, both KTZ enantiomersshowed nonlinearity with increasing racemic doses in rat,presumably to a combination of saturation of some meta-bolic pathways and plasma protein binding.

ACKNOWLEDGMENTS

DAH is a recipient of a studentship from the govern-ment of Egypt.

LITERATURE CITED

1. Vanden Bossche H, Koymans L, Moereels H. P450 inhibitors of usein medical treatment: focus on mechanisms of action. Pharmacol Ther1995;67:79–100.

2. Sheets JJ, Mason JI. Ketoconazole: a potent inhibitor of cytochrome P-450-dependent drug metabolism in rat liver. Drug Metab Dispos1984;12:603–606.

3. Pasanen M, Taskinen T, Iscan M, Sotaniemi EA, Kairaluoma M, Pel-konen O. Inhibition of human hepatic and placental xenobiotic mono-oxygenases by imidazole antimycotics. Biochem Pharmacol 1988;37:3861–3866.

4. Schuster I. The interaction of representative members from twoclasses of antimycotics—the azoles and the allylamines—with cyto-chromes P-450 in steroidogenic tissues and liver. Xenobiotica 1985;15:529–546.

5. Korashy HM, Shayeganpour A, Brocks DR, El-Kadi AO. Induction ofcytochrome P450 1A1 by ketoconazole and itraconazole but not fluco-nazole in murine and human hepatoma cell lines. Toxicol Sci 2007;97:32–43.

6. Velikinac I, Cudina O, Jankovic I, Agbaba D, Vladimirov S. Compari-son of capillary zone electrophoresis and high performance liquidchromatography methods for quantitative determination of ketocona-zole in drug formulations. Farmaco 2004;59:419–424.

7. Rodrigues AD, Lewis DF, Ioannides C, Parke DV. Spectral and kineticstudies of the interaction of imidazole anti-fungal agents with microso-mal cytochromes P-450. Xenobiotica 1987;17:1315–1327.

8. Rodrigues AD, Gibson GG, Ioannides C, Parke DV. Interactions of im-idazole antifungal agents with purified cytochrome P-450 proteins.Biochem Pharmacol 1987;36:4277–4281.

9. Abel S, Russell D, Taylor-Worth RJ, Ridgway CE, Muirhead GJ.Effects of CYP3A4 inhibitors on the pharmacokinetics of maraviroc inhealthy volunteers. Br J Clin Pharmacol 2008;65 (Suppl 1):27–37.

10. Fan Y, Rodriguez-Proteau R. Ketoconazole and the modulation of mul-tidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1drug transport models. Xenobiotica 2008;38:107–129.

11. Elsherbiny ME, El-Kadi AO, Brocks DR. The metabolism of amioda-rone by various CYP isoenzymes of human and rat, and the inhibitoryinfluence of ketoconazole. J Pharm Pharmaceut Sci 2008;11:147–159.

12. Huang YC, Colaizzi JL, Bierman RH, Woestenborghs R, Heykants J.Pharmacokinetics and dose proportionality of ketoconazole in normalvolunteers. Antimicrob Agents Chemother 1986;30:206–210.

13. Daneshmend TK, Warnock DW. Clinical pharmacokinetics of sys-temic antifungal drugs. Clin Pharmacokinet 1983;8:17–42.



14. Baxter JG, Brass C, Schentag JJ, Slaughter RL. Pharmacokinetics ofketoconazole administered intravenously to dogs and orally as tabletand solution to humans and dogs. J Pharm Sci 1986;75:443–447.

15. Remmel RP, Amoh K, Abdel-Monem MM. The disposition and phar-macokinetics of ketoconazole in the rat. Drug Metab Dispos1987;15:735–739.

16. Matthew D, Brennan B, Zomorodi K, Houston JB. Disposition of azoleantifungal agents. I. Nonlinearities in ketoconazole clearance andbinding in rat liver. Pharm Res 1993;10:418–422.

17. Sjoberg P, Ekman L, Lundqvist T. Dose and sex-dependent disposi-tion of ketoconazole in rats. Arch Toxicol 1998;62(2/3):177–180.

18. Rotstein DM, Kertesz DJ, Walker KA, Swinney DC. Stereoisomers ofketoconazole: preparation and biological activity. J Med Chem1992;35:2818–2825.

19. Jamali F, Mehvar R, Pasutto FM. Enantioselective aspects of drugaction and disposition: therapeutic pitfalls. J Pharm Sci 1989;78:695–715.

20. Brocks DR, Jamali F. Stereochemical aspects of pharmacotherapy.Pharmacotherapy 1995;15:551–564.

21. Dilmaghanian S, Gerber JG, Filler SG, Sanchez A, Gal J. Enantioselec-tivity of inhibition of cytochrome P450 3A4 (CYP3A4) by ketocona-zole: testosterone and methadone as substrates. Chirality 2004;16:79–85.

22. Brocks DR. Drug disposition in three dimensions: an update on ste-reoselectivity in pharmacokinetics. Biopharm Drug Dispos 2006;27:387–406.

23. Lu H. Stereoselectivity in drug metabolism. Expert Opin Drug MetabToxicol 2007;3:149–158.

24. Hamdy DA, Brocks DR. A stereospecific high-performance liquidchromatographic assay for the determination of ketoconazole enan-tiomers in rat plasma. Biomed Chromatogr 2008;22:542–547.

25. Barakat MM, El-Kadi AO, du Souich P. L-NAME prevents in vivothe inactivation but not the down-regulation of hepatic cytochromeP450 caused by an acute inflammatory reaction. Life Sci 2001;69:1559–1571.

26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure-ment with the Folin phenol reagent. J Biol Chem 1951;193:265–275.

27. Schuhmacher J, Buhner K, Witt-Laido A. Determination of the freefraction and relative free fraction of drugs strongly bound to plasmaproteins. J Pharm Sci 2000;89:1008–1021.

28. Davies B, Morris T. Physiological parameters in laboratory animalsand humans. Pharm Res 1993;10:1093–1095.

29. Sekar VJ, Lefebvre E, De Pauw M, Vangeneugden T, HoetelmansRM. Pharmacokinetics of darunavir/ritonavir and ketoconazole follow-ing co-administration in HIV-healthy volunteers. Br J Clin Pharmacol2008;66:215-221.

30. Farid NA, Payne CD, Small DS, Winters KJ, Ernest CS II, Brandt JT,Darstein C, Jakubowski JA, Salazar DE. Cytochrome P450 3A inhibi-tion by ketoconazole affects prasugrel and clopidogrel pharmacoki-netics and pharmacodynamics differently. Clin Pharmacol Ther 2007;81:735–741.

31. Engels FK, Loos WJ, Mathot RA, van Schaik RH, Verweij J. Influenceof ketoconazole on the fecal and urinary disposition of docetaxel. Can-cer Chemother Pharmacol 2007;60:569–579.

32. Heel R. Ketoconazole: pharmacological profile. In: Levine HB, editor.Ketoconazole in the management of fungal disease. Balgowlah, Aus-tralia: Adis Press; 1982. p 55–76.

33. Gascogaine EW, Barton GJ, Micheals M, Meuldermans W, HeykantsJ. The kinetics of ketoconazole in animals and man. Clin Res Rev1981;1:177–187.

34. Gharavi N, Sattari S, Shayeganpour A, El-Kadi AO, Brocks DR. Thestereoselective metabolism of halofantrine to desbutylhalofantrine inthe rat: evidence of tissue-specific enantioselectivity in microsomalmetabolism. Chirality 2007;19:22–33.

35. von Moltke LL, Granda BW, Grassi JM, Perloff MD, VishnuvardhanD, Greenblatt DJ. Interaction of triazolam and ketoconazole in P-glyco-protein-deficient mice. Drug Metab Dispos 2004;32:800–804.

36. Babovic-Vuksanovic D, Donaldson MD, Gibson NA, Wallace AM. Haz-ards of ketoconazole therapy in testotoxicosis. Acta Paediatr 1994;83:994–997.

37. Sonino N, Boscaro M, Paoletta A, Mantero F, Ziliotto D. Ketoconazoletreatment in Cushing’s syndrome: experience in 34 patients. ClinEndocrinol (Oxf) 1991;35:347–352.

38. Paine MF, Schmiedlin-Ren P, Watkins PB. Cytochrome P-450 1A1expression in human small bowel: interindividual variation and inhibi-tion by ketoconazole. Drug Metab Dispos 1999;27:360–364.

39. Shayeganpour A, El-Kadi AO, Brocks DR. Determination of theenzyme(s) involved in the metabolism of amiodarone in liver andintestine of rat: the contribution of cytochrome P450 3A isoforms.Drug Metab Dispos 2006;34:43–50.



nonlinear stereoselective pharmacokinetics of ketoconazole in rat after administration of racemate

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