J. vet. Pharmacol. Therap. 31, 230239, doi: 10.1111/j.1365-2885.2008.00953.x.

Evaluation of the interaction between ivermectin and albendazole following

their combined use in lambs
L. ALVAREZ* , 
A. LIFSCHITZ*

,

C. ENTROCASSO 
J. MANAZZA 
L. MOTTIER* , 
B. BORDA

G. VIRKEL* ,  &
C. LANUSSE* , 
*Laboratorio de Farmacologa, Facultad de
Ciencias Veterinarias, Universidad Nacional
del Centro de la Provincia de Buenos Aires
(UNCPBA), Campus Universitario, Tandil,
Argentina; Consejo Nacional de
Investigaciones Cientficas y Tecnicas
(CONICET), Argentina; Instituto Nacional
de Tecnologa Agropecuaria (INTA),
Estacion Experimental Balcarce, Balcarce,
Argentina

Alvarez, L., Lifschitz, A., Entrocasso, C., Manazza, J., Mottier, L., Borda, B.,
Virkel, G., Lanusse, C. Evaluation of the interaction between ivermectin and
albendazole following their combined use in lambs. J. Vet. Pharmacol. Therap.
31, 230239.
Mixtures of drugs from different chemical families have been proposed as a valid
strategy to delay the development of anthelmintic resistance. The current work
summarizes the outcome of the evaluation of the plasma disposition kinetics of
albendazole (ABZ) and ivermectin (IVM) administered either alone or coadministered to lambs infected with gastrointestinal (GI) nematodes resistant to
both anthelmintic molecules. Thirty six (36) Corriedale lambs naturally infected
with multiple resistant GI nematodes were allocated into six treatment groups:
(a) ABZ intravenous (ABZIV); (b) IVMIV; (c) ABZIV + IVMIV; (d) ABZ
intraruminal (IR); (e) IVM subcutaneous (SC) and (f) ABZIR + IVMSC. Plasma
samples were collected over 15 days post-treatment and analysed by HPLC.
The estimated pharmacokinetic (PK) parameters were statistically compared
using parametric and non-parametric statistical tests. The presence of IVM did
not affect the plasma disposition kinetics of ABZ and its metabolites after the i.v.
administration. However, the ABZ sulphoxide (ABZSO) area under the
concentration vs. time curve (AUC) was significantly lower (P < 0.01) after
the intraruminal (i.r.) administration of ABZ alone compared to that obtained
for the combined treatment with IVM [subcutaneous (s.c.) injection]. The IVM
plasma AUC obtained after its i.v. co-administration with ABZ was 88% higher
(P < 0.05) compared to the treatment with IVM alone. Any marked difference
on IVM PK parameters was observed between the treatments ABZ + IVM and
IVM alone injected subcutaneously. The data obtained here indicate that the
co-administration of ABZ and IVM does not induce an adverse kinetic
interaction. This type of pharmacology-based evaluation of drug interactions
is becoming highly relevant as drug combinations are now widely used as an
alternative to control resistant helminth parasites in livestock.
(Paper received 15 November 2007; accepted for publication 9 February 2008)
C. Lanusse, Laboratorio de Farmacologa, Departamento de Fisiopatologa, Facultad
de Ciencias Veterinarias, UNCPBA, Campus Universitario, (7000) Tandil, Argentina. E-mail: clanusse@vet.unicen.edu.ar

INTRODUCTION
Nematode infection control in livestock has been largely based
on the over-use of broad spectrum antiparasitic drugs. Therefore,
the anthelmintic resistance of sheep and cattle nematodes is an
increasing economic problem in many countries (Kaplan, 2004;
Wolstenholme et al., 2004). In this context, mixtures of drugs
from different chemical families have been proposed as a valid
strategy to delay the development of resistance (Anderson et al.,
1988).
Albendazole (ABZ) is a benzimidazole methylcarbamate
anthelmintic compound effective against lungworms and gas230

trointestinal (GI) nematodes, tapeworms and liver flukes (Campbell, 1990; McKellar & Scott, 1990). The intrinsic anthelmintic
action of benzimidazole compounds on the parasite relies on a
progressive disruption of basic cell functions as a result of their
binding to parasite tubulin and depolimerization of microtubules
(Lacey, 1990). Ivermectin (IVM), a member of the macrocyclic
lactones antiparasitic drugs, exhibits a broad-spectrum of
activity against GI and lung nematodes (Benz et al., 1989) as
well as against ectoparasites of domestic animals (McKellar &
Benchaoui, 1996). IVM acts on ligand-gated channels, including
glutamate and GABA-gated chloride channels, which participate
in nematode feeding, reproduction and locomotion (Feng et al.,
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

Albendazole and ivermectin co-administration in lambs 231

2002; Yates et al., 2003). In an attempt to overcome the

potential of the development of resistance to the available
anthelmintic chemical groups, several pharmaceutical formulations combining either two or three chemical entities have been
developed. Several preparations combining benzimidazole and
macrocyclic lactone compounds are available in the veterinary
pharmaceutical market (i.e. ABZ + IVM + levamisole or oxfendazole + IVM + levamisole). The rationale behind using combined anthelmintic preparations is based on the fact that
individual worms may have a lower degree of resistance to a
multiple component formulation (each chemical with different
mode of action) compared with that observed when a single
anthelmintic compound is used. However, potential pharmacokinetic (PK) and or pharmacodynamic (PD) interactions between components may occur and need to be addressed.
The most common PK interactions include enzyme induction
or inhibition, competition for transporter proteins and protein
binding. Evaluation of drug kinetic interactions in ruminant
species is scarce. Some information on the kinetic behaviour of
ivermectin and closantel co-administered to cattle has been
reported (Cromie et al., 2006). However, evaluation of drug-drug
kinetic interactions between benzimidazole and macrocyclic
lactones has not been assessed. Potential PK interactions
between co-administered anthelmintics should be understood
before any practical use can be advised. This study reports the
outcome of the evaluation of the plasma disposition kinetics of
ABZ and IVM administered either alone or co-administered to
lambs infected with GI nematodes resistant to both anthelmintic
molecules. The work reported here is complementary to a clinical
efficacy trial addressed to characterize the clinical efficacy of the
ABZ plus IVM combination against GI resistant nematodes in
lambs.

MATERIAL AND METHODS

Animals
Thirty six (36) female Corriedale lambs (78 month old,
27.3 4.3 kg), naturally infected with resistant GI nematodes
were involved in this trial. The experimental lambs were selected
from a farm where the failure of ABZ and IVM to control GI
nematodes had been previously demonstrated by the fecal egg
counts reduction test (FECRT). The selection of the animals was
based on worm egg per gram counts (epg). On day )1, all lambs
were checked for epg counts, ear tagged and the individual body
weights were recorded. Experimental animals had an average of
2028 1111 epg ranging from 600 to 4860. Animals were
allocated in a paddock and fed on a lucerne white and red clover
pasture during the experiment and for 20 days before starting
the PK study. All the animals had free access to water. Animal
procedures and management protocols were approved by the
Ethics Committee according to the Animal Welfare Policy (act
087 02) of the Faculty of Veterinary Medicine, Universidad
Nacional del Centro de la Provincia de Buenos Aires (UNCPBA),
Tandil, Argentina (http://www.vet.unicen.edu.ar).
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

Chemicals
Standards of ABZ, ABZ-sulphoxide (ABZSO), ABZ-sulphone
(ABZSO2), and oxibendazole (OBZ) used as internal standard,
were obtained from Schering Plough (Kenilworth, NJ, USA). IVM
pure reference standard was purchased from Sigma Chemical
Company (Saint Louis, MO, USA). The commercial formulation
of ABZ was provided by Pfizer Animal Health, Argentina
(Valbazen, 10% suspension) and IVM by Merial, Argentina
(Ivomec, 1% injectable solution). ABZ solution for intravenous
injection was prepared as a 2% (w v) solution in propylene
glycol dimethyl sulphoxide (80 20) (Anedra, Buenos Aires,
Argentina).
Experimental design, treatments and sampling
All parasitized lambs were randomly allocated into six experimental groups (n = 6). Experimental animals received the
following treatments: ABZIV: ABZ (2% solution) intravenously
(i.v.) administered at a dose rate of 3.8 mg kg; IVMIV: IVM
(Ivomec, Merial, 1% solution) administered by the i.v. route at
0.2 mg kg; ABZIV + IVMIV: ABZ (2% solution, 3.8 mg kg) plus
IVM (Ivomec, Merial, 1% solution, 0.2 mg kg), both administered by the i.v. route (two consecutive injections); ABZIR: ABZ
(Valbazen, Pfizer, 10% suspension) intraruminally (i.r.) administered at a dose rate of 3.8 mg kg; IVMSC: IVM (Ivomec,
Merial, 1% solution) subcutaneously (s.c.) administered at the
dose rate of 0.2 mg kg; IVMSC + ABZIR: ABZ (Valbazen, Pfizer,
10% suspension, 3.8 mg kg) plus IVM (Ivomec, Merial, 1%
solution, 0.2 mg kg), administered by the i.r. and s.c. routes,
respectively.
Blood samples were taken from the jugular vein before
administration (time 0) and at 5, 15 and 30 min, 1, 3, 6, 9,
12, 24 and 48 h, and 4, 6 and 10 days following the i.v.
treatments. After the i.r. or s.c. treatments, jugular blood
samples were collected before administration (time 0) and at 1,
3, 6, 9, 12, 18, 24, 36, 48 h and at 4, 6, 10 and 15 days posttreatment. In all the experimental groups, blood samples were
collected using 10 mL heparinised Vacutainers tubes (Becton
Dickinson, NJ, USA). Plasma was separated by centrifugation at
2000 g for 15 min, placed into plastic tubes and frozen at
)20 C until analysis by high performance liquid chromatography (HPLC).
Analytical procedures
ABZ metabolites analysis
Sample clean up: ABZ, ABZSO and ABZSO2 were extracted using
disposable C18 columns (RP-18, 100 mg, Strata, Phenomenex,
CA, USA). Ten (10) lL of OBZ (50 lg mL) was added to 500 lL
of plasma in a glass test tube. Spiked samples were placed into a
C18 column (preconditioned with 0.5 mL of methanol followed
by 0.5 mL water) in a vacuum system (Lichrolut, Merck,
Germany). Samples were washed (2 mL of water) and then
eluted with 2 mL of HPLC grade methanol. After elution, all
samples were concentrated to dryness in a vacuum concentrator

232 L. Alvarez et al.

(Speed-Vac, Savant, MN, USA) and then reconstituted with

300 lL of mobile phase.
HPLC analysis: Experimental and spiked plasma samples (used
for validation) were analyzed by HPLC (Shimadzu 10 A-HPLC
System, Kyoto, Japan) with a UV detector set at 292 nm.
Fifty (50) lL of each previously extracted sample was injected
and the analytes eluted (flow 1.2 mL min) from the analytical
column (5 lm, 250 mm 4.6 mm, C18 column, Phenomenex
Selectosil, CA, USA) by a binary gradient previously described
(Alvarez et al., 1999). The compounds were identified by the
retention times of pure reference standards. Retention times for
ABZSO, ABZSO2, OBZ and ABZ were 5.85, 7.26, 10.01 and
11.24 min, respectively. There was no interference of endogenous compounds in the chromatographic determinations.
Plasma calibration curves for each analyte were constructed
by least squares linear regression analysis, giving a correlation
coefficient (r) between 0.9987 and 0.9995. Mean absolute
recovery percentages for concentrations ranging between 0.25
and 5 lg mL (n = 6) were 77.3 (ABZSO), 85.5 (ABZSO2) and
86.7% (ABZ) with coefficient of variation (CV) of 4.9, 2.5 and
7.8%, respectively. The precision of the method (intra- and interassay) was determined by analyzing plasma samples (n = 6)
fortified with OBZ and metabolites at three different concentrations (0.25, 1 and 5 lg mL). The CV for the intra and interassay precision ranged from 4.37 to 7.44%. The limit of
detection (LOD) was estimated according to the following
equation (Snyder et al., 1997):
LOD A=B SD  3
where A is the baseline threshold at the retention time of each
compound (n = 6) in spiked plasma samples, B the peak area of
the internal standard (OBZ), and SD the standard deviation obtained from A. The LOD obtained were 0.003, 0.002 and
0.002 lg mL for ABZSO, ABZSO2 and ABZ, respectively. The
limit of quantification (LOQ) was defined as the lowest measured
concentration with a CV <20% an accuracy of 20% and a
absolute recovery 70%. The LOQ obtained for the three molecules assayed was 0.1 lg mL. Values below LOQ were not included in the PK analysis.
IVM analysis
Sample clean up and derivatization: The extraction of IVM (22,23
dehydro-avermectin B1a), from spiked and experimental plasma
samples was carried out following the well established technique
(Alvinerie et al., 1993; slightly modified by Lifschitz et al., 1999).
Basically, 1 mL-aliquot of plasma sample was combined with
10 ng of the internal standard compound (abamectin) and then
mixed with 1 mL of acetonitrile-water (4:1). After mixing for
20 min, the solvent-sample mixture was centrifuged at 2000 g
during 15 min. The supernatant was manually transferred into
a tube that was then placed on the appropriate rack of an Aspec
XL sample processor (Gilson, Villiers Le Bel, France). The
supernatant was injected to a Supelclean LC18 cartridge
(Supelco, Bellfonte, PA, USA), previously conditioned by passing
2 mL methanol and 2 mL deionized water. The cartridge was
flushed with 1 mL of water and 1 mL of water methanol (4:1).

The compounds were eluted with 1.5 mL of methanol and

concentrated to dryness under a stream of nitrogen. The resuspension was carried out with 100 lL of a solution of
N-methylimidazole (Sigma Chemical, St. Louis, MO, USA) in
acetonitrile (1:1) (De Montigny et al., 1990). Derivatization was
initiated by adding 150 lL of trifluoroacetic anhydride (Sigma
Chemical, St Louis, MO, USA) solution in acetonitrile (1:2). After
completion of the reaction (<30 sec), an aliquot (100 lL) of this
solution was injected directly into the chromatograph.
HPLC analysis: IVM concentrations were determined by HPLC
using a Shimadzu 10 A HPLC system with autosampler
(Shimadzu Corporation, Kyoto, Japan). HPLC analysis was
undertaken using a reverse phase C18 column (Phenomenex,
5 lm, 4.6 mm 250 mm) and an acetic acid 0.2% in
water methanol acetonitrile (3.8 40 56.2) mobile phase at a
flow rate of 1.5 mL min at 30 C. IVM was detected using a
fluorescence detector (Shimadzu, RF-10 Spectrofluorometric
detector, Kyoto, Japan), readings at 365 nm (excitation wavelength) and 475 nm (emission wavelength). IVM concentrations
were determined by the internal standard method using the
Class LC 10 Software version 1.2 (Shimadzu Corporation, Kyoto,
Japan) on an IBM compatible AT computer. The peak area ratios
were considered to calculate the IVM concentrations in spiked
(validation) and experimental plasma samples. There was no
interference of endogenous compounds in the chromatographic
determinations. The solvents (Baker, Phillipsburg, NJ, USA) used
during the extraction and drug analysis were HPLC grade.
A complete validation of the analytical procedures used for
extraction and quantification of IVM was performed before
starting analysis of the experimental samples obtained during
the PK trial. Calibration curves in the range between 0.1
5 ng mL and 5100 ng mL were prepared for each compound.
Calibration curves were established using least squares linear
regression analysis and correlation coefficients (r) and CV
calculated. Linearity was established to determine the IVM
concentrations detector responses relationship. Percentages of
IVM recovery from plasma were obtained in the range between
0.1 and 50 ng mL. The inter-assay precision of the extraction
and chromatography procedures was estimated by processing
replicate aliquots (n = 6) of pooled sheep plasma samples
containing known IVM concentrations (0.2, 10 and 50 ng mL)
on different working days. The LOD and LOQ were calculated
similarly as described for ABZ metabolites. Concentration values
below the LOQ were not considered for the kinetic analysis of
experimental data. The linear regression lines for IVM analyzed
showed correlation coefficients to 0.998. The mean recovery of
IVM from plasma was in a range between 75 and 80%. The inter
assay precision of the analytical procedures obtained after HPLC
analysis of IVM on different working days showed CV between
3.26 and 7.71%. The LOQ was established at 0.1 ng mL.
Pharmacokinetic analysis of the data
The concentration versus time curves for ABZ metabolites and
IVM in plasma for each individual animal after the different
treatments was fitted with PK Solution 2.0 (Summit research
services, CO, USA). The following equation (Notari, 1987) was
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

Albendazole and ivermectin co-administration in lambs 233

where: Cp = concentration in plasma at time t after administration (lg mL); B = concentration at time zero extrapolated
from the elimination phase (lg mL); e = base of the natural
logarithm; = terminal slope (h)1); and k is the slope obtained
by feathering, which represents either the first order absorption
rate constant (kab) or first order metabolite formation rate constant (kfor) (h)1). The elimination half-life (T el) and absorption
(T ab) or metabolite formation half-lives (T for) were calculated as ln2 and ln2 k, respectively. The peak concentration
(Cmax) and time to peak concentration (Tmax) were displayed
from the plotted concentration-time curve of each analyte.
The area under the concentration time-curve (AUC) was calculated by the trapezoidal rule (Gibaldi & Perrier, 1982) and further
extrapolated to infinity by dividing the last experimental concentration by the terminal slope (). The mean residence time
(MRT) was determined as AUMC AUC (Perrier & Mayersohn,
1982) where AUMC is the area under the curve of the product of
time and the plasma drug concentration vs.time from zero to
infinity (Gibaldi & Perrier, 1982), and AUC is as defined above.
The data points generated for ABZ or IVM in plasma after its
i.v. administration were best-fitted to a two-compartment model:
Cp Aeat  Bebt
where A and B are the primary and secondary disposition
intercepts; a and are the primary and secondary disposition
rate constant (h)1); and Cp was the plasma concentration of ABZ
or IVM at time t. The distribution and elimination half-lives were
calculated as ln 2 divided by the rate constants. The estimated
plasma concentration of ABZ parent drug at zero time (C0) after
its i.v. administration was the sum of the extrapolated zero-time
concentrations of the coefficient A and B. Estimation of the
volume of the central compartment (Dose C0) and microconstants were also obtained. Total body clearance (ClB) was calculated by:
ClB Dose=AUC
The volume of distribution (Vdarea) was estimated by the
following equation:
Vdarea Dose=AUCb
Statistical analysis of the data
The PK parameters and concentration data are reported as
arithmetic mean SD; however, the half-lives (formation and
elimination) and MRT, are presented as harmonic means SD.
Parametric (Students t-test) and or nonparametric (MannWhitney and Wilcoxon) tests were used for the statistical
comparison of the PK data obtained from the different experimental groups. A value of P < 0.05 was considered statistically
significant. The statistical analysis was performed using the
Instat 3.0 Software (Graph Pad Software, CA, USA).
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

The mean ( SD) plasma concentrations of ABZ (a) and ABZSO

(b) after the i.v. administration of ABZ either alone or coadministered with IVM are shown in Fig. 1. Table 1 summarizes
the plasma PK parameters for ABZ and ABZSO obtained after the
i.v. administration of ABZ either alone (ABZIV) or co-administered with IVM (ABZIV + IVMIV) to parasitized lambs. After its
i.v. administration, ABZ was detected in plasma up to 3 h posttreatment. Its main metabolic product found in plasma (ABZSO)
was detected between 5 min and 24 h post-treatment. The
sulphone metabolite reached a Cmax of 0.35 0.08 (ABZIV)
and 0.32 0.10 (ABZIV + IVMIV) lg mL at 9.5 and 10 h posttreatment, respectively. The presence of IVM did not affect the
plasma disposition kinetics of ABZ metabolites (Table 1) after
the i.v. administration.

(a)
10

ABZ plasma
concentrations (g/mL)

Cp Bebt  Bekt

RESULTS

Intravenous treatment
ABZ alone
ABZ + IVM

0.1

5 min

15 min

30 min

3h

1h

Time
(b)

ABZSO
Intravenous treatment
ABZ alone
ABZ + IVM

ABZSO plasma
concentrations (g/mL)

used to describe the biexponential concentration-time curves for

ABZSO, ABZSO2 and IVM after the i.r. and s.c. treatments:

0
0

10

15

20

25

Time (h)
Fig. 1. Comparative mean (SD) plasma concentration profiles (n = 6)
for (a) albendazole (ABZ), and (b) ABZ-sulphoxide (ABZSO), after the
administration of ABZ either alone (3.8 mg kg) or co-administered with
ivermectin (0.2 mg kg), both given by the intravenous route to
parasitized lambs.

234 L. Alvarez et al.

(ABZIV)

Pharmacokinetic
parameters
C0 (lg mL)
Cmax (lg mL)
Tfor (h)*
AUC0-t
(lgh mL)
Tel (h)*
MRT (h)*
Vdarea (L kg)
ClB (L h kg)

(ABZIV + IVMIV)

ABZ

ABZSO

ABZ

ABZSO

5.08 2.40

1.70 0.68

3.22 0.71
0.10 0.08
30.2 5.31

6.60 4.55

2.03 1.10

2.96 0.64
0.14 0.04
33.9 6.65

0.29
0.39
1.44
2.28

7.34 0.95
10.4 1.45

0.32
0.41
1.20
2.20

9.31 1.64
13.2 2.45

0.83
0.60
1.16
0.89

0.38
0.19
0.51
1.02

Table 1. Plasma pharmacokinetic parameters

(mean SD) for albendazole (ABZ) and its
ABZ sulphoxide (ABZSO) metabolite obtained
after the intravenous administration of ABZ
(3.8 mg kg) to parasitized lambs either alone
or co-administered with ivermectin
(0.2 mg kg)

*Time-based parameters calculated as harmonic means. Different from the ABZIV treatment at
P < 0.05.
C0, concentration at time 0; Cmax, peak plasma concentration; Tfor, metabolite formation half
life; AUC0-t, area under the plasma concentration vs. time curve from 0 to the detection time; Tel,
elimination half-life; MRT, mean residence time (obtained by non-compartmental analysis of the
data); Vdarea, apparent volume of distribution (area method); ClB, total body clearance.

The plasma concentration profiles of IVM observed after its i.v.

administration alone or co-administered with ABZ in lambs are
shown in Fig. 2. The PK parameters obtained for IVM in lambs,
after its administration by the i.v. or s.c. route, either alone or coadministered with ABZ, are shown in Table 2. After the i.v.
administration, IVM plasma levels were measured up to 10 days
post-treatment. Conversely to that observed for ABZ-related
metabolites, the plasma disposition kinetics of IVM was modified
by the presence of ABZ metabolites after the i.v. treatment. A
significantly (P < 0.05) higher AUC value was obtained for the
ABZIV + IVMIV treatment (AUC = 210.3 80.6 ngday mL),
compared to that observed in the IVMIV group
(AUC = 112.3 37.4 ngday mL). No statistical differences
between both treatments were observed among other PK
parameters, including C0, T el, MRT, ClB and Vdarea (Table 2).

DISCUSSION

1000
Intravenous treatment

IVM plasma
concentrations (ng/mL)

The comparative mean ABZSO and IVM plasma concentrations profiles measured after the administration of ABZ (i.r.)
alone, IVM (s.c.) alone or after their co-administration to lambs
are shown in Figs 3 & 4. The plasma PK parameters obtained for
ABZSO and ABZSO2 after the i.r. administration of ABZ to lambs
alone or co-administered with IVM are shown in Table 3. ABZ
administered by the i.r. route did not affect the plasma PK
behavior of IVM administered by the s.c. route. However, the
presence of IVM induced changes in the plasma levels of ABZSO.
The ABZSO AUC value increased 42% after administration of the
combination ABZIR + IVMSC (19.8 2.55 lgh mL), compared
to the treatment with ABZIR alone (28.2 3.72 lgh mL).
Furthermore, the MRT and Tfor values obtained for ABZSO2
resulted longer after the ABZIR + IVMSC treatment compared to
ABZIR administration (Table 3).

IVM alone
IVM + ABZ

100

10

0.1
0

10

Time (days)
Fig. 2. Comparative mean (SD) plasma concentration profiles (n = 6)
for ivermectin (IVM) obtained after its administration (0.2 mg kg)
either alone or co-administered with albendazole (ABZ, 3.8 mg kg),
both given by the intravenous route to parasitized lambs.

The interaction between co-administered drugs may induce

changes on the PK behavior of either molecule. The overall
disposition kinetics and pattern of tissue residues are often
modified when drug-drug interactions occur. Both a PK and or a
PD interaction may be established following the combined use of
ABZ and IVM in ruminant species. The kinetic assessment of a
potential interaction undertaken in the current work is useful to
characterize the pharmacological basis of the combination of
both anthelmintic compounds. ABZ was rapidly depleted from
the bloodstream following its i.v. administration to lambs, being
detected up to only 3 h post-treatment. The metabolites ABZSO
(active metabolite) and ABZSO2 (inactive metabolite) appeared
rapidly (5 min) in the bloodstream after the i.v. administration of
ABZ. This disposition pattern agrees with those previously
reported in sheep (Galtier et al., 1991; Alvarez et al., 1999), after
the intravascular administration of ABZ. A rapid and efficient
microsomal biotransformation, and an extensive ABZ tissue
distribution (Vd = 1.44 1.16 L kg, ABZIV), account for the
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

Albendazole and ivermectin co-administration in lambs 235

Table 2. Plasma pharmacokinetic parameters
(mean SD) for ivermectin (IVM) obtained
after its intravenous or subcutaneous
administration to parasitized lambs
(0.2 mg kg) either alone or co-administered
with albendazole (3.8 mg kg)

IVM treatments

Pharmacokinetic
parameters
C0 (ng mL)
Cmax (ng mL)
Tmax (days)
AUC0-t (ngday mL)
Tel (days)*
MRT (days)*
Vdarea (L kg)
ClB (L day kg)

(IVMIV)
281.2

112.3
1.35
1.25
3.85
1.97

(IVMIV + ABZIV)

32.6

314.2

210.3
1.45
1.51
2.44
1.15

37.4
0.19
0.33
1.30
0.72

(IVMSC)

94.8

21.3
2.80
131.1
2.67
5.38

80.6
0.42
0.36
1.20
0.69

13.3
1.5
70.5
1.21
1.19

(IVMSC + ABZIR)

18.2
1.80
139.7
4.15
6.64

5.71
1.20
28.6
1.11
1.26

*Time-based parameters calculated as harmonic means. Different from the IVMIV treatment at
P < 0.05.
C0, concentration at time 0; Cmax, peak plasma concentration; Tfor, metabolite formation half
life; AUC0-t, area under the plasma concentration vs. time curve from 0 to the detection time; Tel,
elimination half-life; MRT, mean residence time (obtained by non-compartmental analysis of the
data); Vdarea, apparent volume of distribution (area method); ClB, total body clearance.

Subcutaneous treatment
IVM alone
IVM + ABZ

Intraruminal treatment
ABZ alone
ABZ + IVM

ABZSO

IVM plasma
concentrations (ng/mL)

ABZSO plasma
concentrations (g/mL)

1.5

1.0

0.5

10

0.0
0

10

15

20

25

30

35

Time (h)
Fig. 3. Comparative mean (SD) plasma concentration profiles (n = 6)
for albendazole sulphoxide (ABZSO) obtained after the administration
of albendazole (ABZ) by the intraruminal route (3.8 mg kg) either alone
or co-administered with ivermectin (IVM) given by the subcutaneous
route (0.2 mg kg) to parasitized lambs.

observed fast depletion from the bloodstream. IVM was detected

in the bloodstream up to 10 days after its i.v. administration.
This extended IVM plasma detection is related to its high
lipophilicity and affinity for fatty tissues, low metabolism and
extensive enterohepatic recycling, which prolong the organic
elimination of the drug (Lanusse et al., 1997; Lifschitz et al.,
2000). IVM is excreted in high concentration in the bile of sheep
(Bogan & McKellar, 1988), which constitutes the primary route
of excretion for this compound. The estimated Vd for IVM after
its i.v. administration was 3.85 1.30 L kg (IVMIV), which
correlates with its well characterized extensive tissue distribution
(Lifschitz et al., 2000). Low metabolic rate and extended
enterohepatic recycling account for the low total body clearance
(1.97 0.72 L day kg) obtained for IVM intravenously injected
to lambs.
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

10

12

14

16

Time (days)
Fig. 4. Comparative mean (SD) plasma concentration profiles (n = 6)
for ivermectin (IVM) obtained after its subcutaneous administration
(0.2 mg kg) either alone or co-administered with albendazole (ABZ)
given by the intraruminal route (3.8 mg kg) to parasitized lambs.

No significant PK changes were observed for ABZ metabolites

after its i.v. co-administration with IVM in lambs. Only the
ABZSO Tel resulted significantly (P < 0.05) longer after the coadministration of ABZIV + IVMIV compared with that observed
after the i.v. administration of ABZ alone (Table 1). Nevertheless,
the observed 26% longer T el does not seem to have any
clinical relevance (Entrocasso et al., in press). Conversely, an
increase on IVM plasma concentrations was observed in the
presence of ABZ, after the i.v. co-administration of both
compounds. The enhanced IVM plasma concentrations observed
after the i.v. co-administration of both anthelmintics resulted in
an increase in the AUC value (87%). No other changes on the PK
disposition after the i.v. administration were observed.
Consistently with kinetic data previously obtained in sheep
(Marriner & Bogan, 1980; Hennessy et al., 1989; Lanusse et al.,
1995), ABZSO and ABZSO2 were the main metabolites recovered

236 L. Alvarez et al.

Pharmacokinetic
parameters
Tfor (h)*
Cmax (lg mL)
Tmax (h)
AUC0-t (lgh mL)
Tel (h)*
MRT (h)*

ABZSO
(ABZIR)
2.25
1.08
9.50
19.8
14.1
27.1

1.33
0.22
2.26
2.55
7.30
9.13

ABZSO2

(ABZIR + IVMSC)
4.30
1.22
14.0
28.2
9.82
20.9

0.54
0.37
5.90
3.72
3.22
3.76

(ABZIR)
1.88
0.39
24.0
6.25
1.93
20.1

0.58
0.23
0.00
2.75
0.65
0.97

(ABZIR + IVMSC)
2.69
0.33
30.0
7.88
2.73
25.7

0.44
0.10
6.57
1.92
0.50
2.15

Table 3. Effects of ivermectin (IVM) on the

disposition of ABZ metabolites. Plasma pharmacokinetic parameters (mean SD) for
albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) obtained after the
intraruminal (i.r.) administration of albendazole (ABZ) to lambs (3.8 mg kg) either alone
or co-administered with ivermectin
(0.2 mg kg) by the subcutaneous (s.c.) route

*Time-based parameters calculated as harmonic means. Different from the ABZIR treatment at
P < 0.05.
Tfor, metabolite formation half life; Cmax, peak plasma concentration; Tmax, time to the Cmax;
AUC0-t, area under the plasma concentration vs. time curve from 0 to the detection time; Tel,
elimination half-life; MRT, mean residence time (obtained by non-compartmental analysis of the
data).

in plasma after the i.r. administration of ABZ, which has been

related to a first-pass oxidation occurring mainly in the liver.
ABZSO was the main metabolite recovered in plasma for a period
of 36 h (Fig. 3) post-treatment, accounting for either 76 (ABZIR)
or 78% (ABZIR. + IVMSC) of the total analytes found in sheep
plasma after the i.r. administration of ABZ. After s.c. administration of IVM to lambs, a Cmax value of 21.3 13.3 ng mL
was obtained at 2.8 1.5 days post-treatment (IVMSC)
(Table 2). These results agree with that previously reported for
IVM administered by the s.c. route to sheep (Marriner et al.,
1987; Imperiale et al., 2004). Unlike the observations after the
i.v. treatments, the presence of IVM (s.c. injection) modified the
plasma PK behavior of ABZSO and ABZSO2. The ABZSO plasma
AUC was significantly (P < 0.05) higher (>42%) following the
ABZ + IVM parenteral co-administration compared to that
observed after ABZ i.r. alone treatment (Table 3). Additionally,
longer MRT and T for values were observed for ABZSO2 in the
presence of IVM (Table 3). Oppositely, ABZ did not modify the
plasma disposition of IVM after their co-administration by the i.r.
and s.c. routes, respectively.
From the results obtained in the current trial, it is evident that
some type of PK interaction between ABZ and IVM occurs after
their coadministration to lambs. This interaction determines that
a) higher IVM plasma concentrations were measured after its coadministration with ABZ (both administered by the i.v. route)
and, b) higher ABZSO plasma availability was obtained after the
i.r. administration of ABZ co-administered with IVM given
subcutaneously. Although it is unclear at which level
ABZ metabolites and IVM may interact, two possible explanations would help to explain the observed PK changes: 1) IVMinduced inhibition of ABZ metabolism, or 2) drug to drug
interaction via drug efflux transporter-mediated mechanisms.
Albendazole is extensively metabolized in the sheep liver
microsomal fraction; the flavin-mono-oxygenase (FMO) (Galtier
et al., 1986) and cytochrome P-450 (CYP) enzymatic systems
(Souhaili-El Amri et al., 1987) are primarily involved in ABZ
sulphoxidation to ABZSO. Recently, it has been demonstrated
that FMO-mediated sulphoxidation accounted for up to 60% of
ABZSO production from ABZ, whilst the CYP contributed with
40% in sheep liver microsomes (Virkel et al., 2004). A relatively

high CYP3A activity (measured as 6-tetosterone hydrolase

activity) was described in ovine liver microsomes (Szotakova
et al., 2004). Since CYP3A is the most important drug-metabolising CYP, its involvement on ABZ metabolism could be
expected. In fact, it has been suggested that CYP 3A4 may be
the key contributor to ABZSO formation (Rawden et al., 2000).
On the other hand, ABZSO undergoes a second irreversible
oxidative step (sulphonation) which forms the inactive ABZSO2
metabolite (Galtier et al., 1991), in which CYP1A is mainly
involved (Souhaili-El Amri et al., 1988; Delatour et al., 1991).
A low rate of IVM metabolism under in vitro conditions has been
reported using rat liver microsomes (Zeng et al., 1996). It is
estimated that only a 8.5% of the total parent drug detected in
cattle plasma is related to IVM metabolites (Lanusse et al., 1997).
The main enzymatic system involved on IVM metabolism
includes CYP3A and 1A in rats (Zeng et al., 1996) and CYP3A4
in humans (Zeng et al., 1998).
The co-administration of fenbendazole with piperonyl butoxide (PB), a potent inhibitor of the CYP mediated oxidation, in
sheep and goats, markedly affected the PK disposition of
fenbendazole metabolites, potentiating their nematodicidal
activity against benzimidazole-resistant strains of Teladorsagia
circumcincta (Benchaoui & McKellar, 1996). Similarly, methimazole a metabolic inhibitor of FMO, potentiates the efficacy of
netobimin (pro-drug of ABZ) against mixed GI nematodes in
cattle (Lanusse & Prichard, 1992). It could be hypothesized that
in the current trial IVM may have interfered with both, ABZ
sulphoxidation and ABZSO sulphonation by mean of a CYP 3A
and 1A competition inhibition. Such a possible metabolic
interaction between both anthelmintic drugs does not seem to
achieve great practical relevance.
Other possible explanation for the observed ABZ and IVM PK
interaction could be based in a drug-drug interaction via a
transporter s mediated drug efflux mechanism. As part of the
ABC superfamily, P-glycoprotein (Pgp) is a transmembrane
protein located in the apical side of cells that participate in the
ATP-dependant efflux of a broad range of structurally and
functionally unrelated compounds out of the cell (Gerlach et al.,
1986). Pgp is physiologically expressed in different organs tissues including adrenals, kidneys, liver (biliary canalicular
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

Albendazole and ivermectin co-administration in lambs 237

surface of hepatocytes), small intestine (epithelial cells) and blood

brain barrier (Balayssac et al., 2005). Pgp plays a key role in the
protection of the organism against ingested toxins and contribute to the biliar, urinary and intestinal elimination of different
unrelated compounds. IVM is largely excreted in bile and feces as
the parent drug, with less than 2% excreted in the urine (Chiu
et al., 1990). IVM has been described as a specific Pgp substrate
(Didier & Loor, 1996; Pouliot et al., 1997) which is actively
secreted from the rat intestine (Laffont et al., 2002). Besides, the
co-administration of IVM with loperamide (a Pgp modulatingagent) resulted in changes on the pattern of bile-fecal excretion,
which accounted for an enhanced IVM availability in rats
(Lifschitz et al., 2004). Recently, IVM was proposed as a
multidrug resistance protein 1 and 2 (MRP 1 and 2) substrate
(Lespine et al., 2006). Additionally, it is known that ABZSO is
actively secreted into the intestinal lumen (Redondo et al.,
1999), probably because of a combination of passive diffusion
and active transport. Pgp, MRP2 and the breast cancer
resistance protein (BCRP) have been proposed as the main
candidate proteins involved on ABZSO intestinal efflux transport
(Merino et al., 2003). A highly efficient transport of ABZSO by
murine BCRP1 has been described (Merino et al., 2005). In
contrast, ABZ parent drug does not seem to interact with Pgp
(Merino et al., 2002, 2005; Dupuy et al., 2006), MRP2 or
BCRP1 (Merino et al., 2005).
ABZ-sulphoxide is mainly eliminated by urine, although biliar
excretion (free and conjugated) in sheep has been described
(Hennessy et al., 1989; Alvarez et al., 1999). In fact, high
intestinal concentrations of ABZSO were detected in sheep
(Alvarez et al., 1999) and cattle (Lanusse et al., 1993) after the
i.r. administration of ABZ or netobimin, respectively. The relative
involvement of the biliary and intestinal excretion mechanisms for
ABZ metabolites and IVM in sheep needs to be elucidated.
However, since both compounds (at least ABZSO and IVM) have
been indicated as Pgp substrates, an interaction at this level should
be considered. IVM competition on ABZSO efflux (at intestinal or
biliar level) may help to explain the significantly higher ABZSO
AUC value observed in lambs treated with ABZIR + IVMSC. There
was no change in the ABZSO plasma concentrations after the
intravenous co-administration of IVM and ABZ. In agreement
with our findings, similar results were reported by Merino et al.
(2003), using verapamil (a Pgp inhibitor), which did not have a
clear effect on the ABZSO intestinal elimination after their i.v. coadministration in rats; however, after the oral administration of
ABZ plus verapamil, a significantly increase on ABZSO availability
was observed both in rats and sheep.
The existence of a synergistic effect of metabolic enzymes and
efflux transporters at the intestinal level has been proposed
(Suzuki & Sugiyama, 2000). The CYP system and Pgp may act
synergistically in reducing the bioavailability of their substrates
after oral administration. In the case of ABZ, after being taken up
by the enterocytes, some of the parent drug suffers a CYPmediated (mainly CYP3A or CYP1A) first intestinal oxidative
step. The ABZSO formed may be eliminated from the cell into the
intestinal lumen via Pgp. Drug molecules (ABZ or ABZSO) which
escape intestinal metabolic conversion or efflux, would be
 2008 The Authors. Journal compilation  2008 Blackwell Publishing Ltd

absorbed via the portal vein system. The effect of IVM on either
ABZ metabolism or ABZSO efflux in the intestine after the i.r.
administration of ABZ, may enhance the ABZ ABZSO concentrations reaching the liver. A similar interaction between ABZ
and IVM (both administered orally) has been suggested (Merino
et al., 2003), where enhanced ABZSO concentration was
attributed to a combined effect on metabolism and drug efflux
transporter interactions. Since no significant differences in the
plasma disposition of ABZ metabolites were observed after the i.v.
combined treatment, an interaction at the intestinal level
between ABZ and IVM would be more important after i.r.
administration of ABZ, when intestinal absorption is critical for
the resultant ABZSO systemic availability. Finally, it is likely that
other transporter protein (s) different to Pgp may be involved on
ABZ efflux at the intestinal wall. If this should be the case, any
IVM-mediated efflux competition could result in enhanced ABZ
systemic absorption. Further in vivo in vitro investigation is
necessary to confirm interactions between antiparasitic compounds and cellular transporter proteins in ruminant species.
Some follow up work on the assessment of the potential PD
interaction of IVM and ABZ (clinical efficacy implications) are
currently undergoing in our lab. This type of pharmacologybased evaluation of drug interactions are becoming highly
relevant since drug combinations are now widely used as an
alternative to control resistant helminth parasites in livestock.

ACKNOWLEDGMENTS
This work was partially supported by Fundacion Antorchas,
(Project 14248-127), CONICET (PIP 6489), and Agencia
Nacional de Promocion Cientfica y Tecnica (ANPCyT) (PICT
08-13763), all from Argentina. The authors appreciate the
collaboration of Ing. Solanet (INTA Balcarce) on the animal
phase of the reported trial.

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