Paper 172 Disc

J. vet. Pharmacol. Therap. 22, 27±34, 1999. PHARMACOKINETICS

Bioequivalence of ivermectin formulations in pigs and cattle

A . L IF SC H IT Z * Lifschitz, A., Pis, A., Alvarez, L., Virkel, G., Sanchez, S., Sallovitz, J., Kujanek, R.,
Lanusse, C. Bioequivalence of ivermectin formulations in pigs and cattle. J. vet.
A. PIS*
Pharmacol. Therap. 22, 27±34.
L. ALVAREZ*
G . V IR K E L * The vehicle in which endectocide compounds are formulated plays a relevant
S . SAN CH E Z * role in their absorption kinetics and resultant systemic availability. The
J. S A L LO V IT Z * pharmaceutical bioequivalence and comparative plasma disposition kinetics of
ivermectin (IVM), following the subcutaneous administration of two injectable
R. K U J A N E K { &
formulations to pigs and cattle were investigated using parallel experimental
C. LANUSSE* designs. Sixteen parasite-free male Duroc Jersey-Yorkshire crossbred pigs (90±
110 kg) (Expt 1) and 16 parasite-free male Holstein calves (100±120 kg) (Expt
*Laboratorio de FarmacologõÂa, Departamento
2) were divided into two groups and treated subcutaneously at either 300 (pigs)
de FisiopatologõÂa, Facultad de Ciencias
or 200 (calves) mg/kg with two different propylene glycol/glycerol formal (60:
Veterinarias, Universidad Nacional del
40) based IVM formulations; in both experiments pigs or calves in Group A
Centro, Campus Universitario, 7000 Tandil,
Argentina; {Bayer Argentina, S.A., 1605 received the test (IVM-TEST) formulation and those in Group B were treated
Munro, Buenos Aires, Argentina with the reference formulation (IVM-CONTROL). Heparinized blood samples
Part of the data included in this article was were taken from 0 h up to either 20 (pigs) or 30 (calves) days post-treatment
Ahed
presented at the 7th International Congress of and plasma was extracted, derivatized and analysed by high performance liquid
Bhed
Ched
the European Association for Veterinary chromatography (HPLC) using fluorescence detection. Early detection of IVM
Pharmacology and Toxicology held in Madrid, (12 h) with a peak plasma concentration (Cmax) between 33 and 39 ng/mL was
Dhed
Spain, July 1997. observed in pigs. The drug was detected in plasma up to 20 days post-
Ref marker
Fig marker administration of either formulation, resulting in elimination half-lives between
Table mar- 3.47 and 3.80 days. There were no differences between the IVM-TEST and
ker IVM-CONTROL formulations in the kinetic parameters (except tmax) obtained in
Ref end pigs. IVM was detected in plasma between 12 h and 30 days post-
Ref start administration of both formulations under investigation in cattle. The plasma
disposition kinetics of IVM in calves was similar following treatment with both
formulations. Cmax values (between 40.5 and 46.4 ng/mL) were achieved at 2
days post-administration of both formulations. None of the estimated kinetic
parameters were statistically different between drug formulations. The
injectable IVM formulations investigated were bioequivalent after their sub-
cutaneous administration to both pigs and calves at recommended dose rates.
(Paper received 27 March 1998; accepted for publication 12 August 1998)
Dr Carlos E. Lanusse, Area FarmacologõÂa, Departamento de FisiopatologõÂa, Facultad
de Ciencias Veterinarias, Universidad Nacional del Centro, Campus Universitario
(7000) Tandil, Argentina. (E-mail: clanusse@vet.unicen.edu.ar)

INTRODUCTION
Ivermectin (IVM) exhibits a broad-spectrum of activity against
gastrointestinal and lung nematodes (Egerton et al., 1981) as
well as against external parasites of domestic animals (Campbell
et al., 1983). Since 1981, IVM has been released in over 60
countries for use in cattle, sheep, goats, horses, swine, dogs, cats,
camels, reindeer, bison and humans (Shoop et al., 1995). IVM is
commercially available as injectable and pour-on formulations
for use in cattle. Injectable and premix formulations are also
available for pigs. IVM is highly effective against the adult,
developing and hypobiotic larvae of most gastrointestinal
nematodes, lungworms and many arthropods in cattle (McKellar
& Benchaoui, 1996). A high degree of efficacy of IVM against the
majority of adult and larval forms of gastrointestinal parasites in
pigs has been demonstrated (Benz et al., 1989).
Ivermectin, a semisynthetic derivative of the avermectin
family, is the generic name given to a mixture of two
chemically modified avermectins containing at least 80% of
22±23 dihydroavermectin B1a and less than 20% 22±23

#1999 Blackwell Science Ltd 27


28 A. Lifschitz et al.
dihydroavermectin B1b (Fisher & Mrozik, 1989). IVM is a large
and highly lipophilic molecule that dissolves in most organic
solvents. It is relatively insoluble in water despite possessing
two sugar rings containing two hydroxyl groups (Jackson,
1989). Owing to its high lipophilicity, IVM distributes largely
to, and is eliminated slowly from, the body compartments
(McKellar & Benchaoui, 1996; Lanusse et al., 1997). The
plasma pharmacokinetic be-haviour of the drug differs accord-
ing to the route of administration, formulation and animal
species (Fink & Porras, 1989).
The close relationship between disposition kinetics and clinical
efficacy for antiparasitic compounds is now well documented
(Lanusse & Prichard, 1993; Baggot & McKellar, 1994). The
characterization of the plasma bioavailability and disposition
kinetics of IVM can be used to predict and optimize its ecto-
endoparasiticide efficacy. The vehicle in which the endectocide
compounds are formulated may play a relevant role in their
absorption kinetics and plasma availability (Lo et al., 1985;
Lanusse et al., 1997). Small differences in formulation and in the
resultant kinetic behaviour of IVM may account for important
changes to its efficacy against the most difficult to control
internal and external parasites in livestock. Pharmaceutical
technology has been applied to develop different drug formula-
tions and delivery systems to optimize the pharmacological
potency of IVM and other endectocide molecules currently
available. Alternative IVM formulations for use in several
different species have been introduced to the market or are
under development since the expiration of the original patent for
the first approved IVM formulation (Ivomec1, MSD AgVet).
The goal of the studies reported here was to evaluate the
bioequivalence and comparative disposition of two injectable
formulations of IVM administered subcutaneously to pigs and
cattle, using parallel experimental designs. The results reported
in this article contribute to further understanding of the plasma
disposition kinetics of IVM in both species. The outcome of these
trials ratifies the usefulness of the parallel design to evaluate
pharmaceutical bioequivalence of veterinary drugs when cross-
over designs are not recommended, such as in the case of drugs
with prolonged half-lives (Toutain & Koritz, 1997).
MATERIALS AND METHODS
Experimental design
The bioequivalence of two IVM preparations, formulated in a
propylene glycol/glycerol formal (60: 40) vehicle for subcutaneous
administration, was evaluated in pigs (Expt 1) and calves (Expt 2).
Experiment 1: Ivermectin bioequivalence in pigs
Animals: The trial was conducted in 16 parasite-free male Duroc
Jersey-Yorkshire crossbred pigs weighing 90±110 kg. The health
of the animals was monitored prior to and throughout the
experimental period. Animals were housed in individual pens
and fed a commercially produced 35% protein/grain (3: 1)
Paper 172 Disc
concentrate diet. Experimental animals had free access to water.
The experiments were conducted following internationally
accepted animal welfare guidelines.
Treatments: Pigs were randomly allocated into two groups of
eight animals each. Treatments were given as follows:
Group A: experimental animals were treated with the
ivermectin test formulation (10 mg/mL) (IVM-TEST) provided
by Bayer Argentina S.A. (Buenos Aires, Argentina) (Batch
LDB601). The treatment was given by subcutaneous injection in
the neck immediately behind the ear at 300 mg/kg body weight.
This formulation was considered as the test product in the
current bioequivalence trial.
Group B: experimental animals were treated with a commer-
cially available formulation of ivermectin (Ivomec1, MSD Agvet,
NJ, USA) (Batch A0352Z) by subcutaneous injection at 300 mg/
kg body weight in the neck immediately behind the ear. This
preparation was considered as the reference (control) product in
the trial (IVM-CONTROL).
Experiment 2: Ivermectin bioequivalence in cattle
Animals: The study was conducted in 16 parasite-free male
Holstein calves weighing 100±120 kg. The health of the animals
was monitored prior to and throughout the experimental period.
Animals were fed ad libitum with lucerne hay and concentrate
ration. They had free access to water.
Treatments: Calves were randomly allocated into two groups of
eight animals each. Treatments were given as follows:
Group A: experimental animals were treated with the
ivermectin test formulation (10 mg/mL) (IVM-TEST) provided
by Bayer Argentina S.A (Batch LDB601). The treatment was
given by subcutaneous injection in the shoulder area at 200 mg/
kg body weight. This formulation was considered as the test
product in the bioequivalence trial.
Group B: experimental animals were treated with a commer-
cially available formulation of ivermectin (Ivomec1, MSD Agvet)
(Batch A0352Z) by subcutaneous injection in the shoulder area
at 200 mg/kg body weight. This preparation was considered as
the reference (control) product in the trial (IVM-CONTROL).
Sampling: Heparinized blood samples were taken in both
experiments at 0, 12, 24 h and at 2, 3, 4, 5, 7, 9, 11, 15 and
20 days post-treatment; two extra samples on days 25 and 30
post-treatment were obtained from treated calves. Blood samples
were centrifuged at 2000 g for 20 min and the recovered plasma
was kept in labelled vials at ±20 8C until analysed. The samples
were analysed within 30 days of their collection.
Analytical procedures
Chemical extraction and derivatization
The extraction of IVM from spiked and experimental samples was
carried out following adaptations of the technique described by
De Montigny et al. (1990) and Alvinerie et al. (1993, 1995). The
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34

adaptation introduced to that methodology included the use of
abamectin as internal standard to quantify IVM plasma
concentrations. Basically, 1 mL-aliquot of plasma sample was
combined with 100 mL of internal standard (abamectin) (100
ng/mL) and then mixed with 1 mL of acetonitrile-water (80: 20).
After mixing for 20 min, the solvent-sample mixture was
centrifuged at 2000 g during 15 min. The supernatant was
injected to a Supelclean LC18 cartridge (Supelco, Bellfonte, PA,
USA) previously conditioned by passing 2 mL of methanol and 2
mL deionized water. The cartridge was flushed with 2 mL of
water/methanol (3:1). The analytes were eluted with 1 mL of
methanol and concentrated to dryness under a stream of
nitrogen. The reconstitution was done using 100 mL of a
solution of N-methylimidazole (Sigma Chemical, St Louis, MO,
USA) in acetonitrile (1: 1). Derivatization was initiated adding
150 mL of trifluoroacetic anhydride (Sigma Chemical, St Louis,
MO, USA) solution in acetonitrile (1: 2). A fluorescent IVM-
derivative is formed by acetylation and dehydration reactions
when the drug reacts with trifluoroacetic anhydride in the
presence of N-methylimidazole (De Montigny et al., 1990). After
completion of the reaction (5 30 s), an aliquot (100 mL) of this
solution was injected directly into a high performance liquid
chromatograph (HPLC).
Drug analysis
IVM concentrations were determined by HPLC using a Shimadzu
10 A HPLC system (Shimadzu Corporation, Kyoto, Japan). HPLC
analysis was undertaken using a reverse phase C18 column
(Supelco, 3 mm, 4.6 mm 6 150 mm) and an acetonitrile/
methanol/water (60/35/5) mobile phase at a flow rate of 1.5
mL/min. Fluorescence detection was conducted with a Shimadzu
RF-10 Spectrofluorometric detector (Kyoto, Japan) reading at an
excitation wavelength of 365 nm and an emission wavelength of
475 nm. 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
486 computer. The IVM/abamectin peak area ratio was obtained
to estimate IVM concentrations in spiked (validation) and
experimental samples. There was no interference of endogenous
compounds in the chromatographic determinations. The sol-
vents (Baker, Phillipsburg, NJ, USA) used during the extraction
and drug analysis were HPLC grade.
Validation procedures
A complete validation of the analytical procedures used for
extraction and quantification of IVM was performed before
analysis of experimental samples from the bioequivalence trials.
IVM (Batch # 95051 2BER01, purity 97.5%) and abamectin
(Batch 9505 25BER01, purity 97.4%) pure reference standards
provided by Bayer Argentina S.A., were used to prepare
calibration curves in the range between 0.25 and 10 ng/mL
and 10±100 ng/mL in pig and calf plasma, respectively. The
validation of the analytical method included the determination of
the linearity of the detector responses, recovery percentages and
limits of detection and quantification.
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34
Paper 172 Disc
Ivermectin in pigs and cattle 29
The analyte was identified with the retention times of IVM
pure reference standards. The linearity was determined by
injection of spiked IVM standards in plasma at different
concentrations (triplicate determinations). Calibration curves
were constructed using the least squares linear regression
analysis and correlation coefficients (r) and coefficient of
variations (CV) were calculated. Drug recovery was established
by comparison of the detector responses (peak areas) obtained
from IVM fortified plasma samples and those of the direct
standards prepared in methanol carried through the derivatiza-
tion process, using abamectin as internal standard. Percentages
of IVM recovery from plasma samples were obtained in the range
between 0.25 and 50 ng/mL. The mean percentage of recovery
and the coefficient of variation (CV) were calculated. The CV was
obtained as the ratio between the standard deviations and the
mean recovery values (Bolton, 1984) of at least three
determinations. The limit of drug detection was established with
injection of plasma blanks fortified with the internal standard
and measuring the baseline noise at the retention time of the
IVM peak. The mean baseline noise at the IVM retention time
plus three standard deviations was defined as the detection limit.
The mean baseline noise plus 10 standard deviations was defined
as the theoretical quantification limit.
Pharmacokinetic and statistical analyses of the data
The plasma concentration vs. time curves obtained after each
treatment in each individual animal were fitted with the
PKCALC computer program (Shumaker, 1986) coupled to an
augmented copy of the stripping program ESTRIP (Brown &
Manno, 1978). Pharmacokinetic parameters were determined
using a model-independent method (Notari, 1987). The peak
concentration (Cmax) and time to peak concentration (tmax) were
read from the plotted concentration±time curve in each
individual animal. The area under the concentration±time
curves (AUC) were calculated by the trapezoidal rule (Gibaldi &
Perrier, 1982) and further extrapolated to infinity by dividing
the last experimental concentration by the terminal slope (b).
Statistical moment theory was applied to calculate the mean
plasma residence time (MRT) for IVM, as follows:
AUMC
MRT ˆ
AUC
where AUC is as defined previously and AUMC is the area under
the curve of the product of time and drug concentration vs. time
from zero to infinity (Gibaldi & Perrier, 1982).
The pharmacokinetic parameters are reported as mean + SEM.
Mean pharmacokinetic parameters for IVM obtained after the
administration of both formulations in both species were
statistically compared by analysis of variance (ANOVA) using
Satistical Analysis Software (SAS). The normal distribution of the
data was confirmed using the Bartlett test. The kinetic
parameters used for the bioequivalence comparison between
formulations (Cmax, AUC) were compared by ANOVA after log
transformation (Steinijans et al., 1992). Mean values were
considered significantly different at P 5 0.05.

30 A. Lifschitz et al.
RESULTS
The analytical procedures including chemical extraction, deri-
vatization and HPLC analysis of IVM were validated adequately.
The linear regression lines for IVM in plasma in the range 0.25±
10 ng/mL and 10±100 ng/mL showed high correlation
coefficients for both pigs (from 0.994 to 0.999) and cattle (from
0.998 to 0.999), respectively.
The mean recovery of IVM from pigs and cattle plasma was
87.1 and 93.2%, respectively. The limits of detection of the
analytical techniques were 0.17 ng/mL (pigs) and 0.05 ng/mL
(cattle). The theoretical quantification limits of IVM in plasma
were 0.25 ng/mL (pigs) and 0.10 ng/mL (cattle). The results of
the validation of the analytical technique to determine IVM in
plasma obtained from pigs and calves are summarized in Table 1.
Ivermectin was detected in plasma between 12 h and 20 days
post-administration of either formulation in pigs. The compara-
tive mean plasma concentration of IVM obtained after the
subcutaneous administration of the two formulations to pigs are
shown in Fig. 1. The mean pharmacokinetic parameters for IVM
obtained after the administration of IVM-TEST and IVM-
CONTROL formulations to pigs are compared in Table 2.
Table 1. Validation of the analytical methodology used to determine
ivermectin concentrations in pig and calf plasma
Pig Calf
Limit of detection (ng/mL) 0.17 0.05
Limit of quantification (ng/mL) 0.25 0.10
Recovery (%) 87.1 (7.02) 93.2 (3.35)
Linearity (r)
(0.25±10 ng/mL) 0.994 0.999
(10±100 ng/mL) 0.999 0.998
Coefficient of variation (%) 7.63 3.58
The validation results were obtained as defined in the analytical
methodology section. Values in brackets represent coefficient of variation
(CV) for the recovery assays. r: coefficient of correlation obtained from
the linear regression lines in the range between 0.25 and 10 ng/mL and
10±100 ng/mL, respectively.
Fig. 1. Mean plasma (+ SEM) concentrations of ivermectin (IVM)
obtained after subcutaneous administration of the IVM-CONTROL (±.±)
and IVM-TEST (±!±) formulations (300 mg/kg) to pigs.
Paper 172 Disc
Table 2. Comparison of the plasma kinetic parameters for ivermectin
obtained after subcutaneous administration of the IVM-TEST and IVM-
CONTROL formulations to pigs at 300 mg/kg
FORMULATION
Kinetic Parameters IVM-TEST IVM-CONTROL P-value
Cmax (ng/mL) 33.3 + 4.10 39.6 + 3.84 0.290
tmax (days) 2.75 + 0.43 0.94 + 0.16 0.002
AUC0±20d (ng.d/mL) 162 + 22.6 127 + 6.89 0.282
AUCtotal (ng.d/mL) 165 + 21.6 132 + 7.23 0.241
AUMCtotal (ng.d2/mL) 995 + 108 763 + 42 0.229
t‰el (days) 3.47 + 0.50 3.80 + 0.19 0.324
MRT (days) 6.29 + 0.63 5.78 + 0.18 0.481
Values are expressed as mean + SEM (n = 8).
Cmax: peak plasma concentration; tmax: time to peak plasma concentra-
tion; AUC0±20d: area under the concentration vs. time curve between
drug administration and 20 days post-treatment; AUCtotal: area under
the concentration vs. time curve extrapolated to infinity; AUMCtotal: area
under the first moment of the concentration vs. time curve extrapolated
to infinity; t‰el: elimination half-life; MRT: mean residence time.
The AUC0±20 days, AUCtotal, AUMC, Cmax, MRT and t‰el
obtained for IVM-TEST and IVM-CONTROL formulations in pigs
were not statistically different. The peak plasma concentration
for the IVM-TEST formulation was achieved significantly later
(P 5 0.002) than that of the IVM-CONTROL formulation. In
fact, the only difference observed between experimental groups
was that after the administration of the IVM-TEST formulation,
the disposition of IVM was characterized by a slightly higher
plasma concentration profile from day 2 to day 7 post-treatment,
resulting in a mean Cmax value of 33.3 + 4.10 ng/mL achieved
at 2.75d post-administration. The highest plasma concentration
was achieved between 12 h and 2 days post-treatment
(Cmax = 39.6 + 3.84 ng/mL; tmax = 0.94d h) following admin-
istration of the IVM-CONTROL.
Thereafter, the plasma profiles of IVM declined, following a
very similar pattern, to a mean concentration between
0.62 + 0.16 ng/mL (IVM-TEST) and 0.69 + 0.10 ng/mL
(IVM-CONTROL) at 20 days post-treatment. IVM elimination
half-lives (3.47 and 3.80 days) and mean residence times (6.29
and 5.78 days) were similar between both formulations.
Ivermectin was detected in plasma between 12 h and 30 days
post-administration of both formulations under investigation to
cattle. The comparative mean plasma concentrations of IVM
obtained after the subcutaneous administration of the two
formulations to cattle are shown in Fig. 2. Mean pharmacoki-
netic parameters for IVM obtained after the administration of
IVM-TEST and IVM-CONTROL formulations to cattle are
compared in Table 3. The overall disposition kinetics of IVM
was similar following treatment with both formulations. None of
the kinetic parameters calculated were statistically different
between drug formulations. The comparison of the mean Cmax
and AUC values obtained for IVM following its subcutaneous
administration to pigs and calves at recommended dose rates, is
shown in Fig. 3.
The statistical comparison (ANOVA), after log transformation of
the data, for AUC and Cmax and the bioequivalence range
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34
 Paper 172 Disc

Ivermectin in pigs and cattle 31

Fig. 2. Mean plasma (+ SEM) concentrations of ivermectin (IVM)

Fig. 3. Comparative ivermectin peak plasma concentrations (Cmax) and
obtained after subcutaneous administration of the IVM-CONTROL (±.±)
area under the plasma concentration vs. time curves (AUC) obtained
and IVM-TEST (±!±) formulations (200 mg/kg) to calves.
after its subcutaneous administration (IVM-CONTROL formulation) to
pigs (300 mg/kg) ( ) and calves (200 mg/kg) ( ). Values express
means with n = 8. These dose-dependent parameters were statistically
different between species (P 5 0.001) when they were normalised by the
administered dose rate.

Table 3. Comparison of the plasma kinetic parameters obtained for

ivermectin after subcutaneous administration of the IVM-TEST and IVM-
CONTROL formulations to calves at 200 mg/kg
obtained for IVM-TEST and IVM-CONTROL after their subcuta-
FORMULATION neous administration to pigs and cattle are shown in Table 4.
Kinetic Parameters IVM-TEST IVM-CONTROL P-value

Cmax (ng/mL) 40.5 + 2.72 46.4 + 3.88 0.239

tmax (days) 2.00 + 0.50 2.12 + 0.14 0.590 DISCUSSION
AUC0±30d (ng.d/mL) 239 + 19.6 263 + 23.0 0.541
AUCtotal (ng.d/mL) 244 + 20.1 266 + 23.3 0.492 Understanding the pharmacokinetic behaviour of broad-spec-
AUMCtotal (ng.d2/mL) 1530 + 244 1452 + 181 0.802 trum antiparasitic drugs and the factors modulating that
t‰el (days) 6.30 + 0.81 5.50 + 0.39 0.359 behaviour is highly important for maximizing their clinical
MRT (days) 6.15 + 0.48 5.39 + 0.25 0.211
efficacy (Lanusse & Prichard, 1993). The pharmacokinetic
Values are expressed as mean + SEM (n = 8). Cmax: peak plasma behaviour of IVM in pigs has not been extensively investigated.
concentration; tmax: time to peak plasma concentration; AUC 0±30d: area The available information on the plasma profiles of IVM in pigs
under the concentration vs. time curve between drug administration and (Fink & Porras, 1989; Scott & McKellar, 1992) was obtained
30 days post-treatment; AUCtotal: area under the concentration vs. time
under experimental conditions different from those used in the
curve extrapolated to infinity; AUMCtotal: area under the first moment of
the concentration vs. time curve extrapolated to infinity; t‰el: elimina-
current trial. Thus, besides the characterization of the bioequi-
tion half-life; MRT: mean residence time. valence of both formulations under study, the results of the
current trial may contribute to the further understanding of the
plasma disposition kinetics of this endectocide molecule in pigs.
In agreement with a previous report (Scott & McKellar, 1992),
Table 4. Bioequivalence between IVM-
Kinetic Log Transformed Value Ratio* P-value
TEST and IVM-CONTROL formulations
Parameters IVM-TEST IVM-CONTROL mT/ mR

Cmax (ng/mL) 1.50 1.58 0.95 0.276

PIGS AUC0±20d (ng.d/mL) 2.18 2.10 1.04 0.246
AUCtotal (ng.d/mL) 2.19 2.11 1.04 0.284
Cmax (ng/mL) 1.60 1.66 0.96 0.256
CATTLE AUC0±30d (ng.d/mL) 2.37 2.41 0.98 0.547
AUCtotal (ng.d/mL) 2.38 2.41 0.99 0.510

(*) mT and mR represent the values of the kinetic parameter for the test and reference (control)
formulations, respectively, in pigs and cattle. Two pharmaceutical formulations are bioequivalent when
the ratio mT/mR for Cmax and AUC between test and reference formulations result between 0.80 and 1.25
(Steinijans et al., 1992).

#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34


32 A. Lifschitz et al.
there were individual variations in the plasma profile of IVM in
the present experiment. The mean Cmax of both formulations in
our trial (between 33.3 and 39.6 ng/mL) was slightly greater
compared to that reported by those authors (28.4 ng/mL); the
tmax obtained for the IVM-CONTROL formulation was similar.
The mean IVM AUC values obtained after the administration of
both IVM-TEST and IVM-CONTROL formulations in the current
trial were greater than those reported by those authors, which
may be associated with the longer sampling period used in our
trial. The mean elimination half-lives of IVM after the admin-
istration of the two formulations were 3.47 and 3.80 days,
suggesting that the drug is cleared slowly from pigs, being
detectable in plasma for up to 20 days post-treatment.
The use of different experimental conditions may be one of
the key factors underlying the reported differences on kinetic
behaviour. In the current trial, the disposition of IVM in pigs
was characterized with a sampling period that lasted for 20
days post-treatment, which is longer than in the above
referenced studies. The sensitivity of the analytical methodol-
ogy used to quantify IVM permitted the detection of the drug in
plasma of treated animals over 20 days post-treatment.
Additionally, the pigs used in the current trial were heavier
(90±110 kg) than those used by Scott & McKellar (1992) (18±
26 kg), which may have some influence in the pharmacokinetic
behaviour of this lipophilic molecule. A greater proportion of fat
in body composition may facilitate longer IVM persistence in
the body, accounting for changes in the drug disposition.
Differences in the bioavailability and half-life of IVM have been
reported following oral and subcutaneous administration to
pigs (Fink & Porras, 1989). Administration by the parenteral
route produced slower drug absorption but an overall higher
drug availability in plasma.
As demonstrated by Lo et al. (1985), Wicks et al. (1993) and
Lanusse et al. (1997), the absorption rate of endectocide
molecules in cattle is markedly influenced by their pharmaceu-
tical preparations. Following parenteral administration, the low
solubility of IVM in water and its deposition in subcutaneous
tissue favour a slow absorption from the injection site providing
a prolonged residence in the bloodstream. In the current trial,
the slow absorption of IVM resulted in a delayed tmax (between 2
and 2.12 days) after subcutaneous administration of IVM-TEST
and IVM-CONTROL to calves. The clinical efficacy of an
antiparasitic compound relies on its disposition kinetics and
pattern of plasma/tissue exchange in the host (Lanusse &
Prichard, 1993). IVM is a highly lipophilic molecule that is
extensively distributed into tissues (Bogan & McKellar, 1988;
Chiu et al., 1990). The distribution into adipose tissue is
particularly relevant and may act as a drug reservoir, that
contributes to the long residence of the drug in the bloodstream.
The mean plasma elimination half-life of IVM obtained in this
trial after the administration of the two formulations (5.50 and
6.30 days) reflects the long persistence of IVM in the body. The
IVM concentration profiles in plasma reflect those achieved in
different tissues, including those where target parasites are
located. The disposition kinetic results obtained for IVM in cattle
in the current trial were similar to those previously obtained in
Paper 172 Disc
our laboratory (Lifschitz et al., 1997) using calves of the same
breed, age and weight. It is important to note that higher IVM
plasma AUC values have been reported, using different cattle
breeds and much longer sampling periods (Lanusse et al., 1997).
In the current trial to evaluate the pharmaceutical bioequiva-
lence of the two formulations, the sampling period was extended
up to 30 days post-treatment, the period considered relevant to
correlate IVM plasma concentration with the ecto-endoparasiti-
cide activity of the drug.
The comparison of the IVM disposition kinetics in both species
was interesting. When the drug was given by subcutaneous
injection at recommended dose rates, the Cmax and AUC values
obtained were higher in calves than in pigs (Fig. 3). Those dose-
dependent parameters were statistically different (P 5 0.001)
between species, after their normalization by the administered
dose rate. Differences in body composition may account for
changes in the pattern of IVM tissue distribution. A more
extensive distribution and deposit of IVM in adipose tissue in pigs
compared to cattle, may account for the lower plasma
availability obtained in this species.
To minimize the different variables which may affect drug
plasma concentrations, bioequivalence studies should be
conducted in healthy animals that are as homogeneous as
possible with respect to age, weight and sex. This is
particularly important for studies conducted as parallel design
(Toutain & Koritz, 1997). Bioequivalence of different formula-
tions of the same active molecule comprises equivalence with
respect to the rate and extent of drug absorption (Steinijans &
Hauschke, 1990). The basic assumption underlying this
definition is that equivalent rates and extent of absorption
will lead to essentially the same drug plasma concentration±
time profiles and therefore, to the same therapeutic or toxic
effects (Toutain & Koritz, 1997).
The area under the concentration curve (AUC) is of interest as
it reflects the extent to which the active drug is absorbed and is
independent of the rate of the absorption process. The
pharmacokinetic parameters used to reflect the rate of drug
absorption are the maximum plasma concentration (Cmax) and
the time at which that occurs following drug administration
(tmax) (Toutain & Koritz, 1997). The lack of statistical differences
between test and reference formulations in these three variables
is sufficient to support bioequivalence. However, if the AUC and
Cmax values for test and reference formulations were not
statistically different, the plasma concentrations profiles are very
likely to be similar. The evaluation of the tmax parameter is less
important and less reliable (Gettinby, 1994). This may be due to
the fact that measurements are restricted to the sampled time
points, so the data are less accurate and more variable (Gettinby,
1994). This is in agreement with the Food and Drug Adminis-
tration (1996), which has established the Cmax and AUC as the
pivotal kinetic parameters to determine bioequivalence. These
kinetic parameters were not significantly different between the
IVM-TEST and IVM-CONTROL formulations in either species in
the current trials.
The present bioequivalence study was carried out using a
parallel design. In this type of design, the two groups of animals
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34

are treated in parallel, whereas in the cross-over design each
formulation is administered to the same animals after a washout
period. The washout period is usually set at least 10 times the
terminal half-life of the active drug (Food & Drug Administration,
1996; Toutain & Koritz, 1997). Given the long residence time of
IVM in adipose tissue, an extremely long washout period would
have been necessary to run a cross-over design in the current
trials. Animal growth and age differences between the first and
second experimental phases would have worked against
standardized experimental conditions to evaluate IVM bioequi-
valence between formulations.
For determining whether two pharmaceutical formulations
are bioequivalent, regulatory agencies have set a statistical
criterion. The criterion specifies that the mean AUC and Cmax
values of the test formulation should not be more than 20%
different from the corresponding mean values of the reference
formulation (Steinijans et al., 1992). The Guidelines on
Bioequivalence from the European Committee for Proprietary
Medicinal Products specifies that the bioequivalence range,
obtained as the ratio between the mean AUC and Cmax values,
for the test and reference formulations, should fall between 0.80
and 1.25 (Committee for Proprietary Medicinal Products, 1991).
This can be expressed as 0.80 5 mT/mR 5 1.25, where mT and mR
denote the pharma-cokinetic parameters for test and reference
formulations, respectively. In order to reach harmonization, the
FDA criterion has been changed from (0.80, 1.20) to (0.80,
1.25) (Steinijans et al., 1992).
The logarithmic transformation of the AUC and Cmax values is
recommended (Steinijans et al., 1992). The use of logarithmic
transformation normalizes the distribution of the parameter,
stabilizes its variance and ensures the additivity of the statistical
model (Steinijans & Hauschke, 1990; Toutain & Koritz, 1997).
The ratio mT/mR for Cmax and AUC for the formulations evaluated
in the current trial were between 0.80 and 1.25, which confirms
the lack of statistical differences between both formulations in
both species (Table 4).
In conclusion, the results reported in this article contribute to
the further understanding of the plasma disposition kinetics of
IVM in pigs and cattle. The usefulness of the parallel design to
evaluate pharmaceutical bioequivalence of veterinary drugs
with prolonged half-lives was ratified. The pivotal bioequivalence
parameters (Cmax and AUC) obtained after administration of the
IVM-TEST and IVM-CONTROL formulations in either species
were not statistically different. Thus, the IVM formulations
investigated were bioequivalent after their subcutaneous admin-
istration to pigs and cattle at recommended dose rates.
ACKNOWLEDGMENTS
The technical advice of Dr Michel Alvinerie (INRA, Toulouse,
France) for the development of the analytical techniques is
gratefully acknowledged. Adrian Lifschitz is a recipient of a
fellowship from the Consejo Nacional de Investigaciones
CientõÂ ficas y TeÂcnicas (CONICET), Argentina. The financial
support received from Bayer Argentina SA is acknowledged.
#1999 Blackwell Science Ltd, J. vet. Pharmacol. Therap. 22, 27±34
Paper 172 Disc
Ivermectin in pigs and cattle 33
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