Ketoconazole increases the plasma levels of ivermectin in sheep

Michel Alvinerie, Jacques Dupuy, Solange Kiki-Mvouaka,Jean-Francois Sutra, Anne Lespine *

INRA-Toulouse, UR66 Laboratoire de Pharmacologie-Toxicologie, 180 chemin de Tournefeuille,

BP 3, F-31931 Toulouse Cedex 9, France

Received 6 March 2008; received in revised form 12 June 2008; accepted 16 June 2008

Abstract

The parasiticide ivermectin and the antifungal drug ketoconazole are drugs that interact with P-glycoprotein. We have tested the

ability of ketoconazole at a clinical dose to modify the pharmacokinetics of ivermectin in sheep. Lacaune lambs were administered

with a single oral dose of ivermectin alone at 0.2 mg/kg (n = 5) or in combination with a daily oral dose of ketoconazole (10 mg/kg)

given for 3 days before and 2 days after the ivermectin (n = 5). The plasma kinetics of ivermectin and its metabolite were followed

over 15 days by HPLC analysis. Co-administration of ketoconazole induced higher plasma concentrations of ivermectin, leading to

a substantial increase in the overall exposure of the animals to the drug. Ketoconazole did not reduce the production of the main

ivermectin metabolite but it may rather act by inhibiting P-glycoprotein, and thus increasing the absorption of ivermectin. The use of

a P-gp reversing agent such as ketoconazole could be useful tool to optimize antiparasitic therapy in the face of the worldwide

development of anthelmintic resistance.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Macrocyclic lactone; Ketoconazole; P-glycoprotein; Drug–drug interaction; Resistance; Sheep

www.elsevier.com/locate/vetpar

Available online at www.sciencedirect.com

Veterinary Parasitology 157 (2008) 117–122

1. Introduction

Parasitic infections continue to play a significant role

in limiting the ability of livestock to reach full

productivity. At the present time, ivermectin (IVM), a

potent parasiticide belonging to the macrocyclic lactone

(ML) family of compounds, remains of major

importance for the treatment of both internal and

external parasites (McKellar and Benchaoui, 1996).

Their remarkable broad spectrum of activity and their

safety profile put these drugs at the cornerstone of

modern anthelmintic therapy in livestock. Several

million humans are also treated with ivermectin for

the control of onchocerciasis and lymphatic filiarisis

* Corresponding author. Tel.: +33 561285387; fax: +33 561285710.

E-mail address: lespine@toulouse.inra.fr (A. Lespine).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.06.017

(Molyneux et al., 2003). The success of ivermectin and

other MLs has also been due to their broad spectrum of

activity, safety and effectiveness against benzimida-

zole-, levamisole-, and pyrantel-resistant strains of

parasites, whose emergence severely restricted the fight

against parasites in the late 1970s (Molyneux et al.,

2003). Now, however, after 25 years of intensive use of

MLs, resistance to them has also appeared, initially in

small ruminants and now in cattle (Kaplan, 2004).

The final concentration and duration of an anthelmin-

tic in the host’s systemic circulation and finally in the

parasite is a key determinant of the efficacy of that drug.

Among the factors contributing to the loss of drug

efficacy and emergence of resistance are under-dosing

and the low bioavailability of some formulations. Due to

their role of the active efflux of xenobiotics out of the

organism, P-gp and probably other ABC transporter

M. Alvinerie et al. / Veterinary Parasitology 157 (2008) 117–122118

proteins play a key role in modulating the bioavailability

of macrocyclic lactones in both host and parasite. P-gp

appears to be the ABC transporter playing the major role

in IVM transport (Pouliot et al., 1997) and body

disposition (Schinkel et al., 1994). As it is located on

the blood–brain barrier, P-gp protects mammals against

the penetration of IVM into the brain and its subsequent

neurotoxicity (Kwei et al., 1999; Mealey et al., 2001;

Roulet et al., 2003; Schinkel et al., 1994). P-gp is also

present on the surface of the intestinal epithelium and the

bile canaliculi, and thus contributes to the high faecal

elimination of MLs (Laffont et al., 2002). In addition, P-

gp homologue gene selection is one of the mechanisms

involved in avermectin resistance in trichostrongylid

nematodes (Prichard and Roulet, 2007). By expelling the

drug out of the parasite, the presence of P-gp homologues

certainly plays a key role in reducing drug activity, and

favouring the development of resistance to MLs

(Sangster et al., 1999; Xu et al., 1998). Given its central

role in controlling the efflux of ivermectin, P-gp has

emerged as a key pharmacological target and the co-

administration of P-gp inhibitors has become a challen-

ging strategy for the improvement of drug action.

Ketoconazole (KTZ), an imidazole derivative, is an

orally active, broad-spectrum systemic antifungal agent

used in veterinary medicine. Ketoconazole and its

derivatives are well known inhibitors of cytochrome

P4503A activity in human hepatic microsomes (Bourrie

et al., 1996; Maurice et al., 1992). In addition, it has

been shown that ketoconazole co-administration

increased the AUC of several P-gp substrate drugs in

rats (Kageyama et al., 2005). Itraconazole, another

member of the imidazole family, has been also shown to

increase ivermectin bioavailability in plasma and to

reduce the intestinal secretion of the drug by inhibiting

P-gp in the rat (Ballent et al., 2006), sheep (Ballent

et al., 2007) and monkey (Ward et al., 2004). In

addition, it has been shown recently that ketoconazole

modifies the disposition of ivermectin in dogs by

reducing the clearance of the drug (Hugnet et al., 2007).

The interaction of ivermectin and ketoconazole in

vivo at therapeutic doses has never been explored in

ruminants. The aim of the study was to examine the

impact of the co-administration of ketoconazole and

ivermectin on the disposition of the ivermectin in sheep.

2. Materials and methods

2.1. Materials

Ketoconazole was purchased as tablets (Nizoral1)

from Janssen Pharmaceutica (Issy les Moulineaux,

France). Ivermectin (Oramec1) was obtained from

Merial (Lyon, France). All other chemicals used as

reagents were of analytical and high-performance liquid

chromatographic (HPLC) grade. The ivermectin meta-

bolite, 3-O-desmethyl ivermectin, was kindly provided

by Merial (Iselin, USA).

2.2. Animals

Ten female Lacaune lambs (UAC, L’Isle en Dodon,

France) weighing 29.1 � 2.0 kg were bought from a

breeder. The animals were raised on a zero-grazing

system and were free of trichostrongyle infections at the

beginning of the experiment. The sheep were housed in

concrete pens and were fed hay ad libitum and

commercial concentrates once a day (DP Nutrition).

No other treatment had been given during the 90 days

prior to the study and the sheep were fasted for 12 h

before the experiment. All procedures involving the

animals were approved by the local and institutional

animal use and care committee.

2.3. Study design

The animals were allocated into two groups (IVM

and KTZ-IVM) of five. The IVM group received a

single administration of ivermectin orally (Oramec,

Merial) at a dosage of 0.2 mg/kg. Lambs from the KTZ-

IVM group received ivermectin (similar administration

than IVM group) combined with repeat doses of

ketoconazole orally at a dose of 10 mg/kg per day as

tablets dissolved in water at 40 mg/ml (10 ml per

animal). The treatment was given 1 h after each

morning meal and repeated over 5 days (3 days before

and 2 days after the ivermectin administration).

2.4. Blood sampling

Blood samples (1 ml) were collected from the

cephalic vein before the administration of ivermectin

and at 1, 2, 4, 8, 24, 32 h, and 2, 3, 4, 6, 8, 10, 13, 15 days

post-administration. Plasma was immediately separated

from whole blood by centrifugation for 10 min at

1500 � g and stored at �18 8C prior to analysis.

2.5. Determination of plasma ivermectin

concentration

Ivermectin was analyzed in plasma by high-

performance liquid chromatography (HPLC) with

automated solid phase extraction and fluorescence

detection according to a previously described method

M. Alvinerie et al. / Veterinary Parasitology 157 (2008) 117–122 119

Fig. 1. Ivermectin concentration vs. time curves in lamb plasma

following oral administration of ivermectin. (A) Ivermectin concen-

tration; (B) metabolite concentration. Ivermectin was administered

orally at a single dose of 0.2 mg kg�1 alone (-*-) or with ketoco-

nazole given orally over 5 days (3 days before and 2 days after the

ivermectin administration) at repeat dose of 10 mg/kg per day (-&-).

Data are the mean � S.D. of five animals in each group.

(Lifschitz et al., 2000). Briefly, plasma samples were

diluted in acetonitrile, mixed for 20 min, sonicated for

10 min (Ultrasound Bath) and centrifuged at 2000 � g

for 2 min. The supernatant obtained was submitted to an

automated phase extraction and placed on a Benchmate

apparatus (BenchMate II, Zymark, Hopkinton, MA,

USA). The fluorescent derivative was obtained by

dissolving the eluent in N-methylimidazole and trifluor-

oacetic anhydride (Aldrich, Milwaukee, WI, USA)

solutions in acetonitrile. The chromatographic conditions

consisted of a mobile phase of 2% acetic acid, methanol,

acetonitrile (4:32:64, v/v/v) pumped at a flow rate of

1.5 ml min�1 through a Supelcosil C18, 3 mm column

(150 mm � 4.6 mm) (Supelco, Bellefonte, PA, USA).

Fluorescence detection (Detector RF 551, Shimadu,

Kyoto, Japan) was at 365 nm excitation and 475 nm

emission wavelengths. For the validation of the analytical

method used, calibration graphs in the range 0.1–

50 ng ml�1 were prepared using pooled drug-free plasma

and taken through the procedure. The regression line of

the plot of peak area against drug concentration showed a

correlation coefficient of 0.995. The mean recovery of the

extraction from plasma was 95%. The inter-assay

precision showed a coefficient of variation of 4.7%

and the limit of quantification was 0.05 ng ml�1.

2.6. Pharmacokinetic analysis

The ivermectin concentrations were analyzed using a

compartmental approach with the KineticaTM (Version

4.2) computer program (InnaPhase, Philadelphia, USA).

The following triexponential equation was fitted to

the plasma concentration vs. time data using a program

adapted from Multi (Yamakoa et al., 1981):

Ct ¼ Ae�at1 þ Ae�bt

2 � Ae�kat3

In which A1, A2 and A3 are the intercepts, C the plasma

concentration at time t, ka is the estimated first-order

rate constant of ivermectin absorption and a and b are

the first-order rate constants of ivermectin distribution

and elimination, respectively.

The data for the ivermectin metabolite were

subjected to non-compartimental analysis using the

statistical moment approach (Perrier and Mayersohn,

1982). The terminal (elimination) (t1/2b), absorption

half-life (t1/2ka) and half-life of distribution (t1/2a) were

calculated as ln 2/b, ln 2/ka and ln 2/a, respectively.

The areas under the plasma concentration–time curves

(AUC) from time zero to the last time with a measurable

concentration were calculated using the linear trape-

zoidal rule. The mean residence time (MRT) was

calculated using the linear trapezoidal rule without

extrapolation to infinity, using the formula:

MRT = AUMC/AUC, where AUMC is the area under

the moment curve and AUC the area under the plasma

concentration time curves, as previously defined. The

peak concentration (Cmax) and time to reach peak

concentration (Tmax) were read from the concentration–

time curves plotted for each animal.

2.7. Statistical analysis

The mean pharmacokinetic parameters for the two

groups were statistically compared with the non-

parametric Mann–Whitney test using the Statview

statistics program (Version 5.0). Mean values were

considered significantly different at p < 0.05.

3. Results

No adverse effects of the treatment were observed in

the two groups throughout the experimental period. The

graph of the mean plasma concentrations vs. time of

ivermectin after oral administration in the two groups is

shown in Fig. 1. In both groups, ivermectin was detected

in the plasma between 1 h and 15 days after

M. Alvinerie et al. / Veterinary Parasitology 157 (2008) 117–122120

Table 1

Influence of ketoconazole co-administration on pharmacokinetic

parameters of ivermectin in sheep

Parameters Ivermectin alone Ivermectin +

ketoconazole

Cmax (ng/ml) 5.4 � 2.3 10.6 � 3.6*

Tmax (d) 0.62 � 0.24 0.71 � 0.23

t1/2ka (d) 0.21 � 0.11 0.26 � 0.14

t1/2a (d) 0.96 � 0.20 0.93 � 0.27

t1/2b (d) 3.35 � 2.34 3.30 � 2.53

Cl/F (ml/min) 11.6 � 8.0 5.2 � 1.3

AUC(0-Clast) (ng d/ml) 15.9 � 8.1 28.0 � 8.18MRT(0-Clast) (d) 2.6 � 0.4 2.36 � 0.2

* p < 0.05, 8 p < 0.07 when compared to ivermectin alone, Cmax,

observed peak plasma concentration; Tmax, time to reach Cmax; t1/2ka,

absorption half-life; t1/2a, distribution half-life; t1/2b, terminal half-life

of elimination; MRT, mean residence time; AUC, area under the

plasma concentration vs. time curve. Values are mean � S.D. Values

are significantly different when p < 0.05.

Fig. 2. Effect of ketoconazole on the ivermectin or metabolite area of

the plasma concentration vs. time (AUC) in the IVM group and KTZ-

IVM group. Data are the mean � S.D. of five animals in each group.

*Significantly different when compared with the IVM group,

p < 0.05.

Table 2

Influence of ketoconazole co-administration on pharmacokinetic

parameters of ivermectin metabolite in sheep

Parameters Ivermectin alone Ivermectin +

ketoconazole

AUC(0-Clast) (ng d/ml) 0.96 � 0.63 1.73 � 0.83

Cmax (ng/ml 0.29 � 0.14 0.51 � 0.27

Tmax (d) 1.17 � 0.24 1.41 � 0.25

MRT(0-Clast) (d) 2.25 � 0.59 2.40 � 0.60

Cmax, observed peak plasma concentration; Tmax, time to reach Cmax;

t1/2b, half-life of elimination; MRT, mean residence time; AUC, area

under the plasma concentration vs. time. Values are mean � S.D.

Values are significantly different when p < 0.05.

administration. Concomitant administration of ketoco-

nazole-induced substantial increases in plasma con-

centrations of ivermectin resulting in a significant

increase in the Cmax (1.75-fold, p < 0.05). In parallel,

the AUC of ivermectin concentration in plasma was

twofold increased when Ketoconazole was co-adminis-

tered, increase which was close to statistical signifi-

cance ( p < 0.07). Data for the main pharmacokinetic

parameters describing the disposition of ivermectin in

both groups are shown in Table 1. The mean MRT was

unchanged whatever the treatment (2.57 days for IVM

vs. 2.34 days for KTZ-IVM) and the apparent clearance

(Cl/F) tended to be decreased (NS).

A metabolite of ivermectin was detected and

quantified in the sheep plasma. By comparison with

previous studies in goats and pigs, this compound was

identified as being the 3-O-desmethyl ivermectin

(Alvinerie et al., 1994; Chiu et al., 1987). The AUC

plot for the metabolite in both groups is shown in Fig. 2.

In our study, this metabolite was measured in the

plasma over 6 days post-treatment and accounted for

6% of the AUC of ivermectin in both groups, and its

concentration vs. time profiles closely paralleled those

of ivermectin in both groups. Although in the

ivermectin + ketoconazole group the metabolite con-

centration was higher with Cmax and AUC which tended

to be higher (NS, Table 2), the ratio of ivermectin/

metabolite concentrations was similar in both groups

varying between 60 and 10 throughout the kinetics.

Similarly, the ratio of the AUCs of the parent

ivermectin vs. the metabolite was similar in both

groups (16.6 and 16.2 for ivermectin alone vs.

ivermectin + ketoconazole).

4. Discussion

The purpose of this study was to investigate the

effect of multiple oral dosing of ketoconazole at

therapeutic doses on the pharmacokinetics of ivermec-

tin in sheep. The oral administration of IVM co-

administered with KTZ resulted in markedly higher

IVM plasma concentrations, leading to a substantial

increase in the overall exposure of the animal to the

antiparasitic drug compared with the IVM treatment

alone. In the ketoconazole group, there was a good

agreement between the increase in the AUC and the

Cmax that are parameters of exposure to ivermectin. The

KTZ-induced reduction of the IVM efflux by P-gp at the

level of cells lining the small intestine wall may have led

to higher absorption and the subsequent increase in the

systemic availability. Indeed, this is in agreement with

the significant role of P-gp in IVM transport through the

intestinal wall that has been well demonstrated (Ballent

et al., 2006, 2007; Griffin et al., 2005; Laffont et al.,

2002). By contrast to what was observed in dogs

(Hugnet et al., 2007), ketoconazole did not change

elimination processes of ivermectin. This is may be

M. Alvinerie et al. / Veterinary Parasitology 157 (2008) 117–122 121

related to a lower persistence of ketoconazole in

polygastric. The significant increase in the plasma

levels of ivermectin shows that ketoconazole increased

the exposure of the animals to the drug mainly by

increasing drug absorption.

Since ketoconazole is also known to inhibit both

CYP3A and the drug efflux transporter P-glycoprotein,

the increase in elimination pathways might also

contribute to the increase in the exposure of the sheep

to ivermectin. While the apparent clearance (Cl/F) of

ivermectin tended to be lower in the group receiving the

drug combination, this was not statistically significant.

Due to the inhibitory effect on CYP3A, the adminis-

tration of ketoconazole might be expected to decrease

the production of the ivermectin metabolite, thus

explaining the increase in ivermectin in the systemic

circulation. Indeed, the co-administration of ketoco-

nazole increases the bioavailability of highly metabo-

lized drugs such as quinidine (Kuroha et al., 2004),

mefloquine (Ridtitid et al., 2005), fexofenadine

(Ogasawara et al., 2007), quetiapine (Grimm et al.,

2006) and everolimus (Kovarik et al., 2005) in humans.

In the present experiment, a metabolite was identified

as 3-O-desmethyl ivermectin, which has been pre-

viously reported as the main ivermectin metabolite in

goats (Alvinerie et al., 1994) and pigs (Chiu et al.,

1987). This is the first report describing such metabolite

in sheep. Nevertheless, the concentration of this

metabolite was low in both experimental groups, never

exceeding 6% of the total ivermectin in plasma,

confirming that ivermectin is a poorly metabolized

drug as previously shown in several animal species

(Chiu et al., 1987). Moreover, the ratio of parent

ivermectin to metabolite concentration in plasma is

similar in the two groups. These results clearly

demonstrate that the increase in the exposure to

ivermectin observed in animals receiving both iver-

mectin and ketoconazole was not due to the inhibition

of ivermectin biotransformation.

The results suggest that the plasma and tissue

distribution of ivermectin was strongly influenced by P-

gp activity as previously shown (Kwei et al., 1999). The

strategic distribution of P-gp on the biliary canicular

membrane of hepatocytes and on the apical surface of

enterocytes means that this ABC-transporter is a key

player in both the biliary and the intestinal secretion of

ivermectin. Indeed, the disposition of ivermectin in the

intestinal fluid and tissue has been shown to be

significantly modified in the presence of P-gp mod-

ulators under both in vitro and in vivo conditions

(Ballent et al., 2006; Laffont et al., 2002). Subsequently,

enhanced ivermectin bioavailability in monogastric as

well as in polygastric animals has also been observed in

vivo with the concurrent administration of ivermectin

with other P-gp inhibitors such as verapamil, a well-

known competitive substrate for the P-gp drug-binding

site, in rats (Alvinerie et al., 1999) and sheep (Molento

et al., 2004) and with itraconazole in rats (Ballent et al.,

2006) and sheep (Ballent et al., 2007) or with

loperamide in rats (Lifschitz et al., 2004).

Our results thus provide a mechanistic explanation

for the increase in the systemic concentration of

ivermectin in sheep treated with ketoconazole: keto-

conazole does not affect the production of ivermectin

metabolite or the elimination processes in our experi-

mental conditions, but increases the absorption of the

parent drug through its potential to inhibit the activity of

P-gp that normally limits the absorption of ivermectin at

the intestinal level.

In conclusion, considering the observed increase in

the systemic bioavailability of ivermectin induced by

ketoconazole and the well-established correlation

between ivermectin plasma profiles and those achieved

in the tissues where parasites are located (Lifschitz

et al., 2000), the use of P-gp reversing agents such as

ketoconazole could be useful tools to optimize

antiparasitic therapy in the face of the worldwide

development of anthelmintic resistance. Further work to

evaluate the clinical relevance of this drug combination

is ongoing by using an optimized larval feeding

inhibition assay with the concomitant addition of

ketoconazole and ivermectin, in order to evaluate the

increase insusceptibility to ivermectin of resistant

nematodes.

Acknowledgement

This work was supported by the European Project

PARASOL CT-200X-022851.

References

Alvinerie, M., Dupuy, J., Eeckhoutte, C., Sutra, J.F., 1999. Enhanced

absorption of pour-on ivermectin formulation in rats by co-admin-

istration of the multidrug-resistant-reversing agent verapamil.

Parasitol. Res. 85, 920–922.

Alvinerie, M., Tardieu, D., Sutra, J., D, Bojensen, G., Galtier, P., 1994.

Metabolic profile of ivermectin in goats: an ‘‘in vivo’’ and ‘‘in

vitro’’ evaluation (ed.) 1994. EAVPT International Congress,

Edinburgh, Scotland. August 7–11, 1994.

Ballent, M., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 2006.

Modulation of the P-glycoprotein-mediated intestinal secretion of

ivermectin: in vitro and in vivo assessments. Drug Metab. Dispos.

34, 457–463.

Ballent, M., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 2007.

Involvement of P-glycoprotein on ivermectin kinetic behaviour in

M. Alvinerie et al. / Veterinary Parasitology 157 (2008) 117–122122

sheep: itraconazole-mediated changes on gastrointestinal disposi-

tion. J. Vet. Pharmacol. Ther. 30, 242–248.

Bourrie, M., Meunier, V., Berger, Y., Fabre, G., 1996. Cytochrome

P450 isoform inhibitors as a tool for the investigation of metabolic

reactions catalyzed by human liver microsomes. J. Pharmacol.

Exp. Ther. 277, 321–332.

Chiu, S.H., Taub, R., Sestokas, E., Lu, A.Y., Jacob, T.A., 1987.

Comparative in vivo and in vitro metabolism of ivermectin in

steers, sheep, swine, and rat. Drug Metab. Rev. 18, 289–302.

Griffin, J., Fletcher, N., Clemence, R., Blanchflower, S., Brayden, D.J.,

2005. Selamectin is a potent substrate and inhibitor of human and

canine P-glycoprotein. J. Vet. Pharmacol. Ther. 28, 257–265.

Grimm, S.W., Richtand, N.M., Winter, H.R., Stams, K.R., Reele, S.B.,

2006. Effects of cytochrome P450 3A modulators ketoconazole

and carbamazepine on quetiapine pharmacokinetics. Br. J. Clin.

Pharmacol. 61, 58–69.

Hugnet, C., Lespine, A., Alvinerie, M., 2007. Multiple oral dosing of

ketoconazole increases dog exposure to ivermectin. J. Pharm.

Pharm. Sci. 10, 311–318.

Kageyama, M., Namiki, H., Fukushima, H., Ito, Y., Shibata, N.,

Takada, K., 2005. In vivo effects of cyclosporin A and ketoco-

nazole on the pharmacokinetics of representative substrates for P-

glycoprotein and cytochrome P450 (CYP) 3A in rats. Biol. Pharm.

Bull. 28, 316–322.

Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary

importance: a status report. Trends Parasitol. 20, 477–481.

Kovarik, J.M., Beyer, D., Bizot,M.N., Jiang,Q., Shenouda, M., Schmou-

der, R.L., 2005. Blood concentrations of everolimus are markedly

increased by ketoconazole. J. Clin. Pharmacol. 45, 514–518.

Kuroha, M., Shirai, Y., Shimoda, M., 2004. Multiple oral dosing of

ketoconazole influences pharmacokinetics of quinidine after intra-

venous and oral administration in beagle dogs. J. Vet. Pharmacol.

Ther. 27, 355–359.

Kwei, G.Y., Alvaro, R.F., Chen, Q., Jenkins, H.J., Hop, C.E., Keohane,

C.A., Ly, V.T., Strauss, J.R., Wang, R.W., Wang, Z., Pippert, T.R.,

Umbenhauer, D.R., 1999. Disposition of ivermectin and cyclos-

porin A in CF-1 mice deficient in mdr1a P-glycoprotein. Drug

Metab. Dispos. 27, 581–587.

Laffont, C.M., Toutain, P.L., Alvinerie, M., Bousquet-Melou, A.,

2002. Intestinal secretion is a major route for parent ivermectin

elimination in the rat. Drug Metab. Dispos. 30, 626–630.

Lifschitz, A., Virkel, G., Sallovitz, J., Sutra, J.F., Galtier, P., Alvinerie,

M., Lanusse, C., 2000. Comparative distribution of ivermectin and

doramectin to parasite location tissues in cattle. Vet. Parasitol. 87,

327–338.

Lifschitz, A.L., Virkel, G.L., Sallovitz, J.M., Pis, A., Imperiale, F.A.,

Lanusse, C.E., 2004. Loperamide modifies the tissue disposition

kinetics of ivermectin in rats. J. Pharm. Pharmacol. 56, 61–67.

Maurice, M., Pichard, L., Daujat, M., Fabre, I., Joyeux, H., Domergue,

J., Maurel, P., 1992. Effects of imidazole derivatives on cyto-

chromes P450 from human hepatocytes in primary culture.

FASEB J. 6, 752–758.

McKellar, Q.A., Benchaoui, H.A., 1996. Avermectins and milbemy-

cins. J. Vet. Pharmacol. Ther. 19, 331–351.

Mealey, K.L., Bentjen, S.A., Gay, J.M., Cantor, G.H., 2001. Ivermec-

tin sensitivity in collies is associated with a deletion mutation of

the mdr1 gene. Pharmacogenetics 11, 727–733.

Molento, M.B., Lifschitz, A., Sallovitz, J., Lanusse, C., Prichard, R.,

2004. Influence of verapamil on the pharmacokinetics of the

antiparasitic drugs ivermectin and moxidectin in sheep. Parasitol.

Res. 92, 121–127.

Molyneux, D.H., Bradley, M., Hoerauf, A., Kyelem, D., Taylor, M.J.,

2003. Mass drug treatment for lymphatic filariasis and onchocer-

ciasis. Trends Parasitol. 19, 516–522.

Ogasawara, A., Kume, T., Kazama, E., 2007. Effect of oral ketoco-

nazole on intestinal first-pass effect of midazolam and fexofena-

dine in cynomolgus monkeys. Drug Metab. Dispos. 35, 410–418.

Perrier, D., Mayersohn, M., 1982. Noncompartmental determination

of the steady-state volume of distribution for any mode of admin-

istration. J. Pharm. Sci. 71, 372–373.

Pouliot, J.F., L’Heureux, F., Liu, Z., Prichard, R.K., Georges, E., 1997.

Reversal of P-glycoprotein-associated multidrug resistance by

ivermectin. Biochem. Pharmacol. 53, 17–25.

Prichard, R.K., Roulet, A., 2007. ABC transporters and beta-tubulin in

macrocyclic lactone resistance: prospects for marker develop-

ment. Parasitology 134, 1123–1132.

Ridtitid, W., Wongnawa, M., Mahatthanatrakul, W., Raungsri, N.,

Sunbhanich, M., 2005. Ketoconazole increases plasma concentra-

tions of antimalarial mefloquine in healthy human volunteers. J.

Clin. Pharm. Ther. 30, 285–290.

Roulet, A., Puel, O., Gesta, S., Lepage, J.F., Drag, M., Soll, M.,

Alvinerie, M., Pineau, T., 2003. MDR1-deficient genotype in

Collie dogs hypersensitive to the P-glycoprotein substrate iver-

mectin. Eur. J. Pharmacol. 460, 85–91.

Sangster, N.C., Bannan, S.C., Weiss, A.S., Nulf, S.C., Klein, R.D.,

Geary, T.G., 1999. Haemonchus contortus: sequence heterogene-

ity of internucleotide binding domains from P-glycoproteins. Exp.

Parasitol. 91, 250–257.

Schinkel, A.H., Smit, J.J., van Tellingen, O., Beijnen, J.H., Wagenaar,

E., van Deemter, L., Mol, C.A., van der Valk, M.A., Robanus-

Maandag, E.C., te Riele, H.P., et al., 1994. Disruption of the

mouse mdr1a P-glycoprotein gene leads to a deficiency in the

blood–brain barrier and to increased sensitivity to drugs. Cell 77,

491–502.

Ward, K.W., Stelman, G.J., Morgan, J.A., Zeigler, K.S., Azzarano,

L.M., Kehler, J.R., McSurdy-Freed, J.E., Proksch, J.W., Smith,

B.R., 2004. Development of an in vivo preclinical screen model to

estimate absorption and first-pass hepatic extraction of xenobio-

tics. II. Use of ketoconazole to identify P-glycoprotein/CYP3A-

limited bioavailability in the monkey. Drug Metab. Dispos. 32,

172–177.

Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R., Prichard,

R., 1998. Ivermectin resistance in nematodes may be caused by

alteration of P-glycoprotein homolog. Mol. Biochem. Parasitol.

91, 327–335.

Yamakoa, K., Tanigavara, K., Nokaguana, K., Unot, T., 1981. A

pharmacokinetic analysis program (multi) for microcomputer. J.

Pharmacobiol. Dyn. 4, 879–885.